Seroprevalence of Scrub Typhus, Typhus, and Spotted Fever Among Rural and Urban Populations of Northern Vietnam.

AbstractRickettsial infections are recognized as important causes of fever throughout southeast Asia. Herein, we determined the seroprevalence to rickettsioses within rural and urban populations of northern Vietnam. Prevalence of individuals with evidence of prior rickettsial infections (IgG positive) was surprisingly low, with 9.14% (83/908) testing positive to the three major rickettsial serogroups thought to circulate in the region. Prevalence of typhus group rickettsiae (TG)-specific antibodies (6.5%, 58/908) was significantly greater than scrub typhus group orientiae (STG)- or spotted fever group rickettsiae (SFG)-specific antibodies (P < 0.05). The majority of TG seropositives were observed among urban rather than rural residents (P < 0.05). In contrast, overall antibody prevalence to STG and SFG were both very low (1.1%, 10/908 for STG; 1.7%, 15/908 for SFG), with no significant differences between rural and urban residents. These results provide data on baseline population characteristics that may help inform development of Rickettsia serological testing criteria in future clinical studies.

Genotypic comparison of five isolates of Rickettsia prowazekii by multilocus sequence typing.

Genetic traits of five Rickettsia prowazekii isolates, including the first from Africa and North America, and representatives from human and flying squirrels were compared using multilocus sequence typing. Four rickettsial genes encoding 17 kDa genus-common antigen (17 kDa gene), citrate synthase (gltA), OmpB immunodominant antigen (ompB) and 120 kDa cytoplasmic antigen (sca4) were examined. Sequence identities of 17 kDa gene and gltA were 100% among the isolates. Limited sequence diversity of ompB (0.02-0.11%) and sca4 (0.03-0.20%) was enough to distinguish the isolates, and evaluation of the combined four genes provided a method to easily differentiate R. prowazekii from other rickettsiae.

Insight into the virulence of Rickettsia prowazekii by proteomic analysis and comparison with an avirulent strain.

Rickettsia prowazekii, an obligate intracellular Gram-negative bacterium, is the etiologic agent of epidemic typhus. We analyzed the proteome of the virulent Breinl strain of R. prowazekii purified from infected egg yolk sacs. Total proteins from purified R. prowazekii Breinl strain were reduced by dithiothreitol, alkylated by iodoacetic acid and digested with trypsin followed by analysis with an integrated two-dimensional liquid chromatography and mass spectrometry system (2D-LC/MS/MS). A comparison was made using previously analyzed proteome of the Madrid E strain and current analysis of the Breinl strain. For Breinl 251 proteins were identified, representing 30% of the total protein-encoding genes, using a shotgun 2D-LC/MS/MS proteomic approach. This result is identical to that of Madrid E strain. Among the identified proteins, 33 from Breinl and 37 from Madrid E have an unknown function. A methyltransferase, RP028/RP027, whose gene is mutated in the avirulent Madrid E strain but not in the virulent Breinl strain, was only detectable in the Breinl strain, consistent with the genetic mutation in Madrid E. This result suggests the possible relationship between this gene product and the virulence of the strains.

Evidence for louse-transmitted diseases in soldiers of Napoleon's Grand Army in Vilnius.

BACKGROUND: Many soldiers in Napoleon's Grand Army died of infectious diseases during its retreat from Russia. Because soldiers were commonly infested with body lice, it has been speculated that louse-borne infectious diseases, such as epidemic typhus (caused by Rickettsia prowazekii), were common. METHODS: We investigated this possibility during recent excavations of a mass grave of Napoleon's soldiers in Vilnius, Lithuania. Segments of 5 body lice, identified morphologically and by polymerase chain reaction (PCR) amplification and sequencing, were found in earth from the grave that also contained fragments of soldiers' uniforms. RESULTS: DNA of Bartonella quintana (the agent of trench fever) was identified by PCR and sequencing in 3 of the lice. Similarly, PCR and sequencing of dental pulp from the remains of 35 soldiers revealed DNA of B. quintana in 7 soldiers and DNA of R. prowazekii in 3 other soldiers. CONCLUSIONS: Our results show that louse-borne infectious diseases affected nearly one-third of Napoleon's soldiers buried in Vilnius and indicate that these diseases might have been a major factor in the French retreat from Russia.

Multispacer typing of Rickettsia prowazekii enabling epidemiological studies of epidemic typhus.

Currently, there is no tool for typing Rickettsia prowazekii, the causative agent of epidemic typhus, currently considered a potential bioterrorism agent, at the strain level. To test if the multispacer typing (MST) method could differentiate strains of R. prowazekii, we amplified and sequenced the 25 most variable intergenic spacers between the R. prowazekii and R. conorii genomes in five strains and 10 body louse amplicons of R. prowazekii from various geographic origins. Two intergenic spacers, i.e., rpmE/tRNA(fMet) and serS/virB4, were variable among tested R. prowazekii isolates and allowed identification of three and two genotypes, respectively. When the genotypes obtained from the two spacers were combined, we identified four different genotypes. MST demonstrated that several R. prowazekii strains circulated in human body lice during an outbreak of epidemic typhus in Burundi. This may help to discriminate between natural and intentional outbreaks. Our study supports the usefulness of MST as a versatile method for rickettsial strain genotyping.

AutoFACT: an automatic functional annotation and classification tool.

BACKGROUND: Assignment of function to new molecular sequence data is an essential step in genomics projects. The usual process involves similarity searches of a given sequence against one or more databases, an arduous process for large datasets. RESULTS: We present AutoFACT, a fully automated and customizable annotation tool that assigns biologically informative functions to a sequence. Key features of this tool are that it (1) analyzes nucleotide and protein sequence data; (2) determines the most informative functional description by combining multiple BLAST reports from several user-selected databases; (3) assigns putative metabolic pathways, functional classes, enzyme classes, GeneOntology terms and locus names; and (4) generates output in HTML, text and GFF formats for the user's convenience. We have compared AutoFACT to four well-established annotation pipelines. The error rate of functional annotation is estimated to be only between 1-2%. Comparison of AutoFACT to the traditional top-BLAST-hit annotation method shows that our procedure increases the number of functionally informative annotations by approximately 50%. CONCLUSION: AutoFACT will serve as a useful annotation tool for smaller sequencing groups lacking dedicated bioinformatics staff. It is implemented in PERL and runs on LINUX/UNIX platforms. AutoFACT is available at http://megasun.bch.umontreal.ca/Software/AutoFACT.htm.

Use of highly variable intergenic spacer sequences for multispacer typing of Rickettsia conorii strains.

By use of the nearly perfectly colinear genomes of Rickettsia conorii and Rickettsia prowazekii, we compared the usefulness of three types of sequences for typing of R. conorii isolates: (i) 5 variable coding genes comprising the 16S ribosomal DNA, gltA, ompB, and sca4 (gene D) genes, which are present in both genomes, and the ompA gene, which is degraded in R. prowazekii; (ii) 28 genes degraded in R. conorii but intact in R. prowazekii, including 23 split and 5 remnant genes; and (iii) 27 conserved and 25 variable intergenic spacers. The 4 conserved and 23 split genes as well as the 27 conserved intergenic spacers each had identical sequences in 34 human and 5 tick isolates of R. conorii. Analysis of the ompA sequences identified three genotypes of R. conorii. The variable intergenic spacers were significantly more variable than conserved genes, split genes, remnant genes, and conserved spacers (P < 10(-2) in all cases). Four of the variable intergenic spacers (dksA-xerC, mppA-purC, rpmE-tRNA(fMet), and tRNA(Gly)-tRNA(Tyr)) had highly variable sequences; when they were combined for typing, multispacer typing (MST) identified 27 different genotypes in the 39 R. conorii isolates. Two batches from the same R. conorii strain, Malish (Seven), with different culture passage histories were found to exhibit the same MST type. MST was more discriminatory for strain genotyping than multiple gene sequencing (P < 10(-2)). Phylogenetic analysis based on MST sequences was concordant with the geographic origins of R. conorii isolates. Our study supports the usefulness of MST for strain genotyping. This tool may be useful for tracing a strain and identifying its source during outbreaks, including those resulting from bioterrorism.

Proteome analysis of Madrid E strain of Rickettsia prowazekii.

Rickettsia prowazekii, an obligate intracellular Gram-negative bacterium, is the etiologic agent of epidemic typhus. The threat of typhus as a biological weapon lies in its stability in the dried louse feces and in its infection by inhalation of an aerosol. Consequently, it is listed as a select agent and warrants more research to understand its pathogenesis. Although the genomic DNA sequence of strain Madrid E has been completed, the actual expression of the individual protein has not been investigated. In order to provide a global view of the expressed protein profile, the whole cell lysate of purified rickettsia (Madrid E strain) was reduced, alkylated, and digested with trypsin. The total digest was characterized by a two-dimensional liquid chromatography mass spectrometry system and analyzed with a modified version of the ProteomeX workstation. A total of 252 proteins out of 834 predicted protein-coding genes were identified, 238 proteins were identified by the detection of at least two unique peptides. Only 14 proteins were identified by the detection of one unique peptide in all three separate analyses. Among the 238 proteins identified by multiple unique peptides, 230 proteins were found in at least two of three separate analyses. The reproducible and convenient methodology and the information described here have provided a foundation for future proteome study of various R. prowazekii strains with different virulence.

Real-time PCR duplex assay for Rickettsia prowazekii and Borrelia recurrentis.

Rickettsia prowazekii, the etiologic agent for epidemic typhus, and Borrelia recurrentis, the etiologic agent of relapsing fever, both utilize the same vector, the human body louse (Pediculus humanus), to transmit human disease. We have developed an assay to detect both bacterial pathogens in a single tube utilizing real-time PCR. Assays for both agents are specific. The R. prowazekii and B. recurrentis assays do not detect nucleic acid from R. typhi, R. canada, or any of eight spotted fever rickettsiae. In addition they did not react with Neorickettsia risticii, N. sennetsu, Franciscella persica, Bartonella quintana, Legionella pneumophila, Proteus mirabilis, Salmonella enterica, Escherichia coli, and Staphylococcus aureus. Moreover, the B. recurrentis assay did not detect B. duttonii, B. coriaceae, B. afzelii, B. garinii, B. hermsii, or B. burgdorferi nucleic acid. Both assays detected repeatedly only R. prowazekii or B. recurrentis either when tested alone or together in one test tube.

Isolation and characterization of the dnaA gene of Rickettsia prowazekii.

The dnaA gene encoding the initiator protein of DNA replication was isolated from the obligate intracellular bacterium, Rickettsia prowazekii. Comparison of the deduced amino acid sequence of R. prowazekii DnaA with other bacterial DnaA proteins revealed extensive similarity. However, the rickettsial sequence is unique in the number of basic lysine residues found within a highly conserved portion of the putative DNA binding region, suggesting that the rickettsial protein may recognize a DNA sequence that differs from the consensus DnaA box sequence identified in other bacteria. Consensus DnaA box sequences, found upstream of many bacterial dnaA genes, were not identified upstream of rickettsial dnaA gene. In addition, gene organization within this region differed from that of other bacteria. The putative start of transcription of the rickettsial dnaA gene was localized to a site 522 nucleotides (nt) upstream of the DnaA start codon.

Ancestral divergence of Rickettsia bellii from the spotted fever and typhus groups of Rickettsia and antiquity of the genus Rickettsia.

The eubacterial genus Rickettsia belongs to the alpha subgroup of the phylum Proteobacteria. This genus is usually divided into three biotypes on the basis of vector host and antigenic cross-reactivity characteristics. However, the species Rickettsia bellii does not fit into this classification scheme; this organism has characteristics common to both the spotted fever group and the typhus group biotypes and also exhibits some unique features. Sequences of the 16S rRNA and 23S rRNA genes from Rickettsia rickettsii (spotted fever group), Rickettsia prowazekii (typhus group), and R. bellii were studied to determine the position of R. bellii in the rickettsial classification scheme. The 23S rRNA gene sequences described in this paper are the first 23S rRNA sequences reported for any member of the Rickettsiaceae. The 23S rRNA gene contains substantially more phylogenetic information than is contained in the 16S rRNA sequences, and the 23S rRNA gene sequence has diverged about 1.9 times faster in the three Rickettsia species which we studied. Taken together, the molecular data obtained from the two genes indicate that R. bellii is not a member of either the spotted fever group or the typhus group; rather, this organism appears to be the product of a divergence which predates the separation of the genus into the spotted fever group and the typhus group. Consequently, different combinations of the ancestral characteristics retained by R. bellii have been retained in the more derived lineages of the genus.(ABSTRACT TRUNCATED AT 250 WORDS)

Determination of genome size and restriction pattern polymorphism of Rickettsia prowazekii and Rickettsia typhi by pulsed field gel electrophoresis.

Pulsed field gel electrophoresis (PFGE) of SmaI, MluI and SalI digested DNA was used to estimate genome size and perform restriction fragment length polymorphism analysis for Rickettsia prowazekii and Rickettsia typhi. We concluded that the genome of R. prowazekii and R. typhi consisted of a single chromosomal DNA. The total length of DNA of R. prowazekii was 1,106 +/- 54 kb and of R. typhi was 1,133 +/- 44 kb. It was possible to differentiate two strains of R. prowazekii, Breinl and EVir, by PFGE analysis after SalI digestion. Restriction fragment length polymorphism analysis did not reveal intraspecies differences between three human isolates and one Xenopsilla cheopis isolate of R. typhi.

Antigenic relationships among the rickettsiae of the spotted fever and typhus groups.

Using immunoblots to analyze antigenic relationships among the pathogenic spotted fever and typhus group rickettsiae, I found that the rickettsial lipopolysaccharide (LPS) was a group-specific antigen. All the rickettsiae examined had 135-kDa and 58-kDa protein antigens. The spotted fever rickettsiae and Rickettsia canada had, in addition, 190-kDa protein antigens which were antigenic analogs of previously described protective antigens of R. conorii and R. rickettsii.

Flying squirrel-associated Rickettsia prowazekii (epidemic typhus rickettsiae) characterized by a specific DNA fragment produced by restriction endonuclease digestion.

The DNA from flying squirrel-associated Rickettsia prowazekii was characterized by using a specific DNA fragment produced by digestion with the enzyme BamHI. The DNA fragment was cloned into a plasmid vector and used to readily distinguish between available human- and flying squirrel-associated R. prowazekii DNAs derived from crude cytoplasmic extracts.

Biochemical characteristics of typhus group rickettsiae with special attention to the Rickettsia prowazekii strains isolated from flying squirrels.

Six strains of Rickettsia prowazekii, two derived from human infections and four isolated from flying squirrels, two strains of R. typhi, and the single available strain of R. canada, were characterized by several biochemical procedures. The electrophoretic patterns on polyacrylamide gels of rickettsial proteins solubilized by sodium dodecyl sulfate revealed several species differences, but strains of the same species appeared to have identical patterns. Cytoplasmic fractions of the rickettsiae were examined for enzymatic activities and for polyacrylamide gel isoelectric focusing patterns. Some species differences were encountered in the activities or ratios of activities of glutamate-oxaloacetate transaminase, glutamate dehydrogenase, and malate dehydrogenase. When polyacrylamide gels were stained for malate dehydrogenase after electrophoresis, a single band became apparent with single extracts or mixtures of two strains of R. prowazekii, but two bands were seen with mixtures of a strain of R. prowazekii and one of R. typhi. The isoelectric focusing patterns of the soluble proteins revealed numerous species differences, especially between R. canada and the other two species, and a few differences among the strains of R. prowazekii. The patterns of the two human strains, Breinl and E(R), differed in at least one location, and both differed from the flying squirrel strains in the displacement of one band. One of the flying squirrel strains, GvF-16, contained a protein band not seen in the other five strains. Despite these minor differences, a striking similarity was revealed by all the biochemical tests performed between the R. prowazekii strains of human and flying squirrel origin.