Biological and genetic characterization of Rickettsia sibirica strains isolated in the endemic area of the north Asian tick typhus.

Restriction fragment length polymorphism (RFLP) analysis of polymerase chain reaction-amplified gene fragments was used to characterize 24 isolates of spotted fever group rickettsiae previously identified as Rickettsia sibirica from their serologic properties. These strains were obtained in Russia between 1946 and 1991 from humans and different species of Ixodid ticks. The RFLP analysis was performed using amplified DNA products obtained with a genus-specific primer pair derived from the R. prowazekii citrate synthase gene and two group-specific primer pairs from the R. rickettsii 190-kD and 120-kD surface protein antigen genes followed by Alu I, Pst I, and Rsa I restriction endonuclease digestions. Although some differences were detected in biological characteristics among the examined strains, only a single R. sibirica genotype was found with these molecular tools of identification.

Genotype Characterization of the Bacterium Expressing the Male-Killing Trait in the Ladybird Beetle Adalia bipunctata with Specific Rickettsial Molecular Tools.

Volume 61, no. 4, p. 1433, Results, line 22: "RsaI" should read "AluI." Figure 3 legend, lines 5 and 6: "RsaI-digested" should read "AluI-digested." [This corrects the article on p. 1431 in vol. 61.].

Interaction of Rickettsia prowazekii strains of different virulence with white rat macrophages.

The growth of mildly pathogenic strain E, its virulent revertant EVir, and prototype virulent strain Breinl of Rickettsia prowazekii in peritoneal macrophage cultures of outbread white rats (WR) was evaluated by light microscopy and bioassay in chick embryos (CE). Macrophage cultures infected with strain E were characteristic by limited number of infected cells, poor or moderate accumulation of rickettsiae in individual cells, poor or nil spread of infectious process during first 7 days of infection, and the death of rickettsiae in cultures as determined by the bioassay in CE. Moreover, rickettsiae were not determined in 20.7% of infected macrophage cultures by either microscopic or bioassay methods. In contrast, the growth of virulent strains EVir and Breinl was characteristic by higher proportion of infected cells, considerable accumulation of rickettsiae, and intensive spread of infectious process within 5-7 days post infection (p.i.). However, the intensity of infectious process in macrophage cultures was less expressed with strain EVir than with strain Breinl.

Serologic response to rickettsial antigens in patients with Astrakhan fever.

Astrakhan fever is a new spotted fever group (SFG) rickettsiosis. Sera of patients with Astrakhan fever have been examined by microimmunofluorescence and western immunoblotting to determine the serologic responses to the Astrakhan strain and to R. conorii M-1 strain and the Israelian isolate of SFG rickettsiae. The serologic response to specific rickettsial agent and to Israelian isolate has been found to be similar, but was different of that to R. conorii. Immunoglobulin G (IgG) and IgM antibodies were detected in most sera and were directed against the lipopolysaccharide. Only one of tested sera contained IgG antibodies which also recognized high molecular weight proteins.

Genotype characterization of the bacterium expressing the male-killing trait in the ladybird beetle Adalia bipunctata with specific rickettsial molecular tools.

The male-killing ladybird beetle (LB) bacterium (AB bacterium) was analyzed with specific rickettsial molecular biology tools in the LB Adalia bipunctata strains. Eight phenotype-positive LB strains showing mortality of male embryos were amplified with rickettsial genus-specific primers from the gene for citrate synthase (CS) and the gene for a 17-kDa protein and spotted fever group-specific primers from the gene for the 120-kDa outer membrane protein (ompB). The specificity of amplification was confirmed by Southern hybridization and the absence of the above-listed gene products in three phenotype-negative LB strains. Restriction polymorphism patterns of three examined amplicons from the CS gene, 17-kDa-protein gene, and ompB gene were identical among the eight phenotype-positive LB strains and were unique among all known rickettsiae of the spotted fever and typhus groups. Amplified fragments of the CS genes of the AB bacterium, Rickettsia prowazekii Breinl, Rickettsia typhi Wilmington, Rickettsia canada 2678, and Rickettsia conorii 7 (Malish) were sequenced. The greatest differences among the above-listed rickettsial and AB bacterium CS gene sequences were between bp 1078 and 1110. Numerical analysis based on CS gene fragment sequences shows the close relationships of the AB bacterium to the genus Rickettsia. Expanding of knowledge about rickettsial arthropod vectors and participation of rickettsiae in the cytoplasmic maternal inheritance of arthropods is discussed.

Astrakhan fever rickettsiae: antigenic and genotypic analysis of isolates obtained from human and Rhipicephalus pumilio ticks.

Two spotted fever group rickettsia strains, A-108 and A-167, were isolated from the hemolymph of Rhipicephalus pumilio ticks collected in the Astrakhan region of Russia, which is area endemic for Astrakhan fever. These tick isolates were compared with a strain isolated from a patient suffering from Astrakhan fever and with reference spotted fever group rickettsiae strains. New tick isolates and the human strain were identical in their serologic, antigenic, and genetic characteristics by several methods: microimmunofluorescence, protein gel electrophoresis with immunoblotting, polymerase chain reaction followed by restriction endonuclease fragment length polymorphism analysis, and pulsed-field gel electrophoresis (PFGE). Astrakhan fever rickettsiae were found to be serologically and antigenically similar to Israeli spotted fever rickettsiae. Both of them probably belong to a single Rickettsia conorii pathotype complex. Only PFGE pattern analysis could clearly discriminate Astrakhan fever rickettsiae from other isolates.

Studies of Rickettsia prowazekii antigens in immunoblotting with specific sera of infected white mice.

Five strains of Rickettsia prowazekii different in origin, biological and genetic properties were compared in protein and LPS patterns by the polyacrylamide gel electrophoresis and in antigenic properties by immunoblotting with specific sera of infected white mice. Three virulent strains Breinl, G and Katsinjan had identical protein patterns and differed from isogenic pair of strains E and EVir in the electrophoretic properties of 29-30 kDa proteins. Silver-strained LPS patterns were different in five compared strains. Strain G and strain Katsinjan had the longest O-chaines of LPS. Polyclonal mouse antisera contained specific antibodies which mainly directed against LPS and 25-60 kDa proteins. Strains E and EVir were identical in all performed immunoblotting reactions and separated from three virulent strains. Out of virulent strains, whole cell antigen of strain Katsinjan and LPS antigen of strain G had different reactions in comparison with correspondent antigen of the standard strain Breinl.

Purification of rickettsial cultures contaminated by mycoplasmas.

An experimental biological model is proposed for purification of rickettsiae-infected cell cultures from mycoplasma contamination using intravenous mouse infection and subsequent passages on Vero cell monolayers. Mycoplasma-free rickettsial cultures were obtained in mouse brain or spleen suspension within 3 hrs or 3 days, respectively, after mouse inoculation. Ten strains of spotted fever group rickettsiae were purified from mycoplasmas by this procedure.

Serological response of patients suffering from primary and recrudescent typhus: comparison of complement fixation reaction, Weil-Felix test, microimmunofluorescence, and immunoblotting.

Microimmunofluorescence and Western immunoblotting were compared with the classical complement fixation reaction and the Weil-Felix test to study the serological responses of patients to Rickettsia prowazekii and both Proteus vulgaris OX19 and OX2 during primary and recrudescent typhus infections. The serological response to R. prowazekii was found to be similar during primary and recrudescent typhus, and all sera examined contained antibodies to the same R. prowazekii cell structures. Immunoglobulin G (IgG) and IgM were found to be the dominant anti-R. prowazekii immunoglobulins in all sera tested and were found to be directed against the 100-kDa protein and the lipopolysaccharide. IgA antibodies, when present, were mainly against the 100-kDa protein. For P. vulgaris, IgG antibodies recognized the proteins and lipopolysaccharides of both OX19 and OX2 serotypes; IgM antibodies were directed against the P. vulgaris OX2 lipopolysaccharide. In addition, donor blood sera, which were negative by microimmunofluorescence, were found to contain IgG immunoglobulins reacting with R. prowazekii protein antigens of 135, 60, and 47 kDa by western immunoblotting.

Genotypic characterization of rickettsiae by DNA probes generated from Rickettsia prowazekii DNA.

Southern blot analysis of HindIII-cleaved rickettsial DNA was used for genotypic characterization of the typhus group (TG) species (R. prowazekii, R. typhi, R. canada) and a few species of the spotted fever group (SFG) rickettsiae (R. sibirica, R. conorii, R. akari). Four different DNA probes were employed. PBH11 and PBH13 probes were morphospecific HindIII fragments of R. prowazekii DNA. MW218 probe contained the gene for 51 K antigen and MW264 probe contained the citrate synthase gene of R. prowazekii. All the probes hybridized with the tested TG and SFG rickettsial DNAs, forming from 1 to 5 bands, but they did not with R. tsutsugamushi or C. burnetii DNAs. All the probes demonstrated specific hybridization patterns with TG species and R. akari. PBH11, PBH13 and MW264 probes clearly distinguished R. sibirica and R. conorii from the other tested rickettsiae, but not from each other. However, these two species differed slightly with MW218 probe. Several strains of each species were analyzed in this way and except for strains of R. conorii identical intraspecies patterns were obtained. These data lead us to consider the obtained hybridization patterns as criteria for genotypic identification.

Genomic identification of Rickettsia slovaca among spotted fever group rickettsia isolates from Dermacentor marginatus in Armenia.

Restriction fragment length polymorphism (RFLP) analysis of polymerase chain reaction (PCR) amplified genes was used for genomic identification of Armenian isolates of the Spotted fever group (SFG) rickettsiae with unclear taxonomic position. Analysis was performed by using one genus-specific primer pair derived from R. prowazekii citrate synthase gene and two species-specific primer pairs derived from R. rickettsii genes for 190 K and 120 K antigens following AluI, PstI and RsaI digestion of amplicons. All tested rickettsial SFG Armenian isolates from Dermacentor marginatus were identified as R. slovaca. The geographic distribution and genetic homogeneity of R. slovaca strains are discussed.

Genotypic and biological characteristics of non-identified strain of spotted fever group rickettsiae isolated in Crimea.

A strain of rickettsiae, designated Crimea-108, was isolated from ticks Dermacentor marginatus in the Crimea in 1977. Its immunobiological characteristics involve low pathogenicity for experimental animals, moderate infectivity for chick embryos, and antigenic relatedness to spotted fever group (SFG) rickettsiae (R. sibirica, R. conorii, R. akari), especially to R. sibirica. The genotypic characterization of the strain Crimea-108 was carried out in comparison with SFG and typhus group rickettsiae by using restriction fragment length polymorphism (RFLP) analysis and DNA-probe hybridization. The marked similarity was detected between DNA restriction patterns of the strains Crimea-108, R. sibirica and R. conorii, but each of them besides comigrating fragments had specific ones. Genotypic analysis of the strain Crimea-108, the SFG and typhus group rickettsiae by three independent DNA probes, based on R. prowazekii DNA, gave unique hybridization patterns for the Crimea-108 strain with all probes. The obtained data show that the Crimea-108 isolate does not belong to the species of R. sibirica, R. conorii, R. akari. The strain Crimea-108 is a novel strain of SFG rickettsiae for the Crimea region.

Identification of Rickettsia prowazekii using the polymerase chain reaction.

Polymerase chain reaction (PCR) was used to identify Rickettsia prowazekii, the etiologic agent of epidemic typhus. For the PCR, Thermus thermophilus thermostable DNA polymerase was applied with buffer containing a relatively low Mg2+ concentration (1.5-2 mM with dNTP's at 250 microM each). A primer pair used to amplify a 448-base-pair (bp) fragment of R. prowazekii genome was synthesized on the basis of the DNA sequence of gene rpa14/16, coding for a precursor of the mature polypeptides of molecular weight (Mr) 14,000 and/or 16,000 (16kD) from R. prowazekii strain E. For determining the specificity of the primer pair, purified genomic DNAs of 16 rickettsial and 10 other bacterial strains were used.

Proteinic and genomic identification of spotted fever group rickettsiae isolated in the former USSR.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), restriction fragment length polymorphism of polymerase chain reaction-amplified genes (RFLP-PCR), and pulsed-field gel electrophoresis (PFGE) were used to identify 25 isolates of spotted fever group rickettsia collected in the former USSR. Six Rickettsia akari isolates which were identical to the MK reference strain from the American Type Culture Collection were found. Also, 14 isolates were found to be Rickettsia sibirica and identical to reference strain 246. Two of three isolates previously considered as atypical, low-pathogenic strains of R. sibirica, were found to be strains of Rickettsia slovaca. The third, strain S, was similar in its RFLP-PCR profile to "R. africae" sp. nov. (proposed name for a rickettsia pathogenic for human beings in southern Africa) but in its SDS-PAGE and PFGE profiles was unique among spotted fever group rickettsiae. Strain M-1 was confirmed as a genetic variant of Rickettsia conorii. The Astrachan isolate, the causative agent of a tick-bite rickettsiosis at the North of the Caspian Sea, showed a previously described RFLP-PCR profile identical to that of the Israeli tick typhus rickettsia, but its SDS-PAGE and PFGE profiles different from those of the other strains tested.

Protein antigens of genetically related Rickettsia prowazekii strains with different virulence.

The protein antigens of two distinct lines of genetically related strains, namely the nonpathogenic strain E and its virulent revertant EVir and of the standard virulent strain Breinl were compared in SDS-PAGE and immunoblot assay using typhus patient sera and immune rabbit sera. No differences in the polypeptide pattern as detected in SDS-PAGE were found between strain E and EVir; the Breinl strain differed in a 30 kD protein. The high immunogenicity of the protein antigens of E, EVir and Breinl strains was demonstrated by immunoblot assay with human sera, which did not show any differences between the strains studied. Immunoblot analysis with immune rabbit sera to the strain E, EVir, and Breinl showed differences in immunological response to the 70 kD and 60 kD polypeptides of low virulent strain E and those of virulent strains EVir and Breinl.

Cloning and expression in Escherichia coli of the 37-, 14-, and/or 16-kilodalton antigens genes from Rickettsia prowazekii strain E.

A gene bank of Rickettsia prowazekii strain E constructed in the phage vector lambda EMBL4 was screened for antigen production with anti-R. prowazekii serum. One of the immunoreactive clones, grown at 37 degrees C exhibited the expression of at least two antigens of molecular weight (M(r)) 37 kD and 14 kD. Subcloning and further analysis revealed that the antigens (polypeptides) of Mr 37, 14, and/or 16 kD apparently represent structural units of the 138 kD complex antigen. Assembly of the above mentioned polypeptides was found to be thermosensitive as it took place at 30 degrees C but not at 37 degrees C and resulted in an oligomeric structure of M(r) 138 kD. The nucleotide sequence of the gene coding for a precursor of the mature polypeptides of Mr 14 and/or 16 kD was determined.

Restriction fragment length polymorphism of the DNA of typhus group rickettsiae.

The DNA of 10 strains of Rickettsia prowazekii, 5 strains of Rickettsia typhi and 1 strain of Rickettsia canada was investigated by restriction fragment length polymorphism analysis. Interspecies differences were characterized by a great number of noncomigrating bands. Using the endonuclease HindIII and PstI fragments comigration as a quantitative criterion, genetic similarity coefficient was calculated for the pair Rickettsia prowazekii/Rickettsia typhi-32.0%, for Rickettsia prowazekii/Rickettsia canada-22.7%, and for Rickettsia typhi/Rickettsia canada-23.5%. Intraspecies differences expressed are very subtle and concern 1-2 noncomigrating fragments. The investigated strains of Rickettsia prowazekii and Rickettsia typhi can be divided into 2 groups without any correlation to the source and period of isolation, or to strain passage history.

Isolation and partial characterization of the M(r) 100 kD protein from Rickettsia prowazekii strains of different virulence.

The major M(r) 100 kD protein (protein I) from the standard virulent R. prowazekii strain Breinl, from the nonvirulent strain E and its virulent revertant EVir were isolated by chromatography and characterized. Purified protein I from the three strains of different virulence and origin had the same physico-chemical and antigenic properties, protected guinea pigs against infection with the virulent strain Breinl and induced the production of antibodies, which neutralized the toxic and haemolytic activities of R. prowazekii. The amino acid composition of protein I as determined for the three above mentioned strains was similar. Modified residues of Lys, Asn, and/or Gln were found in protein I. Protein I from virulent strains Breinl and EVir differed from that of nonpathogenic strain E by the quantity of N epsilon-Me-Lys and N epsilon-Me3-Lys, but all had the same total amount of Lys and its derivatives. It may be suggested that a difference may exist in the processing of the protein I of nonpathogenic strain E and of virulent strains of R. prowazekii.

The use of molecular hybridization to evaluate the divergence of some altered Rickettsia prowazekii strains.

Molecular DNA/DNA hybridization was used to investigate the degree of divergence of different Rickettsia prowazekii strains, namely strain Breinl, the vaccine strain E, its spontaneous erythromycin-resistant mutant, nitrosoguanidine-induced rifampicin-resistant mutant and a variant of strain E with increased virulence upon mouse lung passaging. Hybridization of highly polymerised rickettsial DNAs was carried out on nitrocellulose filters with in vitro labelled fragments of reference DNA of the Breinl strain. Nucleotide composition of the strains was also studied. The results obtained suggest the high degree of homology of nucleotide sequences in DNAs of R. prowazekii strains under study; the existing differences found are within the intraspecies range.