[Determination of Ehrlichia genome size by pulse gel electrophoresis]. / Opredelenie razmera genoma patogennykh érlikhii metodom pul's-gel'-élektroforeza.

Ehrlichia infections are more and more common in the USA and Europe. The genetics and genome organization of Ehrlichia are little studied due to great difficulties in cultivating these bacteria. Pulse gel electrophoresis was first used to determine the sizes of a genome of 3 representatives of the genus Ehrlichia. The sizes of a genome was established for E. sennetsu (881 kb), for E. risticii (867 kb), E. chaffeensis (1,236 kb).

New Rickettsiae in ticks collected in territories of the former soviet union.

Dermacentor nuttallii from Siberia, Rhipicephalus sanguineus from Crimea, and Rh. pumilio from the Astrakhan region were infected with Rickettsia sibirica (12%), R. conorii (8%), and the Astrakhan fever agent (3%), respectively. Three new Rickettsiae of the R. massiliae genogroup were identified in ticks by 16S rDNA, gltA, and ompA sequencing.

Determination of the genome size of Ehrlichia spp., using pulsed field gel electrophoresis.

Ehrlichiae are obligatory intracellular, Gram-negative bacteria which belong to the alpha subclass of the phylum Proteobacteria and are responsible for infectious diseases of humans. Little is known about genetics and genomic organization of Ehrlichia spp. The genome sizes of four representatives of the genus Ehrlichia were determined for the first time by pulsed field gel electrophoresis. The sizes for E. sennetsu, E. risticii, E. chaffeensis (strain Arkansas and strain 91HE17), and the HGE agent were 878.5 kb, 880.3 kb, 1225.8 kb, 1262.3 kb and 1494 kb respectively.

Citrate synthase gene comparison, a new tool for phylogenetic analysis, and its application for the rickettsiae.

Using PCR and an automated laser fluorescent DNA sequencer, we amplified and sequenced a 1,234-bp fragment of the citrate synthase-encoding gene (gltA) of 28 bacteria belonging to the genus Rickettsia. Comparative sequence analysis showed that most of the spotted fever group (SFG) rickettsiae belonged to one of two subgroups. The first subgroup included Rickettsia massiliae, strain Bar 29, Rickettsia rhipicephali, "Rickettsia aeschlimanni," and Rickettsia montana, which have been isolated only from ticks. The second subgroup was larger and included the majority of the human pathogens and also rickettsiae isolated only from ticks; the members of this subgroup were strain S, Rickettsia africae, "Rickettsia monglotimonae," Rickettsia sibirica, Rickettsia parkeri, Rickettsia conorii, Rickettsia rickettsii, the Thai tick typhus rickettsia, the Israeli tick typhus rickettsia, the Astrakhan fever rickettsia, "Rickettsia slovaca," and Rickettsia japonica. The sequence analysis also showed that the tick-borne organisms Rickettsia helvetica and Rickettsia australis and the mite-borne organism Rickettsia akari were associated with the SFG cluster, that Rickettsia prowazekii and Rickettsia typhi, two representatives of the typhus group, clustered together, and that Rickettsia canada; Rickettsia bellii, and the AB bacterium probably represent three new groups. We compared the phylogenetic trees inferred from citrate synthase gene sequences and from 16S ribosomal DNA (rDNA) sequences. For rickettsial phylogeny, the citrate synthase approach was more suitable, as demonstrated by significant bootstrap values for all of the nodes except those in the larger subgroup defined above. We also compared phylogenetic analysis results obtained in a comparison of the sequences of both genes for all of the representatives of the domain Bacteria for which the gltA sequence was determined. We believe that comparison of gltA sequences could be a complementary approach to 16S rDNA sequencing for inferring bacterial evolution, especially when unstable phylogenetic models are obtained from ribosomal sequences because of high levels of sequence similarity between the bacteria studied.

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.

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.

[Characteristics of DNA restriction fragment length polymorphisms of C. burnetti strains, isolated from areas of Russia]. / Kharakteristika polimorfizma dlin restriktsionnykh fragmentov DNK shtammov C. burnetii, vydelennykh na territorii Rossii.

The structural heterogeneity in Coxiella burnetii chromosomal DNA isolated in the European part of Russia from people, agricultural animals, and ticks has been studied. It is compared with the one of the European strains Henzerling and M44, the only genetically characterized strains up to date. The digestion of the total DNA by the restriction endonucleases BamHI, PstI, XhoI resulted in obtaining two types of restriction patterns. The ones for Henzerling and M44 differed from the restriction patterns of the Russian strains, while the latter proved to be identical. The obtained data are in proof of genetical homogeneity in the Russian group of strains. The group is different from the genomic group including Henzerling and M44. The fact is in proof of the genetical heterogeneity of the European population of coxiellae.

[Genotypic characteristics of Rickettsia sibirica strains]. / Genotipicheskaia kharakteristika shtammov Rickettsia sibirica.

Six Rickettsia sibirica strains isolated in Siberia and Far East (Primorje) from various sources (patient, ticks D. nuttali, D. silvarum, H. concinna) at different time (1940-1980) were studied by the RFLP and DNA probe hybridization techniques. All studied strains were found to have the identical profiles of migrating fragments in restrictograms got by using a set of endonucleases (EcoRI, PstI, PvuII, Bg1I, XbaI, HindIII, MspI) and similar zones of hybridization with a DNA probe derived from Rickettsia prowazekii DNA. The obtained data point to a close similarity between the genomes of investigated Rickettsia sibirica strains. Long-term isolation of the genetically similar Rickettsia sibirica strains testifies to their constant circulation, thus apparently determining the stability of epidemiologic manifestation of tick-borne typhus fever of Northern Asia in the central part of its area (Siberia, Far East).

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.

[The rickettsial genome studied by DNA restriction analysis]. / Izuchenie genoma rikketsii metodom restriktsionnogo analiza DNK.

The restriction analysis of 6 Rickettsia prowazekii strains with the use of 8 restrictases (Cfr13I, EcoRI, HindIII, MSpI, MvaI, PstI, XhoI, BamHI) has been carried out. In the presence of considerable homology in the restriction pictures of DNA in these strains some differences in 1-2 fragments within the range of 8,000-20,000 nucleotide pairs have been established. The strains under study have been divided into two groups according to the character of differences in their restrictograms: the group of virulent typing strain Breinl (Breinl, G. Anan'ev) and the group of strain E with low pathogenicity (E, EVir, Katsinian). Differences in the restrictograms of DNA do not correlate with the virulence of R. prowazekii strains and the areas of their isolation.

Restriction endonuclease analysis of the DNA of Rickettsia prowazekii vaccine strain E and its revertant.

The DNA of Rickettsia prowazekii vaccine strain E was analysed by restriction analysis with 17 endonucleases in comparison with its virulent revertant - Evir and the virulent reference strain Breinl. The DNA of cloned and uncloned strains showed identical restriction endonuclease patterns. In spite of stable differences in virulence, strains E and Evir displayed a totally identical DNA cleavage pattern indicating the absence of marked structural differences between their genomes. On the other hand 9 endonucleases showed differences in the restrictograms of the DNA strain Breinl as compared with strains E and Evir.

[The library of Rickettsia prowazekii genes]. / Biblioteka genov rikketsii Provacheka.

The DNA of Rickettsia provazekii strain E was cleaved by PstI restriction endonuclease under the conditions of partial restriction. The fragments were inserted into the PstI site of pBR325 and cloned in this plasmid. E. coli strain HB101 was used as a recipient for cloning. 880 clones sensitive to ampicillin and resistant to tetracycline were selected from 5120 transformants. The cloning of rickettsial DNA has been confirmed by the blot hybridization technique. Analysis of individual and net probes of the hybrid DNA by gel electrophoresis makes it possible to conclude that 90% of the selected clones harbour hybrid plasmids, the size of the cloned fragments rangers from 0.9 to 10.4 Kb, the obtained library of clones contains 70% of the whole genome of Rickettsia provazekii.

[Use of the immunofluorescence reaction for evaluating the immunological transformation in those inoculated with a chemical typhus vaccine]. / Ispol'zovanie reaktsii immunofliuorestsentsii dlia otsenki immunologicheskoi perestroiki y privitykh khimicheskoi sypnotifoznoi vaktsinoi.

The results of the serological examination of persons immunized with chemical typhus vaccine (CTV) are presented. The examination was carried out by means of the complement fixation test (CFT), the passive hemagglutination test (PHAT), the toxin neutralization test (TNT) and the immunofluorescence test (IFT). The acetone-fixed live culture of Rickettsia prowazekii, strain Breinl, served as antigen in IFT. If persons immunized with CTV showed positive titers in CFT, TNT and PHAT, the results of IFT were highly correlated with the CFT titers. In 6-12 months after immunization with CTV the titers of CFT, TNT and PHAT became negative, while the IFT titers remained positive for several subsequent years.

Persistence of Rickettsia prowazekii with different initial biological properties in infected cotton rats.

Evidence has been obtained indicating the association of certain biological properties of Rickettsia prowazekii with their capacity for persistence. Highly virulent rickettsial cultures inducing increased levels of complement-fixing antibodies were shown to cause a high percentage of rickettsial carriers in infected cotton rats. Within an interval of 133-189 days after inoculation with the virulent Breinl strain, rickettsial carrier-state was still demonstrable in 20% of the animals. The vaccine strain E of R. prowazekii had a low capacity for persistence in cotton rats, because 64 days after inoculation all animals were found free of rickettsiae.