Properties in culture and persistence in cotton rats of the Rickettsia prowazekii vaccine strain E and its mutants.

Cultural properties and the capacity for persistence were studied in spontaneous erythromycin-resistant (E errSM), in induced erythromycin-resistant (E errI) mutants and in a virulent revertant (E Vir) of the vaccine strain E, as compared with parent vaccine strain E and standard virulent strain Breinl of Rickettsia prowazekii. Cultural properties of the strains were found to differ in passages in chick embryos (CE) and cultures of FL cells. Multiplication indices in CE of mutant E errI were significantly lower than those of other strains (E, E errSM, E Vir, Breinl). The multiplication rate in FL cells was found to be high in strains E errSM, Breinl, E Vir, being much lower in strains E errI and E. The capacity of the virulent revertant E Vir to persist in cotton rat (CR) was higher as compared with that of standard strain Breinl and significantly higher than that of the parent strain E. Low level carrier state of rickettsia was registered in CR infected with the mutant E errI.

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.

Methods for purification of Rickettsia prowazekii separated from the host tissue: a step-by-step comparison.

Two methods for purification of Rickettsia prowazekii strains E, E Vir, and Breinl grown in chick embryo yolk sacs are described. These methods combine either differential centrifugation or sucrose mix, centrifugation through sucrose cushion, 10 mmol/l MgCl2 treatment, filtration through a glass filter AP-20 and 2 cycles of verografin discontinuous density gradient centrifugation. The purification procedure including sucrose mix allowed to recover about 38-42% biologically active rickettsiae, a yield which was by 10% higher than that obtained by the method beginning at differential centrifugation. The rickettsiae free of host cell components preserved their infectious activity. The obtained biomass was suitable for immunological and biological characterization of Rickettsia prowazekii and for isolation of its total DNA.

[Cloning and expression in Escherichia coli of three Rickettsia prowazekii genes, coding outer membrane proteins]. / Klonirovanie i ékspressiia v Escherichia coli trekh genov Rickettsia prowazekii, kodiruiushchikh belki vneshnei membrany.

Rickettsia prowazekii (virulent Breinl strain) random genomic DNA fragments were cloned in the lambda gt11 expression vector by using non-palindromic adaptors. Several immunoreactive clones were selected after screening 20,000 individual recombinant plaques with human convalescent serum. Some recombinants synthesized the complete 60 K protein, and others synthesized beta-galactosidase fusion polypeptides containing epitopes of 134 K protein of the R. prowazekii outer membrane. The amplified genomic library was screened with monospecific antibodies directed against abundant 31 K and 29.5 K outer membrane proteins. Several recombinant clones expressing full or part of 29.5 K polypeptide, and none expressing 31 K polypeptide were revealed. The serum of a patient convalescing from epidemic typhus did not react in western blot with recombinant 29.5 K protein.

[Biochemical and genetical study of Rickettsia]. / Biokhimicheskoe i geneticheskoe izuchenie rikketsii.

The review deals with the phenomenology in the studies on characteristics of surface antigenic and immunogenic structures of Rickettsia, their cellular membranes, the processes of metabolic cooperation and interaction with the host cells, and the structure of Rickettsia genome. The data on active antigenic and immunogenic proteins distribution in inner and outer membranes and on osmotically active functioning cellular membrane, including the specific substrate carriers, are discussed. The materials, are presented on the specific ADP-ATP transport system, slightly different from the mitochondrial one, in evidence that Rickettsia utilize ATP in two pathways: endogenous and exogenous. The metabolic regulatory processes, controlled by adenine nucleotides are discussed that could be used as a means of fitting to constantly changing conditions of Rickettsia ecological niche. The Rickettsia deficiency in AMP catabolism enzyme could be used for allosteric-regulation of citrate synthase, the key enzyme in the Krebs cycle. The data on the mol mass of Rickettsia DNA (1 x 10(9)) and the characteristics of plasmids are presented. In conclusion new data on molecular cloning of Rickettsia genes in vector plasmids and the restriction analysis of specific DNA sequences are discussed.

[Methods of isolation and polypeptide composition of membrane fractions of Rickettsia prowazekii]. / Metody polucheniia i polipeptidnyi sostav membrannykh fraktsii rikketsii Provacheka.

The methods of cell lysis by lysozyme in tris-EDTA-sucrose with the consequent disruption of spheroplasts by the osmotic shock were used to obtain the total membranes from the intact or temperature-inactivated Rickettsia prowazekii. Detergents solubilization methods were used for analysis of outer membrane proteins. Sarcosyl insoluble material is shown to contain the main 134, 31, 29.5 and 25 Kd proteins, the minor 78, 60, 42, 17 Kd proteins, while the mixture of both membranes possess a more complex composition. Treatment of total membranes by the 2% octylglycoside results in elimination of the 31 Kd polypeptide. Inactivated Rickettsia can be used for isolation of the outer layer proteins diminishing the risk of working with this pathogenic microorganism.

[Homology between the ribosomal and RNA-polymerase genes of E. coli and vaccine strain E of Rickettsia prowazekii]. / K voprosu o gomologii mezhdu ribosomnymi i RNK-polimeraznymi genami E. coli i vaktsinnogo shtamma E rikketsii Provacheka.

The pIB52 plasmid contains the ribosomal rp1A, J, K, L and RNA-polymerase genes rpo C, B of Escherichia coli. No homology has been found between the plasmid used as a molecular probe and the chromosomal DNA from Rickettsia provazekii in the blot hybridization experiments.

[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.

[Electrophoretic and immunochemical characteristics of proteins of Rickettsia prowazekii strains of various virulence]. / Elektroforeticheskaia i immunokhimicheskaia kharakteristika belkov shtamma Rickettsia prowazekii razlichnoi virulentnosti.

PAAG-electrophoresis of the isogenic pair of Rickettsia prowazekii strains E and Evir lysates demonstrate the similarity in polypeptide tracks. The different electrophoretic mobility of the Mr 30 Kd protein from these strains as compared with the mobility of analogous protein from the standard virulent Breinl strain is registered. In immunoblot experiments the specific rabbit antiserums obtained on the 30th day of infection with the Breinl, E or Evir strains demonstrate the presence of the different main antigens 60 Kd or 70 Kd. The difference evidently reflects the specificity of development of two forms of infection by the strains having different virulence. The surface tris-soluble antigens of Rickettsia prowazekii have the similar polypeptide contents and immunochemical properties. The main component of tris-soluble antigens Mr 130 Kd protein is not strain specific having the common thermolabile epitope.

[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).

[Rickettsia conorii and prowazekii plaque study in a chick fibroblast cell culture]. / Izuchenie bliashek rikketsii Konori i Provacheka v kul'ture kletok kurinykh fibroblastov.

The results of the study of plaques formed by R. conorii (strain M 1) and R. prowazeki (strain E and erythromycin-resistant strain E) in chick fibroblast cell culture are presented. In this study the tissue monolayer was inoculated with rickettsiae suspended in various media, and media of different composition were used in the nutrient cover and for cell cultivation. The maximum plaque formation was observed under the following conditions: the monolayer of chick fibroblasts (seeding density was not less than 375,000 cells per 1 sq. cm) was grown in medium 199 with 5-10% of fresh fetal or calf serum and inoculated with rickettsiae suspended in heart-brain infusion; the nutrient cover was prepared on the basis of Seakem agarose (USA) and contained medium 199 (without antibiotics) and 10% of fresh fetal or calf serum. In these conditions R. conorii formed plaques 2 mm in diameter, the first plaques being observed on day 6, and most of them on days 7-9; the both strains of R. prowazeki formed plaques 1 mm in diameter, the first plaques being observed on days 8-9, and most of them on days 10-13.

[Serological demonstration of the detection of tick-borne rickettsial disease in Astrakhan Province]. / Serologicheskie dokazatel'stva vyiavleniia zabolevaniia kleshchevym rikketsiozom v Astrakhanskoi oblasti.

Starting from 1978, noncontagious febrile diseases of unclear etiology, accompanied by pronounced headache, roseolous-papular eruptions, prolonged convalescence period, are registered in May-September in Astrakhan Province. These diseases can be effectively treated with chrolamphenicol. In 11 out of 12 sera obtained from such patients the complement fixation test with the antigens of rickettsiae causing tick-borne spotted fever, epidemic typhus, as well as Coxiella burnetii antigen, revealed the presence of antibodies (in 8 sera) only to the antigens of rickettsiae causing tick-borne spotted fever (R. akari, R. conorii, R. sibirica), or the titers of antibodies to these antigens were greater (1 serum), equal and lower (2 sera) in comparison with those of the antigens of rickettsiae causing epidemic typhus. The dynamics and values of antibody titers in 7 patients with the antigens of three rickettsial species of the tick-transmitted biotype indicated that the disease was related to tick-borne spotted fever.

[Production of the mutant E strain of Rickettsia prowazekii by mutagen exposure]. / Poluchenie mutantov shtamma E rikketsii Provatseka pri mutagennom vozdeistvii.

The possibility of obtaining the mutants of R. prowazekii, strain E, by exposing these organisms to the action of N-methyl-N-nitro-N-nitrosoguanidine was studied; this substance, used in doses of 5-10 micrograms, showed a mutagenic effect on rickettsiae suspended in physiological saline, after their exposure for 10-20 minutes at 37 degrees C. The mutants thus obtained proved to be resistant to erythromycin and rifampicin and were characterized by heterogeneity in the degree and stability of their antibiotic resistance. The effectiveness of selection was increased if mutagen-treated rickettsiae were selected after the first passage in chick embryos. The induced mutants differed from the original rickettsial strain by their lower infectiosity for chick embryos.

[Luminescent-serologic analysis of the antigenic interrelationship between R. canada and classic representatives of rickettsiae of the typhus group]. / Liuminestsentno-serologicheskii analiz antigennykh vzaimosviazei R. canada i klassicheskikh predstavitelei rikketsii sypnotifoznoi gruppy.

The results of studying the antigenic relationships of R. canada, a new Rickettsia species, and classical Rickettsia species of the typhus group are presented. The study was carried out by luminescent serological analysis with the use of corpuscular antigens and the live infectious agent cultures. R. canada and Rickettsiae of the typhus group were similar in their antigenic structures; this, however, could be revealed only in the study of the live cultures of the infectious agents. The study of corpuscular antigens revealed unilateral relationship: R. prowazeki antigen could be detected with homologous and heterologous sera, R. canada antigen with homologous serum only. In the CFT and the agglutination test corpuscular R. canada antigen reacted with homologous and heterologous sera. The study of the live cultures of the infectious agents revealed that different R. prowazeki and R. typhi strains vary in the degree of their similarity to R. canada.

[The sensitivity of a number of cell cultures to different strains of Rickettsia prowazekii]. / Chuvstvitel'nost' riada kletochnykh kul'tur k razlichnym shtammam Rickettsia prowazekii.

The sensitivity of some cell cultures to different R. prowazekii strains (strain E with low pathogenicity, virulent strain Breinl, strains ERifRI and EVir) has been studied with a view to the selection of an adequate culture for growing these strains and the study of their biological properties. Experiments on titration in cells have revealed that 6- to 7-day primary and secondary irradiated quail fibroblasts and human amnion cells FL show the maximum sensitivity to all strains under study, comparable to that of chick embryos. The sensitivity of 6- to 7-day primary and secondary irradiated chick fibroblasts is faintly pronounced, and 24-hour chick and quail fibroblasts are still less sensitive. Cells FL have shown high sensitivity to strain E and mutant ERifRI in prolonged subculturing for 140 and 63 days (the term of observation) respectively after a single inoculation.

[Antibiotic sensitivity of the erythromycin-resistant strain E of Rickettsia prowazekii cultured in the vector's body]. / Izuchenie chuvstvitel'nosti k antibiotikam ustoichivogo k éritromitsinu shtamma E Rickettsia prowazekii pri kul'tivirovanii v organizme perenoschika.

Experiments on the laboratory cultures of lice infected by Weigl's method revealed that the spontaneous, erythromycin-resistant mutant of R. prowazekii strain E, adapted to the vector's organism, retained its resistance to erythromycin during 50 successive passages without the maintenance concentrations of this antibiotic. The above strain remained sensitive to tetracycline and levomycetin. Its level of sensitivity to the latter antibiotics was similar to that of R. prowazekii strains cultivated in the vector's organism for a long time.

[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.

[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.

[Inactivation of concentrated biomasses of Rickettsia prowazekii]. / Inaktivatsiia kontsentrirovannykh biomass Rickettsia prowazekii.

The methods used for the sparing inactivation of highly concentrated R. prowazekii biomass and for the decrease of its infectious activity are described. These methods are recommended for use in experiments in the field of molecular biology, as well as for disinfection of different materials contaminated with rickettsiae. As conditions for complete inactivation, incubation at 50 degrees C for 1 hour without chemical disinfectants, treatment with 0.5% phenol solution at 30 degrees C for 12 hours and with 0.1% formaldehyde solution at 4 degrees C for 24 hours have been selected. Treatment with 0.5% phenol solution at 36 degrees C for 1 hour or incubation at 45 degrees C without the use of disinfectants ensures an essential decrease in the infectivity of the material if the work with viable infective agents is necessary. Ultraviolet irradiation for 1.5 hours and exposure to the action of 0.1-0.5% sodium azide are less effective.