Biological properties of antibiotic-resistant mutants of the mildly pathogenic Rickettsia prowazekii E strain

The use of R. prowazekii strain E with low pathogenicity as live vaccine against exanthematous typhus is limited by its high specific reactogenicity, which is probably due to the reversion of the virulence of the strain. One of the approaches to the stabilization of the avirulent properties of strain E is obtaining its mutants with stable decreased pathogenic properties. The article presents the data on the infectious properties of R. prowazekii antibiotic-resistant strain E mutants obtained in earlier experiments, in respect of chick embryos and laboratory animals, as well as the capacity of this strain for producing immunity to challenge with R. prowazekii virulent strain in guinea pigs. The study has revealed that the erythromycin-resistant mutant of R. prowazekii strain E, induced by nitrosoguanidine (NG), has lower infective capacity for chick embryos, guinea pigs, cotton rats and white mice. The infective capacity of the NG-induced rifampicin-resistant and spontaneous erythromycin-resistant mutants of R. prowazekii strain E is similar to the infective capacity of the initial strain. The rifampicin-resistant and spontaneous erythromycin-resistant mutants of R. prowazekii strain E possess immunogenicity similar to that of the initial strain E, and the NG-induced erythromycin-resistant mutant possesses lower, but sufficiently pronounced immunogenicity despite its decreased infective capacity for guinea pigs.     

Use of a method of immunoradiometric analysis for detecting the persistence of Rickettsiae and their antigens in an experimental infection in cotton rats

An indirect modification of the radioimmunoassay (RIA) was shown to be highly sensitive in the determination of the minimal amounts of R. prowazekii and their antigens in the organs and tissues of infected animals. RIA proved to be effective in the determination of the antigens in the blood in cases of R. prowazekii persistence in infected animals. The data obtained in these investigations point to good prospects for using RIA in the study of persistence of microorganisms and their antigens.     

Antibiotic sensitivity of the erythromycin-resistant strain E of Rickettsia prowazekii cultured in the vector's body

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.     

Establishment of a replicating plasmid in Rickettsia prowazekii

Rickettsia prowazekii, the causative agent of epidemic typhus, grows only within the cytosol of eukaryotic host cells. This obligate intracellular lifestyle has restricted the genetic analysis of this pathogen and critical tools, such as replicating plasmid vectors, have not been developed for this species. Although replicating plasmids have not been reported in R. prowazekii, the existence of well-characterized plasmids in several less pathogenic rickettsial species provides an opportunity to expand the genetic systems available for the study of this human pathogen. Competent R. prowazekii were transformed with pRAM18dRGA, a 10.3 kb vector derived from pRAM18 of R. amblyommii. A plasmid-containing population of R. prowazekii was obtained following growth under antibiotic selection, and the rickettsial plasmid was maintained extrachromosomally throughout multiple passages. The transformant population exhibited a generation time comparable to that of the wild type strain with a copy number of approximately 1 plasmid per rickettsia. These results demonstrate for the first time that a plasmid can be maintained in R. prowazekii, providing an important genetic tool for the study of this obligate intracellular pathogen.     

A murine model of infection with Rickettsia prowazekii: implications for pathogenesis of epidemic typhus

Epidemic typhus remains a major disease threat, furthermore, its etiologic agent, Rickettsia prowazekii, is classified as a bioterrorism agent. We describe here a murine model of epidemic typhus that reproduced some features of the human disease. When BALB/c mice were inoculated intravenously with R. prowazekii (Breinl strain), they survived but did not clear R. prowazekii infection. Immunohistological analysis of tissues and quantitative PCR showed that R. prowazekii was present in blood, liver, lungs and brain 1 day after infection and persisted for at least 9 days. Importantly, infected mice developed interstitial pneumonia, with consolidation of the alveoli, hemorrhages in lungs, multifocal granulomas in liver, and hemorrhages in brain, as seen in humans. Circulating antibodies directed against R. prowazekii were detected at day 4 post-infection and steadily increased for up to 21 days, demonstrating that R. prowazekii lesions were independent of humoral immune response. R. prowazekii-induced lesions were associated with inflammatory response, as demonstrated by elevated levels of inflammatory cytokines including interferon-gamma, tumor necrosis factor and the CC chemokine RANTES in the lesions. We concluded that the BALB/c mouse strain provides a useful model for studying the pathogenic mechanisms of epidemic typhus and its control by the immune system.     

Reduction of ribonucleotides by the obligate intracytoplasmic bacterium Rickettsia prowazekii.

Rickettsia prowazekii, an obligate intracellular parasitic bacterium, was shown to have a ribonucleotide reductase that would allow the rickettsiae to obtain the deoxyribonucleotides needed for DNA synthesis from rickettsial ribonucleotides rather than from transport. In the presence of hydroxyurea, R. prowazekii failed to grow in mouse L929 cells or SC2 cells (a hydroxyurea-resistant cell line), which suggested that R. prowazekii contains a functional ribonucleotide reductase. This enzymatic activity was demonstrated by the conversion of ADP to dADP and CDP to dCDP, using (i) a crude extract of Renografin-purified R. prowazekii that had been harvested from infected yolk sacs and (ii) high-performance liquid chromatographic analysis. The rickettsial ribonucleotide reductase utilized ribonucleoside diphosphates as substrates, required magnesium and a reducing agent, and was inhibited by hydroxyurea. ADP reduction was stimulated by dGTP and inhibited by dATP. CDP reduction was stimulated by ATP and adenylylimido-diphosphate and inhibited by dATP and dGTP. These characteristics provided strong evidence that the rickettsial enzyme is a nonheme iron-containing enzyme similar to those found in mammalian cells and aerobic Escherichia coli.     

Flying squirrels and their ectoparasites: disseminators of epidemic typhus

Information gathered during the past decade indicates that the eastern flying squirrel, Glaucomys volans, is a zoonotic reservoir of Rickettsia prowazekii - causative agent of louse-borne (epidemic) typhus. The sporadic cases o f typhus that have occurred in the USA in association with flying squirrels provide evidence that flying squirrels can transmit R. prowazekii infection to humons. Strains of R. prowazekii, isolated from flying squirrels multiply readily in human body lice, but flying squirrel lice, although readily infected, are very host specific and tend not to bite humans. It may be that the infection is spread to humans in infective ectoporasite faeces aerosolized when the flying squirrels groom themselves. As Joseph McDade emphasizes in this article, current concepts of typhus epidemiology and control must be re-evaluated to take into account this zoonotic aspect.     

Immunogenicity of a chemical typhus vaccine and of a corpuscular radioantigen obtained from Rickettsia prowazekii

The protective activity of chemical typhus vaccine and R. prowazekii corpuscular radioantigen (CRA) was studied. Guinea pigs were immunized with doses of 32 and 48 antigenic units. Antibody production was assayed in the complement fixation test. On days 7, 15, 21, 30 and 60 after immunization the animals were challenged with R. prowazekii introduced in an amount of 10(5) minimum embryonal infective doses (MEID). On day 30 some of the animals were challenged with 10(3) MEID of R. typhi. The results demonstrated that both preparations were highly immunogenic and capable of protecting most of the animals from 10(5) MEID of R. prowazekii. Immunity developed earlier after immunization with CRA. The guinea pigs immunized with CRA, purified in percoll density gradient, and challenged with 10(3) MEID of R. typhi on day 30 showed a high level of cross immunity. In all control animals high fever and periorchitis were observed.     

The molar ratio of σ73 to core polymerase in the obligate intracellular bacterium, Rickettsia prowazekii

In the obligate Intracellular parasitic bacterium, Rickettsia prowazekii, the molar ratio of σ⁷³ to core RNA polymerase, that is, the degree of saturation of the core polymerase by the catalytically active sigma factor, was very low. This ratio was determined from the radioactivity in rickettsial RNA polymerase immuno-precipitated from crude extracts of infected L929 cells in which the parasite was exponentially growing. If we assume that, as Is true for the σ subunit, in R. prowazekii and Escherichia coli the β’and β subunits of the RNA polymerase have similar methionine and cysteine contents (the radiolabelled amino acids), the molar ratio of σ⁷³ to core polymerase in R prowazekii would be 0.1. This is in striking contrast to E. Coli where the ratio is typically 0.4. it remains to be established whether this low sigma saturation results In a limitation of active RNA polymerase in R. prowazekii and contributes to its slow growth.     

Isolation and characterization of the Rickettsia prowazekii recA gene.

The recA gene has been isolated from Rickettsia prowazekii, an obligate intracellular bacterium. Comparison of the amino acid sequence of R. prowazekii RecA with that of Escherichia coli RecA revealed that 62% of the residues were identical. The highest identity was found with RecA of Legionella pneumophila, in which 69% of the residues were identical. Amino acid residues of E. coli RecA associated with functional activities are conserved in rickettsial RecA, and the R. prowazekii recA gene complements E. coli recA mutants for UV light and methyl methanesulfonate sensitivities as well as recombinational deficiencies. The characterized region upstream of rickettsial recA did not contain a sequence homologous to an E. coli LexA binding site (SOS box), suggesting differences in the regulation of the R. prowazekii recA gene.     

Combined action of penicillin, tetracycline and rifampicin on R. sibirica and R. prowazekii in experiments on chick embryos

The effect of combinations of penicillin, tetracycline and rifampicin on R. prowazekii (the causative agent of typhus) and R. sibirica (the causative agent of tick-borne rickettsiosis of the North Asia) was studied. It was shown that tetracycline and penicillin used in combination had a summation effect on both R. sibirica and R. prowazekii. The dose of each antibiotic was 2 times lower than the doses of the antibiotics used alone. However, R. sibirica was less sensitive to this combination than R. prowazekii: the minimum rickettsiocidic doses of the combination were 0.5 mg of tetracycline + 10000 units of penicillin per embryo with respect to R. sibirica and 0.1 mg of tetracycline + 10000 units of penicillin per embryo with respect to R. prowazekii. The combinations of rifampicin with penicillin or tetracycline in the concentrations used had no rickettsiocidic effect on either R. sibirica or R. prowazekii. However, it should be noted that these combinations had a synergistic action and provided a rickettsiostatic effect on R. prowazekii: the dose of rifampicin in its combination with penicillin was decreased 10 times and in the combination of rifampicin with tetracycline the doses of both rifampicin and tetracycline were decreased 10 times. Still, penicillin even in a dose of 20000 units per embryo had only a rickettsiostatic effect on R. sibirica and R. prowazekii.     

Isolation and characterization of the gene coding for the major sigma factor of Rickettsia prowazekii DNA-dependent RNA polymerase.

The gene coding for the major sigma factor of Rickettsia prowazekii, an obligate intracellular parasitic bacterium, has been isolated utilizing an oligodeoxyribonucleotide as a probe to a conserved region of major sigma factors. Nucleotide sequence analysis revealed an open reading frame of 1905 bp that could encode a protein of 635 amino acids (aa) with a calculated molecular size of 73 kDa (sigma 73). R. prowazekii sigma 73 displayed extensive homology with major sigma factors from a variety of eubacteria. Comparison of the major sigma factors from Escherichia coli and R. prowazekii revealed 44.9% aa identity. R. prowazekii sigma 73 produced in E. coli minicells migrated as a 85-kDa protein when analyzed by sodium dodecyl sulfate-polyacrylamide-gel electrophoresis. This anomalous migration is characteristic of eubacterial major sigma factors and agrees with the migration noted for the purified rickettsial sigma protein. Despite a similarity to the E. coli sigma 70 encoded by rpoD, R. prowazekii sigma 73 did not complement E. coli rpoD temperature-sensitive mutants.     

Action of tetracycline and rifampicin on Rickettsia prowazekii and Rickettsia sibirica

The effect of tetracycline and rifampicin on R. prowazekii, strain Breinl and R. sibirica, strain X1 was studied in the experiments with chick embryos exposed to the antibiotic mixture with the infection material. It was shown that tetracycline in doses of 0.1 and 1 mg/embryo had the rickettsiostatic and rickettsiocidic effects respectively on R. sibirica. Rifampicin had only the rickettsiostatic effect in a dose of 0.1 mg/embryo and no rickettsiocidic effect even in a dose of 2 mg/embryo. Higher doses were toxic for 100 per cent of the embryos. The rickettsiostatic and rickettsiocidic effects of tetracycline on R. prowazekii were evident in doses of 0.05 and 0.1 mg/embryo, respectively. Rifampicin in a dose of 0.05 mg/embryo had both the rickettsiostatic and the rickettsiocidic effects on R. prowazekii. Therefore, rifampicin was more active with respect to R. prowazekii and tetracycline was more active with respect to R. sibirica. In addition, R. sibirica was more resistant to both tetracycline and rifampicin as compared to R. prowazekii.     

Experimental Infection of the Cotton Rat Sigmodon hispidus with Rickettsia rickettsii

Studies of experimental infection of the cotton rat, Sigmodon hispidus, with the virulent Sheila Smith (R type) and the avirulent Si 7 (U type) strains of Rickettsia rickettsii were undertaken to evaluate the role of this native wild mammal in the ecology of Rocky Mountain spotted fever. The Sheila Smith strain, which was highly lethal for guinea pigs, was nonpathogenic for cotton rats. Serial passage of the R-type strain in the cotton rat did not alter the virulence of the agent for cotton rats or guinea pigs. The U-type strain, which was originally recovered from a wild cotton rat, could not be maintained beyond the first passage in this animal host. Rickettsemia in the cotton rat occurred over a 24-hr period after inoculation of the virulent strain but was detected only 1 hr after inoculation of the avirulent strain. The short period of rickettsemia suggests that the cotton rat probably is not an important reservoir of R. rickettsii. Specific complement-fixing antibodies developed rapidly after infection with either strain, but the antibodies evoked by the R strain attained higher titers and persisted longer. Cotton rats previously infected with the Sheila Smith strain developed rickettsemia after reinfection with the same strain, even though relatively high levels of antibody were still present.     

Survey for zoonotic rickettsial pathogens in northern flying squirrels, Glaucomys sabrinus, in California

Epidemic typhus, caused by Rickettsia prowazekii, is maintained in a southern flying squirrel (Glaucomys volans) sylvatic cycle in the southeastern United States. The northern flying squirrel (Glaucomys sabrinus) has not been previously associated with R. prowazekii transmission. A second rickettsial pathogen, Anaplasma phagocytophilum, infects dusky-footed woodrats (Neotoma fuscipes) and tree squirrels in northern California. Because northern flying squirrels or their ectoparasites have not been tested for these rickettsial pathogens, serology and polymerase chain reaction (PCR) were used to test 24 northern flying squirrels for R. prowazekii and A. phagocytophilum infection or antibodies. Although there was no evidence of exposure to R. prowazekii, we provide molecular evidence of A. phagocytophilum infection in one flying squirrel; two flying squirrels also were seropositive for this pathogen. Fleas and ticks removed from the squirrels included Ceratophyllus ciliatus mononis, Opisodasys vesperalis, Ixodes hearlei, Ixodes pacificus, and Dermacentor paramapertus.     

Monoclonal antibodies to the species-specific and group antigens of Rickettsia prowazekii]

A number of hybridomas to different R. prowazekii determinants were obtained by the hybridization of spleen cells of BALB/c mice immunized with R. prowazekii corpuscular and soluble antigens. Some of the monoclonal antibodies (McAb) reacted with R. prowazekii thermolabile species-specific protein and did not react with R. typhi antigens (McAb of batches B4/4 and A-D3). McAb C5/2 and A3/2 reacted with the group thermostable antigen, common for R. prowazekii and R. typhi. McAb to the species-specific thermolabile antigen belonged to IgG2a. The McAb thus obtained permit the identification of R. prowazekii and R. typhi and the solution of the problem of the intragroup differentiation of rickettsiae belonging to the typhus group.     

Transformation of Rickettsia prowazekii to erythromycin resistance encoded by the Escherichia coli ereB gene

Rickettsia prowazekii, the etiologic agent of epidemic typhus, is an obligate, intracytoplasmic, parasitic bacterium. Recently, the transformation of this bacterium via electroporation has been reported. However, in these studies identification of transformants was dependent upon either selection of an R. prowazekii rpoB chromosomal mutation imparting rifampin resistance or expression of the green fluorescent protein and flow cytometric analysis. In this paper we describe the expression in R. prowazekii of the Escherichia coli ereB gene. This gene codes for an erythromycin esterase that cleaves erythromycin. To the best of our knowledge, this is the first report of the expression of a nonrickettsial, antibiotic-selectable gene in R. prowazekii. The availability of a positive selection for rickettsial transformants is an important step in the characterization of genetic analysis systems in the rickettsiae.     

Tuberculin skin test and interferon-gamma release assay values are associated with antimicrobial peptides expression in? polymorphonuclear cells during latent tuberculous infection

It has been reported that patients with progressive tuberculosis (TB) express abundant amounts of the antimicrobial peptides (AMPs) cathelicidin (LL-37) and human neutrophil peptide-1 (HNP-1) in circulating cells, whereas latent TB infected donors showed no differences when compared with purified protein derivative (PPD) and QuantiFERON^®-TB Gold (QFT)-healthy individuals. The aim of this study was to determine whether LL-37 and HNP-1 production correlates with higher tuberculin skin test (TST) and QFT values in TB household contacts. Twenty-six TB household contact individuals between 26-58 years old TST and QFT positive with at last two years of latent TB infection were recruited. AMPs production by polymorphonuclear cells was determined by flow cytometry and correlation between TST and QFT values was analysed. Our results showed that there is a positive correlation between levels of HNP-1 and LL-37 production with reactivity to TST and/or QFT levels. This preliminary study suggests the potential use of the expression levels of these peptides as biomarkers for progression in latent infected individuals.     

Seroimmunologic monitoring of microorganisms of Rickettsia and Bartonella species microorganisms in the Moscow region

Serological study of 788 blood sera, taken from residents of the Moscow region was conducted using antigens of microorganisms of the genera Rickettsia and Bartonella. The first group under examination consisted of 355 patients with diagnosed diseases of nonreckettsial nature. The second group includes 433 healthy adults working at a meat processing and packing factory. The main method used for sera survey was the indirect immunofluorescence test. In the sera taken from the first group of subjects specific antibodies to R. prowazekii, R. typhi, B. quintana, B. henselae antigens were detected in 2.3%, 5.1%, 4.0% and 2.9% of serum samples respectively. In the serum samples taken from the second group the proportion of antibodies to R. prowazekii, R. typhi, B. quintana, B. henselae antigens was different: 0.5%, 3.3%, 1.7% and 4.0% respectively. In total, specific antibodies to R. typhi and B. henselae prevailed over specific antibodies to R. prowazekii and B. quintana twofold.     

Biochemical and immunochemical study of antigen preparations of Rickettsia prowazekii

The biochemical and immunochemical study of the qualitative composition of R. prowazekii antigenic preparations, isolated and purified by different methods, indicates that these preparations contain high-molecular polypeptide 3 (133600 D), morphologically linked with the cell membrane of R. prowazekii. The method of adsorption chromatography on calcium phosphate permits obtaining specific rickettsial antigens with a greater degree of purification from ballast admixtures than the methods of acid precipitation and gel filtration on Sephadex G-200.