Biomedical subjects

D Raoult

Publications and source records attributed to D Raoult.

At least 55 records · Page 3Linked to original sources

PubMed1994

Differentiation among spotted fever group rickettsiae species by analysis of restriction fragment length polymorphism of PCR-amplified DNA.

Restriction fragment length polymorphism (RFLP) analysis of PCR-amplified genes was used to study spotted fever group (SFG) rickettsiae, extending the previous work of Regnery et al. (R.L. Regnery, C.L. Spruill, and B.D. Plikaytis, J. Bacteriol. 173:1576-1589, 1991). Twenty-six strains of SFG rickettsia were studied, including several recognized species which have never been studied (R. parkeri, R. helvetica, and R. japonica) as well as strains which are not currently classified. Two previously used primer pairs derived from the R. prowazekii citrate syntase gene and the R. rickettsii 190-kDa protein antigen gene were studied, as were primer pairs obtained from the R. rickettsii 120-kDa protein antigen gene. By using three amplifications and three enzyme digestions, it was possible to differentiate between almost all of the known SFG rickettsia species and to differentiate between several strains of the R. conorii complex. Two human pathogens, "R. africae" and the Israeli tick typhus rickettsia, were first separated by using BG-12 pair primer amplification and then RsaI restriction endonuclease digestion. The proposed simplified model of identification may be useful in studying the geographical distributions of SFG rickettsiae.

Animals↗

PubMed1994

Isolation and characterization by immunofluorescence, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, western blot, restriction fragment length polymorphism-PCR, 16S rRNA gene sequencing, and pulsed-field gel electrophoresis of Rochalimaea quintana from a patient with bacillary angiomatosis.

Rochalimaea quintana was isolated from the blood of a French human immunodeficiency virus-infected patient with bacillary angiomatosis. The isolate showed the typical growth characteristics of Rochalimaea species and was inert when typical biochemical testing was used. The purpose of the present work was to characterize and compare this new isolate with reference strains of R. quintana, Rochalimaea vinsonii, and Rochalimaea henselae by using immunofluorescence, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), Western blot (immunoblot), restriction fragment length polymorphism-PCR of the citrate synthase gene, 16S rRNA gene sequencing, and pulsed-field gel electrophoresis. SDS-PAGE, Western blot, restriction fragment length polymorphism-PCR with TaqI enzyme, and 16S rRNA gene sequencing could differentiate the three Rochalimaea species and allowed characterization of the French isolate as R. quintana. However, identification of the Rochalimaea isolate to the species level was more easily obtained by immunofluorescence with specific murine antisera. Pulsed-field gel electrophoresis allowed differentiation of the French R. quintana isolate from R. quintana Fuller and may serve as an epidemiological tool.

AIDS-Related Opportunistic Infections↗

PubMed1994

Chronic Q fever in hemodialysis patients.

We describe two cases of chronic Q fever in hemodialysis patients (HD). In the first case, we discovered chronic Q fever when looking for the cause of an unexplained fever. In the second case, Q fever was diagnosed in a patient who complained of an unexplained shoulder arthritis. To our knowledge these are the first reported cases of chronic Q fever in HD.

Adult↗

PubMed1994

Isolation and identification of a rickettsial strain related to Rickettsia massiliae in Greek ticks.

Adult ticks were collected in a rural area of central Greece in order to isolate and identify rickettsiae. A hemolymph test using Gimenez staining was used for detection, while simultaneous isolation was performed using the shell-vial technique. Serologic, antigenic, and genomic characterization of the isolates was achieved by microimmunofluorescence, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting, the polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP), and pulsed-field gel electrophoresis (PFGE), respectively. Although none of the 242 collected ticks was positive by the hemolymph test, one rickettsial isolate, designated GS, was obtained by the shell-vial technique. This isolate originated from a female Rhipicephalus sanguineus. Microimmunofluorescence serologic typing by the method of Philip and others demonstrated that GS belongs to the same serotype as the recently isolated Rickettsia massiliae (Mtu1). Protein analysis by SDS-PAGE and immunoblotting by Western blot revealed similar profiles between the two rickettsiae. Using Alu I, Rsa I, and Pst I restriction endonucleases in PCR-RFLP analysis, GS and R. massiliae were found to possess identical restriction sites. However, PFGE showed differences when the two genomes were digested with Bss HII and Sma I restriction endonucleases, in spite of their equal size. In conclusion, the first rickettsial isolation in Greece was found to be antigenically identical and genotypically close to the French isolate R. massiliae, despite small differences showed by PFGE.

Animals↗

PubMed1994

Identification of rickettsiae from ticks collected in the Central African Republic using the polymerase chain reaction.

Spotted fever rickettsiosis have been identified on the African continent since their historical description in 1909. However, only Rickettsia conorii and R. africae have been described in Africa, and the current techniques for the detection of rickettsiae in ticks are difficult to apply in large field studies. We report here a preliminary study using genomic amplification by the polymerase chain reaction followed by restriction fragment length polymorphism (PCR-RFLP) analysis directly on 310 crushed ticks (Rhipicephalus, Amblyomma, and Haemaphysalis species) collected in 1985 in the Central African Republic. Among 310 specimen tested, 21.6% were positive. The rate of infection ranged from 0% to 64.3%, depending on the tick species. Based on PCR-RFLP, five different rickettsiae profiles were found: R. conorii and R. africae, previously known in Africa, R. rhipicephali, which has never been described in Africa, and two isolates identical to R. massiliae and Mtu5, previously obtained from Rh. turanicus in southern France. This work shows that PCR-RFLP is a powerful tool to study tick collections, and that it is applicable to samples from developing countries. Further work is needed to confirm the identification of the rickettsiae found in this work, using traditional identification procedures.

Animals↗

PubMed1994

Identification of spotted fever group rickettsiae isolated from Dermacentor marginatus and Ixodes ricinus ticks collected in Switzerland.

When 155 ticks collected in different regions of Switzerland were tested by the hemolymph test, 10.3% were found to contain spotted fever group rickettsiae. Six rickettsial isolates were made from Dermacentor marginatus ticks and three were made from Ixodes ricinus ticks. The polymerase chain reaction followed by restriction fragment length polymorphism analysis showed that the Dermacentor ticks were infected with Rickettsia length polymorphism analysis showed that the Dermacentor ticks were infected with Rickettsia slovaca and the Ixodes ticks were infected with a spotted fever group rickettsia. Microimmunofluorescence serologic type, sodium dodecyl sulfate-polyacrylamide gel electrophoresis of proteins, and Western blot assay with polyclonal mouse antisera confirmed the results and determined that the Ixodes were infected with R. helvetica, the only previously described Swiss rickettsia. However, an additional new strain that could not be isolated was detected in one I. ricinus by hemolymph test and provisionally characterized by enzymatic restriction of its amplified DNA.

Animals↗

PubMed1994

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.

Animals↗

PubMed1994

Further evidence for the efficacy of imidocarb dipropionate in the treatment of Ehrlichia canis infection.

Three dogs experimentally infected with Ehrlichia canis developed thrombocytopaenia and high antibody titres to E. canis in indirect fluorescent antibody tests. One dog also became leukopaenic. At Weeks 6 and 8 post-infection, the dogs were treated with imidocarb dipropionate (5 mg kg-1 subcutaneously) and a further dose was administered at Week 12 (5 mg kg-1 intramuscularly). Twelve weeks after the last treatment (post-treatment), all dogs had normal platelet counts which persisted for a further 10 weeks until the end of the experiment. The leukopaenia resolved 20 weeks post-treatment. Although antibody titres (< 1/5) to E. canis could not be detected prior to infection, titres of 1/2 560 to 1/5 120 developed by Week 6. By Week 8 post-treatment titres began to decline and by the end of the experiment were 5- to 6-fold serum dilutions lower (1/80 to 1/320). Sub-inoculation experiments 18 weeks post-treatment, failed to cause disease or stimulate antibody responses in susceptible dogs. Serology and sub-inoculation studies on 2 dogs experimentally infected with E. canis but not treated with imidocarb dipropionate, showed that these animals remained infected for the duration of the experiment. The results of these experiments confirm that imidocarb dipropionate is effective in the treatment of canine ehrlichiosis.

Animals↗

PubMed1994

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.

Animals↗

PubMed1994

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.

Animals↗

PubMed1994

A new pathogenic spotted fever group rickettsia from Africa.

A spotted fever group (SFG) rickettsia was isolated in Zimbabwe from a patient with tick-bite, fever, headache and regional lymphadenopathy. A further six isolates were obtained from Amblyomma hebraeum ticks collected in Zimbabwe. These human and tick isolates were indistinguishable from each other, and from an Ethiopian SFG rickettsia, by microimmunofluorescence (MIF), sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), Western blotting and polymerase chain reaction followed by restriction fragment length polymorphism analysis (PCR-RFLP). They were, however, distinguishable from other SFG ricketsiae by MIF serotyping and in the case of the most closely related serotypes, Rickettsia conorii and the Israeli SFG rickettsia, by SDS-PAGE and Western blot. PCR-RFLP failed to distinguish between the Zimbabwean and Israeli SFG rickettsia, though each of these gave different digestion products from R. conorii. The Zimbabwean human and tick isolates and the Ethiopian SFG rickettsiae therefore represent a previously undescribed rickettsial serotype which apparently is pathogenic in human beings. It is proposed that the new serotype be named the agent of African tick-bite fever in order to distinguish it from R. conorii, which until now has been recognized as the only SFG rickettsia to infect man in Africa.

Animals↗

PubMed1993

Phylogenic homogeneity of Coxiella burnetii strains as determinated by 16S ribosomal RNA sequencing.

DNA coding for the 16S rRNA of six strains of the obligate intracellular bacterium Coxiella burnetii was directly amplified from lysed host cells using the polymerase chain reaction. The amplification product was sequenced using a linear-PCR procedure and compared with other published 16S rRNA sequences. The results of this analysis confirm the position of C. burnetii in the gamma subgroup of the proteobacteria. The data show that all of the C. burnetii strains are highly related (> 99%) on the basis of 16S rRNA sequences although they had different geographic origins and phenotypic characteristics. The data support a phylogenetic homogeneity of the genus Coxiella with only one species which is C. burnetii.

Base Sequence↗

PubMed1993

Demonstration of Rickettsia conorii-induced endothelial injury in vivo by measuring circulating endothelial cells, thrombomodulin, and von Willebrand factor in patients with Mediterranean spotted fever.

The endothelial cell (EC) is the primary target for Rickettsia conorii (RC) in Mediterranean spotted fever (MSF). Clinical manifestations such as thrombosis and vasculitis are mediated by pathologic changes localized in blood vessels. To study the in vivo endothelial injury induced by RC, markers of endothelial damage, including circulating EC (CEC), plasmatic thrombomodulin (TM), and von Willebrand factor (vWF), were investigated in 12 patients with MSF. CEC were counted in whole blood by a new immunomagnetic separation assay using a specific anti-EC antibody, S-Endo 1. Plasmatic TM and vWF antigens were measured by enzyme-linked immunosorbent assay. High levels of CEC and cell fragments were found in patients with a severe or malignant form of MSF. Sequential studies of CEC showed a decrease from 162 +/- 454 cells/mL before treatment to 6 +/- 7 cells/mL during treatment and recovery. Mean plasma TM and vWF levels that were also elevated before therapy (TM, 106 +/- 27 ng/mL; vWF, 420% +/- 164%) decreased progressively (TM, 55 +/- 43 ng/mL; vWF, 148% +/- 26%) during treatment. The measurement of cellular and molecular markers of vascular damage such as CEC, plasmatic TM, and vWF contributes to the definition of the Rickettsia-induced endothelial injury in vivo.

Antibodies, Monoclonal↗

PubMed1993

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.

DNA Restriction Enzymes↗

PubMed1993

Proposed tests for the routine identification of Rochalimaea species.

A study was conducted to establish tests for the routine identification of Rochalimaea species. Strains used were reference strains of Rochalimaea vinsonii and Rochalimaea quintana, and a type strain and six human isolates of Rochalimaea henselae. Rochalimaea species were confirmed to be gram-negative, oxidase-negative, non-motile, urease-negative, indole-negative, catalase-negative, glucose-nonfermenting organisms which failed to grow on MacConkey agar. Further testing of the organisms in a commercial identification system with the addition of hemin (100 micrograms/ml) to the medium revealed biochemical reactivity of the organisms not previously observed. The Voges-Proskauer reaction, tests for hydrolysis of hippurate and esculin, leucine arylamidase activity and the lactose test allowed identification and differentiation of the three species. Rochalimaea henselae was the only species with a positive lactose test and Rochalimaea quintana was the only species with a positive Voges-Proskauer reaction. Further studies are needed to confirm the validity of these tests for identification of Rochalimaea species.

Bacterial Typing Techniques↗

PubMed1993

Confirmation that Rickettsia helvetica sp. nov. is a distinct species of the spotted fever group of rickettsiae.

We propose the name Rickettsia helvetica sp. nov. for a rickettsial serotype of unknown pathogenicity isolated in 1979 in Switzerland from Ixodes ricinus ticks and designated the Swiss agent. The growth characteristics and the results of microimmunofluorescence serologic typing, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blotting (immunoblotting) with specific mice sera, and a polymerase chain reaction followed by restriction fragment length polymorphism analysis confirmed previously reported preliminary findings which suggested that this rickettsia, to which a name was given provisionally, does represent a new member of the spotted fever group of rickettsiae. The type strain is C3 (Reference Center for Rickettsioses, Marseille, France).

Animals↗

PubMed1993

In vitro susceptibilities of spotted fever group rickettsiae and Coxiella burnetti to clarithromycin.

The in vitro bacteriostatic activity of clarithromycin, a new macrolide derivative, against Rickettsia rickettsii, Rickettsia conorii, and "Rickettsia israeli" was determined by the plaque assay and the dye uptake assay. Both bacteriostatic and bactericidal activities of clarithromycin against the Nine Mile, Q212, Priscilla, and ME9 strains of Coxiella burnetti were evaluated by using three cell culture systems. Clarithromycin showed improved antibacterial activity compared with that of erythromycin. A bacteriostatic activity was obtained at concentrations below the reported maximum concentration of clarithromycin in human serum (about 4 micrograms/ml) for all tested rickettsiae. MICs ranged from 1 to 2 micrograms/ml for the three Rickettsia species and from 1 to 4 micrograms/ml for the C. burnetti strains. No bactericidal activity against C. burnetti was obtained when clarithromycin was used at 4 micrograms/ml.

Animals↗

PubMed1993

Genotypic and antigenic identification of two new strains of spotted fever group rickettsiae isolated from China.

Four isolates of spotted fever group rickettsiae isolated from ticks in China were compared with all known species and strains of spotted fever group rickettsiae by immunofluorescence assay, DNA polymerase chain reaction followed by restriction endonuclease fragment length polymorphism analysis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and Western immunoblot. The Chinese isolates belonged to three types, including a novel serotype which has not been described before. One isolate obtained from tick ova of Dermacentor nuttallii in Inner Mongolia was antigenically and genotypically identical to Rickettsia sibirica. Two isolates obtained from Dermacentor sinicus collected from Beijing were identical, different from other members of spotted fever group rickettsiae but apparently closely related to R. sibirica. HA-91, a strain isolated from Hyalomma asiaticum bv. kozlovi olenew, was antigenically and genotypically unique among spotted fever group rickettsiae, and we feel that data presented here should prompt consideration of it as a new species on the basis of current rickettsial taxonomy.

Animals↗