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In vitro activities of telithromycin (HMR 3647) against Rickettsia rickettsii, Rickettsia conorii, Rickettsia africae, Rickettsia typhi, Rickettsia prowazekii, Coxiella burnetii, Bartonella henselae, Bartonella quintana, Bartonella bacilliformis, and Ehrlichia chaffeensis.

In vitro activities of telithromycin compared to those of erythromycin against Rickettsia spp., Bartonella spp., Coxiella burnetii, and Ehrlichia chaffeensis were determined. Telithromycin was more active than erythromycin against Rickettsia, Bartonella, and Coxiella burnetii, with MICs of 0.5 microg/ml, 0.003 to 0.015 microg/ml, and 1 microg/ml, respectively, but was inactive against Ehrlichia chaffeensis.

Anti-Bacterial Agents↗

Serological evidence of infection with Rickettsia typhi and Rickettsia felis among the human population of Catalonia, in the northeast of Spain.

Murine typhus (MT) is a cause of fever of intermediate duration in the south of Spain. Rickettsia typhi has been described as the MT etiological agent. Rickettsia felis produces an infection similar to MT. The aim of the study is to determine their seroprevalence in humans in Catalonia. Antibodies to Rickettsia typhi and Rickettsia felis from 217 serum samples were examined by indirect immunofluorescence assay (IFA). Age, gender, residence area, contact with animals, and occupation were surveyed. Rickettsia typhi and Rickettsia felis seroprevalences were 8.8% and 3.2%, respectively. Rickettsia typhi was present in 7.6% of the samples in urban, 8.5% in semirural, and in 21.4% in rural areas, whereas Rickettsia felis was present in 3.5% in urban, 1.7% in semirural, and 7.1% in rural area. The only statistically significant association observed was that between Rickettsia felis seropositivity and age. Our data seem to indicate the presence of Rickettsia typhi and Rickettsia felis in humans in Catalonia.

Adolescent↗

Structural properties of lipopolysaccharides from Rickettsia typhi and Rickettsia prowazekii and their chemical similarity to the lipopolysaccharide from Proteus vulgaris OX19 used in the Weil-Felix test.

The lipopolysaccharides (LPSs) isolated from typhus group (TG) rickettsiae Rickettsia typhi and Rickettsia prowazekii were characterized by chemical analysis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by silver staining. LPSs from two species of TG rickettsiae contained glucose, 3-deoxy-D-manno-octulosonic acid, glucosamine, quinovosamine, phosphate, and fatty acids (beta-hydroxylmyristic acid and heneicosanoic acid) but not heptose. The O-polysaccharides of these LPSs were composed of glucose, glucosamine, quinovosamine, and phosphorylated hexosamine. Resolution of these LPSs by their apparent molecular masses by SDS-PAGE showed that they have a common ladder-like pattern. Based on the results of chemical composition and SDS-PAGE pattern, we suggest that these LPSs act as group-specific antigens. Furthermore, glucosamine, quinovosamine, and phosphorylated hexosamine were also found in the O-polysaccharide of the LPS from Proteus vulgaris OX19 used in the Weil-Felix test, suggesting that they may represent the antigens common to LPSs from TG rickettsiae and P. vulgaris OX19.

Antigens, Bacterial↗

Isolation of species-specific protein antigens of Rickettsia typhi and Rickettsia prowazekii for immunodiagnosis and immunoprophylaxis.

A simple procedure for the selective isolation of the protective species-specific protein antigens (SPAs) of Rickettsia typhi and Rickettsia prowazekii was developed to permit use of the SPAs in the immunodiagnosis and immunoprophylaxis of typhus infections. Although the SPAs were readily extracted from lysozyme- or detergent-treated rickettsiae, as measured by rocket immunoelectrophoresis, other polypeptides were also present, as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In contrast, both water and seven buffers, each at a 10 mM concentration and pH 7.6, were nearly equally effective in the selective release of the SPAs from whole cells by extraction for 30 min at 45 degrees C. High-ionic-strength buffers and MgCl2 abolished this SPA release, thus suggesting that divalent cations were important in the binding of the SPAs to the cell envelope. The efficacy of the dilute buffer extraction procedure for isolation of large amounts of SPAs was tested by further characterization of the supernatants obtained by centrifugation (200,000 x g) of two successive tris-(hydroxymethyl)aminomethane-hydrochloride buffer (Tris) extracts. With this procedure, between 10 and 15 mg of SPA was obtained from 100 mg of purified rickettsiae. Although low-molecular-weight ribonucleic acid fragments were released into the Tris extracts in significant amounts, only the SPAs were detected, in significant quantities, as measured by polyacrylamide gel electrophoresis and rocket immunoelectrophoresis. The Tris extracts contained the same major and minor SPA polypeptides as those observed previously in SPA preparations obtained by extensive diethylaminoethyl-cellulose column chromatography, but the Tris SPAs were more satisfactory antigens in an enzyme-linked immunosorbent assay.

Antigens, Bacterial↗

Evidence of infection in humans with Rickettsia typhi and Rickettsia felis in Catalonia in the Northeast of Spain.

Murine typhus is a cause of fever of intermediate duration in the south of Spain, where antibodies against Rickettsia typhi and Rickettsia felis were observed in humans. This study presents the first report from the northeast of Spain. Human serum samples were tested by serological test. R. typhi and R. felis seroprevalences were 8.8% and 3.2%, respectively.

Age Distribution↗

Seroprevalence of Rickettsia typhi and Rickettsia conorii infection among rodents and dogs in Egypt.

A serological survey of 1813 rodent and 549 dog sera, collected from 1979 to 1986 from animals in 16 Egyptian Governorates were tested for antibody to Rickettsia typhi and Rickettsia conorii by the indirect fluorescent antibody test. Only three of 82 (4%) sera from Rattus rattus collected near Aswan had antibody to R. conorii. The prevalence of R. typhi antibody in dog sera was only 0.4% (n = 549) while 25% (n = 547) of Rattus norvegicus and 11% (n = 1138) of R. rattus had measurable antibodies. Among the other rodents, antibody was demonstrated in only 2% (n = 45) of Arvicanthis spp., and 1% (n = 83) of Acomys spp. Collectively, rodents captured in the Nile Delta had a higher prevalence (mean 24% (n = 787] than those captured in the Nile Valley (mean 4% (n = 650]. Antibody to R. typhi was detected in rodents collected in all port cities: ismailiya, 13%; Port Said, 9%; Suez, 9%; Safaga, 16%; Quseir, 32% and Alexandria, 34%. These data showed evidence of R. typhi infection among rodents in widespread geographic localities of Egypt and suggested that infected rodents may be a source of human infections.

Animals↗

Development of quantitative real-time PCR assays to detect Rickettsia typhi and Rickettsia felis, the causative agents of murine typhus and flea-borne spotted fever.

Rickettsia typhi and Rickettsia felis are the etiologic agents of murine typhus and flea-borne spotted fever, respectively. We have constructed two quantitative real-time polymerase chain reaction (qPCR) assays to detect these pathogenic rickettsiae. The qPCR assays were developed utilizing unique sequences of the R. typhi and R. felis outer membrane protein B genes (ompB) to design the specific primers and molecular beacon probes. The assays were found to be species-specific and did not yield false-positive reactions with nucleic acid from other rickettsiae, orientiae, neorickettsiae or unrelated bacteria. In addition, the assays were sensitive enough to detect three target sequence copies per reaction and were capable of detecting R. typhi and R. felis nucleic acid in the cat flea, Ctenocephalides felis. These results demonstrate that two sensitive and specific qPCR assays have been successfully developed to detect and enumerate R. typhi and R. felis.

Animals↗

Seroepidemiological study of Rickettsia felis, Rickettsia typhi, and Rickettsia conorii infection among the population of southern Spain.

Rickettsia typhi and Rickettsia conorii, the etiologic agents of, respectively, murine typhus and Mediterranean spotted fever, are recognized as frequent causes of fever of intermediate duration in southern Spain; in addition, in recent years Rickettsia felis has been detected in potential vectors in this area. Nevertheless, limited data exist regarding the actual prevalence of past infection due to these three pathogens. In the present study, the prevalence of past infection due to R. felis, R. typhi, and R. conorii was determined in a representative population of southern Spain during 2002. In addition, the possible risk factors associated with exposure to these pathogens were investigated. An epidemiological survey was completed by all subjects included in the study. Serum samples were tested by indirect immunofluorescence assay. The prevalence of past infection due to R. felis, R. typhi, and R. conorii among the 504 total subjects was 6.5, 3.8 and 8.7%, respectively. In multivariate analysis, infection due to R. felis was independently associated with a high-risk occupation (one that required working outdoors in nature, close contact with domestic animals, or potential contact with rodents) (OR=5.8; 95%CI 2.1-15.6), while infection due to R. typhi was associated with older age (factor of 1.04 [95%CI 1.008-1.068]) and frequent insect bites (OR=10.3; 95%CI 2.3-45.5). Two factors were associated with infection due to R. conorii: a high-risk occupation (OR=9.3; 95%CI 3.7-23.2), and participation in outdoor activities (OR=7.2; 95%CI 1.4-38.5). The results confirm the widespread prevalence of past infection due to R. felis, R. typhi, and R. conorii in the population of southern Spain.

Adolescent↗

Mapping of monoclonal antibody binding sites on CNBr fragments of the S-layer protein antigens of Rickettsia typhi and Rickettsia prowazekii.

The 120 kDa surface protein antigens (SPAs) of typhus rickettsiae lie external to the outer membrane in regular arrays and chemically resemble the S-layer proteins of other bacteria. These proteins elicit protective immune responses against the rickettsiae. In order to study the immunochemistry of these proteins, purified SPAs from Rickettsia typhi and Rickettsia prowazekii were fragmented with CNBr. The fragments were separated by SDS-PAGE and were recovered on PVDF membrane following electroblotting. The origin of eight major fragments from R. prowazekii and seven major fragments from R. typhi was determined by automated N-terminal amino acid sequencing and by comparison with the DNA sequence encoding R. prowazekii SPA. The cleavage patterns and protein sequences of the two proteins differed significantly. CNBr fragments corresponding to the C-terminus (amino acid 1372-1612 of the deduced sequence from encoding gene spaP) were not present in both SPAs. This suggests that the corresponding C-terminal region was not synthesized or was removed during SPA translocation to the cell surface. Modified amino acids were detected in each protein. Eighteen monoclonal antibodies selected for varied reactivity with both native and denatured SPA proteins could be classified into eight different types based on western blot analysis of the CNBr fragments. Six of the monoclonal antibody types reacted predominantly with a single region of the SPAs. Two types of antibodies bound to several CNBr fragments which contained both limited sequence similarity and modified amino acids either of which might account for the multisite binding of these antibodies.

Amino Acid Sequence↗

Partial purification and characterization of the major species-specific protein antigens of Rickettsia typhi and Rickettsia prowazekii identified by rocket immunoelectrophoresis.

Species-specific antigens from Rickettsia typhi and Rickettsia prowazekii were readily solubilized by French pressure cell extraction or sonication of Renografin density gradient-purified rickettsiae and were identified by rocket immunoelectrophoresis. As measured by quantitative rocket immunoelectrophoresis, the species-specific typhus rocket antigens (STRAs) appeared to be proteins; they were denatured by heating at 56 degrees C for 30 min but not by 50 degrees C treatment, and they were sensitive to pronase and trypsin but were not affected by periodate oxidation, glycosidases of various specificities, phospholipase A, or lipase. STRAs from both R. typhi and R. prowazekii were separated from common antigens by DE52 column chromatography of 100,000-X-g supernatant fractions of rickettsial extracts. The purified STRAs were characterized by crossed immunoelectrophoresis, by polyacrylamide gel electrophoresis on Davis and sodium dodecyl sulfate gels, and by an enzyme-linked immunosorbent assay. The two purified STRAs were proteins with similar native electrophoretic mobilities in agarose and polyacrylamide gels, and these proteins had similar polypeptide patterns on sodium dodecyl sulfate gels. Most of the STRA activity migrated as a single protein band on sodium dodecyl sulfate-polyacrylamide and Davis polyacrylamide gels, although minor protein bands with STRA activity were also detected. The major STRA proteins constituted 10 to 15% of the total cellular protein of R. typhi and R. prowazekii. According to sensitive enzyme-linked immunosorbent assay titrations, the STRA of R. prowazekii had substantial cross-reactivity with rabbit antiserum prepared against R. typhi, as shown also by rocket immunoelectrophoresis, whereas the STRA of R. typhi reacted only very weakly with antiserum prepared against R. prowazekii according to the enzyme-linked immunosorbent assay and not at all according to rocket immunoelectrophoresis.

Antigens, Bacterial↗

Metabolism of Rickettsia typhi and Rickettsia akari in irradiated L cells.

L cells that had been exposed to 3,000 r of (60)Co the previous day were used to study the growth and metabolism of Rickettsia typhi and R. akari. Viable (unirradiated) L cells were used to study the effect of rickettsial infection on host-cell metabolism. Monolayers were infected with a rickettsial multiplicity of 1.2 and given Eagle's minimal essential medium containing 25 mmN-2-hydroxyethylpiperazine-N'-2'-ethanesulfonic acid buffer and 10% calf serum. At various intervals, cycloheximide (2 mug/ml) was added to one set of cultures, to inhibit eukaryotic protein and deoxyribonucleic acid (DNA) metabolism; phosphate-buffered saline (PBS) was added to another set. After 1 hr, the cultures received a mixture of 15 (14)C-labeled amino acids or adenine-8-(14)C. The cultures were harvested 16 hr later and were tested for incorporation of labeled carbon into the fraction precipitated by cold trichloroacetic acid. Viable cells were exposed to thymidine-2-(14)C for 2-hr periods. Infectivity of R. typhi increased to a peak of 150 to 400 hemolytic units/culture on day 4; the titer remained approximately the same on days 5 and 6, and declined rapidly on day 7. Total amino acid incorporation was about the same in infected and uninfected cultures up to day 6, but metabolic activity was reduced to a negligible level on day 7 in infected cells. Cycloheximide-resistant activity was higher in the infected cultures, with a peak equivalent to one-half the total activity at day 4 to 5. Total as well as cycloheximide-resistant adenine incorporation was higher in the infected cells between days 3 and 5 after infection, with a peak at day 3 to 4. Somewhat similar results were obtained with R. akari, except that the cycle of infection and of cycloheximide-resistant activity proceeded and was completed more rapidly. (14)C-DNA of both rickettsiae was isolated from infected cultures that had received labeled adenine. With labeled thymidine, which was not incorporated by the rickettsiae, it was shown that R. typhi and R. akari differ considerably in their effects on the host cell. R. typhi elicited moderate inhibition, whereas R. akari infection led to a complete inhibition of thymidine incorporation by the third day, at the time of highest rickettsial activity. It is concluded that rickettsiae have the necessary enzymes for protein and nucleic acid synthesis, but, thus far, these enzymes have been activated or induced only in an intracellular environment.

Adenine↗

Identification and molecular analysis of the gene encoding Rickettsia typhi hemolysin.

Rickettsia typhi, the causative agent of murine typhus, grows directly within the host cell cytoplasm, accumulating a large number of progeny, and eventually lyses the cells. Typhus group rickettsiae (R. typhi and R. prowazekii) adhere to and lyse human and sheep erythrocytes. However, the molecular mechanism underlying erythrocyte lysis by R. typhi has not been defined. Here we describe the cloning and nucleotide sequence analysis of the gene (tlyC) encoding a hemolysin from R. typhi. DNA sequence analysis of R. typhi tlyC revealed an open reading frame of 912 bp, which encodes a protein of 304 amino acids with a predicted molecular mass of 38 kDa. To associate the R. typhi tlyC gene product with hemolytic activity, we performed complementation studies with hemolysin-negative Proteus mirabilis WPM111 (a HpmA(-) mutant of BA6163) transformed with R. typhi tlyC or R. typhi GFPuv-tlyC constructs. We demonstrated that the cloned tlyC gene conferred a hemolytic phenotype on an otherwise nonhemolytic mutant of P. mirabilis. The availability of the cloned R. typhi tlyC will permit further characterization and definition of its role in rickettsial virulence.

Amino Acid Sequence↗

Seroepidemiology of Rickettsia typhi, spotted fever group rickettsiae, and Coxiella burnetti infection in pregnant women from urban Tanzania.

Immunofluorescent antibody (IFA) testing was performed on sera drawn from 150 pregnant women in the port city of Dar es Salaam, Tanzania. Prevalence of antibodies to Rickettsia typhi was 28%, higher than in any of the 12 other African countries in which serosurveys using IFA testing have been performed. Seroprevalence of antibodies to spotted fever group rickettsiae antigens was 25.3%, comparable with that found in other sub-Saharan countries endemic for Amblyomma ticks. Only 4.7% of women were seropositive for Coxiella burnetii.

Adolescent↗

Rickettsial antibody prevalence in southern Israel: IgG antibodies to Coxiella burnetii, Rickettsia typhi, and spotted fever group rickettsiae among urban- and rural-dwelling and Bedouin women.

A retrospective serological survey was carried out using sera obtained from women at childbirth in the southern desert region of Israel to determine exposure experience to three rickettsial agents: Coxiella burnetii, Rickettsia typhi, and spotted fever group rickettsiae. Using the indirect fluorescent antibody method for determining IgG antibodies, it was found that about 40% of all sera examined demonstrated antibodies to one or more rickettsiae. Bedouin women appeared to be at greater risk of having antibodies to C. burnetii and spotted fever group rickettsiae than did Jewish residents of Beersheba, agricultural settlements, and development towns. The residents of development towns appeared to be at lower risk of developing antibodies to spotted fever group rickettsiae than did other populations sampled. Possible reasons for these differences are discussed.

Antibodies, Bacterial↗

Antibodies to Orientia tsutsugamushi, Rickettsia typhi and spotted fever group rickettsiae among febrile patients in rural areas of Malaysia.

A serosurvey was conducted in 1995-97 among 1596 febrile patients from 8 health centres in Malaysia for antibodies against Orientia tsutsugamushi (OT), Rickettsia typhi (RT) and TT118 spotted fever group rickettsiae (SFGR) by using an indirect immunoperoxidase assay. A total of 51.4% patients had antibody against at least 1 of those rickettsiae. Antibody to SFGR was most prevalent (42.5%), followed by RT (28.1%) and OT (24.9%). The seroprevalences of antibodies to SFGR, RT or OT alone were 12.4, 3.6 and 4.3%, respectively. Antibodies against more than 1 species of rickettsiae were presence in 31.1% of the patients, suggesting the possibility of co-infection, previous exposures or serological cross-reactivities. Seroprevalence of the various rickettsiae varied according to locality, with SFGR antibodies being the most prevalent in most areas. There was no significant association of prevalence of rickettsial antibody with gender. The seroprevalence of OT, SFGR and RT increased with patient age but an increase of antibody titre with age was not significant. Those working in the agricultural sectors had significantly higher seroprevalence of OT, SFGR and RT than those not related with agricultural activities. Scrub typhus remains a public health problem with an estimated annual attack rate of 18.5%. Tick typhus and murine typhus as shown in this serosurvey appear much more widespread than scrub typhus in this country.

Adolescent↗

Seroepidemiologic survey of Orientia tsutsugamushi, Rickettsia typhi, and TT118 spotted fever group rickettsiae in rubber estate workers in Malaysia.

The seroprevalence of Orientia tsutsugamushi, Rickettsia typhi, and TT118 spotted fever group (SFG) rickettsiae in 300 rubber estate workers in Slim River, Malaysia was determined in December 1996 and March 1997. In December, which was the wet season, 23.3%, 3.0%, and 57.3% of the population had antibodies detected against the three rickettsiae, respectively. The highest seropositive rate of 40% was detected for single infection with SFG rickettsiae, followed by a rate of 15.3% for both O. tsutsugamushi and SFG rickettsiae among the rubber estate workers. Subjects less than 21 years old had a lower seroprevalence of SFG rickettsiae compared with the other age groups. Indians had a higher seroprevalence of O. tsutsugamushi compared with other ethnic groups. Rubber tappers had a higher seroprevalence of SFG rickettsiae compared with other occupational groups. During the dry season in March 1997, there was a significant increase in the seroprevalence of R. typhi. The seroconversion rates for IgM against O. tsutsugamushi, R. typhi, and SFG rickettsiae were 5.7%, 12.3%, and 15.1%, respectively, during the four-month period. Significant variations of antibody titers towards the three rickettsiae was noted among subjects who were bled twice. This suggests a significant and continual exposure of rubber estate workers to the three rickettsiae.

Adolescent↗

Antibody prevalence of Orientia tsutsugamushi, Rickettsia typhi and TT118 spotted fever group rickettsiae among Malaysian blood donors and febrile patients in the urban areas.

The seroprevalence of Orientia tsutsugamushi (OT), Rickettsia typhi (RT) and TT118 spotted fever group rickettsiae (SFGR) among blood donors and febrile Malaysian patients in the urban areas was determined. Of the 240 blood donors, 5.4%, 9.2% and 1.7% had either present or previous exposure to OT, RT and SFG rickettsiae, respectively. Patients admitted to an urban hospital had high seroprevalences of OT (43.5%) and RT (22.9%), as compared to SFGR (11.6%). Antibody levels suggestive of recent infections of scrub typhus, murine typhus and tick typhus were detected in 16.8%, 12.7% and 8.2% of patients respectively. No significant difference was noted in the distribution of rickettsial antibodies among urban patients from 2 geographical locations. However, the serologic patterns of rickettsial infection in the urban areas were different form those of rural areas.

Adolescent↗

Rickettsia typhi infection in childhood.

UNLABELLED: Rickettsia typhi infection (murine typhus) is generally underdiagnosed in childhood, as clinical presentations are often non-specific. We present the manifestations in nine children hospitalized in the Department of Paediatrics of the University Hospital, Heraklion, Crete, over a 3-y period from 1998 to 2000. Titres > 1:400 for IgM and >1:960 for IgG and/or a fourfold increase in a second sample were considered strongly suggestive of acute infection. Children presented with prolonged fever, hepatosplenomegaly and lymphadenopathy. Five children presented with a rash. Unusual manifestations included aseptic meningitis and Kawasaki-like presentation. Laboratory findings included anaemia, leucopenia, and thrombocytopenia. Three children were treated with appropriate antibiotic regimens and all nine had a complete recovery. CONCLUSION: Rickettsia typhi infection should be considered in the differential diagnosis of children residing in or returning from Southern Europe countries who present with prolonged fever, rash and lymphadenopathy.

Adolescent↗