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Molecular identification of Rickettsia typhi and R. felis in co-infected Ctenocephalides felis (Siphonaptera: Pulicidae).

Rickettsia typhi and R. felis, 2 closely related rickettsial species, often have been identified in cat fleas, Ctenocephalides felis (Bouché) from the same geographical location. However, no fleas have been found to be naturally infected concurrently with both rickettsial species. To examine whether one rickettsial species can develop simultaneously with another species in the same flea host, cat fleas, naturally infected with R. felis, were allowed to feed on blood containing R. typhi (Ethiopian strain, 10(7) PFU/ml). Experimental controls consisted of uninfected cat fleas, fleas infected with only R. typhi; and fleas naturally infected with only R. felis. After 9 d at 28 degrees C, the fleas were examined by PCR amplification and subsequent restriction digest analysis and dot blot hybridization of PCR products. Results from these studies demonstrated that R. felis and R. typhi are capable of co-existing in the same flea host. Subsequent isolation of R. typhi from dually infected fleas by tissue culture indicated that R. typhi was viable and capable of being maintained in fleas naturally infected with R. felis. As more studies confirm the presence of R. felis and R. typhi in domestic pets and peridomestic vertebrates in urban areas, the ability of the individual cat fleas, which live on these animals, to support both rickettsial species could be an epidemiologically important consideration.

Animals↗

Molecular and functional analysis of the Rickettsia typhi groESL operon.

The groESL operon from an obligate, intracellular, Gram-negative bacterium Rickettsia typhi, the etiologic agent of murine typhus, was cloned and sequenced. The sequence analysis of 2229 bp of the groESL operon reveals two open reading frames of 288 nucleotides (groES) and 1653 nucleotides (groEL) separated by 20 nucleotides. The deduced amino acid sequence of R. typhi GroES and GroEL shows a high degree of identity with other bacterial GroES and GroEL. Reverse transcriptase-polymerase chain reaction and Northern blot analysis indicated that both groES and groEL are transcribed as a single mRNA. The transcriptional start point at 81 nucleotides upstream of the groES start codon was determined by primer extension. The promoter analysis shows no regulatory CIRCE element as it is known for many Gram-positive and Gram-negative bacteria. However, it contains the sequence similar to the putative sigma(70)-dependent promoter and lacks the -35 sequence of the putative sigma(32)-dependent promoter. Complementation assay by R. typhi groESL in a temperature sensitive Escherichia coli groEL mutant restored significant growth ability at non-permissive temperature.

Amino Acid Sequence↗

Separation of viable Rickettsia typhi from yolk sac and L cell host components by renografin density gradient centrifugation.

Rickettsia typhi cultivated in the yolk sac of chicken embryos or in L cells irradiated 7 days previously was separated from host cell components by two cycles of Renografin density gradient centrifugation. Preliminary steps involved differential centrifugation and centrifugation over a layer of 10% bovine plasma albumin of infected yolk sac suspensions, or trypsinization and passage through filters of wide porosity of infected L cell suspensions. Rickettsial preparations obtained by these methods appeared to be free from host cell components while retaining high levels of hemolytic activity, egg infectivity, and capacity to catabolize glutamate. Average yields were 3.3 mg of rickettsial protein per yolk sac or 0.44 mg per 16-oz (ca. 475-ml) L cell culture. Extracts from these two preparations displayed malate dehydrogenase activity of electrophoretic mobility identical to each other but quite different in migration patterns from the corresponding host cell enzymes. This method of separation of rickettsiae from host cell constituents appears to be particularly well suited for the study of rickettsial enzymatic activity.

Animals↗

Suppression of Rickettsia typhi transmission in fleas maintained on murine typhus-immune rats.

The presence of host antibodies against Rickettsia typhi in the hemolymph of Xenopsylla cheopis fed on immune rats was studied using an indirect immunofluorescent antibody (IFA) test and enzyme-linked immunosorbent assay (ELISA). The time course experiment revealed that antibody to R. typhi appears in the flea hemolymph after 18 hr exposure to host and persists for 24 hr. IFA tests utilizing specific antisera to rat IgG (Fc) and rat IgG (Fab) fragments, indicate that some unaltered rat IgG to R. typhi were present in the hemolymph of immune fed fleas. Host antibody to R. typhi was detected on the surface of the rickettsiae in the flea hemolymph and gut contents by IFA. Maintenance of infected fleas on immune host had no significant effect on the establishment and subsequent growth of rickettsiae within the fleas. However, 19-22 day infected fleas maintained on immune hosts failed to transmit R. typhi to baby rats.

Animals↗

Characterization of the gene encoding the protective paracrystalline-surface-layer protein of Rickettsia prowazekii: presence of a truncated identical homolog in Rickettsia typhi.

The DNA sequence of the gene encoding the protective surface protein antigen (SPA) of Rickettsia prowazekii has been determined. The open reading frame of 4836 nucleotides with promoter and ribosome-binding site is present on a 10.1-kilobase EcoRI fragment. The encoded carboxyl terminus of the 169-kDa protein contains a potential transmembrane region and hydrophilic regions with many lysine and arginine residues potentially accessible to proteolytic cleavage. Because the rickettsia-derived SPA has an estimated molecular mass of only 120 kDa and does not contain several predicted large carboxyl-region CNBr fragments, the SPA product appears to be processed by the rickettsiae. Eight other CNBr fragments were identical in sequence to those predicted from the encoded gene. A complementary 8.7-kilobase EcoRI fragment of Rickettsia typhi DNA was cloned. This fragment lacked a 1433-base-pair region that included the promoter, ribosome-binding site, and the initial 1162 base pairs of the open reading frame encoding the R. prowazekii SPA but had a 3674-base-pair region identical with the remainder of the R. prowazekii SPA gene sequence.

Amino Acid Sequence↗

Adenine nucleotide degradation by the obligate intracellular bacterium Rickettsia typhi.

Adenosine 5'-triphosphate (ATP) was catabolized by whole cells and cell-free extracts of Rickettsia typhi to adenosine 5'-diphosphate (ADP) and then to adenosine 5'-monophosphate (AMP), the end product of ATP catabolism under the experimental conditions used. The only intermediate of the pathway from ATP to AMP which was identified by thin-layer chromatography and quantitated by the (14)C content was ADP, whereas products such as adenine, adenosine, hypoxanthine, inosine, and inosine 5'-monophosphate were not detected. The enzymes which could be theoretically responsible for the catabolism or the anabolism of AMP were not detected by standard assay procedures. Most importantly, 5'-nucleotidase or nonspecific phosphatase and AMP nucleosidase activities were undetectable under a variety of experimental conditions. Although these two enzymes remove AMP from the adenylate pool in other cells, they are apparently nonfunctional in R. typhi. The biosynthesis of ATP was initiated by adenylate kinase because no adenine phosphoribosyltransferase or adenosine kinase could be detected. Furthermore, AMP was transported intact without prior dephosphorylation. These observations suggest that for R. typhi the in vivo activity of adenine nucleotide interconversion was limited to the nucleotides, with AMP being the end product of ATP catabolism, and that the salvage of purine bases and nucleosides was not an essential feature of purine metabolism. These results elucidate the findings of a previous study which showed that in the absence of glutamate as a source of energy, the adenylate energy charge of resting cells of R. typhi is drastically lowered by the high proportion of AMP.

ATP Synthetase Complexes↗

Prevalence of antibodies to Coxiella burnetti, Rickettsia conorii, and Rickettsia typhi in seven African countries.

The prevalences of antibodies reactive with Coxiella burnetti, Rickettsia conorii, and Rickettsia typhi were determined by indirect fluorescent antibody testing of sera from seven African countries. The seroprevalences of antibodies reactive with C. burnetti and R. conorii in countries from North Africa were similar to those reported from southern Europe. In countries of sub-Saharan Africa, the seroprevalence of antibodies reactive with C. burnetti varied greatly; the generally higher figures for West Africa, where stock breeding is prominent, suggested that domestic animals might be the main reservoirs of infection. The prevalence of antibodies to the recently described species Rickettsia africae was higher than that reported for spotted fever-group rickettsiae from elsewhere in the world and paralleled the distribution of Amblyomma species. Western blot results suggested that the antibodies detected were more likely to be reactive with R. africae than with R. conorii, the main vector of which (Rhipicephalus species) rarely feeds on humans. The seroprevalences of antibodies reactive with R. typhi were higher in coastal regions, where Rattus norvegicus--the natural host of the vector Xenopsylla--is more prevalent, than in inland areas.

Africa↗

Evidence of Rickettsia typhi and the potential for murine typhus in Jayapura, Irian Jaya, Indonesia.

Murine typhus (etiologic agent: Rickettsia typhi) is endemic to Indonesia, especially on the highly populated island of Java. A survey of rodents from Irian Jaya, the eastern-most province of Indonesia, indicated striking geographic variation in risk factors associated with murine typhus. Murid rodents (n = 112) collected from two villages in the Arso district of northeastern Irian Jaya, were found to be free of ectoparasites normally associated with transmission of R. typhi (i.e., Xenopsylla cheopis). All rodents (n = 72) tested by enzyme-linked immunosorbent assay were negative for antibodies to R. typhi, whereas 12.5% (9/72) were positive for antibodies to Orienta tsutsugamushi. In contrast, both Rattus norvegicus and R. rattles (combined n = 87) from the harbor area of the provincial capital, Jayapura, were infested with flea ectoparasites. X. cheopis was found on 31 (35.6%) of the live-captured rodents. Serum samples from nine of 82 rodents contained antibodies reactive to R. typhi (11.0%). These data show for the first time that rodents exposed to R. typhi are well established in Jayapura, and that some of these rodents harbor fleas potentially capable of transmitting murine typhus and plague.

Animals↗

Lack of association between Kawasaki syndrome and infection with Rickettsia conorii, Rickettsia typhi, Coxiella burnetii or Ehrlichia phagocytophila group.

BACKGROUND: The etiology of Kawasaki syndrome (KS) is unknown. Rickettsiae, intracellular microorganisms that invade the vascular endothelium, might cause KS. OBJECTIVES: To investigate whether there is an association between KS and infection with Rickettsia conorii, Rickettsia typhi, Coxiella burnetii or Ehrlichia phagocytophila group. METHODS: All children who were diagnosed with KS at the University of Athens Second Department of Pediatrics from December, 1999, through November, 2000, were prospectively studied. Paired serum specimens were obtained from all patients and antibody titers against R. conorii, R. typhi, C. burnetii and E. phagocytophila group were assessed by microimmunofluorescence assay. RESULTS: Eleven children with a median age of 2.5 years were included in the study. A 15-month-old child had a 4-fold rise of antibody titers against C. burnetii, which is indicative of acute Q fever. The patient had a history of recent exposure to possible sources of C. burnetii. The remaining patients tested negative for the presence of antibodies against R. conorii, R. typhi, C. burnetii and E. phagocytophila group. CONCLUSIONS: Our study does not provide serologic evidence that KS is the result of infection with R. conorii, R. typhi, C. burnetii or E. phagocytophila group. It is suggested that C. burnetii may cause a KS-like illness in young children.

Boutonneuse Fever↗

Cloning and sequence analysis of the gene encoding the crystalline surface layer protein of Rickettsia typhi.

The nucleotide sequence of the gene (slpT) encoding the crystalline surface layer protein (SLP) of Rickettsia typhi was determined. The slpT gene consists of 4935 bp coding for a 1645-amino-acid (aa) protein containing a predicted signal peptide at the N terminus. The size of the predicted SLP exceeds the observed size (135 kDa) on SDS-PAGE. The N-terminal aa sequence of the 32-kDa protein of R. typhi reported by Hackstadt et al. [Infect. Immun. 60 (1992) 159-165] was found in the C-terminal portion of the deduced aa sequence, suggesting that the product of slpT is processed into the mature SLP and the 32-kDa protein.

Amino Acid Sequence↗

Prevalence of Rickettsia conorii and Rickettsia typhi infections in the population of northern Greece.

Seroepidemical surveys concerning the prevalence of Rickettsia conorii and Rickettsia typhi have never been studied in northern Greece. We examined 1,584 sera samples from residents of northern Greece for the detection of antibodies to R. conorii and to R. typhi by means of immunofluorescence assay. In addition, we compared the prevalence of rickettsial infections among the demographic variables of sex, age, occupation, and area of residence. Antibodies to R. conorii and R. typhi were found in 125 (7.9%) and 31 (2.0%) of the examined subjects, respectively. The prevalence of antibodies to R. conorii correlated with increasing age and was statistically higher in men. Farmers had significantly higher prevalence of antibodies to both species of rickettsiae studied versus other professions. Residents of rural areas showed a statistically higher prevalence for R. conorii versus urban residents, although this difference was not demonstrated for R. typhi. We also detected differences in the prevalence of rickettsial infections among the different prefectures. Our data show the wide distribution of R. conorii in northern Greece and indicate the presence of R. typhi.

Adolescent↗

Development of a new epitope tag recognized by a monoclonal antibody to Rickettsia typhi.

The epitope recognized by a mouse monoclonal antibody (MAb) to the crystalline surface layer protein of Rickettsia typhi, SRT10, was mapped to 10 amino acid residues (SRTag TFIGAIATDT). The oligonucleotide sequence covering the epitope recognized by SRT10 was inserted into a mammalian expression vector together with multiple cloning sites. When the SRTag was fused in frame to the coding region of the NCC27/CLIC1 gene and expressed in mammalian cells, the MAb SRT10 could detect the tagged protein by immunoblotting, immunocytochemistry, and immunoprecipitation. In addition to the SRT-NCC27/CLIC1, SRT10 could detect N-terminal-tagged MEF2D and C-terminal-tagged CD4 by immunocytochemistry. We suggest that this specific recognition of the SRTag by SRT10 is generally applicable to cellular and molecular biology research that requires the expression and detection of fusion proteins.

Amino Acid Sequence↗

Prevalence of antibodies to Rickettsia typhi in an area of the center of Spain.

It was the aim of the present work to evaluate Rickettsia typhi antibody prevalence in 'Comunidad Autónoma de Madrid' (CAM), an area in the centre of Spain. A total of 640 serum samples from the general population were tested by an indirect immunofluorescence assay. Specific R. typhi antibodies were found in 44 (6.8%) of the samples. The present survey results demonstrate the occurrence of R. typhi human infections in this area.

Adult↗

[Meningitis revealing Rickettsia typhi infection].

INTRODUCTION: Neurological manifestations are rarely observed in murine typhus. We present a case of meningitis caused by Rickettsia typhi. EXEGESIS: We report a case of Tunisian 57-year-old woman admitted for suspicion of meningitis. Clinical examination revealed fever at 39,5 degrees C and nuchal rigidity. There were no focal neurologic signs, cutaneous rash or eschar. Lumbar puncture showed clear cerebrospinal fluid containing normal glucose, 0,48 g/l protein and 30 WBC (78% lymphocyte). Gram-stained smear and culture were negative. Serology confirmed the diagnosis. The patient was initially treated by ampicillin 12 g daily but remained febrile. Retinal lesions were detected on ophthalmic examination, suggesting rickettsial infection. Clinical outcome was good after 7-day treatment with oral ciprofloxacin 1,5 g daily. The mean follow-up was six months. CONCLUSION: Murine typhus is an endemic zoonosis. Neurological manifestations were uncommon. An ophthalmic examination is recommended if rickettsiosis was suspected.

Anti-Infective Agents↗

Epidemiological study of Rickettsia typhi infection in two provinces of the north of Spain: analysis of sera from the general population and sheep.

Data relating to Rickettsia typhi infection in Spain are scarce. A serological survey of 383 serum samples (184 males, 199 females) from the general population and 120 sera from sheep were studied by immunofluorescence assay (IFA). The human serum samples were collected from the general population of Palencia and Burgos provinces, and sheep serum samples were collected from Palencia province. Of the 383 human serum samples studied, 29 were positive for antibodies against R. typhi (7.5%) No statistical differences were found according to age, sex, origin (rural vs. urban) or geographic distribution, but a significant difference was observed related to occupation. In addition, 69 serums were found positives for R. typhi in sheep samples (57.5%). Our results indicated the circulation of R. typhi infectious for humans and sheep in Palencia and Burgos provinces. This study indicates that sheep may be infected with R. typhi, and that animals can, therefore be used as indicators of the presence of this organism.

Adolescent↗