Detection of murine typhus infection in fleas by using the polymerase chain reaction.

Polymerase chain reaction (PCR) amplification of DNA was used to detect the etiologic agent of murine typhus, Rickettsia typhi, in experimentally infected adult fleas. A primer pair derived from the 17-kilodalton antigen sequence of typhus and spotted fever group rickettsiae was used to amplify a 434-base-pair (bp) fragment of the genome of the murine typhus rickettsiae. The amplified 17-kilodalton protein antigen-specific sequence was detected in ethidium bromide-stained agarose gels in individual fleas as early as 2 days after exposure to rickettsemic rats (two of six tested). The 434-bp sequence was not detected in uninfected control fleas. A dot hybridization assay used to detect the 434-bp fragment was also specific and about 100-fold more sensitive than the agarose gel PCR assay. Since the PCR assay employed a boiled extract of triturated fleas, both PCR and an antigen capture enzyme-linked immunosorbent assay (ELISA) could be performed on the same individual flea homogenate. The ELISA identified 12 infected fleas out of 29 randomly selected fleas, compared with 14 specimens which were positive by PCR. The PCR assay detected rickettsiae in samples in which no viable rickettsiae were detected by plaque assay. Like the ELISA, the PCR assay sensitivity was due in part to its suitability for detecting small numbers of both live and dead R. typhi in fleas.     

Identification of a novel rickettsial infection in a patient diagnosed with murine typhus.

Identification of ELB agent-infected fleas and rodents within several foci of murine typhus in the United States has prompted a retrospective investigation for this agent among human murine typhus patients. This agent is a recently described rickettsia which is indistinguishable from Rickettsia typhi with currently available serologic reagents. Molecular analysis of the 17-kDa antigen gene and the citrate synthase gene has discriminated this bacterium from other typhus group and spotted fever group rickettsiae. Current sequencing of its 16S ribosomal DNA gene indicates a homology of 98.5% with R. typhi and 99.5% with R. rickettsii. Through a combination of restriction fragment length polymorphism and Southern hybridization analysis of rickettsia-specific PCR products, one of five tested patient blood samples was shown to be infected with ELB while R. typhi infections were confirmed in the remaining samples. This is the first reported observation of a human infection by the ELB agent and underscores the utility of PCR-facilitated diagnosis and discrimination of these closely related rickettsial infections.     

Isolation, cultivation, and partial characterization of the ELB agent associated with cat fleas.

ELB rickettsiae from cat flea homogenates were recovered in tissue culture cells following sequential passage through laboratory rats and the yolk sacs of embryonated chicken eggs. Seven days after inoculation of ELB from the infected yolk sacs, Vero cells and L929 cells were observed to contain intracellular bacteria as demonstrated by Diff Quik and indirect immunofluorescence assay staining. The rickettsial and ELB identity of the cultured agent was confirmed by PCR detection of the 16S rRNA and citrate synthase genes and PCR-restriction fragment length polymorphism analysis of the 17-kDa conserved rickettsial antigen gene. The ELB rickettsiae induced plaques in Vero cells on day 11 postinfection. Rat anti-ELB serum reacted at 1:4,096 to cultured ELB and had lower reactivity to Rickettsia typhi Wilmington (1:1,024), Rickettsia akari Kaplan (1:512), and Rickettsia australis JC (1:64). Spotted fever group polyclonal sera also exhibited lower reactivity to ELB than to the homologous antigen. Coomassie blue-stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis profiles of the ELB isolate and two R. typhi strains were identical.     

Rickettsial infections of fleas collected from small mammals on four islands in Indonesia

Ectoparasites were sampled from small mammals collected in West Java, West Sumatra, North Sulawesi, and East Kalimantan, Indonesia, in 2007-2008 and were screened for evidence of infection from bacteria in the Rickettsaceae family. During eight trap nights at eight sites, 208 fleas were collected from 96 of 507 small mammals trapped from four orders (379 Rodentia; 123 Soricomorpha; two Carnivora; three Scandentia). Two species of fleas were collected: Xenopsylla cheopis (n = 204) and Nosopsyllus spp. (n = 4). Among the 208 fleas collected, 171 X. cheopis were removed from rats (Rattus spp.) and 33 X. cheopis from shrews (Suncus murinus). X. cheopis were pooled and tested for DNA from rickettsial agents Rickettsia typhi, Rickettsia felis, and spotted fever group rickettsiae. R. typhi, the agent of murine typhus, was detected in X. cheopis collected from small mammals in West Java and East Kalimantan. R. felis was detected in X. cheopis collected from small mammals in Manado, North Sulawesi. R. felis and spotted fever group rickettsiae were detected in a pool of X. cheopis collected from an animal in East Kalimantan. Sixteen percent of the X. cheopis pools were found positive for Rickettsia spp.; four (10.8%) R. typhi, one (2.7%) R. felis, and one (2.7%) codetection of R. felis and a spotted fever group rickettsia. These data suggest that rickettsial infections remain a threat to human health across Indonesia.     

Prevalence of vectors of the spotted fever group Rickettsiae and murine typhus in a Bedouin town in Israel

A survey of the vectors of spotted fever group Rickettsiae and of murine typhus was carried out in Rahat, a Bedouin town in the Negev Desert, where the diseases are endemic. Houses with known cases of spotted fever group Rickettsiae or murine typhus were compared with those without reported clinical cases. A neighboring Jewish community, Lehavim, where no cases of spotted fever group Rickettsiae and murine typhus were reported in recent years, was used as a control. In the houses of patients with spotted fever group Rickettsiae in Rahat, an average of 7.4 times more ticks were found than in control houses. Out of 190 ticks isolated from sheep and goats or caught by flagging in Rahat, 90% were Rhipicephalus sanguineus (Latreille), 7.9% Rhipicephalus turanicus Pomerantzev, and 2.1% were Hyalomma sp. In the houses of patients with murine typhus, three times more rats were caught and, on the average, each rat was infested with 2.2 times more fleas than rats in the control houses. Out of 323 fleas collected from 35 Norwegian rats (Rattus norvegicus Berkenhout), 191 were Xenopsylla cheopis Rothschild and 132 Echidnophaga murina Tiraboschi. Thus, there was a six to seven times higher probability of encountering a tick or flea vector where infections had occurred than in control houses in Rahat. The percentage of rats seropositive to Rickettsia typhi was similar in study and control households (78.3 and 76.2, respectively). In the control settlement, Lehavim, only three Mus musculus L. were caught, which were not infested with ectoparasites and their sera were negative for murine typhus. Out of 10 dogs examined in this settlement, 15 R. sanguineus and eight specimens of the cat flea (Ctenocephalides felis felis Bouché) were isolated. No rats were caught in this settlement. These data indicate that there is a correlation among the density of domestic animals, their ectoparasites, and the incidence of spotted fever group Rickettsiae and murine typhus in Rahat.     

Detection and characterization of mouse monoclonal antibodies to epidemic typhus rickettsiae.

A solid-phase immunofluorometric assay was used to detect mouse monoclonal antibodies to epidemic typhus rickettsiae, Rickettsia prowazekii (the immunizing antigen), and to murine typhus rickettsiae, Rickettsia typhi, a related antigen. Of the 649 hybridoma cultures obtained, 628 contained antibodies either to R. prowazekii or to both R. prowazekii and R. typhi. A total of 72 cultures were cloned by limiting dilution and yielded 137 antibody-producing clones. Of these, 104 produced antibodies specific for R. prowazekii, 22 produced antibodies that reacted with R. prowazekii and R. typhi, and 11 produced antibodies that reacted with R. prowazekii, R. typhi, and R. canada. The immunoglobulin isotypes of the mouse monoclonal antibodies produced were identified by a related indirect immunofluorometric assay technique with fluorescein isothiocyanate-conjugated antisera specific for each isotype. Antibodies were also evaluated by indirect fluorescent antibody tests, and antibodies from selected clones were found to neutralize rickettsial toxic activity in mice.     

Serological differentiation of murine typhus and epidemic typhus using cross-adsorption and Western blotting

Differentiation of murine typhus due to Rickettsia typhi and epidemic typhus due to Rickettsia prowazekii is critical epidemiologically but difficult serologically. Using serological, epidemiological, and clinical criteria, we selected sera from 264 patients with epidemic typhus and from 44 patients with murine typhus among the 29,188 tested sera in our bank. These sera cross-reacted extensively in indirect fluorescent antibody assays (IFAs) against R. typhi and R. prowazekii, as 42% of the sera from patients with epidemic typhus and 34% of the sera from patients with murine typhus exhibited immunoglobulin M (IgM) and/or IgG titers against the homologous antigen (R. prowazekii and R. typhi, respectively) that were more than one dilution higher than those against the heterologous antigen. Serum cross-adsorption studies and Western blotting were performed on sera from 12 selected patients, 5 with murine typhus, 5 with epidemic typhus, and 2 suffering from typhus of undetermined etiology. Differences in IFA titers against R. typhi and R. prowazekii allowed the identification of the etiological agent in 8 of 12 patients. Western blot studies enabled the identification of the etiological agent in six patients. When the results of IFA and Western blot studies were considered in combination, identification of the etiological agent was possible for 10 of 12 patients. Serum cross-adsorption studies enabled the differentiation of the etiological agent in all patients. Our study indicates that when used together, Western blotting and IFA are useful serological tools to differentiate between R. prowazekii and R. typhi exposures. While a cross-adsorption study is the definitive technique to differentiate between infections with these agents, it was necessary in only 2 of 12 cases (16.7%), and the high costs of such a study limit its use.     

Detection of Rickettsia rickettsii and Bartonella henselae in Rhipicephalus sanguineus ticks from California

Sixty-two questing adult Rhipicephalus sanguineus (Latreille) ticks were collected by direct removal from blades of turfgrass and adjacent concrete walkways at a suburban home in Riverside County, CA, and tested for the presence of Rickettsia, Bartonella, and Ehrlichia DNA. Polymerase chain reaction (PCR) was used to amplify fragments of the 17-kDa antigen gene and the rOmpA gene of the spotted fever group rickettsiae. One male tick contained R. rickettsii DNA; its genotype differed from R. rickettsii isolates found in Montana and Arizona that cause Rocky Mountain spotted fever and from Hlp#2 and 364D serotypes. One male tick and one female tick contained B. henselae DNA. No Ehrlichia platys or Ehrlichia canis DNAs were detected using nested PCR for their 16S rRNA genes. These findings extend the area where Rickettsia rickettsii may be vectored by Rh. sanguineus. Rh. sanguineus also may be infected with Bartonella henselae, a human pathogen that is typically associated with fleas and causes cat scratch disease.     

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.     

Rickettsia felis: a new species of pathogenic rickettsia isolated from cat fleas.

A flea-borne rickettsia, previously referred to as ELB, has been implicated as a cause of human illness. Using sequence data obtained from a fragment of the citrate synthase gene, we compared ELB, Rickettsia australis, R. rickettsii, and R. akari with the louse-borne R. prowazekii. We tallied 24 base pair differences between ELB and R. prowazekii and 25 between R. rickettsii and R. prowazekii; there were 30 base pair differences between R. australis and R. prowazekii and 29 between R. akari and R. prowazekii. We observed 32 differences between Rickettsia typhi and ELB. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analyses of ELB, with typing sera against R. typhi indicate that ELB surface antigens are more closely related to the flea-borne R. typhi than to the mite-borne R. akari. On the basis of the results of citrate synthase gene sequence comparisons, as well as previous comparisons with 16S rRNA and 17-kDa-protein gene segments, we found that ELB is sufficiently genetically distinct from other rickettsiae to be designated a new species, Rickettsia felis.     

Heterogeneity of CD4-positive human T-cell clones which recognize the surface protein antigen of Rickettsia typhi

Immunity to the typhus group of rickettsiae is largely dependent on the effector function of several classes of T lymphocytes, including those which produce gamma interferon. Since the surface protein antigen (SPA) derived from typhus group rickettsiae has been shown to be an effective immunogen in animal models, human T-cell clones specific for the SPA of Rickettsia typhi were isolated and tested for their antigenic specificity, as well as for their ability to produce gamma interferon. Eighteen CD4-positive clones specific for the SPA of R. typhi exhibited considerable diversity in their response to the SPAs derived from two strains of Rickettsia prowazekii and from Rickettsia canada. The vast majority of clones also recognized the SPAs from R. prowazekii but not from R. canada. Two heteroclitic clones demonstrated significantly higher proliferative responses to the SPAs derived from one or both of the R. prowazekii strains than to the SPA of R. typhi, and one clone demonstrated a significantly higher response to the SPA of R. typhi than to the other SPAs. All 18 clones produced gamma interferon in response to SPA stimulation. We conclude that the SPAs from typhus group rickettsiae can elicit both a diverse T-cell response in humans and the efficient stimulation of gamma interferon-mediated immunity.     

Studies on the growth of Bartonella henselae in the cat flea (Siphonaptera: Pulicidae)

Two out of three pools of cat fleas, Ctenocephalides felis (Bouche), that were fed Bartonella henselae-positive cat blood for 3 d and then bovine blood for 3 d, were polymerase chain reaction (PCR) positive for B. henselae. In a second experiment, three cats were inoculated with a streptomycin-resistant strain of B. henselae. After the cats were inoculated, caged cat fleas were fed on the cats during three different periods, and then pooled and transferred to noninfected recipient cats. In the first trial, the bacteria in the flea feces were below level of detection when the fleas were transferred from the infected cats to the recipient cat. After the fleas had fed on the recipient cat for 6 d, a bacteria level of 4.00 x 10(3) CFU/ mg was detected in the flea feces. Subsequently, the bacteria level increased for 4 d and then declined. In another experiment, the bacteria level in the flea feces was 1.80 x 10(3) CFU/mg at 2 h after collection and 3.33 x 10(2) CFU/mg at 72 h after collection. These data indicated that this strain of B. henselae can persist in flea feces in the environment for at least 3 d, and that B. henselae can multiply in the cat flea.     

Establishment of a novel endothelial target mouse model of a typhus group rickettsiosis: evidence for critical roles for gamma interferon and CD8 T lymphocytes

A mouse model of typhus rickettsiosis that reproduces the hematogenous dissemination to the critical target organs, including brain, lungs, heart, and kidneys, primary endothelial and, to a lesser degree, macrophage intracellular rickettsial infection, and typical vascular-based lesions of louse-borne typhus and murine typhus was established. Intravenous inoculation of C3H/HeN mice with Rickettsia typhi caused disease with a duration of the incubation period and mortality rate that were dependent on the infective dose of rickettsiae. Lethal infection was associated with high concentrations of R. typhi in the lungs and brain, despite a brisker humoral immune response to the rickettsiae than in the sublethal infection. Gamma interferon and CD8 T lymphocytes were demonstrated to be crucial to clearance of the rickettsiae and recovery from infection in experiments in which specific monoclonal antibodies were administered to deplete these components. Death of animals depleted of gamma interferon or CD8 T lymphocytes was associated with overwhelming rickettsial infection demonstrated by titers of infectious rickettsiae and by immunohistochemistry. An effective antirickettsial immune response was associated with elevated serum concentrations of IL-12 on Day 5 and increased secretion of IL-12 by concanavalin-A-stimulated spleen cells on Day 5. Evidence for transient suppression of the immune response consisted of marked reduction in the secretion of IL-2 and IL-12 by concanavalin-A-stimulated spleen cells on Days 10 and 15. This model offers excellent opportunities for study of attenuation and pathogenetic mechanisms of typhus rickettsiae, which are established biologic weapons of potential use in bioterrorism.     

Detection of Rickettsia tsutsugamushi in Experimentally infected mice by PCR.

We developed a rapid procedure for the detection of Rickettsia tsutsugamushi DNA by the PCR technique. The primer pair used for the PCR was designed from the DNA sequence of the gene encoding a 120-kDa antigen, which was proven to be group specific by immunoblot analysis with mouse hyperimmune sera against various rickettsial strains. This PCR method was able to detect up to 10 ag of plasmid DNA (pKT12). Specific PCR products were obtained with DNAs from R. tsutsugamushi Kato, Karp, Gilliam, TA716, TA1817, and Boryong, but not with DNAs from other rickettsiae, such as R. prowazekii, R. typhi, R. akari, and strain TT118. In a study with experimentally infected mice, the PCR method could detect rickettsial DNA from 2 days after inoculation (DAI), whereas serum antibody against R. tsutsugamushi could be detected from 6 to 8 DAI by an immunofluorescence test. Although clinical manifestations subsided after 14 DAI, rickettsial DNA in blood samples could be detected by PCR for up to 64 DAI. These results suggest that this PCR method can be applied to the early diagnosis of scrub typhus and can also be used to detect the residual rickettsiae after clinical symptoms subside.     

Mechanisms of immunity in typhus infections. V. Demonstration of Rickettsia mooseri-specific antibodies in convalescent mouse and human serum cytophilic for mouse peritoneal macrophages.

Antibodies in both mouse and human Rickettsia mooseri (Rickettsia typhi) convalescent serum that were cytophilic for mouse macrophages were demonstrated by the rosette technique. Mouse peritoneal macrophages, passively sensitized with early and late serum from mice with a sublethal infection of R. mooseri, were washed and exposed to rickettsiae. Rosettes of rickettsiae were found around macrophages, maintained at 4 degrees C, which had been sensitized with immune serum (direct sensitization of macrophages), but no rosettes were found around macrophages sensitized with serum from normal mice. When the macrophages were maintained at 34 degrees C after addition of the rickettsiae, phagocytosis of rickettsiae occurred, indicating one probable role for cytophilic antibodies in typhus infections. If the rickettsiae were mixed with serum from infected mice, washed, and then added to macrophages (indirect sensitization of macrophages), more rosettes were found around the macrophages than around directly sensitized macrophages. The presence of mouse immunoglobulin G on the macrophage surface was also shown by staining living sensitized macrophages with rabbit fluroescein-conjugated anti-mouse immunoglobulin G.     

Development and characterization of high-titered, group-specific fluorescent-antibody reagents for direct identification of rickettsiae in clinical specimens.

Rabbits were inoculated with purified antigen preparations of Coxiella burnetii and representative species of the spotted fever and typhus groups of rickettsiae. Their antibody responses were monitored by complement fixation tests; high-titered antisera were fractionated with ammonium sulfate and then labeled with fluorescein isothiocyanate by the dialysis method. The conjugates had homologous 3+ staining titers of 1:256 to 1:2,048 and did not exhibit nonspecific staining. The Rickettsia rickettsii, R. conorii, and R. akari conjugates reacted only with rickettsiae of the spotted fever group; the R. canada, R. prowazekii, and R. typhi conjugates were specific for the typhus group rickettsiae; and the C. burnetii conjugate stained only homologous organisms. One of these conjugates (R. rickettsii) is currently being used to identify rickettsiae in clinical specimens and has already proven its value as a diagnostic tool.     

Characterization of the Madrid E strain of Rickettsia prowazekii purified by renografin density gradient centrifugation.

The avirulent Madrid E strain of Rickettsia prowazekii cultivated in chicken yolk sacs could be purified successfully with a Renografin density gradient method developed previously for Rickettsia typhi. Recovery during purification, viability, and lack of contamination with host cell components were similar for the two species, although yields of R. prowazekii per yolk sac were lower. Purified typhus rickettsiae provided satisfactory antigens in the complement fixation, Ouchterlony double-diffusion, and microagglutination tests. The retention of the typhus soluble group antigen during purification was readily demonstrated by complement fixation tests. However, removal of the soluble group antigen by ether treatment was not always adequate for the demonstration of type-specific particulate antigens. Heat-killed R. prowazekii cells gave higher serum microagglutination titers than untreated or formalized cells, a difference was noted for R. typhi cells. Although the protein profiles of whole cells and extracts of R. typhi and R. prowazekii on sodium dodecyl sulfate-polyacrylamide gels were relatively similar, a small but reproducible, difference in the electrophoretic mobilities of their malate dehydrogenases was detected. Purification of typhus rickettsiae on Renografin gradients has no apparent adverse effects on their metabolic or antigenic properties.     

Distribution of cat fleas (Siphonaptera: Pulicidae) on the cat

A total of 3,382 cat fleas, Ctenocephalidesfelis (Bouche), was taken from 164 of the 200 stray cats examined. It was observed that cat fleas preferred specific areas on the cat. A significantly higher mean number of fleas was found on the area of head plus neck than on the ventral part of the body. More specifically, the mean number of fleas was highest on both of the neck and dorsal areas. However, in terms of the density of fleas, the neck had more fleas than the dorsal area did. The fewest fleas were found infesting the legs and tail. Distribution of fleas on the cat may well be explained by the various grooming patterns of the cat, and the knowledge of flea distribution may be valuable for application of on-animal flea control procedures.     

Experimental transmission of Bartonella henselae by the cat flea.

Bartonella henselae is an emerging bacterial pathogen, causing cat scratch disease and bacillary angiomatosis. Cats bacteremic with B. henselae constitute a large reservoir from which humans become infected. Prevention of human infection depends on elucidation of the natural history and means of feline infection. We studied 47 cattery cats in a private home for 12 months to determine the longitudinal prevalence of B. henselae bacteremia, the prevalence of B. henselae in the fleas infesting these cats, and whether B. henselae is transmitted experimentally to cats via fleas. Vector-mediated transmission of B.henselae isolates was evaluated by removing fleas from the naturally bacteremic, flea-infested cattery cats and transferring these fleas to specific-pathogen-free (SPF) kittens housed in a controlled, arthropod-free University Animal Facility. B. henselae bacteremia was detected in 89% of the 47 naturally infected cattery cats. A total of 132 fleas were removed from cats whose blood was simultaneously cultured during different seasons and were tested individually for the presence of B. henselae DNA by PCR. B. henselae DNA was detected in 34% of 132 fleas, with seasonal variation, but without an association between the presence or the level of bacteremia in the corresponding cat. Cat fleas removed from bacteremic cattery cats transmitted B. henselae to five SPF kittens in two separate experiments; however, control SPF kittens housed with highly bacteremic kittens in the absence of fleas did not become infected. These data demonstrate that the cat flea readily transmits B. henselae to cats. Control of feline infestation with this arthropod vector may provide an important strategy for the prevention of infection of both humans and cats.     

Expression of green fluorescent protein in Rickettsia conorii

Rickettsiae are obligate intracellular class III pathogens for which genetic manipulation has only recently been shown to be feasible. Such experiments were restricted to the typhus group rickettsiae, namely R. typhi and R. prowazekii. Here we report the first genetic manipulation of Rickettsia conorii, the bacterial agent responsible for the Mediterranean spotted fever. A gene encoding a variant of the green fluorescent protein under the control of the sterically repressed promoter (srp) from E. coli was integrated into the genome of this bacteria and detected by FACS analysis.