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 rickettsiaspecific 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.
The recent discovery of cat fleas (Ctenocephalidesfelis) infected with a typhuslike rickettsia (designated the ELB agent) raises the question of whether similar rickettsial infections exist in wild cat flea populations. We verified the presence of the ELB agent and Rickettsia typhi in urban and suburban areas of Los Angeles, Calif. Opossums trapped in close proximity to the residences of human murine typhus cases in Los Angeles county and other areas within the city of Los Angeles were tested for the presence of typhus group rickettsiae by the polymerase chain reaction (PCR). The presence of rickettsiae in the spleen tissues of three opossums (n = 9) and in 66 opossum fleas (n = 205) was determined by PCR and was verified by dot blot and Southern transfer hybridization. Further analysis of the amplified PCR products generated by a series of primer pairs derived from either the 17-kDa antigen gene or the citrate synthase gene revealed that both R. typhi and the ELB agent were present in the tested samples. Dual infection was not noted in the samples; however, the fleas were infected with either R. typhi or the ELB agent. The presence of the ELB agent in the cat flea population may have implications for public health. Whether this agent is responsible for the mild cases of human murine typhus in urban and suburban areas of Los Angeles or in other endemic foci remains to be determined.
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.
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.
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