Rickettsia felis in Ctenocephalides spp. fleas, Brazil

Abstract

In June 2000, suspected cases of Brazilian spotted fever (BSF) occurred in Coronel Fabriciano Municipality, Minas Gerais State, Brazil. Pooled fleas collected near two fatal cases contained rickettsial DNA. The nucleotide sequence alignment of the 391-bp segment of the 17-kDa protein gene showed that the products were identical to each other and to the R. felis 17-kDa gene, confirming circulation of R. felis in Brazil.

Figure 1

Map of Brazil and Minas Gerais State, showing Coronel Fabriciano municipality.

Figure 2

Detection of the Rickettsia specific 17-kDa gene by polymerase chain reaction amplification in DNA extracted from ticks and fleas. The vectors were first placed in 1.5-mL microcentrifuge tubes containing 200 µL of 10 mM phosphate-buffered saline, pH 7.4, and were crushed with a micropestle. The suspensions were lysed in 0.5% sodium dodecyl sulfate and incubated with 100 µg/mL proteinase K at 37°C for 1 hour in the case of fleas or overnight in the case of ticks. The lysed suspensions were extracted twice with an equal volume of phenolchloroform, followed by a single chloroform extraction. The extracted DNA was amplified with primer 1 (5′-GCTCTTGCAACTTCTATGTT-3′) and primer 2 (5′-CATTGTTCGTCAGGTTGGCA-3′) as described by Webb et al. for amplification of a 434-bp fragment from the rickettsial 17-kDa protein gene. PCR was performed at 30 cycles for 1 minute at 94°C, 5 minutes at 48°C, and 2 minutes at 72°C. The PCR products were then separated by electrophoresis in 1% agarose gel and stained with ethidium bromide. Lanes 1-3: DNA from cat fleas, Lanes 4-6: DNA from dog fleas, Lane 7: 17- kDa gene Rickettsia felis DNA (Positive Control), Lanes 8-14: DNA from ticks.