Emerging Infectious Disease ISSN: 1080-6059

Rickettsia felis in Ctenocephalides spp. Fleas, Brazil

Riva P. Oliveira*, Márcio A.M. Galvão*

, Claudio L. Mafra*, Chequer B. Chamone†, Simone B. Calic†, Sergio U. Silva‡, and David H. Walker§

Author affiliations: *Universidade Federal de Ouro Preto, Minas Gerais State, Brazil; †Fundação Ezequiel Dias, Minas Gerais State, Brazil; ‡Secretaria Municipal de Saúde de Coronel Fabriciano, Minas Gerais State, Brazil; §University of Texas Medical Branch, Galveston, Texas, USA;

Main Article

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. (10) 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.

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. (10) 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.

Main Article

Top of Page

The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the U.S. Department of Health and Human Services, the Public Health Service, the Centers for Disease Control and Prevention, or the authors' affiliated institutions. Use of trade names is for identification only and does not imply endorsement by any of the groups named above.