Adult females of R. sanguineus infected with R. rickettsii were used in this study, which was performed in the Laboratory of Parasitary Diseases of the Department of Preventive Veterinary Medicine and Animal Health - VPS, Veterinary Medicine and Zootechny College of USP - University of Sao Paulo, SP, Brazil, under the supervision of Prof. Dr. Marcelo Bahia Labruna.
Two phases of infestation were necessary for the experiment, in accordance to Piranda et al. . In the first phase, six guinea pigs were divided in two groups, the control group with three individuals (C1, C2, C3) and the infected group also with three individuals (I1, I2, I3). In each guinea pig of the infected group a 3 mL solution containing brains (n°: 7/9/2009) and liver (n°: 1-16/03/09) of guinea pigs positive for R. rickettsii and brain-heart infusion (BHI) was inoculated intraperitoneally after asepsis in the abdominal region according to the protocol described by Labruna et al. . The temperature of the guinea pigs was measured daily during the whole feeding period in order to confirm the infection. After the feeding period the fully engorged nymphs were placed in a biochemical oxygen demand (BOD) incubator at 27°C, remaining there for 48 days. During this period the nymphs completed ecdysis, reaching the adult phase.
In the second phase of infection, six other guinea pigs were used, being divided into two groups (control and infected). The individuals from the control group were infested by adult R. sanguineus from the control groups of the first feeding period, while the individuals from the infected group were infested by R. sanguineus adults from the infected group of the first feeding period. As in the first phase, the temperature of the guinea pigs was measured daily for confirmation of infection.
Analysis of the ticks
Females of R. sanguineus, which had fed for 5 days and the fully engorged females were prepared for the hemolymph test to confirm the infection and the ovaries were removed to be processed for histology. Ten females from the control group and 10 from the infected group were kept in a freezer at -20°C, for the extraction of DNA and performance of PCR according to the protocol described by Labruna et al. , for the confirmation of infection by R. rickettsii.
The performance of the test followed the protocol described by Burgdorfer , where the distal portion of one of the front legs of the ticks was cut with scissors and one or two drops of hemolymph were placed onto a glass slide previously cleaned and de-greased. The slides were then fixed at room temperature, stained by the method of Gimenez  and examined and photographed using a Leica photomicroscope, in the Histology Laboratory of the Biology Department of the Biosciences Institute of UNESP campus Rio Claro (SP), Brazil.
For the performance of histological techniques the ovaries were removed and fixed in 4% paraformaldehyde and in 10% neutral-buffered formaldehyde solution (pH 7-7.4) and acetone in the proportion 9:1 for 1 hour at room temperature and for 30 minutes at 4°C. The material was then dehydrated in increasing concentrations of ethanol (70%, 80%, 90% and 95%), for 15 minutes each, transferred to embedding resin and sectioned with a microtome in 3 μm-thick sections, which were collected on glass slides, rehydrated in distilled water for 1 minute and stained with solution containing Giemsa (8 g), Glycerol (500 mL) and Methanol (buffered pH 6.8, 1000 mL), dissolved in a buffered solution of NaOH4 (1:50), for 40 minutes, and washed in buffer. After drying, the slides were diaphanized in xylol, mounted in synthetic Canada balsam and covered with a coverslip. The material was observed and photographed using a Leica photomicroscope in the Histology Laboratory of the Biology Department of the Biosciences Institute of UNESP campus Rio Claro (SP), Brazil.