Mem Inst Oswaldo Cruz, Rio de Janeiro, 91(3) May/Jun 1996
Original Article

Primary Isolation of Spotted Fever Group Rickettsiae from Amblyomma cooperi Collected from Hydrochaeris hydrochaeris in Brazil

Elba Regina Sampaio de Lemos
+, Heloísa Helena Barbosa MellesII, Sílvia ColomboII, Raimundo Diogo MachadoI, José Rodrigues Coura, Maria Angélica Arpon GuimarãesI, Selênio R SanseverinoI, Aline MouraI

Departamento de Medicina Tropical, Instituto Oswaldo Cruz, Av. Brasil 4365, 21045-900 Rio de Janeiro, RJ , Brasil
IDepartamento de Virologia, Instituto de Microbiologia, Universidade Federal do Rio de Janeiro, Cidade Universitária, 21941-590 Rio de Janeiro, RJ, Brasil
IISetor de Rickettsia do Serviço de Virologia, Instituto Adolpho Lutz, Av. Dr. Arnaldo 355, São Paulo, SP, Brasil

Page: 273-275
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This paper reports the first isolation of a spotted fever group rickettsia from anu00a0Amblyomma cooperiu00a0ixodid collected from a capybara (Hydrochaeris hydrochaeris) in an endemic area of spotted fever in the County of Pedreira, State of São Paulo, Brazil. Isolation was performed in Vero cell culture and submitted to immunofluorescence, using antibody from Rickettsia rickettsii-positive human serum.

Since humans are accidental victims of spotted fever, a tick-borne rickettsiosis, the epidemiology of this exanthematic febrile disease depends primarily on the species and ecology of the tick vector involved (Brezina et al. 1973, Burgdorfer 1975). In Brazil, most human cases of this rickettsiosis described have been limited to the southeastern region states where dozens of confirmed cases have occurred in the last two decades (Gonçalves et al. 1981, Lemos 1991, Souza et al. 1991, Dietz et al. 1992, Melles et al. 1992). In Brazil, Amblyomma cajennense is considered the most important ixodid vector and although the studies performed by the 1950's identified other arthropod vectors as potential vector, naturally infected ticks were rarely reported (Dias 1938, Dias & Martins 1939, Travasssos & Vallejo-Freire 1944-1945, Magalhães 1952, Aragão & Martins 1961, McDade & Newhouse 1986).

Considering the limited available information on this rickettsiosis in Brazil, principally in relation to the vectors involved in perpetuating it in foci, we designed a project in an endemic area in order to expand knowledge on this zoonosis. This paper provides partial results of attempts at isolating rickettsiae from ticks collected in the County of Pedreira, State of São Paulo.



Three female A. cooperi tick specimens were collected from a capybara (Hydrochaeris hydrochaeris) captured at the workers' settlement of the Nadir Figueiredo factory located in the County of Pedreira (22°44'21" S, 46°54'27" W) in October 1994. After cleaning with 3% hydrogen peroxide, 10% formaldehyde solution, 70% alcohol, and sterile distilled water, the ixodidae were stored in test tubes containing Snyder solution at -70°C until the moment of inoculation in cell culture.

The initial inoculum was prepared with the triturated ticks in 1.0 ml of BHI (brain-heart infusion). The suspension was centrifuged at 700 X g for 10 min at O°C and 0.2 ml of the supernatant was inoculated in a confluent monolayer of Vero cells on circular slides adapted to the flat-bottomed tubes. Prior to inoculation, the cell cultures were maintained with Eagle growth medium without antimicrobials for 24-48 hr. The tubes containing the inocula were centrifuged at 1500-1800 X g at 25°-30°C for 1 hr. After decantation of the inoculum, 1.0 ml of modified Eagle minimum medium was added containing 5% bovine fetal serum, 10 mg/ml of gentamicin, 100 mg/ml of vancomycin, and 2.5 mg/ml of amphotericin B. Following incubation for five days at 37°C, the second passage was performed with one of the three tubes into new cell culture tubes, using growth medium with one third the dosage of antimicrobials used in the previous stage. Assessment of the inoculated cell cultures for presence of infection was performed on the slides from the other two tubes, using Giménez stain (Elisberg & Bozeman 1979) and the immunofluorescence reaction prepared with standard Rickettsia rickettsii-positive human serum.



A rickettsia strain from the spotted fever group was isolated, and despite the absence of a cytopathic effect on the culture cells, the immunofluorescence reaction and Giménez stain were considered positive. In the immunofluorescence reaction, we were able to observe the presence of microorganisms in the form of intracellular bacteria, including intranuclear ones, and a fluorencent intensity of 4 + (Fig.).

The presence of a rickettsia strain from the spotted fever group isolated from A. cooperi suggests that this rickettsia, as yet unidentified, is prevalent at the endemic region in the County of Pedreira. The fact that this tick species almost exclusively infests the capybara, a very abundant animal in the region, leads us to believe that this animal is important in perpetuating rickettsiosis there.

Further information will be obtained subsequently when we evaluate the isolate genetically, correlating it with other strains isolated in Brazil.



To Mayor's Office and population of Pedreira, particularly Mr Adauto Cavalcanti, for their cooperation in the field work; Prof. Nicolau M Serra-Freire for helping identify the ixodes, and Mrs Lina Cristina for her technical assistance.



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+Corresponding author. Fax: 55-21-280.3740
Received 30 November 1995
Accepted 28 February 1996
Supported by CNPq and FIOCRUZ.

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