MEM INST OSWALDO CRUZ, RIO DE JANEIRO, 91(3) May/Jun 1996
PAGES: 273-275 DOI: Full paper
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

Abstract

This paper reports the first isolation of a spotted fever group rickettsia from an Amblyomma cooperi ixodid 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.

 

MATERIALS AND METHODS

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.

 

RESULTS AND DISCUSSION

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.

 

ACKNOWLEDGMENTS

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.

 

REFERENCES

Aragão H, Fonseca F 1961. Notas de Ixodologia. VIII. Lista e chave para os representantes da fauna ixodológica brasileira.Mem Inst Oswaldo Cruz 59: 115-148.

Brezina R, Murray ES, Tarizzo ML, Bögel K 1973. Rickettsiae and rickettsial diseases. Bull WHO 49: 433-442.

Burgdorfer W 1975. A review of Rocky Moutain spotted fever (tick-borne typhus), its agent, and its tick vectors in the United States. J Med Entomol 12: 269-278.

Dias E 1938. Depositários naturaes e transmissores da febre maculosa brasileira. Brasil Médico 52: 269-272.

Dias E, Martins AV 1939. Spotted fever in Brazil. A summary. Am J Trop Med 19: 103-108.

Dietz R, Reis JA, Teixeira L, Peçanha P, Falqueto A, Sexton DJ, Corey GR 1992. Febre maculosa brasileira: descrição do primeiro surto da doença no Espírito Santo. An. XXVIII Congresso da Sociedade Brasileira de Medicina Tropical, Programa Resumos p. 29.

Elisberg BL, Bozeman FM 1979. The rickettsiae, p. 1061-1108. In EH Lennet, Diagnostic Procedure, Am Publ Health Assoc.

Gonçalves AJR, Lopes PFA, Melo JCP, Pereira AA, Pinto AMM, Lazera MS, Sousa MLS, Teixeira CRU, Oliveira JC, Duarte F 1981. Rickettsioses a propósito de quatro casos diagnosticados no Rio de Janeiro de febre maculosa. F Méd (Br) 82: 127-134.

Lemos ERS 1991. Aspectos epidemiológicos da riquetsiose do grupo da febre maculosa em uma área endêmica do Estado de Minas Gerais, Brasil. Msc thesis, Instituto Oswaldo Cruz, Rio de Janeiro, 159 pp.

Magalhães O 1952. Contribuição para o conhecimento das doenças do grupo do tifo exantemático. Monog Inst Oswaldo Cruz, Rio de Janeiro, no. 6.

McDade JE, Newhouse VF 1986. Natural history of Rickettsia rickettsii. Ann Rev Microbiol 40: 287-309.

Melles HHB, Colombo S, Silva MV 1992. Febre maculosa: isolamento de rickettsia em amostra de biópsia de pele. Rev Inst Med Trop São Paulo 34: 37-41.

Souza MAA,Paula AB, Primo JDA, Ramos EP, Magalhães SA, Perpétuo AC 1991. Febre maculosa brasileira em Virginópolis - Minas Gerais. VI Congresso Brasileiro de Infectologia, Programa Anais, p. 68.

Travasssos J, Vallejo-Freire A 1944-1945. Criação artificial de Amblyomma cajennense para o preparo da vacina contra a febre maculosa. Mem Inst Butantan 18: 145-235.

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

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