Memórias do Instituto Oswaldo Cruz Fundação Oswaldo Cruz, Fiocruz ISSN: 1678-8060 EISSN: 1678-8060 Vol. 91, Num. 3, 1996, pp. 273-275

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

Elba Regina Sampaio de Lemos/^+, Heloisa Helena Barbosa Melles**, Silvia Colombo**, Raimundo Diogo Machado*, Jose Rodrigues Coura, Maria Angelica Arpon Guimar es*, Selenio R Sanseverino*, Aline Moura*

Departamento de Medicina Tropical, Instituto Oswaldo Cruz, Av. Brasil 4365, 21045-900 Rio de Janeiro, RJ , Brasil *Departamento de Virologia, Instituto de Microbiologia, Universidade Federal do Rio de Janeiro, Cidade Universitaria, 21941-590 Rio de Janeiro, RJ, Brasil **Setor de Rickettsia do Servico de Virologia, Instituto Adolpho Lutz, Av. Dr. Arnaldo 355, S o Paulo, SP, Brasil

Code Number: OC96054
Size of Files:
    Text: 9K
    Graphics:

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 S o Paulo, Brazil. Isolation was performed in Vero cell culture and submitted to immunofluorescence, using antibody from Rickettsia rickettsii-positive human serum.

Key words: Rickettsia - Amblyomma cooperi - isolation - capybara (Hydrochaeris hydrochaeris) - Brazil

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 (Goncalves 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, Magalh es 1952, Arag o & 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 S o 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^o44'21" S, 46^o54'27" 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 ug/ml of gentamicin, 100 ug/ml of vancomycin, and 2.5 ug/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 Gimenez 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 Gimenez 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

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

Brezina R, Murray ES, Tarizzo ML, Boel 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. Depositarios naturaes e transmissores da febre maculosa brasileira. Brasil Medico 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, Pecanha P, Falqueto A, Sexton DJ, Corey GR 1992. Febre maculosa brasileira: descric o do primeiro surto da doenca no Espirito 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.

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

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

Magalh es O 1952. Contribuic o para o conhecimento das doencas do grupo do tifo exantematico. 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 biopsia de pele. Rev Inst Med Trop S o Paulo 34: 37-41.

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

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

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

Figure: Evidence of rickettsial multiplication in Vero cell culture after inoculation of triturated ticks - three female Amblyomma cooperi (indirect immunofluorescent staining, magnification, X 400).

Copyright 1996 Fundacao Oswaldo Cruz

The following images related to this document are available:

Photo images