Mem Inst Oswaldo Cruz, Rio de Janeiro, 92(4) Jul/Ago 1997
Rickettsiae-infected Ticks in an Endemic Area of Spotted Fever in the State of Minas Gerais, Brazil
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 Universitária, 21941-590 Rio de Janeiro, RJ, Brasil
A study on tick-borne rickettsiosis was developed in the county of Santa Cruz do Escalvado, State of Minas Gerais, Brazil, where a clinical case of the disease, confirmed by necropsy, had been reported. Of the 1,254 ticks collected, 1,061 belonged to theu00a0Amblyommau00a0genus, 57 to the Rhipicephalus sanguineusu00a0species, 81 tou00a0Boophilus microplus, and 46 tou00a0Anocentor nitens. The hemolymph test associated with Giménez staining showed that 18 of the 221u00a0A. cajennense specimens, 1 of the 16u00a0R. sanguineus, 1 of the 22u00a0B. microplus, 3 of theu00a0A. nitens, and 1 of theu00a0A. ovaleu00a0contained rickettsia-like microorganisms. Only 3u00a0A. cajennenseu00a0ticks were positive under direct immunofluorescence.u00a0A. cajennenseu00a0was the only species found on humans.
In Brazil, Amblyomma cajennense is considered the most important vector for Rickettsia rickettsii, the causal agent for Brazilian spotted fever. This tick species is widely distributed in Brazil and is frequently found infesting human beings.
Although few studies have been performed on this subject, there has been an increase in case reports of this rickettsiosis in southeastern Brazil, including the states of Minas Gerais, São Paulo, Rio de Janeiro, and Espírito Santo (Gonçalves et al. 1977, 1981, Sampaio et al. 1988, Souza et al. 1991, Sexton et al. 1993).
This paper reports the prevalence of rickettsia-infected ticks in an endemic area where a fatal clinical case confirmed by necropsy had been reported (Lemos et al. 1994). A five-year-old child died after a tick bite presenting fever, chills, widespread maculopapular rash, and encephalopathy.
MATERIALS AND METHODS
The village of Patrimônio, from where the index case came, and other eigth localities, were selected in the county of Santa Cruz do Escalvado (20o13'36" S, 42o49'24" W), Minas Gerais, using as reference the geographical coordinates. It was thus possible to show areas with different geographic characteristics, in addition to differences in soil manegment and population density. The human population was higher in Patrimônio, São José de Vargem Alegre, Merengo, Zito Soares and Soberbo than in other villages chosen for this study (Fig.).
Patrimônio (1) and São José de Vargem Alegre - Sites with human settlements, where crop fields have been abandoned and turned into pasture, with virtually no farming activity other than cattle-raising.
Empreitada (3), Merengo (2), and Gambá (7) - Sites along Rio Doce with various crop areas (corn, sugar cane, beans), where slashing and burning is common.
Zito Soares (4), Pedreira (5), and Facão (8) - Sites away from the banks of Rio Doce, with numerous abandoned crop fields and clumps of primary forest growth.
Soberbo (6) - A site near Rio Doce with no crop or pasture areas. The vegetation of this area consisted of secondary vegetation.
With the help of a veterinarian, 1,254 ticks were colected during seven field visits at 2-3 months intervals from July 1989 to November 1990. Feeding ticks were removed from theirs hosts (human and domestic animals) and free-living ticks were colected from vegetation, near human settlements, using a flannel cloth.
After taxonomic identification (Rohr 1909, Aragão & Fonseca 1961) only a group of ticks was submitted to hemolimph test (Burgdorfer 1970) because the other, mainly the immature stages, was carried to taxonomic confirmation in the Departament of Entomology at the Oswaldo Cruz Institute.
The ticks were disinfected with 4% formaldehyde solution, 3% hydrogen peroxide, and 70% alcohol and washed in sterile distilled water for 5 min.
For each live tick, two slides were prepared with the hemolymph, fixed in acetone for 15 min. The first was to investigate rickettsia-like microorganisms using Giménez staining, and the second to perform the direct immunofluorescence test with R. rickettsii-positive human serum and fluorescein.
The tick species collected in this study and their distribution in relation to locations and food sources are shown in Tables I and II. Of the 1,254 ticks collected from animals, human beings, and vegetation, A. cajennense was the predominant species.
All the ticks taken from human beings belonged to the nymphal stage of A. cajennense and came from Patrimônio.
Of the 1,254 ticks collected, only 514 were submitted to the hemolymph test with Giménez staining, and of these, 24 displayed intracellular particles in the hemocytes. These rickettsia-like particles were observed invading the nucleus of the hemocyte in some specimens, suggesting spotted fever group rickettsiae (Table III).
The species displaying positive results under Giménez staining were: A. cajennense (18); A. nitens (3); R. sanguineus (1); B. microplus (1); and A. ovale (1).
Prevalence of infection in the ticks as determined by Giménez staining was 8.1%, considering that hemocytes were not observed in 218 ticks, meaning that the latter were not included in computing the results of this analysis (Table III).
Only three specimens of A. cajennense (adults) prepared with Giménez staining reacted to the conjugate with antibody for R. rickettsii, and all had been collected at Patrimônio.
The ixodid fauna in this study presented only five species. A. cajennense, although also found in equines, bovines, and the vegetation, was the only species found in human beings. This finding stresses the importance of this species in spotted fever ecology in Brazil, a fact that has been well documented by various Brazilian researchers (Fonseca 1932, Monteiro & Fonseca 1932, Moreira & Magalhães 1937, Dias & Martins 1939, Magalhães & Moreira 1939, Travassos & Vallejo-Freire 1944-45, Magalhães 1952).
A. cajennense was recovered in all studied areas but most of them were found in Patrimônio. It may probably be due to the great number of abandoned horses and dogs, the lack of control of tick infestation and the predominance of abandoned fields in this village.
Although they were not observed feeding on human beings, B. microplus, A. nitens, A. ovale, and R. sanguineus (the latter known as a vector for spotted fever caused by R. conori in Europe) may be implicated in maintaining spotted fever group rickettsiae in nature.
The infection frequency based on direct immunofluorescence was 1% for the 269 ticks examined, a result similar to that observed by Magnarelli et al. (1981) in Connecticut, USA. However, the various studies on prevalence of infection in ticks display wide variation, with prevalences ranging from 0.14% to 13.5%, based on the areas studied or even the period within a given area (Burgdorfer et al. 1974, Sexton et al. 1976, Benach et al. 1977, Feng et al. 1980, Lane et al. 1981, Taylor et al. 1986).
Magnarelli et al. (1981) observed that the number of infected ticks detected by direct immuno-fluorescence did not vary when they compared those collected from what were considered endemic areas for the human disease and locations where spotted fever had never been reported. This may apparently invalidate the importance of infection in ticks as an indicator of rickettsial activity; however, we should not overlook that the presence of human cases is merely a biological accident, undoubtedly a poor and unreal indicator of precise spotted fever group rickettsiosis distribution in nature. Although the presence of infected ticks is necessary, it is not sufficient to produce human cases; presence of the latter basically provides a point of departure for studying the disease's epidemiology and ecology, in addition to fostering improved epidemiological surveillance.
To Dr Bernardino C Albuquerque for discussing this study during the planning stage. To Prof. Rubens P Mello for assistance in tick identification. To Nilma P Gusmão for technical assistance, and Prof. Nicolau M Serra-Freire for discussing the manuscript.
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