Rickettsia species infecting Amblyomma cooperi ticks from an area in the state of São Paulo, Brazil, where Brazilian spotted fever is endemic

Abstract

Owing to the potential role of the tick Amblyomma cooperi in the enzootic cycle of Rickettsia rickettsii, the etiologic agent of Brazilian spotted fever (BSF), this study evaluated infection by Rickettsia species in A. cooperi ticks collected from an area in Brazil where BSF is endemic. Among a total of 40 A. cooperi adult ticks collected in an area of BSF endemicity in the state of São Paulo, PCR analysis detected DNA of Rickettsia bellii in 16 ticks (40%), and 3 other ticks (7.5%) were positive for a previously unidentified spotted-fever-group (SFG) rickettsia. Cultivation in Vero cell cultures by the shell vial technique with individual A. cooperi ticks resulted in two isolates of R. bellii and one isolate genotypically characterized as an SFG rickettsia. The two R. bellii isolates were established in Vero cell cultures in the laboratory and were confirmed to be R. bellii by molecular analysis of the gltA and 17-kDa protein-encoding genes and by electron microscopic analysis. The SFG rickettsial isolate could not be stably passaged in cell culture in the laboratory, but molecular analysis of early passages suggested that it was closely related to Rickettsia parkeri, Rickettsia africae, and Rickettsia sibirica. These results do not support the role of A. cooperi in the ecology of R. rickettsii in the area studied, but they add two more species of rickettsiae to the poorly developed list of species occurring in ticks in South America.

FIG. 1.

Neighbor-joining phylogram based on partial ompA sequences, showing the phylogenetic placement of strain COOPERI among SFG rickettsial species. Levels of bootstrap support (>50%) for phylogenetic groupings are given. Percentages of difference between taxa are indicated by the scale of the drawing. Bar, 1% difference.

FIG. 2.

Neighbor-joining phylogram based on partial 17-kDa sequences showing the phylogenetic placement of strain COOPERI and R. bellii isolate Ac25 among rickettsial species. Levels of bootstrap support (>50%) for phylogenetic groupings are given. Percentages of difference between taxa are indicated by the scale of the drawing. Bar, 1% difference.

FIG. 3.

Neighbor-joining phylogram based on partial gltA sequences showing the phylogenetic placement of strain COOPERI among validated rickettsial species. Levels of bootstrap support (>50%) for phylogenetic groupings are given. Percentages of difference between taxa are indicated by the scale of the drawing. Bar, 0.5% difference.

FIG. 4.

Electron photomicrograph of two adjacent cells containing several intracytosolic rickettsiae (arrows). Note the prominent electron-lucent “halos” surrounding each rickettsia. Arrowheads mark the borders between the cells. Bar, 1.0 μm.

FIG. 5.

Electron photomicrograph of an intracytosolic rickettsia in the process of binary fission. Rickettsiae possess a characteristic gram-negative morphology, with an electron-lucent “halo” or slime layer (arrowheads) adjacent to the cell wall (small arrow) and a cytoplasmic membrane (large arrow) separated from the cell wall by the periplasmic space. Bar, 0.5 μm.

FIG. 6.

A filamentous rickettsia (marked by an asterisk), with associated slime layer (arrowheads), measuring 3.0 μm before extending out of the plane of section (arrow). Bar, 1.0 μm.

FIG. 7.

Rickettsial cytoplasmic membrane (large solid arrow) A higher magnification of the cell wall revealed a cell wall membrane with an inner leaflet (arrowheads) that is slightly thicker than the outer leaflet (small solid arrow), and an associated electron-lucent slime layer (asterisk) adjacent to the outer cell wall. A thin, electron-dense layer on the outer leaflet of the cell wall are morphologically consistent with a microcapsular layer (open arrows). Bar, 100 nm.