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.

Surveys of horse serum are a useful method of surveillance for Brazilian spotted fever in areas where humans are exposed to Amblyomma cajennense ticks.

In serum samples obtained from all the healthy humans, horses, dogs, and donkeys present on three farms in the Pedreira Municipality, an endemic area for Brazilian spotted fever, an indirect immunofluorescence assay (IFA) detected antibodies against Rickettsia rickettsii in 17 (77.3%) horses, 5 (31.3%) dogs (titers ranging from 64 to 4,048), and none of 4 donkeys or 50 humans. Five canine and eight equine sera with high antibody titers to R. rickettsii were also tested by IFA against R. bellii, R. akari, and R. africae antigens. Sera from two horses and two dogs that showed similar high antibody titers against two rickettsial antigens were evaluated after cross-absorption. Sera from seven horses and two dogs contained antibodies specific for R. rickettsii, and one dog serum had antibodies against a Rickettsia species very closely related to R. africae. The latter may have been caused by infection with the recently identified COOPERI strain.

This study investigated rickettsial infection in animals, humans, ticks, and fleas collected in five areas of the state of São Paulo. Eight flea species (Adoratopsylla antiquorum antiquorum, Ctenocephalides felis felis, Polygenis atopus, Polygenis rimatus, Polygenis roberti roberti, Polygenis tripus, Rhopalopsyllus lugubris, and Rhopalopsyllus lutzi lutzi), and five tick species (Amblyomma aureolatum, Amblyomma cajennense, Amblyomma dubitatum, Ixodes loricatus, and Rhipicephalus sanguineus) were collected from dogs, cats, and opossums. Rickettsia felis was the only rickettsia found infecting fleas, whereas Rickettsia bellii was the only agent infecting ticks, but no animal or human blood was shown to contain rickettsial DNA. Testing animal and human sera by indirect immunofluorescence assay against four rickettsia antigens (R. rickettsii, R. parkeri, R. felis, and R. bellii), some opossum, dog, horse, and human sera reacted to R. rickettsii with titers at least four-fold higher than to the other three rickettsial antigens. These sera were considered to have a predominant antibody response to R. rickettsii. Using the same criteria, opossum, dog, and horse sera showed predominant antibody response to R. parkeri or a very closely related genotype. Our serological results suggest that both R. rickettsii and R. parkeri infected animals and/or humans in the studied areas.Key words: Rickettsia -spotted fever -fleas -ticks -opossum -domestic animals Rickettsiae are obligate intracellular bacteria that have been classically divided into two groups: the typhus group (TG), composed of Rickettsia prowazekii and Rickettsia typhi, which are associated with lice and fleas, respectively; and the spotted fever group (SFG), which includes more than 20 valid species, mostly associated with ticks (e.g., Rickettsia rickettsii, Rickettsia parkeri), and at least one species associated with fleas (Rickettsia felis). Other species, such as Rickettsia bellii and Rickettsia canadensis, both associated with ticks, have been considered a distinct group sharing antigenic, genetic and ecological characters with both TG and SFG rickettsiae (Parola et al. 2005b). Since all SFG Rickettsia share common outer membrane antigens, conventional serological assays employing a single Rickettsia antigen (e.g., R. rickettsii) allow detecting a SFG infection, but they do not identify the Rickettsia species responsible for the infection, even in the presence of high titers (La Scola & Raoult 1997). For a more specific serological diagnosis, it is recommended techniques such as testing serum against all known Rickettsia species present in a given area, or serum cross-absorption tests (La Scola & Raoult 1997, Parola et al. 2005b).For many years, SFG rickettsiosis in the Americas was thought to be caused only by R. rickettsii, transmitted by ticks (Weiss & Moulder 1984). This rickettsia has been reported in several countries from North, Central, and South America causing an acute, highly lethal febrile disease receiving different names according to the country...

The present study evaluated the rickettsial infection among dogs living in the rural and urban areas of Monte Negro, state of Rondônia, western Brazilian Amazon. Canine sera were tested by the indirect immunofluorescence assay (IFA) using six rickettsial antigens: Rickettsia bellii, Rickettsia amblyommii, Rickettsia rhipicephali, Rickettsia rickettsii, Rickettsia parkeri, and Rickettsia felis. While the first three Rickettsia species are known to occur in the study site, the latter three species are known to occur in southeastern Brazil. For each serum, end point titer reacting with each Rickettsia antigen was determined. Serum showing for a Rickettsia species titer at least fourfold higher than that observed for any other Ricketttsia species was considered homologous to the first Rickettsia species or to a very closely related genotype. A total of 164 rural and 153 urban dogs were tested. Overall, 19 (11.6%) and 6 (3.9%) dogs from rural and urban areas, respectively, reacted positively to at least one Rickettsia species. In the rural area, three sera showed titers to R. parkeri at least four-fold higher than any of the other five antigens. These sera were considered to be homologous to R. parkeri or a very closely related genotype. Using the same criteria, two rural sera were considered homologous to R. amblyommii, two other rural sera to R. rhipicephali, and one urban serum to R. parkeri. Because dogs living in the rural area of Monte Negro are commonly infested by the same tick species infesting humans, they indeed serve as sentinels for human rickettsial diseases. Thus, humans living in Monte Negro are likely to be infected by at least three Rickettsia species: R. parkeri, R. amblyommii, and R. rhipicephali. While R. parkeri is a known human pathogen, further studies are required to verify the potential role of R. amblyommii and R. rhipicephali as human pathogens.

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BackgroundUntil recently, Amblyomma cajennense (Fabricius, 1787) was considered to represent a single tick species in the New World. Recent studies have split this taxon into six species. While the A. cajennense species complex or A. cajennense (sensu lato) (s.l.) is currently represented by two species in Brazil, A. cajennense (sensu stricto) (s.s.) and Amblyomma sculptum Berlese, 1888, their geographical distribution is poorly known.MethodsThe distribution of the A. cajennense (s.l.) in Brazil was determined by morphological examination of all lots of A. cajennense (s.l.) in two large tick collections of Brazil, and by collecting new material during three field expeditions in the possible transition areas between the distribution ranges of A. cajennense (s.s.) and A. sculptum. Phylogenetic analysis inferred from the ITS2 rRNA gene was used to validate morphological results. Morphological description of the nymphal stage of A. cajennense (s.s.) is provided based on laboratory-reared specimens.ResultsFrom the tick collections, a total 12,512 adult ticks were examined and identified as 312 A. cajennense (s.s.), 6,252 A. sculptum and 5,948 A. cajennense (s.l.). A total of 1,746 ticks from 77 localities were collected during field expeditions, and were identified as 249 A. cajennense (s.s.), 443 A. sculptum, and 1,054 A. cajennense (s.l.) [these A. cajennense (s.l.) ticks were considered to be males of either A. cajennense (s.s.) or A. sculptum]. At least 23 localities contained the presence of both A. cajennense (s.s.) and A. sculptum in sympatry. DNA sequences of the ITS2 gene of 50 ticks from 30 localities confirmed the results of the morphological analyses. The nymph of A. cajennense (s.s.) is morphologically very similar to A. sculptum.ConclusionOur results confirmed that A. cajennense (s.l.) is currently represented in Brazil by only two species, A. cajennense (s.s.) and A. sculptum. While these species have distinct distribution areas in the country, they are found in sympatry in some transition areas. The current distribution of A. cajennense (s.l.) has important implications to public health, since in Brazil A. sculptum is the most important vector of the bacterium Rickettsia rickettsii, the etiological agent of Brazilian spotted fever.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-016-1460-2) contains supplementary material, which is available to authorized users.

The present study provides a rickettsial serosurvey in 25 dogs and 35 humans in an endemic area for Brazilian spotted fever in the State of São Paulo, where the tick Amblyomma aureolatum is the main vector. Testing canine and human sera by indirect immunofluorescence against four Rickettsia antigens (R. rickettsii, R. parkeri, R. felis and R. bellii) showed that 16 (64%) of canine sera and 1 (2.8%) of human sera reacted to at least one of these rickettsial antigens with titers <FONT FACE=Symbol>³</FONT> 64. Seven canine sera and the single reactive human serum showed titers to R. rickettsii at least four times those of any of the other three antigens. The antibody titers in these 7 animals and 1 human were attributed to stimulation by R. rickettsii infection. No positive canine or human serum was attributed to stimulation by R. parkeri, R. felis, or R. bellii. Our serological results showed that dogs are important sentinels for the presence of R. rickettsii in areas where the tick A. aureolatum is the main vector of Brazilian spotted fever.