Two for the price of one: Co-infection with Rickettsia bellii and spotted fever group Rickettsia in Amblyomma (Acari: Ixodidae) ticks recovered from wild birds in Brazil.

The bacterium Rickettsia bellii has been detected in 25 species of ticks in the American continents, but its pathogenic potential is considered as undetermined. A possible role for this species in the phenomenon of transovarial exclusion of pathogenic members of the spotted fever group (SFG) of Rickettsia has been suggested and co-infections with pathogenic species have been reported infrequently in both North and South America. Traditional methods for the molecular detection of rickettsial agents in ticks focus largely on the identification of sequences found in SFG Rickettsia, an approach that may overlook the presence of co-infections with R. bellii. Two novel, species-specific polymerase chain reaction (PCR) assays, targeting the genes encoding the surface cell antigen (Sca), autotransporter proteins sca9 and sca14, were developed and validated for the detection of R. bellii using 150 Amblyomma ticks collected from wild birds in Brazil. Co-infection of R. bellii infected ticks was evaluated using a novel PCR assay targeting the ompA sequence characteristic of SFG Rickettsia. Preliminary species-level identification was achieved by restriction fragment length polymorphism (RFLP) analysis and subsequently confirmed by sequencing of amplicons. Nine out of seventy-three Amblyomma longirostre and one of two Amblyomma calcaratum ticks were shown to be co-infected with R. bellii and Rickettsia amblyommatis, while two out of sixty-seven Amblyomma sp. haplotype Nazaré ticks were recorded as co-infected with R. bellii and the Rickettsia parkeri-like bacterium, strain ApPR. Interestingly, our data represent the first records of R. bellii in association with A. calcaratum and Amblyomma sp. haplotype Nazaré. The novel PCR-RFLP systems reported herein, provide an alternative, rapid and cost-efficient (relative to strategies based on sequencing or real-time PCR), approach to evaluate rickettsial co-infection of ticks, a potentially significant phenomenon that has most likely been underestimated to date.