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The current study evaluated parasitism of wild birds by ticks in a fragment of Atlantic Forest in the state of Minas Gerais, Brazil, and examined the ticks for rickettsial agents. Birds were captured during 2014 and 2015 and ticks were identified by sequencing fragments of the 16S and 12S ribosomal DNA. Among 260 birds representing 19 families and 52 species, a total of 69 (26.5%) were found to be infested by larvae (LL) and/or nymphs (NN) of Amblyomma longirostre(Koch, 1844) (45 LL, 4 NN), Amblyomma calcaratumNeumann, 1899 (9 LL, 15 NN), Amblyomma nodosumNeumann, 1899 (2 NN), Amblyomma parkeriFonseca and Aragão, 1952 (21 LL), Amblyommasp. haplotype Nazaré (77 LL), and Haemaphysalis leporispalustris(Packard, 1869) (17 LL, 1 NN). The use of PCR and sequencing of the rickettsial genes gltA, htrA, ompAand ompB, revealed the presence of “CandidatusRickettsia amblyommii” in A. longirostre(13/49; 26%) and Rickettsia parkeri(strain ApPR) in both A. parkeri(1/21; 5%) and haplotype Nazaré (42/77; 55%) ticks. In addition, we detected Rickettsia rhipicephaliin 31 (40%) of the 77 haplotype Nazaré ticks. This is the first record of this rickettsial agent in a species of the genus Amblyomma. The pathogenic potential of this bacterium is undetermined, but the unprecedented association with Amblyommaticks may represent a cause for concern for public and/or animal health.

It is already known that the beagle breed of domestic dogs produces semiochemicals capable of repelling the brown dog tick, Rhipicephalus sanguineussensu lato (s.l.). With a view to discovering new non-host semiochemicals as tick repellents, we compared the semiochemicals produced by a putative tick-resistant breed of dog, miniature pinscher, with known tick-resistant (beagle) and tick-susceptible (English cocker spaniel) breeds. Two non-host compounds produced by beagles, i.e. 2-hexanone and benzaldehyde, were shown to be present in samples collected from all three breeds. Furthermore, two compounds, 6-methyl-5-hepten-2-one and 1,2,4-trimethylbenzene, were found in higher amounts in samples collected from miniature pinscher dogs. The mean amounts of benzaldehyde, 2-hexanone and 1,2,4-trimethylbenzene were similar for beagles and miniature pinschers (P> 0.05) and higher than the means observed for cocker spaniels (P< 0.05), whereas the mean amount of 6-methyl-5-hepten-2-one produced by miniature pinschers was significantly higher (P< 0.05) than for the other breeds of dogs. In Petri-dish assays with adult R. sanguineuss.l., 6-methyl-5-hepten-2-one was repellent for all observation periods evaluated for the two highest concentrations (0.100 and 0.200 mg.cm−2, P< 0.01). The obtained results support our hypothesis that miniature pinschers are a tick-resistant dog breed and agree with previous observations of miniature pinschers being the breed least parasitized by ticks. Furthermore, the non-host semiochemical 6-methyl-5-hepten-2-one has potential to be developed for use as a repellent for the protection of susceptible dogs from R. sanguineuss.l. ticks.

The bacterium Rickettsia belliihas 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 Rickettsiahas 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. belliiusing 150 Amblyommaticks collected from wild birds in Brazil. Co-infection of R. belliiinfected ticks was evaluated using a novel PCR assay targeting the ompAsequence 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 longirostreand one of two Amblyomma calcaratumticks were shown to be co-infected with R. belliiand Rickettsia amblyommatis, while two out of sixty-seven Amblyommasp. haplotype Nazaré ticks were recorded as co-infected with R. belliiand the Rickettsia parkeri-like bacterium, strain ApPR. Interestingly, our data represent the first records of R. belliiin association with A. calcaratum and Amblyommasp. 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.