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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.

Recent studies have reported several larvae of an unidentified Amblyommaspecies on passerine birds in Atlantic rainforest fragments in southeastern Brazil. These larvae yielded a unique 16S rRNA haplotype designated as Amblyommasp. haplotype Nazaré, which showed nucleotide identity levels of 91% to Amblyomma parkeriFonseca & Aragão, 1952 and 88% to Amblyomma longirostre(Koch, 1844). Herein, we describe Amblyommasp. haplotype Nazaré as a new species, Amblyomma romarioin. sp. Martins, Luz & Labruna, through a formal description of the male and female adult stages. Amblyomma romarioiis morphologically and genetically most closely related to A. parkeri, A. longirostreand Amblyomma geayiNeumann, 1899. Among males, the rectangular basis capituli and rounded coxa I spurs separates A. romarioifrom A. parkeri, A. longirostre,and A. geayi,which have basis capituli triangular or slightly hexagonal, and pointed coxa I spurs. Among females, the V-shaped genital aperture and coxa I rounded spurs of A. romarioicontrasts to the U-shaped genital aperture and coxa I pointed spurs in A. parkeri, A. longirostre,and A. geayi.Larvae of A. romarioihave been collected on 24 species of passerines. The few records of nymphs and adults were on the black-fronted titi monkey Callicebus nigrifrons(Spix, 1823). The current distribution of A. romarioiis restricted to the Brazilian Atlantic rainforest, southeastern Brazil, in areas with altitude between 363 and 1600 m, within the distribution of C. nigrifrons. We discuss ecological features of Amblyomma romarioi,comparatively to A. parkeri, A. longirostreand A. geayi.The present study increases the Brazilian tick fauna to 74 species.

Rickettsia parkerisensu stricto (s.s.) is an emerging human pathogen in the Americas. Comprehension of the etiology of R. parkeriinfections in South America is complicated by the existence of genetic variants (Atlantic rainforest, NOD and Parvitarsum) of this species that are associated with specific groups of Amblyommaticks. The rickettsial bacterium strain ApPR was first reported in Amblyomma parkeriticks in Southern Brazil in 2012 and was considered, based on sequencing of fragments of the gltA, htrA, ompAand ompBgenes, to represent yet another genetic variant of R. parkeri. In the current work, a multi-locus phylogenetic analysis employing additional genes and intragenic regions was performed using DNA extracted from (a) larvae of A. parkeriand Amblyommaspecies haplotype Nazaré ticks collected from wild birds, (b) a nymph of Amblyommasp. haplotype Nazaré recovered from a monkey (Callicebus nigrifons), representing the first report of that tick parasitizing a non-human primate and (c) from a cultured isolate of ApPR, isolated from colony-reared adults of Amblyomma geayi. Phylogenetic inference performed using Maximum-likelihood (ML), Maximum Parsimony (MP) and Bayesian (B) methods, consistently placed strain ApPR outside the New World R. parkericomplex and instead grouped it in proximity to the Old World species Rickettsia africaeand Rickettsia sibirica. Estimates of evolutionary divergence provided additional support for the inferred phylogenetic relationship. Given the clear evolutionary distance between strain ApPR and R. parkeriwe propose the recognition of “CandidatusRickettsia paranaensis”.