Arlei Marcili

A new genotype of Trypanosoma cruzi associated with bats evidenced by phylogenetic analyses using SSU rDNA, cytochrome b and Histone H2B genes and genotyping based on ITS1 rDNA.

We characterized 15 Trypanosoma cruzi isolates from bats captured in the Amazon, Central and Southeast Brazilian regions. Phylogenetic relationships among T. cruzi lineages using SSU rDNA, cytochrome b, and Histone H2B genes positioned all Amazonian isolates into T. cruzi I (TCI). However, bat isolates from the other regions, which had been genotyped as T. cruzi II (TC II) by the traditional genotyping method based on mini-exon gene employed in this study, were not nested within any of the previously defined TCII sublineages, constituting a new genotype designated as TCbat. Phylogenetic analyses demonstrated that TCbat indeed belongs to T. cruzi and not to other closely related bat trypanosomes of the subgenus Schizotrypanum, and that although separated by large genetic distances TCbat is closest to lineage TCI. A genotyping method targeting ITS1 rDNA distinguished TCbat from established T. cruzi lineages, and from other Schizotrypanum species. In experimentally infected mice, TCbat lacked virulence and yielded low parasitaemias. Isolates of TCbat presented distinctive morphological features and behaviour in triatomines. To date, TCbat genotype was found only in bats from anthropic environments of Central and Southeast Brazil. Our findings indicate that the complexity of T. cruzi is larger than currently known, and confirmed bats as important reservoirs and potential source of T. cruzi infections to humans.

Trypanosoma cruzi in Brazilian Amazonia: Lineages TCI and TCIIa in wild primates, Rhodnius spp. and in humans with Chagas disease associated with oral transmission ☆

In this study, we provide phylogenetic and biogeographic evidence that the Trypanosoma cruzi lineages T. cruzi I (TCI) and T. cruzi IIa (TCIIa) circulate amongst non-human primates in Brazilian Amazonia, and are transmitted by Rhodnius species in overlapping arboreal transmission cycles, sporadically infecting humans. TCI presented higher prevalence rates, and no lineages other than TCI and TCIIa were found in this study in wild monkeys and Rhodnius from the Amazonian region. We characterised TCI and TCIIa from wild primates (16 TCI and five TCIIa), Rhodnius spp. (13 TCI and nine TCIIa), and humans with Chagas disease associated with oral transmission (14 TCI and five TCIIa) in Brazilian Amazonia. To our knowledge, TCIIa had not been associated with wild monkeys until now. Polymorphisms of ssrDNA, cytochrome b gene sequences and randomly amplified polymorphic DNA (RAPD) patterns clearly separated TCIIa from TCIIb-e and TCI lineages, and disclosed small intra-lineage polymorphisms amongst isolates from Amazonia. These data are important in understanding the complexity of the transmission cycles, genetic structure, and evolutionary history of T. cruzi populations circulating in Amazonia, and they contribute to both the unravelling of human infection routes and the pathological peculiarities of Chagas disease in this region.

Comparative phylogeography of Trypanosoma cruzi TCIIc: New hosts, association with terrestrial ecotopes, and spatial clustering☆

We characterized 28 new isolates of Trypanosoma cruzi IIc (TCIIc) of mammals and triatomines from Northern to Southern Brazil, confirming the widespread distribution of this lineage. Phylogenetic analyses using cytochrome b and SSU rDNA sequences clearly separated TCIIc from TCIIa according to terrestrial and arboreal ecotopes of their preferential mammalian hosts and vectors. TCIIc was more closely related to TCIId/e, followed by TCIIa, and separated by large distances from TCIIb and TCI. Despite being indistinguishable by traditional genotyping and generally being assigned to Z3, we provide evidence that TCIIa from South America and TCIIa from North America correspond to independent lineages that circulate in distinct hosts and ecological niches. Armadillos, terrestrial didelphids and rodents, and domestic dogs were found infected by TCIIc in Brazil. We believe that, in Brazil, this is the first description of TCIIc from rodents and domestic dogs. Terrestrial triatomines of genera Panstrongylus and Triatoma were confirmed as vectors of TCIIc. Together, habitat, mammalian host and vector association corroborated the link between TCIIc and terrestrial transmission cycles/ecological niches. Analysis of ITS1 rDNA sequences disclosed clusters of TCIIc isolates in accordance with their geographic origin, independent of their host species.

Survey of ticks (Acari: Ixodidae) and their Rickettsia in an Atlantic rain forest reserve in the state of São Paulo, Brazil.

ABSTRACT The current study investigated the occurrence of ticks and their rickettsiae in the Serra do Mar State Park, which encompasses one of the largest Atlantic rain forest reserves of Brazil. From July 2008 to June 2009, a total of 2,439 ticks (2,196 free living and 243 collected on hosts) was collected, encompassing the following 13 species: Amblyomma aureolatum (Pallas), Amblyomma brasiliense Aragão, Amblyomma dubitatum Neumann, Amblyomma fuscum Neumann, Amblyomma incisum Neumann, Amblyomma longirostre (Koch), Amblyomma naponense (Packard), Amblyomma nodosum Neumann, Amblyomma ovale Koch, Haemaphysalis juxtakochi Cooley, Ixodes aragaoi Fonseca, Ixodes loricatus Neumann, and Rhipicephalus sanguineus (Latreille). Ticks were submitted to polymerase chain reaction assays targeting portions of the rickettsial genes gltA and ompA. Polymerase chain reaction products were DNA sequenced and compared with corresponding sequences available in GenBank. Rickettsia bellii, a rickettsia of unknown pathogenicity, was detected in one A. aureolatum, one A. ovale, and three A. incisum specimens. At least 8.8% (3/34) of the free-living A. ovale ticks, 13.6% (8/59) of the A. ovale ticks collected from dogs, and 1.9% (1/54) of the R. sanguineus (Latreille) ticks were found to be infected by Rickettsia sp strain Atlantic rain forest, a novel strain that has been shown to cause an eschar-associated spotted fever in the state of São Paulo. Our results suggest that A. ovale is the vector of Rickettsia sp strain Atlantic rain forest in the state of São Paulo.

Trypanosoma rangeli isolates of bats from Central Brazil: genotyping and phylogenetic analysis enable description of a new lineage using spliced-leader gene sequences.

Trypanosoma rangeli infects several mammalian orders but has never confidently been described in Chiroptera, which are commonly parasitized by many trypanosome species. Here, we described trypanosomes from bats captured in Central Brazil identified as T. rangeli, T. dionisii, T. cruzimarinkellei and T. cruzi. Two isolates, Tra643 from Platyrrhinus lineatus and Tra1719 from Artibeus planirostris were identified as T. rangeli by morphological, biological and molecular methods, and confirmed by phylogenetic analyses. Analysis using SSU rDNA sequences clustered these bat trypanosomes together with T. rangeli from other hosts, and separated them from other trypanosomes from bats. Genotyping based on length and sequence polymorphism of PCR-amplified intergenic spliced-leader gene sequences assigned Tra1719 to the lineage A whereas Tra643 was shown to be a new genotype and was assigned to the new lineage E. To our knowledge, these two isolates are the earliest T. rangeli from bats and the first isolates from Central Brazil molecularly characterized. Rhodnius stali captured for this study was found infected by T. rangeli and T. cruzi.

Coxiella symbiont in the tick Ornithodoros rostratus (Acari: Argasidae)

In the present study, the presence of tick-associated bacteria and protozoa in Ornithodoros rostratus ticks (adults, nymphs, and eggs) from the Pantanal region of Brazil were determined by molecular detection. In these ticks, DNA from protozoa in the genera Babesia and Hepatozoon, and bacteria from the genera Rickettsia, Borrelia, Anaplasma, and Ehrlichia were not detected. Conversely, all tested ticks (100%) yielded PCR products for 3 Coxiella genes (16S rRNA, pyrG, cap). PCR and phylogenetic analysis of 3 amplified genes (16S rRNA, pyrG, cap) demonstrated that the agent infecting O. rostratus ticks was a member of the genus Coxiella. This organism grouped with Coxiella symbionts of other soft tick species (Argasidae), having different isolates of C. burnetii as a sister group, and these 2 groups formed a clade that grouped with another clade containing Coxiella symbionts of hard tick species (Ixodidae). Analysis of tick mitochondrial 16S rRNA gene database composed mostly of tick species previously shown to harbor Coxiella symbionts suggests a phylogenetic congruence of ticks and their Coxiella symbionts. Furthermore, these results suggest a very long period of coevolution between ticks and Coxiella symbionts and indicates that the original infection may have occurred in an ancestor common to the 2 main tick families, Argasidae (soft ticks) and Ixodidae (hard ticks). However, this evolutionary relationship must be confirmed by more extensive testing of additional tick species and expanded populations.

Phylogeographical, ecological and biological patterns shown by nuclear (ssrRNA and gGAPDH) and mitochondrial (Cyt b) genes of trypanosomes of the subgenus Schizotrypanum parasitic in Brazilian bats.

The genetic diversity and phylogeographical patterns of Trypanosoma species that infect Brazilian bats were evaluated by examining 1043 bats from 63 species of seven families captured in Amazonia, the Pantanal, Cerrado and the Atlantic Forest biomes of Brazil. The prevalence of trypanosome-infected bats, as estimated by haemoculture, was 12.9%, resulting in 77 cultures of isolates, most morphologically identified as Trypanosoma cf. cruzi, classified by barcoding using partial sequences from ssrRNA gene into the subgenus Schizotrypanum and identified as T. cruzi (15), T. cruzi marinkellei (37) or T. cf. dionisii (25). Phylogenetic analyses using nuclear ssrRNA, glycosomal glyceraldehyde 3-phosphate dehydrogenase (gGAPDH) and mitochondrial cytochrome b (Cyt b) gene sequences generated three clades, which clustered together forming the subgenus Schizotrypanum. In addition to vector association, bat trypanosomes were related by the evolutionary history, ecology and phylogeography of the bats. Trypanosoma cf. dionisii trypanosomes (32.4%) infected 12 species from four bat families captured in all biomes, from North to South Brazil, and clustered with T. dionisii from Europe despite being separated by some genetic distance. Trypanosoma cruzi marinkellei (49.3%) was restricted to phyllostomid bats from Amazonia to the Pantanal (North to Central). Trypanosoma cruzi (18.2%) was found mainly in vespertilionid and phyllostomid bats from the Pantanal/Cerrado and the Atlantic Forest (Central to Southeast), with a few isolates from Amazonia.

Geographical distribution of Amblyomma cajennense (sensu lato) ticks (Parasitiformes: Ixodidae) in Brazil, with description of the nymph of A. cajennense (sensu stricto)

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.

Description of a New Species of Bat-Associated Argasid Tick (Acari: Argasidae) from Brazil

abstract:  A new species of argasid tick (Acari: Argasidae) is described from immature and adult specimens collected from several localities in Brazil. A complete morphological account is provided for all postembryonic life stages, i.e., larva, nymph, female, and male. Ornithodoros cavernicolous n. sp. is the 113th in the genus. Morphologically, the new species shares common features, e.g., presence of well-developed cheeks and legs with micromammillate cuticle, with other bat-associated argasid ticks included in the subgenus Alectorobius. In particular, the new species is morphologically related to Ornithodoros azteci Matheson, with which it forms a species group. Phylogenetic analysis based on the 16S rRNA gene sequences supports the placement of the new species within a large clade that includes other New World bat-associated argasids. However, the new species seems to represent an independent lineage within the genus Ornithodoros.

Rickettsial Infection in Ticks (Acari: Ixodidae) Collected on Birds in Southern Brazil

ABSTRACT The aim of the study was to evaluate rickettsial infection in ticks from wild birds of the Semidecidual and Atlantic Rainforest remnants of three municipalities of the State of Paraná, southern Brazil. Overall, 53 larvae and nymphs collected from birds were checked for the presence of Rickettsia DNA by molecular tests. Five tick species were tested: Amblyomma aureolatum (Pallas), Amblyomma calcaratum Neumann, Amblyomma longirostre (Koch), Amblyomma ovale Koch, and Amblyomma parkeri Fonseca and Aragão. A. longirostre ticks were infected with the spotted fever group agents Rickettsia amblyommii strain AL (32.3% infection rate) and Rickettsia parkeri strain NOD (5.9% infection rate). A new rickettsial genotype was detected in the tick A. parkeri (50% infection rate), which had never been reported to be infected by rickettsiae. Through phylogenetic analysis, this new genotype, here designated as strain ApPR, grouped in a cluster composed by different strains of Rickettsia africae, Rickettsia sibirica, and R. parkeri. We consider strain ApPR to be a new genotype of R. parkeri. This study reports for the first time rickettsial infection in ticks from birds in southern Brazil. The role of migrating birds in the dispersal of these rickettsial strains should be considered in ecological studies of spotted fever group agents in Brazil.