Thiago F. Martins

Nymphs of the genus Amblyomma (Acari: Ixodidae) of Brazil: descriptions, redescriptions, and identification key.

Together with the larval stage, the nymphal stage of ticks of the genus Amblyomma are the most aggressive ticks for humans entering areas inhabited by wildlife and some domestic animals in Brazil. However, due to the absence of morphological descriptions of the nymphal stage of most Brazilian Amblyomma species, plus the lack of an identification key, little or nothing is known about the life history of Amblyomma spp. nymphs in the country. In the present study, morphological description of the nymphal stage, illustrating important external characters through scanning electron microscopy, is provided for nymphs of 15 Amblyomma species that occur in Brazil, for which the nymphal stage had never been described: A. aureolatum, A. auricularium, A. calcaratum, A. coelebs, A. fuscum, A. humerale, A. incisum, A. latepunctatum, A. naponense, A. nodosum, A. ovale, A. pacae, A. pseudoconcolor, A. scalpturatum, A. varium. In addition, the nymphal stage of 12 Amblyomma species, which had been previously described, are redescribed: A. brasiliense, A. cajennense, A. dissimile, A. dubitatum, A. longirostre, A. oblongoguttatum, A. parkeri, A. parvum, A. romitii, A. rotundatum, A. tigrinum, A. triste. The descriptions and redescriptions totalized 27 species. Only 2 species (A. geayi, A. goeldii) out of the 29 Amblyomma species established in Brazil are not included in the present study. A dichotomous identification key is included to support taxonomic identification of the nymphal stage of 27 Amblyomma species established in Brazil.

In vitro isolation from Amblyomma ovale (Acari: Ixodidae) and ecological aspects of the Atlantic rainforest Rickettsia, the causative agent of a novel spotted fever rickettsiosis in Brazil.

Recently, a novel human rickettsiosis, namely Atlantic rainforest spotted fever, was described in Brazil. We herein report results of a survey led around the index case in an Atlantic rainforest reserve in Peruibe municipality, southeastern Brazil. A Rickettsia parkeri-like agent (Rickettsia sp. Atlantic rainforest genotype) and Ricketsia bellii were isolated from adult Amblyomma ovale ticks collected from dogs. Molecular evidence of infection with strain Atlantic rainforest was obtained for 30 (12.9%) of 232 A. ovale adult ticks collected from dogs. As many as 88.6% of the 35 examined dogs had anti-Rickettsia antibodies, with endpoint titres at their highest to R. parkeri. High correlation among antibody titres in dogs, A. ovale infestations, and access to rainforest was observed. Amblyomma ovale subadults were found predominantly on a rodent species (Euryoryzomys russatus). From 17 E. russatus tested, 6 (35.3%) displayed anti-Rickettsia antibodies, with endpoint titres highest to R. parkeri. It is concluded that Atlantic rainforest genotype circulates in this Atlantic rainforest area at relatively high levels. Dogs get infected when bitten by A. ovale ticks in the forest, and carry infected ticks to households. The role of E. russatus as an amplifier host of Rickettsia to A. ovale ticks deserves investigation.

Epidemiology of Brazilian spotted fever in the Atlantic Forest, state of São Paulo, Brazil

The tick-borne bacterium Rickettsia rickettsii is the aetiological agent of Brazilian spotted fever (BSF). The present study evaluated tick infestations on wild and domestic animals, and the rickettsial infection in these animals and their ticks in 7 forest areas adjacent to human communities in the São Paulo Metropolitan Area (SPMA). The results were compared to ecological traits of each sampled area. Two main tick species, Amblyomma aureolatum and Rhipicephalus sanguineus, were collected from dogs. The major ticks found on small mammals and birds were Ixodes loricatus and Amblyomma longirostre, respectively. Both anti-R. rickettsii antibodies and R. rickettsii-infected ticks were detected on dogs from only 2 areas in the southern part of the SPMA, which were considered to be endemic for BSF; the remaining 5 areas were considered to be non-endemic. Ecologically, the BSF-endemic areas clearly differed from the non-endemic areas by the presence of significantly more degraded forest patches in the former. The present results corroborate historical observations that have indicated that all human cases of BSF in the SPMA were contracted in the southern part of this metropolitan area. However, not all forest patches in the southern part of the SPMA were shown to be associated with BSF endemism.

Acquisition and transmission of Hepatozoon canis (Apicomplexa: Hepatozoidae) by the tick Amblyomma ovale (Acari: Ixodidae)

The present study aimed to evaluate under controlled conditions the acquisition of Hepatozoon canis by Amblyomma ovale after feeding on infected dogs, and the subsequent induction of infection in uninfected dogs that ingested the experimentally infected ticks. Two H. canis naturally infected dogs were infested with A. ovale adult ticks derived from an uninfected laboratory tick colony. After feeding, two A. ovale females presented H. canis oocysts in the hemolymph at the first and fourth days after removal of ticks from dogs. The oocysts had an average size of 244.34 microm x 255.46 microm. Three uninfected dogs were fed with ticks previously fed on the infected dogs. Only one dog became infected 32 days after oral inoculation, presenting circulating gametocytes, parasitemia less than 1%, and positive PCR confirmed to be H. canis by DNA sequencing. The results obtained indicated A. ovale ticks as potential vector of H. canis in rural areas of Brazil.

Seroprevalence and risk factors to Ehrlichia spp. and Rickettsia spp. in dogs from the Pantanal Region of Mato Grosso State, Brazil.

Sera of 320 dogs from urban and rural areas of a Pantanal region of Brazil were evaluated for rickettsial (Rickettsia rickettsii, R. parkeri, R. amblyommii, R. rhipicephali, R. felis, and R. bellii) and ehrlichial (Ehrlichia canis) infection by the immunofluorescence assay (IFA). Risk factors for ehrlichiosis or rickettsiosis were also evaluated. Positive reaction against Ehrlichia spp. was detected in 227 (70.9%) dogs, 119 (74.3%) from an urban area and 108 (67.5%) from rural areas (P>0.05). For Rickettsia spp., 152 (47.5%) dogs were positive, 31 (19.3%) from urban and 121 (75.6%) from rural areas (P<0.05). Highest anti-Rickettsia spp. endpoint titers were observed for R. amblyommii, suggesting homologous reactions to this agent or a very closely related organism. While most of the urban dogs were found parasitized by the tick Rhipicephalus sanguineus, infestations on rural dogs were predominated by Amblyomma cajenennse. Rickettsial infection was significantly higher (P<0.05) in rural dogs, in dogs with hunting practice, or in A. cajennense-infested dogs.

Comparative Susceptibility of Larval Stages of Amblyomma aureolatum, Amblyomma cajennense, and Rhipicephalus sanguineus to Infection by Rickettsia rickettsii

Abstract The current study compared the susceptibility of larval stages of Amblyomma cajennense (F.), Amblyomma aureolatum (Pallas), and Rhipicephalus sanguineus (Latreille) to infection by a Brazilian strain of Rickettsia rickettsii. Guinea pigs experimentally infected by R. rickettsii were simultaneously infested by larvae of the three tick species. Recovered engorged larvae were allowed to molt to nymphs and held in an incubator at 23°C and 85–90% RH. Subsequent flat nymphs were tested for rickettsial infection by polymerase chain reaction (PCR). Concomitant infestations with sibling ticks on noninfected guinea pigs (control) were done. While 10–60% of the A. cajennense nymphs were shown to be infected by R. rickettsii, both A. aureolatum and R. sanguineus were highly susceptible to R. rickettsii, since 80–100% of their nymphs were shown to be infected in the corresponding trials. Most of the engorged larvae (≈70–95%), regardless of being infected or not, successfully molted to nymphs. Mortality rates for engorged larvae tended to be statistically similar (P > 0.05) for ticks recovered from R. rickettsii-infected and noninfected guinea pigs, within each tick species. The only exceptions were the significantly higher mortalities (P < 0.05) for engorged A. cajennense larvae recovered from two infected guinea pigs. Therefore, A. cajennense was less susceptible to R. rickettsii infection than A. aureolatum and R. sanguineus, while feeding on rickettsemic guinea pigs. These two later species were similarly highly susceptible.

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.

Experimental infection of Amblyomma aureolatum ticks with Rickettsia rickettsii.

We experimentally infected Amblyomma aureolatum ticks with the bacterium Rickettsia rickettsii, the etiologic agent of Rocky Mountain spotted fever (RMSF). These ticks are a vector for RMSF in Brazil. R. rickettsii was efficiently conserved by both transstadial maintenance and vertical (transovarial) transmission to 100% of the ticks through 4 laboratory generations. However, lower reproductive performance and survival of infected females was attributed to R. rickettsii infection. Therefore, because of the high susceptibility of A. aureolatum ticks to R. rickettsii infection, the deleterious effect that the bacterium causes in these ticks may contribute to the low infection rates (<1%) usually reported among field populations of A. aureolatum ticks in RMSF-endemic areas of Brazil. Because the number of infected ticks would gradually decrease after each generation, it seems unlikely that A. aureolatum ticks could sustain R. rickettsii infection over multiple successive generations solely by vertical transmission.

Pesquisa de Rickettsia spp em carrapatos Amblyomma cajennense e Amblyomma dubitatum no Estado de São Paulo

The presence of rickettsial infection was surveyed in 3,545 Amblyomma cajennense ticks and 2,666 Amblyomma dubitatum ticks. Using the hemolymph test, polymerase chain reaction and isolation of Rickettsia in cell cultures, all of the Amblyomma cajennense were negative, whereas 634 (23.8%) of the Amblyomma dubitatum ticks were shown to be infected with Rickettsia bellii.

Experimental Infection of the Opossum Didelphis aurita by Rickettsia felis, Rickettsia bellii, and Rickettsia parkeri and Evaluation of the Transmission of the Infection to Ticks Amblyomma cajennense and Amblyomma dubitatum

This work evaluated the infection of opossums (Didelphis aurita) by Rickettsia felis, Rickettsia bellii, and Rickettsia parkeri and their role as amplifier hosts for horizontal transmission to Amblyomma cajennense and/or Amblyomma dubitatum ticks. Infection in D. aurita was induced by intraperitoneal inoculation with R. felis (n = 4 opossums), R. bellii (n = 4), and R. parkeri (n = 2). Another group of six opossums were inoculated intraperitoneally with Leibovitz-15 sterile culture medium, representing the uninfected groups (n = 2 opossums simultaneously to each infected group). Opossum blood samples collected during the study were used for DNA extraction, followed by real-time polymerase chain reaction targeting the rickettsial gene gltA, hematology, and detection of Rickettsia spp.-reactive antibodies by indirect immunofluorescence assay. Opossums were infested with uninfected A. cajennense and/or A. dubitatum for 30 days postinoculation (DPI). Flat ticks molted from ticks fed on opossums were allowed to feed on uninfected rabbits, which were tested for seroconversion by immunofluorescence assay. Samples of flat ticks were also tested by real-time polymerase chain reaction. Inoculated opossums showed no clinical abnormalities. Antibodies to Rickettsia spp. were first detected at the second to fourth DPI, with detectable titers until the 150th DPI. Rickettsemia was detected only in one opossum inoculated with R. parkeri, at the eighth DPI. Only one A. cajennense tick (2.0%) previously fed on a R. parkeri-inoculated opossum became infected. None of the rabbits infested with opossum-derived ticks seroconverted. The study demonstrated that R. felis, R. bellii, and R. parkeri were capable to produce antibody response in opossums, however, with undetectable rickettsemia for R. felis and R. bellii, and very low rickettsemia for R. parkeri. Further studies must be done with different strains of these rickettsiae, most importantly the strains that have never gone through in vitro passages.