Amália R.M. Barbieri

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.

First Report of the Isolation and Molecular Characterization of Rickettsia amblyommii and Rickettsia felis in Central America

During 2010, 15 adult ticks, identified as Amblyomma cajennense, were collected from horses in Cahuita and Turrialba districts, whereas 7 fleas, identified as Ctenocephalides felis, were collected from a dog in San Jose city, Costa Rica. In the laboratory, three A. cajennense specimens, two from Cahuita and one from Turrialba, were individually processed for rickettsial isolation in cell culture, as was a pool of seven fleas. Rickettsiae were successfully isolated and established in Vero cell culture from the three ticks and from a pool of seven fleas in C6/36 cell culture. The three tick isolates were genotypically identified as Rickettsia amblyommii, and the flea isolate was identified as Rickettsia felis through DNA sequencing of portions of the rickettsial genes gltA, ompA, and ompB of each isolate. In addition, other seven ticks were shown to contain rickettsial DNA. Polymerase chain reaction products of at least two of these ticks were sequenced and also showed to correspond to R. amblyommii. Overall, 66.7% (10/15) of the A. cajennense adult ticks were found to be infected with rickettsiae. This is the first report of a successful isolation in cell culture of R. amblyommii and R. felis from Central America.

Experimental infection of the tick Amblyomma cajennense, Cayenne tick, with Rickettsia rickettsii, the agent of Rocky Mountain spotted fever.

In the laboratory, Amblyomma cajennense (Acari: Ixodidae) (Fabricius) larvae, nymphs and adults were exposed to Rickettsia rickettsii by feeding on needle‐inoculated animals, and thereafter reared on uninfected guinea pigs or rabbits. Regardless of the tick stage that acquired the infection, subsequent tick stages were shown to be infected (confirming transstadial and transovarial transmissions) and were able to transmit R. rickettsii to uninfected animals, as demonstrated by serological and molecular analyses. However, the larval, nymphal and adult stages of A. cajennense were shown to be partially refractory to R. rickettsii infection, as in all cases, only part of the ticks became infected by this agent, after being exposed to rickettsemic animals. In addition, less than 50% of the infected engorged females transmitted rickettsiae transovarially, and when they did so, only part of the offspring became infected, indicating that vertical transmission alone is not enough to maintain R. rickettsii in A. cajennense for multiple generations. Finally, the R. rickettsii‐infected tick groups had lower reproductive performance than the uninfected control group. Our results indicate that A. cajennense have a low efficiency to maintain R. rickettsii for successive generations, as R. rickettsii‐infection rates should decline drastically throughout the successive tick generations.

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.

Epidemiology of Rickettsia sp. strain Atlantic rainforest in a spotted fever-endemic area of southern Brazil

The present study was performed in Vila Itoupava, an area of the state of Santa Catarina, southern Brazil, in which a tick-borne spotted fever illness has been endemic since 2003. Notably, both the etiological agent and the vector of these spotted fever cases remain unknown. During January 2011, humans, domestic dogs, and their ticks were sampled in households that are typically surrounded by highly preserved Atlantic rainforest fragments. Ticks collected from dogs were Amblyomma ovale (34% prevalence), Amblyomma aureolatum (18.9%), and Rhipicephalus sanguineus (3.8%). A total of 7.8% (6/77) A. ovale and 9.3% (4/43) A. aureolatum were infected by Rickettsia sp. strain Atlantic rainforest, a Rickettsia parkeri-like agent recently shown to cause spotted fever illness in southeastern Brazil. Overall, 67.3% (35/52) of the dogs were seroreactive to spotted fever group rickettsiae, mostly with highest endpoint titers to R. parkeri. Among humans, 46.7% (7/15) reacted serologically to rickettsiae at low to moderate endpoint titers. Because canine seroreactivity to R. parkeri was strongly associated with frequent contact with forests (the preferred habitat for A. ovale and A. aureolatum), it is concluded that sampled dogs have been infected by strain Atlantic rainforest through the parasitism of these tick species. The present study provides epidemiological evidence that the spotted fever in the study area has been caused by Rickettsia sp. strain Atlantic rainforest, transmitted to humans by either A. ovale or A. aureolatum. Further studies encompassing direct diagnostic methods on clinical specimens from patients are needed to confirm the above epidemiological evidence.

Borrelia burgdorferi sensu lato Infecting Ticks of the Ixodes ricinus Complex in Uruguay: First Report for the Southern Hemisphere

The Borrelia burgdorferi sensu lato (s.l.) group comprises genetically related spirochetes, mostly associated with tick species belonging to the Ixodes ricinus complex in the Northern Hemisphere. The present study evaluated borrelial infection in the tick Ixodes pararicinus, which is the only representative species of the I. ricinus complex in Uruguay. A total of 137 I. pararicinus ticks were collected from deer, cattle, or vegetation in 2 Uruguayan Departments. A part of these ticks was tested directly by PCR targeting the borrelial gene flagellin (fla), whereas another part of the ticks was inoculated into Barbour-Stoenner-Kelly (BSK)-H medium in an attempt to isolate Borrelia. Overall, Borrelia infection was detected in 9 males and 1 nymphal tick pool. These ticks were found to be infected by unique fla haplotypes, which were shown through phylogenetic analysis to represent possibly 2 new B. burgdorferi s.l. genospecies, 1 associated with B. bissettii, the other phylogenetically closest to B. americana. These results were reinforced by PCR and DNA sequencing analyses of portions of 2 additional borrelial genes, rrfA-rrlB intergenic spacer region (IGS) and 16S rDNA (rrs). Weekly examinations of BSK cultures by dark-field microscopy failed to demonstrate live Borrelia through a 100-day incubation period. However, Borrelia DNA was detected by fla-PCR in culture media from 2 vials up to 90 days after inoculation. To the best of our knowledge, this is the first report of B. burgdorferi s.l. infecting ticks in South America.

Rickettsia bellii infecting Amblyomma sabanerae ticks in El Salvador

Abstract Four Amblyomma sabanerae ticks collected from a turtle (Kinosternon sp.) in San Miguel, El Salvador, were found by molecular analysis to be infected by Rickettsia bellii. We provide the first report of Rickettsia bellii in Central America, and the first report of a Rickettsia species in El Salvador.

Environmental infestation and rickettsial infection in ticks in an area endemic for Brazilian spotted fever

Brazilian spotted fever (BSF), caused by Rickettsia rickettsii, is endemic in the municipality of Americana, southeastern Brazil, where the disease is transmitted by the tick Amblyomma cajennense. This study evaluated the tick fauna and rickettsial infection in free-living ticks that were captured monthly using dry ice traps in areas endemic for BSF in Americana, from July 2009 to June 2010. Two tick species were captured: A. cajennense (6,122 larvae; 4,265 nymphs; 2,355 adults) and Amblyomma dubitatum (7,814 larvae; 3,364 nymphs; 1,193 adults). The immature stages of A. cajennense and A. dubitatum had similar distribution through the 12-month period, with larvae of both species collected in highest numbers between April and July, and nymphs between June and October. The highest numbers of A. cajennense adults were collected between October and December, whereas A. dubitatum adults were collected in relatively similar numbers throughout the 12-month period. Rickettsial infection was evaluated by means of PCR in 1,157 A. cajennense and 1,040 A. dubitatum ticks; only 41 (3.9%) A. dubitatum were found to be infected by Rickettsia bellii. The present study showed that the areas of Americana that are endemic for BSF are characterized by high environmental burdens of A. cajennense and A. dubitatum.

Borrelia infection in Ixodes pararicinus ticks (Acari: Ixodidae) from northwestern Argentina.

The aim of this work was to describe for the first time the presence of Borrelia burgdorferi sensu lato infecting ticks in Argentina. Unfed specimens of Ixodes pararicinus collected from vegetation in Jujuy Province were tested for Borrelia infection by PCR targeting the gene flagellin (fla), the rrfA-rrlB intergenic spacer region (IGS) and the 16S rDNA (rrs) gene. One male and one female of I. pararicinus collected in Jujuy were found to be positive to Borrelia infection with the three molecular markers tested. Phylogenetically, the Borrelia found in I. pararicinus from Jujuy belongs to the B. burgdorferi s.l complex, and it was similar to one of the genospecies detected in I. aragaoi from Uruguay. Also, this genospecies is closely related to two genospecies known from USA, Borrelia americana and the Borrelia sp. genospecies 1. The epidemiological risk that implies the infection with Borrelia in I. paracinus ticks from Argentina appears to be low because the genospecies detected is not suspected of having clinical relevance and there are no records of Ixodes ticks biting humans in the southern cone of South America. Further studies are needed to assess accurately if there is risk of borreliosis transmitted by ticks in South America.

Novel piroplasmid and Hepatozoon organisms infecting the wildlife of two regions of the Brazilian Amazon

During 2009–2012, wild animals were sampled in two areas within the Amazon biome of Brazil, in the states of Mato Grosso and Pará. Animal tissues and blood were molecularly tested for the presence of Piroplasmida (genera Babesia, Theileria, Cytauxzoon) or Hepatozoon DNA. Overall, 181 wild animals comprising 36 different species (2 reptiles, 5 birds, and 29 mammals) were sampled. The following Piroplasmida agents were detected: Cytauxzoon felis in one ocelot (Leopardus pardalis), Theileria cervi in two red brocket deer (Mazama americana), Theileria spp. in three nine-banded-armadillos (Dasypus novemcinctus), one agouti (Dasyprocta sp.), and four lowland pacas (Cuniculus paca), Babesia spp. in one common opossum (Didelphis marsupialis) and one white-lipped peccary (Tayassu pecari). The following Hepatozoon agents were detected: Hepatozoon sp. (possibly Hepatozoon caimani) in three spectacled caimans (Caiman crocodilus), Hepatozoon felis in an ocelot (Leopardus pardalis), and Hepatozoon spp. in one scorpion mud turtle (Kinosternon scorpioides) and one lowland paca (Cuniculus paca). Phylogenetic analyses inferred by the 18S rRNA gene partial sequences supported these results, highlighting at least five novel Piroplasmida agents, and two novel Hepatozoon agents. This study screened the presence of tick-borne protozoa in a number of wildlife species from the Amazon for the first time. Our results indicate that a variety of genetically distinct Piroplasmida and Hepatozoon organisms circulate under natural conditions in the Amazonian wildlife.