Adriano Pinter

Isolation of Rickettsia rickettsii and Rickettsia bellii in Cell Culture from the Tick Amblyomma aureolatum in Brazil

Abstract:  Brazilian spotted fever (BSF) is a highly lethal disease caused by Rickettsia rickettsii. In the present study, rickettsial infection was evaluated in 669 Amblyomma aureolatum adult ticks collected from naturally infested dogs in Taiaçupeba, a BSF‐endemic area in the state of São Paulo. Ten (1.49%) ticks were infected with Rickettsia bellii, and 6 (0.89%) ticks were infected with R. rickettsii. Both Rickettsia species were isolated and established in Vero cell cultures. The Rickettsia isolates were characterized by molecular analyses, sequencing fragments of different rickettsial genes. Our results suggest that A. aureolatum is an important vector of R. rickettsii in Brazil.

Ticks (Acari: Ixodida) on wild carnivores in Brazil

The present study reports field data of ticks infesting wild carnivores captured from July 1998 to September 2004 in Brazil. Additional data were obtained from one tick collection and from previous published data of ticks on carnivores in Brazil. During field work, a total of 3437 ticks were collected from 89 Cerdocyon thous (crab-eating-fox), 58 Chrysocyon brachyurus (maned-wolf), 30 Puma concolor (puma), 26 Panthera onca (jaguar), 12 Procyon cancrivorus (crab-eating-raccoon), 4 Speothos venaticus (bush-dog), 6 Pseudalopex vetulus (hoary-fox), 6 Nasua nasua (coati), 6 Leopardus pardalis (ocelot), 2 Leopardus tigrinus (oncilla), 1 Leopardus wiedii (margay), 1 Herpailurus yagouaroundi (jaguarundi), 1 Oncifelis colocolo (pampas-cat), 1 Eira barbara (tayara), 1 Galictis vittata (grison), 1 Lontra longicaudis (neotropical-otter), and 1 Potus flavus (kinkajou). Data obtained from the Acari Collection IBSP included a total of 381 tick specimens collected on 13 C. thous, 8 C. brachyurus, 3 P. concolor, 10 P. onca, 3 P. cancrivorus, 4 N. nasua, 1 L. pardalis, 1 L. wiedii, 4 H. yagouaroundi, 1 Galictis cuja (lesser grison), and 1 L. longicaudis. The only tick infested-carnivore species previously reported in Brazil, for which we do not present any field data are Pseudalopex gymnocercus (pampas fox), Conepatus chinga (Molina’s hog-nosed skunk), and Conepatus semistriatus (striped hog-nosed skunk). We report the first tick records in Brazil on two Felidae species (O. colocolo, H. yagouaroundi), two Canidae species (P. vetulus, S. venaticus), one Procyonidae species (P. flavus) and one Mustelidae (E. barbara). Tick infestation remains unreported for 5 of the 26 Carnivora species native in Brazil: Oncifelis geoffroyi (Geoffroy’s cat), Atelocynus microtis (short-eared dog), Pteronura brasiliensis (giant otter), Mustela africana (Amazon weasel), and Bassaricyon gabbii (olingo). Our field data comprise 16 tick species represented by the genera Amblyomma (12 species), Ixodes (1 species), Dermacentor (1 species), Rhipicephalus (1 species), and Boophilus (1 species). Additional 5 tick species (3 Amblyomma species and 1 species from each of the genera Ixodes and Ornithodoros) were reported in the literature. The most common ticks on Carnivora hosts were Amblyomma ovale (found on 14 host species), Amblyomma cajennense (10 species), Amblyomma aureolatum (10 species), Amblyomma tigrinum (7 species), Amblyomma parvum (7 species), and Boophilus microplus (7 species).

Rickettsia infection in five areas of the state of São Paulo, Brazil

This study investigated rickettsial infection in animals, humans, ticks, and fleas collected in five areas of the state of São Paulo. Eight flea species (Adoratopsylla antiquorum antiquorum, Ctenocephalides felis felis, Polygenis atopus, Polygenis rimatus, Polygenis roberti roberti, Polygenis tripus, Rhopalopsyllus lugubris, and Rhopalopsyllus lutzi lutzi), and five tick species (Amblyomma aureolatum, Amblyomma cajennense, Amblyomma dubitatum, Ixodes loricatus, and Rhipicephalus sanguineus) were collected from dogs, cats, and opossums. Rickettsia felis was the only rickettsia found infecting fleas, whereas Rickettsia bellii was the only agent infecting ticks, but no animal or human blood was shown to contain rickettsial DNA. Testing animal and human sera by indirect immunofluorescence assay against four rickettsia antigens (R. rickettsii, R. parkeri, R. felis, and R. bellii), some opossum, dog, horse, and human sera reacted to R. rickettsii with titers at least four-fold higher than to the other three rickettsial antigens. These sera were considered to have a predominant antibody response to R. rickettsii. Using the same criteria, opossum, dog, and horse sera showed predominant antibody response to R. parkeri or a very closely related genotype. Our serological results suggest that both R. rickettsii and R. parkeri infected animals and/or humans in the studied areas.

Serosurvey of Rickettsia spp. in dogs and humans from an endemic area for Brazilian spotted fever in the State of São Paulo, Brazil

The present study provides a rickettsial serosurvey in 25 dogs and 35 humans in an endemic area for Brazilian spotted fever in the State of São Paulo, where the tick Amblyomma aureolatum is the main vector. Testing canine and human sera by indirect immunofluorescence against four Rickettsia antigens (R. rickettsii, R. parkeri, R. felis and R. bellii) showed that 16 (64%) of canine sera and 1 (2.8%) of human sera reacted to at least one of these rickettsial antigens with titers >0r= 64. Seven canine sera and the single reactive human serum showed titers to R. rickettsii at least four times those of any of the other three antigens. The antibody titers in these 7 animals and 1 human were attributed to stimulation by R. rickettsii infection. No positive canine or human serum was attributed to stimulation by R. parkeri, R. felis, or R. bellii. Our serological results showed that dogs are important sentinels for the presence of R. rickettsii in areas where the tick A. aureolatum is the main vector of Brazilian spotted fever.

Prevalence of Ehrlichia canis (Rickettsiales: Anaplasmataceae) in Dogs and Rhipicephalus sanguineus (Acari: Ixodidae) Ticks from Brazil

Abstract The current study evaluated the prevalence of Ehrlichia canis Donatien and Lestoquard in domestic dogs, Canis familiaris L., and Rhipicephalus sanguineus (Latreille) (Acari: Ixodidae) ticks from different areas of Brazil. In Monte Negro County (state of Rondônia, Brazilian western Amazon), the indirect immunofluorescence assay detected E. canis-reactive antibodies (titer ≥40) in 58/153 (37.9%) and 40/161 (24.8%) dogs from the urban and rural areas, respectively. These values were significantly different between the two areas. Ticks from a household in the urban area of Monte Negro, and from households in three other localities (162–165 adult ticks per household) in the state of São Paulo (SP) were tested by polymerase chain reaction (PCR) targeting an erlichial dsb gene fragment. The prevalence of infected ticks (given as minimal infection rate) was 2.3, 6.2, and 3.7% for populations 1 (Monte Negro), 2 (Jundiaí, SP), and 3 (São Paulo I, SP), respectively, which were statistically similar. In contrast, no infected tick was detected in population 4 (São Paulo II, SP). DNA sequences were determined for some of the PCR products generated from ticks and dogs from populations 1–3, being all identical to each other and to available sequences of E. canis in GenBank. These results reinforce previous records of E. canis-infecting dogs in Brazil. Natural infection of R. sanguineus ticks by E. canis is reported for the first time in Brazil, where this tick is the commonest species infesting dogs. RESUMO O presente estudo avaliou a prevalência da infecção por Ehrlichia canis Donatien e Lestoquard em cães domésticos, Canis familiaris L., e carrapatos Rhipicephalus sanguineus (Latreille) (Acari: Ixodidae) em diferentes áreas do Brasil. Pela Reação de Imunofluorescência Indireta (RIFI) (título ≥40), foram detectados anticorpos reativos para E. canis em 37.9% de 153 cães da área urbana e 24.8% de 161 cães da área rural do município de Monte Negro, estado de Rondônia, Amazônia brasileira. Carrapatos R. sanguineus (162 a 165 carrapatos adultos por população) de uma residência da área urbana de Monte Negro e de outras três localidades do estado de São Paulo foram testados por PCR para um fragmento do gene dsb de Ehrlichia. A prevalência de carrapatos infectados [avaliada como Prevalência Mínima de Infecção (PMI)] foi de 2.3, 6.2, e 3.7% para as populações 1 (Monte Negro), 2 (Jundiaí, SP), e 3 (São Paulo I, SP), respectivamente, sendo estes resultados similares. Nenhum carrapato infectado foi detectado na população 4 (São Paulo II, SP). Os produtos da PCR de alguns carrapatos e cães das populações 1, 2 e 3 foram seqüenciados, sendo as seqüências obtidas idênticas entre si e à seqüência de E. canis disponível no GenBank. Estes resultados reforçam estudos anteriores que relataram a infecção por E. canis em cães do Brasil, contudo relata pela primeira vez no Brasil a infecção natural por E. canis em carrapatos R. sanguineus, tido como o principal carrapato de cães no país.

Ticks (Acari: Ixodidae) associated with domestic dogs in Franca region, São Paulo, Brazil.

Ticks on 140 domestic dogs from both urban and rural areas of Franca region in São Paulo state were identified with 102 dogs from urban areas and 38 from rural areas. Of urban dogs, 27.5% were infested exclusively by Rhipicephalus sanguineus ticks. Of the rural dogs, 36.8% were infested with the following tick species: R. sanguineus, Boophilus microplus, Amblyomma ovale and A. cajennense. Mixed infestations included a dog hosting A. cajennense and A. ovale and another with B. microplus and R. sanguineus. The most intense infestations were detected on urban dogs. Hemolymph tests of these ticks performed to detect rickettsial or Borrelia bacteria yielded negative results.

Ecology, biology and distribution of spotted-fever tick vectors in Brazil

Spotted-fever-caused Rickettsia rickettsii infection is in Brazil the major tick-borne zoonotic disease. Recently, a second and milder human rickettsiosis caused by an agent genetically related to R. parkeri was discovered in the country (Atlantic rainforest strain). Both diseases clearly have an ecological background linked to a few tick species and their environment. Capybaras (Hydrochoerus hydrochaeris) and Amblyomma cajennense ticks in urban and rural areas close to water sources are the main and long-known epidemiological feature behind R. rickettsii-caused spotted-fever. Unfortunately, this ecological background seems to be increasing in the country and disease spreading may be foreseen. Metropolitan area of São Paulo, the most populous of the country, is embedded in Atlantic rainforest that harbors another important R. rickettsii vector, the tick Amblyomma aureolatum. Thus, at the city–forest interface, dogs carry infected ticks to human dwellings and human infection occurs. A role for R. rickettsii vectoring to humans of a third tick species, Rhipicephalus sanguineus in Brazil, has not been proven; however, there is circumstantial evidence for that. A R. parkeri-like strain was found in A. ovale ticks from Atlantic rainforest and was shown to be responsible for a milder febrile human disease. Rickettsia-infected A. ovale ticks are known to be spread over large areas along the Atlantic coast of the country, and diagnosis of human infection is increasing with awareness and proper diagnostic tools. In this review, ecological features of the tick species mentioned, and that are important for Rickettsia transmission to humans, are updated and discussed. Specific knowledge gaps in the epidemiology of such diseases are highlighted to guide forthcoming research.

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.

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.

Ticks collected on birds in the state of São Paulo, Brazil

The present study reports a collection of Amblyomma spp. ticks in birds from several areas of the state of São Paulo, Brazil. A total of 568 tick specimens (404 larvae, 164 nymphs) were collected from 261 bird specimens. From these ticks, 204 (36%) specimens (94 larvae, 110 nymphs) were reared to the adult stage, being identified as Amblyomma longirostre (94 larvae, 90 nymphs), Amblyomma calcaratum (13 nymphs), Amblyomma nodosum (2 nymphs), and Amblyomma cajennense (5 nymphs). Additionally, 39 larvae reared to the nymphal stage and 8 nymphs that died before reaching the adult stage were identified as A. longirostre according to peculiar characters inherent to the nymphal stage of this species: scutum elongate, and hypostome pointed. The remaining 271 larvae and 46 nymphs were identified as Amblyomma sp. Ticks were collected from 51 species of birds distributed in 22 bird families and 6 orders. The order Passeriformes constituted the vast majority of the records, comprising 253 (97%) out of the 261 infested birds. Subadults of A. longirostre were identified from 35 species of Passeriformes, comprising 11 families (Cardinalidae, Dendrocolaptidae, Fringillidae, Furnariidae, Parulidae, Pipridae, Thamnophilidae, Thraupidae, Turdidae, Tyrannidae, and Vireonidae), and from 1 species of a non-passerine bird, a puffbird (Bucconidae). Subadults of A. calcaratum were identified from 5 species of Passeriformes, comprising 5 families (Cardinalinae, Conopophagidae, Pipridae, Thamnophilidae and Turdidae). Subadults of A. nodosum were identified from 2 species of Passeriformes, comprising two bird families (Thamnophilidae and Pipridae). Subadults of A. cajennense were identified from 2 species of non-passerine birds, belonging to 2 different orders (Ciconiiformes: Threskiornithidae, and Gruiformes: Cariamidae). Birds were usually infested by few ticks (mean infestation of 2.2 ticks per bird; range: 1–16). Currently, 82 bird species are known to be infested by immature stages of A. longirostre, with the vast majority [74 (90%)] being Passeriformes. Our results showed that Passeriformes seems to be primary hosts for subadult stages of A. longirostre, A. calcaratum, and A. nodosum. However, arboreal passerine birds seem to be the most important hosts for A. longirostre whereas ground-feeding passerine birds seem to be the most important for both A. calcaratum and A. nodosum. In contrast, the parasitism of birds by subadults of A. cajennense has been restricted to non-passerine birds.