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.

Rickettsia bellii in ticks Amblyomma varium Koch, 1844, from birds in Peru

Amazonian birds were caught and examined for the presence of ectoparasites in the Allpahuayo Mishana National Reserve near Iquitos, Peru, from 13 to 16 August 2011. A total of 40 birds representing 16 species were examined. Two birds (5%) were infested with 2 larvae of Amblyomma varium Koch, 1844, and one nymph of A. calcaratum Neumann, 1899. The 2 larvae of A. varium were infected with Rickettsia bellii. This is the first report of R. bellii in A. varium and also the first record of this rickettsia in Peru. In addition, an immature A. calcaratum is reported from Peru for the first time. (c) 2012 Elsevier GmbH. All rights reserved.

A Third Amblyomma Species and the First Tick-Borne Rickettsia in Chile

During November 2010, three ticks were collected from three dogs living in the rural area of Arica, northern Chile. Morphological analyses of the ticks in the laboratory revealed that they were most similar to Amblyomma maculatum Koch and Amblyomma triste Koch. However, because of unique metatarsal spurs, neither of the Chilean specimens could be assigned with certainty to A. maculatum or A. triste, based on external morphology. The mitochondrial 16S rRNA gene partial sequences obtained from two Chilean specimens were 99.5% identical to A. triste from Uruguay, and 99.0% identical to A. maculatum from the United States. Through phylogenetic analysis inferred from partial 16S rRNA sequences, the Chilean specimens were classified as A. triste. Molecular analyses also showed that one of the three Chilean ticks was infected by Candidatus 'Rickettsia andeanae'. These findings extend the geographical distribution of A. triste to Chile, where no tick-associated rickettsia had been reported previously.

Rickettsia bellii infecting Amblyomma sabanerae ticks in El Salvador

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.

Distribution of Rickettsia rickettsii in ovary cells of Rhipicephalus sanguineus (Latreille1806) (Acari: Ixodidae)

Abstract Background Considering the fact that the dog tick, Rhipicephalus sanguineus, has a great potential to become the vector of Brazilian Spotted Fever (BSF) for humans, the present study aimed to describe the distribution of the bacterium Rickettsia rickettsii, the etiological agent of BSF, in different regions of the ovaries of R. sanguineus using histological techniques. The ovaries were obtained from positive females confirmed by the hemolymph test and fed in the nymph stage on guinea pigs inoculated with R. rickettsii. Results The results showed a general distribution of R. rickettsii in the ovary cells, being found in oocytes in all stages of development (I, II, III, IV and V) most commonly in the periphery of the oocyte and also in the cytoplasm of pedicel cells. Conclusions The histological analysis of the ovaries of R. sanguineus infected females confirmed the presence of the bacterium, indicating that the infection can interfere negatively in the process of reproduction of the ticks, once alterations were detected both in the shape and cell structure of the oocytes which contained bacteria.

Natural blood feeding and temperature shift modulate the global transcriptional profile of Rickettsia rickettsii infecting its tick vector

Rickettsia rickettsii is an obligate intracellular tick-borne bacterium that causes Rocky Mountain Spotted Fever (RMSF), the most lethal spotted fever rickettsiosis. When an infected starving tick begins blood feeding from a vertebrate host, R. rickettsii is exposed to a temperature elevation and to components in the blood meal. These two environmental stimuli have been previously associated with the reactivation of rickettsial virulence in ticks, but the factors responsible for this phenotype conversion have not been completely elucidated. Using customized oligonucleotide microarrays and high-throughput microfluidic qRT-PCR, we analyzed the effects of a 10 degrees C temperature elevation and of a blood meal on the transcriptional profile of R. rickettsii infecting the tick Amblyomma aureolatum. This is the first study of the transcriptome of a bacterium in the genus Rickettsia infecting a natural tick vector. Although both stimuli significantly increased bacterial load, blood feeding had a greater effect, modulating five-fold more genes than the temperature upshift. Certain components of the Type IV Secretion System (T4SS) were up-regulated by blood feeding. This suggests that this important bacterial transport system may be utilized to secrete effectors during the tick vector's blood meal. Blood feeding also up-regulated the expression of antioxidant enzymes, which might correspond to an attempt by R. rickettsii to protect itself against the deleterious effects of free radicals produced by fed ticks. The modulated genes identified in this study, including those encoding hypothetical proteins, require further functional analysis and may have potential as future targets for vaccine development.

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. (c) 2012 Elsevier GmbH. All rights reserved.

A surrogate life cycle of Amblyomma ovale Koch, 1844

As ticks spend most of their time off-host, the environment is a major selective force of these parasites. In fact, human impact on landscapes has favored a minority of tick species which became well-known pests. However, this is an ongoing process and novel pests may arise. We herein report a surrogate life cycle of a neotropical tick species. Amblyomma ovate, and which may be related to an increased risk of human rickettsiosis. Under natural conditions, adults of this tick species feed on carnivores and exhibit non-nidicolous ambush behavior, whereas larvae and nymphs feed on small rodents and birds. In an anthropized spot within an Atlantic rainforest reserve of Brazil, an A. ovate population exhibited a nidicolous behavior with all 3 tick stages feeding on the dog. This dog's infestation was outstandingly high, and it displayed the highest anti-Rickettsia titers and harbored Rickettsia-infected ticks. (c) 2012 Elsevier GmbH. All rights reserved.

Brazilian spotted fever: Real-time PCR for diagnosis of fatal cases

Suspicion of Brazilian spotted fever (BSF) should occur in endemic regions upon surveillance of the acute febrile icteric hemorrhagic syndrome (AFIHS). However, limitations associated with currently available laboratory tests pose a challenge to early diagnosis, especially in fatal cases. Two real-time PCR (qPCR) protocols were evaluated to diagnose BSF in 110 fatal AFIHS cases, collected in BSF-endemic regions in 2009-2010. Of these, 24 were positive and 86 negative by indirect immunofluorescence (IFA) assay (cutoff IgG and/or IgM >= 128). DNA from these samples was used in the qPCR protocols: one to detect Rickettsia spp. (Citrate synthase gene) and another to determine spotted fever group (SFG) Rickettsia species (OmpA gene). Of the 24 IFA-positive samples, 5 (21%) were positive for OmpA and 9 (38%) for citrate synthase. In the IFA-negative group (n = 86), OmpA and citrate synthase were positive in 23 (27%) and 27 (31%), respectively. These results showed that the 2 qPCR protocols were about twice as sensitive as the IFA test alone (93% concordance). In conclusion, qPCR is a sensitive method for the diagnosis of fatal BSF cases and should be considered for routine surveillance of AFIHS in places like Brazil, where spotted fever-related lethality is high and other endemic diseases like dengue and leptospirosis can mislead diagnosis. (C) 2012 Elsevier GmbH. All rights reserved.

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

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 Parana, 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 Aragao. 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.

Rickettsial Infection in Ticks Collected from Road-Killed Wild Animals in Rio de Janeiro, Brazil

During 2008D2010, ticks were collected from road-killed wild animals within the Serra dos Orgaos National Park area in the state of Rio de Janeiro, Brazil. In total, 193 tick specimens were collected, including Amblyomma dubitatum Neumann and Amblyomma cajennense (F.) from four Hydrochoerus hydrochaeris (L.), Amblyomma calcaratum Neumann and A. cajennense from four Tamandua tetradactyla (L.), Amblyomma aureolatum (Pallas) and A. cajennense from five Cerdocyon thous L., Amblyomma longirostre (Koch) from one Sphiggurus villosus (Cuvier), Amblyomma varium Koch from three Bradypus variegatus Schinz, and A. cajennense from one Buteogallus meridionalis (Latham). Molecular analyses based on polymerase chain reaction targeting two rickettsial genes (gltA and ompA) on tick DNA extracts showed that 70.6% (12/17) of the A. dubitatum adult ticks, and all Amblyomma sp. nymphal pools collected from capybaras were shown to contain rickettsial DNA, which after DNA sequencing, revealed to be 100% identical to the recently identified Rickettsia sp. strain Pampulha from A. dubitatum ticks collected in the state of Minas Gerais, Brazil. Phylogenetic analysis with concatenated sequences (gltA-ompA) showed that our sequence from A. dubitatum ticks, referred to Rickettsia sp. strain Serra dos Orgaos, segregated under 99% bootstrap support in a same cluster with Old World rickettsiae, namely R. tamurae, R. monacensis, and Rickettsia sp. strain 774e. Because A. dubitatum is known to bite humans, the potential role of Rickettsia sp. strain Serra dos Orgaos as human pathogen must be taken into account, because both R. tamurae and R. monacencis have been reported infecting human beings.

Epidemiology of Brazilian spotted fever in the Atlantic Forest, state of Sao 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 Sao 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.

Exposure of small mammals to ticks and rickettsiae in Atlantic Forest patches in the metropolitan area of Recife, North-eastern Brazil

Between December 2007 and March 2009, small mammals were captured in 6 Atlantic Forest patches in Brazil. We assessed tick-host associations and whether they differ among forest strata, sites, seasons, and host age classes or between sexes. Moreover, we assessed the exposure of animals to Rickettsia spp. In total, 432 animals were captured and 808 ticks were found on 32-9% of them. Significant differences were found among host species, collection sites, and forest strata; microhabitat preference was a strong risk factor for tick infestation. The highest tick density rates were recorded in forest fragments settled in rural areas; 91.3% of the ticks were collected from animals trapped in these forest fragments. A high prevalence (68.8%) of antibodies to Rickettsia spp. was detected among animals. This study suggests that disturbed Atlantic Forest fragments provide an environment for ticks and small mammals, which are highly exposed to rickettsiae. It also indicates that forest patches settled in rural areas are usually associated with higher small mammal diversity as well as with higher tick density rates.

Rickettsial infection in Amblyomma cajennense ticks and capybaras (Hydrochoerus hydrochaeris) in a Brazilian spotted fever-endemic area

Brazilian spotted fever (BSF), caused by the bacterium Rickettsia rickettsii, is the deadliest spotted fever of the world. In most of the BSF-endemic areas, capybaras (Hydrochoerus hydrochaeris) are the principal host for the tick Amblyomma cajennense, which is the main vector of BSF. In 2012, a BSF case was confirmed in a child that was bitten by ticks in a residential park area inhabited by A. cajennense-infested capybaras in Itú municipality, southeastern Brazil. Host questing A. cajennense adult ticks were collected in the residential park and brought alive to the laboratory, where they were macerated and intraperitoneally inoculated into guinea pigs. A tick-inoculated guinea pig that presented high fever was euthanized and its internal organs were macerated and inoculated into additional guinea pigs (guinea pig passage). Tissue samples from guinea pig passages were also used to inoculate Vero cells through the shell vial technique. Infected cells were used for molecular characterization of the rickettsial isolate through PCR and DNA sequencing of fragments of three rickettsial genes (gltA, ompA, and ompB). Blood serum samples were collected from 172 capybaras that inhabited the residential park. Sera were tested through the immunofluorescence assay using R. rickettsii antigen. A tick-inoculated guinea pig presented high fever accompanied by scrotal reactions (edema and marked redness). These signs were reproduced by consecutive guinea pig passages. Rickettsia was successfully isolated in Vero cells that were inoculated with brain homogenate derived from a 3rd passage-febrile guinea pig. Molecular characterization of this rickettsial isolate (designated as strain ITU) yielded DNA sequences that were all 100% identical to corresponding sequences of R. rickettsii in Genbank. A total of 83 (48.3%) out of 172 capybaras were seroreactive to R. rickettsii, with endpoint titers ranging from 64 to 8192. A viable isolate of R. rickettsii was obtained from the tick A. cajennense, comprising the first viable R. rickettsi isolate from this tick species during the last 60 years. Nearly half of the capybara population of the residential park was seroreactive to R. rickettsii, corroborating the findings that the local A. cajennense population was infected by R. rickettsii.

New records and human parasitism by Ornithodoros mimon (Acari: Argasidae) in Brazil

The bat tick Ornithodoros mimon Kohls, Clifford & Jones is currently known by only few reports in Bolivia, Uruguay, Argentina, and the state of São Paulo in southeastern Brazil. Here, we expand the distribution of O. mimon in Brazil to the states of Minas Gerais (southeastern region), Goiás (central-western), Pernambuco, and Rio Grande do Norte (northeastern). Ticks were collected on human dwellings, where there had been repeated complains of tick bites on persons during the night. Tick bites were generally followed by intense inflammatory reactions that lasted for several weeks at the bite site. Bats and opossums were reported to inhabit the attic of the infested houses. In addition, a free-ranging opossum (Didelphis albiventris Lund) trapped in Rio Grande do Norte was found infested by argasid larvae. Based on morphological and/or molecular analysis, all ticks were identified as O. mimon. From one of the sites (Tiradentes, state of Minas Gerais), 20 field-collected nymphs were tested by a battery of polymerase chain reaction protocols targeting tick-borne microorganisms of the genera Babesia, Hepatozoon, Rickettsia, Borrelia, Anaplasma, Ehrlichia, and Coxiella; no tick specimen was found infected by any of these microorganism genera. The current study expands northwards the distribution of O. mimon, which has been shown to be very harmful to humans because of the intense inflammatory response that usually occurs after tick bites.