Molecular and Serological Survey of the Cat-Scratch Disease Agent (Bartonella henselae) in Free-Ranging Leopardus geoffroyi and Leopardus wiedii (Carnivora: Felidae) From Pampa Biome, Brazil.

The genus Bartonella comprises emerging bacteria that affect humans and other mammals worldwide. Felids represent an important reservoir for several Bartonella species. Domestic cats are the main reservoir of Bartonella henselae, the agent of cat scratch disease (CSD). It can be transmitted directly by scratches and bites from infected cats and via cat fleas. This study aims to investigate the circulation of Bartonella spp. in free-ranging Neotropical wild felids from Southern Brazil using serological and molecular methods. In this study, 53 live-trapped free-ranging wild felids were sampled, 39 Leopardus geoffroyi and 14 Leopardus wiedii, from five municipalities in the Rio Grande, do Sul state, southern Brazil. All captured animals were clinically healthy. Two blood samples of L. geoffroyi were positive, by PCR, for the presence of B. henselae DNA. Conversely, none of L. wiedii blood samples were positive when tested using PCR. Indirect immunofluorescence assay (IFA) showed that 28% of serum samples of wild felids were reactive (seropositive) for B. henselae by immunofluorescence, with titers ranging from 64 to 256. The results presented here provide the first evidence of a Bartonella-enzootic cycle involving L. geoffroyi and L. wiedii, which may account for the spillover of the emerging zoonotic pathogen B. henselae for the indigenous fauna in Southern Brazil.

A tick cell line as a powerful tool to screen the antimicrobial susceptibility of the tick-borne pathogen Anaplasma marginale.

Anaplasma marginale is the causative agent of the severe bovine anaplasmosis. The tick Rhipicephalus microplus is one of the main vectors of A. marginale in tropical and subtropical regions of the world. After the tick bite, the bacterium invades and proliferates within the bovine erythrocytes leading to anemia, impairment of milk production and weight loss. In addition, infection can cause abortion and high mortality in areas of enzootic instability. Immunization with live and inactivated vaccines are employed to control acute bovine anaplasmosis. However, they do not prevent persistent infection. Consequently, infected animals, even if immunized, are still reservoirs of the bacterium and contribute to its dissemination. Antimicrobials are largely employed for the prophylaxis of bovine anaplasmosis. However, they are often used in sublethal doses which may select pre-existing resistant bacteria and induce genetic or phenotypic variations. Therefore, we propose a new standardized in vitro assay to evaluate the susceptibility of A. marginale strains to different antimicrobials. This tool will help health professionals to choose the more adequate treatment for each herd which will prevent the selection and spread of resistant strains. For that, we initially evaluated the antimicrobial susceptibility of two field isolates of A. marginale (Jaboticabal and Palmeira) infecting bovines. The least susceptible strain (Jaboticabal) was used for the standardization of an antimicrobial assay using a culture of Ixodes scapularis-derived tick cell line, ISE6. Results showed that enrofloxacin (ENRO) at 0.25, 1 or 4 µg/mL and oxytetracycline (OTC) at 4 or 16 µg/mL are the most efficient treatments, followed by OTC at 1 µg/mL and imidocarb dipropionate (IMD) at 1 or 4 µg/mL. In addition, this proposed tool has technical advantages compared to the previously established bovine erythrocyte culture. Thereby, it may be used to guide cattle farmers to the correct use of antimicrobials. The choice of the most suitable antimicrobial is essential to eliminate persistent infections, prevent the spread of resistant strains and help controlling of bovine anaplasmosis.

Detection of Rangelia vitalii (Piroplasmida: Babesiidae) in asymptomatic free-ranging wild canids from the Pampa biome, Brazil.

Canine rangeliosis is a tick-borne disease caused by the protozoan Rangelia vitalii, which has only been reported in South America. With this knowledge, we hypothesized that neotropical foxes could act as asymptomatic natural carriers of R. vitalii. To test this, we captured 44 free-ranging foxes and investigated the presence of R. vitalii DNA, and whether the infected animals presented any clinical findings or hematological changes. Eight foxes (18%), seven Cerdocyon thous (7/27-25%), and one Lycalopex gymnocercus (1/17-5%) were positive for R. vitalii. All foxes were clinically healthy and showed no hematological abnormalities. Thus, we propose that neotropical canids, particularly C. thous, could be the natural carriers of R. vitalii.

Clinical Findings Associated with Ornithodoros brasiliensis Tick Parasitism in Travelers, Southern Brazil.

We report a series of clinical cases associated with parasitism by the Ornithodoros brasiliensis tick in a group of travelers in the Caxias do Sul municipality, Southern Brazil. These cases draw attention to underdiagnosed noninfectious syndromes caused by ticks with restricted local distributions.

Molecular survey of Rickettsia spp. in the Neotropical deer tick Haemaphysalis juxtakochi from Brazilian Pampa.

Spotted fever (SF) is a tick-borne disease associated with Rickettsia spp.. In the Pampa biome, Southern Brazil, cases of SF seem to be strongly linked with the practice of hunting wild animals. An investigation of rickettsiae in tick species found on wild animals could provide more information regarding the rickettsiosis enzootic cycle. The aim of this study is to describe the results of a molecular survey of Rickettsia spp. in the Neotropical deer tick, Haemaphysalis juxtakochi Cooley, 1946 (Acari: Ixodidae), from the Brazilian Pampa. Ticks were obtained from 14 road-killed gray brocket deer, Mazama gouazoubira (Artiodactyla: Cervidae), found in nine different municipalities of Rio Grande do Sul state, Southern Brazil. Ticks were processed individually to obtain genomic DNA, and then Rickettsia spp. was investigated using a set of PCR reactions that amplified the rickettsial fragments of the gltA, ompA, and htrA genes. Of the 24 tick samples tested, DNA of Rickettsia parkeri sensu stricto (s.s.) was found in 11 H. juxtakochi specimens collected in two different areas of the Brazilian Pampa. This is the first report of R. parkeri s.s. (the main agent associated with SF in the Uruguayan, Argentinian, and Brazilian Pampa) in H. juxtakochi ticks. These findings indicate that R. parkeri s.s. may be much more common and widely distributed in the Pampa biome than previously assumed. Moreover, H. juxtakochi ticks and gray brocket deer could participate in the potential spillover of R. parkeri s.s. from endemic to non-endemic areas in the South American Pampa.

DETECTION OF BARTONELLA SP. IN DEER LOUSE FLIES (LIPOPTENA MAZAMAE) ON GRAY BROCKET DEER (MAZAMA GOUAZOUBIRA) IN THE NEOTROPICS.

Louse flies or deer keds, Lipoptena spp., are widespread in Neotropical cervids, but the vector-borne pathogens of louse flies had only been previously reported in the Northern hemisphere. This is the first report of Bartonella spp. in deer louse flies (Lipoptena mazamae) in the neotropics collected from gray brocket deer ( Mazama gouazoubira ) in Southern Brazil. DNA from Bartonella sp. was detected in all 429 L. mazamae collected from 11 road-killed gray brocket deer. The same sequences of DNA of Bartonella spp. were identified in samples. Gray brocket deer are abundant in Brazil, so Bartonella-infected Lipoptena spp. may be widely distributed in the neotropics.

Rotational and selective protocols using acaricides to control a multi-resistant strain of Rhipicephalus microplus under field conditions in Southern Brazil.

The cattle tick, Rhipicephalus microplus, causes severe economic losses to beef and dairy production in several countries. The control of the cattle tick is based mostly on chemical acaricides. Currently, there are reports of tick populations resistant to all six classes of acaricides available on the market. The aim of this work was to evaluate two protocols for the control of the cattle tick, R. microplus, under field conditions. For that, an acaricide rotational-based approach and selective (partial)-based protocols were tested against a multi-resistant tick strain. Eighteen Brangus cattle were divided into three experimental groups: Group 1, a rotational-based approach aiming at six treatments per year (spring-spring period) using four different classes of acaricide (IPV6 protocol); Group 2, a selective (partial) and rotational-based protocol, in which only half of the animals with the highest tick counts were treated (IPV7S protocol); and Group 3 (non-treated). The protocols were able to maintain low tick infestation during the 57 weeks of study. The two experimental groups showed lower tick counts than the non-treated group, but not between them. Weekly efficacy of the experimental protocols reached 95% in Group 1 and 88% in Group 2. Herein, we proposed two protocols of cattle tick control (IPV6 and IPV7S) for use in multi-resistant tick populations in Southern Brazil.

Mechanisms of amitraz resistance in a Rhipicephalus microplus strain from southern Brazil.

Amitraz is one of the most used acaricides for the control of ticks of domestic animals, however, extensive use of this active ingredient has favored the development of resistant populations of Rhipicephalus microplus worldwide. The possible mechanisms of metabolic and/or target-site alterations mechanisms of amitraz resistance were investigated in a Brazilian field population of R. microplus (São Gabriel strain). Bioassays with the synergists piperonylbutoxide, triphenylphosphate and diethyl-maleate were used to evaluate the metabolic mechanisms involved. Target-site insensitivity was investigated by amplification and sequencing of a fragment of the octopamine/tyramine (OCT/TYR) receptor gene. Piperonylbutoxide synergism (synergism ratio = 2.8) indicated the participation of the P450 pathway in the detoxification of amitraz. Previously reported single nucleotide polymorphisms that confer amino acid changes in the OCT/TYR receptor, threonine to proline (T8P) and leucine to serine (L22S), were found in the amitraz-resistant strain but not in the susceptible reference strain. The results suggest that amitraz resistance in the studied strain is multi-factorial and may result from cytochrome P450 detoxification and mutations in octopamine receptors.

Transcriptomic Analysis of Salivary Glands of Ornithodoros brasiliensis Aragão, 1923, the Agent of a Neotropical Tick-Toxicosis Syndrome in Humans.

Tick salivary glands produce and secrete a variety of compounds that modulate host responses and ensure a successful blood meal. Despite great progress made in the identification of ticks salivary compounds in recent years, there is still a paucity of information concerning salivary molecules of Neotropical argasid ticks. Among this group of ticks, considering the number of human cases of parasitism, including severe syndromes and hospitalization, Ornithodoros brasiliensis can be considered one of the major Neotropical argasid species with impact in public health. Here, we describe the transcriptome analysis of O. brasiliensis salivary glands (ObSG). The transcriptome yielded ~14,957 putative contigs. A total of 368 contigs were attributed to secreted proteins (SP), which represent approximately 2.5% of transcripts but ~53% expression coverage transcripts per million. Lipocalins are the major protein family among the most expressed SP, accounting for ~16% of the secretory transcripts and 51% of secretory protein abundance. The most expressed transcript is an ortholog of TSGP4 (tick salivary gland protein 4), a lipocalin first identified in Ornithodoros kalahariensis that functions as a leukotriene C4 scavenger. A total of 55 lipocalin transcripts were identified in ObSG. Other transcripts potentially involved in tick-host interaction included as: basic/acid tail secretory proteins (second most abundant expressed group), serine protease inhibitors (including Kunitz inhibitors), 5' nucleotidases (tick apyrases), phospholipase A2, 7 disulfide bond domain, cystatins, and tick antimicrobial peptides. Another abundant group of proteins in ObSG is metalloproteases. Analysis of these major protein groups suggests that several duplication events after speciation were responsible for the abundance of redundant compounds in tick salivary glands. A full mitochondrial genome could be assembled from the transcriptome data and confirmed the close genetic identity of the tick strain sampled in the current study, to a tick strain previously implicated in tick toxicoses. This study provides novel information on the molecular composition of ObSG, a Brazilian endemic tick associated with several human cases of parasitism. These results could be helpful in the understanding of clinical findings observed in bitten patients, and also, could provide more information on the evolution of Neotropical argasids.

Molecular characterization of bacterial communities of two neotropical tick species (Amblyomma aureolatum and Ornithodoros brasiliensis) using rDNA 16S sequencing.

Ticks are one of the main vectors of pathogens for humans and animals worldwide. However, they harbor non-pathogenic microorganisms that are important for their survival, facilitating both their nutrition and immunity. We investigated the bacterial communities associated with two neotropical tick species of human and veterinary potential health importance from Brazil: Amblyomma aureolatum and Ornithodoros brasiliensis. In A. aureolatum (adult ticks collected from wild canids from Southern Brazil), the predominant bacterial phyla were Proteobacteria (98.68%), Tenericutes (0.70%), Bacteroidetes (0.14%), Actinobacteria (0.13%), and Acidobacteria (0.05%). The predominant genera were Francisella (97.01%), Spiroplasma (0.70%), Wolbachia (0.51%), Candidatus Midichloria (0.25%), and Alkanindiges (0.13%). The predominant phyla in O. brasiliensis (adults, fed and unfed nymphs collected at the environment from Southern Brazil) were Proteobacteria (90.27%), Actinobacteria (7.38%), Firmicutes (0.77%), Bacteroidetes (0.44%), and Planctomycetes (0.22%). The predominant bacterial genera were Coxiella (87.71%), Nocardioides (1.73%), Saccharopolyspora (0.54%), Marmoricola (0.42%), and Staphylococcus (0.40%). Considering the genera with potential importance for human and animal health which can be transmitted by ticks, Coxiella sp. was found in all stages of O. brasiliensis, Francisella sp. in all stages of A. aureolatum and in unfed nymphs of O. brasiliensis, and Rickettsia sp. in females of A. aureolatum from Banhado dos Pachecos (BP) in Viamão municipality, Brazil, and in females and unfed nymphs of O. brasiliensis. These results deepen our understanding of the tick-microbiota relationship in Ixodidae and Argasidae, driving new studies with the focus on the manipulation of tick microbiota to prevent outbreaks of tick-borne diseases in South America.

Genomic analysis on Brazilian strains of Anaplasma marginale.

Anaplasma marginale is a vector-borne pathogen that causes a disease known as anaplasmosis. No sequenced genomes of Brazilian strains are yet available. The aim of this work was to compare whole genomes of Brazilian strains of A. marginale (Palmeira and Jaboticabal) with genomes of strains from other regions (USA and Australia strains). Genome sequencing of Brazilian strains was performed by means of next-generation sequencing. Reads were mapped using the genome of the Florida strain of A. marginale as a reference sequence. Single nucleotide polymorphisms (SNPs) and insertions/deletions (INDELs) were identified. The data showed that two Brazilian strains grouped together in one particular clade, which grouped in a larger American group together with North American strains. Moreover, some important differences in surface proteins between the two Brazilian isolates can be discerned. These results shed light on the evolutionary history of A. marginale and provide the first genome information on South American isolates. Assessing the genome sequences of strains from different regions is essential for increasing knowledge of the pan-genome of this bacteria.

Serosurvey of antibodies against zoonotic pathogens in free-ranging wild canids (Cerdocyon thous and Lycalopex gymnocercus) from Southern Brazil.

The expansion of urbanization on natural areas is increasing contact between human populations with wild animals. Wild carnivores can act as sentinel hosts or environmental health indicators. Thus, the aim of this work was to investigate the exposure of two major species of wild canids from Southern Brazil to selected pathogens. For that, we live-trapped free-ranging Cerdocyon thous and Lycalopex gymnocercus in five localities and determined the frequency of animals with antibodies against Toxoplasma gondii, Trypanosoma cruzi, Leishmania infantum, Neospora caninum, and Leptospira spp. Among the canids sampled, 23% (12/52) (95%CI: 13-36%) had antibodies against T. gondii, with titers ranging from 64 to 512. For T. cruzi, 28% (15/52) (95%CI: 18-42%) of sampled canids were seropositive, with titers ranging from 8 to 64. Concerning the protozoan pathogen N. caninum, a total of 5% (3/52) (95%CI: 2-15%) of wild canids had antibodies against it. None of the sampled canids showed the presence of antibodies against L. infantum. On the other hand, 44% (23/52) (95%CI: 31-57%) of the wild canids showed antibodies against Leptospira spp. The set of results presented here, show that free-ranging neotropical wild canids are exposed and have antibodies against to T. gondii, T. cruzi, Leptospira spp., and to a lesser degree to N. caninum. We found no evidence of L. infantum circulation among the studied populations. These results highlight some of the major pathogens which may represent risks for populations of these wild canids. DATA AVAILABILITY STATEMENT: The data that support the findings of this study are available from the corresponding author upon reasonable request.

Molecular detection and phylogenetic relationship of Haemosporida parasites in free-ranging wild raptors from Brazil.

The order Haemosporida is widely distributed parasitizing members of the Aves class. In birds of prey, infection with Plasmodium spp. parasites varies from an apathogenic form to a clinical syndrome. However, studies on Haemosporida in raptors from the neotropical region are scarce. The aim of this study was to investigate natural infection by Plasmodium spp., Haemoproteus spp. and Leucocytozoon spp. in free-ranging wild raptors from southern Brazil. For this, we sampled 206 individuals of 21 species: 94 live-trapped Southern Caracaras (Caracara plancus) and 112 raptors from other species that were brought to rehabilitation centers. The presence of infection was investigated using a nested-PCR for Haemosporida performed on blood samples. Overall, 56 out of 206 birds were positive for Plasmodium spp./Haemoproteus spp. Twenty-two percent (21/94) of the C. plancus samples were positive. Of the 112 wild raptors rescued, 31% (35/112) of those belonging to 15 other species tested positive. No sample was positive for Leucocytozoon spp. Herein, we demonstrated nine lineages of Haemosporidian parasites (eight Plasmodium sp. and one Haemoproteus sp.) in free-living species of Brazilian birds of prey, being six of them potential novel lineages. It suggests that information currently available on South-American haemosporidian from these birds greatly underestimate the potential lineage diversity in this region.

Ticks, mites, fleas, and vector-borne pathogens in free-ranging neotropical wild felids from southern Brazil.

The study of vectors and vector-borne diseases associated with ectoparasites from free-living Neotropical small wild felids is scarce, and the few existing studies on this theme focused on the genus Panthera, Paleartic species or in captive animals. For this reason, the aim of this study was to identify the diversity of ectoparasites and potentially associated Rickettsia and Bartonella species in free-ranging neotropical wild cats collected in the State of Rio Grande do Sul, southern Brazil. A total of 82 ticks, 10 fleas and 22 mites were collected from 18 Geoffroy's cats (Leopardus geoffroyi), 11 margays (Leopardus wiedii), two southern tiger cats (Leopardus guttulus), two jaguarundis (Herpailurus yagouaroundi), three ocelots (Leopardus pardalis) and two pumas (Puma concolor). We identified four tick species: Rhipicephalus microplus and three species from genus Amblyomma, the most frequent being Amblyomma aureolatum; three flea species corresponding to Ctenocephalides felis, Xenopsylla cheopis, and Pulex irritans; and one mite of genus Eutrombicula. In ectoparasites we found DNA of Rickettsia parkeri and Rickettsia asembonensis in ticks and DNA of Bartonella clarridgeiae, Bartonella sp. and R. asembonensis in fleas. Our results highlight the evidences of vectors and vector-borne agents in wild animals, and their potential wide distribution in the Pampa biome and the southernmost portion of the Atlantic Rainforest in Brazil.

Rickettsia parkeri in the Pampa biome of southern Brazil: Isolation, molecular characterization, and serological evidence of canine infection.

In this work we performed a field study in a spotted fever-endemic area of the Pampa biome in Rio Grande do Sul, the southernmost state of Brazil (Rosário do Sul municipality), with the aim to evaluate the serological status of dogs for spotted fever group (SFG) rickettsiae, to attempt the isolation rickettsiae from A. tigrinum ticks, and to further perform a multi-genic molecular characterization of the rickettsial isolate. For this purpose, we collected blood serum samples and ticks from 18 rural dogs during 2016-2017. By immunofluorescence assay, all 18 dogs (100%) reacted serologically to SFG rickettsiae, with highest endpoint titers to R. parkeri, suggesting that at least part of these dogs have been infected by R. parkeri. Among 23 ticks collected from the dogs (13 A. tigrinum and 10 Rhipicephalus sanguineus sensu stricto), 46% (6/13) A. tigrinum contained DNA of R. parkeri, which was successfully isolated in Vero cell culture from one of these ticks. Partial sequences of five genes (gltA, ompA, virB4, dnaA, dnaK) and 3 intergenic spacers (mppA-purC, rrl-rrf-ITS, rpmE-tRNAfMet) were generated from this rickettsial isolate, and were all 100% identical to corresponding sequences of R. parkeri s.s. We provide the first report of a viable isolate of R. parkeri from A. tigrinum, confirming that this isolate belongs to the strain R. parkeri s.s., as it has already been demonstrated for other two tick species of the Amblyomma maculatum species complex (A. maculatum and Amblyomma triste).

Detection of Bartonella sp. and a novel spotted fever group Rickettsia sp. in Neotropical fleas of wild rodents (Cricetidae) from Southern Brazil.

The Neotropical region shows a great diversity of fleas, comprising more than 50 genera. The importance of the study of fleas is linked to their potential role as disease vectors. The aim of this study is to investigate the presence of Rickettsia spp. and Bartonella spp. in Neotropical fleas collected from wild rodents in Southern Brazil. From 350 rodents captured, 30 were parasitized by fleas. A total of 61 fleas belonging to two genera and six different species were collected (Craneopsylla minerva minerva, Polygenis occidentalis occidentalis, Polygenis platensis, Polygenis pradoi, Polygenis rimatus, and Polygenis roberti roberti). In 13 % of fleas of three different species (C. minerva, P. platensis, and P. pradoi) Rickettsia sp. DNA was found. Phylogenetic analysis of concatenated sequences of gltA, htrA, and ompA genes showed that Rickettsia sp. found in rodent fleas (referred as strain Taim) grouped together with Spotted Fever Group Rickettsia. In reference to Bartonella spp., five genotypes were identified in seven fleas of two species (C. minerva and P. platensis) and in five rodent spleens. Also, 207 frozen samples of wild rodents were screened for these pathogens: while none was positive for Rickettsia spp.; five rodent spleens were PCR-positive for Bartonella spp.. Herein, we show the detection of potential novel variants of Bartonella sp. and Rickettsia sp. in fleas collected of wild rodents from Southern Brazil. Further studies are needed to fully characterize these microorganisms, as well as to improve the knowledge on the potential role of Neotropical flea species as diseases vectors.

Records of ticks on humans in Rio Grande do Sul state, Brazil.

More than seventy tick species have been reported in Brazil. Despite the emergence of tick-borne diseases in Neotropical region, there are still limited data available on tick species parasitizing humans in Brazil. Rio Grande do Sul is the southernmost state of Brazil, comprising the only part of Brazilian territory inside the Pampa biome, as well as the transition between subtropical and temperate zones. Here, we report on human parasitism by ticks in Rio Grande do Sul state between 2004 and 2017. Seventy cases of human parasitism by ticks were recorded, with a total of 81 tick specimens collected. These included 11 tick species belonging to three genera of Ixodidae (hard-ticks), Amblyomma, Haemaphysalis and Rhipicephalus; and one genus of Argasidae, Ornithodoros. The most prevalent tick species associated to cases of human parasitism were Amblyomma parkeri (24%), Rhipicephalus sanguineus sensu lato (22%), Amblyomma aureolatum (15%) and Amblyomma ovale (12%). A spatial analysis showed two major hot spots of human parasitism by ticks in Rio Grande do Sul state. The findings of this study highlight the need for permanent monitoring of human parasitism by ticks in order to provide a better understanding of tick and tick-borne disease eco-epidemiology, and the early identification of potential cases of tick-borne diseases, particularly in spotted fever endemic regions.

Evaluation of the vector competence of six ixodid tick species for Rangelia vitalii (Apicomplexa, Piroplasmorida), the agent of canine rangeliosis.

Rangelia vitalii is the etiologic agent of canine rangeliosis, a severe piroplasmosis that affects domestic dogs in Brazil, Uruguay and Argentina. While R. vitalii is one of the most pathogenic tick-borne pathogens for dogs in the world, its tick vector has remained unknown. The present study evaluated the vector competence of Rhipicephalus sanguineus sensu lato (both tropical and temperate species), Amblyomma aureolatum, Amblyomma ovale, Amblyomma tigrinum, and Amblyomma sculptum for R. vitalii. These six tick species were selected for the study because they comprise the main tick species infesting dogs within the distribution area of canine rangeliosis in South America. Acquisition feeding of the above six tick species was performed on domestic dogs showing clinical signs of canine rangeliosis, after being experimentally infected through intravenous inoculation or infestation with R. vitalii-infected ticks. Thereafter, engorged ticks were evaluated for transstadial and transovarial passages of R. vitalii through molecular analysis after molting or oviposition and egg hatching. The resultant ticks were evaluated for their competence to transmit R. vitalii to susceptible dogs. Among the six tick species, only A. aureolatum was able to acquire and perpetuate R. vitalii by transstadial and transovarial passages, as demonstrated by >5% infection rates of ticks after hatching or molting. When exposed to transmission feeding, only A. aureolatum ticks were competent to transmit R. vitalii to dogs, which became severely ill, and the results confirmed by molecular methods and blood smear examination to have acquired rangeliosis. Results of the present study, coupled with epidemiological data, indicate that A. aureolatum is a natural vector of R. vitalii. Our results also indicate that R. vitalii is the first Piroplasmorida agent to be transovarially transmitted in Amblyomma ticks.

Rickettsia parkeri in Amblyomma dubitatum ticks in a spotted fever focus from the Brazilian Pampa.

Spotted fever is an acute febrile illness, which is considered severely underreported and misdiagnosed in the Brazilian Pampa, caused by tick-borne Rickettsiae. Here, we report an eco-epidemiological investigation of Rickettsia spp. in ticks from a spotted fever focus in Toropi, southern Brazil. Ticks were collected from capybara carcasses and processed individually to obtain genomic DNA. Rickettsia was investigated using PCR that amplified the rickettsial fragments of the gltA, ompA and htrA genes. DNA from Rickettsia parkeri was found in four of 14 Amblyomma dubitatum ticks collected from capybara carcasses in Toropi and the nearby municipality of Quevedos. We also tested 210A. dubitatum ticks obtained from road-killed capybaras of other localities from the Pampa biome; none of them were positive for Rickettsiae. Thus, in Rio Grande do Sul, two Rickettsia species can be potentially associated to spotted fever: Rickettsia sp. strain Atlantic Rainforest, associated with Amblyomma ovale ticks in the Atlantic Rainforest biome, and R. parkeri, associated both with Amblyomma tigrinum and A. dubitatum ticks in the Pampa biome. Our results reinforce that R. parkeri may be the agent associated with spotted fever in the Brazilian Pampa.