Density and behavior of capybara (Hydrochoerus hydrochaeris) ticks (Acari: Ixodidae) Amblyomma sculptum and Amblyomma dubitatum with notes on Rickettsia bellii infection: Assessing human exposure risk.

In several urban and peri­urban areas of Brazil, populations of Amblyomma sculptum and Amblyomma dubitatum ticks are maintained by capybaras (Hydrochoerus hydrochaeris). In some of these areas, this host and these tick species are associated with Brazilian spotted fever (BSF), a lethal human disease caused by the bacterium Rickettsia rickettsii. In this work, we evaluated the risk of human exposure to these tick species using four collection techniques to discern host-seeking behavior. The study was carried out in 10 urban sites inhabited by capybaras in Uberlândia, a BSF-free municipality in southeastern Brazil. Ticks were collected in areas of 400 m2 at each site and at three seasons. Within the same municipality, the distance and speed of A. sculptum nymphs moving towards the CO2 traps were evaluated. In a sample of ticks Rickettsia DNA was investigated. During the study period, 52,953 ticks were collected. Among these, 83.4 % were A. sculptum (1,523 adults, 10,545 nymphs and 32,104 larvae) and 16.6 % were A. dubitatum (464 adults, 2,153 nymphs and 6,164 larvae). An average annual questing tick density of 4.4/m² was observed, with the highest density recorded at one site in autumn (31.8/m²) and the lowest in summer at another site (0.03/m²). The visual search yielded the highest proportion of A. sculptum larvae, constituting 47 % of the total and 63.6 % of all A. sculptum larvae. In contrast, CO2 traps collected a greater proportion of nymphs and adults of A. sculptum ticks. In the case of A. dubitatum, the CO2 trap was the most efficient technique with 57.7 % of captures of this species, especially of nymphs (94.5 % of captures) and adults (97.8 % of captures). Ticks' ambush height on vegetation (9 to 77 cm), observed by visual search 30 times, yielded a total of 20,771 ticks. Of these, 28 (93 %) were A. sculptum ticks, with only two (7 %) identified as A. dubitatum ticks. Among A. sculptum ticks, the nymph was the most attracted stage to humans and larva in the case of A. dubitatum. Amblyomma sculptum adults and nymphs were significantly more attracted to humans than those of A. dubitatum, but A. dubitatum larvae were significantly more attracted than the same stage of A. sculptum. The maximum distance and speed of horizontal displacement for A. sculptum nymphs were five meters and 2.0 m/h, respectively. The only species of Rickettsia detected in ticks, exclusively in A. dubitatum, was R. bellii. Importantly, it was observed that the higher the proportion of A. sculptum in the community of ticks, the lower the rate of infection of A. dubitatum by R. bellii. In conclusion, host-seeking behavior differed between the two tick species, as well as between stages of the same species. A greater restriction of A. dubitatum ticks to the soil was observed, while larvae and nymphs of A. sculptum dispersed higher in the vegetation. The behavior presented by A. sculptum provides greater opportunities for contact with the hosts, while A. dubitatum depends more on an active search for a host, the hunter behavior. Taken together, these observations show that a human being crossing an area infested with A. sculptum and A. dubitatum ticks will have almost exclusive contact with A. sculptum larvae and/or nymphs. Humans in a stationary position (sitting, lying or immobile) are exposed to both tick species, but they are more attractive to adults and mainly nymphs of A. sculptum compared to the corresponding stages of the tick A. dubitatum. The negative effect of A. sculptum on A. dubitatum infection by R. bellii deserves further studies.

Anaplasma species infecting questing ticks in the Iberá wetlands ecoregion, Argentina.

The present study evaluated the presence of Anaplasma species in questing ticks from six sites with opposing land usage (i.e., protected natural areas or livestock establishments) within the Iberá wetlands ecoregion in Argentina. The ticks were determined as Amblyomma dubitatum (n = 15,096), Rhipicephalus microplus (n = 399), Amblyomma triste (n = 134), Haemaphysalis juxtakochi (n = 5), and Amblyomma tigrinum (n = 1). Using a real-time PCR assay targeting the 16S rRNA gene, Anaplasma sp. was detected in A. dubitatum samples (one nymph, three nymph pools and one larvae pool) and one R. microplus larvae pool. The overall minimum infection rate (MIR) for Anaplasma sp. in questing A. dubitatum nymphs was 0.169% (0.175% in protected natural areas and 0% in livestock establishments). For R. microplus, overall Anaplasma sp. MIR was 0.25% (0.52% in protected natural areas and 0% in livestock establishments). Phylogenetic analysis positioned the Anaplasma sp. from A. dubitatum in the same clade as Anaplasma odocoilei, whereas the Anaplasma sp. from R. microplus was related to Anaplasma platys. In conclusion, these results support a possible role of A. dubitatum in the ecology of the Anaplasma agent reported to infect capybaras in the region.

Molecular detection of Babesia spp. and Rickettsia spp. in coatis (Nasua nasua) and associated ticks from midwestern Brazil.

Procyonids are reservoirs of many zoonotic infectious diseases, including tick-borne pathogens. The role of coatis (Nasua nasua) in the epidemiology of piroplasmids and Rickettsia has not been fully addressed in Brazil. To molecularly study these agents in coatis and associated ticks, animals were sampled in two urban areas in Midwestern Brazil. Blood (n = 163) and tick (n = 248) DNA samples were screened by PCR assays targeting the 18S rRNA and gltA genes of piroplasmids and Rickettsia spp., respectively. Positive samples were further molecularly tested targeting cox-1, cox-3, ß-tubulin, cytB, and hsp70 (piroplasmid) and ompA, ompB, and htrA 17-kDa (Rickettsia spp.) genes, sequenced and phylogenetically analyzed. All coatis' blood samples were negative for piroplasmids, whereas five pools of ticks (2%) were positive for two different sequences of Babesia spp.. The first from Amblyomma sculptum nymphs was close (i.e., ≥ 99% nucleotide identity) to a Babesia sp. previously found in capybaras (Hydrochoerus hydrochaeris); the second from Amblyomma dubitatum nymphs and Amblyomma spp. larvae was identical (100% nucleotide identity) to a Babesia sp. detected in opossums (Didelphis albiventris) and associated ticks. Four samples (0.8%) were positive by PCR to two different Rickettsia spp. sequences, being the first from Amblyomma sp. larva identical to Rickettsia belli and the second from A. dubitatum nymph identical to Rickettsia species from Spotted Fever Group (SFG). The detection of piroplasmids and SFG Rickettsia sp. highlights the importance of Amblyomma spp. in the maintenance of tick-borne agents in urban parks where humans and wild and domestic animals are living in sympatry.

Comparing scapular morphology of Amblyomma sculptum and Amblyomma dubitatum nymphs allows a fast and practical differential diagnosis of ticks in highly infested areas with dominance of these two species.

Rickettsia rickettsii infection causes the highest human fatality rate among all Rickettsia species of the world and is endemic in Southeast Brazil. In this part of the country most human spotted fever cases are related to unnaturally high populations of ticks, usually a mix of two species, Amblyomma dubitatum Neumann and Amblyomma sculptum Berlese and their local host, capybara (Hydrochoerus hydrochaeris). At the same time, an increase in the number of SF notifications as well as its urbanization was observed, and a better characterization of disease epidemiology is mandatory for control measures and to halt its expansion. It was recently noticed in southeast Brazil that SF endemic areas were characterized by overgrowth populations of A. sculptum and decreased populations of A. dubitatum. Hence, characterization of areas with potential to endemicity, eco-epidemiological studies and control measures may rely on the evaluation of A. sculptum/A. dubitatum rate. However, in potentially endemic scenarios, discriminating the two tick species, particularly the nymphs considered the main vector stage, is a challenge in face of several hundreds to thousands of ticks that should be examined for quantitative studies. We herein present additional morphological features to an existing identification key for Amblyomma nymphs that considerably diminishes the labor to distinguish nymphs of these two tick species. Specifically, the oval-shaped scutum of A. dubitatum and a conspicuous scapula of A. sculptum are prominent discriminating features.

Diversity and Seasonal Dynamics of Ticks on Ring-Tailed Coatis Nasua nasua (Carnivora: Procyonidae) in Two Urban Areas from Midwestern Brazil.

Understanding the diversity and ecology of ectoparasites in wild animals is essential for surveillance of vector-borne diseases. Coatis (Nasua nasua) easily adapt to anthropized areas, favoring close contact with domestic animals and humans, with the possibility of exchange of ectoparasites and pathogens. The present study aimed to identify the diversity of ticks parasitizing coatis from forest urban areas of midwestern Brazil, to evaluate the seasonal dynamics of ticks during the seasons of the year, and to assess the correlation between tick species and gender and age of the sampled coatis. For this purpose, 103 coatis were captured in two Conservation areas, both located in Campo Grande city, Mato Grosso do Sul state, Midwestern Brazil. The animals' entire body was inspected for the presence of ectoparasites, and ticks were removed for taxonomic identification. In total, 168 captures were performed in both areas during the observational study considering the first capture and recaptures. In total, 2242 ticks were collected: 838 Amblyomma larvae, 1241 A. sculptum nymphs, and 150 A. dubitatum nymphs. Thirteen adult ticks were identified as three males and five females of A. sculptum and two males and three females of A. ovale. While a quantity of Amblyomma larvae was observed in the first months of the year (January, April and May), Amblyomma nymphs showed a higher quantity during the months of July, August, October and November. No statistical difference was observed when comparing mean intensity and prevalence of Amblyomma larvae, nymphs of A. sculptum and A. dubitatum between the two sampled areas, males vs. females and immature vs. mature animals. In conclusion, three tick species were identified parasitizing coatis from forested urban fragments in midwestern Brazil, namely A. dubitatum nymphs, A. sculptum adults and nymphs, and A. ovale adults. Coatis from anthropized areas seem to present tick species diversity lower than those from natural areas. The lack of statistical difference regarding mean intensity and prevalence of Amblyomma larvae and nymphs between males vs. females and immature vs. mature animals might have reflected the gregarious behavior of coatis, since adult males live together with females and offspring outside and inside the mating season, forming large groups of individuals.

Detection of an Undescribed Babesia sp. in Capybaras and Amblyomma Ticks in Central-Western Brazil.

Capybaras (Hydrochoerus hydrochaeris) are the largest rodents on Earth. While capybaras are hosts for various tick species, there is limited information regarding the tick-borne pathogens they can carry. We investigated the presence of piroplasmids and Ehrlichia spp. in capybaras and their associated ticks in two peri-urban areas in Goiás state, central-western Brazil. Blood samples collected from 23 capybaras were used to investigate the presence of piroplasmids and Ehrlichia spp. in stained-blood smears and by PCR. Ticks collected from the capybaras were identified morphologically and also tested using PCR for the same pathogens. A total of 955 ticks were collected, including 822 (86.1%) Amblyomma sculptum, 132 (13.8%) Amblyomma dubitatum, and one (0.1%) unidentified larva of Amblyomma sp. Neither the capybaras nor ticks were positive for Ehrlichia spp. However, a stained-blood smear examination revealed the presence of ring-stage and pyriform-shaped merozoites in the erythrocytes of one (4.4%) capybara. In the same way, 47.8% (11/23) and 19.9% (36/181) of blood samples and ticks, respectively, were positive for piroplasmids in the PCR. We successfully sequenced a partial 18S rRNA gene fragment of four samples (two capybaras, one A. sculptum, and one A. dubitatum), and the phylogenetic reconstruction disclosed that the organism reported in the present study clusters within the genus Babesia. Further research is required for a formal delineation of this species (designated as Babesia sp. strain Capybara) and to investigate the hypothesis of A. dubitatum and A. sculptum ticks being vectors.

Rickettsial infection in free-ranging capybaras (Hydrochoerus hydrochaeris) and their ticks (Acari, Ixodidae) in the Caatinga and Atlantic forest biomes, Northeastern Brazil.

Caatinga and Atlantic Forest are Brazilian biomes threatened by anthropogenic disturbances, which are a serious threat to biodiversity and favor the emergence of zoonotic pathogens. Capybara populations are increasingly present in urban and rural areas due to the loss of their natural habitats. There are no studies on the rickettsial infection in these rodents in Northeastern Brazil, although there are case records of Brazilian Spotted Fever (BSF) in this region. In this context, we evaluated rickettsial infection in capybaras and their ticks in four areas of the Atlantic Forest and Caatinga biomes in Northeastern Brazil. Blood sera from 21 capybaras were tested by indirect immunofluorescence assay (IFA) using Rickettsia rickettsii, R. parkeri, and R. amblyommatis antigens. Blood samples and ticks (Amblyomma sp., Amblyomma dubitatum and Amblyomma sculptum) were tested by PCR, targeting a fragment of the rickettsial gltA gene and R. bellii primers. Overall, 15 out of 21 capybaras (71.4%) reacted positively (titer ≥64) to at least one of the three Rickettsia antigens tested, with final titers ranging from 64 to 1024. No rickettsial DNA was found in capybara blood samples. Rickettsial DNA was detected in 22 of 31 adult tick pools using gltA gene, being all nine pools of A. sculptum and 16 of the 19 of A. dubitatum, but all samples were negative to R. bellii specific primers. All attempts to sequence the amplicon were unsuccessful, indicating a possible low rickettsial load in the tick samples. The present study reports for the first time the serological evidence of Rickettsia spp. infection in free-ranging capybaras and suggest the rickettsial infection in A. sculptum and A. dubitatum in the Caatinga and Atlantic Forest biomes in Northeastern Brazil.

'Candidatus Mycoplasma haematohydrochoerus', a novel hemoplasma species in capybaras (Hydrochoerus hydrochaeris) from Brazil.

Three different species of hemoplasmas have been described in rodents, Mycoplasma coccoides, 'Candidatus Mycoplasma haemomuris' and 'Candidatus Mycoplasma haemosphiggurus'. Additionally, potentially novel hemoplasma species have been detected in wild rodents from Brazil, including capybaras (Hydrochoerus hydrochaeris). Capybaras are the largest rodent in the world and are well adapted to live within close proximity to humans, which increases the risk to spread of zoonotic pathogens. Herein, we investigate the occurrence and genetic diversity of hemoplasmas infecting free-ranging capybaras from southern Brazil. Blood samples and ticks from 17 capybaras were collected. Packed cell volume and total plasma protein were measured, DNA was extracted, and further screened by species-specific and pan-hemoplasma PCR assays targeting the 16S rRNA gene of hemoplasmas. Sixteen out of 17 (94.12%; 95% CI: 73.02-98.95%) were anemic. Only one young female was hypoproteinemic. All capybaras were infested by adults and nymphs of Amblyomma dubitatum ticks. Using the PCR assay targeting the 16S rRNA gene of M. coccoides, 13/17 (76.47%; 95% CI: 52.74-90.44%) capybaras were positive for hemoplasmas. When DNA samples were tested by the pan-hemoplasma PCR, 16/17 (94.12%; 95% CI: 73.02-98.95%) animals were positive. One out of 11 (9.09%) adult ticks salivary glands tested positive for hemoplasma by the pan-hemoplasma PCR assay. Sequencing and phylogenetic analysis of the 16S and 23S rRNA gene fragments confirmed that animals were infected by a novel hemotropic Mycoplasma sp. previously reported in capybaras from Brazil. Additionally, sequencing and phylogenetic analysis of the 23S rRNA gene from three hemoplasma-positive capybaras samples from a previous study performed in midwestern Brazil also confirm our findings. Based on phylogenetic and Neighbor-Net network analysis of the 16S rRNA and 23S rRNA genes, the name 'Candidatus Mycoplasma haematohydrochoerus' is proposed for this novel organism.

Seasonal dynamics and rickettsial infection in free-living Amblyomma dubitatum in the Atlantic forest biome in north-eastern Brazil.

The genus Amblyomma is the most representative tick genus in Brazil and some species act as vectors of pathogenic organisms to animals and humans. Information on the seasonal dynamics of Amblyomma spp. as well as on rickettsial organisms infecting these ticks in some regions in Brazil is still fragmentary. Herein, we investigated the seasonal dynamics and rickettsial infections in Amblyomma dubitatum ticks collected in the Atlantic forest biome in north-eastern Brazil. Using carbon dioxide traps, ticks were collected monthly for two consecutive years. In total, 15,789 ticks were collected: 69 females (0.4%), 116 males (0.7%), 1,067 nymphs (6.8%), and 14,537 larvae (92.1%). All nymphs, females and males were identified as A. dubitatum, whereas larvae were identified as Amblyomma spp. Larvae were more frequent in summer (77% of the larvae collected), whereas nymphs were collected with similar frequency in summer (32.8%), autumn (30.0%) and spring (28.4%). Adults were more frequent in spring (47.6%). A total of 648 ticks (485 nymphs, 60 females, and 103 males) were tested by PCR for the gltA gene of Rickettsia spp. and 87 (13.4%; 95% CI: 10.9-16.3%) were positive. A consensus sequence (size, 350 bp) of 66 gltA gene sequences indicate that the organism detected herein is similar to Rickettsia tamurae, Rickettsia monacencis and Rickettsia sp. strain Pampulha. One of these positive samples was also positive for the ompA gene of spotted fever group rickettsiae, but attempts to sequence the amplicon were not successful. We also tested this sample by a PCR targeting the rickettsial htrA gene, but no amplification product could be detected. This study indicates that A. dubitatum may be a common tick in areas where capybaras are present in north-eastern Brazil, occurring during the whole year. It also suggests the circulation of a spotted fever group rickettsia in this A. dubitatum population, whose identity has yet to be determined.

Closing the Gaps to Understand the Tick Transmission of Anaplasma marginale among Giant Anteaters (Myrmecophaga tridactyla) in Argentina.

Anaplasma marginale, a well-known cattle pathogen of tropical and subtropical world regions, has been previously molecularly characterized in a giant anteater (Myrmecophaga tridactyla) from Corrientes, Argentina. Ticks or other hematophagous arthropod involved in the wild transmission cycle remained unknown. The aim of the present study was to analyze the simultaneous occurrence of A. marginale in blood samples and ticks from giant anteaters from Corrientes in order to investigate if ticks could be relevant in the transmission among these mammals. Blood samples from 50 giant anteaters collected in different years and 26 ticks Amblyomma dubitatum and A. sculptum were studied through the molecular amplification of two unequivocal species-specific genes from A. marginale: msp5 and msp1ß. Twenty five giant anteaters and tick organs (salivary glands, gut and oviduct) from 11 ticks tested positive to the A. marginale DNA amplification. The further molecular characterization through MSP1a tandem repeats analysis revealed the presence of genotypes circulating among giant anteaters that had been previously identified in cattle blood samples from the same geographical region. These results confirm the presence of A. marginale in giant anteaters in Corrientes and suggests that A. dubitatum and A. sculptum ticks could be involved in the transmission among giant anteaters. Future studies will determine the role of these tick species in the wild transmission cycle in the study area and the eventual connection with the domestic cycle.

Mapping Brazilian spotted fever: Linking etiological agent, vectors, and hosts.

Brazilian spotted fever (BSF) is a highly lethal disease in southeastern Brazil. BSF is caused by the bacterium Rickettsia rickettsii and is transmitted by the bites of the tick of the genus Amblyomma. The spatial distribution of BSF risk areas is not well known in the country given the complexity of the transmission cycle. This study used the ecological niche modeling (ENM) approach to anticipate the potential distribution of the etiological agent (Rickettsia rickettsii), vectors (Amblyomma sculptum and A. dubitatum), and hosts (Hydrochoerus hydrochaeris, Didelphis aurita, and D. marsupialis) of BSF in Brazil. We compiled occurrence records for all vectors, hosts, and BSF from our own field surveillance, online repositories, and literature. ENM identified BSF risk areas in southeastern and southern Brazil, and anticipated other dispersed suitable areas in the western, central, and northeastern coast regions of Brazil. Tick vectors and mammalian hosts were confined to these same areas; however, host species showed broader suitability in northern Brazil. All species ENMs performed significantly better than random expectations. We also tested the BSF prediction based on 253 additional independent cases identified in our surveillance; the model anticipated 251 out of 253 of these independent cases. Background similarity tests comparing the ENMs of R. rickettsii, tick vectors, and mammalian hosts were unable to reject null hypotheses of niche similarity. Finally, we observed close coincidence between independent BSF cases, and areas suitable for combinations of vectors and hosts, reflecting the ability of these model pairs to anticipate the distribution of BSF cases across Brazil.

The Dynamics of Ticks and Capybaras in a Residential Park Area in Southeastern Brazil: Implications for the Risk of Rickettsia rickettsii Infection.

The bacterium Rickettsia rickettsii causes Brazilian spotted fever (BSF), a highly lethal disease that is transmitted by Amblyomma sculptum ticks in areas where capybaras (Hydrochoerus hydrochaeris) are the tick's major hosts. In this study, we evaluated the expansion of a capybara population in a residential park in São Paulo state, and the implications of such expansion to the occurrence of ticks and BSF. The capybara population was quantified during 2004-2013. In 2012, there was a BSF human case in the area, culminating in the complete fencing of the residential park and the official culling of all capybaras. Quantification of ticks in the environment was performed by dry ice traps from 2005 to 2018. Domestic dogs in 2006-2011 and capybaras in 2012 were serologically tested for the presence of anti-R. rickettsii antibodies. Our results show that capybara numbers increased ≈5 times from 2004 (41 capybaras) to 2012 (230 capybaras). Dry ice traps collected A. sculptum and Amblyomma dubitatum. The number of A. dubitatum adult ticks was generally higher than A. sculptum adults during 2005-2006; however, during 2012-2013, A. sculptum outnumbered A. dubitatum by a large difference. During 2016-2018 (after capybara culling), the number of both species fell close to zero. The low numbers of A. sculptum adult ticks during 2005-2006 coincided with relatively low capybara numbers (<80). Thereafter, in 2012, we counted the highest numbers of both A. sculptum ticks and capybaras (230 animals). All 40 canine blood samples were seronegative to R. rickettsii, in contrast to the 48.3% seropositivity (83/172) among capybaras. Our results support that the emergence of BSF in the residential park was a consequence of the increase of the local capybara population, which in turn, provided the increment of the A. sculptum population. Culling the entire capybara population eliminated the risks of new BSF cases.

Free-living ticks in a cerrado fragment, state of Mato Grosso do Sul, Brazil / Carrapatos de vida livre em fragmento de cerrado, no Estado de Mato Grosso do Sul, Brasil

The fatal cases of spotted fever reported in some Brazilian municipalities are generally associated with the presence of ticks and capybaras (Hydrochoerus hydrochaeris). The urban forest fragments where this rodent is present are of great concern. We investigated the diversity of free ticks as well as the occurrence of ticks infected by Rickettsia in one of these fragments, in Campo Grande city, Mato Grosso do Sul state, Brazil. From winter 2012 until the autumn of 2015, using CO2 traps, we collect 1219 ticks, of which 1045 belonged to the species Amblyomma sculptum and 174 belonged to A. dubitatum. Both species were more abundant in spring (September to December) and summer (December to March). Rickettsial DNA was not detected in any of the specimens collected; however, one of the species found, A. sculptum is considered the main vector of Brazilian Spotted Fever, thus deserving constant vigilance by the public health organs.

Casos fatais de febre maculosa que foram relatados em alguns municípios brasileiros geralmente estão associados à presença de carrapatos e capivaras (Hydrochoerus hydrochaeris). Os fragmentos florestais urbanos onde este roedor está presente são de grande preocupação. Investigou-se aqui a diversidade de carrapatos livres e a ocorrência de carrapatos infectados por riquétsias em um desses fragmentos em Campo Grande, Mato Grosso do Sul. Desde o inverno de 2012 até o outono de 2015, usando armadilhas de CO2, foram coletados 1219 carrapatos, dos quais 1045 pertenciam à espécie Amblyomma sculptum e 174 pertenciam a A. dubitatum. Ambas as espécies foram mais abundantes na primavera (Setembro a Dezembro) e no verão (Dezembro a Março) . O DNA riquetsial não foi detectado em nenhum dos espécimes coletados; entretanto, uma das espécies encontradas, o A. sculptum é considerado o principal vetor da Febre Maculosa Brasileira, merecendo assim vigilância constante pelos Orgãos de Saúde Pública.

Influence of microhabitat use and behavior of Amblyomma sculptum and Amblyomma dubitatum nymphs (Acari: Ixodidae) on human risk for tick exposure, with notes on Rickettsia infection.

Brazilian spotted fever (BSF) is a potentially lethal human disease caused by Rickettsia rickettsii transmitted by ticks, including Amblyomma sculptum. However, in Southeast Brazil, where most BSF cases occur, capybaras are key hosts for both A. sculptum and Amblyomma dubitatum. We therefore compared the risk of human exposure to these ticks at a non-endemic anthropogenic site in Southeast Brazil where both tick species are maintained by capybaras and occur at high abundance. Cloth dragging, human baits and CO2 traps were used to assess tick abundance and risk for human exposure. The two tick species displayed profound differences in behavior and microhabitat use. Notably, A. sculptum but not A. dubitatum quested for hosts openly from vegetation (ambush behavior) and infested human baits. Furthermore, A. dubitatum was more aggregated at a specific site whereas A. sculptum was more widespread along differing and drier microhabitats. Adults and nymphs of both species were infected with Rickettsia bellii. Overall, the results indicate that even though both species co-existed in the same area, A. sculptum posed a greater risk for biting humans and thus also for transmitting tick-borne pathogens.

New records of Rickettsia bellii-infected ticks in Brazil / Novos relatos de carrapatos infectados por Rickettsia bellii no Brasil

This study investigated the occurrence of rickettsial infection in ticks collected from wild animals in two areas of Brazil. Amblyomma dubitatum ticks were collected from a capybara (Hydrochoerus hydrochaeris) in Guarda-Mor municipality, state of Minas Gerais, and Amblyomma pseudoconcolor ticks were collected from a six-banded armadillo (Euphractus sexcinctus) in Corumbá municipality, state of Mato Grosso do Sul. Attempts to isolate rickettsia in Vero cell culture were performed with one A. dubitatum tick and one A. pseudoconcolor tick, which were previously shown by the hemolymph test to contain Rickettsia-like structures within their hemocytes. Rickettsiae were successfully isolated in Vero cell culture from the two tick species. The two isolates were identified as Rickettsia bellii, since gltA partial sequences were 99.9%-100% identical to corresponding sequences of R. bellii in GenBank. While there have been several previous reports of R. bellii infecting A. dubitatum ticks, we provide the first report for A. pseudoconcolor, which increases to 25 the number of R. bellii-infected tick species in the American continent.(AU)

O presente trabalho investigou a ocorrência de infecção por Rickettsia em carrapatos coletados em animais selvagens de duas áreas do Brasil. Carrapatos da espécie Amblyomma dubitatum foram coletados de uma capivara (Hydrochoerus hydrochaeris) no município de Guarda-Mor, Minas Gerais, enquanto exemplares da espécie Amblyomma pseudoconcolor foram coletados de um tatu-peba (Euphractus sexcinctus) do município de Corumbá, Mato Grosso do Sul. Tentativas para isolar Rickettsia em cultura de células Vero foram realizadas com um exemplar de A. dubitatum e um de A. pseudoconcolor, que foram previamente positivos no teste de hemolinfa com estruturas semelhantes a Rickettsia visualizadas em seus hemócitos. Rickettsia foram isoladas com sucesso em culturas de células Vero a partir das duas espécies de carrapatos. Os dois isolados foram identificados como Rickettsia bellii, uma vez que suas sequências parciais do gene gltA foram 99,9-100%, idênticas a sequências de R. bellii do GenBank. Embora haja vários relatos anteriores de R. bellii infectando A. dubitatum, este é o primeiro relato em A. pseudoconcolor, aumentando para 25 o número de espécies de carrapatos infectadas por R. bellii no continente americano.(AU)

First report of Amblyomma dubitatum (Acari: Ixodidae) parasitizing the crab-eating raccoon, Procyon cancrivorus (Carnivora: Procyonidae)

In October 2015, a tick nymph was collected from a male Procyon cancrivorus found dead at the roadside of the National Road 119 near Mercedes city, Corrientes province, Argentina. The nymph collected was morphologically and molecularly identified as Amblyomma dubitatum and it was found infected with the alpha-proteobacterium Rickettsia bellii. To the best of our knowledge, this report constitutes the first record of the A. dubitatum - P. cancrivorus association.

En octubre de 2015, una ninfa de garrapata fue colectada sobre un ejemplar macho de Procyon cancrivorus hallado muerto a la vera de la Ruta Nacional 119 en las cercanías de la ciudad de Mercedes, provincia de Corrientes, Argentina. La ninfa colectada fue identificada morfológica y molecularmente como Amblyomma dubitatum, y la misma se encontró infectada con la alfa-proteobacteria Rickettsia bellii. Este reporte constituye el primer registro de la asociación A. dubitatum P. cancrivorus.

Epidemiological surveillance of capybaras and ticks on warning area for Brazilian spotted fever.

AIM: The vulnerability of tropical developing countries to the emerging disease constitutes a critical phenomenon in which the invasion of wild niches by human hosts, contributes to expansion of zoonotic diseases, such as the Brazilian spotted fever (BSF). This study performed a diagnosis of species occurrence of their hosts (Hydrochoerus hydrochaeris) and vectors (Amblyomma sculptum and Amblyomma dubitatum) on the warning area for this reemerging disease in Brazil. MATERIALS AND METHODS: The study was conducted in a warning area for BSF in the city of Americana, São Paulo state. The occurrence of capybaras was registered by use of binoculars and GPS equipment and 24 acarological researches were performed through 180 CO2 traps. Samples of adult ticks were dissected for salivary glands removal, DNA extraction, and evaluation by polymerase chain reaction (PCR) being tested by initial gltA-PCR, ompA-PCR, and Rickettsia bellii-specific PCR, with the positive samples subjected to sequencing. RESULTS: Eleven clusters of capybaras (total of 71 individuals), were observed along the riparian of Ribeirão Quilombo and 7,114 specimens of A. sculptum and 7,198 specimens of A. dubitatum were collected in this same area. About 568 samples of adult ticks were dissected for salivary glands removal, DNA extraction and evaluation by gltA-PCR, with results of 1.94% (11/568) of positive samples. Results for the initial gltA-PCR indicated none positive sample to Rickettsia species into A. sculptum and 11 positive samples to A. dubitatum. These samples were negative to the ompA-PCR and positive to the Rickettsia bellii-specific PCR protocol and subjected to DNA sequencing, whose result indicated 100% similarity to Rickettsia bellii. The distribution of tick species A. sculptum and A. dubitatum was configured regarding to the biotic potential of the riparian areas, measuring the risks for BSF in peri-urban areas of Americana. CONCLUSION: These results confirmed a status of epidemiological warning with a strong association of the amplifiers hosts of Rickettsia and tick vectors for the transmission of BSF to humans in this region.

Spotted fever group Rickettsia in Amblyomma dubitatum tick from the urban area of Campo Grande, Mato Grosso do Sul, Brazil.

Rickettsia infection of each tick was evaluated by the hemolymph test and polymerase chain reaction (PCR) targeting gltA and ompA genes. All hemolymph tests were negative and PCR of one A. dubitatum detected both Rickettsia genes. Sequence of ompA exhibited a 99% identity with Rickettsia parkeri and R. africae and a 98% identity with R. sibirica. Rickettsia of the spotted fever group in A. dubitatum is described for the first time in an urban area within the municipality of Campo Grande in the state of Mato Grosso do Sul (MS), Brazil. This finding reinforces the importance of more detailed studies to determine the role of A. dubitatum in the transmission of spotted fever agents.

First molecular detection of Rickettsia parkeri in Amblyomma tigrinum and Amblyomma dubitatum ticks from Uruguay.

Rickettsia parkei is the etiological agent of spotted fever in Uruguay, where is transmitted to humans by the tick Amblyomma triste. In the present study, ticks were collected from capybaras (Hydrochoerus hydrochaeris) and domestic dogs during 2011-2012 in different parts of Uruguay. Three out of 11 (27.3%) Amblyomma dubitatum ticks collected from capybaras, and 4 out of 6 (66.7%) Amblyomma tigrinum ticks collected from dogs were shown by molecular analyses to be infected by Rickettsia parkeri strain Maculatum 20. Until the present work, A. triste was the only tick species that was found infected by R. parkeri in Uruguay. This is the first report of R. parkeri infecting these two tick species in Uruguay, expanding the current distribution of this rickettsial pathogen in the country.

Experimental infection with Rickettsia rickettsii in an Amblyomma dubitatum tick colony, naturally infected by Rickettsia bellii.

Amblyomma dubitatum engorged females, naturally infected by Rickettsia bellii, were used to establish a laboratory colony. Larvae, nymphs, and adults were exposed to two strains of Rickettsia rickettsii by feeding on needle-inoculated guinea pigs, and thereafter reared on uninfected guinea pigs. After acquisition feeding, engorged larvae and nymphs molted to nymphs and adults, respectively, which were shown to be infected (confirming transstadial perpetuation), and were able to transmit both strains of R. rickettsii to uninfected animals, as demonstrated by clinical, serological, and molecular analyses. However, the larval, nymphal, and adult stages of A. dubitatum showed to be only partially susceptible to R. rickettsii infection, since in all cases, only part of the ticks became infected by this agent, after being exposed to rickettsemic animals. While transovarial transmission of R. rickettsii was inefficient in the A. dubitatum engorged females of the present study, 100% of these females passed R. bellii transovarially. Because it has been reported that a primary infection by a Rickettsia species would preclude transovarial transmission of a second Rickettsia species, it is likely that the ineffectiveness of A. dubitatum to perpetuate R. rickettsii by transovarial transmission was related to its primary infection by R. bellii; however, it could also be related to unknown factors inherent to A. dubitatum. The relevance of A. dubitatum as a natural vector of R. rickettsii to humans or animals is discussed.