New insights into the molecular phylogeny, biogeographical history, and diversification of Amblyomma ticks (Acari: Ixodidae) based on mitogenomes and nuclear sequences.

BACKGROUND: Amblyomma is the third most diversified genus of Ixodidae that is distributed across the Indomalayan, Afrotropical, Australasian (IAA), Nearctic and Neotropical biogeographic ecoregions, reaching in the Neotropic its highest diversity. There have been hints in previously published phylogenetic trees from mitochondrial genome, nuclear rRNA, from combinations of both and morphology that the Australasian Amblyomma or the Australasian Amblyomma plus the Amblyomma species from the southern cone of South America, might be sister-group to the Amblyomma of the rest of the world. However, a stable phylogenetic framework of Amblyomma for a better understanding of the biogeographic patterns underpinning its diversification is lacking. METHODS: We used genomic techniques to sequence complete and nearly complete mitochondrial genomes -ca. 15 kbp- as well as the nuclear ribosomal cluster -ca. 8 kbp- for 17 Amblyomma ticks in order to study the phylogeny and biogeographic pattern of the genus Amblyomma, with particular emphasis on the Neotropical region. The new genomic information generated here together with genomic information available on 43 ticks (22 other Amblyomma species and 21 other hard ticks-as outgroup-) were used to perform probabilistic methods of phylogenetic and biogeographic inferences and time-tree estimation using biogeographic dates. RESULTS: In the present paper, we present the strongest evidence yet that Australasian Amblyomma may indeed be the sister-group to the Amblyomma of the rest of the world (species that occur mainly in the Neotropical and Afrotropical zoogeographic regions). Our results showed that all Amblyomma subgenera (Cernyomma, Anastosiella, Xiphiastor, Adenopleura, Aponomma and Dermiomma) are not monophyletic, except for Walkeriana and Amblyomma. Likewise, our best biogeographic scenario supports the origin of Amblyomma and its posterior diversification in the southern hemisphere at 47.8 and 36.8 Mya, respectively. This diversification could be associated with the end of the connection of Australasia and Neotropical ecoregions by the Antarctic land bridge. Also, the biogeographic analyses let us see the colonization patterns of some neotropical Amblyomma species to the Nearctic. CONCLUSIONS: We found strong evidence that the main theater of diversification of Amblyomma was the southern hemisphere, potentially driven by the Antarctic Bridge's intermittent connection in the late Eocene. In addition, the subgeneric classification of Amblyomma lacks evolutionary support. Future studies using denser taxonomic sampling may lead to new findings on the phylogenetic relationships and biogeographic history of Amblyomma genus.

ATP-sensitive inward rectifier potassium channels regulate secretion of pro-feeding salivary proteins in the lone star tick (Amblyomma americanum).

Understanding the physiological and molecular regulation of tick feeding is necessary for developing intervention strategies to curb disease transmission by ticks. Pharmacological activation of ATP-gated inward rectifier potassium (KATP) channels reduced fluid secretion from isolated salivary gland and blood feeding in the lone star tick, Amblyomma americanum, yet the temporal expression pattern of KATP channel proteins remained unknown. KATP channels were highly expressed in type II and III acini in off-host stage and early feeding phase ticks, yet expression was reduced in later stages of feeding. We next assessed KATP channel regulation of the secreted proteome of tick saliva. LC-MS/MS analysis identified 40 differentially secreted tick saliva proteins after exposure to KATP activators or inhibitors. Secretion of previously validated tick saliva proteins that promote tick feeding, AV422, AAS27, and AAS41 were significantly reduced by upwards of 8 log units in ticks exposed to KATP channel activators when compared to untreated ticks. Importantly, activation of KATP channels inhibited tick feeding and vice versa for KATP channel inhibitors. Data indicate KATP channels regulate tick feeding biology by controlling secretion of pro-feeding proteins that are essential during early feeding phases, which provides insights into physiological and molecular regulation of tick feeding behavior.

Crude saliva of Amblyomma cajennense sensu stricto (Acari: Ixodidae) reduces locomotor activity and increases the hemocyte number in the females of Aedes aegypti (Diptera: Culicidae).

Aedes aegypti are vector insects of arboviruses such as dengue, Zika, and chikungunya. All available vector control methods have limited efficacy, highlighting the urgent need to find alternative ones. Evidence shows that arachnids like ticks are sources of biologically active compounds. Moreover, chemical modulation of the locomotor and immune systems of vector insects can be used to control arbovirus transmission. The present study evaluated the effectiveness of crude saliva of female Amblyomma cajennense sensu stricto (s.s.) ticks in reducing locomotor activity and inducing an immune response in Ae. aegypti females. Additionally, the study evaluated the protein constitution of tick saliva. For this purpose, the crude saliva obtained from several semi-engorged A. cajennense females was used. A volume of 0.2 nL of crude tick saliva was administered to mosquitoes by direct intrathoracic microinjection. The effect of the tick's saliva on the locomotor activity of the mosquito was observed using Flybox, a video-automated monitoring system, and the hemolymph hemocyte levels were quantified by reading slides under a light microscope. The protein concentration of the crude tick saliva was 1.27 µg/µL, and its electrophoretic profile indicates the presence of proteins with a molecular weight ranging between ∼17 and 95 kDa. Microplusins, ixodegrins, cystatin, actins, beta-actin, calponin, albumin, alpha-globulins, and hemoglobin were the main proteins identified by proteomics in the saliva of A. cajennense. The microinjected saliva had low toxicity for Ae. aegypti females and significantly reduced their locomotor activity, especially in the transition between the light and dark phases. The crude tick saliva did not change the period and rhythmicity of the circadian cycle. The tick saliva significantly increased the number of hemocytes two days after injection and reduced it after five days. These results suggest that further evaluation of the biological properties of tick saliva proteins against Ae. aegypti would be of interest.

Tick innate immune responses to hematophagy and Ehrlichia infection at single-cell resolution.

Introduction: Ticks rely on robust cellular and humoral responses to control microbial infection. However, several aspects of the tick's innate immune system remain uncharacterized, most notably that of the immune cells (called hemocytes), which are known to play a significant role in cellular and humoral responses. Despite the importance of hemocytes in regulating microbial infection, our understanding of their basic biology and molecular mechanisms remains limited. Therefore, we believe that a more detailed understanding of the role of hemocytes in the interactions between ticks and tick-borne microbes is crucial to illuminating their function in vector competence and to help identify novel targets for developing new strategies to block tick-borne pathogen transmission. Methods: This study examined hemocytes from the lone star tick (Amblyomma americanum) at the transcriptomic level using the 10X genomics single-cell RNA sequencing platform to analyze hemocyte populations from unfed, partially blood-fed, and Ehrlichia chaffeensis-infected ticks. The functional role of differentially expressed hemocyte markers in hemocyte proliferation and Ehrlichia dissemination was determined using an RNA interference approach. Results and discussion: Our data exhibit the identification of fourteen distinct hemocyte populations. Our results uncover seven distinct lineages present in uninfected and Ehrlichia-infected hemocyte clusters. The functional characterization of hemocytin, cystatin, fibronectin, and lipocalin demonstrate their role in hemocyte population changes, proliferation, and Ehrlichia dissemination. Conclusion: Our results uncover the tick immune responses to Ehrlichia infection and hematophagy at a single-cell resolution. This work opens a new field of tick innate immunobiology to understand the role of hemocytes, particularly in response to prolonged blood-feeding (hematophagy), and tick-microbial interactions.

Febre maculosa brasileira na cidade de Jacareí, Vale do Paraíba, SP: descrição dos componentes da paisagem / Brazilian Spotted Fever in the city of Jacareí, Vale do Paraíba region, São Paulo State: description of landscape components

A Febre Maculosa Brasileira (FMB) é uma zoonose infecciosa, febril, aguda e exantemática, de notificação compulsória no Brasil, que tem como agente patogênico bactérias da espécie Rickettsia rickettsii, que são transmitidas por alguns carrapatos do gênero Amblyomma. No Brasil, a maior incidência de casos ocorre no Estado de São Paulo, onde há casos em locais de grande urbanização, e apresenta alta taxa de letalidade que pode variar de 30% à 60% dependendo da região do Estado. A cidade de Jacareí está na região do Vale do Paraíba, onde alguns municípios identificaram em seus territórios casos humanos da doença. No período de 2014 a 2020 ocorreram quatro casos confirmados de FMB com Local Provável de Infecção (LPI) no município de Jacareí, SP, com taxa de letalidade de 100 %. O estudo tem como objetivo descrever a ocorrência de Febre Maculosa Brasileira no Município de Jacareí nesse período. Para essa finalidade, foram selecionadas cinco áreas para esse estudo epidemiológico com propósito descritivo, retrospectivo, e com abordagem ecológica. O estudo foi baseado na revisão dos relatórios de investigações epidemiológicas do banco de dados da Diretoria de Vigilância em Saúde do Município de Jacareí. Os casos humanos descritos tem predomínio de indivíduos do sexo masculino na faixa etária de 50 a 69 anos associados a atividades recreativas e ocupacionais praticadas em áreas urbanas ou periurbanas de mata ciliar ou próximo a estas. As caracteristicas do perfil paisagístico comuns para as áreas estudadas foram determinados por: cobertura vegetal nativa e espelho d'água com alta fragmentação, locais de ocupação e deslocamento humano presentes em 30,4% da área considerada em média, presença de hospedeiros primários, presença de animais sentinela sororreagentes e relato de parasitismo humano. As notificações de parasitismo humano devem ser implementadas no município para uma posterior pesquisa em campo e mapeamento de novos locais possivelmente infestados, e a sinalização com placas informativas das áreas infestadas deve ser mantida e constantemente atualizada. A realização de atividades educativas deve estar voltada para perfil epidemiológico dos indivíduos considerados vulneráveis, e aos profissionais de saúde informando sobre ambientes predispostos para ocorrência da FMB.

Brazilian Spotted Fever (BSF) is an infectious, febrile, acute and exanthematic zoonosis, of compulsory notification in Brazil, which has Rickettsia rickettsii bacterium as its pathogenic agent, which are transmitted by some ticks species of the genus Amblyomma. In Brazil the highest incidence of cases occurs in the State of São Paulo where there are cases in places of great urbanization, and it has a high lethality rate that can vary from 30 % to 60 % depending on the region of the State. The city of Jacareí is located in the Vale do Paraiba region, São Paulo, Brazil. In this region some municipalities have identified human cases of the disease within their territories. In the period between 2014 and 2020 there were four human cases of the disease that had its Probably Local of Infection in the municipality of Jacareí, with a lethality rate of 100%. We aim to describe the occurrence of BSF in the Jacareí municipality in that timeframe. For this goal, five areas were selected for this epidemiological study, with a descriptive, retrospective purpose, and with an ecological approach. The study was based on a review of epidemiological investigation reports from the Jacareí Health Surveillance Directorate database. The described human cases predominantly affect males in the age range of 50 to 69 years, associated with recreational and occupational activities carried out in urban or peri-urban areas near riparian forests. The common landscape characteristics found in the studied areas were determined as follows: native vegetation cover and water mirrors with high fragmentation, human occupation and mobility present in 30.4% of the considered area on average, presence of primary hosts, presence of seropositive sentinel animals, and reports of human parasitism. Notifications of human parasitism should be implemented in the municipality for subsequent field research and mapping of potentially infested areas. Signage with informative plaques in infested areas should be maintained and regularly updated. Educational activities should focus on the epidemiological profile of vulnerable individuals and inform healthcare professionals about environments predisposed to Brazilian Spotted Fever occurence.

Distribuição espacial de carrapatos (Acari: Ixodidae), no período de 2018 a 2021, no município de Jacareí/SP - Região Metropolitana do Vale do Paraíba / Spatial distribution of ticks (Acari: Ixodidae), from 2018 to 2021, in the municipality of Jacareí/SP - Metropolitan Region from the Paraíba Valley

A Febre Maculosa Brasileira - FMB é doença exantemática, causada pela bactéria Rickettsia rickettsii e transmitida pela picada de carrapatos do gênero Amblyomma infectados. A Região Metropolitana do Vale do Paraíba e Litoral Norte - RMVPLN é área de risco para transmissão da doença, com registro de 45 casos e 12 óbitos no período de 2007 a 2021, sendo 5 (11,11%) e 4 (33,33%) respectivamente os números do município de Jacareí/SP. Avaliou-se o risco de exposição da população humana do município de Jacareí/SP nas áreas com presença do vetor, mapeadas a partir de amostras de carrapatos coletados no ambiente. Os locais de coleta de amostra ambiental com presença do vetor no município de Jacareí/SP no período de 2018 a 2021 foram georreferenciados e caracterizados, de forma a permitir comparação de fatores para avaliação de risco à saúde. As características geográficas indicam alternativas de abrigo, alimentação e reprodução tanto para o vetor quanto para os hospedeiros. Observou-se que as amostras ambientais do período analisado têm predomínio da espécie Amblyomma sculptum (86,42%), com a maioria dos espécimes coletados em áreas de travessia de pessoas (38,89%) e de lazer (36,11%). Pelo mapa de calor o ponto com maior densidade de amostragem a cada ano está situado próximo ao Rio Paraíba do Sul, sugerindo maior risco nas áreas adjacentes ao rio. A presença de capivaras (Hydrochoerus hydrochaeris) na região favorece a manutenção da doença. A avaliação dos dados aponta risco nas áreas adjacentes ao Rio Paraíba do Sul, em especial nas áreas de lazer.

Brazilian Spotted Fever - FMB is an exanthematic disease caused by the bacterium Rickettsia rickettsii and transmitted by the bite of infected ticks of the genus Amblyomma. The Metropolitan Region of Vale do Paraíba and North Coast - RMVPLN is a risk area for transmission of the disease, with a record of 45 cases and 12 deaths in the period from 2007 to 2021, of which 5 (11.11%) and 4 (33.33 %) respectively the numbers of the municipality of Jacareí/SP. The risk of exposure of the human population in the municipality of Jacareí/SP in areas with the presence of the vector, mapped from samples of ticks collected in the environment, was evaluated. The environmental sample collection sites with the presence of the vector in the municipality of Jacareí/SP in the period from 2018 to 2021 were georeferenced and characterized, to allow comparison of factors for health risk assessment. Geographic characteristics indicate alternatives for shelter, food, and reproduction for both the vector and the hosts. It was observed that environmental samples collected during the study period have a predominance of the species Amblyomma sculptum (86,42%), with most specimens collected in areas crossed by people (38,89%) and leisure (36,11%). According to the heat map, the point with the highest sampling density each year is located close to the Paraíba do Sul River, suggesting a greater risk in areas adjacent to the river. The presence of capybaras (Hydrochoerus hydrochaeris) in the region favors the maintenance of the disease. The evaluation of the data points to risk in the areas adjacent to the Paraíba do Sul River, especially in the leisure areas.

Dermal secretion physiology and thermoregulation in the lone star tick, Amblyomma americanum.

Ticks are hematophagous ectoparasites that transmit a wide range of pathogens. The lone star tick, Amblyomma americanum, is one of the most widely distributed ticks in the Midwest and Eastern United States. Lone star ticks, as other three-host ixodid ticks, can survive in harsh environments for extended periods without a blood meal. Physiological mechanisms that allow them to survive during hot and dry seasons include thermal tolerance and water homeostasis. Dermal fluid secretions have been described in metastriate ticks including A. americanum. We hypothesized that tick dermal secretion in the unfed tick plays a role in thermoregulation, as described in other hematophagous arthropods during blood feeding. In this study, we found that physical contact with a heat probe at 45 °C or high environmental temperature at ∼50 °C can trigger dermal secretion in A. americanum and other metastriate ticks in the off-host period. We demonstrated that dermal secretion plays a role in evaporative cooling when ticks are exposed to high temperatures. We find that type II dermal glands, having paired two cells and forming large glandular structures, are the source of dermal secretion. The secretion was triggered by an injection of serotonin, and the serotonin-mediated secretion was suppressed by a pretreatment with ouabain, a Na/K-ATPase blocker, implying that the secretion is controlled by serotonin and the downstream Na/K-ATPase.

Establishment and multiapproach characterization of Amblyomma sculptum (Acari: Ixodidae) cell line (ASE-14) from Brazil.

The establishment and characterization of the ASE-14 cell line derived from embryos of Amblyomma sculptum is described here. Primary cultures were started, and after 60 days of culturing a confluent monolayer was formed and the first subculture was then carried out. After this, new subcultures were carried out every 4 weeks. Cryopreservation of cells was successful only after the 14th subculture. We compared the chromosomes of the ASE-14 cell line with those of parental ticks. Cytogenetic analysis revealed occurrences of variable and increased diploid numbers in the ASE-14 cell line in comparison with adult ticks, probably through polyploidization events, chromosome fusions and translocations, which allowed generation of cells with distinct diploid numbers. Confirmation of the origin of the A. sculptum cell line was obtained through conventional PCR and sequencing of a fragment of the mitochondrial 16S rRNA gene. In addition, no DNA from Anaplasma marginale, Anaplasma spp., Babesia/Theileria spp., Bartonella spp., Coxiella spp., Ehrlichia canis, Mycoplasma spp. or Rickettsia spp. was detected in the cells through PCR assays. Cytological analyses were performed using live phase contrast microscopy and cytocentrifuge smears stained with Giemsa, while periodic acid-Schiff and bromophenol blue staining techniques were used to detect polysaccharides and protein, respectively. In conclusion, a new cell line derived from embryos of A. sculptum was generated and characterized in this study. The ASE-14 cell line was deposited in the Tick Cell Biobank at the University of Liverpool, and in the Tick Cell Biobank South America Outpost at the Oswaldo Cruz Foundation (FIOCRUZ). The ASE-14 cell line is an important addition to the existing panel of tick cell lines and can be used as a tool for advancing research in various areas of the virology, bacteriology, biology and control of this tick.

Investigation of the parasitoid Ixodiphagus hookeri (Hymenoptera: Encyrtidae) in Amblyomma sculptum (Acari: Ixodidae) ticks in the municipality of Salto, São Paulo, Brazil

The presence of capybaras (Hydrochoerus hydrochaeris) in urban and periurban areas has caused increased numbers of cases of Brazilian spotted fever. With the aim of investigating the presence of the parasitoid Ixodiphagus hookeri in Amblyomma sculptum ticks in the municipality of Salto, state of São Paulo, samples were collected monthly from 14 sites. Thirty samples were placed in containers for observation of the emergence of microhymenopterans and 88 samples were subjected to molecular testing to identify the presence of I. hookeri DNA. Neither dissections nor observation of emergence indicated any presence of I. hookeri larvae in ticks. Samples subjected to polymerase chain reaction (PCR) amplification of the mCOX I region of I. hookeri did not reveal its presence, although fragments corresponding to mRNA 16S of Amblyomma sculptum ticks were amplified in all samples tested.

Diversity of tick species (Acari: Ixodidae) in military training areas in Southeastern Brazil / Diversidade de espécies de carrapatos (Acari: Ixodidae) em áreas de instrução militar na região Sudeste do Brasil

Abstract Tick-borne pathogens belong to one of the two main groups of occupational biohazards, and occupational exposure to such agents puts soldiers at risk of zoonotic infections, such as those caused by rickettsiae. There are few studies on acarological fauna and occupational risk in military areas in Brazil. Thus, the present study aimed to analyze the diversity of ticks present in the military training areas of municipalities in the Southeast Region of Brazil. The ticks were collected from the selected areas using the dragging and flagging techniques as well as by visual detection on the operators' clothing, and environmental information was also recorded. A total of ten species were collected from the 66 surveyed areas, belonging to five genera and nine species: Amblyomma sculptum, Amblyomma dubitatum, Amblyomma brasiliense, Amblyomma longirostre, Amblyomma aureolatum, Dermacentor nitens, Rhipicephalus spp., Ixodes spp. and Haemaphysalis spp. The frequent presence of tick species in military training areas along with traces and sightings of wild animals, most commonly capybaras (Hydrochoerus hydrochaeris), in most of the studied areas, indicates high levels of exposure of the military to tick vectors of spotted fever group rickettsiae and the possible occurrence of infections among the troops.

Resumo As doenças transmitidas por carrapatos estão entre os dois principais grupos de riscos biológicos ocupacionais. Tal exposição ocupacional de militares os colocam sob maior risco de adquirirem doenças zoonóticas, como infecções rickettsiais, entre outras. No Brasil, há raros estudos sobre fauna acarológica e o risco ocupacional em áreas militares. Neste sentido, o presente estudo teve por objetivo analisar a diversidade de carrapatos presentes em áreas de treinamento militar de municípios da região Sudeste do Brasil. Os carrapatos foram coletados nas áreas através de arrasto de flanela, bandeiramento e/ou coleta nas vestimentas do operador, sendo registradas também informações ambientais. Das 66 áreas pesquisadas, foram coletados 9.374 carrapatos, com uma diversidade de cinco gêneros, distribuídos em dez espécies: Amblyomma sculptum, Amblyomma dubitatum, Amblyomma brasiliense, Amblyomma longirostre, Amblyomma aureolatum, Amblyomma spp., Dermacentor nitens, Rhipicephalus spp., Ixodes spp. e Haemaphysalis spp. A presença frequente de espécies de carrapatos nas áreas de instrução militar, associada aos vestígios e à presença de animais silvestres, mais comumente de capivaras na maioria das áreas estudadas, evidencia uma grande exposição dos militares a carrapatos vetores de rickettsias do Grupo da Febre Maculosa e a possível ocorrência de casos humanos nas tropas.

Avaliação da competência de cães domésticos como hospedeiros amplificadores da bactéria Rickettsia rickettsii para carrapatos Amblyomma aureolatum e modelagem espacial ecológica preditiva para ocorrência de Febre Maculosa Brasileira no Estado de São Paulo / Evaluation of domestic dogs as competent amplifier hosts of the bacterium Rickettsia rickettsii for Amblyomma aureolatum ticks and predictive spatial ecological modeling for the occurrence of Brazilian Spotted Fever in the State of São Paulo

A Febre Maculosa Brasileira (FMB) é uma zoonose transmitida por vetores que tem como agente patogênico, bactérias da espécie Rickettsia rickettsii. Na Região Metropolitana de São Paulo (RMSP), o vetor incriminado na transmissão é o carrapato Amblyomma aureolatum, que depende de características ambientais típicas do bioma Mata Atlântica para sobreviver. Cães domésticos são os principais hospedeiros do estágio adulto do carrapato em áreas de matriz urbana próxima a áreas de mata fragmentada, e participam do ciclo da doença ao carrear carrapatos infectados da mata para o ambiente antrópico. Além disso, são hospedeiros amplificadores do agente e contribuem para o aumento do número de carrapatos infectados na natureza. Com o objetivo de elucidar as lacunas em relação a FMB na RMSP, o presente estudo testou a hipótese de que cães domésticos atuariam como hospedeiros amplificadores da bactéria R.rickettsii para carrapatos da espécie A. aureolatum após um segundo contato com o agente. Os resultados mostraram que apesar de apresentarem uma resposta imunológica, com aumento no título de anticorpos, os cães não foram capazes de gerar novas linhagens de carrapatos infectados. Esse estudo determinou, por meio de modelagem espacial, quais as áreas mais ou menos propensas a apresentarem casos de FMB levando em consideração as condições necessárias para ocorrência do vetor A. aureolatum e os aspectos ecológicos que facilitam a interação do vetor com seus hospedeiros no ambiente. As variáveis ambientais de altitude e temperatura mostraram-se de maior influência para a distribuição do vetor enquanto o aumento do perímetro de borda e redução de áreas de mata favorecem a ocorrência de casos de FMB. Este estudo traz novos dados sobre a ecoepidemiologia da Febre Maculosa Brasileira no Estado de São Paulo e chama atenção para a importância dos cães domésticos no ciclo da doença que ocorre na Região Metropolitana do estado. Ações de prevenção com foco na população de cães se mostram de extrema importância para a redução da incidência da doença e melhoria das condições de saúde da população.

Brazilian Spotted Fever (BSF) is a vector-borne zoonosis whose agent is the bacteria Rickettsia rickettsii. In the São Paulo Metropolitan Region (SPMR), the vector responsible for the disease transmission is the tick Amblyomma aureolatum, which depends on environmental characteristics of the Atlantic Forest biome to survive. Domestic dogs are the main hosts of the adult stage of the tick in areas of urban matrix close to fragmented forest, and participate in the disease cycle by carrying infected ticks from the forest to the anthropic environment. Furthermore, they can amplify the bacteria and contribute to increase the number of infected ticks in nature. In order to elucidate the gaps in relation to BSF in the SPMR, this study tested the hypothesis that dogs could act as amplifying hosts of R. rickettsii for A. aureolatum ticks after a second contact with the agent. The results showed that the dogs could not generate new infected tick strains despite an immune response with increased antibody titers. This study also determined, through spatial modeling, which areas are more likely to present cases of BSF, taking into account the necessary conditions for A. aureolatum occurrence and the ecological aspects that facilitate the interaction of the vector with its hosts in the environment. The environmental variables of altitude and temperature showed to have the greatest influence on vector distribution, while the increase in edge perimeter and decrease in forest cover favored the occurrence of BSF. This study brings new data on the ecoepidemiology of Brazilian Spotted Fever in the State of São Paulo and draws attention to the importance of domestic dogs in the disease cycle in the Metropolitan Region of the state. Prevention actions focused on dog population are extremely important to reduce disease incidence and improve the human population's health conditions.

Avaliação soroepidemiológica de cães expostos a Rickettsia parkeri e correlação com a estrutura da paisagem no litoral do estado de São Paulo / Serosurvey on dogs for Rickettsia parkeri and correlation with landscape structure in São Paulo state coast (Brazil)

A febre maculosa causada por Rickettsia parkeri é a segunda riquetsiose de maior importância no Brasil. No estado de São Paulo, o agente é transmitido pelo carrapato Amblyomma ovale, que ocorre em número elevado em regiões costeiras de Mata Atlântica. Até o presente momento, não foi estabelecido como a estrutura da paisagem pode afetar a dispersão de carrapatos infectados por R. parkeri e, portanto, dinâmica de transmissão da doença. Propõe-se avaliar a relação entre a sorologia de cães domésticos expostos a R. parkeri e a estrutura da paisagem em áreas de comunidades adjacentes à Mata Atlântica no litoral paulista. Em um estudo transversal, foram selecionadas cinco áreas localizadas nos municípios de Cubatão, Peruíbe, São Sebastião, Cananéia e Ilhabela e, em cada área, amostras de soro de 30 cães foram colhidas e submetidas a reação de imunofluorescência indireta para detecção do título máximo de anticorpos anti-R. parkeri. Spearman foi usado como teste de correlação entre a sorologia e os componentes principais extraídos das métricas de paisagem em cada área de estudo. A análise da estrutura da paisagem evidenciou dois componentes principais não correlacionados que, somados, explicam 94,77% da variabilidade entre as áreas, em que o primeiro componente está relacionado à conectividade e o segundo componente está relacionado à quantidade de borda. A soroprevalência das áreas variou de 16,7% a 83,3%. A exposição de cães domésticos a R. parkeri está associada positivamente com a conectividade de habitat para A. ovale em áreas estudadas do litoral do estado de São Paulo. Não foi evidenciada associação entre a sorologia e a quantidade de borda na paisagem. É recomendado que as ações de vigilância epidemiológica para febre maculosa por R. parkeri sejam priorizadas em áreas com maior conectividade florestal, juntamente com a conscientização da posse responsável de cães.

Spotted fever caused by Rickettsia parkeri is the second most important rickettsiosis in Brazil. In São Paulo state, this agent is transmitted by the Amblyomma ovale tick, highly prevalent in coastal Atlantic Rainforest. Until the present moment, it is not known how landscape structure could affect A. ovale dispersion, and therefore, R. parkeri transmission dynamics. It is proposed to evaluate the seroepidemiology from R. parkeri exposed dogs and its correlation with landscape structure in São Paulo state coast. A transversal study was conducted to evaluate five different areas from communities nearby Atlantic Rainforest (municipalities of Cubatão, Peruíbe, São Sebastião, Cananéia and Ilhabela). Within each area, blood samples from 30 dogs were collected for serologic testing by indirect immunofluorescence assay. Correlation test Spearman was used between serology data and principal components extracted from landscape structure metrics in each area. Principal components analysis pointed two principal uncorrelated components which, together, explain 94,77% of the variability between landscape metrics. First component is related to landscape connectivity, and the second is related to edge abundance. It was found a seroprevalence from 16,7% to 83,3% in the different areas. A positive association between antibodies anti-R. parkeri found in dogs and landscape connectivity (p=0) was demonstrated. There was no association between serologic testing and edge abundance in the study areas. It is recommended that R. parkeri rickettsiosis surveillance to be priorized in connected forest areas, and responsible dog ownership must be encouraged.

Repellent and acaricidal activity of coconut oil fatty acids and their derivative compounds and catnip oil against Amblyomma sculptum.

This study was carried out aiming to evaluate the repellent and acaricidal activity of major ingredient compounds from coconut oil including their methyl ester derivatives and catnip oil against nymphs and larvae of Amblyomma sculptum. Repellent candidates, coconut oil free fatty acids (coconut FFA mainly C12, C10 and C8 acid); lauric acid (C12 acid); capric acid (C10 acid); methyl laurate; methyl caprate and 10 % each of C12, C10 and C8 acid (1:1:1) in lavender oil formulation (CFA in lavender formula) and catnip oil (Nepeta cataria), were screened using a Petri dish bioassay to assess repellency. Catnip oil, methyl caprate, methyl laurate, and CFA in lavender formulation repelled ticks strongly (P < 0.05) at almost all times evaluated, with an average of 77.8-100% repellency. Some candidate repellents with consistent strong repellence observed were selected for further evaluation, with coconut CFA in lavender formula showing a repellency lasted up to 7 days, while those of catnip oil and methyl caprate were active for 4 and 3 days, respectively. For the acaricide test, five concentrations (2.5; 5; 10; 15 and 20 mg/mL) were evaluated using the larval packet test. Only CFA in lavender formula and two methyl esters showed acaricidal activity, with methyl laurate presenting the strongest toxicity at 15 mg/mL concentration, which was effective against more than 93 % of the tested larvae. Catnip oil caused no mortality of A. scultptum larvae in all concentrations tested.

Two Atypical Cases of Tick Bites: A Fully Engorged Tick and Multiple Ticks.

Ticks have a cosmopolitan distribution and, as such, are also found in Japan. Ticks are typically ectoparasites of wild animals, however, humans can also be bitten when visiting environments inhabited by ticks. Herein, we describe two cases with atypical tick bites. Case 1 was an elderly Japanese male patient who presented with a fully engorged tick measuring 20 × 17 × 8 mm; it is rare for ticks to attain a length of 20 mm. Case 2 was an elderly Japanese female with severe dementia who presented with multiple tick bites, which is rare, after going missing for 6 days before being found in a densely wooded area. Ticks are responsible for the transmission of many infectious agents, such as bacteria, viruses and parasites. The National Institute of Infectious Diseases and the Ministry of Health, Labour and Welfare regularly inform citizens of the risks posed by tick bites. However, the tick bites could not be prevented in our patients. Further edification about tick bites, tick-borne diseases, and their prevention are considered necessary in Japan.

Efficacy of a novel topical combination of esafoxolaner, eprinomectin and praziquantel against Amblyomma americanum in cats.

Esafoxolaner, a purified enantiomer of afoxolaner with insecticidal and acaricidal properties, is combined with eprinomectin and praziquantel in NexGard® Combo, a novel topical endectoparasiticide product for cats. The efficacy of this novel formulation was assessed in two experimental studies against induced infestations with Amblyomma americanum, a tick species of major importance, highly prevalent in a large southeastern quarter of the United States. In each study, 10 cats were randomly allocated to a placebo control group and 10 cats to a novel formulation treated group. Infested cats were treated topically once at the minimum recommended dose. Both studies were designed to test curative efficacy on existing infestation, 72 h after treatment, and to test preventive efficacy, 72 h after subsequent weekly (Study #1) or fortnightly (Study #2) infestations for one month. For each infestation, all cats were infested with 50 unfed adult A. americanum. At each tick count, in both studies, at least 8 in 10 placebo control cats were infested with 13 (26%) or more live ticks, demonstrating adequate infestation throughout the studies. Curative efficacy of the novel formulation was 99% in both studies; preventive efficacy was 92% and 100% for at least one month.

TITLE: Efficacité d'une nouvelle association topique d'esafoxolaner, d'éprinomectine et de praziquantel contre Amblyomma americanum chez le chat. ABSTRACT: L'esafoxolaner, un énantiomère purifié d'afoxolaner aux propriétés insecticides et acaricides, est associé à l'éprinomectine et au praziquantel dans NexGard® Combo, un nouvel endectoparasiticide topique pour chats. L'efficacité de cette nouvelle formulation a été évaluée dans deux études expérimentales contre les infestations induites par Amblyomma americanum, une espèce de tique d'importance majeure, très répandue dans un grand quart sud-est des États-Unis. Dans chaque étude, dix chats ont été répartis au hasard dans un groupe témoin placebo et dix chats dans un groupe traité par une nouvelle formulation. Les chats infestés ont été traités une fois par voie topique à la dose minimale recommandée. Les deux études ont été conçues pour tester l'efficacité curative sur une infestation existante, 72 heures après le traitement, et pour tester l'efficacité préventive, 72 heures après des infestations hebdomadaires (étude n° 1) ou bimensuelles (étude n° 2) pendant un mois. Pour chaque infestation, tous les chats étaient infestés par 50 A. americanum adultes non nourris. À chaque décompte de tiques, dans les deux études, au moins 8 chats sur 10 du groupe témoin placebo étaient infestés de 13 (26 %) ou plus tiques vivantes, ce qui démontre une infestation adéquate tout au long des études. L'efficacité curative de la nouvelle formulation était de 99 % dans les deux études, l'efficacité préventive était de 92 % et 100 % pendant au moins un mois.

Parasitism of a capybara (Hydrochoerus hydrochaeris) by Amblyomma nodosum Neumann, 1899 and Amblyomma humerale Koch, 1844 in an Amazon forest area, Rondônia, Brazil / [Parasitismo por Amblyomma nodosum Neumann, 1899 e Amblyomma humerale Koch, 1844 em capivara (Hydrochoerus hydrochaeris) em área de Floresta Amazônica, Rondônia, Brasil]

Dois carrapatos adultos, sendo um Amblyomma nodosum e um Amblyomma humerale foram encontrados parasitando ativamente uma capivara (Hydrochoerus hydrochaeris) proveniente da região central do estado de Rondônia, Brasil, na zona rural do município de Ji-Paraná, localizado na Amazônia ocidental. Poucos trabalhos científicos relatam a presença de A. humerale em capivaras. Assim, esta é a primeira vez que A. nodosum é relatado parasitando essa espécie animal. Por não se tratar de um hospedeiro animal conhecido para A. nodosum ou comum para A. humerale, esses achados podem sugerir uma nova relação entre hospedeiros para eles. Portanto, são necessários mais estudos acerca da ecologia desses parasitas, principalmente por serem potenciais transmissores de patógenos de importância em medicina veterinária e saúde pública.(AU)

Ectoparasites infesting animals living in close contact with human beings: a real trouble for One Health perspective? / [Ectoparasitos infestando animais que vivem em contato próximo com seres humanos: um problema real para a perspectiva Saúde Única?]

The number of domestic animals living with human beings is rapidly increasing in parallel with an enhanced risk of transmission of their parasites and the pathogens they might carry. The aim of this study was to assess the occurrence of hematophagous arthropods infesting domestic animals from Northeastern Brazil and to remark the implications of their occurrence on the epidemiology and control of selected veterinary and human diseases. From January 2017 to April 2019, ectoparasites infesting domestic cats, dogs and horses were collected for their respective hosts and identified. Overall, ectoparasites were sampled from 86 domestic animals, living in different anthropic settings. A total of 401 specimens (344 ticks and 57 fleas) were collected from different hosts [i.e., 10 (2.49%), 96 (23.94%) and 295 (73.57%) from cats, dogs and horses, respectively]. Two flea (i.e., Ctenocephalides canis and Ctenocephalides felis) and 5 tick species (i.e., Amblyomma ovale, Amblyomma sculptum, Dermacentor nitens, Rhipicephalus microplus and Rhipicephalus sanguineus sensu lato) were identified. This study provides data on the ectoparasite fauna infesting domestic animals from Northeastern Brazil. The diagnosis and treatment of these parasites should not be underestimated, considering the role that hematophagous arthropods display as vectors of pathogens of medical and veterinary concern.(AU)

O número de animais domésticos infestados por ectoparasitos vivendo em estreito contato com seres humanos está aumentando, elevando o risco de infecção pelos patógenos transmitidos por vetores. Objetivou-se neste estudo avaliar a ocorrência de ectoparasitos infestando animais do nordeste do Brasil e discutir as implicações desse parasitismo na epidemiologia e no controle de doenças de importância médico-veterinária. De janeiro de 2017 a abril de 2019, artrópodes foram coletados de gatos, cães e cavalos, e identificados morfologicamente. Ectoparasitos foram removidos de 86 animais infestados provenientes de áreas urbanas e rurais. Foram identificados 401 espécimes (344 carrapatos e 57 pulgas) coletados de diferentes hospedeiros (10 (2,49%), 96 (23,94%) e 295 (73,57%) de gatos, cães e cavalos, respectivamente. Duas espécies de pulgas (Ctenocephalides canis e Ctenocephalides felis) e cinco espécies de carrapatos (Amblyomma ovale, Amblyomma sculptum, Dermacentornitens, Rhipicephalus microplus e Rhipicephalus sanguineus sensu lato) foram identificadas. Este estudo fornece informações sobre a ectofauna de animais domésticos do nordeste do Brasil. Portanto, o diagnóstico e o tratamento desses parasitos não devem ser subestimados, considerando que os artrópodes aqui relatados podem veicular patógenos de importância médico-veterinária.(AU)

Molecular Diagnosis and Prevalence of Trypanosoma vivax (Trypanosomatida: Trypanosomatidae) in Buffaloes and Ectoparasites in the Brazilian Amazon Region.

Trypanosoma vivax Ziemann is a parasite that affects both wild and domestic ungulates and is transmitted mechanically via tabanids and other blood-sucking insects in the Americas. A total of 621 blood samples from water buffaloes (Bubalus bubalis (Linnaeus) (Artiodactyla: Bovidae), and 184 ectoparasite samples (Amblyomma cajennense (Fabricius) sensu stricto and Rhipicephalus (Boophilus) microplus (Canestrini) (Acari: Ixodidae), and Haematopinus tuberculatus (Burmeister) (Phthiraptera: Haematopinidae)) were obtained from 60 farms in the State of Pará, Brazilian Amazon. Twelve buffalo blood samples (1.89%) and 11 ectoparasites (6%) were positive for T. vivax based on the cathepsin L-like gene. All sequences were 99% similar to T. vivax from northeastern Brazil (EU753788) in amplified PCR assays on each of the hosts tested.

Phylogenies from mitochondrial genomes of 120 species of ticks: Insights into the evolution of the families of ticks and of the genus Amblyomma.

The evolution and phylogenetic relationships of the ticks at both the family and genus levels are contested. The genus Amblyomma and its subgenera are in a state of flux; moreover, the relationships among the three tick families are controversial due to conflicting phylogenetic support for different arrangements of the three families of living ticks. With 18 newly sequenced mitochondrial (mt) genomes of ticks included, we executed the largest mt genome phylogenetic study of ticks so far. Phylogenetic trees were inferred from one sea spider mt genome, one horseshoe crab, five mite mt genomes and 146 tick mt genomes from 120 species: 153 mt genomes in total. Sixteen phylogenetic trees were inferred from 10 datasets using both maximum likelihood and Bayesian inference methods. We describe the first novel mt gene-arrangement for the metastriate Ixodidae in Amblyomma (Africaniella) transversale. Also, three unusual partial 16S rRNA gene inserts were found in the mt genome of Haemaphysalis (Alloceraea) kitaokai: we consider the possible role of past genome translocation events in the formation of these inserts. Our phylogenies revealed evidence that: (i) the genus Amblyomma is polyphyletic with respect to Amblyomma (Africaniella) transversale; (ii) the subgenus Aponomma is apparently embedded in the genus Amblyomma; (iii) Haemaphysalis (Segalia) parva and Haemaphysalis (Alloceraea) kitaokai form a clade to the exclusion of other Haemaphysalis species; and (iv) the phylogenetic position of the family Nuttalliellidae is unstable among phylogenies from different datasets.

Perfil Epidemiológico da Febre Maculosa no Brasil / Epidemiological Profile of Spotted Fever in Brazil

Introdução: A Febre Maculosa é uma doença febril aguda, causada pela bactéria Rickettsia rickettsii e transmitida pela picada de carrapatos infectados. Atualmente, ocorre um aumento da incidência e das mortes pela doença em diversas áreas do Brasil, o que pode significar um surto e a necessidade de alerta. Metodologia: Estudo quantitativo, descritivo, transversal, que embasou sua busca nas plataformas de notificações brasileiras (DATASUS e SINAN) e boletins epidemiológicos no período de 2007 a 2019. Resultados: O número de casos e a mortalidade demonstraram valores maiores nos meses correspondentes à fase de ninfa do carrapato vetor, com incidência maior na zona rural, na região sudeste e durante o lazer. Além disso, a faixa etária mais atingida foi de 40 a 59 anos, com predominância da população masculina. Os valores maiores apresentados nas zonas rurais, assim como grupo etário e sexo se devem às atividades realizadas que expõem mais os indivíduos ao ambiente natural dos vetores, à sazonalidade dos carrapatos e à presença da Mata Atlântica. Nota-se a presença de grande subnotificação no Brasil. Conclusão: Constata-se a necessidade de desenvolver de políticas de prevenção e disseminar informações sobre o ciclo da doença, manifestações clínicas, prevenção e tratamento.

Introduction: Spotted Fever is an acute febrile disease, caused by the bacteria Rickettsia rickettsii and transmitted by the bite of infected ticks. Currently, there is an increase in the incidence and deaths from the disease in several areas of Brazil, which may mean an outbreak and the need for alert. Methodology: Quantitative, descriptive, cross-sectional study that based its search on the Brazilian notification platforms (DATASUS and SINAN) and newsletters. Results: The number of cases and mortality showed higher values in months corresponding to the vector tick nymph phase (from June to September), with a higher incidence in countryside, in southeast region and at moments of leisure practice. In addition, the most affected age group was 40 to 59 years old, in male population. Higher values presented in countryside, as well as age group and sex are due to activities that expose individuals more to natural environment of vectors, the seasonality of the ticks and the presence of the Atlantic Forest. In addition, there is a large underreporting in Brazil. Conclusion: It´s necessary to develop prevention policies, as well as disseminate information about disease cycle, clinical manifestations, prevention and treatment.