Sculptum Ticks Research Articles

Babesia bigemina (smith and Kilbourne, 1893) detection in Amblyomma sculptum (Berlese, 1888) ticks in the Mato Grosso do Sul state, Brazil.

Ticks parasitize various hosts, including humans, and are known to transmit pathogens that can be harmful not only to animals but also to humans. To evaluate the possible presence of pathogens in ticks, we aimed to collect and identify tick fauna specimens in Lagoa Comprida Municipal Natural Park, an anthropogenic urban area located in Aquidauana, Mato Grosso do Sul, Brazil. A total of 1216 ticks, of which 51.2% were Amblyomma sculptum, 1.2% were Amblyomma dubitatum, and 41% were Amblyomma spp. were collected. These results show that the prevalence of A. sculptum is significantly higher than that of A. dubitatum across all four seasons. Molecular analyses revealed positive samples for the genus Babesia, including the confirmation of Babesia bigemina in an A. sculptum specimen, marking the first record of this relationship. This unexpected finding demands greater attention and deeper analysis in the context of the epidemiology of tick-borne diseases.

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 peri‑urban 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.

Survey of Rickettsia spp. in ticks (Acari: Ixodidae) infesting opossums (Didelphis albiventris) and capybaras (Hydrochoerus hydrochaeris) from an urban park in southeastern Brazil.

Ticks are hematophagous arthropods and, during feeding, may transmit pathogens to vertebrate hosts, including humans. This study aimed to investigate the presence of Rickettsia spp. in ticks collected between 2010 and 2013 from free-ranging capybaras (Hydrochoerus hydrochaeris) and opossums (Didelphis albiventris) that inhabit Sabiá Park in Uberlândia, Brazil. Overall, 1,860 ticks were collected: 1,272 (68.4%) from capybaras (487 of the species Amblyomma sculptum, 475 adults and 12 nymphs; 778 Amblyomma dubitatum, 727 adults and 51 nymphs; and seven larva clusters of the genus Amblyomma); and 588 (31.6%) from opossums (21 A. sculptum, one adult and 20 nymphs; 79 A. dubitatum, all nymphs; 15 Ixodes loricatus, 12 adults and three nymphs; 457 Amblyomma sp. larva clusters; 15 Ixodes sp. larva clusters; and one Argasidae larva cluster). Out of 201 DNA samples tested for the presence of Rickettsia spp. DNA using polymerase chain reaction (PCR) 12 showed amplification of a gtlA gene segment that was specific to Rickettsia bellii, a bacterium non-pathogenic to humans. As there has been a report showing serological evidence of infections caused by Rickettsia species of the spotted fever group (SFG) in capybaras and opossums in the park, including Rickettsia rickettsii, the etiological agent of Brazilian spotted fever, and considering the presence of A. sculptum ticks, which are aggressive to humans, as well as these vertebrate hosts, which are amplifiers of R. rickettsii, it is important to monitor the presence of SFG rickettsiae in the Sabiá Park, which is visited daily by thousands of people.

Structural and functional properties of the Kunitz-type and C-terminal domains of Amblyomin-X supporting its antitumor activity.

Amblyomin-X is a Kunitz-type FXa inhibitor identified through the transcriptome analysis of the salivary gland from Amblyomma sculptum tick. This protein consists of two domains of equivalent size, triggers apoptosis in different tumor cell lines, and promotes regression of tumor growth, and reduction of metastasis. To study the structural properties and functional roles of the N-terminal (N-ter) and C-terminal (C-ter) domains of Amblyomin-X, we synthesized them by solid-phase peptide synthesis, solved the X-Ray crystallographic structure of the N-ter domain, confirming its Kunitz-type signature, and studied their biological properties. We show here that the C-ter domain is responsible for the uptake of Amblyomin-X by tumor cells and highlight the ability of this domain to deliver intracellular cargo by the strong enhancement of the intracellular detection of molecules with low cellular-uptake efficiency (p15) after their coupling with the C-ter domain. In contrast, the N-ter Kunitz domain of Amblyomin-X is not capable of crossing through the cell membrane but is associated with tumor cell cytotoxicity when it is microinjected into the cells or fused to TAT cell-penetrating peptide. Additionally, we identify the minimum length C-terminal domain named F2C able to enter in the SK-MEL-28 cells and induces dynein chains gene expression modulation, a molecular motor that plays a role in the uptake and intracellular trafficking of Amblyomin-X.

A Kunitz-type inhibitor from tick salivary glands: A promising novel antitumor drug candidate.

Salivary glands are vital structures responsible for successful tick feeding. The saliva of ticks contains numerous active molecules that participate in several physiological processes. A Kunitz-type factor Xa (FXa) inhibitor, similar to the tissue factor pathway inhibitor (TFPI) precursor, was identified in the salivary gland transcriptome of Amblyomma sculptum ticks. The recombinant mature form of this Kunitz-type inhibitor, named Amblyomin-X, displayed anticoagulant, antiangiogenic, and antitumor properties. Amblyomin-X is a protein that inhibits FXa in the blood coagulation cascade and acts via non-hemostatic mechanisms, such as proteasome inhibition. Amblyomin-X selectively induces apoptosis in cancer cells and promotes tumor regression through these mechanisms. Notably, the cytotoxicity of Amblyomin-X seems to be restricted to tumor cells and does not affect non-tumorigenic cells, tissues, and organs, making this recombinant protein an attractive molecule for anticancer therapy. The cytotoxic activity of Amblyomin-X on tumor cells has led to vast exploration into this protein. Here, we summarize the function, action mechanisms, structural features, pharmacokinetics, and biodistribution of this tick Kunitz-type inhibitor recombinant protein as a promising novel antitumor drug candidate.

Surgical sterilization of free-ranging capybaras (Hydrochoerus hydrochaeris): "Passos Nunes" uterine horn ligature.

Capybaras are the primary hosts of Amblyomma sculptum tick, vectors of Rickettsia rickettsia bacteria, and the zoonotic agent of Brazilian Spotted Fever (BSF). In this context, contraceptive methods have been suggested for population control in order to reduce the number of free-ranging capybaras cohabiting with humans in urban and rural areas and acting as disease amplifiers. To maintain the group's expected behavior and social hierarchy, sterilization techniques that preserve the gonads are recommended. On 126 female capybaras in the Brazilian state of São Paulo, a new surgical technique named “Passos Nunes” uterine horn ligature was performed after adequate general anesthesia. It achieved effective surgical sterilization, with an incision length of about 3 cm in the periumbilical linea alba, cranial to the pubis. After entering the abdominal cavity, the urinary bladder is pulled laterally to access the uterine horns and the cervix. The uterine horn is folded up, forming a strap; the distal portion of the strap is ligated and its distal end sectioned. The exact process is performed on the opposite horn. After the surgical procedure, the musculature is sutured in a sultan pattern and the subcutaneous tissue with a horizontal mattress pattern. The skin is sutured in a separate simple format, using nylon 2.0 for all steps. The wide exposure of the uterine horns facilitates the confirmation of pregnancy, allowing the surgeon to choose between salpingo hysterectomy or ligature of the uterine horns. The present study presents a new technique of surgical sterilization that can be used in female free-ranging mammals in which maintenance of the gonads is recommended, and births of offspring should not occur.

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.

Oxidative and local histopathological response on skin wound of horses due to Amblyomma sculptum tick parasitism

Amblyomma sculptum is frequently observed parasitizing horses, responsible for economic losses, damage to the host’'s skin and transmission of pathogens. The oxidative stress profile and inflammatory mechanisms involved in this parasitism remain poorly studied. Thus, this study aimed to assess the histopathological changes and oxidative profile responses of horses in the attachment site of A. sculptum to find variations that indicate resistance and susceptibility between the breeds to this tick, based on the hypothesis that resistant animals have a greater inflammatory response and lesser number of attached ticks. We analyzed female horses of two breeds, Mangalarga Marchador and Breton Postier, naturally infested by Amblyomma sculptum. The ticks were counted and full-thickness excisional skin wounds of 10 mm were made on the perineal region on the attachment site of partially engorged females for histological and biochemical analyzes. The occurrence of the tick on the skin caused an increase in cellularity, inflammatory infiltrate, mast cells, pyknotic nuclei, and changes in the fibrous components of the matrix. The negative correlation observed between tick infestation and inflammatory response indicated that animals with greater inflammatory response tend to have less tick infestation. The oxidative stress markers, MDA, PCN and NO not present great variation; however, between the antioxidant enzymes levels, SOD was higher in tick attachment of Breton Postier skin, this may mean that these animals had higher oxidative enzymatic activity and consequently less tissue damage, while the GST dropped in the attachment sites compared to the control, which may indicate that animals were in a state of significant oxidative stress or raises the question of the possibility of enzymatic sequestration by ticks. No significant differences were found in the resistance of the two breeds since most of the analyzes varied due to the presence or absence of the tick attached to the skin. We draw attention to the importance of studying characteristics of the animal's antioxidant responses to the tick and the action of tick saliva on antioxidant enzymes and ROS because these characteristics are interdependent with the inflammatory response.

Spotted fever outbreak with cases and deaths in the same family in a municipality in south eastern Brazil, 2019

Background: Brazilian Spotted Fever (BSF) is a highly lethal hemorrhagic febrile infectious disease whose etiological agent is the bacterium Rickettsia rickettsii, transmitted by the Amblyomma sculptum tick. Between May and June/2019 there was an outbreak of the disease in the municipality of Contagem, state of Minas Gerais, with previous identification of Rickettsia rickettsia in 2017. The objectives were to investigate the outbreak, describe cases and deaths and raise hypotheses for illness. Methods and materials: Was conducted a descriptive study, with a semi-structured questionnaire from May to June/2019. The population was individuals attended or notified by Contagem municipality as a possible exposed to the probable site of infection. Suspected Cases were individual with fever, headache, myalgia who reported tick bite and/or contact with domestic and/or wild animals and/or visited known area with transmission of BSF from April 28 to June/02, 2019. Confirmed cases were individuals with laboratory confirmation for BSF by Polymerase Chain Reaction. Results: Among the 10 individuals that attended to the suspected case definition, five were confirmed and four died (80% lethality). Median age of the cases were 40 years, four male and all had fever, myalgia and headache; three manifested rash and petechiae; four participated together in a weeding activity and three related tick bite. All deaths happened in the same family and had dengue as the first diagnostic hypothesis; three of these had first health assistance in a private medium complexity service and a seven days median between symptoms and death. Conclusion: There was a high lethality outbreak, with cases and deaths in the same family, possibly related to a weeding activity in a preserved forest area where cases developed all classic symptoms of the disease. It's recommended cleaning and maintenance routines in public spaces with grass and vegetation; local professionals training for the need to investigate BSF in the health assistance routine; investigation and diagnostic capacity increase for BSF and intensification of educational actions aimed in the transmission mode, disease control and prevention.

Capybaras (Hydrochoerus hydrochaeris) as amplifying hosts of Rickettsia rickettsii to Amblyomma sculptum ticks: Evaluation during primary and subsequent exposures to R. rickettsii infection

Rickettsia rickettsii is the agent of Brazilian spotted fever (BSF), the most lethal tick-borne disease in the western hemisphere. Amblyomma sculptum is the main vector of R. rickettsii in southeastern Brazil. Capybaras act as primary hosts for this tick species, and as amplifying hosts for R. rickettsii, creating new infected lineages of A. sculptum in BSF-endemic areas. In a recent study, we demonstrated that capybaras successively exposed to R. rickettsii-infected A. sculptum ticks developed a mean rickettsemic period of 9.2 days during primary infection, but no rickettsemia during subsequent expositions, when capybaras were immune to R. rickettsii. During the primary and subsequent infections, capybaras were also infested with uninfected A. sculptum ticks. These infestations compose the present study, which aimed to evaluate: (i) if either rickettsemic or non-rickettsemic capybaras could serve as infection sources of R. rickettsii for A. sculptum larvae and nymphs; (ii) the vector competence of the subsequent nymphs and adults (molted from the engorged ticks collected from capybaras); and (iii) if there were R. rickettsii-acquisition by uninfected ticks co-feeding with infected ticks on immune capybaras (without rickettsemia). Through experimental infection of capybaras with R. rickettsii via tick feeding, simulating a natural condition, we demonstrated that primarily infected capybaras developed rickettsemia that resulted in successful acquisition feeding of larvae and nymphs of A. sculptum, since part of these ticks maintained R. rickettsii transstadially, and the resultant molted ticks (either nymphs or adults) successfully transmitted the bacterium by feeding on susceptible rabbits. Contrastingly, all rabbit infestations with ticks derived from acquisition feeding on R. rickettsii-immune capybaras (including when acquisition ticks fed in direct contact with donor ticks) evidenced absence of R. rickettsii transmission due to lack of clinical signs and antibody response in those rabbits. Our results indicate that capybaras could serve as R. rickettsii-amplifying hosts for A. sculptum ticks only during the capybara’s primary infection, but not during subsequent infections on immune capybaras. Finally, the probable co-feeding nonsystemic transmission of R. rickettsii seems to be irrelevant in the context of BSF epidemiology, in areas where capybaras are incriminated as main amplifying hosts of R. rickettsii for A. sculptum ticks.

Wild carnivores, domestic dogs and ticks: shared parasitism in the Brazilian Cerrado.

The increase of contact between natural and rural areas is prominent in Brazil, due to agricultural activities and concern with the environmental conservation. In this context, domestic animals, wild fauna and humans are exposed to mutual exchange of parasites, microorganisms and diseases. We studied tick parasitism of wild carnivores and domestic dogs, and the environmental of questing ticks, in extensive cattle ranch areas intermingled with natural vegetation, and in a natural reserve, both in a region of Cerrado biome, Midwestern Brazil. From 2008 to 2015, we inspected 119 wild carnivores from nine species, and collected six tick species (Amblyomma sculptum, Amblyomma ovale, Amblyomma dubitatum, Amblyomma tigrinum, Dermacentor nitens and Rhipicephalus microplus). The most numerous and infested hosts were Cerdocyon thous, Lycalopex vetulus, Chrysocyon brachyurus, Puma concolor and Conepatus amazonicus. From 139 domestic dogs, we collected A. sculptum, Rhipicephalus sanguineus and R. microplus. From vegetation, samplings resulted in A. sculptum, A. dubitatum, A. ovale, Amblyomma rotundatum and R. microplus, with dominance of A. sculptum. Domestics and wild animals presented high overlapping of infestations by A. sculptum, a generalist and anthropophilic tick species. This tick is the most important vector of the Brazilian spotted fever, a lethal human disease. This fact elicits attention and requires efforts to monitor the presence of pathogens vectored by ticks circulating in this type of agroecosystem, including in other regions of the Brazil, because the most of the natural vegetation remaining have been increasingly immersed in pastures and agricultural matrix.

Clinical and serological evaluation of capybaras (Hydrochoerus hydrochaeris) successively exposed to an Amblyomma sculptum-derived strain of Rickettsia rickettsii

Brazilian spotted fever (BSF), caused by Rickettsia rickettsii, is the most lethal tick-borne disease in the western hemisphere. In Brazil, Amblyomma sculptum ticks are the main vector. Capybaras (Hydrochoerus hydrochaeris), the largest living rodents of the world (adults weighing up to 100 Kg), have been recognized as amplifying hosts of R. rickettsii for A. sculptum in BSF-endemic areas; i.e., once primarily infected, capybaras develop bacteremia for a few days, when feeding ticks acquire rickettsial infection. We conducted experimental infections of five capybaras with an A. sculptum-derived strain of R. rickettsii and performed clinical and bacteremia evaluation during primary and subsequent infections. Bacteremia was detected in all capybaras during primary infection, but not in subsequent infections. All animals seroconverted to R. rickettsii (titres range: 64–32,768), and remained seropositive throughout the study. Primary infection resulted in clinical spotted fever illness in four capybaras, of which two had a fatal outcome. Subsequent infections in seropositive capybaras resulted in no clinical signs. Capybaras developed a sustained immune response that prevented a second bacteremia. This condition may imply a high reproduction rate of capybaras in BSF-endemic areas, in order to continuously generate capybaras susceptible to bacteremia during primary infection.

Serological survey of Rickettsia in equids from Vale do Paraíba, São Paulo, Brazil, and their tick identification and molecular investigation of Rickettsia

Brazilian spotted fever is a serious and lethal illness for humans and is caused by the Rickettsia rickettsii bacteria. In the state of São Paulo/SP (Brazil), the etiological agent of this disease is transmitted by the Amblyomma sculptum tick. It was already shown that horses infected with this bacteria produce a strong immune response and could be important sentinels for the detection of the disease in a proper region. The present investigation performed a serological survey in horses from five farms of Vale do Paraíba, São Paulo state, Brazil, searching for antibodies against, Rickettsia rickettsii, Rickettsia parkeri, Rickettsia amblyommatis, Rickettsia rhipicephali, and Rickettsia bellii. In each farm, ticks were also collected that were taxonomically identified and examined by real-time PCR for Rickettsia spp DNA. Blood samples were collected from 206 horses, and 334 ticks were picked up from these animals from January to December 2017. Eighty ticks wereA. sculptum and 254 Dermacentor nitens. Of the blood samples, 7.3% seroconverted to Rickettsia spp. Of these, 0.97% had a positive serological response to R. bellii. None of the 80 A. sculptum ticks were positive through real-time PCR for Rickettsia spp. Although there was no detection of ticks infected by Rickettsia spp in five farms of Paraíba Valley, the horses presented serological positive reactions against this agent. Thus, further large studies should be conducted in the area targeting hosts and vectors to generate data for control measures of the transmission of Brazilian spotted fever.

Rickettsia rickettsii (Rickettsiales: Rickettsiaceae) Infecting Amblyomma sculptum (Acari: Ixodidae) Ticks and Capybaras in a Brazilian Spotted Fever-Endemic Area of Brazil.

The bacterium Rickettsia rickettsii is the agent of Brazilian spotted fever (BSF), a highly fatal disease that is transmitted in Brazil mainly by the tick Amblyomma sculptum Berlese, which uses capybaras (Hydrochoerus hydrochaeris Linnaeus) as major hosts. In 2015, we captured nine capybaras in a BSF-endemic area of southeastern Brazil. From each capybara, we collected blood sera that were tested through the immunofluorescence assay using Rickettsia spp. antigens, and A. sculptum ticks, processed for isolation of R. rickettsii through guinea pig inoculation. All capybaras (100%) were seroreactive to Rickettsia spp., with highest titers to R. rickettsii. A total of 166 A. sculptum ticks were macerated and inoculated into nine guinea pigs, from which only one presented high fever and seroconverted to R. rickettsii. Blood from this febrile animal was inoculated into additional guinea pigs (guinea pig passages), which also became febrile and seroconverted, or when euthanized during the fever period, their internal organs (spleen, lung) were shown to contain R. rickettsii DNA. The present rickettsial isolate has been maintained cryopreserved as infected guinea pig organs. There was at least one R. rickettsii-infected tick among the 166 macerated ticks, giving a minimal infection rate of 0.6% (1/166). This infection rate is within the range of previous studies, which reported that only 0.05% to at most 1.28% A. sculptum ticks were infected by R. rickettsii in other BSF-endemic areas. These low infection rates support the low incidence of BSF, despite of A. sculptum being the most frequent human-biting tick in southeastern Brazil.

Species richness and seasonal dynamics of ticks with notes on rickettsial infection in a Natural Park of the Cerrado biome in Brazil

This study evaluated the species richness and seasonal dynamics of ticks and rickettsial agents infecting ticks in the largest natural Reserve of the Cerrado biome of Brazil, the Grande Sertão Veredas National Park. During 2012–2014, a total of 9531 host-seeking ticks were collected by dry ice traps and dragging, whereas 1563 ticks were collected from small mammals, and 1186 ticks from domestic animals. Overall, the following 12 tick species were identified: Amblyomma auricularium, Amblyomma dubitatum, Amblyomma naponense, Amblyomma ovale, Amblyomma parvum, Amblyomma sculptum, Amblyomma tigrinum, Amblyomma triste, Dermacentor nitens, Rhipicephalus microplus, Rhipicephalus sanguineus sensu lato, and Ornithodoros mimon. The three most abundant tick species, A. sculptum, A. parvum, and A. triste, are likely to develop one generation per year, with adults predominating between spring and autumn, and immature ticks during autumn-winter. Small mammals seem to be important hosts for immature stages of A. parvum, and A. triste, but not for A. sculptum. Molecular analyses revealed the presence of the human pathogen Rickettsia parkeri in 10% of the A. triste ticks, whereas two agents of unknown pathogenicity, Rickettsia bellii and ‘Candidatus Rickettsia andeanae’ were found in 7 and 5%, respectively, of the A. parvum ticks. A fourth rickettsial agent, Rickettsia amblyommatis, was found in a single A. sculptum tick. Several Vero cell-established isolates of R. parkeri and R. bellii were obtained from A. triste and A. parvum, respectively. Serological analyses of small mammals suggest that they have been infected by R. parkeri and R. bellii, possibly via natural infestations by A. triste and A. parvum, respectively. Because the Park has suffered low anthropic alterations, our results should provide baseline data that shall be used for future comparisons with other Cerrado areas with higher degree of anthropic changes.

Ticks as potential vectors of Mycobacterium leprae: Use of tick cell lines to culture the bacilli and generate transgenic strains.

Leprosy is an infectious disease caused by Mycobacterium leprae and frequently resulting in irreversible deformities and disabilities. Ticks play an important role in infectious disease transmission due to their low host specificity, worldwide distribution, and the biological ability to support transovarial transmission of a wide spectrum of pathogens, including viruses, bacteria and protozoa. To investigate a possible role for ticks as vectors of leprosy, we assessed transovarial transmission of M. leprae in artificially-fed adult female Amblyomma sculptum ticks, and infection and growth of M. leprae in tick cell lines. Our results revealed M. leprae RNA and antigens persisting in the midgut and present in the ovaries of adult female A. sculptum at least 2 days after oral infection, and present in their progeny (eggs and larvae), which demonstrates the occurrence of transovarial transmission of this pathogen. Infected tick larvae were able to inoculate viable bacilli during blood-feeding on a rabbit. Moreover, following inoculation with M. leprae, the Ixodes scapularis embryo-derived tick cell line IDE8 supported a detectable increase in the number of bacilli for at least 20 days, presenting a doubling time of approximately 12 days. As far as we know, this is the first in vitro cellular system able to promote growth of M. leprae. Finally, we successfully transformed a clinical M. leprae isolate by inserting the reporter plasmid pCHERRY3; transformed bacteria infected and grew in IDE8 cells over a 2-month period. Taken together, our data not only support the hypothesis that ticks may have the potential to act as a reservoir and/or vector of leprosy, but also suggest the feasibility of technological development of tick cell lines as a tool for large-scale production of M. leprae bacteria, as well as describing for the first time a method for their transformation.

Seasonal dynamics, geographical range size, hosts, genetic diversity and phylogeography of Amblyomma sculptum in Argentina

The aim of this work was to generate knowledge on ecological aspects of Amblyomma sculptum in Argentina, such as seasonal dynamics, geographical range size, hosts, genetic diversity and phylogeography. Adult and immature A. sculptum ticks were collected in different localities of Argentina to know the geographical range size and hosts. The genetic diversity of this tick was studied through analyses of 16S rDNA sequences. To describe the seasonal dynamics, free-living ticks were monthly collected from October 2013 to October 2015. A. sculptum shows a marked ecological preference for Chaco Húmedo eco-region and “Albardones” forest of the great rivers in the wetlands in the Chaco Biogeographical Province, and for Selvas Pedemontanas and Selva Montana in the Yungas Biogeographical Province. This species has low host specificity, and it has large wild and domestic mammals as principal hosts to both immature and adult stages. Amblyomma sculptum is characterized by a one-year life cycle. Larvae peak in early winter, nymphs peaked during mid-spring, and adults during late summer and mid-summer. The genetic divergence was low and the total genetic variability was attributable to differences among populations. This fact could be associated to stochastics process linked to micro-habitat variations that could produce a partial restriction to gene flow among populations. The geographic regions do not contribute much to explain the A. sculptum population genetic structure, with an ancestral haplotype present in most populations, which gives rise to the rest of the haplotypes denoting a rapid population expansion.

Anti-neoplastic activity of Amblyomma sculptum, Amblyomma parvum and Rhipicephalus sanguineus tick saliva on breast tumor cell lines

Cancer is one of the most troubling diseases and is becoming increasingly common. Breast cancer has a high cure rate when diagnosed early, but when diagnosed late, treatment is frequently painful, devastating and unsuccessful. The search for new treatments that are more effective and less harmful has led to several substances and biomolecules from plants and animals with potential anti-tumor activity. Within this context, ticks have emerged as an excellent source of new molecules with a wide array of therapeutic properties. Various molecules in tick saliva have immunomodulatory, anticoagulant, anti-inflammatory and anti-tumor effects across different tumor cell lines. Our study evaluates the effect of saliva from three widespread and important tick species in Brazil (Amblyomma sculptum, Amblyomma parvum and Rhipicephalus sanguineus) on MCF-7, MDA-MB-231 breast cancer cell lines and on the non-neoplastic MCF-10A cell line. We found that tick saliva from all three tick species showed cytotoxicity to tumor cells (MCF-7, MDA-MB-231) but not to the non-tumor cells (MCF-10A). Morphological changes on the surface of MCF-7 and MDA-MB-231 tumor cells did not occur on the MCF-10A cells. We also demonstrated that tumor cells die by apoptosis induced by caspase-3 and caspase 7 activity, suggesting that intrinsic pathway apoptosis may be triggered by tick saliva. These changes were not observed in MCF10A cells, which remained broadly unchanged even after exposure to diverse types of saliva. These results suggest that tick saliva from these tick species is a source of molecules, or biomolecules, useful for the potential source for the development of new breast cancer drugs.

Internalization and intracellular trafficking of an antitumor molecule

Amblyomin‐X is a proteasome inhibitor that was identified through the transcriptome analysis of the salivary gland from Amblyomma sculptum tick. This recombinant protein triggers apoptosis in different tumor cell lines, and promotes regression of tumor growth and reduction of metastasis. Recent studies have indicated endocytosis mechanism could be associated with pro‐apoptotic activity of this protein. Here, by using confocal microscopy and high content analysis (ImageXpress Micro Confocal High‐Content, Molecular Devices), we found that Amblyomin‐X (Alexa Fluor 488 conjuaged) preferentially co‐localize with Alexa Fluor 555 Cholera Toxin Subunit B, caveolin‐1 and 19S proteasome subunit in human tumor cells (SK‐MEL‐28 and MIA PaCa‐2). Remarkably, we identified Amblyomin‐X co‐immunoprecipitates with caveolin‐1 and Rab‐11. Also, we observed an alteration in Ambyomin‐X trafficking in human tumor cells transfected with GFP‐Rab5 mutants (Rab5:Q79L and Rab5:N133I). Taken all results together, we can suggest that Amblyomin‐X internalization occurs via lipid raft pathway and intracellular trafficking involved of caveosomes and early and recycling endosomes. Then, Amblyomin‐X reaches proteasome system, a possible intracellular destination.Support or Funding InformationSupported by CAPES, Fapesp, BNDES and União Química FarmacêuticaThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

C‐terminal domain is responsible for a Kunitz‐type inhibitor uptake by tumor cells

Amblyomin‐X is a recombinant Kunitz‐type FXa inhibitor that was identified through the transcriptome analysis of the salivary gland from Amblyomma sculptum tick. This protein triggers apoptosis in different tumor cell lines, and promotes regression of tumor growth and reduction of metastasis. In this regard, recently arose question about it structure‐fuction. Thereby, we investigated the effects of two truncated (synthetic) regions of Amblyomin‐X, which we termed Kunitz and C‐terminal domains. Here, using MTT and FXa chromogenic activity assay, we found that both domains have no cytotoxicity in human tumor cells (SK‐MEL‐ 28 and MIA PaCa‐2) and only Kunitz domain inhibit FXa slightly. Also, through confocal microscopy and high content analysis (ImageXpress Micro Confocal High‐Content, Molecular Devices), we observed C‐terminal domains (Alexa Fluor 488 conjugated) uptake by tumor cells previously than Amblyomin‐X (whole protein), but with similar intracellular trafficking and negligible nuclear translocation. Besides, circular dichroism analysis shown that Kunitz domain has quite a structure of α‐helix and β‐sheet, whereas the C‐terminal domain has the great part of random structure. Taken all results together, we can suggest that the C‐terminal domain is responsible for Amblyomin‐X uptake and could be an unusual cell penetrating peptide.Support or Funding InformationSupported by BNDES, Fapesp and União Química FarmacêuticaThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.