Search

Opossums are synanthropic marsupials able to interchange among wild, peri‑urban and urban environments, playing an epidemiologically important role as hosts for emerging pathogens and ectoparasites of relevance in public health. The present study aimed to detect and molecularly characterize vector-borne agents in a population of common opossums (Didelphis marsupialis) from the Island of São Luís do Maranhão, northeastern Brazil. Of the 45 animals analyzed, one (2.22%) was positive in the nested PCR assay based on the 18S rRNA gene of piroplasmids. The obtained sequence was phylogenetically positioned in a clade containing sequences of Babesia sp. previously detected in Didelphis aurita, Didelphis albiventris and associated ticks from Brazil. Eight (17.77%) samples were positive in PCR for Ehrlichia spp. based on the dsb gene; four samples were sequenced and positioned into a new clade, sister to E. minasensis and Ehrlichia sp. clade detected in Superorder Xenarthra mammals. No samples tested positive in the screening PCR assays based on the 16S rRNA gene of Anaplasma spp. Two samples were positive in the qPCR for Bartonella spp. based on the nuoG gene. Seven animals (15.56%) were positive in the nPCR based on the 16S rRNA gene of hemoplasmas. Of these, three were positive in a PCR based on the 23S rRNA gene. The phylogenies based on both 16S rRNA and 23S rRNA genes corroborated to each other and positioned the sequences in the same clade of hemoplasmas previously detected in D. aurita and D. albiventris sampled in Brazil. Finally, three (6.66%) animals were positive in the PCR for Hepatozoon spp.; the obtained 18S rRNA sequence was positioned into the H. felis clade.The present study showed, for the first time, the circulation of piroplasmids, Hepatozoon spp., Ehrlichia spp., hemoplasmas and Bartonella spp. in D. marsupialis sampled in northeastern Brazil, with description of putative novel genotypes of Ehrlichia and Hepatozoon and copositivity by different vector-borne agents. The present work consolidates the "South American Marsupialia" piroplasmid clade, adding one more genotype of Babesia sp. to this clade., Competing Interests: Declaration of Competing Interest The authors declared no conflict of interest. The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:, (Copyright © 2023 Elsevier B.V. All rights reserved.)

Ticks are vectors for several pathogens, including bacteria belonging to the Rickettsia genus, such as Rickettsia rickettsii and Rickettsia parkeri, the causative agents of spotted fever. The aim of the present study was to investigate the tick species richness and rickettsial agents associated with wild birds captured in the Humaita Forest Reserve, Acre, in the Western Amazon region. Wild birds were captured with ornithological nets for visual inspection with the purpose of collecting ticks, which were identified through morphological analyses and molecular tests for several genes (12S rDNA, 16S rDNA, gltA, ompA, and sca4). A total of 607 wild birds were captured, 12% of which were parasitized by 268 ticks of the Amblyomma genus, with new host-parasite associations reported for Amblyomma calcaratum, Amblyomma geayi, Amblyomma longirostre, Amblyomma naponense, Amblyomma nodosum, and Amblyomma varium. Of the total ticks collected, 113 were tested for the presence of rickettsial DNA fragments, with 19 testing positive for R. parkeri in A. geayi, Rickettsia tamurae-like in Amblyomma sp., and Rickettsia amblyommatis in A. geayi, A. longirostre, and Amblyomma sp. We detected R. tamurae-like in Amblyomma larvae for the first time in the Western Brazilian Amazon biome, and registered spotted fever group rickettsiae, although the relevance of the detected species in a public health context should be further explored in South America, as well as new host-parasite interactions in this underexplored region., Competing Interests: Declaration of Competing Interest The authors declare that there is no conflict of interest regarding the publication of this paper., (Copyright © 2023 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Visceral Leishmaniasis is a serious public health problem and dogs are considered to be the main source of infection in urban areas. In Brazil, this disease is present in all regions, but with high concentration of cases in the Northeast, and the state of Maranhão is considered to be an endemic region. The aim of this study was to conduct an epidemiological, spatial, molecular and serological survey on Leishmania infantum among domestic dogs in the municipality of Belágua, Maranhão. Blood samples were collected from dogs and questionnaires were applied to their owners to obtain epidemiological data and risk factors relating to this zoonosis in the region. The coordinates of the dogs' homes were obtained to produce a disease risk map. Serological diagnoses were made using the indirect immunofluorescence reaction (IFAT) and the dual-path platform chromatographic immunoassay test (DPP®) (Bio-Manguinhos/FIOCRUZ, Brazil). A molecular investigation was undertaken using the polymerase chain reaction (PCR). Georeferencing was performed using the global positioning system (GPS) and cases of canine visceral leishmaniasis in the municipality were spatially represented and analyzed using QGIS version 3.16.6 (QGIS Development Team, 2021). A total of 205 blood samples were collected, of which 122 (59.51%) were seroreactive for L. infantum through IFAT, while the DPP test showed 84 reactive samples (40.97%). IFAT and DPP detected 16 positive animals simultaneously. One sample that was seroreactive through IFAT was also positive through PCR. In the clinical evaluation, it was observed that among the seropositive dogs, 112 (91.80%) were symptomatic and 10 (8.20%) were asymptomatic. In the spatial analysis, the Kernel density estimator enabled determination of the place at greatest risk of occurrence of the disease. The areas with the highest concentrations of cases were in districts with large quantities of precarious housing and lack of basic sanitation. This was the first report on the occurrence of L. infantum among dogs in the municipality of Belágua. The results show that canine visceral leishmaniasis is well dispersed in this municipality, thus putting at risk the human population., Competing Interests: Declaration of Competing Interest None., (Copyright © 2023. Published by Elsevier B.V.)

Platynosomosis is a parasitic disease caused by a trematode of the genus Platynosomum, a bile duct and gallbladder fluke that has been described in captive neotropical primates (New World primates; NWPs) and causes high morbidity and variable mortality. Although it is a major concern for ex-situ conservation of these animals, there are only a few studies of platynosomosis in free-ranging NWPs. Therefore, the aim of this study was to characterize platynosomosis in a free-ranging population of marmosets (Callithrix spp) from the Brazilian Atlantic Forest, focusing on the epidemiological and pathological aspects of the disease. A total of 1,001 marmosets were evaluated and on the basis of clinicoepidemiological data, histopathology, histochemistry and immunohistochemistry, we concluded that Platynosomum spp infection has a prevalence of 8.9% (confidence interval: 7.3-10.8%) in free-ranging marmosets, with a higher frequency in the Metropolitan Region of Rio de Janeiro. Infection was associated with fibrosing and proliferative cholangiohepatitis associated with biliary lithiasis (3.0% of cases) and secondary bacterial infections (14.6% of cases)., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Following decades of riparian buffer zone (RBZ) studies there remains a need to look across individual site data for collective evidence on the site-specific pollution mitigation and river water quality. We explored primary study evidence on runoff, sediment, P, N, coliforms and pesticides using complimentary styles of metadata interpretation. A quantitative assessment of pollution retention (75 studies, 474 data rows) derived relationships for retention versus width, including significant covariates of clay particle size and buffer slope for sediment, total and dissolved P. Total N and coliforms related to texture and slope but were independent of width. Other factors across pollutants were inconsistently reported. With limitations on quantitative studies a second approach examined factor significance (formal testing versus inferred; 93 studies) across source pressure, transport/physical, vegetation and soil biogeochemical factors on pollution effectiveness. The RBZ evidence showed considerable disagreement and bias in shorter term study implications on longer-term processes. Screening for stronger evidence by study number and agreement left fifteen factors informing on at least one pollutant, whereas only rainfall intensity, preferential deposition, tree planting, soil infiltration remained addressing three or more pollutants. Key messages were that: data complexities, from short-term trapping in upper buffer edges and so-called 'negative effectiveness' associated with internal recycling and/or errors in constraining mass inputs for dissolved pollutant and subsurface transport require careful interpretation; RBZ intervention and study durations were limited compared to effect times (particularly vegetation management and changing soil conditions); factors affect pollutants with particulate and dissolved phases differently and must be understood to limit RBZ pollution swapping. Buffer functioning is highly site-specific. To understand this better attention should be given to revisiting studies of vegetation management to extend timeframes, wider study of belowground (soil biogeochemical and transport) processes and studies should document site contexts across source pressures, riparian hydrological, soil and vegetation factors., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Trypanosoma cruzi, the etiological agent for Chagas disease, is widely distributed in the Americas. Its hosts are humans and wild and domestic mammals, and its vectors are triatomine insects. Studies have indicated that domestic dogs are sentinel animals in the epidemiology of Chagas disease in endemic regions, including states in the Legal Amazon region of Brazil. In São Luís, the capital of Maranhão, a non-endemic state, the existence of a domestic cycle involving domestic rats has been proven, along with a wild cycle maintained by didelphids. However, no studies on T. cruzi infection in domestic animals in this locality have been conducted. The aim of this study was to investigate occurrence of T. cruzi in dogs living in the Itaqui Bacanga district of São Luís, Maranhão, by means of serological and molecular tests. Blood samples were obtained from 330 dogs and structured epidemiological questionnaires were applied to their keepers. These samples were used in the indirect immunofluorescent antibody test (IFAT), enzyme-linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR). Fisher's exact test was used for statistical calculations with the aim of identifying risk factors. Out of the 330 animals, 105 (31.8%) were reactive in IFAT, 46 (13.0%) in ELISA and 20 (6.0%) in both serological tests. The results were not significant (p > 0.05) when submitted to statistical analysis for the studied variables. From PCR, 58 samples (17.5%) were found to be positive and, of these, one (0.3%) showed similarity to T. cruzi after sequencing. These data demonstrate that dogs were exposed to and infected by T. cruzi. Thus, they can be considered sentinel animals for Chagas disease in the locality studied, which signals that there is a need for epidemiological surveillance actions., (Copyright © 2021 Elsevier B.V. All rights reserved.)

We studied communities of small mammals and their ticks in endemic (E) and non-endemic (NE) areas for Brazilian spotted fever (BSF), aiming to infer if diversity parameters of parasites and hosts could be related to occurrence and prevalence of rickettsial infection, especially Rickettsia rickettsii. We compared E and NE areas in human-modified landscapes (HMLs) and natural areas (BIO) with no report of BSF cases. Composition and equitability were important components of diversity explaining differences among areas. The marsupial Didelphis albiventris was dominant in HMLs, but not in natural areas, and this opossum was the main host for the tick Amblyomma sculptum, principal vector of R. rickettsii, especially in E areas. Communities of ticks were dominated by A. sculptum, followed by Amblyomma dubitatum in E areas. In NE areas, this dominance was inverted, with more A. dubitatum than A. sculptum infesting small mammals, but the numbers of ticks were much lower than in E areas. Composition and abundance of ticks in natural areas were very dissimilar from HMLs, with the lowest tick burdens. Didelphis albiventris in E areas presented higher seroprevalence and endpoint titres against R. rickettsii than in other areas. At least three Rickettsia species, non-pathogenic to humans, were detected in natural areas (Rickettsia bellii, Rickettsia amblyommatis and 'Candidatus Rickettsia andeanae'), and only one non-pathogenic species in HMLs (R. bellii). Our results suggest that higher diversity of ticks, hosts and rickettsiae could be relevant factors in buffering the effect in BSF occurrence. Particularly for D. albiventris, its importance has to be quantified in further studies considering the epidemiological scenario of BSF., (Copyright © 2021 Elsevier GmbH. All rights reserved.)

Worldwide herbicide use in agriculture, whilst safeguarding yields also presents water quality issues. Controlling factors in agricultural catchments include both static and dynamic parameters. The present study investigated the occurrence of herbicides in streams and groundwater in two meso-scale catchments with contrasting flow controls and agricultural landuse (grassland and arable land). Using a multi-dimensional approach, streams were monitored from November 2018 to November 2019 using Chemcatcher® passive sampling devices and groundwater was sampled in 95 private drinking water wells. The concentrations of herbicides were larger in the stream of the Grassland catchment (8.9-472.6 ng L -1 ) dominated by poorly drained soils than in the Arable catchment (0.9-169.1 ng L -1 ) dominated by well-drained soils. Incidental losses of herbicides during time of application and low flows in summer caused concentrations of MCPA, Fluroxypyr, Trichlorpyr, Clopyralid and Mecoprop to exceeded the European Union (EU) drinking water standard due to a lack of dilution. Herbicides were present in the stream throughout the year and the total mass load was higher in winter flows, suggesting a persistence of primary chemical residues in soil and sub-surface environments and restricted degradation. Losses of herbicides to the streams were source limited and influenced by hydrological conditions. Herbicides were detected in 38% of surveyed drinking water wells. While most areas had concentrations below the EU drinking water standard some areas with well-drained soils in the Grassland catchment, had concentrations exceeding recommendations. Individual wells had concentrations of Clopyralid (619 ng L -1 ) and Trichlorpyr (650 ng L -1 ). Despite the study areas not usually associated with herbicide pollution, and annual mass loads being comparatively low, many herbicides were present in both surface and groundwater, sometimes above the recommendations for drinking water. This whole catchment assessment provides a basis to develop collaborative measures to mitigate pollution of water by herbicides., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

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

The Brazilian state of Maranhão is located in a transition area of the Amazon and Cerrado biomes, where there is a rich fauna of vertebrates. This study aimed to update the list of the ticks occurring in Maranhão, through a compilation of literature records and examination of three tick collections, plus the addition of unpublished collections of ticks from road-killed animals during recent years. Our results indicate that the tick fauna of Maranhão includes 26 species: seven in the family Argasidae (Antricola guglielmonei, Argas miniatus, Ornithodoros cavernicolous, Ornithodoros hasei, Ornithodoros mimon, Ornithodoros rietcorreai and Ornithodoros rudis); and 19 in the family Ixodidae (Amblyomma auricularium-provisional, Amblyomma cajennense sensu stricto, Amblyomma calcaratum, Amblyomma dissimile, Amblyomma geayi, Amblyomma longirostre, Amblyomma naponense, Amblyomma nodosum, Amblyomma oblongoguttatum, Amblyomma ovale, Amblyomma parvum, Amblyomma rotundatum, Amblyomma sculptum, Amblyomma triste, Dermacentor nitens, Ixodes luciae, Haemaphysalis juxtakochi, Rhipicephalus microplus, and Rhipicephalus sanguineus sensu lato). Eleven of the above species are reported for the first time in Maranhão. We consider previous reports of Ornithodoros talaje and Amblyomma pseudoconcolor in Maranhão as misidentification with O. hasei and A. auricularium, respectively. Until 1958, only 11 tick species were reported in Maranhão, with no additional reports until 2009. During 2010-2019, 15 additional species were reported. We also highlight the importance of ticks for public and veterinary health in Maranhão., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Information about human tick bites in Brazil is mostly anecdotal. Published information is typically restricted to single tick infestation episodes and does not address human exposure occurring on a daily basis in natural, rural, or green urban areas. We present a comprehensive 2-yr study on human parasitism during a broad survey on ticks within a Brazilian savannah reserve. Overall, 439 tick bites were recorded from the following species: Amblyomma sculptum (n = 331 bites), Amblyomma parvum (n = 64), Amblyomma auricularium (n = 3), Rhipicephalus microplus (n = 2), Dermacentor nitens (n = 1) and 38 Amblyomma spp. ticks. Nymphs accounted for most tick bites (n = 292, 66.5 % of all bites) and these were overwhelmingly A. sculptum (92.8 % of nymphal bites). The main adult tick biting humans was A. parvum (n = 61, 50 % of all adult tick bites) followed closely by A. sculptum (n = 58, 47.5 % of all adult tick bites). Winter was the season with the highest percentage of tick bites (39.7 % of all bites), followed by spring (32.4 %); summer (18.9 %) and autumn (9 %). Amblyomma sculptum adult bites peaked in summer whereas nymphal bites occurred primarily in winter and spring. Amblyomma parvum adult bites peaked in spring and summer. The most common tick attachment sites included the waist, legs and belly, but A. parvum adults were recorded from the head of humans as well. A noteworthy observation was the lack of human parasitism by Amblyomma triste, the third most prevalent species in the environment. This tick species is a frequent human biter in both Uruguay and Argentina where it transmits a pathogen, Rickettsia parkeri, to humans., (Copyright © 2019 Elsevier GmbH. All rights reserved.)

Visceral leishmaniasis is a zoonotic disease caused by Leishmania infantum for which dogs are the main reservoir. In South America, presence of this disease is expanding along with increasing dispersion of its principal vector, the sand-fly Lutzomyia longipalpis. Feline leishmaniasis is an emerging disease in domestic cats, but epidemiological studies in endemic areas of the Amazon region of Brazil are scarce and the role of cats as reservoirs of L. infantum has been debated. The aim of this study was to investigate L. infantum infection in cats living in the Amazon biome region, using serological and molecular methods. A total of 105 cats were subjected to clinical examination and blood samples were taken for immunofluorescent-antibody (IFAT) serological evaluation, to determine anti-Leishmania antibody titers. Conventional PCR and Sanger's sequencing targeting L. infantum chitinase and Leishmania species ribosomal internal transcribed spacer (ITS-1) encoding genes were performed on conjunctival swabs from these cats. Seropositivity was detected in 32 animals (30.48%), thus confirming that contact between these cats and the parasite was occurring. PCR followed by amplicon sequencing showed that three samples (2.86%) were positive for a chitinase gene and six (5.71%) were positive for the ITS-1 gene. Parasite-positive diagnoses presented a statistically significant association with free access to the streets (p = 0.0111), cohabitation with dogs affected previously by VL (p = 0.0006) and absence of backyard cleaning and garbage collection (p = 0.00003). These results emphasize that cats should be included in epidemiological surveys of leishmaniasis, especially in endemic areas, if not as the reservoir host (unproven), at least as a "sentinel host" that is useful for revealing situations of endemic circulation of L. infantum. Moreover, in these areas, feline leishmaniasis needs to be considered in the differential diagnosis among domestic cats presenting alopecia, rarefied hair, lacerations and ulcerative dermatitis., (Copyright © 2019 Elsevier B.V. All rights reserved.)

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., (Copyright © 2018 Elsevier GmbH. All rights reserved.)

Mechanisms to detoxify aluminium (Al) is a hot topic for cultivated plants. However, little information is known about the mechanisms used by native plants to deal with Al-toxicity. In Cerrado, some generalist mistletoe species, such as Passovia ovata (Pohl ex DC.) Kuijt and Struthanthus polyanthus Mart. can parasitize Al-accumulating and Al-excluding plant species without any clear symptoms of toxicity and mineral deficiency, while Psittacanthus robustus (Mart.) Marloth, a more specialist mistletoe, seems to be an Al-dependent species, parasitizing only Al-accumulating hosts. Here we (i) characterized the forms and compartmentalization of Al in leaves of P. robustus; (ii) compared Ca and Al leaf concentration, and leaf concentration of organic acids and polyphenols between facultative Al-accumulating (P. ovata and S. polyanthus) and Al-dependent (P. robustus) mistletoe species infecting Miconia albicans (Sw.) Steud. (Al-accumulating species). P. robustus chelated Al 3+ with oxalate and stored it in the phloematic and epidermic leaf tissues. Leaf Ca and Al concentration did not differ among species. Leaf oxalate concentration was higher in the Al-dependent species. Concentrations of citrate and phenolic compounds were higher in the leaves of the facultative Al-accumulating species. These results show that facultative Al-accumulating and Al-dependent species use different mechanisms to detoxify Al. Moreover, this is the first report on a mistletoes species (P. robustus) with a potential calcifuge behaviour in Cerrado., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Rangelia vitalii is the etiologic agent of canine rangeliosis, a severe piroplasmosis that affects domestic dogs in Brazil, Uruguay and Argentina. While R. vitalii is one of the most pathogenic tick-borne pathogens for dogs in the world, its tick vector has remained unknown. The present study evaluated the vector competence of Rhipicephalus sanguineus sensu lato (both tropical and temperate species), Amblyomma aureolatum, Amblyomma ovale, Amblyomma tigrinum, and Amblyomma sculptum for R. vitalii. These six tick species were selected for the study because they comprise the main tick species infesting dogs within the distribution area of canine rangeliosis in South America. Acquisition feeding of the above six tick species was performed on domestic dogs showing clinical signs of canine rangeliosis, after being experimentally infected through intravenous inoculation or infestation with R. vitalii-infected ticks. Thereafter, engorged ticks were evaluated for transstadial and transovarial passages of R. vitalii through molecular analysis after molting or oviposition and egg hatching. The resultant ticks were evaluated for their competence to transmit R. vitalii to susceptible dogs. Among the six tick species, only A. aureolatum was able to acquire and perpetuate R. vitalii by transstadial and transovarial passages, as demonstrated by >5% infection rates of ticks after hatching or molting. When exposed to transmission feeding, only A. aureolatum ticks were competent to transmit R. vitalii to dogs, which became severely ill, and the results confirmed by molecular methods and blood smear examination to have acquired rangeliosis. Results of the present study, coupled with epidemiological data, indicate that A. aureolatum is a natural vector of R. vitalii. Our results also indicate that R. vitalii is the first Piroplasmorida agent to be transovarially transmitted in Amblyomma ticks., (Copyright © 2018 Elsevier GmbH. All rights reserved.)

Vernonia sensu lato is the largest and most complex genus of the tribe Vernonieae (Asteraceae). The tribe is chemically characterized by the presence of sesquiterpene lactones and flavonoids. Over the years, several taxonomic classifications have been proposed for Vernonia s.l. and for the tribe; however, there has been no consensus among the researches. According to traditional classification, Vernonia s.l. comprises more than 1000 species divided into sections, subsections and series (sensu Bentham). In a more recent classification, these species have been segregated into other genera and some subtribes were proposed, while the genus Vernonia sensu stricto was restricted to 22 species distributed mainly in North America (sensu Robinson). In this study, species from the subtribes Vernoniinae, Lepidaploinae and Rolandrinae were analyzed by UHPLC-UV-HRMS followed by multivariate statistical analysis. Data mining was performed using unsupervised (HCA and PCA) and supervised methods (OPLS-DA). The HCA showed the segregation of the species into four main groups. Comparing the HCA with taxonomical classifications of Vernonieae, we observed that the groups of the dendogram, based on metabolic profiling, were in accordance with the generic classification proposed by Robinson and with previous phylogenetic studies. The species of the genera Stenocephalum, Stilpnopappus, Strophopappus and Rolandra (Group 1) were revealed to be more related to the species of the genus Vernonanthura (Group 2), while the genera Cyrtocymura, Chrysolaena and Echinocoryne (Group 3) were chemically more similar to the genera Lessingianthus and Lepidaploa (Group 4). These findings indicated that the subtribes Vernoniinae and Lepidaploinae are non-chemically homogeneous groups and highlighted the application of untargeted metabolomic tools for taxonomy and as indicators of species evolution. Discriminant compounds for the groups obtained by OPLS-DA were determined. Groups 1 and 2 were characterized by the presence of 3',4'-dimethoxyluteolin, glaucolide A and 8-tigloyloxyglaucolide A. The species of Groups 3 and 4 were characterized by the presence of putative acacetin 7-O-rutinoside and glaucolide B. Therefore, untargeted metabolomic approach combined with multivariate statistical analysis, as proposed herein, allowed the identification of potential chemotaxonomic markers, helping in the taxonomic classifications., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

In South America, early reports from more than 50 years ago incriminated Ornithodoros rudis as vector of Borrelia venezuelensis, an agent of tick-borne relapsing fever (TBRF). Herein we report the rediscovery of O. rudis by means of morphological, biological and molecular analyses, which also comprise the first report of this tick species in Brazil. Phylogenetic analysis using partial fragments of mitochondrial 16S rRNA gene suggested that O. rudis forms a monophyletic group with Ornithodoros erraticus. By using laboratory rodents as hosts, we isolated a relapsing fever Borrelia from an infected O. rudis female. Phylogenetic analysis inferred from the rrs, flaB, and glpQ genes of Borrelia spp. placed the spirochete harbored by O. rudis closely related to Borrelia turicatae. Until further genetic evidence is not obtained we are referring to this O. rudis spirochete as B. venezuelensis. This is the first in vitro isolation of a TBRF Borrelia from South America. The presence of O. rudis in Brazil should not be overlooked, since this tick has been historically implicated in human cases of TBRF in Colombia, Panama, and Venezuela. This study provides new reports of O. rudis and B. venezuelensis after decades of scientific silence on these agents., (Copyright © 2018 Elsevier GmbH. All rights reserved.)

The genus Rickettsia comprises obligatory intracellular bacteria, well known to cause zoonotic diseases around the world. The present work aimed to investigate the occurrence of Rickettsia spp. in wild animals, domestic dogs and their respective ectoparasites in southern Pantanal region, central-western Brazil, by molecular and serological techniques. Between August 2013 and March 2015, serum, whole blood and/or spleen samples were collected from 31 coatis, 78 crab-eating foxes, seven ocelots, 42 dogs, 110 wild rodents, and 30 marsupials. Serum samples from canids, felids, rodents and marsupials were individually tested by indirect fluorescent antibody test (IFAT) in order to detect IgG antibodies to Rickettsia rickettsii, Rickettsia parkeri and Rickettsia amblyommatis. DNA samples from mammals and ectoparasites were submitted to a multiplex qPCR assay in order to detect and quantify spotted fever group (SFG) and typhus group (TG) rickettsiae and Orientia tsutsugamushi. Positive samples in qPCR assays were submitted to conventional PCR assays targeting gltA, ompA, ompB and htrA genes, followed by sequencing and phylogenetic analyses. The ticks collected (1582) from animals belonged to the species Amblyomma sculptum, Amblyomma parvum, Amblyomma ovale, Amblyomma tigrinum, Rhipicephalus (Boophilus) microplus, Rhipicephalus sanguineus sensu lato and Amblyomma auricularium. Overall, 27 (64.2%) dogs, 59 (75.6%) crab-eating foxes and six (85.7%) ocelots were seroreactive (titer≥64) to at least one Rickettsia species. For 17 (40.4%) dogs, 33 (42.3%) crab-eating foxes, and two (33.3%) ocelots, homologous reactions to R. amblyommatis or a closely related organism were suggested. One hundred and sixteen (23.5%) tick samples and one (1.2%) crab-eating fox blood sample showed positivity in qPCR assays for SFG Rickettsia spp. Among SFG Rickettsia-positive ticks samples, 93 (80.2%) belonged to A. parvum, 14 (12%) belonged to A. sculptum species, three (2.5%) belonged to A. auricularim, and six (5.2%) were Amblyomma larval pools. Thirty samples out of 117 qPCR positive samples for SFG Rickettsia spp. also showed positivity in cPCR assays based on gltA, htrA and/or ompB genes. The Blast analyses showed 100% identity with 'Candidatus Rickettsia andeanae' in all 30 sequences obtained from gltA, htrA and/or ompB genes. The concatenated phylogenetic analysis based on gltA and 17-kDa htrA genes grouped the Rickettsia sequences obtained from tick samples in the same clade of 'Candidatus Rickettsia andeanae'. The present study revealed that wild and domestic animals in southern Pantanal region, Brazil, are exposed to SFG rickettsiae agents. Future studies regarding the pathogenicity of these agents are necessary in order to prevent human cases of rickettsiosis in Brazilian southern Pantanal., (Copyright © 2017 Elsevier GmbH. All rights reserved.)

The present study was performed in a non-endemic area for spotted fever (SF) in Imperatriz microregion, state of Maranhão, Brazil. Blood samples and ectoparasites were collected from 300 dogs of the Imperatriz microregion. Canine serum samples were tested individually by indirect immunofluorescence assay (IFA), using five Rickettsia isolates from Brazil. Antibodies reactive to at least one of the five species of Rickettsia were detected in 1.6% of the dogs (5/300). These sera were considered reactive to Rickettsia rickettsii and Rickettsia amblyommatis or very closely related species. The ticks (Acari: Ixodidae), identified as Rhipicephalus sanguineus sensu lato (Latreille), and the fleas, identified as Ctenocephalides felis, were tested by polymerase chain reaction (PCR) for detection of rickettsial DNA. More than 78% (83/106) of the C. felis fleas were found to be infected with Rickettsia species using gltA as rickettsial PCR targets, whereas no evidence of Rickettsia spp. was found in R. sanguineus s. l. Genetic analysis based on genes gltA, htrA and ompB showed that the detected strain, is most closely related to Rickettsia asembonensis (formerly Candidatus Rickettsia asemboensis). The present study is the first report of a R. asembonensis related infecting C. felis fleas in Brazil., (Copyright © 2017 Elsevier B.V. All rights reserved.)

A novel rickettsial sequence in the citrate synthase gltA gene indicating a novel Rickettsia species has been detected in 7 out of 4524 Ixodes ricinus ticks examined within several surveys performed in the Czech Republic from 2005 to 2009. This new Candidatus Rickettsia sp. sequence has been found in 2 nymphs feeding on wild birds (Luscinia megarhynchos and Erithacus rubecula), in a male tick from vegetation, and 4 ticks feeding on a dog (3 males, 1 female tick). Portions of the ompA, ompB, sca4, and htrA genes were not amplifiable in these samples. A maximum likelihood tree of rickettsiae based on comparisons of partial amino acid sequences of citrate synthase and nucleotide sequences of 16S rDNA genes and phylogenetic analysis revealed a basal position of the novel species in the proximity of R. bellii and R. canadensis. The novel species has been named 'Candidatus Rickettsia mendelii' after the founder of genetics, Gregor Mendel., (Copyright © 2016 Elsevier GmbH. All rights reserved.)

Rickettsia parkeri rickettsiosis is a vector-borne zoonosis that occurs in some countries of the American continent. Following the first description and determination of the pathogenicity to humans in 2004 in USA, this bacterium has been reported in several South American countries. Human cases have been diagnosed in both Uruguay and Argentina in the past years. This study consisted in a serosurvey of 1000 domestic dogs living in the endemic area of rickettsiosis in Uruguay, where Amblyomma triste is the tick vector. Sera were analyzed by Indirect Immunofluorescence Assay (IFA), against antigens of three different rickettsial species: R. rhipicephali, R. felis and R. parkeri. It was determined that 20.3% of the dogs had antibodies that reacted to at least one of the three species tested, taking as cut off ≥64 titers. Furthermore, 140 of the seropositive dogs (14%) had a titer at least 4 times higher to R. parkeri than those of any of the other species, thus, it was considered that the immune response was stimulated by that species in particular. This is the first serological survey in primary hosts for adults of A. triste in Uruguay, and therefore the first prevalence values are reported. Adult A. triste ticks collected from the environment as well as from dogs were analyzed by PCR in order to confirm the current circulation of the agent in the area. In this matter, two out of 28 ticks from dogs, and 3 out of 53 ticks from the environment were positive, and the corresponding sequence analysis revealed 100% similarity with R. parkeri strain maculatum., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The aim of this study was to analyze the prevalence of rickettsiae in the tree-hole tick Ixodes arboricola in the Czech Republic and Slovakia. During May to September of 2009 and 2013, bird boxes belonging to three different areas were screened for ticks. In total, 454 nestlings and 109 nests of 10 hole-breeding bird species were examined. Ticks were found on Ficedula albicollis, Parus major, Cyanistes caeruleus and Sitta europaea and/or in their nests. In total, 166 ticks (17 nymphs, 10 males and 139 females) were found at 3 areas (arithmetic mean±standard error: 55.3±45.9). All ticks were tested for the presence of Rickettsia species by polymerase chain reaction targeting the rickettsial genes gltA, ompA, ompB and htrA and amplicon sequencing. All individuals except 3 nymphs were infected with 'Candidatus Rickettsia vini'. Multilocus sequence typing showed closest proximity to Rickettsia japonica and Rickettsia heilongjiangensis cluster. The presence of 'Ca. R. vini' is reported for the first time in Slovakia., (Copyright © 2015 Elsevier GmbH. All rights reserved.)

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., (Copyright © 2014 Elsevier GmbH. All rights reserved.)

Canine rangeliosis, caused by the piroplasmid protozoon Rangelia vitalii, is currently recognized as a reemerging disease that affects domestic dogs in Brazil. In the present study, piroplasmid infection was searched in wild canids (20 Cerdocyon thous and 4 Lycalopex gymnocercus) in Brazil. Molecular analysis, based on PCR and DNA sequencing of a portion of the 18S rRNA gene, revealed that 30% (6/20) C. thous were infected by R. vitalii. Blood and bone marrow samples from one of the R. vitalii-infected C. thous were inoculated into a domestic dog, which developed clinical rangeliosis that was confirmed by molecular tests. However, the C. thous donor showed no clinical, hematological or biochemical alterations, even though its R. vitalii infection status was confirmed for at least 80 days. These observations suggest that R. vitalii is not as highly pathogenic for C. thous as it is for domestic dogs. Phylogenetic analysis inferred by the 18S rRNA gene placed R. vitalii embedded in the clade 'Babesia sensu stricto', consisting of a number of species that represent truly the genus Babesia. It is proposed that the species R. vitalii should be transferred to the genus Babesia. The present study expands our knowledge on the natural history of R. vitalii, suggesting that it might have a natural cycle involving the wild canid C. thous. Further studies are needed to confirm that C. thous is a natural reservoir of R. vitalii in Brazil., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Biotransformation of chemicals by microorganisms can be effective in increasing chemical diversity. Some fungi have been described to be useful for the biotransformation of sesquiterpene lactones. Nevertheless, in most cases, only minor or simple transformations of functional groups have been observed. Budlein A is a sesquiterpene lactone found in high amounts in American sunflower-like species of the genus Viguiera (Asteraceae). It shows important biological effects like in vitro and in vivo anti-inflammatory activity, as well as cytotoxicity against cancer cell lines. With the aim to obtain potentially bioactive derivatives of budlein A and taking into account that obtaining semi-synthetic analogues is a very complex task, the capability of soil fungi to promote biotransformation was investigated. In this work, the biotransformation of budlein A by the soil fungi Aspergillus terreus and A. niger affording three unusual sesquiterpenoid derivatives with carbon skeletons is reported. The chemical structures of the compounds were elucidated by 1D and 2D NMR spectrometry and HR-ESI-MS. The stereochemistry and molecular conformation of one derivative was assessed by molecular modeling techniques. The fungal metabolites displayed a reduced cytotoxicity against HL-60 cells when compared to the original natural product. The results show the versatility of microbial-catalyzed biotransformations leading to unusual derivatives., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The CH(2)Cl(2) extract of aerial parts of Eupatorium perfoliatum L. exhibits antiprotozoal activity under in vitro conditions, especially against Plasmodium falciparum (IC(50)=2.7μg/ml). The search for active compounds yielded seven sesquiterpene lactones: Four structurally similar guaianolides, one dimeric guaianolide, and two germacranolides. The guaianolides differ in the degree of oxidation at C-14, ranging from a hydroxyl group up to a free carboxylic acid. The dimeric guaianolide, structurally closely related to the monomers, displays an unusual type of interguaianolide linkage between C-14 and C-4. Except for the germacranolide euperfolitin, all STLs described here were hitherto unknown. Furthermore, the flavonoid aglycones eupafolin, hispidulin, patuletin, and kaempferol were identified in the extract, which, except for kaempferol, have not been described as constituents of E. perfoliatum before. The dimeric guaianolide was shown to be the most active constituent against Plasmodium falciparum (IC(50) = 2.0μM) and was less cytotoxic against rat skeletal myoblasts (IC(50) = 16.2μM, selectivity index of about 8)., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Introduction: This ex vivo study used cone beam computed tomography to evaluate the amount of dentin removal from the distal wall of the mesial canal of human mandibular first molars caused by 4 instruments used to flare the cervical third., Methods: Thirty-two mesial roots were divided into 4 groups prepared by using ProTaper, K3, Gates-Glidden, or LA Axxess. The dentin thickness of the distal cervical wall of mesial canals was measured before and after the preparation by using computed tomography and Adobe Photoshop software., Results: There was no statistically significant difference between the study groups (P > 05)., Conclusions: All the instruments used for cervical preparation seemed to be safe and did not damage the dentin structure of the distal wall of mesial root canals of mandibular molars., (Copyright (c) 2010 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

The dichloromethane crude extract from the roots of Viguiera arenaria (VaDRE) has been employed in an antimicrobial screening against several bacteria responsible for human pathologies. The main diterpenes isolated from this extract, as well as two semi-synthetic pimarane derivatives, were also investigated for the pathogens that were significantly inhibited by the extract (MIC values lower than 100 microg mL(-1)). The VaDRE extract was significantly active only against Gram-positive microorganisms. The compounds ent-pimara-8(14),15-dien-19-oic acid (PA); PA sodium salt; ent-8(14),15-pimaradien-3beta-ol; ent-15-pimarene-8 beta,19-diol; and ent-8(14),15-pimaradien-3beta-acetoxy displayed the highest antibacterial activities (MIC values lower than 10 microg mL(-1) for most pathogens). In conclusion, our results suggest that pimaranes are an important class of natural products for further investigations in the search of new antibacterial agents.

The herbivory activity of the bordered patch larvae (Chlosyne lacinia, Lepidoptera) on leaves of a Brazilian population of Tithonia diversifolia and the antifeedant potential of its leaf rinse extract were investigated. The caterpillars fed only on the adaxial face, where the density of glandular trichomes is very low, and avoided the abaxial face, which contains high levels of trichomes. Deterrent activity against the larvae was observed in leaf discs treated with leaf rinse extract at concentrations of 1-5% of fresh leaf weight. High-performance liquid chromatography (HPLC) analysis indicated that sesquiterpene lactones are the main constituents of the glandular trichomes. Dichloromethane rinse extracts of the leaves and inflorescences were chemically investigated, and 16 compounds were isolated and identified: 14 sesquiterpene lactones, a flavonoid and a diterpenoid. In this study, five sesquiterpene lactones are described for the first time in the genus, including two lactones, one of which has an unusual seco-guaianolide skeleton. Our findings indicate that the caterpillars avoid the sesquiterpene-lactone-rich glandular trichomes, and provide evidence for the antifeedant activity of the dichloromethane leaf rinse extract. In addition, a study of the seasonal variation of the main constituents from the leaf surface throughout a year demonstrated that a very low qualitative but a very high quantitative variation occurs. The highest level of the main metabolite tagitinin C was observed between September and October and the lowest was from March to June, the later corresponding to the period of highest infestation by the larvae.

The research, development and use of natural products as therapeutic agents, especially those derived from plants, have been increasing in recent years. Despite the fact that plants provide a rich source of novel biologically active compounds, only a small percentage have been phytochemically investigated and studied for their medical potential. Viguiera is a genus that belongs to the family Asteraceae and to the sunflower tribe Heliantheae, which is widespread mostly in Mexico and in other areas of the Andes and upland areas of Brazil. A review on the secondary metabolites pointed out that sesquiterpene lactones and diterpenes, of the kaurane and pimarane-type, are the main compounds produced by these plants. Some reports have shown that kaurane- and pimarane-type diterpenes exert several biological activities such as anti-inflammatory action, antimicrobial and antispasmodic activities. Kaurenoic and pimaradienoic acids, which are the main secondary metabolites isolated by our research group from the roots of Viguiera robusta and V. arenaria, respectively, have been evaluated on vascular smooth muscle contractility. We showed that these diterpenoids are able to inhibit the vascular contractility mainly by blocking extracellular Ca(2+) influx. Additionally, in this review we discuss the structure-activity relationship of the diterpenes regarding their inhibitory activity on vascular contractility.

This work describes an application of artificial neural networks on a small data set of sesquiterpene lactones (STLs) of three tribes of the family Asteraceae. Structurally different types of representative STLs from seven subtribes of the tribes Eupatorieae, Heliantheae and Vernonieae were selected as input data for self-organizing neural networks. Encoding the 3D molecular structures of STLs and their projection onto Kohonen maps allowed the classification of Asteraceae into tribes and subtribes. This approach allowed the evaluation of structural similarities among different sets of 3D structures of sesquiterpene lactones and their correlation with the current taxonomic classification of the family. Predictions of the occurrence of STLs from a plant species according to the taxa they belong to were also performed by the networks. The methodology used in this work can be applied to chemosystematic or chemotaxonomic studies of Asteraceae.

In addition to known compounds, the floral parts of Viguiera radula afforded two new sesquiterpene lactones. All compounds were detected in glandular trichomes, which were micromechanically collected from the anther appendages and analyzed by HPLC. Structure identification was performed by NMR and MS techniques., (Copyright 2003 Elsevier Science Ltd.)

ent-Pimara-8(14),15-dien-19-oic acid (PA) isolated from Viguiera arenaria (Asteraceae; Heliantheae) inhibited rat carotid rings contraction induced by phenylephrine (10(-8) mol/l) or potassium chloride (45 mmol/l) at concentration ranging from 5 to 20 microg/ml. This inhibitory effect was not reversible after the removal of this compound from the medium bath., (Copyright 2002 Elsevier Science B.V.)

Five guaianolides and a germacrolide were isolated from the leaf rinse extract of Viguiera gardneri (Asteraceae), together with known compounds. All compounds were detected in glandular trichomes collected from the leaves and were analyzed by HPLC. Structure elucidation was based on the analysis of spectroscopic data. Low energy conformations were obtained by quantum mechanical calculations. Three closely related guaianolides which were isolated as the main compounds were studied for their anti-inflammatory activity using the transcription factor NF-kappaB as molecular target. NF-kappaB DNA binding was inhibited at sesquiterpene lactones concentrations of 10 or 50 microM.

ent-kaurenoic acid (KA) isolated from Viguiera robusta was tested for activity on vascular smooth muscle contactility. Cumulative concentration-response curves were obtained for a stepwise increase (10(-10)-10(-05) mol/l) in the concentration of phenylephrine (Phe), a selective alpha(1)-adrenergic agonist. The effects in the presence of KA (0.2, 2.0 and 20.0 microg/ml), and 90 min after the removal of KA from the medium bath were compared to controls. At 20.0 microg/ml, KA inhibited the in vitro contractility of rat carotid artery elicited by Phe, but had no effect at lower concentrations (0.2 and 2.0 microg/ml). The effect elicited by KA was reversible after 90 minutes.

The extract of the floral parts of Viguiera quinqueremis afforded, in addition to known compounds, six new sesquiterpene lactones as well as a new myoinositol derivative. All compounds were detected in glandular trichomes which were collected micromechanically from the anther appendages and were analyzed by HPLC. Structure identification was performed by 1H NMR measurements including LC NMR and LC MS experiments.