Detection of Mycobacterium tuberculosis in sputum from Suruí Indian subjects, Brazilian Amazon

This investigation aimed at the detection of Mycobacterium tuberculosis (MTB) in the sputum of Suruí Indian subjects from Amazonia, Brazil. Polymerase chain reaction analyses were positive for 12 samples, five of which were also culture-positive (N = 147). Four MTB genotypes were identified, one of which showed resistance to rifampicin and isoniazid. The study also highlighted one village complex as of particular importance, considering the relatively high number of tuberculosis cases reported and of MTB isolates obtained.     

Diversity and three-dimensional structures of the alpha Mcr of the methanogenic Archaea from the anoxic region of Tucuruí Lake, in Eastern Brazilian Amazonia

Methanogenic archaeans are organisms of considerable ecological and biotechnological interest that produce methane through a restricted metabolic pathway, which culminates in the reaction catalyzed by the Methyl-coenzyme M reductase (Mcr) enzyme, and results in the release of methane. Using a metagenomic approach, the gene of the α subunit of mcr (mcrα) was isolated from sediment sample from an anoxic zone, rich in decomposing organic material, obtained from the Tucuruí hydroelectric dam reservoir in eastern Brazilian Amazonia. The partial nucleotide sequences obtained were 83 to 95% similar to those available in databases, indicating a low diversity of archaeans in the reservoir. Two orders were identified - the Methanomicrobiales, and a unique Operational Taxonomic Unit (OTU) forming a clade with the Methanosarcinales according to low bootstrap values. Homology modeling was used to determine the three-dimensional (3D) structures, for this the partial nucleotide sequence of the mcrα were isolated and translated on their partial amino acid sequences. The 3D structures of the archaean Mcrα observed in the present study varied little, and presented approximately 70% identity in comparison with the Mcrα of Methanopyrus klanderi. The results demonstrated that the community of methanogenic archaeans of the anoxic C1 region of the Tucurui reservoir is relatively homogeneous.     

Molecular Characterization of Human Pathogenic Bunyaviruses of the Nyando and Bwamba/Pongola Virus Groups Leads to the Genetic Identification of Mojuí dos Campos and Kaeng Khoi Virus

Background

Human infection with Bwamba virus (BWAV) and the closely related Pongola virus (PGAV), as well as Nyando virus (NDV), are important causes of febrile illness in Africa. However, despite seroprevalence studies that indicate high rates of infection in many countries, these viruses remain relatively unknown and unstudied. In addition, a number of unclassified bunyaviruses have been isolated over the years often with uncertain relationships to human disease.

Methodology/Principal Findings

In order to better understand the genetic and evolutionary relationships among orthobunyaviruses associated with human disease, we have sequenced the complete genomes for all 3 segments of multiple strains of BWAV (n = 2), PGAV (n = 2) and NDV (n = 4), as well as the previously unclassified Mojuí dos Campos (MDCV) and Kaeng Khoi viruses (KKV). Based on phylogenetic analysis, we show that these viruses populate 2 distinct branches, one made up of BWAV and PGAV and the other composed of NDV, MDCV and KKV. Interestingly, the NDV strains analyzed form two distinct clades which differed by >10% on the amino acid level across all protein products. In addition, the assignment of two bat-associated bunyaviruses into the NDV group, which is clearly associated with mosquito-borne infection, led us to analyze the ability of these different viruses to grow in bat (RE05 and Tb 1 Lu) and mosquito (C6/36) cell lines, and indeed all the viruses tested were capable of efficient growth in these cell types.

Conclusions/Significance

On the basis of our analyses, it is proposed to reclassify the NDV strains ERET147 and YM176-66 as a new virus species. Further, our analysis definitively identifies the previously unclassified bunyaviruses MDCV and KKV as distinct species within the NDV group and suggests that these viruses may have a broader host range than is currently appreciated.     

Cloning, sequencing and antigenic characterization of rVirB9 of Anaplasma marginale isolated from Paraná State, Brazil

Anaplasma marginale, a tick-borne bacterium, causes bovine anaplasmosis responsible for significant economic losses in tropical and subtropical regions worldwide. Various major outer membranes have been described, and VirB9, a type IV secretion system protein, has been recently indicated as a candidate in vaccine development against anaplasmosis. The virB9 gene of an A. marginale strain isolated in Paraná, Brazil, was cloned by polymerase chain reaction and sequenced; its cloning into the pETSUMO vector produced a virB9-SUMO-6x His fusion gene construct. This recombinant clone was over-expressed in Escherichia coli BL21 (DE3), and the expressed fusion protein was solubilized with urea and purified with an Ni-NTA column. This method produced a relatively high yield of rVirB9. The deduced amino acid sequence encoded by VirB9 showed 99% homology to A. marginale isolates from St. Maries. rVirB9 was recognized by serum from cattle immunized with PR1 strain and by bovine sera infected with heterologous strains, showing that rVirB9 has conserved epitopes, which suggests that rVirB9 could be useful for the development of a vaccine against anaplasmosis.     

Cloning, sequencing, expression, and antigenic characterization of rMSP4 from Anaplasma marginale isolated from Paraná State, Brazil

Anaplasmosis is a bovine intraerythrocytic disease caused by the bacterium Anaplasma marginale; it causes significant economic losses in tropical and subtropical regions, worldwide. The msp4 gene of an A. marginale strain isolated in Paran , Brazil, was amplified by PCR and sequenced; its cloning into the pET102/D-TOPO vector produced an msp4-6xHis-V5-HP thioredoxin fusion gene construct. This recombinant clone was over-expressed in Escherichia coli BL21(DE-3); the expressed fusion protein was found almost entirely in the insoluble form (inclusion bodies) in the cell lysate. The inclusion bodies were solubilized with urea and the recombinant protein was purified by Ni-NTA column and dialyzed. This method produced a relatively high yield of rMSP4, which was used to immunize rabbits. The deduced amino acid sequence encoded by MSP4 showed 99% homology to A. marginale isolates from Florida, USA, and from Minas Gerais, Brazil. Both rMSP4 and native MSP4 were recognized by post-immunization rabbit serum, showing that rMSP4 has conserved epitopes. As antigenicity was preserved, rMSP4 might be useful for the development of vaccine against anaplasmosis.     

A novel β-glucosidase isolated from the microbial metagenome of Lake Poraquê (Amazon,Brazil)

The Amazon region holds most of the biological richness of Brazil. Despite their ecological and biotechnological importance, studies related to microorganisms from this region are limited. Metagenomics leads to exciting discoveries, mainly regarding non-cultivable microorganisms. Herein, we report the discovery of a novel β-glucosidase (glycoside hydrolase family 1) gene from a metagenome from Lake Poraquê in the Amazon region. The gene encodes a protein of 52.9 kDa, named AmBgl-LP, which was recombinantly expressed in Escherichia coli and biochemically and structurally characterized. Although AmBgl-LP hydrolyzed the synthetic substrate p-nitrophenyl-β-d-glucopyranoside (pNPβG) and the natural substrate cellobiose, it showed higher specificity for pNPβG (k_cat/K_m = 6 s⁻¹·mM⁻¹) than cellobiose (k_cat/K_m = 0.6 s⁻¹·mM⁻¹). AmBgl-LP showed maximum activity at 40 °C and pH 6.0 when pNPβG was used as the substrate. Glucose is a competitive inhibitor of AmBgl-LP, presenting a K_i of 14 mM. X-ray crystallography and Small Angle X-ray Scattering were used to determine the AmBgl-LP three-dimensional structure and its oligomeric state. Interestingly, despite sharing similar active site architecture with other structurally characterized GH1 family members which are monomeric, AmBgl-LP forms stable dimers in solution. The identification of new GH1 members by metagenomics might extend our understanding of the molecular mechanisms and diversity of these enzymes, besides enabling us to survey their industrial applications.     

Virulence and molecular characterization of Toxoplasma gondii isolated from goats in Ceará, Brazil

One hundred and sixty-nine fragments of heart muscle collected from goats in the State of Ceará, Brazil, were analyzed by mouse bioassay with the aim of isolating Toxoplasma gondii. Two T. gondii isolates, named G1 and G2, were obtained and were characterized by PCR-RFLP. In addition, their virulence was evaluated by experimental inoculation into BALB/c mice. The G1 isolate presented high virulence leading to 100% mortality of mice after inoculations with 10¹, 10², and 10³ tachyzoites. The G2 isolate presented low virulence and none of the doses tested lead to mortality of mice. The PCR-RFLP analysis showed that the two isolates are recombinants of the types I/III. However, they differ in the haplotype combination for the genotypes analyzed.     

β-Globin gene cluster haplotypes in Yanomama Indians from the Amazon region of Brazil

Summary Six polymorphic restriction enzyme sites in the beta-globin gene cluster were investigated in Yanomama Indians from the Amazon region of Brazil, using the polymerase chain reaction (PCR) technique. Four haplotypes were identified; the haplotype frequency distribution is similar to those reported for Polynesians, Micronesians and most Asian populations.     

Does the composition of Scarabaeidae (Coleoptera) communities reflect the extent of land use changes in the Brazilian Amazon?

Scarab beetles (Coleoptera: Scarabaeidae) have been used to investigate the effects of environmental disturbances on forest structure and diversity. This group is recognized as sensitive to habitat perturbations and ecosystem changes. Here we examine the effects of anthropogenic impacts on Scarabaeidae composition, testing the following hypotheses: (1) Scarab beetle communities react to land use disturbances with predictable trends, (2) disturbed habitats are able to retain only a part of the Scarab beetle community of native forests or late secondary forests; (3) habitats largely differ in terms of species richness, taxonomic diversity and ecological composition, supporting exclusive and indicator species. We selected areas of native forest, agriculture, pasture for extensive livestock and secondary forests in different stages of regeneration. Our results show that the Scarabaeidae species were not indifferent to the gradient of structural changes represented by the studied areas. In fact, their patterns of habitat preference reveals communities more abundant and diverse in pristine habitats. In contrast, disturbed habitats, dominated by agricultural activities and pasture, indicated clear detrimental effects on the abundance of all forest Scarab beetle specialists. On the other hand, the generalist species, mainly associated with open environments, seemed to be favoured by the prevailing conditions induced by agricultural activities. Overall, the composition of the Scarab beetle communities is variable and sensitive to those structural gradients and, therefore, capable of responding as useful ecological indicators for assessing the extent of land use change or degradation.     

Spatial and Temporal Patterns of Diversity and Distribution of the Upper Juruá River Fish Community (Brazilian Amazon)

We studied spatial and temporal patterns in fish species composition and diversity at the upper Juruá River located in the west Brazilian Amazon. We collected with gillnet 822 fishes belonging to 90 species in the main Juruá River, its tributaries and the floodplain lakes during wet and dry seasons. Fish abundance and species richness were greater in the dry season. During that season, fishes may be concentrated due to the low water level, being caught more easily by gillnets. There has been a trend towards a greater fish biomass caught in lakes. This might be associated with a greater environmental stability as lakes may be less subject to large variations in water level. The fish communities differed between the two seasons and between lakes and the lotic environments (main river and tributaries). Fish species from the family Curimatidae were most abundant in the lakes, while Pimelodus spp. and Hypostomus spp. predominated in the main Juruá River. Seasonal variations in fish communities may be related to differences in the migratory behavior among fish species. Such spatial and temporal patterns influencing fish community structure at the Upper Juruá Extractive Reserve must be accounted for in management and conservation strategies.     

Selenium in sediment and food webs of the Tapajós River basin (Brazilian Amazon) and its relation to mercury

BackgroundWe investigated Se levels along the Tapajós River basin - which is an important tributary of the Amazon River - and the possible antagonistic effect of Se in Hg availability. This is the first study to investigate Se transfer from abiotic to biotic compartments and along the food chain in aquatic ecosystems of the Amazon basin.MethodsSe concentrations were measured in superficial sediment (n = 29), plankton (n = 28) and fishes (n = 121) along two stretches of the Tapajós River basin (Tap_up/mi and Tap_low), comprising approximately 500 km with different hydrological characteristics.ResultsSe concentrations in sediment were significantly higher in the Tap_low (345−664 μg kg⁻¹) than in the Tap_up/mi (60−424 μg kg⁻¹). The seasonal flooding of the Amazon River probably helps to carry selenium-rich sediment to the Tapajós mouth (Tap_low stretch). We suggest that Se in sediment could decrease the bioavailability of Hg resulting in lower MeHg concentrations in fish, as observed in the Tap_low (45−934 μg kg⁻¹). Sediment and plankton were positively correlated in relation to their Se concentrations (r = 0.62; p = 0.001) suggesting that sediment can possibly be the main source of Se to plankton. Our data indicate Se uptake by primary consumers, as noted in phytoplankton levels. The decrease of Se concentrations along the food chain was also noteworthy.ConclusionThis work elucidates some aspects of Se biogeochemistry in the Amazon basin and shows its importance regarding Hg cycles in aquatic ecosystems.     

Late Quaternary environmental changes in the Lagoa da Curuça region (eastern Amazonia, Brazil) and evidence of Podocarpus in the Amazon lowland

Late-glacial and Holocene vegetational and climate dynamics and human impact of the eastern Amazon rain forest in the coastal region of eastern Pará State in northern Brazil have been studied in two sedimentary recores from Lagoa da Curu?a. Seven radiocarbon dates provide the chronology for two pollen and charcoal records starting from 11,700 and 9440 uncal B. P. to present. Dense and high diverse terra firme Amazon rain forest in the coastal area shows that there was no savanna during the Late-glacial period. The occurrence of Podocarpus trees in the coastal region during the Late-glacial indicate wet and markedly colder conditions at that time. A possible habitat for Pleistocene populations of Podocarpus trees which are now also evident from several other Amazon regions is discussed. There were no rain forest fires between 11,700 and 10,840 uncal B. P. The increase of charcoal particles in the lake deposits after this period suggest an early Amerindian occupation of the coastal region. Received March 26, 2001 / Accepted August 8, 2001     

Ultrastructural characterization of the ovarian stages of the worker bee, Apis mellifera mellifera Linné (Hymenoptera, Apidae)

The classification of the ovarian stages remains still vague. The ultrastructure of the basal follicle made it possible to solve this problem. From the sixth to the eighth day of the worker bee's adult life, the length of the ovariole hardly exceeds 2 mm. The basal oocytes of stages 1 and 2 are characterized by the presence of many ranges of granules encircled by the dilated cylinders of the granular endoplasmic reticulum. These cylinders decondensate the proteinic granules and the lipidic globules generating multivesicular bodies. The basal oocyte, at the third stage of development, measures 0.35+/-0.05 mm in length. This stage of the previtellogenesis' end is characterized by the appearance, in the ooplasm, of many tubules, whose dilated end constitutes a lysosome. At the beginning of the second week, vitellogenesis starts. Stage 4 is defined by the appearance, in the ooplasm, of the yellow granules in relationship to the tubular formations. At the 14th to 16th days of the adult life, 25+/-7.1% of the worker bees reach the end vitellogenesis, or stage 5. The ovariole, about 7 mm in length, contains 11 follicles. This stage 5 is distinguished by the synthesis from the vitelline membrane, the regression of the size of yellow granules and the accessory cores. The sixth stage or oocyte maturation is reached by 12.5+/-3.5% of the workers aged 18 to 20 days. It is defined by the presence of the chorion, the dissolution of the yellow granules, the disappearance of the accessory nuclei and the total degeneration of the follicular cells.     

Molecular differentiation of species of the genus Zungaro (Siluriformes, Pimelodidae) from the Amazon and Paraná-Paraguay River basins in Brazil

Fish species of the Zungaro genus (Siluriformes, Pimelodidae) are amongst the largest migratory fish in Latin America and have considerable economic importance for commercial fishing in Brazil. However, natural populations of this large catfish are experiencing a severe decline. There are significant taxonomical inconsistencies for this fish. Two geographically separated species of the fish were initially described, one endemic in the Amazon and another in the Paraná-Paraguay River basins. A taxonomic review had recently proposed that there is only one Zungaro species in Brazil, based on morphological data. We made a molecular study of Zungaro populations in an attempt to solve taxonomical inconsistencies and to analyze genetic diversity in natural populations of this genus. We analyzed two regions of the mitochondrial DNA (the control region and the ATPase 6 gene region) of individuals sampled from the Paraná-Paraguay River and Amazon River basins. Analyses based on p-distances and maximum likelihood phylogenetic models showed a genetic difference between populations corresponding to different species. Genetic differentiation between Zungaro populations was at the same level as that observed between other Siluriformes species, using the same DNA sequences. We conclude that Zungaro species of the Paraná-Paraguay River basin do not belong to the same species found in the Amazon basin. This finding has a significant implication for conservation of this fish, given that populations are disappearing at a high rate in the Paraná-Paraguay River basin, mainly due to impoundments.     

A new species of the Neotropical loricariid Hypostomus cochliodon group (Hypostominae) from the lower Rio Tapajós basin,Brazilian Amazon

A new species of Hypostomus with spoon-shaped teeth is described from the Rio Cupari basin, a right margin affluent of the lower Rio Tapajós, Pará State, Brazil. The new species inhabits rocky bottom areas in the main channel of Rio Cupari and its tributaries. The new species can be distinguished from all its congeners, except from the Hypostomus cochliodon group, by having six to eight spoon-shaped teeth, dentaries angled at less than 80° and the absence of a notch between the hyomandibular and the metapterygoid. The new species is distinguished from all species of the H. cochliodon group by its unique colour pattern, containing dark vermiculations on head and anterior portion of the trunk, and by the absence of medial buccal papillae.     

Chagas disease: from bush to huts and houses. Is it the case of the Brazilian Amazon?

Two of the major problems facing the Amazon - human migration from the other areas and uncontrolled deforestation - constitute the greatest risk for the establishment of endemic Chagas disease in this part of Brazil. At least 18 species of triatomines had been found in the Brazilian Amazon, 10 of them infected with Trypanosoma cruzi, associated with numerous wild reservoirs. With wide-range deforestation, wild animals will perforce be driven into other areas, with tendency for triatomines to become adapted to alternative food sources in peri and intradomicilies. Serological surveys and cross-sectional studies for Chagas disease, carried out in rural areas of the Rio Negro, in the Brazilian Amazon, showed a high level of seropositivity for T. cruzi antibodies. A strong correlation of seroreactivity with the contact of gatherers of pia?ava fibers with wild triatomines could be evidenced.     

Purification and functional characterization of the first stilbene glucoside-specific β-glucosidase isolated from Lactobacillus kimchi

This study aimed to develop viable enzymes for bioconversion of resveratrol-glucoside into resveratrol. Out of 13 bacterial strains tested, Lactobacillus kimchi JB301 could completely convert polydatin into resveratrol. The purified enzyme had an optimum temperature of 30–40 °C and optimum pH of pH 5.0 against polydatin. This enzyme showed high substrate specificities towards different substrates in the following order: isorhaponticin >> polydatin >> mulberroside A > oxyresveratrol-3-O-glucoside. Additionally, it rarely hydrolyzed astringin and desoxyrhaponticin. Based on these catalytic specificities, we suggest this enzyme be named stilbene glucoside-specific β-glucosidase. Furthermore, polydatin extracts from Polygonum cuspidatum were successfully converted to resveratrol with a high yield (of over 99%). Stilbene glucoside-specific β-glucosidase is the first enzyme isolated from lactic acid bacteria capable of bio-converting various stilbene glucosides into stilbene.     

Recognition of the main geobotanical features along the Bragança mangrove coast (Brazilian Amazon Region) from Landsat TM and RADARSAT-1 data

Orbital remote sensing data were assessedfor mapping of the main geomorphologicaland vegetation units of the Bragançapeninsula (North Brazil), which belongs toa vast though sparsely mapped mangrovecoast. Wide wetland environmentscharacterize the region with extremevariations in extension, temporalevolution, and spatial complexity. Thiswork was based on different digitalprocessing techniques separately applied toLandsat TM and RADARSAT-1 images. Inaddition, both data were merged on apixel-to-pixel basis using algorithms,which rendered an enhancement of thecoastal landforms and a betterdiscrimination of wetlands and landformtypes. The interpretation of the integratedproduct, aided by field validation, made itpossible to significantly extend existingmapping. Nineteen geobotanical units wereidentified: estuarine channels, submergedsand banks, sandflats, old estuarinesandbanks, mudflats, ebb-tidal deltas,barrier-beach ridges, coastal dunes,chenier sand ridges, young intertidalmangroves, intertidal and supratidalmangroves, outer and inner marshes, fluvialflood plains, coastal plateau, degraded andregenerated mangroves, and an artificiallagoon. The digital integration of Fine RADARSAT-1 and Landsat TM data sensitivelyhighlighted geobotanical coastal features,providing a useful tool for a synopticanalysis of their natural and man-drivenchanges.     

Molecular characterization of the Fe‑hydrogenase gene marker in Trichomonas gallinae isolated from birds in Riyadh,Saudi Arabia

Trichomonas gallinae causes avian oropharyngeal trichomonosis. This pathogen affects a large number of bird species and may cause substantial economic losses to the poultry industry. Al-Azizia poultry market in Riyadh, Saudi Arabia is among the largest poultry markets in the Arabian Gulf. Birds traded in this market may be exposed to a variety of T. gallinae strains. Genetic diversity of T. gallinae among birds in the market was examined using Fe‑hydrogenase gene sequences. These sequences were amplified by PCR for twenty-nine isolates of T. gallinae from four different avian species, including 21 feral pigeons, one common mynah, three chickens, and four turkeys. Sequence analysis showed ten variant gene sequences. Nine sequences comprise a new subtype, including A(KSAF1), C(KSAF1) and C(KSAF3) with 34.48% (n = 10), 6.90% (n = 2), 6.90% (n = 2) of the isolates, respectively. Analyses also showed an additional five new sequences (KSAF1.1., KSAF2, KSAF13, KSAF14, KSAF15), representing 17.24% of the isolates. Subtype II (KSAF) was found in four feral pigeons (13.80%). To our knowledge, this report is the first to describe genotypes of T. gallinae from pigeons in Saudi Arabia using Fe‑hydrogenase gene sequences for subtyping. Subtype analysis infers the presence of multiple genotypes of T. gallinae in Saudi avian populations.