Detection of Paracoccidioides spp. in environmental aerosol samples

Taking into account that paracoccidioidomycosis infection occurs by inhalation of the asexual conidia produced by Paracoccidioides spp. in its saprobic phase, this work presents the collection of aerosol samples as an option for environmental detection of this pathogen, by positioning a cyclonic air sampler at the entrance of armadillo burrows. Methods included direct culture, extinction technique culture and Nested PCR of the rRNA coding sequence, comprising the ITS1-5.8S-ITS2 region. In addition, we evaluated one armadillo (Dasypus novemcinctus) as a positive control for the studied area. Although the pathogen could not be isolated by the culturing strategies, the aerosol sampling associated with molecular detection through Nested PCR proved the best method for discovering Paracoccidioides spp. in the environment. Most of the ITS sequences obtained in this investigation proved to be highly similar with the homologous sequences of Paracoccidioides lutzii from the GenBank database, suggesting that this Paracoccidioides species may not be exclusive to mid-western Brazil as proposed so far. © 2013 ISHAM.

Acidovorax citruli: genetic analyses and protocol for its detection in seeds

Bacterial fruit blotch of cucurbits (BFB), caused by the seed borne Gramnegative bacterium Acidovorax citrulli is a serious threat to cucurbit industry worldwide. Since late 1980`s after devastating outbreaks in watermelon fields in southern United States, BFB has spread worldwide and has been reported in other cucurbit crops such as melon, pumpkin, cucumber and squash. To date, there is evidence for the existence of at least two genetically and pathogenically distinct populations of A. citrulli. In Brazil, the first report of BFB was in 1991, in a watermelon field in São Paulo. Although widespread in the country, BFB has been a major problem to melon production. More precisely, BFB has caused significant yield losses to melon production in northeastern Brazil, which concentrates > 90% of the country`s melon production. Despite the management efforts and the recent advances in A. citrulli research, BFB is still a continuous threat to the cucurbit industry, including seed producers, growers and transplant nurseries. To better understand the population structure of A. citrulli strains in Brazil, and to provide a basis for the integrated management of BFB, we used pulsed-field gel electrophoresis (PFGE), multilocus sequence analysis (MLSA) of housekeeping and virulence-associated genes and pathogenicity tests on different cucurbit seedlings to characterize a Brazilian population of A. citrulli strains from different hosts and regions. Additionally, we conducted for the first time a comparative analysis of the A. citrulli group I and II population at genomic level and showed that these two groups differ on their genome sizes due to the presence of eight DNA segments, which are present in group II and absent in group I genomes. We also provide the first evidence to suggest that temperature might be a driver in the ecological adaptation of A. citrulli populations under nutrient-rich or -depleted conditions. Finally, in order to improve the routine detection of A. citrulli on melon seedlots, we designed a new primer set that is able to detect the different Brazilian haplotypes, thus minimizing the risk of false-negatives on PCR-based seed health testing.

Molecular and serological detection of tick-borne pathogens in dogs from an area endemic for Leishmania infantum in Mato Grosso do Sul, Brazil

Patógenos transmitidos por carrapatos atingem uma variedade de hospedeiros vertebrados. Para identificar os agentes patogênicos transmitidos por carrapatos entre cães soropositivos para Leishmania infantum no município Campo Grande-MS, foi realizado um estudo sorológico e molecular para a detecção de Ehrlichia canis, Anaplasma platys e Babesia vogeli em 60 amostras de soro e baço, respectivamente. Adicionalmente, foi realizado o diagnóstico confirmatório de L. infantum por meio de técnicas sorológicas e moleculares. Também foi realizado o alinhamento e análise filogenética das sequências para indicar a identidade das espécies de parasitas que infectam esses animais. Anticorpos IgG anti-Ehrlichia spp., anti-B. vogeli e anti-L. infantum foram detectados em 39 (65%), 49 (81,6%) e 60 (100%) dos cães amostrados, respectivamente. Vinte e sete (45%), cinquenta e quatro (90%), cinquenta e três (88,3%), dois (3,3%) e um (1,6%) cães mostraram-se positivos na PCR para E. canis, Leishmania spp., Leishmania donovani complex, Babesia sp. e Anaplasma sp., respectivamente. Após o seqüenciamento, os amplicons mostraram 99% de similaridade com isolados de E. canis, B. vogeli e A. platys e Leishmania chagasi. Os resultados deste estudo indicaram que os cães soropositivos para L. infantum de Campo Grande, MS, são expostos a vários agentes transmitidos por carrapatos, e, portanto, devem ser incluídos no diagnóstico diferencial em cães com suspeita clínica de leishmaniose.

Detection of Shiga toxigenic (STEC) and enteropathogenic (EPEC) Escherichia coli in dairy buffalo

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Comparative analysis of two techniques for detection and segmentation of moving bodies

This paper makes a comparative analysis of results produced by the application of two techniques for the detection and segmentation of bodies in motion captured in images sequence, namely: 1) technique based on the temporal average of the values of each pixel recorded in N consecutive image frames and, 2) technique based on historical values associated with pixels recorded in different frames of an image sequence.

Sequential sampling of soybean and beans seeds for Sclerotinia sclerotiorum detection (Lib.) DeBary

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Detection of chromosomal bla(CTX-M-2) in diverse Escherichia coli isolates from healthy broiler chickens

The rise of extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli in food-producing animals is a growing concern for public health. We investigated ESBL producers isolated from broiler chickens in Brazil and characterized 19 CTX-M-2-producing E.coli. The ISCR1 was detected upstream of the chromosome-located gene bla(CTX-M-2), associated with sul-1 type integron structure. CTX-M-2-producing E.coli exhibited different PFGE-types and phylogenetic groups, showing a non-clonal dissemination. The sequence types found (ST93, ST155 and ST2309) have been associated with humans and animals worldwide. Herein, we report the chromosomal location of bla(CTX-M-2) on E.coli, highlighting the risks of multidrug-resistant bacteria in food-producing animals.

Molecular detection and phylogenetic analysis of bovine astrovirus in Brazil

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Detection of bla(CTX-M)-type genes in complex class 1 integrons carried by Enterobacteriaceae isolated from retail chicken meat in Brazil

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Change detection as support for monitoring the dynamic of land around reservoir

The change detection technique was used in this study to provide preliminary information on the dynamics of land cover in the region over the western basin of the Tiete River. This area is characterized by sequence of reservoirs and intense agricultural activity, triggering series negative effects. One of the impacts is contamination and proliferation of aquatic organisms in these aquatics environments, increased by release of nutrients from human activities. This work was possible to observe a large switching classes and secondary vegetation bare land, probably related to agricultural activity.

Venomics of the Australian eastern brown snake (Pseudonaja textilis): detection of new venom proteins and splicing variants

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Development of an Electrochemical Insulin Sensor Based on a High Affinity DNA Sequence Found in the Insulin-linked Polymorphic Region

The detection of pertinent biomarkers has the potential provide an early indication of disease progression before considerable damage has been incurred. A decrease in an individual’s sensitivity to insulin, which may be quantified as the ratio of insulin to glucose in the blood after a glucose pulse, has recently been reported as an early predictor of insulin-dependent diabetes mellitus. Routine measurement of insulin levels is therefore desirable in the care of diabetes-prone individuals. A rapid, simple, and reagentless method for insulin detection would allow for wide-spread screenings that provide earlier signs of diabetes onset. The aim of this thesis is to develop a folding-base electrochemical sensor for the detection of insulin. The sensor described herein consists of a DNA probe immobilized on a gold disc electrode via an alkanethiol linker and embedded in an alkanethiol self-assembled monolayer. The probe is labeled with a redox reporter, which readily transfers electrons to the gold electrode in the absence of insulin. In the presence of insulin, electron transfer is inhibited, presumably due to a binding-induced conformational or dynamic change in the DNA probe that significantly alters the electron-tunneling pathway. A 28-base segment of the insulin-linked polymorphic region that has been reported to bind insulin with high affinity serves as the capture element of the DNA probe. Three probe constructs that vary in their secondary structure and position of the redox label are evaluated for their utility as insulin-sensing elements on the electrochemical platform. The effects of probe modification on secondary structure are also evaluated using circular dichroism spectroscopy.

Detection Methods for the Genus Lysobacter and the Species Lysobacter enzymogenes

Strains of Lysobacter enzymogenes, a bacterial species with biocontrol activity, have been detected via 16S rDNA sequences in soil in different parts of the world. In most instances, however, their occurrence could not be confirmed by isolation, presumably because the species occurred in low numbers relative to faster-growing species of Bacillus or Pseudomonas. In this study, we developed DNA-based detection and enrichment culturing methods for Lysobacter spp. and L. enzymogenes specifically. In the DNA-based method, a region of 16S rDNA conserved among Lysobacter spp. (L4: GAG CCG ACG TCG GAT TAG CTA GTT), was used as the forward primer in PCR amplification. When L4 and universal bacterial primer 1525R were used to amplify DNA from various bacterial species, an 1100-bp product was found in Lysobacter spp. exclusively. The enrichment culturing method involved culturing soils for 3 days in a chitin-containing broth amended with antibiotics. Bacterial strains in the enrichment culture were isolated on yeast-cell agar and then identified by 16S rDNA sequence analysis. A strain of L. enzymogenes added to soils was detected at populations as low as 102 and 104 CFU/g soil by PCR amplification and enrichment culturing, respectively. In a survey of 58 soil samples, Lysobacter was detected in 41 samples by PCR and enrichment culture, out of which 6 yielded strains of Lysobacter spp. by enrichment culture. Among isolated strains, all were identified to be L. enzymogenes, with the exception of a strain of L. antibioticus. Although neither method alone is completely effective at detecting L. enzymogenes, they are complementary when used together and may provide new information on the spatial distribution of the species in soil.

Detection of dengue virus in sera of Brazilian blood donors

BACKGROUND: Dengue is the most important arboviral disease in the world. Dengue viruses (DENVs) have produced huge outbreaks in Brazil in the past 25 years with more than 5 million reported cases. During these epidemics, asymptomatic individuals infected with DENV could donate blood and serve as a source of virus dissemination in the community. Here, we studied the circulation of DENV in healthy individuals during an epidemic outbreak. STUDY DESIGN AND METHODS: The study included 500 serum samples from healthy blood donors collected at the Hemotherapy Center of Ribeirao Preto, Brazil, during a dengue outbreak. The presence of DENV RNA in the serum samples was screened by real-time reverse transcriptionpolymerase chain reaction (PCR). The virus serotype was determined by a heminested PCR procedure. A partial fragment of the NS5 gene sequence was used for phylogenetic analysis. RESULTS: DENV RNA was detected in the serum sample of 2 of 500 (0.4%) individuals. Both of them were infected with DENV-3 Genotype III, a virus that has been circulating in Brazil in the past decade. CONCLUSION: Individuals with asymptomatic DENV infection can be blood donors and serve as a source of virus dissemination in the community. Further studies are needed to determine the risk of recipient infection by DENV as a result of transfusion in Brazil, especially during epidemic periods.

Multi-planet extrasolar systems - detection and dynamics

20 years after the discovery of the first planets outside our solar system, the current exoplanetary population includes more than 700 confirmed planets around main sequence stars. Approximately 50% belong to multiple-planet systems in very diverse dynamical configurations, from two-planet hierarchical systems to multiple resonances that could only have been attained as the consequence of a smooth large-scale orbital migration. The first part of this paper reviews the main detection techniques employed for the detection and orbital characterization of multiple-planet systems, from the (now) classical radial velocity (RV) method to the use of transit time variations (TTV) for the identification of additional planetary bodies orbiting the same star. In the second part we discuss the dynamical evolution of multi-planet systems due to their mutual gravitational interactions. We analyze possible modes of motion for hierarchical, secular or resonant configurations, and what stability criteria can be defined in each case. In some cases, the dynamics can be well approximated by simple analytical expressions for the Hamiltonian function, while other configurations can only be studied with semi-analytical or numerical tools. In particular, we show how mean-motion resonances can generate complex structures in the phase space where different libration islands and circulation domains are separated by chaotic layers. In all cases we use real exoplanetary systems as working examples.