A novel system for large-scale gene expression analysis: bacterial colonies array

In the present work, we report the use of bacterial colonies to optimize macroarray technique. The devised system is significantly cheaper than other methods available to detect large-scale differential gene expression. Recombinant Escherichia coli clones containing plasmid-encoded copies of 4,608 individual expressed sequence tag (ESTs) were robotically spotted onto nylon membranes that were incubated for 6 and 12 h to allow the bacteria to grow and, consequently, amplify the cloned ESTs. The membranes were then hybridized with a beta-lactamase gene specific probe from the recombinant plasmid and, subsequently, phosphorimaged to quantify the microbial cells. Variance analysis demonstrated that the spot hybridization signal intensity was similar for 3,954 ESTs (85.8%) after 6 h of bacterial growth. Membranes spotted with bacteria colonies grown for 12 h had 4,017 ESTs (87.2%) with comparable signal intensity but the signal to noise ratio was fivefold higher. Taken together, the results of this study indicate that it is possible to investigate large-scale gene expression using macroarrays based on bacterial colonies grown for 6 h onto membranes.

Molecular characterization of bacterial populations of different soils

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

Occurrence of anti-Neospora caninum antibodies in Brazilian cervids kept in captivity

Neospora caninum is a coccidian parasite that causes disease in captive and domesticated animals and has been found in wild animals such as cervids. Sera from 150 cervids of the genus Mazama, were collected from 31 captive herds and 16 zoos from different Brazilian regions and analyzed by indirect fluorescent antibody test for anti-N. caninum antibodies. Positive reactions were found in 42% (63) of the samples and the titers varied from 50 to 51,200. of the 86 cervids from the captive herds, 38 (44.2%) had anti N. caninum antibodies and of the 64 samples from the zoo, 25 (39.1%) were positive. No significant difference (p > 0.05) was found for the occurrence values observed between the animals from captive herds and zoos as well as within the values documented for each one of the species analyzed. Therefore, the results indicate that the agent is prevalent from cervids in captivity in Brazil. (c) 2005 Elsevier B.V. All rights reserved.

Environmental effect on the occurrence of anti-Neospora caninum antibodies in pampas-deer (Ozotoceros bezoarticus)

Sera obtained from pampas-deer (Ozotoceros bezoarticus) captured in two different Brazilian environments were analyzed for the presence of anti-Neospora caninum antibodies by the indirect fluorescent antibody test (IFAT). Samples were collected from 23 animals from a savanna area in the National Park of Emas, in the state of Goias, Brazil. This area is surrounded by cultivated lands and allows very little contact between wild and domestic animals. Another batch of samples was collected from 16 animals from the Pantanal region, in the state of Mato Grosso, Brazil. This area is a flood plain where domestic animals have intensive contact with cervids. The 39 samples were analyzed (IFAT >= 1:50), and the values for the occurrences found in the animals from each region were compared by the test for comparison of two proportions. of the 39 cervids examined, 38.46% (15) had anti-N. caninum antibodies. Three (13%) of the 23 samples from the National Park of Emas. and 12 (75%) of the 16 samples from the Pantanal were positive, with significant differences between regions (p < 0.001). These results suggested that the presence of domestic animals, mainly dogs and cattle, may be responsible for the greater occurrence of N. caninum in the Pantanal cervids.Thus, as a recommendation of the Conservation Units that care for the pampas-deer, attention should be taken to carefully monitor the flow of diseases between the domestic animals and this species. (c) 2005 Elsevier B.V. All rights reserved.

Analysis of the serological variability of Lettuce mosaic virus using monoclonal antibodies and surface plasmon resonance technology

A panel of 19 monoclonal antibodies (mAbs) was used to study the immunological variability of Lettuce mosaic virus (LMV), a member of the genus Potyvirus, and to perform a first epitope characterization of this virus. Based on their specificity of recognition against a panel of 15 LMV isolates, the mAbs could be clustered in seven reactivity groups. Surface plasmon resonance analysis indicated the presence, on the LMV particles, of at least five independent recognition/ binding regions, correlating with the seven mAbs reactivity groups. The results demonstrate that LMV shows significant serological variability and shed light on the LMV epitope structure. The various mAbs should prove a new and efficient tool for LIVIV diagnostic and field epidemiology studies.

Grapevine bacterial canker in the State of São Paulo, Brazil: detection and eradication

Sintomas do cancro bacteriano da videira na variedade Red Globe foram observados em agosto de 2009 em pomar de Tupi Paulista, Estado de São Paulo, Brasil, e o agente causal Xanthomonas campestris pv. viticola foi identificado por meio de testes patológicos e moleculares. O procedimento de erradicação foi adotado e aproximadamente 4.700 plantas foram destruídas. Um levantamento realizado nas regiões produtoras do Estado de São Paulo não encontrou nenhum outro pomar contaminado, e essa espécie bacteriana é considerada ausente neste estado.

Comparative Studies of the (Anti) Mutagenicity of Baccharis dracunculifolia and Artepillin C by the Bacterial Reverse Mutation Test

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

Yersinia enterocolitica O : 3 isolated from patients with or without reactive arthritis induces polyclonal activation of B cells and autoantibody production in vivo

The mechanisms by which arthritis-provoking pathogens such as Yersinia enterocolitica interact with the human immune system to produce inflammatory synovitis are not well known. One of the immunomodulating mechanisms used against these pathogens is the polyclonal activation of lymphocytes. In this study, we investigated the extent of the B-lymphocyte activation induced in mice by a strain of Y. enterocolitica O:3 (FCF 526) isolated from a patient with arthritis, and compared it with two other strains, a virulent one (FCF 397[+]) isolated from a patient without arthritis and its plasmidless isogenic pair (FCF397[-]). Also we investigated the production of autoantibodies in mice infected with these different strains. SPF Swiss mice were infected intravenously with a suspension of Y. enterocolitica . Spleen cells were taken on days 7, 14, 21 and 28 after infection and the number of cells secreting nonspecific and specific antibodies of IgG 1 , IgG 2a , IgG 2b , IgG 3 , IgM and IgA isotypes were determined by the ELISPOT technique. The presence of autoantibodies in mouse serum was investigated by the dot-blot assay. The pattern of infection of the three bacterial strains were almost the same. We observed a general increase in the number of nonspecific Ig-secreting cells with all three strains, and the greatest increases observed were in the IgG 2a and IgG 3 isotypes. Only a small fraction of the immunoglobulins detected were antibacterial, suggesting that the rest resulted from polyclonal B cell activation. The strain isolated from the patient with arthritis (FCF526) induced the greatest production of autoantibodies, coinciding with the period in which the greatest activation of nonspecific B lymphocytes was seen. There were no signs of arthritis or inflammation in the joints of the infected animals. Based on our results, we were unable to determine whether there is an association between the arthritogenic capability of Y. enterocolitica and polyclonal activation of B cells.

Antibodies to yeast Sm motif 1 cross-react with human Sm core polypeptides

Two regions common to all UsnRNP core polypeptides have been described: Sm motif 1 and Sm motif 2. Rabbits were immunized with a 22 amino-acid peptide containing one segment of Sm motif 1 (YRGTLVSTDNYFNLQL-NEAEEF, corresponding to residues 11-32) from yeast F protein. After immunization, the rabbit sera contained antibodies that not only reacted specifically with the peptide from yeast F protein but also cross-reacted with Sm polypeptides from mammals; that is, with purified human U1snRNPs. The results suggest that the peptide used and human Sm polypeptides contain a common feature recognized by the polyclonal antibodies. A large collection of human systemic lupus erythematosus sera was assayed using the yeast peptide as an antigen source. Seventy per cent of systemic lupus erythematosus sera contain an antibody specificity that cross-reacts with the yeast peptide.

Taenia saginata: Production and characterization of monoclonal antibodies against Taenia saginata metacestode antigens

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

Bacterial sterilization by a dielectric barrier discharge (DBD) in air

Cold atmospheric plasma treatment of microorganisms and living tissues has become a popular topic in modern plasma physics and in medical science. The plasma is capable of bacterial inactivation and noninflammatory tissue modification, which makes it an attractive tool for treatment of skin diseases, open injuries and dental caries. Because of their enhanced plasma chemistry, Dielectric Barrier Discharges (DBDs) have been widely investigated for some emerging applications such as biological and chemical decontamination of media at ambient conditions. Despite the high breakdown voltage in air at atmospheric pressure, the average current of DBD discharges is low. Therefore, a DBD can be applied in direct contact with biological objects without causing any damage. In this work a 60 Hz DBD reactor, which generates cold atmospheric plasma inside Petri dishes with bacterial culture, is investigated. Samples of Staphylococcus aureus, a Gram-positive bacterium and Escherichia coil a Gram-negative bacterium were selected for this study. The bacterial suspensions were evenly spread on agar media planted in Petri dishes. The reactor electrodes were placed outside the Petri dish, thus eliminating the risk of samples microbial contamination. The covered Petri dish with agar medium in it serves as dielectric barrier during the treatment. The plasma processing was conducted at same discharge power (similar to 1.0 W) with different exposure time. Sterilization of E. coil and S. aureus was achieved for less than 20 min. Plasma induced structural damages of bacteria were investigated by Scanning Electron Microscopy. (C) 2010 Elsevier B.V. All rights reserved.

Bacterial cellulose-silica organic-inorganic hybrids

Bacterial cellulose (BC) hydrated membranes present nanometric reticulated structure that can be used as a template in the preparation of new organic-inorganic hybrids. BC-silica hybrids were prepared from BC membranes and tetraethoxysilane, (TEOS) at neutral pH conditions at room temperature. Macroscopically homogeneous membranes were obtained containing up to 66 wt.% of silica spheres, 20-30 nm diameter. Scanning electron micrographs clearly show the silica spheres attached to cellulose microfibrils. By removing the cellulose, the silica spheres can be easily recovered. The new hybrids are stable up to 300 degrees C and display a broad emission band under UV excitation assigned to oxygen-related defects at the silica particles surface. Emission color can be tuned by changing the excitation wavelength.

Leptospira spp. and Toxoplasma gondii antibodies in vampire bats (Desmodus rotundus) in Botucatu region, SP, Brazil

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