In vitro culture, PCR , and nested PCR for the detection of Theileria equi in horses submitted to exercise

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

Detached leaf culture: Viability to evaluate 2,4-D toxicity symptoms in cotton apex leaves

To study the viability of detached leaf culture technique, studies were carried out with detached leaves from cotton apex (true trilobed leaves). The prepared leaves were sprayed with 2,4-D amine and ester, at rates of 10, 30, 70, and 100% of the recommended doses. Detached leaves without herbicide spray were used as controls. Simultaneously, a greenhouse experiment was conducted with the same treatments as used for the detached leaves experiment. Toxicity was measured through a 0-to-5 grading according to the percentage of affected leaf area in the detached leaves experiment or examining the affected rate of whole plant as indicated in the greenhouse. Results showed that the ester form of the herbicide induced earlier and more severe toxicity symptoms in detached leaves and greenhouse grown plants. Positive and significant correlations (p < 0.001) were found between toxicity results obtained at 7 and 14 days after application in detached leaves and greenhouse plants (r = 0.97 and 0.92, respectively). Negative, significant correlations (p < 0.005) were found between the toxicity levels found at 7 and 14 days after application in detached leaves and dry matter of cotton plants grown in the greenhouse (r = -0.92 and -0.92, respectively).

Growth and enzymatic responses of phytopathogenic fungi to glucose in culture media and soil

The effect of inoculation of Aspergillus flavus, Fusarium verticillioides, and Penicillium sp. in Dystrophic Red Latosol (DRL) and Eutroferric Red Latosol (ERL) soils with or without glucose on the total carbohydrate content and the dehydrogenase and amylase activities was studied. The fungal growth and spore production in culture medium with and without glucose were also evaluated. A completely randomized design with factorial arrangement was used. The addition of glucose in the culture medium increased the growth rate of A. flavus and Penicillium sp. but not of F. verticillioides. The number of spores increased 1.2 for F. verticillioides and 8.2 times for A. flavus in the medium with glucose, but was reduced 3.5 times for Penicillium sp. The total carbohydrates contents reduced significantly according to first and second degree equations. The consumption of total carbohydrates by A. flavus and Penicillium sp. was higher than the control or soil inoculated with F. verticillioides. The addition of glucose to soils benefited the use of carbohydrates, probably due to the stimulation of fungal growth. Dehydrogenase activity increased between 1.5 to 1.8 times (p <0.05) in soils with glucose and inoculated with the fungi (except F. verticillioides), in relation to soil without glucose. Amylase activity increased 1.3 to 1.5 times due to the addition of glucose in the soil. Increased amylase activity was observed in the DRL soil with glucose and inoculated with A. flavus and Penicillium sp. when compared to control.

New Culture Medium to Xanthan Production by Xanthomonas campestris pv. campestris

Xanthan is a important biopolymer for commercial purpose and it is produced in two stages by Xanthomonas campestris. In the first one, the bacterium is cultivated in the complex medium enriched in nitrogen and the biomass produced is used as inoculum for the next stage in which the gum is produced in another medium. In this work a new medium for the first stage is proposed in place of currently used YM medium. Different formulated growth media were studied and the correspondent biomass produced was analysed as inoculum for the second stage. The inoculum and gum were produced by batch process in shaker at 27A degrees C in pH 6.0 and at 30A degrees C in pH 7.0, respectively. The gum was precipitated with ethanol (3:1 v/v). The dryed biomass and xathan gum produced were determined by drying in oven at 105 and 40A degrees C, respectively. The viscosity of the fermentation broth and 1% gum solution in water were determined in Brookfield viscometer. The formulated medium presented the increase in gum production (30%), broth (136%) and 1% gum solution viscosity (60%) compared to YM, besides the inferior cost. The results showed the importance of the quality of the inoculum from the first stage of the culture which influenced on the gum viscosity in the second stage.

Vibrational spectroscopy applied to the study of archeological ceramic artifacts from Guarani culture in Brazil

An archeological artifact can be seen as a chronological element, which helps to determine the age of certain society and to understand the thinking, values and the way of life of this society. Thus, the classification of archeological artifacts is one of the approaches used to study the cultural system of antique societies trying to reconstruct their history. The "Centro de Museologia, Antropologia e Argueologia (CEMAARQ)" of the "Unesp Univ Estadual Paulista" in Presidente Prudente, São Paulo state, Brazil, develops projects within this context (identification and preservation). This is the case of the archeological site named "Lagoa São Paulo-02" discovered in 1993 at the margins of the Parana river in the region of Presidente Epitacio city, São Paulo state, Brazil. This site has ceramic fragments of different shapes and sizes that have a strong influence of traces of the Guarani culture, which is one of the Brazilian native populations. These samples were basically characterized via micro-Raman scattering and Fourier transform infrared absorption (FTIR) spectroscopies. The main objective was to identify the pigments used in the manufacture of the ceramic artifacts and to analyze the composition of the ceramic body to understand how the artifacts were made. Three pigments were found: red, black and white. For the red pigment were identified characteristic bands of hematite, an iron oxide found in the red rocks of the river banks that were eroded by water. The black pigment, probably, is due to the use of vegetal charcoal, which is found in nature as the product of burning organic material such as wood. For the white pigment, the FTIR spectra suggested the use of kaolin, either in the ceramic body or in the proper white pigment, due to the presence of the characteristic bands of the kaolinite. Complementary, the additives applied as anti-plastics were identified as charcoal and quartz, being the latter found in the rocks present in the archeological site. (C) 2010 Elsevier B.V. All rights reserved.

The use of transmission electron microscopy and oocyte transfer to evaluate in vitro maturation of equine oocytes in different culture conditions

The current study evaluates the ability of equine oocytes matured in different conditions to undergo nuclear and cytoplasmic maturation.. After oocyte transfer, embryonic development was diagnosed at 1.5 and 90 days of gestation. For each group, immature oocytes obtained from slaughterhouse ovaries were matured in vitro (5 replicates). In experiment I, three different media were tested. HTF:BME, SOFaa, and TCM 199. In experiment 11, the HTF:BME was chosen as maturation medium containing pFSH, eFSH, or eFSH + eGH. Nuclear maturation was estimated after stripping the oocytes and staining with Hoechst 33342. The evaluation of cytoplasmic maturation was performed by transmission electron microscopy. For oocyte transfer, six non-cycling recipient mares were used, and 8 to 15 oocytes were transferred in each mare. In experiment I, the results showed no differences (P > .05) in nuclear maturation (MII) among experimental groups. The percentage of MII was 29.3 ( +/- 9.6), 23.4 ( +/- 8.4), and 13.5 ( +/- 12.4) for HTF:BME, SOF, and TCM, respectively. In experiment II, all media tested were efficient in inducing metaphase II. Also, no statistical differences (P > .05) were observed in percentages of nuclear maturation rates when porcine (37.1 +/- 22.4) or equine (25.8 +/- 8.2) FSH were used, or when eFSH + eGH was added to HTF:BME (29.4 +/- 12.3). The analysis of cytoplasmic morphology of oocytes cultured in TCM 199 and SOFaa showed signs of incomplete cytoplasmic maturation and premature cortical reaction. Meanwhile, oocytes cultured in HTF:BME medium presented cytoplasmic characteristics similar to those described by others for in vivo-matured oocytes. The addition of eFSH to the HTF:BME medium resulted in an improvement of cytoplasmic morphology. After oocyte transfer, two mares became pregnant, one from pFSH group and one from eFSH+eGH group. These results indicate that although in vitro matured equine oocytes are capable of fertilization and embryonic development, the percentage of competent oocytes is still low.

Comparison of conventional PCR, quantitative PCR, bacteriological culture and the Warthin Starry technique to detect Leptospira spp. in kidney and liver samples from naturally infected sheep from Brazil

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

Nutritional composition, phenolic compounds, nitrate content in eatable vegetables obtained by conventional and certified organic grown culture subject to thermal treatment

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

Callus cell culture of Pothomorphe umbellata (L.) under stress condition leads to high content of peroxidase enzyme

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

Antibiosis associated to Lactobacillus acidophilus growth .2. Optimization of some culture parameters in whey and soy milk medium

Whey supplemented with soy milk has been used as a low-cost alternative in the growth of Lactobacillus acidophilus for the production of antimicrobial compounds. Response Surface Metodology has been employed in order to study the effects of initial pH, incubation temperature and soy milk rate for supplementation in the production of antimicrobial substance. It has been observed that both tested microrganisms used (S. aureus and E. coli) were inhibited by antimicrobial substance produced by L. acidophilus. The results obtained with E. coli inhibition did not follow the employed statistical model. on the other hand, when the tested microorganism S. aureus was used, the best inhibition results have been obtained when L. acidophilus was incubated at 36.80 degrees C in whey with 5.6 initial pH and 31,90% (v/v) rate supplemented with soy milk. The analysed antimicrobial substances were nor acids neither hidrogen peroxid.

Bacterial Pathogens Related to Chronic Suppurative Otitis Media in Individuals With Cleft Palate: Bacteriological Culture and Polymerase Chain Reaction

Objective: To characterize the microbial etiology of chronic suppurative otitis media comparing the methods of classical bacteriological culture and polymerase chain reaction.Design/Setting/Patients: Bacteriological analysis by classical culture and by molecular polymerase chain reaction of 35 effusion otitis samples from patients with cleft lip and palate attending the Hospital for Rehabilitation of Craniofacial Anomalies of the University of Sao Paulo, Bauru, Brazil.Interventions: Collection of clinical samples of otitis by effusion through the external auditory tube.Main Outcome Measure: Otolaryngologic diagnosis of chronic suppurative otitis media.Results: Positive cultures were obtained from 83% of patients. Among the 31 bacterial lineages the following were isolated. In order of decreasing frequency: Pseudomonas aeruginosa (54.9%), Staphylococcus aureus (25.9%), and Enterococcus faecalis (19.2%). No anaerobes were isolated by culture. The polymerase chain reaction was positive for one or more bacteria investigated in 97.1% of samples. Anaerobe lineages were detected by the polymerase chain reaction method, such as Fusobacterium nucleatum, Bacteroides fragilis, and Peptostreptococcus anaerobius.Conclusions: Patients with cleft lip and palate with chronic suppurative otitis media presented high frequency of bacterial infection in the middle ear. The classical bacteriological culture did not detect strict anaerobes, whose presence was identified by the polymerase chain reaction method.

Evaluation of Three Formulations of Culture Media for Isolation of Brucella spp. regarding Their Ability to Inhibit the Growth of Contaminating Organisms

Three culture media (Brucella agar, Farrell medium, and CITA) were compared for their effectiveness in inhibiting contamination and for isolating Brucella spp. One hundred lymph nodes from pigs (n = 50) and wild boars (n = 50) with lymphadenitis were collected in slaughterhouses in the State of Sao Paulo and were assessed on these three selective media for Brucella spp. All of the samples were negative for Brucella spp. on the three culture media. On the agar medium, fungal (70 plates) and Gram-positive bacterial (59 plates) contaminants were observed; in the CITA medium, the absence of fungal and Gram-positive bacteria on 15 plates was observed; no bacterial or fungal growth was observed on the Farrell media. The results demonstrated that the CITA and Farrell media inhibited the growth of contaminants better than the Brucella agar.

Control of microorganisms of oral health interest with Arctium lappa L. (burdock) extract non-cytotoxic to cell culture of macrophages (RAW 264.7)

Objectives: To evaluate the antimicrobial activity of Arctium lappa L. extract on Staphylococcus aureus, S. epidermidis, Streptococcus mutans, Candida albicans, C. tropicalis and C. glabrata. In addition, the cytotoxicity of this extract was analyzed on macrophages (RAW 264.7).Design: By broth microdilution method, different concentrations of the extract (250-0.4 mg/mL) were used in order to determine the minimum microbicidal concentration (MMC) in planktonic cultures and the most effective concentration was used on biofilms on discs made of acrylic resin. The cytotoxicity A. lappa L. extract MMC was evaluated on RAW 264.7 by MTT assay and the quantification of IL-1 beta and TNF-alpha by ELISA.Results: The most effective concentration was 250 mg/mL and also promoted significant reduction (log(10)) in the biofilms of S. aureus (0.438 +/- 0.269), S. epiderrnidis (0.377 +/- 0.298), S. mutans (0.244 +/- 0.161) and C. albicans (0.746 +/- 0.209). Cell viability was similar to 100%. The production of IL-beta was similar to the control group (p > 0.05) and there was inhibition of TNF-alpha (p < 0.01).Conclusions: A. lappa L. extract was microbicidal for all the evaluated strains in planktonic cultures, microbiostatic for biofilms and not cytotoxic to the macrophages. (C) 2014 Elsevier Ltd. All rights reserved.

Green tea polyphenol epigallocatechin-3-gallate and cranberry proanthocyanidins act in synergy with cathelicidin (LL-37) to reduce the LPS-induced inflammatory response in a three-dimensional co-culture model of gingival epithelial cells and fibroblasts

Objectives: The human antimicrobial peptide cathelicidin (LL-37) possesses anti-inflammatory properties that may contribute to attenuating the inflammatory process associated with chronic periodontitis. Plant polyphenols, including those from cranberry and green tea, have been reported to reduce inflammatory cytokine secretion by host cells. In the present study, we hypothesized that A-type cranberry proanthocyanidins (AC-PACs) and green tea epigallocatechin-3-gallate (EGCG) act in synergy with LL-37 to reduce the secretion of inflammatory mediators by oral mucosal cells. Methods: A three-dimensional (3D) co-culture model of gingival epithelial cells and fibroblasts treated with non-cytotoxic concentrations of AC-PACs (25 and 50 mg/ml), EGCG (1 and 5 mg/ml), and LL-37 (0.1 and 0.2 mM) individually and in combination (AC-PACs + LL-37 and EGCG + LL-37) were stimulated with Aggregatibacter actinomycetemcomitans lipopolysaccharide (LPS). Multiplex ELISA assays were used to quantify the secretion of 54 host factors, including chemokines, cytokines, growth factors, matrix metalloproteinases (MMPs), and tissue inhibitors of metalloproteinases (TIMPs). Results: LL-37, AC-PACs, and EGCG, individually or in combination, had no effect on the regulation of MMP and TIMP secretion but inhibited the secretion of several cytokines. ACPACs and LL-37 acted in synergy to reduce the secretion of CXC-chemokine ligand 1 (GRO-a), granulocyte colony-stimulating factor (G-CSF), and interleukin-6 (IL-6), and had an additive effect on reducing the secretion of interleukin-8 (IL-8), interferon-g inducible protein 10 (IP-10), and monocyte chemoattractant protein-1 (MCP-1) in response to LPS stimulation. EGCG and LL-37 acted in synergy to reduce the secretion of GRO-a, G-CSF, IL-6, IL-8, and IP-10, and had an additive effect on MCP-1 secretion. Conclusion: The combination of LL-37 and natural polyphenols from cranberry and green tea acted in synergy to reduce the secretion of several cytokines by an LPS-stimulated 3D coculture model of oral mucosal cells. Such combinations show promising results as potential adjunctive therapies for treating inflammatory periodontitis.