Yeasts as sustainable biocontrol agents against ochratoxigenic Aspergillus species and in vitro optimization of ochratoxin A detoxification.

AIMS: The aims of this study were to evaluate the potential of Hanseniaspora opuntiae, Meyerozyma caribbica, and Kluyveromyces marxianus for in vitro biocontrol of Aspergillus ochraceus, A. westerdijkiae, and A. carbonarius growth, the ochratoxin A (OTA) effect on yeast growth, and yeast in vitro OTA detoxification ability using an experimental design to predict the combined effects of inoculum size, incubation time, and OTA concentration. METHODS AND RESULTS: Predictive models were developed using an incomplete Box-Behnken experimental design to predict the combined effects of inoculum size, incubation time, and OTA concentration on OTA detoxification by the yeasts. The yeasts were able to inhibit fungal growth from 13% to 86%. Kluyveromyces marxianus was the most efficient in inhibiting the three Aspergillus species. Furthermore, high OTA levels (100 ng ml-1) did not affect yeast growth over 72 h incubation. The models showed that the maximum OTA detoxification under optimum conditions was 86.8% (H. opuntiae), 79.3% (M. caribbica), and 73.7% (K. marxianus), with no significant difference (P > 0.05) between the values predicted and the results obtained experimentally. CONCLUSION: The yeasts showed potential for biocontrol of ochratoxigenic fungi and OTA detoxification, and the models developed are important tools for predicting the best conditions for the application of these yeasts as detoxification agents.

Development of Indirect Competitive Enzyme-Linked Immunosorbent Assay to Detect Fusarium verticillioides in Poultry Feed Samples.

Fumonisins are a group of toxic secondary metabolites that are produced by Fusarium verticillioides which are associated with poultry health hazard and great economic losses. The objective of the present study was to develop an immunological method to detect F. verticillioides in poultry feed samples. An indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) based on a polyclonal antibody against 67 kDa protein of the F. verticillioides 97K exoantigen was developed to detect this fungus. Antibody anti-67 kDa protein showed cross-reactivity against F. graminearum (2⁻7%) and F. sporotrichioides (10%), but no or low cross-reactivity against Aspergillus sp. and Penicillium sp. exoantigens. The detection limit for the 67 kDa protein of F. verticillioides was 29 ng/mL. Eighty-one poultry feed samples were analyzed for Fusarium sp. count, 67 kDa protein of F. verticillioides and fumonisin concentrations. Eighty of the 81 feed samples (98.6%) showed Fusarium sp. contamination (mean 6.2 x 104 CFU/g). Mean 67 kDa protein and fumonisin concentration in the poultry feed samples was 21.0 µg/g and 1.02 µg/g, respectively. The concentration of 67 kDa protein, as determined by ic-ELISA correlated positively (p < 0.05) with fumonisin levels (r = 0.76). These results suggest that this ic-ELISA has potential to detect F. verticillioides and predict fumonisin contamination in poultry feed samples.

Real-time PCR-based method for rapid detection of Aspergillus niger and Aspergillus welwitschiae isolated from coffee.

Some species from Aspergillus section Nigri are morphologically very similar and altogether have been called A. niger aggregate. Although the species included in this group are morphologically very similar, they differ in their ability to produce mycotoxins and other metabolites and their taxonomical status has evolved continuously. Among them, A. niger and A. welwitschiae are ochratoxin A and fumonisin B2 producers and their detection and/or identification is of crucial importance for food safety. The aim of this study was the development of a real-time PCR-based method for simultaneous discrimination of A. niger and A. welwitschiae from other species of the A. niger aggregate isolated from coffee beans. One primer pair and a hybridization probe specific for detection of A. niger and A. welwitschiae strains were designed based on the BenA gene sequences, and used in a Real-time PCR assay for the rapid discrimination between both these species from all others of the A. niger aggregate. The Real-time PCR assay was shown to be 100% efficient in discriminating the 73 isolates of A. niger/A. welwitschiae from the other A. niger aggregate species analyzed as a negative control. This result testifies to the use of this technique as a good tool in the rapid detection of these important toxigenic species.

Molecular analysis of Aspergillus section Flavi isolated from Brazil nuts.

Brazil nuts are an important export market in its main producing countries, including Brazil, Bolivia, and Peru. Approximately 30,000 tons of Brazil nuts are harvested each year. However, substantial nut contamination by Aspergillus section Flavi occurs with subsequent production of aflatoxins. In our study, Aspergillus section Flavi were isolated from Brazil nuts (Bertholletia excelsa), and identified by morphological and molecular means. We obtained 241 isolates from nut samples, 41% positive for aflatoxin production. Eighty-one isolates were selected for molecular investigation. Pairwise genetic distances among isolates and phylogenetic relationships were assessed. The following Aspergillus species were identified: A. flavus, A. caelatus, A. nomius, A. tamarii, A. bombycis, and A. arachidicola. Additionally, molecular profiles indicated a high level of nucleotide variation within ß-tubulin and calmodulin gene sequences associated with high genetic divergence from RAPD data. Among the 81 isolates analyzed by molecular means, three of them were phylogenetically distinct from all other isolates representing the six species of section Flavi. A putative novel species was identified based on molecular profiles.

Detection of TT virus in HIV-1 exposed but uninfected individuals and in HIV-1 infected patients and its influence on CD4+ lymphocytes and viral load.

The TT virus (TTV) was detected for the first time in the serum of a patient with post-transfusion hepatitis of unknown origin. TTV was subsequently, also found in the serum of blood donors with no history of blood transfusion. In the present study, the percentage of TTV carriers among HIV-infected and noninfected patients was determined. The study was conducted to evaluate CD4 count and HIV viral load in 100 asymptomatic patients infected with HIV-1, 100 symptomatic patients with AIDS, 100 HIV-1 exposed but uninfected individuals and 100 normal healthy blood donors. In this work, the presence of TTV was investigated by nested-PCR. TTV was detected in 6% of normal donors, 12.5% of HIV-infected individuals and 21% of exposed individuals. The presence of TTV was statistically significant in the HIV-exposed individuals (21/100) compared with blood donors (6/100). Odds ratio=4.16 (95%CI 1.60-10.83). No inter-group relations were found for CD4 and CD8 counts or HIV viral load. In the symptomatic group, patients with TTV presented minor viral load. This work demonstrated that TTV was detected in HIV-exposed individuals and no relation was verified for CD4, CD8 and viral load in the asymptomatic and symptomatic HIV patients.

Plasma malondialdehyde levels and CXCR4 expression in peripheral blood cells of breast cancer patients.

INTRODUCTION: Breast cancer is one of the most common neoplasms in women and is a leading cause of cancer related deaths worldwide. Chemokines and their receptors are involved in the control of lymphocyte traffic, a critical component of systemic immunity. CXCR4 mRNA could be involved in the development of variety of diseases. Lipid peroxidation, the result of nonenzymatic autooxidation of polyunsaturated fatty acids, presents numerous harmful effects on biological systems and has been implicated in diseases like cancer. This study examined CXCR4 mRNA expression in peripheral blood cells and malondialdehyde (MDA) in plasma from blood donors and breast cancer patients. MATERIALS AND METHODS: CXCR4 expression in peripheral blood cells from 59 breast cancer patients and 76 healthy blood donors was analyzed by real-time PCR. Plasma MDA was analyzed using high-performance liquid chromatography (HPLC). CONCLUSION: In all stages, MDA levels in total breast cancer patients (1.41 +/- 0.11) were significantly higher (P < 0.01) than those in healthy subjects (0.34 +/- 0.03). No statistically significant difference occurred between CXCR4 expression in peripheral blood cells from breast cancer patients (1.69 +/- 1.05) and the normal healthy control group (1.8 +/- 0.65). However, stage II samples differed statistically (4.3 +/- 1.72) from control, total cancer patients and stages I, III and IV samples.

Identification of novel temperature-regulated genes in the human pathogen Cryptococcus neoformans using representational difference analysis.

Cryptococcus neoformans is a basidiomycetous fungus and an opportunistic human pathogen that causes infections in both immunocompromised and immunocompetent hosts. The ability to survive and proliferate at the human body temperature is an essential virulence attribute of this microorganism. Representational difference analysis (RDA) was used to profile gene expression in C. neoformans grown at 37 degrees C or 25 degrees C. Contig assembly of 300 high-quality sequenced cDNAs and comparison analysis to the GenBank database led to the identification of transcripts that may be critical for both pathogen-host interactions and responses to either low or high temperature growth. Gene products involved in cell wall integrity, stress response, filamentation, oxidative metabolism, protein targeting and fatty acids metabolism were induced at 37 degrees C. In addition, genes related to chromatin silencing and phospholipid transport were upregulated at 25 degrees C. Therefore, our RDA analysis, comparing saprophytic and host temperature conditions, revealed new genes with potential involvement in C. neoformans virulence.

Analysis of the CC chemokine receptor 5 delta32 polymorphism in a Brazilian population with cutaneous leishmaniasis.

Patients with mucocutaneous leishmaniasis (MCL) show a vigorous T-cell immune response against Leishmania braziliensis. Because the Th response is associated with inflammation, the non-functional CC chemokine receptor 5 (CCR5) may rely in a less severe inflammatory state. The aim of this study was to investigate the CCR5 gene in a Brazilian population with leishmaniasis compared with healthy control subjects and to determine the progression from cutaneous to MCL in the Delta32 allele carriers. Among 100 patients with Montenegro skin test and indirect immunofluorescence assay (IIF) values positive for leishmaniasis, there were 32% women and 68% men. The patients were 89% CCR5/CCR5, 10% CCR5/Delta32, and 1% Delta32/Delta32, while healthy subjects showed a 91% incidence of CCR5/CCR5, 8% of CCR5/Delta32, and 1% of Delta32/Delta32. The CCR5/CCR5 patients (89%) showed a large spectrum of clinical manifestations, where 22.47% had active mucous lesions and 77.53% had cutaneous lesions. In this work, the Delta32 allele carriers (10%) showed only cutaneous manifestations when compared with wild-type individuals. Finally, with regard to the Delta32 allele carriers, a less severe spectrum of clinical manifestations was observed in comparison with wild-type individuals. Although a lack of mucocutaneous lesions was evident among Delta32 allele carriers, the number of individuals studied was small. Therefore, further investigations are needed to elucidate the role of CCR5 in the clinical aspects of leishmaniasis.

Expression of noncoding mRNA in human blood cells activated with synthetic peptide of HIV.

T cells activation includes several steps such as translational events, activation of transcription for different genes, expression of surface molecules, secretion of cytokines, effectors functions. Knowledge has been accumulated on various nontranslatable RNA transcripts that are synthesized. In this context, a member of T cell noncoding transcripts (NTT) has been identified. It has been known that this gene is selectively expressed in activated T cells, as a 17-kb transcript. In this study, we investigate cell activation using RT-PCR to detect NTT. We investigated the expression of IFNgamma mRNA, a cytokine produced by activated blood mononuclear cells treated with HLA-A2 restricted synthetic peptide of HIV (p9) by RT-PCR detecting a fragment of 300 bp. This finding demonstrated that human HLA-A2 blood mononuclear cells have been activated in the presence of synthetic peptide of HIV (p9) and can induce expression of mRNA of NTT and IFNgamma which was confirmed by direct sequencing. For the first time, we have demonstrated an endogenous noncoding human RNA molecule, NTT mRNA, suggesting its implication in the cellular immune response.

Agrobacterium tumefaciens-mediated genetic transformation of the entomopathogenic fungus Beauveria bassiana.

Agrobacterium tumefaciens-mediated transformation (agro-transformation) was successfully applied to the entomogenous fungus Beauveria bassiana. Conidia of B. bassiana were transformed to hygromycin B resistance using the hph gene of Escherichia coli as the selective trait, under the control of a heterologous fungal promoter and the Aspergillus nidulans trpC terminator. The efficiency of transformation was up to 28 and 96 transformants per 10(4) and 10(5) target conidia, respectively, using three distinct vectors. High mitotic stability of the transformants (80-100%) was demonstrated after five successive transfers on a nonselective medium. Abortive transformants were observed for all the hph(r) vectors used. Putative transformants were analysed for the presence of the hph gene by PCR and Southern analysis. The latter analysis revealed the integration of two or more copies of the hph gene in the genome. The agro-transformation method was found to be effective for the isolation of B. bassiana hygromycin resistant transformants and may represent a useful tool for insertional mutagenesis studies in this fungus.

Molecular investigation of the stromal cell-derived factor-1 chemokine in lymphoid leukemia and lymphoma patients from Brazil.

The stromal cell-derived factor-1 (SDF-1) gene contains a common polymorphism, termed SDF1-3'A, in an evolutionarily conserved segment of the 3'-untranslated region (3'-UTR). We compared SDF-1 genotypes in patients diagnosed with lymphoid leukemias and lymphomas. Since the SDF1-3'A variant deletes the MspI restriction site, PCR-restriction fragment length polymorphism (RFLP) analysis was used for identification of genotypes. We identified the heterozygous genotype (3'A/wt) in 38.8% (24/62) of lymphoma patients and in 26.2% (11/42) of lymphoid leukemias. The percentage of 3'A carriers was significantly higher in lymphomas (43.5%) than in lymphoid leukemias (26.2%; P < 0.05). Our study indicates that lymphoma patients from Brazil are more likely to carry the 3'A gene than patients with lymphoid leukemias, suggesting that this polymorphism may be a differential determinant of lymphomas and lymphoid leukemia.