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PubMed1991

Aspergillus fumigatus specific IgE and IgG antibodies for diagnosis of Aspergillus-related lung diseases.

IgE and IgG antibodies against Aspergillus fumigatus were detected by crossed radio immunoelectrophoresis (CRIE) on the sera of seven patients with aspergilloma, six patients with allergic broncho-pulmonary aspergillosis (ABPA) and 25 patients with extrinsic asthma with Aspergillus allergy. IgE-CRIE analysis indicated the presence of A. fumigatus-specific IgE in sera of patients with ABPA and Aspergillus asthma but not of aspergilloma patients. IgG-CRIE showed that both aspergilloma and ABPA patient sera contained high levels of circulating specific IgG antibodies in contrast to sera of Aspergillus asthma patients, which did not show detectable amounts of Aspergillus-specific IgG antibodies. Specific IgE binding could be demonstrated for the major allergens Ag-10 and AG-40 in all ABPA patients, in 80% of Aspergillus asthma patients but not in sera from aspergilloma patients. Specific IgG antibodies directed towards the major allergens could be detected in most of the aspergilloma patients, between 30-70% of the ABPA patients but not in sera from patients with Aspergillus asthma.

Aspergillosis↗
PubMed1999

Sequences from the aspergillopepsin PEP gene of Aspergillus fumigatus: evidence on their use in selective PCR identification of Aspergillus species in infected clinical samples.

In immunodeficient patients, Aspergillus species emerge as circumstantial pathogens. Aspergillus fumigatus is a distant first among the pathogenic aspergilli, which cause deep-seated mycoses. Sequences of the pep gene of A. fumigatus as potential PCR primers, which have not been tested before, were used to identify this species and if possible, differentiate it from other, co-identified, clinically important species of the genus. We present results of the three most promising primer pairs, pep-1/pep-22, pep-15/pep-22 and pep-21/pep32. The second pair was of better specificity when tested with DNA extracted from pure cultures of a multitude of aspergilli, whereas the first co-amplified four clinically significant Aspergillus species. The compatibility of the PCR method with the CTAB DNA extraction protocol varied according to the biological fluid tested and the primer pair used. The first two pairs showed moderate adaptability to the different commercial DNA extraction kits, which were tested in whole blood, spiked with Aspergillus fumigatus hyphae and conidia - as were all the biological fluids used. Restriction of the amplification products with MspI produced distinct patterns for different Aspergillus spp. This approach, as a potential diagnostic tool, seems reliable and sensitive due to its flexibility, speed, low cost, ease of application and selectable breadth of detection.

Aspartic Acid Endopeptidases↗
PubMed1999

Density and molecular epidemiology of Aspergillus in air and relationship to outbreaks of Aspergillus infection.

After five patients were diagnosed with nosocomial invasive aspergillosis caused by Aspergillus fumigatus and A. flavus, a 14-month surveillance program for pathogenic and nonpathogenic fungal conidia in the air within and outside the University Hospital in Rotterdam (The Netherlands) was begun. A. fumigatus isolates obtained from the Department of Hematology were studied for genetic relatedness by randomly amplified polymorphic DNA (RAPD) analysis. This was repeated with A. fumigatus isolates contaminating culture media in the microbiology laboratory. The density of the conidia of nonpathogenic fungi in the outside air showed a seasonal variation: higher densities were measured during the summer, while lower densities were determined during the fall and winter. Hardly any variation was found in the numbers of Aspergillus conidia. We found decreasing numbers of conidia when comparing air from outside the hospital to that inside the hospital and when comparing open areas within the hospital to the closed department of hematology. The increase in the number of patients with invasive aspergillosis could not be explained by an increase in the number of Aspergillus conidia in the outside air. The short-term presence of A. flavus can only be explained by the presence of a point source, which was probably patient related. Genotyping A. fumigatus isolates from the department of hematology showed that clonally related isolates were persistently present for more than 1 year. Clinical isolates of A. fumigatus obtained during the outbreak period were different from these persistent clones. A. fumigatus isolates contaminating culture media were all genotypically identical, indicating a causative point source. Knowledge of the epidemiology of Aspergillus species is necessary for the development of strategies to prevent invasive aspergillosis. RAPD fingerprinting of Aspergillus isolates can help to determine the cause of an outbreak of invasive aspergillosis.

Air Microbiology↗
PubMed2002

Polymerase chain reaction-mediated characterization of molds belonging to the Aspergillus flavus group and detection of Aspergillus parasiticus in peanut kernels by a multiplex polymerase chain reaction.

The Aspergillus flavus group covers species of A. flavus and Aspergillus parasiticus as aflatoxin producers and Aspergillus oryzae and Aspergillus sojae as koji molds. Genetic similarity among these species is high, and aflatoxin production of a culture may be affected by cultivation conditions and substrate composition. Therefore, a polymerase chain reaction (PCR)-mediated method of detecting the aflatoxin-synthesizing genes to indicate the degree of risk a genotype has of being a phenotypic producer was demonstrated. In this study, 19 strains of the A. flavus group, including A. flavus, A. parasiticus, A. oryzae, A. sojae, and one Aspergillus niger, were subjected to PCR testing in an attempt to detect four genes, encoding for norsolorinic acid reductase (nor-1), versicolorin A dehydrogenase (ver-1), sterigmatocystin O-methyltransferase (omt-1), and a regulatory protein (apa-2), involved in aflatoxin biosynthesis. Concurrently, the strains were cultivated in yeast-malt (YM) broth for aflatoxin detection. Fifteen strains were shown to possess the four target DNA fragments. With regard to aflatoxigenicity, all seven aflatoxigenic strains possessed the four DNA fragments, and five strains bearing less than the four DNA fragments did not produce aflatoxin. When peanut kernels were artificially contaminated with A. parasiticus and A. niger for 7 days, the contaminant DNA was extractable from a piece of cotyledon (ca. 100 mg), and when subjected to multiplex PCR testing using the four pairs of primers coding for the above genes, they were successfully detected. The target DNA fragments were detected in the kernels infected with A. parasiticus, and none was detected in the sound (uninoculated) kernels or in the kernels infected with A. niger.

Aflatoxins↗
PubMed1985

[Peracute disseminated course of fatal Aspergillus fumigatus infection in liver failure and corticoid therapy. A case report on the epidemiology, pathogenesis and diagnosis of the systemic course of Aspergillus infections].

Cytostatic-treated persons and cases of severe hepatic failure under corticoid therapy are predisposed to disseminating Aspergillus infections. Constant exposure to Aspergillus spores may result in a fatal Aspergillus infection. The triad of hepatic failure, corticoid therapy and constant exposure to Aspergillus spores is described in a 70-year-old female patient. A painless icterus was clinically diagnosed as non-A non-B hepatitis, with a protracted cholestatic course. She had been treated with an oral corticoid preparation. After leaving the hospital at her own insistance when still in the icteric stage, severe pneumonia due to Aspergillus developed within 14 days; this was confirmed radiologically. The autopsy results showed unexpected infarction, similar to pneumonic foci, in all lobes and dissemination in the myocardium, stomach, kidneys and brain. The liver showed subacute dystrophy. Constant exposure to the conidia of A fumigatus came about as a result of the soil of potted ornamental plants in the patient's living room. The fungus could only be successfully cultured by putting infected tissue particles on Sabouraud dextrose agar; it was not possible by the common method of fractionated streaking.

Aged↗
PubMedDate not supplied

Isolation and sequencing of a new glucoamylase gene from an Aspergillus niger aggregate strain (DSM 823) molecularly classified as Aspergillus tubingensis.

Based on morphological characteristics the taxa included in the Aspergillus aggregate can hardly be differentiated. For that reason the phylogeny of this genus was revised several times as different criteria, from morphological to later molecular, were used. We found, comparing nucleotide sequences of the ITS-region, that the strain Aspergillus niger (DSM 823) which is claimed to be identical to the strains ATCC 10577, IMI 027809, NCTC 7193 and NRRL 2322 can be molecularly classified as Aspergillus tubingensis, exhibiting 100% identity with the A. tubingensis CBS strains 643.92 and 127.49. We amplified, cloned and sequenced a new glucoamylase gene (glaA) from this strain of A. tubingensis (A. niger DSM 823) using primers derived from A. niger glucoamylase G1. The amplified cDNA fragment of 2013 bp contained an open reading frame encoding 648 amino acid residues. The calculated molecular mass of the glucoamylase, deduced from the amino acid sequence, was 68 kDa. The nucleotide sequence of glaA showed 99% similarity with glucoamylases from Aspergillus kawachii and Aspergillus shirousami, whereas the similarity with the glucoamylase G1 from A. niger was 92%

Amino Acid Sequence↗
PubMed2005

Study of Spanish grape mycobiota and ochratoxin A production by Isolates of Aspergillus tubingensis and other members of Aspergillus section Nigri.

The native mycobiota of five grape varieties grown in Spain has been studied. Four (Bobal, Tempranillo, Garnacha, and Monastrell) were red varieties and one (Moscatel) was white. The main fungal genera isolated were Alternaria, Cladosporium, and Aspergillus. The isolation frequency of Aspergillus spp. section Nigri in contaminated samples was 82%. Ochratoxin A (OTA) production was assessed using yeast extract-sucrose broth supplemented with 5% bee pollen. Cultures of 205 isolates from this section showed that 74.2% of Aspergillus carbonarius and 14.3% of Aspergillus tubingensis isolates produced OTA at levels ranging from 1.2 to 3,530 ng/ml and from 46.4 to 111.5 ng/ml, respectively. No Aspergillus niger isolate had the ability to produce this toxin under the conditions assayed. Identification of the A. niger aggregate isolates was based on PCR amplification of 5.8S rRNA genes and its two intergenic spacers, internal transcribed spacer 1 (ITS1) and ITS2, followed by digestion with restriction endonuclease RsaI of the PCR products. The restriction patterns were compared with those from strains of A. niger CECT 2807 and A. tubingensis CECT 20393, held at the Spanish Collection of Type Cultures. DNA sequencing of the ITS1-5.8S rRNA gene-ITS2 region of the OTA-producing isolates of A. tubingensis matched 99 to 100% with the nucleotide sequence of strain A. tubingensis CBS 643.92. OTA determination was accomplished by liquid chromatography with fluorescence detection. OTA confirmation was carried out by liquid chromatography coupled to ion trap mass spectrometry. The results showed that there are significant differences with regard to the isolation frequency of ochratoxinogenic fungi in the different grape varieties. These differences were uncorrelated to berry color. The ability of A. tubingensis to produce OTA and the influence of grape variety on the occurrence of OTA-producing fungi in grapes are described in this report for the first time.

Aspergillus↗
PubMedDate not supplied

Expression of Aspergillus hemoglobin domain activities in Aspergillus oryzae grown on solid substrates improves growth rate and enzyme production.

DNA fragments coding for hemoglobin domains (HBD) were isolated from Aspergillus oryzae and Aspergillus niger. The HBD activities were expressed in A. oryzae by introduction of HBD gene fragments under the control of the promoter of the constitutively expressed gpdA gene. In the transformants, oxygen uptake was significantly higher, and during growth on solid substrates the developed biomass was at least 1.3 times higher than that of the untransformed wild-type strain. Growth rate of the HBD-activity-producing strains was also significantly higher compared to the wild type. During growth on solid cereal substrates, the amylase and protease activities in the extracts of the HBD-activity-producing strains were 30-150% higher and glucoamylase activities were at least 9 times higher compared to the wild-type strain. These results suggest that the Aspergillus HBD-encoding gene can be used in a self-cloning strategy to improve biomass yield and protein production of Aspergillus species.

Amylases↗
PubMed1978

[Presence of sexual forms (cleistothecia and Hülle cells) in a case of human maxillary sinus aspergillosis caused by Aspergillus nidulans in association with Aspergillus fumigatus].

The authors observed, in a biopsic sample from a human maxillary sinus, the coexistence, in parasitic stages, of two different Aspergillus species: A. fumigatus and A. nidulans. After having described the morphologies of each species, in sections on slides, and particularly those of cleistothecia and Hülle-cells for the second, they draw attention to the three interests of such an observation: -- rarety of observing Aspergillus nidulans in a parasitic stage, in spite of its particular thermotolerant character. -- association of two different Aspergillus species in a same sample, in parasitic stages, observed in man for the first time, at least according their informations. -- presence of reproductive sexual forms, observed in vivo for the first time, in an Aspergillus species.

Adult↗
PubMed1989

Immunochemical assay applied to mycotoxin biosynthesis: ELISA comparison of sterigmatocystin production by Aspergillus versicolor and Aspergillus nidulans.

Conventional thin layer and instrumental methods for analyzing mycotoxins and their precursors are time-consuming and make the investigation of mycotoxin biosynthesis particularly difficult. As an alternative, sensitive enzyme-liked immunosorbent assays (ELISAs) can be utilized to analyze for these compounds. In this report, sterigmatocystin production in test tube cultures of Aspergillus versicolor ATCC 18643 and Aspergillus nidulans ATCC 32610 were compared using competitive ELISA. Polyclonal antiserum that was prepared against a sterigmatocystin hemiacetal-bovine serum albumin conjugate exhibited greatest specificity for sterigmatocystin hemiacetal and sterigmatocystin with less reactivity for O-methylsterigmatocystin. The antiserum could be used to detect as little as 50 ng/ml sterigmatocystin in ELISA. Direct ELISA could be performed on diluted culture broth and on mycelial extracts solubilized with N,N-dimethylformamide. Aspergillus versicolor ATCC 18643 produced more sterigmatocystin in SLS medium than in YES medium, and showed maximal levels at between 9 to 12 days incubation. Approximately 75% of sterigmatocystin was detectable in mycelium (254 micrograms/ml culture) compared to the extracellular fraction (87 micrograms/ml culture). Aspergillus nidulans exhibited qualitatively similar patterns of growth and toxigenesis in SLS medium but accumulated maximal levels of only 15 micrograms mycelial sterigmatocystin/ml culture and 5 micrograms extracellular sterigmatocystin/ml broth, respectively.

Aspergillus↗
PubMed1990

Aflatoxin production by entomopathogenic isolates of Aspergillus parasiticus and Aspergillus flavus.

Fourteen isolates of Aspergillus parasiticus and 2 isolates of Aspergillus flavus isolated from the mealybug Saccharicoccus sacchari were analyzed for production of aflatoxins B1, B2, G1, and G2 in liquid culture over a 20-day period. Twelve Aspergillus isolates including 11 A. parasiticus and 1 A. flavus produced aflatoxins which were extracted from both the mycelium and culture filtrate. Aflatoxin production was detected at day 3 and was detected continually for up to day 20. Aflatoxin B1 production was greatest between 7 and 10 days and significantly higher quantities were produced by A. flavus compared to A. parasiticus. Aflatoxin production was not a stable trait in 1 A. parasiticus isolate passaged 50 times on agar. In addition to loss of aflatoxin production, an associated loss in sporulation ability was also observed in this passaged isolate, although it did maintain pathogenicity against S. sacchari. An aflatoxin B1 concentration of 0.16 micrograms/mealybug (14.2 micrograms/g wet wt) was detected within the tissues of infected mealybugs 7 days after inoculation. In conclusion, the ability of Aspergillus isolates to produce aflatoxins was not essential to the entomopathogenic activity of this fungus against its host S. sacchari.

Aflatoxins↗
PubMed2004

PCR-restriction fragment length analysis of aflR gene for differentiation and detection of Aspergillus flavus and Aspergillus parasiticus in maize.

Contamination of food and feedstuffs by Aspergillus species and their toxic metabolites is a serious problem as they have adverse effects on human and animal health. Hence, food contamination monitoring is an important activity, which gives information on the level and type of contamination. A PCR-based method of detection of Aspergillus species was developed in spiked samples of sterile maize flour. Gene-specific primers were designed to target aflR gene, and restriction fragment length polymorphism (RFLP) of the PCR product was done to differentiate Aspergillus flavus and Aspergillus parasiticus. Sterile maize flour was inoculated separately with A. flavus and A. parasiticus, each at several spore concentrations. Positive results were obtained only after 12-h incubation in enriched media, with extracts of maize inoculated with A. flavus (101 spores/g) and A. parasiticus (104 spores/g). PCR products were subjected to restriction endonuclease (HincII and PvuII) analysis to look for RFLPs. PCR-RFLP patterns obtained with these two enzymes showed enough differences to distinguish A. flavus and A. parasiticus. This approach of differentiating these two species would be simpler, less costly and quicker than conventional sequencing of PCR products.

Aspergillus↗
PubMed2005

PCR detection assays for the ochratoxin-producing Aspergillus carbonarius and Aspergillus ochraceus species.

Two PCR assays have been developed to detect Aspergillus carbonarius and Aspergillus ochraceus, considered the main sources of ochratoxin A (OTA) contaminating commodities, particularly grapes, coffee and derivatives, in warm climates. The species specific primers have been designed on the basis of ITS (internal transcribed spacers of rDNA units) sequence comparisons obtained from Aspergillus strains and have been tested in a number of strains from different origins and hosts. These PCR assays, based on multi-copy sequences, are highly sensitive and specific and represent a good tool for an early detection of OTA-producing Aspergillus species and to prevent OTA entering the food chain.

Aspergillus↗
PubMed1990

Restriction fragment length polymorphisms in isolates of Aspergillus fumigatus probed with part of the intergenic spacer region from the ribosomal RNA gene complex of Aspergillus nidulans.

Differences in restriction fragment length polymorphisms (RFLPs) have been detected in isolates of Aspergillus fumigatus. Genomic DNA from 11 isolates was digested with EcoRI, separated by electrophoresis, Southern blotted and probed with DNA from the intergenic spacer or non-transcribed spacer region of the rRNA gene complex of Aspergillus nidulans. Three distinct RFLP patterns were detected which differed from the control patterns observed with A. nidulans, Aspergillus flavus and Aspergillus niger hybridized with the same probe. Furthermore, the differences in RFLP patterns in the A. fumigatus isolates were not detected when probed with DNA coding for the rRNA complex in Saccharomyces cerevisiae. These findings may be of use in the study of the epidemiology and pathogenesis of infections caused by A. fumigatus.

Aspergillus fumigatus↗
PubMed1992

Application of ubiquinone systems and electrophoretic comparison of enzymes to identification of clinical isolates of Aspergillus fumigatus and several other species of Aspergillus.

The ubiquinone systems and electrophoretic comparison of enzymes were used to determine the relatedness among 64 isolates of seven Aspergillus spp. These were 31 clinical and 3 nonclinical isolates of Aspergillus fumigatus Fres., 2 isolates of A. nidulellus Samson & W. Gams, 8 isolates of A. terreus Thom, 4 isolates of A. flavus Link, 1 isolate of A. oryzae (Ahlburg) Cohn, 14 isolates of A. niger van Tieghem, and 1 isolate of A. japonicus Saito. The enzymes glucose 6-phosphate dehydrogenase, lactate dehydrogenase, glutamate dehydrogenase, fumarase, and malate dehydrogenase were examined. The relative mobilities were analyzed numerically. The results were presented as a dendrogram. Isolates from clinical and nonclinical sources within the same species had identical ubiquinone systems and identical or very similar enzyme patterns. In the dendrogram, 64 of the tested isolates were separated into seven major clusters at a 60% similarity level. Each major cluster corresponds to a single species. On the dendrogram, A. fumigatus isolates showed homogeneity, whereas A. niger isolates showed relative heterogeneity; in particular, A. niger MF-24 and the other A. niger isolates were distantly linked to each other. All A. fumigatus isolates had the Q-10 ubiquinone system and formed a single major cluster at a similarity level of 73% or greater. Glucose 6-phosphate dehydrogenase and glutamate dehydrogenase were key enzymes for differentiating all clinical and nonclinical isolates of A. fumigatus from the other Aspergillus spp. Ubiquinone systems and enzyme patterns appear to be objective and useful indicators for use in the precise identification of clinical isolates of Aspergillus spp.

Aspergillus↗
PubMed2001

Identification of two different 14-alpha sterol demethylase-related genes (cyp51A and cyp51B) in Aspergillus fumigatus and other Aspergillus species.

Two cyp51-related genes (cyp51A and cyp51B) encoding 14-alpha sterol demethylase-like enzymes were identified in the opportunistic human pathogen Aspergillus fumigatus. PCR amplification using degenerate oligonucleotides based on conserved areas of cytochrome P450 demethylases of other filamentous fungi and yeasts allowed the cloning and sequencing of two different homologue genes in A. fumigatus. Southern analysis confirmed that both genes hybridized to distinct genomic loci and that both are represented as single copies in the genome. Comparison of the deduced Cyp51A and Cyp51B proteins with the CYP51 proteins from Penicillium italicum, Aspergillus nidulans, Erysiphe graminis, Uncinula necator, Botrytis cinerea, Ustilago maydis, Cryptococcus neoformans, Candida albicans, Saccharomyces cerevisiae, Candida tropicalis, and Candida glabrata showed that the percentages of identity of the amino acid sequences (range, 40 to 70%) were high enough to consider Cyp51A and Cyp51B to be members of the fungal CYP51 family. Fragments from both genes were also cloned from other Aspergillus spp. (A. flavus, A. nidulans, and A. terreus). Phylogenetic analysis showed that, at least in the most pathogenic species of Aspergillus, there are two fungal CYP51 proteins. This is the first report of the existence of two homologue genes coding for 14-alpha sterol demethylase in the fungal kingdom. This finding could provide insights into the azole resistance mechanisms operating in fungi. The primers used here may be useful molecular tools for facilitating the cloning of novel 14-alpha sterol demethylase genes in other filamentous fungi.

Amino Acid Sequence↗
PubMed1994

Circular dichroism studies of acid proteinases from Aspergillus niger and Aspergillus awamori.

Acid proteinases produced by strains of Aspergillus niger and Aspergillus awamori were isolated by means of ethanol precipitation, gel filtration and anion-exchange high resolution chromatography. In each case, the purified proteinase showed a single band in polyacrylamide gel electrophoresis. Their molecular weights were almost identical (approx. 45,000). However, the proteinase from Aspergillus awamori contained 16% of neutral hexoses while the other enzyme (Aspergillus niger) showed negligible amounts of these carbohydrates. Both enzymes displayed circular dichroism spectra that share a number of features with that of penicillopepsin. This suggests that proteinases from Aspergilli possess the structural folding pattern typical of aspartic proteinases. Proteolytic-activity pH optima were different, thus distinguishing one enzyme from another. This variation seems to be related to the particular resistance of the proteinases to acid denaturation, as indicated by changes in their circular dichroism spectra when the pH is decreased.

Aspartic Acid Endopeptidases↗
PubMed1998

Allergy to Aspergillus-derived enzymes in the baking industry: identification of beta-xylosidase from Aspergillus niger as a new allergen (Asp n 14).

BACKGROUND: Aspergillus-derived enzymes are used in dough improvers in bakeries. Some of these enzymes are identified as causing IgE-mediated sensitization in up to 25% of bakers with workplace-related symptoms. OBJECTIVE: The aim of this study was to compare the frequency of sensitization to Aspergillus xylanase, cellulase, and glucoamylase with the sensitization to alpha-amylase (Asp o 2) and to identify IgE-reactive proteins in enzyme preparations. METHODS: Sensitization to Aspergillus-derived enzymes and cross-reactivity were retrospectively studied by enzyme allergosorbent test (EAST) and EAST-inhibition experiments. IgE-reactive proteins were detected by electrophoretic separation and immunoblotting. Liquid chromatography with electrospray ionization mass spectrometry and Edman degradation of tryptic protein fragments were used for the biochemical identification of an unknown IgE-binding protein. RESULTS: Twenty-three percent of 171 tested bakers had specific IgE to alpha-amylase, 8% reacted to glucoamylase, 13% reacted to cellulase, and 11% reacted to xylanase. Xylanase and cellulase preparations, each containing at least 6 different proteins, showed cross-reactivity in the range of 80%. The main IgE-binding protein in the xylanase preparation recognized in 7 of 8 xylanase-positive subjects was a protein of about 105 kd. This protein was identified as beta-xylosidase by peptide mass spectrometric fingerprinting. The identification was confirmed by matching 12 peptide sequences obtained by N-terminal and mass spectrometric sequencing to this protein. CONCLUSIONS: Beta-Xylosidase from Aspergillus niger is an occupational allergen present in currently used baking additives, which causes sensitization in at least 4% of symptomatic bakers. According to the International Union of Immunological Societies nomenclature, we suggest the term Asp n 14 for this allergen.

Adult↗