Fumigatus Culture Filtrate Research Articles

The Aspergillus fumigatus Secretome Alters the Proteome of Pseudomonas aeruginosa to Stimulate Bacterial Growth: Implications for Co-infection

Individuals with cystic fibrosis are susceptible to co-infection by Aspergillus fumigatus and Pseudomonas aeruginosa Despite the persistence of A. fumigatus in the cystic fibrosis lung P. aeruginosa eventually predominates as the primary pathogen. Several factors are likely to facilitate P. aeruginosa colonization in the airways, including alterations to the microbial environment. The cystic fibrosis airways are hypoxic, nitrate-rich environments, and the sputum has higher amino acid concentrations than normal. In this study, significant growth proliferation was observed in P. aeruginosa when the bacteria were exposed to A. fumigatus culture filtrates (CuF) containing a high nitrate content. Proteomic analysis of the A. fumigatus CuF identified a significant number of environment-altering proteases and peptidases. The molecular mechanisms promoting bacterial growth were investigated using label-free quantitative (LFQ) proteomics to compare the proteome of P. aeruginosa grown in the A. fumigatus CuF and in CuF produced by a P. aeruginosa-A. fumigatus co-culture, to that cultured in P. aeruginosa CuF. LFQ proteomics revealed distinct changes in the proteome of P. aeruginosa when cultured in the different CuFs, including increases in the levels of proteins involved in denitrification, stress response, replication, amino acid metabolism and efflux pumps, and a down-regulation of pathways involving ABC transporters. These findings offer novel insights into the complex dynamics that exist between P. aeruginosa and A. fumigatus Understanding the molecular strategies that enable P. aeruginosa to predominate in an environment where A. fumigatus exists is important in the context of therapeutic development to target this pathogen.

E-cadherin mediates adhesion of Aspergillus fumigatus to non-small cell lung cancer cells.

A spergillus fumigatus is a widely distributed microorganism, and recently, A. fumigatus culture filtrate has been shown to trigger apoptotic cell death in several human cancer cell lines, including non-small lung cancer (NSCLC) cells, A549. Nevertheless, the molecular adhesion of A. fumigatus to these cancer cells to trigger cell death remains unknown. Here, we knocked down E-cadherin in A549 cells and examined its effects on A. fumigatus. The blastospores of A. fumigatus were incubated with the complete protein extracts from A549 cells, using an affinity purification procedure. Preliminary exploration of E-cadherin-interacting protein on the surface of Aspergillus fumigates was done by immunoprecipitation and mass spectrometry analysis. We found that the adhesion of the blastospores to A549 cells was significantly reduced by E-cadherin suppression in A549 cells, suggesting that E-cadherin of A549 cells may mediate the surface adhesion of A. fumigatus blastospore. Mass spectrometry (MS) analysis predicted two binding proteins for E-cadherin on A. fumigatus, AfA24A6.130c and XP_747789. Finally, the growth of E-cadherin-depleted A549 cells significantly increased by infection of A. fumigatus in vivo. Thus, our study suggests that E-cadherin mediates adhesion of A. fumigatus to NSCLC cells to trigger cell death and provides molecular evidence for the treatment of NSCLC with controlled A. fumigatus infection.

Chitin enhances serum IgE in Aspergillus fumigatus induced allergy in mice

Aspergillus fumigatus (A. fumigatus) is a ubiquitous fungus that activates, suppresses or modulates the immune response by changing its cell wall structure and by secreting proteases. In this study, we show that chitin acts as an adjuvant in a murine model of A. fumigatus protease induced allergy. The mice were immunised intraperitoneally with A. fumigatus culture filtrate antigen either with or without chitin and were subsequently challenged with the culture filtrate antigen intranasally. Alum was used as an adjuvant control. Compared to alum, chitin induced a weaker inflammatory response in the lungs, measured as the total cell efflux in BAL, EPO and chitinase production. However, chitin enhanced the total IgE, specific IgE and specific IgG1 production as efficiently as alum. Pre-treatment with chitin but not with alum depressed the concentration of the Th2 cytokines IL-4 and IL-13 in BAL fluid. These results shows that chitin, in spite of a reduction of the Th2 cytokine levels in the lungs, enhanced the total and specific IgE production in A. fumigatus culture filtrate induced allergy.

Antibacterial Activities of Stationary Phase Culture Filtrates of Aspergillus fumigates

Our previous studies performed for the development of polymicrobial biofilm showed that simultaneous coculturing of Aspergillus fumigatus conidia or sporelings with Pseudomonas aeruginosa resulted in the killing of A. fumigatus cells whereas hyphae older than 12h were recalcitrant to the bacterial fungicidal activity. Since A. fumigatus produces an array of antimicrobial molecules, we examined the antibacterial activity of A. fumigatus culture filtrates on bacterial growth to examine the possible role of such small molecules minimizing the fungicidal activity of P. aeruginosa by spectrophotometric and colony forming unit assays. Culture filtrates collected from 48h stationary phase cultures inhibited the growth of P. aeruginosa and S. aureus, but not that of Candida albicans. Colony forming unit assay of culture filtrate-treated P. aeruginosa cells showed that the effect was bacteriostatic. Culture filtrate incubated in a boiling water bath for 15min retained its antibacterial activity compared to that of the unboiled culture filtrate. These results show that stationary phase liquid cultures of A. fumigatus produce a heat stable bacteriostatic compound(s) in liquid culture that is released into the growth medium inhibits the growth of P. aeruginosa.

In vitro protease inhibition and cytotoxicity of Aspergillus fumigatus biomolecules secreted under long-term aerated conditions.

The fatality rate of invasive aspergillosis (IA) is still very high, especially in prolonged and untreated pulmonary cases. Aspergillus fumigatus is the main causative agent of IA and investigation of its metabolites could provide valuable insight into virulence factor(s) associated with this organism. We evaluated the A. fumigatus culture filtrate (CF) products generated during short- and long-term aerated and non-aerated conditions and tested for (i) inhibition of cysteine or serine proteases and (ii) cytotoxicity. In addition, the mathematical model was determined using response surface methodology (RSM) to estimate the influence of different fermentation conditions on A. fumigatus CF characteristics, predict enzyme inhibition and make possible correlations with in vivo conditions. Biosynthesis of A. fumigatus low molecular weight proteinaceous products (from 6.4 to 15.4 kDa) was observed after 6 days of growth under aerated and alkaline conditions. Also, only these CFs showed significant reduction in cell lines survival (Caco-2 and WISH 35.6% and 54.6%, respectively). Obtained results provide solid starting point for further studies that would include: (i) detailed chemical characterization of A. fumigatus CF, (ii) activity relationships and in vivo correlation with pathogenicity of prolonged pulmonary IA and (iii) possible use of biomolecules as diagnostic or therapeutic markers.

Aspergillus fumigatus inhibits angiogenesis through the production of gliotoxin and other secondary metabolites

AbstractIn susceptible hosts, angioinvasion by Aspergillus fumigatus triggers thrombosis, hypoxia, and proinflammatory cytokine release, all of which are stimuli for angiogenesis. We sought to determine whether A fumigatus directly modulates angiogenesis. A fumigatus culture filtrates profoundly inhibited the differentiation, migration, and capillary tube formation of human umbilical vein endothelial cells in vitro. To measure angiogenesis at the site of infection, we devised an in vivo Matrigel assay in cyclophosphamide-treated BALB/c mice with cutaneous invasive aspergillosis. Angiogenesis was significantly suppressed in Matrigel plugs implanted in A fumigatus–infected mice compared with plugs from uninfected control mice. The antiangiogenic effect of A fumigatus was completely abolished by deletion of the global regulator of secondary metabolism, laeA, and to a lesser extent by deletion of gliP, which controls gliotoxin production. Moreover, pure gliotoxin potently inhibited angiogenesis in vitro in a dose-dependent manner. Finally, overexpression of multiple angiogenesis mediator–encoding genes was observed in the lungs of cortisone-treated mice during early invasive aspergillosis, whereas gene expression returned rapidly to baseline levels in cyclophosphamide/cortisone-treated mice. Taken together, these results indicate that suppression of angiogenesis by A fumigatus both in vitro and in a neutropenic mouse model is mediated through secondary metabolite production.

Performance Characteristics of a Polyclonal Enzyme Immunoassay for the Quantitation ofHistoplasmaAntigen in Human Urine Samples

An antigen detection assay was prepared with rabbit anti- Histoplasma antibodies to detect and quantitate Histoplasma capsulatum antigen in urine samples. By using a 4-parameter curve fit, the assay calibration ranges from 2 to 1,000 enzyme immunoassay (EIA) units. We compared results for 99 urine samples with those of a reference laboratory, half of which tested positive or equivocal by that reference laboratory. Performance characteristics were further defined by studying the assay linearity, precision, percentage of positive agreement, and percentage of negative agreement. An acceptable correlation with the reference laboratory (R2=0.7174) was obtained with results ranging from less than 2 (negative samples) to 132 EIA units by the new method. Compared with the reference laboratory, the percentage of agreement for positive samples, excluding equivocal samples, was 92% and for negative samples was 98%. Crossreactivity occurred with culture filtrates of Paracoccidioides brasiliensis, Coccidioides immitis, and Blastomyces dermatiditis. No cross-reactivity was observed with Candida albicans or Aspergillus fumigatus culture filtrates. The current EIA for the detection and quantitation of H capsulatum antigen in urine specimens is a valid assay that agrees well with results from an established reference laboratory.

Effect of aeration on gliotoxin production by Aspergillus fumigatus in its culture filtrate

Gliotoxin, one of the mycotoxins produced by Aspergillus fumigatus, has various, potent bioactivities. However, it has not been considered to be a toxic (or virulence) factor because of its slow production. The aim of the present study was to investigate the effects of aeration on the cytotoxicity of A. fumigatus culture filtrate, and to determine the optimal condition for the rapid production of gliotoxin from this fungus. Fungal culture filtrates were made in three different containers under various conditions of aeration and O2 concentration. These filtrates were compared in terms of their cytotoxicity on murine macrophages and analyzed by gas chromatography. The culture filtrate showed high cytotoxicity when it was made under highly aerated conditions, but it was significantly less cytotoxic when prepared under non-aerated conditions. The cytotoxic activity became evident within 15 h of culture at 20% O2, when the fungus had already started producing gliotoxin. The culture filtrates also contained some other as yet unidentified substances that might also to some extent contribute to the cytotoxicity. In light of these results, the authors propose that a highly aerated condition is responsible for the rapid production of gliotoxin, and that gliotoxin might play an important role in the respiratory infection by A. fumigatus, with other toxic substances acting additively or synergistically.

Identification and expression of an allergen Asp f 13 from Aspergillus fumigatus and epitope mapping using human IgE antibodies and rabbit polyclonal antibodies

The Aspergillus genus of fungi is known to be one of the most prevalent aeroallergens. On two-dimensional immunoblotting using patients' sera containing IgE specific for Asp f 13, an allergen with a molecular mass of 33 kDa and a pI of 6.2 was identified. This allergen was also present in A. fumigatus culture filtrates. Furthermore, the sequence of the Asp f 13 cDNA was identical to that for alkaline protease isolated from A. fumigatus and showed 42-49% identity of amino acids with two proteases from P. cyclopium and T. album and with the Pen c 1 allergen from P. citrinum. Asp f 13 coding sequences were expressed in Escherichia coli as a [His](6)-tagged fusion protein which was purified by Ni(2+)-chelate affinity chromatography. Recombinant Asp f 13 was recognized by rabbit polyclonal antibodies against Asp f 13 and by IgE antibodies from subject allergic to A. fumigatus. To identify and characterize the linear epitopes of this allergen, a combination of chemical and enzymatic cleavage and immunoblotting techniques, with subsequent N-terminal sequencing and mass spectrometry, were performed. At least 13 different linear epitopes reacting with the rabbit anti-Asp f 13 antiserum were identified, located throughout the entire molecule. In contrast, IgE from A. fumigatus-sensitive patients bound to three immunodominant epitopes at the C-terminal of the protein.

Identification and expression of an allergen Asp f 13 from Aspergillus fumigatus and epitope mapping using human IgE antibodies and rabbit polyclonal antibodies

The Aspergillus genus of fungi is known to be one of the most prevalent aeroallergens. On two-dimensional immunoblotting using patients' sera containing IgE specific for Asp f 13, an allergen with a molecular mass of 33 kDa and a pI of 6.2 was identified. This allergen was also present in A. fumigatus culture filtrates. Furthermore, the sequence of the Asp f 13 cDNA was identical to that for alkaline protease isolated from A. fumigatus and showed 42-49% identity of amino acids with two proteases from P. cyclopium and T. album and with the Pen c 1 allergen from P. citrinum. Asp f 13 coding sequences were expressed in Escherichia coli as a [His]6-tagged fusion protein which was purified by Ni2+-chelate affinity chromatography. Recombinant Asp f 13 was recognized by rabbit polyclonal antibodies against Asp f 13 and by IgE antibodies from subject allergic to A. fumigatus. To identify and characterize the linear epitopes of this allergen, a combination of chemical and enzymatic cleavage and immunoblotting techniques, with subsequent N-terminal sequencing and mass spectrometry, were performed. At least 13 different linear epitopes reacting with the rabbit anti-Asp f 13 antiserum were identified, located throughout the entire molecule. In contrast, IgE from A. fumigatus-sensitive patients bound to three immunodominant epitopes at the C-terminal of the protein.

Culture filtrates of Aspergillus fumigatus induce different modes of cell death in human cancer cell lines.

Aspergillus fumigatus culture filtrate (CF) has a potent cytotoxic effect on three human cancer cell lines (DLKP, A549 and HEp-2) and initiates cell death by apoptosis but the execution of the apoptotic process is incomplete. DLKP cells treated with A. fumigatus CF demonstrate features associated with apoptosis but cytoplasmic and nuclear fragmentation were not observed and cells ultimately underwent necrosis. The apoptotic process commenced in A549 and HEp-2 cells upon exposure to CF, cell shrinkage was observed but membrane blebbing and apoptotic body formation were not detected and detached cells died by necrosis. In contrast, extensive nuclear fragmentation and apoptotic body formation were evident in DLKP and A549 cells treated with anti-neoplastic agents. This work indicates that A. fumigatus CF is cytotoxic to cancer cells and can initiate apoptosis but that the complete apoptotic pathway is not followed.

Effects of Aspergillus fumigatusculture filtrate on antifungal activity of human phagocytes in vitro

BACKGROUNDAspergillus fumigatus can colonise the airways and the lungs with localised underlying conditions and occasionally invade the surrounding lung tissues even in subjects without systemic predisposing factors, presumably by escaping...

Effects of alkaline protease or restrictocin deficient mutants of Aspergillus fumigatus on human polymorphonuclear leukocytes.

Several substances including proteases and restrictocin have been suggested as candidates for virulence determinants in invasive pulmonary aspergillosis. However, the roles of such substances are not well understood. This study compared the in vitro suppressive effects of Aspergillus fumigatus culture filtrates (ACFs), on the functions of human polymorphonuclear leukocytes (PMNLs), the principal cells in the host defence against aspergillus hyphae, from a clinically isolated wild-type and isogenic mutant strains which lack production of elastolytic alkaline protease (Alp) and/or restrictocin. ACFs were obtained by culturing conidia of each strain in Medium- 199 at 37 degrees C for 5 days. ACFs of the wild-type significantly (p<0.01) suppressed chemotaxis, superoxide anion (O2-) release and PMNL-mediated hyphal damage, compared with the control (Medium-199). ACFs of the mutant strains that lack Alp or restrictocin significantly (p<0.01) suppressed chemotaxis and O2(-)-release, but did not suppress hyphal damage, compared with the control. The wild-type significantly (p<0.01) suppressed chemotaxis of PMNLs compared with the mutant strains lacking Alp or restrictocin, whereas there were no significant differences in suppression of O2(-)-release and hyphal damage by PMNLs. ACF of a mutant strain that lacks both Alp and restrictocin had much less activity, but significantly (p<0.01) suppressed chemotaxis of PMNLs compared with the control. In conclusion, alkaline protease and restrictocin may play roles in the suppressive effect of Aspergillus fumigatus culture filtrates on the functions of human polymorphonuclear leukocytes. Other antiphagocytic substances produced by Aspergillus fumigatus remain to be identified.

Purification of a major allergen, Asp f 2 binding to IgE in allergic bronchopulmonary aspergillosis, from culture filtrate of Aspergillus fumigatus

Most cases of allergic bronchopulmonary aspergillosis (ABPA) are caused by the fungus Aspergillus fumigatus. Successful treatment of this disease depends on early diagnosis with the use of well-characterized and relevant antigens/allergens of the organism. The aim of this study was to purify and characterize relevant proteins from A. fumigatus that could be used in the reliable diagnosis of ABPA. Monoclonal antibodies were raised against A. fumigatus culture filtrate antigens. A Concanavalin A nonbinding protein fraction was purified with use of one of the monoclonal antibody immunoaffinity columns. The purified protein was analyzed on sodium dodecylsulfate-polyacrylamide gel electrophoresis gel and Western blots. The sensitivity and specificity of the purified protein were evaluated by RAST and ELISA with sera from 25 patients with ABPA, from 10 with allergic asthma, and from 10 normal control subjects. The 37 kD Concanavalin A nonbinding protein reacted specifically with IgE antibodies in patients with ABPA. Among the 25 patients with ABPA studied, 96% had IgE antibody against the allergen, whereas none of the subjects with allergic asthma who had positive results on the skin prick test or normal control subjects had a reaction. Both RAST and ELISA results exhibited strong correlation with IgE binding. This allergen exhibited N-terminal sequence identity to a recombinant allergen Asp f 2. A 37 kD protein with complete N-terminal homology to Asp f 2 is a major allergen of A. fumigatus that significantly reacts with IgE antibody in patients with ABPA, but does not elicit reaction in Aspergillus-sensitive subjects with asthma and normal control subjects.

Molecular cloning and expression of a recombinant Aspergillus fumigatus protein Asp f II with significant immunoglobulin E reactivity in allergic bronchopulmonary aspergillosis

The cDNA of Aspergillus fumigatus encoding an allergen was cloned and expressed in Escherichia coli. The 987 bp long cDNA clone expressed a recombinant protein Asp f II of 34 kd. This protein exhibited binding to immunoglobulin E (IgE) in the serum samples from patients with allergic bronchopulmonary aspergillosis (ABPA). The patients with ABPA and central bronchiectasis demonstrated high levels of serum IgE antibodies, whereas patients with ABPA without central bronchiectasis, patients with asthma and Aspergillus skin test reactivity but no evidence of ABPA, and patients with aspergilloma showed only low levels of IgE antibody to Asp f II. In two-dimensional electrophoresis, a native antigen electroeluted from an A. fumigatus culture filtrate antigen preparation showed an isoelectric point and molecular weight similar to that of Asp f II. In a competitive enzyme-linked immunosorbent assay (ELISA), the IgE antibody reactivity of Asp f II with patient serum samples could be significantly inhibited by culture filtrate antigens of A. fumigatus. These results indicate that Asp f II has immunologic reactivities comparable to those of native A. fumigatus antigens. The recombinant Asp f II can be expressed in E. coli in large quantities and should prove useful as a standardized allergen for sensitive and specific immunodiagnosis of ABPA, especially in patients with central bronchiectasis.

Aspergillus culture filtrates and sputum sols from patients with pulmonary aspergillosis cause damage to human respiratory ciliated epithelium in vitro

Aspergillus species frequently colonize lower respiratory tracts and lungs with localized underlying conditions (healed tuberculous cavity, cystic fibrosis, bronchiectasis, etc.) even in subjects without systemic predisposing factors. We investigated the in vitro effects of culture filtrates of Aspergillus species and sputum sols from patients with pulmonary aspergillosis on ciliary beat frequency (CBF) and epithelial integrity of human respiratory ciliated epithelium. Culture filtrates of 25 clinically isolated fungi (16 Aspergillus fumigatus, three Aspergillus niger, one Aspergillus flavus, three Candida albicans, and two Cryptococcus neoformans) were obtained by culturing the fungi in Medium-199 at 37 degrees C for 7 days, and five sputum sols were obtained from patients with pulmonary aspergillosis infected by A. fumigatus. During 6 h experiments using a photometric technique, 14 out of 16 A. fumigatus culture filtrates caused progressive and significant reduction in CBF associated with marked epithelial disruption, whilst the culture filtrates of A. niger and A. flavus caused minor epithelial damage without slowing of CBF, and Medium-199 alone (Control) showed neither epithelial damage nor slowing of CBF. All of the sputum sols also caused significant slowing of CBF as well as epithelial disruption. Culture filtrates of C. albicans and Cr. neoformans had no effects on human respiratory epithelium. We conclude that Aspergillus species, especially A. fumigatus release a factor (or factors) which causes damage to respiratory epithelium and slows CBF, and that these factors may contribute to the colonization of the lower respiratory tracts by the Aspergillus species and may possibly contribute to the further proliferation and spread of the lesions in pulmonary aspergillosis.

Purification and characterization of a fibrinogenolytic serine proteinase from Aspergillus fumigatus culture filtrate

A fibrinogenolytic proteinase has been isolated from Aspergillus fumigatus culture filtrate by ammonium sulfate precipitation followed by successive chromatographics on Sephadex G-75 and immobilized phenylalanine. The purified proteinase exhibited a molecular weight of about 33 kDa. When analysed by SDS-polyacrylamide gels containing co-polymerized fibrinogen, the proteinase appeared as a broad band at the top of the gels, which could correspond to polymerization of the enzyme, as suggested by SDS-PAGE analysis of the unboiled eluate. The isoelectric point was 8.75 and the enzyme was not glycosylated. Proteinase activity was optimum at pH 9 and between 37 and 42°C, although a decrease in activity was observed above 37°C. PMSF and chymostatin markedly inhibited the proteinase activity, and good kinetic constants were obtained for the synthetic substrate, N-Suc-Ala-Ala-Pro-Phe-pNA. These results provide direct evidence that this enzyme belongs to the chymotrypsin-like serine proteinase group.

Use of aspergillus fumigatus culture filtrate fractions in ELISA for the serological diagnosis of allergic aspergillosis.

Summary: Fractions of an Aspergillus fumigatus culture filtrate antigen were compared with the whole antigen in an ELISA for the serodiagnosis of allergic aspergillosis. Fractions were prepared by Sephadex G‐200 chromatography and analyzed by fused‐rocket immunoelectrophoresis against rabbit anti‐A. fumigatus sera and by ELISA against sera from allergic aspergillosis patients. The time‐dependence of antigen and IgG adsorption in the ELISA was determined. Both types of antigen adsorbed rapidly to the solid phase. Adsorption of specific anti‐A. fumigatus IgG to both antigens was rapid and increased with incubation time. Both whole antigen and antigen fractions detected specific IgG in allergic aspergillosis patients. In tests with the whole antigen, all patients with allergic aspergillosis had anti‐A. fumigatus IgG levels higher than those seen in asthmatic patients with precipitins to A. fumigatus. In tests with the antigen fractions, only one serum from the allergic aspergillosis group failed to demonstrate an IgG response higher than that seen in the control group of chronic asthmatics.Zusammenfassung: Fraktionen eines Aspergillus‐fumigatus‐Kulturfiltrat‐Antigens wurden mit dem Gesamtantigen bei der Verwendung des ELISA‐Testes für die Serodiagnose der allergischen Aspergillose verglichen. Die Fraktionen wurden mit der Sephadex G‐200 Chromatographie präpariert. Anschließend wurde eine Analyse durch Immunelektrophorese gegen Anti‐Aspergillus‐fumigatus‐Kaninchenserum und mit dem ELISA‐Test gegen Seren von Patienten mit allergischer Aspergillose durchgeführt. Der zeitliche Ablauf der Antigen‐ und der IgG‐Absorption wurde im ELISA‐Test bestimmt. Beide Antigen‐Typen wurden rasch an die Festphase adsorbiert. Die Adsorption von spezifischem Anti‐Aspergillus‐fumigatus‐IgG erfolgte sehr schnell an beide Antigene und steigerte sich mit der Inkubationszeit. Mit dem Gesamt‐Antigen wie mit Antigen‐Fraktionen ließ sich spezifisches IgG bei dem Patienten mit allergischer Aspergillose nachweisen. Bei Verwendung des Gesamtantigens zeigten alle Patienten mit allergischer Aspergillose höhere Anti‐Aspergillus‐fumigatus‐IgG‐Titer als eine Gruppe von Asthma‐Patienten mit Präzipitinen gegen Aspergillus fumigatus. Bei den Untersuchungen mit den fraktionierten Antigenen war mit der Ausnahme eines Serums ebenfalls ein höherer spezifischer IgG‐Titer in der Gruppe mit allergischer Aspergillose im Vergleich zur Kontrollgruppe chronischer Asthmatiker nachzuweisen.

Characterization of antigens from Aspergillus fumigatus. V. Reactivity in immunodiffusion tests with serums from patients with aspergillosis caused by Aspergillus flavus, A. niger, and A fumigatus.

Antigen-rich mycelial extract (ME) and fractions of Aspergillus fumigatus were compared with culture filtrates of A. fumigatus. A. flavus, and A. niger for their abilities to detect antibody in serums from patients with confirmed aspergillosis caused by these fungi. In double immunodiffusion tests, homologous antigen preparations were more effective than heterologous preparations in detecting precipitins. Mycelial extract of A. fumigatus was more effective than A. fumigatus culture filtrate in detecting antibody in serums from patients with disease caused by A. flavus and A. niger. It did not, however, detect antibody in the serums from approximately half of the patients with heterologous infections.