Immunobiochemical analysis of cross-reactive glutathione-S-transferase allergen from different fungal sources

Clinical observations suggest the presence of cross-reactive allergens. There is a need to identify these cross-reactive allergens to improve the treatment used for allergic disorders. The present study was aimed to identify and characterize a cross-reactive allergenic protein from fungi. Allergen extracts of various fungi viz. Alternaria alternata, Aspergillus fumigatus, Cladosporium herbarum, Curvularia lunata, and Epicoccum purpurascens showed GST enzymatic activity ranging from 0.765 to 1.004 delta340 nm/min/microg where as activity of rGST was 1.123 delta340 nm/min/microg. Immunoblot with GST antibodies showed a band of approximately 26 kDa in all these fungal extracts. Sera of fungal allergy patients showed the presence of IgE antibodies to GST. Rabbit antibodies raised against the fungal extracts reacted with rGST confirming the presence of GST-like protein in these extract. ELISA inhibition using GST antibodies revealed inhibition with C. herbarum, A. alternata, C. lunata, A. fumigatus, and E. purpurascens demonstrating that fungal GST competes for binding to anti-GST. In summary, a GST-like protein was recognized as cross-reactive allergen in these fungal extracts.     

Description of a Novel Panallergen of Cross-Reactivity between Moulds and Foods

The present investigation is undertaken to demonstrate a novel cross-reactivity between aeroallergens (moulds fungi imperfecti) and allergens from foods (spinach and mushroom Agaricus bisporus). We have performed a dual study in vivo and in vitro, in a population of atopic patients. Data from in vivo tests performed with spinach and mushroom have been statistically analysed. To the in vitro assays, mushroom and spinach extracts have been obtained, and sera from moulds allergic patients analysed by means of IgE–immunoblott assays. Inhibition experiments have been also performed to study a possible relation between proteins. Statistical analysis of data showed a relation between allergenicity to moulds (Alternaria alternata, Cladosporium herbarum and/or Aspergillus fumigatus), and positive skin prick tests with mushroom and/or spinach. The immunoblotts performed showed that seven moulds allergic patients had a strong recognition of a protein with a molecular weight of about 30 kD present both in spinach and mushroom extracts, and by means of inhibition assays we could determine that these two proteins were related. This study demonstrates the existence of a new allergen responsible for cross reactivity between moulds and two frequently consumed foods, mushroom and spinach. We conclude that a novel cross-reactive allergen between aeroallergens and foods has been identified.     

Protease-dependent activation of epithelial cells by fungal allergens leads to morphologic changes and cytokine production

Background: Proteases in extracts of Aspergillus fumigatus cause epithelial cell desquamation and release of proinflammatory cytokines. Objective: We sought to assess protease activity in Alternaria alternata , Cladosporium herbarum , and Aspergillus fumigatus extracts and study the ability of these extracts to cause desquamation and release of proinflammatory cytokines from epithelial cells. Methods: Protease activities of the fungal extracts were quantified. Changes with respect to cell morphology, cell desquamation, and cytokine production (IL-6 and IL-8) were measured in the absence and presence of the fungal extracts in an airway-derived epithelial cell line (A549) and primary epithelial nasal cells. Results: Fungal proteases differentially induced morphologic changes, cell desquamation, and production of IL-6 and IL-8 in a dose- and time-dependent fashion. Alternaria alternata extracts induced cell shrinking and cell desquamation and strongly enhanced the production of IL-6 and IL-8 at higher concentrations. Aspergillus fumigatus extracts caused cell shrinking, cell desquamation, and production of IL-6 and IL-8, even at low concentrations. The Aspergillus fumigatus –derived extract grown on collagen medium induced a strong dose-dependent decline in cytokine production at higher concentrations. Cladosporium herbarum extracts did not induce morphologic changes or cell desquamation but enhanced IL-6 and IL-8 productions at higher concentrations. The dependence of these effects on intact protease activity was shown by their abrogation by protease inhibitors. Conclusion: Proteases present in fungal extracts interact with epithelial cells, leading to morphologic changes, cell desquamation, and induction of proinflammatory cytokines. It is proposed that these fungal proteases may activate epithelial cells through a protease-activated receptor type 2–driven mechanism. (J Allergy Clin Immunol 2000;105:1185-93.)     

Pulmonary immune responses to Aspergillus fumigatus in an immunocompetent mouse model of repeated exposures

Abstract

Aspergillus fumigatus is a filamentous fungus that produces abundant pigmented conidia. Several fungal components have been identified as virulence factors, including melanin; however, the impact of these factors in a repeated exposure model resembling natural environmental exposures remains unknown. This study examined the role of fungal melanin in the stimulation of pulmonary immune responses using immunocompetent BALB/c mice in a multiple exposure model. It compared conidia from wild-type A. fumigatus to two melanin mutants of the same strain, Δarp2 (tan) or Δalb1 (white). Mass spectrometry-based analysis of conidial extracts demonstrated that there was little difference in the protein fingerprint profiles between the three strains. Field emission scanning electron microscopy demonstrated that the immunologically inert Rodlet A layer remained intact in melanin-deficient conidia. Thus, the primary difference between the strains was the extent of melanization. Histopathology indicated that each A. fumigatus strain induced lung inflammation, regardless of the extent of melanization. In mice exposed to Δalb1 conidia, an increase in airway eosinophils and a decrease in neutrophils and CD8⁺ IL-17⁺ (Tc17) cells were observed. Additionally, it was shown that melanin mutant conidia were more rapidly cleared from the lungs than wild-type conidia. These data suggest that the presence of fungal melanin may modulate the pulmonary immune response in a mouse model of repeated exposures to A. fumigatus conidia.     

Studies on shared antigenic/allergenic components among fungi

Fungal allergens have been found to be one of the most prevalent aeroallergens in India. Knowledge of shared/unique components among different fungi is necessary for proper diagnosis and treatment of patients allergic to fungi. In the present study, crude extracts (CE) of 11 common fungi (Alternaria alternata, Aspergillus flavus, Asp. fumigatus, Asp. niger, Asp. tamarii, Asp. versicolor, Cladosporium herbarum, Curvularia lunata, Mucor hiemalis, Penicillium citrinum, and Fusarium solani) were characterized by isoelectric focusing (IEF), SDS-PAGE, and immunoblot. On IEF (pI 3–9), the number of protein bands was found to be greatest (46) in M. hiemalis extract. SDS-PAGE exhibited a varied number of bands, generally 18–40, with mol. mass ranging from 14 to 100 kDa. IgG-specific immunoprint using rabbit anti-F. solani CF antibodies demonstrated a mol. mass distribution of shared antigenic proteins of 14–100 kDa in most of the fungi. Shared allergenicity was observed in a number of allergenic proteins in fungal extracts with mol. mass ranging between 14 and 70 kDa on IgE-specific immunoblot using pooled sera of patients allergic to Fusarium. A 45-kDa protein was found to be common among these fungi on immunoblot with patients as well as with rabbit antibodies. F. solani CF extract contained more antigenic/allergenic proteins than F. solani CE. It was concluded that F. solani CF shared several antigenic/allergenic components with CE of other common fungi. This fact needs to be taken into account when fungal extracts are used in diagnosis and immunotherapy of allergic patients.     

Standardization of allergen extracts by inhibition of RAST, skin test, and chemical composition

Five allergen extracts of Dermatophagoides pteronyssinus, Lolium perenne, Alternaria tenuis, Aspergillus fumigatus and Cladosporium herbarum, obtained from four different manufacturers, were examined by inhibition of RAST, content of protein and carbohydrate, contents of phosphorylcholine (Pc) and tridacnin reactive components, and by skin test. Inhibition of RAST was used as a primary method for establishing allergenic potency and demonstrated wide variations for each preparation supplied by the different manufacturers. The extracts also varied widely in protein and carbohydrate content and in the ratio of these parameters, indicating internal heterogeneity. Pc content was significantly related to RAST potency for extracts of A. fumigatus and A. tenuis, suggesting that Pc content may be used as a primary standarization procedure for these extracts. Skin test reactions undertaken at a single concentration did not show any significant variation in weal size between preparations of a given allergen extract. However, of particular importance to practising clinicians is the finding that varying numbers of patients showed negative skin reactions to one preparation of a particular allergen yet were positive to the corresponding preparations supplied by the other companies.     

Fungal antigens as a source of sensitization and respiratory disease in Scottish maltworkers

Mycological and serological studies were carried out as part of a survey of respiratory disease in Scottish maltworkers. 70% of stained sputum smears from 574 workers showed the presence of higher plant cells and/or mycelia, and the spores of common environmental fungi. Penicillium spp. (90/%), Rhizopus stolonifer (48%) and yeasts (53%) were the dominant fungi in 699 sputum cultures, and showed a similar proportional distribution in 327 samples of grain, malt, culms and dusts from fifty-six makings. 57% of 711 men were serologically positive for fungi, 22% for Aspergillus fumigatus, 20% for A. clavatus, 10% for A. niger, 16% for Cladosporium herbarum and over 3% for Rhizopus stolonifer. 6% of 132 men were positive for Penicillium cyclopium. No precipitating antibodies to antigens from Alternaria tenuis, Aureobasidium pullulans, Candida albicans, Geotrichum candidum, Rhodotorula glutinis or Trichoderma viride were detected in tests of forty sera. Sera from the 5.2% of men with symptoms of extrinsic allergic alveolitis showed increased reactivity to mycelial antigens from Aspergillus clavatus. The fungus was cultured from 21 % of maltings, 7% of all environmental samples and from the sputa of 8% of maltworkers.     

Production of melanin pigments in saprophytic fungi in vitro and during infection

Melanins are one of the great natural pigments produced by a wide variety of fungal species that promote fitness and cell survival in diverse hostile environments, including during mammalian infection. In this study, we sought to demonstrate the production of melanin in the conidia and hyphae of saprophytic fungi, including dematiaceous and hyaline fungi. We showed that a melanin‐specific monoclonal antibody (MAb) avidly labeled the cell walls of hyphae and conidia, consistent with the presence of melanin in these structures, in 14 diverse fungal species. The conidia of saprophytic fungi were treated with proteolytic enzymes, denaturant, and concentrated hot acid to yield dark particles, which were shown to be stable free radicals, consistent with their identification as melanins. Samples obtained from patients with fungal keratitis due to Fusarium falciforme, Aspergillus fumigatus, Aspergillus flavus, Curvularia lunata, Exserohilum rostratum, or Fonsecaea pedrosoi were found to be intensely labeled by the melanin‐specific MAb at the fungal hyphal cell walls. These results support the hypothesis that melanin is a common component that promotes survival under harsh conditions and facilitates fungal virulence. Increased understanding of the processes of melanization and the development of methods to interfere with pigment formation may lead to novel approaches to combat these complex pathogens that are associated with high rates of morbidity and mortality.     

Dosages des IgG antiaviaires et antimoisissures sur UniCAP dans l'alvéolite allergique extrinsèque et l'aspergillose bronchopulmonaire allergique

Immunofluorimetric determination of IgG specific antibodies may be realised with UniCAP system (Pharmacia). We have measured IgG titers against birds (budgerigar, parrot, pigeon, canary) , thermophilic actinomycetes (Thermoactinomyces vulgaris, Micropolyspora faeni) and molds (Aspergillus fumigatus and vulgaris, Penecillium sp, Cladosporium herbarum and cladosporoïdes, Stachybotrys atra, Alternaria tenuis) in 12 patients with clinically proven hypersensitivity pneumonitis. We observed elevated IgG in 83% of the subjects using normal values established in our laboratory. On the contrary, in 15 patients for whom diagnostic was only suspected, IgG where normal or not significanty elevated. Mold IgG antibodies interest is limited to inacurate choice of the antigen rather than to the absence of disease. The results must always be interpreted in accordance with clinic. Anti-Aspergillus fumigatus antibodies where realised in 43 patients with asthma or cystic fibrosis. Among six patients with clinical proven allergic bronchopulmonary aspergillosis, only 3 had high IgG titers. Despite of those false negative results, measurement of IgG especially againts birds and Aspergillus fumigatus is an useful diagnostic tool in the diagnosis of pneumopathy caused by fungi or birds.     

The association of lung shadowing with hypersensitivity responses in patients with allergic broncho-pulmonary aspergillosis

The levels of total serum IgE and IgE antibodies to Aspergillus fumigatus allergens were highest during an episode of symptoms associated with fresh pulmonary shadowing and a rise in the direct cosinophil count in patients with allergic bronchopulmonary aspergillosis. Agglutinin titres were greater than those found in the sera of control subjects hut did not always correlate in magnitude with total serum IgE and IgE antibody levels. The highest agglutinin titre was found in the serum of a patient with an aspergilloma in association with allergic aspergillosis. The maximum in vitro histamine release from leucocytes exposed to A. fumigatus extracts or anti-human IgE was independent of the concentration of total IgE or IgL antibodies. The leucocytes appeared to he relatively insensitive to both allergic and reverse allergic histamine release. This was considered to result from either continuous in vivo desensitization of the leucocytes by antigens shed from the fungal hyphae or, to the effect of drugs used in the treatment of (he disease, It was concluded that serial serum IgE measurements may be of value in monitoring the onset of the active phase of this disease.     

Optimisation of a 2-D gel electrophoresis protocol for the human-pathogenic fungus Aspergillus fumigatus

Aspergillus fumigatus is the most important airborne fungal pathogen causing life-threatening infections in immunosuppressed patients. One of the important questions concerning A. fumigatus is the identification of pathogenicity determinants. To obtain a comprehensive overview about the proteins produced at different physiological conditions that are related to the infectious process a proteomic approach has been applied. Here, 2-D gel electrophoresis for filamentous fungi was optimised concerning removal of interfering compounds, protein extraction and separation methods. A trichloroacetic acid-based precipitation method of proteins with their subsequent solubilisation by the use of a combination of CHAPS with a second sulfobetaine detergent gave the best results. The optimised protocol was evaluated by the analysis of the proteomes of A. fumigatus grown on two different carbon sources, i.e., glucose and ethanol. Carbon catabolite repression has not been studied in detail at the protein level in A. fumigatus yet. In addition, growth on ethanol leads to activation of the glyoxylate cycle which was shown to be essential for pathogenesis in bacteria and fungi. In A. fumigatus, differential patterns of enzymes of the gluconeogenesis, glyoxylate cycle and ethanol degradation pathway during growth on glucose and ethanol were observed.     

Genomic analysis of allergen genes in Aspergillus spp: the relevance of genomics to everyday research.

Full genomic sequencing of Aspergillus fumigatus and other genomes has allowed correction of Aspergillus allergen gene sequences and requires revision of Genbank and IUIS sequences of allergens. In addition allergens in other fungal species may be found in the aspergilli. We compared the published sequences of numerous allergens with recently available genome sequences. This analysis suggests that Aspf 56KD, Asp f 15 and Asp f 16 should be removed from the approved allergen list and that Asp f 17 is a larger protein than published. Additionally we propose likely gene candidates for Asp f GST (Afu6g09690) and Asp o lipase (AO090701000644). We suggest that the heat shock allergens should be re-classified: Asp f 12 should be classified as HSP90 (Asp f 12), HSP88 (Mala s 10) and HSP70 (Alt a 3, Cla h 4 and Pen c 19) according to human gene nomenclature. Comparison of fungal allergen databases with genome sequences suggests the presence of a core set of allergen - like proteins in all fungi. We also analysed allergens in the 3 sequenced aspergilli to look for internal homologies and this suggests that multi gene families may produce numerous cross-reactive allergens.     

Molecular cloning of fungal allergens and clinical applications of recombinant allergens in fungal allergy]

A large number of fungi are associated with allergic disorders. There are many problems in using non-standardized fungal extracts for diagnosis of fungal allergy. These problems can be solved by using genetically engineered recombinant allergens. At present, more than 70 fungal allergens have been cloned and sequenced, and the recombinant forms of several of these are commercially available. Measurement of IgE antibodies to these commercially available recombinant allergens could provide the tools useful for characterizing the differential sensitization pattern in relation to a particular disease and the allergenic cross-reactivity among fungi. Only a limited number of recombinant allergens are available at present, thus further studies on the molecular biology of fungal allergens are needed so that more recombinant allergens can be used in the clinical field.     

Rhizosphere fungi of five plants in Egypt

75 species which belong to 25 genera were collected during this investigation. All of these fungi were previously isolated from Egyptian soils and other sources. The total count and the number of genera and species in the rhizosphere soil of the 5 plants were regularly higher than in non-rhizosphere soil. The composition of the rhizosphere fungus flora was considerably influenced by the type and the age of the plant. The most prevalent fungi in the rhizosphere of the test plants after 45, 90, and 150 days were as follows: in Helianthus annuus, Stachybotrys atra and Aspergillus niger; in Chrysanthemum coronarium, Cladosporium herbarum, A. sydowii and Penicillium funiculosum; in Nigella sativa, Fusarium moniliforme and A. sydowii; in Datura innoxia, A. fumigatus and A. terreus; in Hyoscyamus muticus, C. herbarum and A. sydowii; and in soil, P. funiculosum, A. terreus and A. niger. The counts of total fungi and most of the common fungal species, except in the case of Datura innoxia, reached their maxima after 90 days and sharply declined after 150 days.     

Prevalence of precipitating antibodies to different extracts of Aspergillus fumigatus in a North American asthmatic population

This study was carried out to find the prevalence of precipitin reactions in the sera of 200 North American asthmatic subjects. Precipitins were detected by the double diffusion technique using different extracts of Aspergillus fumigatus, including a reference ‘home produced’ extract and five commercial extracts from three different suppliers. In addition, antigenicity of these extracts was assessed by crossed immunoelectrophoresis (XIE). Of the sera, 13.5% reacted to the reference extract and from 2.5 to 12% to the different commercial extracts; 22.5% of the sera reacted to at least one extract. No one serum reacted to all the extracts. Two of fifty-one (4%) non-atopic patients with a negative immediate prick test to A. fumigatus, six of eighty-seven (6.9%) atopic patients with a negative immediate reaction to A. fumigatus, and thirty-seven of sixty two (59%) atopic patients with a positive immediate reaction to A. fumigatus had precipitins to at least one of the extracts used, the skin tests being performed using the A. fumigatus reference extract. The prevalence of precipitin reactions bore a strong correlation with the antigenicity of the extracts by XIE. The same reference extract was also used for specific IgE measurements (Brompton extract, Malo & Paquin, 1979). It was found that patients with precipitins had significantly (P < 0.001) higher specific and total IgE values than patients without precipitins. In the group of patients with positive skin test, those with precipitins had significantly (P<0.05) higher specific IgE values than those without. The authors conclude that different extracts of A, fumigatus should be used to assess the presence of precipitins. The antigenicity of these extracts should also be assayed.     

Evaluation of inhibitory effects of citric and tartaric acids and their combination on the growth of Trichophyton mentagrophytes, Aspergillus fumigatus, Candida albicans, and Malassezia furfur

The present study was conducted to evaluate the effects of citric and tartaric acids and their combination on the growth inhibition of some important pathogenic fungi in vitro. Trichophyton mentagrophytes var. mentagrophytes, Candida albicans, Aspergillus fumigatus, and Malassezia furfur were cultured into specific media, and subsequently, fungal conidia were harvested from the medium surface and counted by hemacytometer method. The antifungal susceptibility test of citric and tartaric acids and their combination against fungi were assayed by broth macrodilution technique. The results demonstrated that citric acid had more fungistatic and fungicidal activities than those of tartaric acid against all pathogenic fungi tested, and its effect on filamentous fungi was higher than that on the yeasts. Antifungal activity of the acid combination was similar to citric acid but higher than tartaric acid alone. Further research is needed to assess the efficacy of citric and tartaric acids as inhibitors of fungal growth in clinical trials, especially in treatment of patients with fungal infections.     

Allergens in Aspergillus fumigatus

Aspergillus fumigatus grown in submerged and surface cultures was extracted, and the extracts were analyzed separately. The submerged extract contained 31.9% protein and 8.3% carbohydrate, while the corresponding values were 17.0% and 33.3% for the surface material. With individual sera from patients with allergic asthma, SDS-PAGE combined with immunoblotting revealed that the submerged extract contained at least six strong IgE-binding components (20, 30, 38, 50, 68, and 90 kDa) in addition to several weak to medium IgE-binding components. The surface extract contained about the same number of IgE-binding components, but only one gave a strong reaction (20 kDa). The allergens present were shown to have pI between 4.5 and 5.6 as demonstrated by isoelectric focusing (IEF) combined with immunoblotting. For identification of A. fumigatus glycoprotein allergens, both extracts were treated with periodate under mild conditions. Two allergens of the submerged extract (90 and 38 kDa) partly lost their IgE-binding ability by this treatment, indicating that these components are glycoproteins and that the carbohydrate moiety is involved in the IgE binding. The IgE-binding ability of the 20-kDa allergen was not influenced by periodate. For assessment of the stability of the two allergen extracts, aqueous solutions were kept at 4°C for 2, 7, and 21 d and then analyzed by SDS-PAGE and immunoblotting. The results showed that most allergens of the submerged extract were partly inactivated after 2 d. After 21 d, only the 20-kDa and 30-kDa components were still able to bind IgE. Similar results were obtained by analyzing the surface extract. When the same experiment was performed on samples in a 50% glycerol solution, the results strongly indicated that glycerol had a stabilizing effect on allergens in both extracts. The enzyme content, estimated by the API ZYM-test, showed that both extracts contained several protein- and carbohydrate-degrading enzymes. The presence of these enzymes may explain the lability of the extracts.     

Automated specific IgE assay with recombinant allergens: evaluation of the recombinant Aspergillus fumigatus allergen I in the Pharmacia CAP System

Background We report the results of a study comparing the recombinant Aspergiilus fumigatus allergen I (rAsp f I) to commercial A. jumigatus extracts in serological assays, Pharmacia CAP System and skin tests. Objective The study was designed to test the feasibility of using recombinant allergens in an automated serology system for determination of allergen-specific IgE. Methods Patients with allergic bronchopulmonary aspergillosis (ABPA), asthmatics with A. fumigatus allergy and control subjects, who included allergic asthmatics without allergy to A. fumigatus and healthy subjects, were investigated. All subjects were characterized with respect to their total IgE level, radio allergosorbent test to A. fumigatus and skin test reactivity to both commercial A. fumigatus extracts and recombinant rAsp f I protein. Results All patients with ABPA (n = 30) showed positive skin test reactions with commercial A. fumigatus extracts, and 24 were sensitized to r Asp f I by the same criterion. The 10 patients with asthma and A. fumigatus allergy showed positive skin reactions to at least one commercial extract, and five reacted to r Asp f I. AU control subjects (H= 19) scored negatively in skin tests to A. fumigatus extracts and r Asp f I, and showed no detectable rAsp f I-specific IgE. ImmunoCAP carrying immobilized r Asp f I were evaluated using sera from all individuals described and the results compared with those obtained with the r Asp f I-specific ELISA for IgE. The data obtained with the two r Asp f I-specific detection systems correlated closely (r= 0.997) and were in perfect agreement with the skin test results. Conclusion The data show that r Asp f I can be used as immobilized allergen in the Pharmacia CAP System indicating the feasibility of using recombinant allergens for an automated serological diagnosis of allergic diseases. However, every recombinant allergen needs to be evaluated individually for its performance if applied to a new diagnostic technology.     

Isolation and immunological characterization of a novel Cladosporium herbarum allergen structurally homologous to the α/β hydrolase fold superfamily

Because the ascomycete Cladosporium herbarum embodies one of the most important, world-wide occurring fungal species responsible for eliciting typical IgE-mediated hypersensitivity reactions ranging from rhinitis and ocular symptoms to severe involvement of the lower respiratory tract, a more comprehensive definition of its detailed allergen repertoire is unquestionably of critical medical as well as therapeutic significance. By screening a C. herbarum cDNA library with IgE antibodies pooled from 3 mold-reactive sera, we were able to identify, clone and affinity-purify a novel allergen candidate (29.9 kDa) exhibiting considerable (three-dimensional) homology to the α/β hydrolase fold superfamily. The latter covers a collection of hydrolytic enzymes of widely differing phylogenetic origin as well as catalytic activity (operating in countless biological contexts) that in general exhibit only little sequence similarity yet show a remarkable conservation of structural topology. Our present study (i) characterizes recombinant non-fusion C. herbarum hydrolase as a natively folded, minor mold allergen that displays a prevalence of IgE reactivity of approximately 17% in our in vitro immunoblot experiments, (ii) proposes the existence of several putative (speculatively cross-reactive) ascomycete orthologues as determined via genome-wide in silico predictions, and (iii) finally implies that C. herbarum hydrolase could be included in forthcoming minimal testing sets when fungal allergy is suspected.     

Susceptibility of adult pigeons and hybrid falcons to experimental aspergillosis

Aspergillosis caused by Aspergillus fumigatus seems to be more prevalent in some avian species than in others. We compared the development of aspergillosis in 8-month-old Gyr-Saker hybrid falcons and 8-month-old pigeons after a single intratracheal inoculation of different dosages of A. fumigatus conidia (10⁷, 10⁵ and 10³). Clinical signs, including vomiting, discoloration of the urates, loss of appetite and dyspnoea, were observed in four out of five falcons and in four out of five pigeons inoculated with 10⁷ A. fumigatus conidia. Necropsy revealed the presence of granulomas in the air sacs and/or lungs in four out of five falcons and in four out of five pigeons in the high dosage group. A. fumigatus was isolated from these granulomas in three falcons and in three pigeons. The presence of fungal hyphae was detected with Periodic acid Shiff reagent staining in three out of five falcons and in three out of five pigeons in the high dosage group. Avian respiratory macrophages were clearly present in and around the fungal granulomas. In the other dosage groups, no granulomas, positive A. fumigatus cultures or fungal hyphae were present, except for one falcon in the middle dosage group in which a sterile granuloma without fungal hyphae was noticed. In conclusion, the study shows that adult falcons and pigeons are susceptible to aspergillosis after inoculation of a single dose of conidia intratracheally.