Indirect Immunodetection of Fungal Fragments by Field Emission Scanning Electron Microscopy

Abstract

dSubmicronic fungal fragments have been observed in in vitro aerosolization experiments. The occurrence of these particles has therefore been suggested to contribute to respiratory health problems observed in mold-contaminated indoor environments. However, the role of submicronic fragments in exacerbating adverse health effects has remained unclear due to limitations associated with detection methods. In the present study, we report the development of an indirect immunodetection assay that utilizes chicken polyclonal antibodies developed against spores from Aspergillus versicolor and high-resolution field emission scanning electron microscopy (FESEM). Immunolabeling was performed with A. versicolor fragments immobilized and fixed onto poly-L-lysine-coated polycarbonate filters. Ninety percent of submicronic fragments and 1-to 2-m fragments, compared to 100% of >2-m fragments generated from pure freeze-dried mycelial fragments of A. versicolor, were positively labeled. In proof-of-concept experiments, air samples collected from moldy indoor environments were evaluated using the immunolabeling technique. Our results indicated that 13% of the total collected particles were derived from fungi. This fraction comprises 79% of the fragments that were detected by immunolabeling and 21% of the spore particles that were morphologically identified. The methods reported in this study enable the enumeration of fungal particles, including submicronic fragments, in a complex heterogeneous environmental sample. P ersonal exposure to fungal aerosols in damp buildings has been associated with respiratory morbidity (1, 2). Fungal spore exposure levels in residential indoor environments seem too low to explain such an association (3). Submicronic fungal fragments observed in vitro during aerosolization experiments with fungal cultures are believed to contribute to the respiratory health problems observed in moldy indoor environments (4, 5). However, this role of submicronic fragments has remained unclear due to limitations associated with their quantification.Airborne fungal particles have been shown to include spores in addition to larger and smaller (submicronic) fragments of spores and hyphae. These fragments may constitute a significant reservoir for antigens, allergens, and toxins in addition to spores. To date, the quantification of submicronic fungal fragments has remained technically challenging in environmental samples due to the lack of adequate detection and enumeration methods (6, 7). In this regard, the evaluation of the exposure burden of fungal submicronic fragments in fungally contaminated environments has been underestimated. In vitro studies that have evaluated the release of submicronic fragments have provided insight into the aerodynamic characteristics as well as the abiotic factors that influence the release of these particles. These laboratory studies of common indoor fungal isolates have shown the need to include the enumeration of submicronic fragments in addition to spores and larger fragments during expos...

Fungal aerosol composition in moldy basements

“…Our AM of outdoor levels were close to outdoor levels reported in Danish strawberry fields: 41 × 10 3 m −3 , but were substantially higher than that of hyphal fragments discernible by light microscopy reported in United Kingdom by Pady and Kramer: 1.4 × 10 3 m −3 , in Canada by Li and Kendrick: 0.11 × 10 3 m −3 and in Southern California (USA) by Delfino and co‐workers: 0.20 × 10 3 m −3 . On the other hand, these levels were far lower than those recorded in occupational settings like during renovation of moldy walls: 600 × 10 3 m −3 , work in grain farming: 3000 × 10 3 m −3 , and both studies from Norway and during seed processing work in Denmark: 360‐870 × 10 3 m −3 . Regarding the overall indoor concentrations of fragments, they were many folds higher than those reported by Li and Kendrick: 0.15 × 10 3 m −3 .…”

“…For details, photographs of the sampling sites are shown in Figure . Stationary sampling was adopted following the procedure described previously but with minor adjustments. Briefly, airborne particles were sampled onto poly‐L‐lysine‐coated polycarbonate filters mounted in open 37‐mm‐diameter standard aerosol SKC cassettes (SKC Inc).…”

“…The filter specimens were then mounted onto a 25‐mm carbon tab attached to a circular supporting metal grid and placed in a 6‐well cell culture plate for downstream treatments. Immunogold labeling of fragments was performed following the procedure previously described . Briefly, free aldehyde binding sites were first quenched with 0.02 M glycine (MERCK) before being blocked with 5% skimmed milk freshly dissolved in Tris‐buffered saline (pH 8, MERCK) and containing 0.05% Tween 20 (TBSTSM).…”

“…While spores were recognized by their morphology, fragments were identified as of fungal origin when labeled with gold particles. Significantly, labeled submicron fragments and larger fragments required at least two and four visible gold particles, respectively, so to distinguish fragments of fungal origin from background staining . The two types of particles were counted in 100 randomly selected fields imaged at 3000× magnification.…”

Bedroom Allergen Exposure Beyond House Dust Mites

“…Allergens are dispersed by airborne spores and fungal fragments, which can be highly variable in size. However, most spores from allergenic genera are smaller than 10 μm and fragments are even smaller in size, allowing them to penetrate deeply into the airways [132, 133]. …”