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, 71 (1), 114-22

Detection of Airborne Stachybotrys Chartarum Macrocyclic Trichothecene Mycotoxins on Particulates Smaller Than Conidia

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Detection of Airborne Stachybotrys Chartarum Macrocyclic Trichothecene Mycotoxins on Particulates Smaller Than Conidia

T L Brasel et al. Appl Environ Microbiol.

Abstract

Highly respirable particles (diameter, <1 microm) constitute the majority of particulate matter found in indoor air. It is hypothesized that these particles serve as carriers for toxic compounds, specifically the compounds produced by molds in water-damaged buildings. The presence of airborne Stachybotrys chartarum trichothecene mycotoxins on particles smaller than conidia (e.g., fungal fragments) was therefore investigated. Cellulose ceiling tiles with confluent Stachybotrys growth were placed in gas-drying containers through which filtered air was passed. Exiting particulates were collected by using a series of polycarbonate membrane filters with decreasing pore sizes. Scanning electron microscopy was employed to determine the presence of conidia on the filters. A competitive enzyme-linked immunosorbent assay (ELISA) specific for macrocyclic trichothecenes was used to analyze filter extracts. Cross-reactivity to various mycotoxins was examined to confirm the specificity. Statistically significant (P < 0.05) ELISA binding was observed primarily for macrocyclic trichothecenes at concentrations of 50 and 5 ng/ml and 500 pg/ml (58.4 to 83.5% inhibition). Of the remaining toxins tested, only verrucarol and diacetylverrucarol (nonmacrocyclic trichothecenes) demonstrated significant binding (18.2 and 51.7% inhibition, respectively) and then only at high concentrations. The results showed that extracts from conidium-free filters demonstrated statistically significant (P < 0.05) antibody binding that increased with sampling time (38.4 to 71.9% inhibition, representing a range of 0.5 to 4.0 ng/ml). High-performance liquid chromatography analysis suggested the presence of satratoxin H in conidium-free filter extracts. These data show that S. chartarum trichothecene mycotoxins can become airborne in association with intact conidia or smaller particles. These findings may have important implications for indoor air quality assessment.

Figures

FIG. 1.
FIG. 1.
Experimental air sampling apparatus. Filtered house air at a flow rate of 30 liters/min (LPM) was passed over cellulose ceiling tile with confluent S. chartarum growth for various periods of time. A total of six gas-drying tubes representing approximately 1,176 cm2 of S. chartarum growth were connected by using polyvinyl chloride (PVC) tubing. Particles were separated and collected on 47-mm-diameter polycarbonate membrane filters with pore sizes of 5.0 μm (filter A), 1.2 μm (filter B), and 0.4 μm (filter C) and later were analyzed for the presence of macrocyclic trichothecenes.
FIG. 2.
FIG. 2.
Scanning electron micrographs of polycarbonate membrane filters following 72 h of sampling from the air sampling apparatus. The filter pores are clearly distinguishable from captured particulate matter (dark round and irregularly shaped bodies, respectively). (A) Filter (pore size, 5.0 μm) with a captured S. chartarum spore. Magnification, ×5,000. (B) The same type of filter with an intact S. chartarum spore lodged in a pore (arrowhead). Magnification, ×2,000. (C) Filter (pore size, 1.2 μm) with no S. chartarum spores but with a significant amount of debris. Magnification, ×2,500. (D and E) Filters (pore size, 0.4 μm) with extremely small captured particulates (arrowheads). Magnification, ×10,000.
FIG. 3.
FIG. 3.
ELISA-based macrocyclic trichothecene standard curve. Equal concentrations of satratoxins G and H, roridin A, and verrucarin A were mixed in methanol, and the preparation was diluted in PBS (500 to 0.1 ng/ml) and tested by using a macrocyclic trichothecene-specific ELISA. Optical densities at 450 nm (OD450) were plotted against toxin concentrations in a power curve. This standard curve was used to estimate macrocyclic trichothecene equivalents for experimental filter extracts. For reference, the trichothecene concentrations, optical densities at 450 nm, and percent inhibition values are indicated. The standard deviations (based on three replicates) for all values are also indicated.
FIG. 4.
FIG. 4.
HPLC chromatograms of filter extracts from 120-h sampling. The retention times (in minutes) are plotted on the x axis. The peak sizes (in milliabsorbance units) are plotted on the y axis. For UV spectrum analyses (insets), wavelengths (in nanometers) are plotted on the x axis. (A) Satratoxin H standard (indicated by an asterisk) at a concentration of 1 ng/μl, with a retention time of 9.777 min. The inset shows the UV spectrum of the toxin with a maximum absorbance near 235 nm. (B, C, and D) Chromatograms of extracts from the 5-, 1.2-, and 0.4-μm-pore-size filters, respectively, that were used in a 120-h sampling experiment. The 5-μm-pore-size filter extract clearly shows the presence of satratoxin H with a retention time of 9.482 min (indicated by an asterisk). The results of UV spectral analyses are also presented (inset). The 1.2- and 0.4-μm-pore-size filter extract chromatograms have major peaks at 9.845 and 9.848 min, respectively, that could be satratoxin H. UV analyses demonstrated that these two peaks were qualitatively similar to the peak for purified satratoxin H.

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