Cultivation and aerosolization of Stachybotrys chartarum for modeling pulmonary inhalation exposure

Abstract

Objective:Stachybotrys chartarum is a hydrophilic fungal species commonly found as a contaminant in water-damaged building materials. Although several studies have suggested that S. chartarum exposure elicits a variety of adverse health effects, the ability to characterize the pulmonary immune responses to exposure is limited by delivery methods that do not replicate environmental exposure. This study aimed to develop a method of S. chartarum aerosolization to better model inhalation exposures. Materials and methods: An acoustical generator system (AGS) was previously developed and utilized to aerosolize and deliver fungal spores to mice housed in a multi-animal nose-only exposure chamber. In this study, methods for cultivating, heat-inactivating, and aerosolizing two macrocyclic trichothecene-producing strains of S. chartartum using the AGS are described. Results and discussion: In addition to conidia, acoustical generation of one strain of S. chartarum resulted in the aerosolization of fungal fragments (<2 µm aerodynamic diameter) derived from conidia, phialides, and hyphae that initially comprised 50% of the total fungal particle count but was reduced to less than 10% over the duration of aerosolization. Acoustical generation of heat-inactivated S. chartarum did not result in a similar level of fragmentation. Delivery of dry, unextracted S. chartarum using these aerosolization methods resulted in pulmonary inflammation and immune cell infiltration in mice inhaling viable, but not heat-inactivated S. chartarum. Conclusions: These methods of S. chartarum growth and aerosolization allow for the delivery of fungal bioaerosols to rodents that may better simulate natural exposure within water-damaged indoor environments.

Keywords: Fungi; acoustical generator; fungal aerosolization; fungal exposure; fungal fragments; inhalation exposure.

Figure 1.

An illustration depicting the acoustical generation system (AGS) used to aerosolize S. chartarum spores. Conidia-laden rice grains are placed on a rubber membrane within the generator where acoustical energy is used to aerosolize the S. chartarum spores. Spores can then be delivered to an exposure chamber where real-time particle size and concentration data are collected using the aerodynamic particle sizer (APS) and DataRAM 4.

Figure 2.

Images presenting the growth of viable S. chartarum IBT 9460 and IBT 7711 on grains of white rice. Individual rice grains were almost entirely covered in sporulation S. chartarum.

Figure 3.

Representative field emission scanning electron microscopy images of viable and heat-inactivated S. chartarum conidia derived from strains IBT 9460 and IBT 7711 illustrating no difference in conidia morphology following heat inactivation. (A) IBT 9460 viable conidia, (B) IBT 9460 heat-inactivated conidia, (C) IBT 7711 viable conidia, (D) IBT 7711 heat-inactivated conidia. Magnification, ×5000.

Figure 4.

Polyacrylamide gel electrophoresis analysis of S. chartarum conidia extracts prior to and after heat-inactivation. MW- molecular weight marker; Lane 1- IBT 9460 viable conidia extract; Lane 2- IBT 9460 heat-inactivated conidia extract; Lane 3- IBT 7711 viable conidia extract; Lane 4- IBT 7711 heat-inactivated conidia extract.

Figure 5.

Aerodynamic particle size distributions following aerosolization of viable and heat-inactivated S. chartarum conidia. The top 2 panels show the viable and heat-inactivated IBT 9460 aerosols captured during the early (≤20 min) and late (≥60 min) stages of aerosolization. The bottom panel shows the aerodynamic particle size distributions following aerosolization of S. chartarum IBT 7711 conidia, which remained constant throughout the course of the aerosolization. The graphs shown here are representative of the size distributions observed over multiple aerosolization experiments and have been normalized based on the particle count concentration observed within the chamber for comparison.

Figure 6.

The aerodynamic particle size distributions of the smaller fragments (0.5-2 μm) derived from viable S. chartarum IBT 9460 after 5 (solid line), 20 (dashed line) and 40 (dotted line) minutes of aerosolization. The size distributions at each time point have been normalized based on the particle count concentration observed within the chamber for comparison.

Figure 7.

Field emission scanning electron microscopy images of fragments derived from viable S. chartarum strain IBT 9460. Fragments appear to be derived from both conidia and hyphae and range in size from <0.5 μm to 2 μm. (A) Magnification, ×100,000; (B) Magnification, ×60,000; (C) Magnification, ×70,000; (D) Magnification, ×45,000; (E) Magnification, ×30,000; (F) Magnification, ×20,000.

Figure 8.

UPLC/MSMS quantification of verrucarol present on polycarbonate filters sampled within the acoustical generator chamber following S. chartarum aerosolization. Verrucarol derived from aerosolized viable and heat-inactivated IBT 9460 and IBT 7711 strains. Based on the number of spores estimated to be deposited on the filter by real-time APS measurements, verrucarol (pmol) per 1 x 10⁶ S. chartarum spores was calculated. Statistically significant differences were observed between strains (p <0.01) but not as a result of heat-inactivation.

Figure 9.

Representative photomicrographs of murine lung sections following exposure to S. chartarum IBT 9460 (top panel) or IBT 7711 (bottom panel). Images shown represent air-only control (n=3), heat-inactivated (n=3/strain), or viable (n=3/strain) S. chartarum exposures. Airway inflammation (indicated by the black arrowheads) was observed following exposure to both viable strains of S. chartarum, but not following exposure to heat-inactivated S. chartarum. Images were captured using a 20X objective.

Figure 10.

Changes in bronchoalveolar lavage cell populations after exposure to S. chartarum IBT 9460 or IBT 7711. Graphs represent the fold change in cell number compared to air-only controls for total BAL cells, B cells, CD4⁺ T cells and CD8⁺ T cells as determined by flow-cytometry. Statistically significant increases in each cell population were observed following exposure to viable, but not heat inactivated S. chartarum IBT 9460 (** represents a p <0.01). Increases, although not significant, were also seen following exposure to viable S. chartarum IBT 7711 (* represents p=0.05).