Fungal Contamination in Public Buildings: Health Effects and Investigation Methods

2. Health Effects of Indoor Molds (Continued)

2.3 Animal Studies

Several experimental studies with animal models exposed to fungal cells, antigens or constituents have found effects similar to those observed in humans in epidemiological studies, such as eosinophilia and increased serum IgE.

• Twenty-four adult albino guinea pigs inhaled daily 8 mg of a Penicillium chrysogenum extract nebulized in phosphate-buffered saline (PBS), and two of them were sacrificed each week (up to 12 weeks). Twelve other guinea pigs were handled the same way, but received only nebulized PBS, and one of them was sacrificed each week. No histopathological lesion was found in control animals throughout the experiment, but interstitial infiltrates appeared in the alveoles of Penicillium-treated animals after four weeks, and granulomas appeared after 10 weeks. Also, specific IgM and IgG antibodies to P. notatum were detectable in Penicillium-treated animals after seven weeks (Alonso et al. 1998).
• A similar experiment carried out using a Rhizopus nigrans extract yielded similar results (i.e. IgG antibodies to R. nigrans in serum after seven weeks, and interstitial infiltrates four weeks and granulomas after 10 weeks in the alveoles of Rhizopus-treated animals) (Alonso et al. 1997).
• Male and female guinea pigs were exposed to aerosols containing either 30 µg/m³ of (1->3)-ß-D-glucan (a fungal cell component), 75 µg/m³ of Escherichia coli lipopolysaccharide (a bacterial endotoxin), both, or the vehicle only (controls). Animals were exposed four hours per day, five days per week for five weeks and then sacrificed. Cell counts were determined in Bronchoalveolar lavage (BAL) fluid and in lung interstitium. Macrophages were increased by endotoxin (BAL fluid ρ<0.001, interstitium ρ<0.05) and by glucan+endotoxin (BAL fluid ρ<0.001, interstitium ρ<0.05), but not by glucan alone. Lymphocytes were increased in BAL fluid by endotoxin (ρ<0.05) and glucan+endotoxin (ρ<0.01), but the highest response was observed with glucan alone (ρ<0.001 in BAL fluid and interstitium). Neutrophils were increased in BAL fluid by endotoxin (ρ<0.001) and glucan+endotoxin (ρ<0.001), but not by glucan. Eosinophils were strongly increased by glucan in both BAL fluids and interstitium (ρ<0.001), slightly increased by glucan+ endotoxin in BAL fluid only (ρ<0.05) and not affected by endotoxin (Fogelmark et al. 2001).
• C57BL/6 mice aged six to eight weeks were sensitized to Aspergillus antigens (100 µg in 50 µl saline) three successive days a week for three weeks, and sacrificed after one, two or three weeks. Control mice were handled the same way, but were administered 50 µl saline instead of the antigen solution. Total cells counts were strongly increased in sensitized mice compared to controls at weeks one, two and three (ρ<0.02 at each week). The proportion of macrophages in BAL cells remained constant at 97% over weeks in control mice, while in Aspergillus-treated mice the proportion of macrophages decreased (34% at week one) and the proportion of lymphocytes, neutrophils and eosinophils increased. The neutrophil counts in BAL fluid reached (62.2±14.4) × 10⁴ cells at week two and (78.2±29.1) × 10⁴ cells at week three in Aspergillus-treated mice, compared to (0.1±0.1) × 10⁴ cells in control mice throughout the study (ρ<0.05) (Wang et al. 1994).
• Groups of six to nine female C57B1/6 mice were inoculated intranasally with 50 µl saline containing either no fungal conidia (control group), 10⁴ nonviable (methanol-treated) conidia or 10⁴ untreated conidia (of which 25±5% were viable) of Penicillium chrysogenum once a week for six weeks, and were sacrificed 24 hours after the last inoculation. P. chrysogenum conidia were isolated from a building affected by building-related illness. Mean total IgE levels in serum were 804 ng/ml (SD 301 ng/ml) in controls, 833 ng/ml (SD 339 ng/ml) in animals treated with non-viable conidia, and 2627 ng/ml (SD 1778 ng/ml) in animals treated with viable conidia. Mean percentage of eosinophils over total peripheral white blood cells were 5.3% (SD 0.8%) in controls, 7.3% (SD 1.7%) in animals treated with non-viable cells, and 10.9% (SD 0.9%) in animals treated with viable conidia. Finally, mean eosinophil counts in BAL were 0.22 (SD 0.44) per 1000 BAL cells in controls, 0.5 (SD 0.55) in animals treated with non-viable conidia, and 14.17 (SD 5.91) in animals treated with untreated conidia. Total serum IgE, peripheral eosinophil count and BAL eosinophil counts were significantly higher in the group treated with viable conidia than in controls (ρ<0.05) (Cooley et al. 2000).

Also, some studies found severe hemorrhagic responses induced by Stachybotrys chartarum spores.

• Four-day-old Sprague-Dawley rat pups were instillated intratracheally with 1.0 to 8.0×10⁵ intact spores (suspended in saline) per gram of body weight, similar suspensions of spores treated with ethanol to remove trichothecene toxins, or with saline only. Animals were sacrificed on their 7th or 12th day of life. Cell counts of macrophages, lymphocytes and neutrophils were increased two-fold, five-fold, and seven-fold, respectively, in the bronchoalveolar lavage fluid of animals treated with 1.1×10⁵ intact spores/g compared to those treated with saline only or with ethanol- treated spores (ρ<0.001 for each cell type). There was no difference between the two latter groups. Hemoglobin concentration in BAL fluids in animals treated with intact spores, ethanol-treated spores and saline were 2.46±0.33 mg/ml, 1.26±0.16 mg/ml and 1.22 mg/ml, respectively; the difference between groups was significant (ρ=0.004). The S. chartarum strain used in this study had been isolated in the water-damaged house of an infant that was part of the Cleveland outbreak (Yike et al. 2002). These findings indicate that mycotoxins (or another constituent removed by the ethanol treatment) may be responsible for inflammatory and hemorrhagic response of the infant lung to S. chartarum.

Other studies with rodents exposed to Stachybotrys chartarum showed effects on lung physiology that may be mediated by different mechanisms.

• Carworth Farms White mice were intranasally instilled with 50 ml saline containing either 10⁷ Cladosporium cladosporioides conidia per ml, 10⁷ Stachybotrys chartarum conidia per ml, or 10^-7 M of isosatratoxin F; another group was untreated (control group). For each treatment, groups of two to four mice were sacrificed 0, 12, 24, 48 and 72 hours post- exposure. None of the mice, regardless of treatment, showed any apparent clinical sign of respiratory distress or sickness. The phospholipid composition of lung surfactant was significantly modified from 12 hours to the end of the experiment following exposure to S. chartarum conidia and isosatratoxin, while following C. cladosorioides exposure small changes were observed at 48 hours post-exposure only (Mason et al. 1998).
• Groups of five Carworth Farms White mice were intranasally instilled with 50 ml saline containing either 1.4×10⁶ Cladosporium cladosporioides conidia per ml, 1.4×10⁶ Stachybotrys chartarum conidia per ml, or 10 µg/ml of isosatratoxin F, or with saline only. Animals were sacrificed after 24 hours. In vitro conversion of a biologically active form of alveolar surfac tant to a biologically inactive form was significantly higher in surfactant of S. chartarum-treated mice than in those of all other groups, including controls; other treatment groups were not different from controls with respect to that end-point (Mason et al. 2001).
• Stachybotrys chartarum spores suspended in saline were instilled into mouse trachea, and mice were sacrificed 24 hours later. Exposure to S. chartarum induced an overall reduction of phospholipid content in alveolar surfactant. The relative distribution of phospholipids across surfactant fraction and the nature of surfactant phospholipids were also altered (McCrae et al. 2001).
• Groups of five to six mice were inoculated intratracheally with 50 ml saline containing either 1.4×10⁶ Cladosporium cladosporioides conidia per ml, 1.4×10⁶ Stachybotrys chartarum conidia per ml, or 0.02 µg/ml of isosatratoxin F, or with saline only. No difference was observed between alveolar type II cells of control, saline-treated or C. cladosporioides-treated animals, while alveolar type II cells from mice treated with either S. chartarum spores or isosatratoxin F showed remarkable ultrastuctural changes compared to controls (Rand et al. 2002).