Hemolysis, toxicity, and randomly amplified polymorphic DNA analysis of Stachybotrys chartarum strains

Abstract

Stachybotrys chartarum is an indoor air, toxigenic fungus that has been associated with a number of human and veterinary health problems. Most notable among these has been a cluster of idiopathic pulmonary hemorrhage cases that were observed in the Cleveland, Ohio, area. In this study, 16 strains of S. chartarum isolated from case (n = 8) or control (n = 8) homes in Cleveland and 12 non-Cleveland strains from diverse geographic locations were analyzed for hemolytic activity, conidial toxicity, and randomly amplified polymorphic DNA banding patterns. In tests for hemolytic activity, strains were grown at 23 degrees C on wet wallboard pieces for an 8-week test period. Conidia from these wallboard pieces were subcultured on sheep's blood agar once a week over this period and examined for growth and clearing of the medium at 37 or 23 degrees C. Five of the Cleveland strains (all from case homes) showed hemolytic activity at 37 degrees C throughout the 8-week test compared to 3 of the non-Cleveland strains. Five of the Cleveland strains, compared to two of the non-Cleveland strains, produced highly toxic conidia (>90 microgram of T2 toxin equivalents per g [wet weight] of conidia) after 10 and 30 days of growth on wet wallboard. Only 3 of the 28 strains examined both were consistently hemolytic and produced highly toxic conidia. Each of these strains was isolated from a house in Cleveland where an infant had idiopathic pulmonary hemorrhage.

Figures

FIG. 1

Appearance of strain 58-06 conidia on SBA after conidia on filters were heat treated (left) or not heat treated (right). After 5 weeks of growth on wallboard, conidia from strain 58-06 were directly blotted onto borosilicate filters. The filter on the left was heat treated at 86°C for 20 min, and the one on the right was held at 23°C. Filters were placed on SBA and incubated at 37°C for 10 days.

FIG. 2

Phylogram of the 11 strains of S. chartarum which were highly toxic and/or consistently hemolytic. The phylogram presented is the most parsimonious tree inferred from the binary data (Table 5) by using the branch-and-bond option in PAUP 3.1. The scale bar represents the distance resulting from one character change. Distance analysis of the data set was performed in PAUP to determine which strain was the least similar. This strain was used as the outgroup in the phylogenetic evaluation. Values appearing above the branches are percentages of 1,000 bootstrap analysis replicates in which the branches were found. Only percentages greater than 50% are shown. A strain number followed by an asterisk indicates a strain that came from an IPH case house in Cleveland.

FIG. 3

Phylogram of the seven strains of S. chartarum which were consistently hemolytic. The phylogram presented is the most parsimonious tree inferred from the binary data (Table 5) by using the branch-and-bond option in PAUP 3.1. The scale bar represents the distance resulting from one character change. Distance analysis of the data set was performed in PAUP to determine which strain was the least similar. This strain was used as the outgroup in the phylogenetic evaluation. Values appearing above the branches are percentages of 1,000 bootstrap analysis replicates in which the branches were found. Only percentages greater than 50% are shown. A strain number followed by an asterisk indicates a strain that came from an IPH case house in Cleveland.

FIG. 4

Phylogram of the seven highly toxic strains of S. chartarum. The phylogram presented is the most parsimonious tree inferred from the binary data (Table 5) by using the branch-and-bond option in PAUP 3.1. The scale bar represents the distance resulting from one character change. Distance analysis of the data set was performed in PAUP to determine which strain was the least similar. This strain was used as the outgroup in the phylogenetic evaluation. Values appearing above the branches are percentages of 1,000 bootstrap analysis replicates in which the branches were found. Only percentages greater than 50% are shown. A strain number followed by an asterisk indicates a strain that came from an IPH case house in Cleveland. Strains 51-06 and 58-02 were significantly different from the other highly toxic strains, as indicated by the 90 on the line connecting to the rest of the strains.