Intranasal exposure to a damp building mould, Stachybotrys chartarum, induces lung inflammation in mice by satratoxin-independent mechanisms.

BACKGROUND: Stachybotrys chartarum is a damp building mould and a potent toxin producer that has been related to serious cases of respiratory health problems. However, the direct link between exposure and health symptoms has not been established. OBJECTIVE: To examine the mechanism by which exposure to spores of satratoxin producing and non-producing S. chartarum strains induce inflammatory responses in murine lungs. METHODS: BALB/c mice were intranasally exposed for 3 weeks to spores of a satratoxin-producing and a non-producing S. chartarum strain. Inflammatory cell infiltration was characterized from bronchoalveolar lavage (BAL) fluid. Cytokine and chemokine mRNA expression in lung tissue was measured with real-time PCR. Bronchial responsiveness to methacholine (MCh) was determined by whole-body plethysmography and serum antibody levels by ELISA. RESULTS: A dose-dependent increase in monocytes, neutrophils and lymphocytes was observed in BAL fluid after intranasal (i.n.) instillation of S. chartarum spores. There was no difference in the BAL between exposure to the satratoxin-producing and the non-producing strains. Infiltration of inflammatory cells was associated with an induction of pro-inflammatory cytokine (IL-1beta, IL-6 and TNF-alpha) and chemokine (CCL3/MIP-1alpha, CCL4/MIP-1beta and CCL2/MCP-1) mRNA levels in the lungs. Interestingly, CXCL5/LIX was the only chemokine that showed significantly higher mRNA levels after exposure to the satratoxin-producing strain compared with the non-producing strain. MCh-induced bronchial responsiveness was not altered significantly after mould instillation. Moreover, no significant increase in total or specific IgE, IgG2a and IgG1 antibody levels were found after S. chartarum exposure. CONCLUSION: These results indicate that lung inflammation induced by i.n. instillations of S. chartarum spores is regulated by the induction of pro-inflammatory cytokines and leucocyte-attracting chemokines. The data also imply that S. chartarum-derived components, other than satratoxins, are mediating the development of this inflammatory response.

Detection of aflatoxins (G(1-2), B(1-2)), sterigmatocystin, citrinine and ochratoxin A in samples contaminated by microbes.

A method is described for the simultaneous determination of common aflatoxins (G1, G2, B1, B2) and their precursor sterigmatocystin, and also citrinine and ochratoxin A. The method was applied to a building material matrix artificially contaminated with mycotoxin-producing fungi. The method includes extraction, sample pre-treatment and reversed-phase HPLC separation with tandem mass spectrometric identification and quantification using electrospray ionisation on a quadrupole ion trap mass analyser (ESI-MS-MS). Aqueous methanol was used in the initial extraction and solvent partitioning and solid phase extraction in the purification of samples. The HPLC separation was run on-line with the ESI-MS-MS detection. The limit of quantification of the procedure was 200 ng for all compounds. Recoveries of the sample pre-treatment varied from 28 to 99%. The average compound- and concentration-dependent accuracy and precision (RSD) were 21 and 113%, respectively. The method includes small sample volumes (approximately 1 g in 20 ml) and few, non-labour intensive, sample treatment steps. It should allow for a high throughput of samples with good prospects of automation.

PCR-based typing of Mycobacterium avium isolates in an epidemic among farmed lesser white-fronted geese (Anser erythropus).

Mycobacterium avium is an important veterinary pathogen causing avian tuberculosis in birds. The aim of the study was to evaluate the genetic relatedness in M. avium isolates from deep tissues of farmed lesser white-fronted geese with avian tuberculosis and in samples from the farm environment. The strains were analyzed by two PCR-based typing methods, inverted repeat (IR) typing and random amplified polymorphic DNA (RAPD) analysis. The primers for the inverted repeats of the insertion sequences IS1245 and IS1311 were used in IR typing, and the RAPD analysis was performed with six primers. Seven of the nine avian strains yielded an identical pattern in the IR typing, but they could be divided into two groups in the RAPD analysis. The remaining two bird isolates had an identical IR pattern (IR cluster II) which they shared with two environmental isolates. However, the RAPD analysis revealed that these environmental isolates had a RAPD pattern (RAPD cluster VI) distinct and different from either of the bird isolates (RAPD clusters II and IV). In all, four M. avium strains were verified as being inducers of avian tuberculosis in birds, and all were distinct from the three environmental strains identified. Thus, the results did not confirm the preliminary idea that a single strain had caused the epidemic. The polymorphism among M. avium strains highlighted the great biodiversity among an M. avium population even in a limited environmental setting during a short time span, and indicated the high susceptibility to avian tuberculosis of lesser white-fronted geese.

Mycotoxins in crude building materials from water-damaged buildings.

We analyzed 79 bulk samples of moldy interior finishes from Finnish buildings with moisture problems for 17 mycotoxins, as well as for fungi that could be isolated using one medium and one set of growth conditions. We found the aflatoxin precursor, sterigmatocystin, in 24% of the samples and trichothecenes in 19% of the samples. Trichothecenes found included satratoxin G or H in five samples; diacetoxyscirpenol in five samples; and 3-acetyl-deoxynivalenol, deoxynivalenol, verrucarol, or T-2-tetraol in an additional five samples. Citrinine was found in three samples. Aspergillus versicolor was present in most sterigmatocystin-containing samples, and Stachybotrys spp. were present in the samples where satratoxins were found. In many cases, however, the presence of fungi thought to produce the mycotoxins was not correlated with the presence of the expected compounds. However, when mycotoxins were found, some toxigenic fungi usually were present, even if the species originally responsible for producing the mycotoxin was not isolated. We conclude that the identification and enumeration of fungal species present in bulk materials are important to verify the severity of mold damage but that chemical analyses are necessary if the goal is to establish the presence of mycotoxins in moldy materials.

Study of toxin production by isolates of Stachybotrys chartarum and Memnoniella echinata isolated during a study of pulmonary hemosiderosis in infants.

A cluster of cases of pulmonary hemosiderosis among infants was reported in Cleveland, Ohio, during 1993 and 1994. These unusual cases appeared only in infants ranging in age from 1 to 8 months and were characterized by pulmonary hemorrhage, which caused the babies to cough up blood. A case-control study identified major home water damage (from plumbing leaks, roof leaks, or flooding) as a risk factor for development of pulmonary hemorrhage in these infants. Because of an interest in the possibility that trichothecene mycotoxins might be involved in this illness, a number of isolates of Stachybotrys chartarum were grown in the laboratory on rice, and extracts were prepared and analyzed both for cytotoxicity and for specific toxins. Two isolates of Memnoniella echinata, a fungus closely related to S. chartarum, were also included in these studies. S. chartarum isolates collected from the homes were shown to produce a number of highly toxic compounds, and the profiles of toxic compounds from M. echinata were similar; the most notable difference was the fact that the principal metabolites produced by M. echinata were griseofulvins.

Effects of intranasal exposure to spores of Stachybotrys atra in mice.

The effects of highly toxic and nontoxic spores of Stachybotrys atra were investigated in mice after six intranasal administrations of 1 x 10(5) and 1 x 10(3) spores in phosphate-buffered saline during a 3-week period. Toxic spores contained the trichothecene mycotoxins, satratoxins G and H, as well as the immunosuppressant stachybotrylactones and -lactams. No trichothecenes were detected in the nontoxic spores, and they contained only minor amounts of stachybotrylactones and -lactams. In mice injected with toxic and nontoxic spores, the platelet count was decreased and leucocyte and erythrocyte counts, hemoglobin concentration, and hematocrit were increased. No IgG antibodies to S. atra were detected in sera of mice exposed intranasally to spores. No histological changes were detected in spleen, thymus, or intestines of mice. The mice receiving 1 x 10(5) toxic spores intranasally developed severe inflammatory changes within both bronchioles and alveoli. Hemorrhage was detected in alveoli. The mice receiving 1 x 10(5) nontoxic spores also developed inflammatory changes in the lungs, but these changes were significantly milder than those in mice receiving toxic spores. The mice receiving 1 x 10(3) toxic spores developed inflammatory changes in the lungs that were less severe than those in the mice receiving 1 x 10(5) toxic spores. No inflammatory changes were detected in the mice receiving 1 x 10(3) of nontoxic spores. The present findings indicate that exposure to S. atra spores containing toxins (satratoxins) can be a significant health risk.

Bacteria, molds, and toxins in water-damaged building materials.

Microbial toxins and eukaryotic cell toxicity from indoor building materials heavily colonized by fungi and bacteria were analyzed. The dominant colonizers at water-damaged sites of the building were Stachybotrys chartarum (10(3) to 10(5) visible conidia cm-2), Penicillium and Aspergillus species (10(4) CFU mg-1), gram-negative bacteria (10(4) CFU mg-1), and mycobacteria (10(3) CFU mg-1). The mycobacterial isolates were most similar to M. komossense, with 98% similarity of the complete 16S rDNA sequence. Limulus assay of water extracts prepared from a water-damaged gypsum liner revealed high contents of gram-negative endotoxin (17 ng mg-1 of E. coli lipopolysaccharide equivalents) and beta-D-glucan (210 ng mg-1 of curdlan equivalents). High-performance liquid chromatography analysis of the methanol extracts showed that the water-damaged gypsum liner also contained satratoxin (17 ng mg-1). This methanol-extracted substance was 200 times more toxic to rabbit skin and fetus feline lung cells than extract of gypsum liner sampled from a non-water-damaged site. The same extract contained toxin(s) that paralyzed the motility of boar spermatozoa at extremely low concentrations; the 50% effective concentration was 0.3 microgram of dry solids per ml. This toxicity was not explainable by the amount of bacterial endotoxin, beta-D-glucan, or satratoxin present in the same extract. The novel in vitro toxicity test that utilized boar spermatozoa as described in this article is convenient to perform and reproducible and was a useful tool for detecting toxins of microbial origin toward eukaryotic cells not detectable in building materials by the other methods.

Experimental lung mycotoxicosis in mice induced by Stachybotrys atra.

Stachybotrys atra is often isolated from building materials in houses with moisture problems. Spores of S. atra can contain mycotoxins which may lead to various symptoms in exposed residents in damp houses. The pathogenesis of S. atra-induced lung diseases has not been elucidated. The purpose of the present study was to investigate lung mycotoxicosis experimentally in mice after an intranasal exposure to spores of S. atra-fungus. One group of mice received one intranasal injection of spores of a toxic strain of S. atra (1 x 10(6) spores) and the other group spores of a less toxic strain. Spores of both strains contained spirolactones and spirolactams while the highly toxic strain contained also trichothecene mycotoxins, satratoxins. The spores containing satratoxins caused severe intra-alveolar, bronchiolar and interstitial inflammation with haemorrhagic exudative processes in the alveolar and bronchiolar lumen. A significant difference was observed in the severity of the lung damage caused by the two strains of S. atra. The spores without satratoxins induced a milder inflammation, so that the toxic compounds of S. atra-spores are most likely responsible for the severity of the lung injury.

Toxigenic molds in water-damaged buildings: dechlorogriseofulvins from Memnoniella echinata.

An investigation of a cluster of cases of pulmonary hemosiderosis in infants in Cleveland, OH, led to the isolation of many isolates of Stachybotrys atra and two isolates of a related toxigenic fungus, Memnoniella echinata. M. echinata produces two cytotoxic trichothecene mycotoxins, trichodermol (1a) and trichodermin (1b), as well as several griseofulvins. Dechlorogriseofulvin (2a) and epidechlorogriseofulvin (2b) were the major compounds isolated. This is the first report of a fungus outside the Penicillium genus producing griseofulvins.

Characterization of the mouse gene for the alpha 1 chain of type XVIII collagen (Col18a1) reveals that the three variant N-terminal polypeptide forms are transcribed from two widely separated promoters.

The mouse gene for the alpha 1 chain of type XVIII collagen (Col18a1) is more than 102 kb and consists of 43 exons. Type XVIII collagen transcripts encode polypeptides that differ with respect to three variant N-terminal noncollagenous domains that are 301 (NC1-301), 517 (NC1-517), or 764 (NC1-764) residues in length. Characterization of genomic clones revealed that the three variant NC1 domains result from the use of two alternative promoters, separated by a distance of 50 kb. The upstream promoter, promoter 1, directs the synthesis of the NC1-301 domain in conjunction with exons 1 and 2, whereas the downstream promoter, promoter 2, directs that of the NC1-517 and NC1-764 domains in conjunction with exon 3, with the latter two variants differing with respect to alternative splicing of the exon 3 sequences. Exons 4-9 encode a portion of the NC1 domain shared by all three polypeptide variants, and exons 9-43 encode the common collagenous and C-terminal noncollagenous sequences. The marked differences previously observed in the expression of variant type XVIII collagen transcripts in mouse tissues thus result from tissue-specific use of these two promoters.

Comparison of detection methods for trichothecenes produced by Fusarium sporotrichioides on fodder and grains at different air humidities.

Growth and toxin production of a highly toxic strain of Fusarium sporotrichioides Sherb were studied on oat and wheat grains and on straw under experimental conditions, in which relative humidity (RH) of air was regulated. The materials were incubated at three different RH levels at a range of 84-100%. F. sporotrichioides grew well on oat and wheat grains at RH 97-100% but grew less well at RH 84-88% and on straw. Toxin production was measured with three biological toxicity tests (cytotoxicity test, dermotoxicity test, and yeast cell toxicity test), with chemical analysis, and T-2 ELISA assay. Cytotoxicity and production of trichothecene mycotoxins were detected in all the samples incubated at all three RH levels. On oat and wheat grains, T-2 toxin, neosolaniol, and diacetoxyscirpenol were found, and on straw T-2 toxin, HT-2 toxin, neosolaniol, and T-2 tetraol were determined. In the T-2 ELISA assay, all material samples were found to contain T-2 toxin. The cytotoxicity test was the most sensitive method for detecting biological toxicity of samples inoculated with fungus. The T-2 ELISA assay and chemical analysis were about equally sensitive to detect T-2 toxin in samples.

Stachybotrys atra Growth and Toxin Production in Some Building Materials and Fodder under Different Relative Humidities.

Growth of Stachybotrys atra and its toxin production on some building materials and in animal fodder were studied at relative humidities ranging from 78 to 100%. Toxins were detected by biological assays and chemical methods. Strong growth of the fungus and presence of macrocyclic trichothecenes, mainly satratoxins G and H, were detected on wallpaper and gypsum boards and in hay and straw at saturation conditions. On pine panels, S. atra grew well, but neither biological toxicity nor production of macrocyclic trichothecenes was observed.

Primary structure of the alpha 1 chain of mouse type XVIII collagen, partial structure of the corresponding gene, and comparison of the alpha 1(XVIII) chain with its homologue, the alpha 1(XV) collagen chain.

We have isolated cDNAs that complete the elucidation of the primary structure of the mouse alpha 1(XVIII) collagen chain, a polypeptide homologous to the alpha 1(XV) collagen chain. The 1315-residue alpha 1(XVIII) chain includes a 25-residue signal peptide, a 301-residue NH2-terminal non-collagenous domain (NC1), a 674-residue collagenous sequence with nine interruptions of 10-24 residues, and a 315-residue COOH-terminal noncollagenous domain (NC11). Seven of the collagenous domains and both flanking noncollagenous domains share homology with the alpha 1(XV) chain. The COOH-terminal noncollagenous domains are unique to the alpha 1(XVIII) and alpha 1(XV) chains, and they contain a homologous beginning, a variable portion, and a highly homologous COOH-terminal half with 4 conserved cysteines. The differences in the collagenous sequences probably preclude the existence of the two chains in the same molecule, however. A 12.5-kilobase pair genomic sequence was found to contain the 12 extreme 3'-exons of the alpha 1(XVIII) gene, covering 40% of the coding sequences. Exons start with either a complete codon or a split codon for the glycines of Gly-Xaa-Yaa repeats, and seven exons completely cover the NC11 domain. Comparison of the sequences encoded by these seven exons with the corresponding region of the alpha 1(XV) gene indicated conserved exon-intron organization, suggesting that the two genes derive from a common ancestor.

Occurrence of Trichothecenes in feed and cereals in Finland.

In 1985 82 samples of feed and food grain were analyzed for trichothecenes deoxynivalenol (DON), nivalenol (NV), diacetoxyscirpenol (DAS), T-2 toxin, HT-2 toxin and fusarenon-X (F-X). Trichothecenes were found in 77 of these samples. The highest amounts were DON 6300 ug/kg and DAS 1680 ug/kg.In 1986, in a corresponding study of 113 samples, trichothecenes were found in 110 samples. A new trichothecene in Finland, 3-acetyldeoxynivalenol (3-AcDON), was identified in 35 of these samples in concentration of 2-211 ug/kg.Analyzing methods were gas chromatography and GC-mass spectrometry. It is characteristic of the feed samples suspected of causing outbreaks in animals in Finland that several trichothecenes are often found in the same sample. As an example of this is a poultry feed with following results: DON 33 ug/kg, 3-AcDON 21 ug/kg, DAS 45 ug/kg, T-2 toxin 40 ug/kg, HT-2 toxin 12 ug/kg.

Variation of F-2 toxin production on different substrates.

The F-2 producing capacity of one Fusarium graminearum strain (strain No. 13) and of three hyphal tip transverse lines (a b and c) isolated from the original strain and of a mixture of these lines (a b and c) was studied in two successive years on different substrates: oats, barley, wheat, grain mixture and wheat bran. In the first year the original strain produced high amounts of F-2 but was heterogenous in toxin production. The F-2 producing capacity of one of the hyphal tip transverse lines (a) was high and that of two lines (b and c) and of the mixture of the three lines (a+b+c) was poor. In the following year the F-2 producing capacity of the fungal cultures had changed: the F-2 producing capacity of the original strain (No. 13) was greatly reduced, that of one hyphal tip transverse line (b) remained poor and that of two hyphal tip transverse lines (a and c) and of the mixture of the lines clearly increased. The F-2 production was changed in all the substrates and in about the same proportion. In general oats was the best substrate for F-2 production. The possible causes of the changed F-2 production are discussed. The estrogenic effect of F-2 produced in different substrates was studied by using as criteria the uterine weight, vaginal opening and liquid content in the uterus of immature female rats. The effect was in direct proportion to the amount of F-2 ingested by the rats. The substrate was without any influence on the physiological effect of F-2, and in this respect our results deviate from some earlier findings.