David C. Straus

Detection of Airborne Stachybotrys chartarum Macrocyclic Trichothecene Mycotoxins on Particulates Smaller than Conidia

ABSTRACT Highly respirable particles (diameter, <1 μm) 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.

Role of salivary protease activity in adherence of gram-negative bacilli to mammalian buccal epithelial cells in vivo.

Serious illness is accompanied by markedly increased susceptibility to colonization of the respiratory tract by gram-negative bacilli and an increase in the number of such organisms which adhere to regional epithelial cells during incubation in vitro. Trypsinization of cells from normal subjects causes a similar increase in bacillary adherence. We studied bacillary adherence to buccal cells in vitro, protease activity of upper respiratory secretions with a fibrin plate technique, and the amount of fibronectin on the surface of buccal cells with a direct radioimmunobinding assay. Among 10 patients seriously ill with acute respiratory failure bacillary adherence to buccal cells and protease activity in secretions were increased compared with controls and cell-surface fibronectin was decreased; all patients were colonized in vivo with gram-negative bacilli. These changes were persistent and 80% of the patients died. Serial determinations were made in eight patients undergoing coronary artery bypass surgery. Following surgery, protease activity and bacillary adherence increased and cell-surface fibronectin decreased; 38% of coronary artery bypass patients became colonized. In these uncomplicated patients the changes observed were transient, largely returning to normal by the third postoperative day. Increased protease activity of secretions and alterations in epithelial cell surfaces as reflected by loss of buccal cell-surface fibronectin occur swiftly after major illness and appear to underlie enhanced cell adherence of bacilli and colonization of the upper respiratory tract. These findings suggest new approaches to the prevention of nosocomial pneumonia.

Detection of airborne Stachybotrys chartarum macrocyclic trichothecene mycotoxins in the indoor environment.

ABSTRACT The existence of airborne mycotoxins in mold-contaminated buildings has long been hypothesized to be a potential occupant health risk. However, little work has been done to demonstrate the presence of these compounds in such environments. The presence of airborne macrocyclic trichothecene mycotoxins in indoor environments with known Stachybotrys chartarum contamination was therefore investigated. In seven buildings, air was collected using a high-volume liquid impaction bioaerosol sampler (SpinCon PAS 450-10) under static or disturbed conditions. An additional building was sampled using an Andersen GPS-1 PUF sampler modified to separate and collect particulates smaller than conidia. Four control buildings (i.e., no detectable S. chartarum growth or history of water damage) and outdoor air were also tested. Samples were analyzed using a macrocyclic trichothecene-specific enzyme-linked immunosorbent assay (ELISA). ELISA specificity was tested using phosphate-buffered saline extracts of the fungal genera Aspergillus, Chaetomium, Cladosporium, Fusarium, Memnoniella, Penicillium, Rhizopus, and Trichoderma, five Stachybotrys strains, and the indoor air allergens Can f 1, Der p 1, and Fel d 1. For test buildings, the results showed that detectable toxin concentrations increased with the sampling time and short periods of air disturbance. Trichothecene values ranged from <10 to >1,300 pg/m3 of sampled air. The control environments demonstrated statistically significantly (P < 0.001) lower levels of airborne trichothecenes. ELISA specificity experiments demonstrated a high specificity for the trichothecene-producing strain of S. chartarum. Our data indicate that airborne macrocyclic trichothecenes can exist in Stachybotrys-contaminated buildings, and this should be taken into consideration in future indoor air quality investigations.

Airborne Microbial Flora in a Cattle Feedlot

ABSTRACT A total of 1,408 cattle held in eight commercial feedlot pens were used to examine the quantity and diversity of microorganisms in cattle feedlot air. The effect of two feeding patterns on the generation of airborne dust and the total numbers of microorganisms was also examined (four feedlot pens/treatment). Microbial samples were collected, and dust particles that were 2.5 μm or less in diameter were measured with a Dustrak monitor during the evening dust peak for 4 days at sites both upwind and downwind of the feedlot pens. An Andersen biological cascade sampler was employed with different medium and incubation combinations for the capture and identification of bacteria and fungi. The results showed that when bacteria were considered, only nonpathogenic gram-positive organisms were recovered. However, gram-negative bacteria may have been present in a viable but nonculturable state. Fungi were recovered in smaller numbers than bacteria, and none of the fungi were pathogenic. The Dustrak results showed that one feeding pattern resulted in cattle behavior that generated levels of downwind dust lower (P = 0.04) than the levels generated by the behavior resulting from the other feeding pattern. However, the Andersen sampler results showed that there were no differences between feeding patterns with regard to the total number or diversity of microorganisms. The disparity may have been due to the different operating principles of the two systems. The overall numbers of microorganisms recovered were lower than those reported in studies of intensively housed farm animals in which similar recovery techniques were used.

Molds, mycotoxins, and sick building syndrome

The following is a review of some of the work we have done since 2004 regarding the importance of molds and their mycotoxins in the phenomenon of sick building syndrome (SBS). In these studies we showed that the macrocyclic trichothecene mycotoxins (MTM) of Stachybotrys chartarum (SC) are easily dissociated from the surface of the organism as it grows and could therefore be consequently spread in buildings as the fungus experiences additional water events. We then showed that SC and Penicillium chrysogenum (PC) colonies remain viable long after a water source has been removed, and the MTM produced by SC remain toxic over extended periods of time. We next showed that PC when inhaled, can release in vivo, a protease allergen that can cause a significant allergic inflammatory reaction in the lungs of mice. We then showed, in a laboratory study, that the MTM of SC can become airborne attached to spores or SC particulates smaller than spores. Following that study, we next showed that the same phenomenon actually occurred in SC infested buildings where people were complaining of health problems potentially associated with SBS. Finally, we were able to demonstrate the presence of MTM in the sera of individuals who had been exposed to SC in indoor environments. This last study was done with enough mold exposed individuals to allow for the statistical significance of SC exposure to be evaluated.

Continually Measured Fungal Profiles in Sick Building Syndrome

Abstract. Buildings with indoor air quality (IAQ) complaints frequently have high airborne concentrations of Penicillium species, while buildings with few IAQ complaints have an indoor air (IDA) fungal ecology similar to outdoor air (ODA), where Cladosporium species is usually the dominant microorganism. These studies compared fungal air profiles, measured continually over 6 h in a documented sick building, in IDA in a room experiencing IAQ problems with fungal profiles measured concurrently in ODA. The dominant species collected at both sites were Penicillium species, Cladosporium species, and Alternaria species. In the IDA, Penicillium species were always the dominant organisms, ranging from 150 to 567 cfu/m3 (89.8–100% of the total fungi). In the ODA, Cladosporium species were dominant in four samples (40.0–70.6%), while Penicillium species were dominant (52.7–79.6%) in two. These data demonstrate that, even though ODA fungal profiles are changing continuously, IDA fungal profiles in “sick” buildings tend to remain unchanged.

Mycotoxin Detection in Human Samples from Patients Exposed to Environmental Molds

The goal of this study was to determine if selected mycotoxins (trichothecenes, aflatoxins, and ochratoxins) could be extracted and identified in human tissue and body fluids from patients exposed to toxin producing molds in their environment. Human urine and methanol extracted tissues and sputum were examined. Trichothecenes were tested using competitive ELISA techniques. Aflatoxins B1, B2, G1, and G2, and ochratoxin A were tested by using immunoaffinity columns and fluorometry. Test sensitivity and specificity were determined. Levels of detection for the various mycotoxins varied from 0.2 ppb for trichothecenes, 1.0 ppb for aflatoxins, and 2.0 ppb for ochratoxins. Trichothecene levels varied in urine, sputum, and tissue biopsies (lung, liver, brain) from undetectable (<0.2 ppb) to levels up to 18 ppb. Aflatoxin levels from the same types of tissues varied from 1.0 to 5.0 ppb. Ochratoxins isolated in the same type of tissues varied from 2.0 ppb to > 10.0 ppb. Negative control patients had no detectable mycotoxins in their tissues or fluids. These data show that mycotoxins can be detected in body fluids and human tissue from patients exposed to mycotoxin producing molds in the environment, and demonstrate which human tissues or fluids are the most likely to yield positive results.

The roles of Penicillium and Aspergillus in sick building syndrome.

Publisher Summary This chapter focuses on the importance of Penicillium spp. and Aspergillus spp. in inducing symptoms consistent with sick building syndrome (SBS) and provides experimental evidence for some of the symptoms caused by these organisms. Penicillium spp. are some of the most commonly isolated molds from contaminated buildings. Penicillium spp. and Aspergillus spp. play a major role in causing many of the allergic and respiratory symptoms seen in people exposed to high levels of spores and allergens from these organisms in contaminated buildings. Although the spores are ubiquitous and too small to filter from most indoor environments, better control of humidity, air handling, and plumbing as well as incorporation of mold-inhibiting building components should decrease the incidence of complaints related to these organisms. Although many allergens have been characterized from various species of Penicillium and Aspergillus and important animal studies have been conducted, much more evidence is needed to determine the exact mechanisms of sensitization to fungal spores and allergens.

Serotyping and Enzyme Characterization of Pasteurella haemolytica and Pasteurella multocida Isolates Recovered from Pneumonic Lungs of Stressed Feeder Calves

Abstract. Ninety-one isolates of Pasteurella multocida (Pm) and 124 of Pasteurella haemolytica (Ph) were recovered from the lungs of calves that died of bovine respiratory tract disease (BRTD). Nine Pm enzyme profiles (A through I) and 9 Ph enzyme profiles (J through R) were determined for the Pasteurella isolates. The Pm isolates were relatively evenly divided among the enzyme profiles, with one exception, profile I. The Ph isolates were not evenly distributed among the profiles. Fifty of the 91 Pm isolates were serotyped. Forty-two Pm isolates were positive for capsule type A, and 8 were untypable. Five somatic type antigen profiles (3; 3,4; 3,7; 3,4,7; and 4) were identified among the 50 serotyped Pm isolates; one isolate was untypable. The Ph isolates were further divided through serotyping and grouped as follows: 74 (60%) Pasteurella haemolytica A1 (PhA1), 12 (10%) PhA2, 4 (3%) PhA5, and 34 (27%) PhA6. Eighty-one percent of the Ph serotypes were clustered in the M and N enzyme profile. The P enzyme profile was almost unique to PhA2 (8 of 12, 67% of PhA2 isolates). Results of this study indicate a need to collect more data on Ph serotypes at the state veterinary diagnostic laboratories.

Effect of Chlorine Dioxide Gas on Fungi and Mycotoxins Associated with Sick Building Syndrome

ABSTRACT The growth of indoor molds and their resulting products (e.g., spores and mycotoxins) can present health hazards for human beings. The efficacy of chlorine dioxide gas as a fumigation treatment for inactivating sick building syndrome-related fungi and their mycotoxins was evaluated. Filter papers (15 per organism) featuring growth of Stachybotrys chartarum, Chaetomium globosum, Penicillium chrysogenum, and Cladosporium cladosporioides were placed in gas chambers containing chlorine dioxide gas at either 500 or 1,000 ppm for 24 h. C. globosum was exposed to the gas both as colonies and as ascospores without asci and perithecia. After treatment, all organisms were tested for colony growth using an agar plating technique. Colonies of S. chartarum were also tested for toxicity using a yeast toxicity assay with a high specificity for trichothecene mycotoxins. Results showed that chlorine dioxide gas at both concentrations completely inactivated all organisms except for C. globosum colonies which were inactivated an average of 89%. More than 99% of ascospores of C. globosum were nonculturable. For all ascospore counts, mean test readings were lower than the controls (P < 0.001), indicating that some ascospores may also have been destroyed. Colonies of S. chartarum were still toxic after treatment. These data show that chlorine dioxide gas can be effective to a degree as a fumigant for the inactivation of certain fungal colonies, that the perithecia of C. globosum can play a slightly protective role for the ascospores and that S. chartarum, while affected by the fumigation treatment, still remains toxic.