The goal of this interagency agreement is to provide support of National Toxicology Program (NTP) hazard identification (or hazard assessment) activities targeted toward the prevention of diseases or adverse effects caused by environmental exposure to chemical or physical agents. These cooperative studies continue to improve the risk assessment process by determining quantitatively what constitutes an adverse health effect on the immune system in humans. These studies evaluate unique cohorts of individuals from professions associated with immune-mediated occupational diseases including asthma, respiratory and contact allergy, chronic beryllium disease, rhinitis, and silicosis. Occupational cohorts are being studied for a number of endpoints including, impact of genetic polymorphisms on inflammatory disease development and clinical outcomes, the role that genetic variations play in environmental and workplace related diseases and identification of unique immunological biomarkers for disease. Traditional methods for assessing fungal exposure have well-documented limitations. For example, fungal assessment based on viable culture generates biases against slow-growing fungi, and several fungi are not capable of growth on artificial culture. Non-viable methods of assessment are also limited to the identification of fungi to the Order level due to the morphologically indiscernible spore morphologies shared by many fungi (ex. Aspergillus, Penicillium, and Paecilomyces). To more completely assess the fungal species to which people are exposed, a fungal ribosomal RNA (rRNA) gene sequencing study was designed to test the hypothesis that fungal bioaerosols in the United States indoor built environments are much more diverse than previously estimated using traditional methods of analysis. In collaboration with Childrens Mercy Hospital (Kansas City, MO) NIOSH will isolate mold specific ribosomal RNA genes from air and dust samples derived from homes participating in the Kansas City Safe and Healthy Homes Partnership Project with the aim of characterizing the complete diversity of fungi. The routine sampling performed in these homes included on-site measurements for specific indoor air quality parameters such as temperature, relative humidity, and carbon dioxide concentration. Measurements were also made to determine air circulation in the target rooms. A composite dust sample from the subject's bedroom and the family vacuum were collected and analyzed by monoclonal immunoassay for the allergens Der f1, Der p1, Fel d1, Can f1, Mus m1 and Bla g2. Total antigen content of Alternaria, Aspergillus, Cladosporium and Penicillium species has also be determined using a previously developed polyclonal assay. Air sampling from at least 4 areas of the home have been examined microscopically for fungal conidia. Two additional samples have been collected using the size-selective NIOSH BC 251 sampler and the Biostage viable sampler. The sampling locations for these samplers included the child's bedroom and a more heavily contaminated area of the house (usually the basement). A Biostage viable sampler was also utilized to collect viable fungal bioaerosols using the same sampling interval and location. Methods have been optimized to extract DNA from fungal particles derived from both dust and air samples. Fungal gene sequences have been obtained for 31 dust samples and 10 aerosol samples. To date, data from dust samples indicate a prevalence of species within the Order Pleosporales (genera commonly detected: Phoma, Pithomyces, Curvularia, Alternaria, Stagnospora, and Epicoccum). Other basidiomycete fungi are also common and detected in dust samples and include species within the genera Cryptococcus, Rhodoturula, Wallemia, and Puccinia (rusts). Interestingly, aerosol samples have yielded many rRNA sequences highly similar to Aspergillus and Penicillium species. Although these genera are typically encountered in viable culture studies, they have been less prevalent in the few fungal rRNA sequencing studies that have been conducted thus far. The genomic DNA extraction method that was optimized during the preliminary study may be more efficient at extracting DNA from the spores of these genera, which are typically small and recalcitrant to cell lysis. A second project is developing species-specific monoclonal antibodies (mAbs) to recombinant fungal antigens. These mAbs will be important for the quantification of species-specific biomarkers of fungal exposure, particularly to those fungi that are being studied by the NTP. Aspergillus terreus has been used as a model organism as its genome has been sequenced. This has made it possible to develop fungal specific proteins using recombinant technologies. A. terreus is an emerging and poorly characterized opportunistic pathogen and an important source of occupational exposure in agricultural and other water-damaged environments. To date, A. terreus terrelysin, has been identified, characterized and based on the sequence reported by NIOSH, annotated in several protein databases. Twelve mAbs were developed and characterized. Native terrelysin was measured in growing hyphae in the first four days of liquid culture, suggesting that terrelysin may be expressed during hyphal aggregration and may function as a biomarker of A. terreus exposure. Cross-reactivity of the terrelysin mAbs was also tested with other closely related species belonging to the genera Aspergillus, Penicillium and Paecilomyces and identified to be specific. NIOSH continues to work with these A. terreus reagents with the aim of developing a standardized immunoassay. Recombinant Chaetomium globosum enolase (rec-CgEnol) has been cloned, expressed, purified and confirmed using mass spectrometry. Three rec-CgEnol specific mAbs have been produced. The specificity of each rec-CgEnol mAb is currently being examined in cross reactivity studies. In addition, conserved fungal allergens derived from the fungal order, Pleosporales, are currently being identified as part of a collaborative study with Johns Hopkins University. Preliminary data demonstrate several candidate allergens that are shared between members of this occupationally relevant fungal order. mAbs to hyphal exoantigens derived from the occupational contaminant, Paecilomyces variotii have also been produced. Current research efforts are aimed at identifying and characterizing these hyphal exoantigens and developing immunoassays that will be used to quantify this occupational contaminant in a collaborative exposure assessment study of Norwegian sawmill workers. A third project is developing an animal model for dry fungal aerosol exposures to identify and study potential human health effects. Current exposure methods rely on liquid installation of fungal spores or extracts, which may not simulate human exposures in mold contaminated indoor environments. An acoustical generator and nose only chamber to be used for the delivery of dry fungal spores and hyphal fragments has been developed and characterized. In characterization studies using A. fumigatus grown on rice, airborne concentrations exceeding 1 X 106 spores could be aerosolized using the chamber. Field emission scanning electron microscopy analysis confirmed that particles aerosolized were amerospores and no hyphal material was observed. Recently, the system has been optimized for the acoustical generation of hyphal fragments and preliminary data demonstrated that hyphal particles of equal aerodynamic diameter could be aerosolized. Aerosols from A. fumigatus, A. terreus, A. vericolor, Paecilomyces variotii, and Stachybotrys chartarum be utilized and viable and non-viable (UV irradiated) fungal aerosols will be compared using the model. These studies will provide unique insight into the innate and adaptive immune responses following inhalation of dry fungal spores and hyphae aerosolized from contaminated building materials. Finally, NIOSH has evaluated allergic sensitization in a cohort of sera from 677 farmers with or without pesticide exposures as part of the NIEHS Agricultural Health Study. Total IgE (tIgE) and mold mix specific IgE (sIgE) was determined using the Phadia ImmunoCap assay. Total IgE greater than 100 kU/L was quantified in 21% of the farmer population. Allergic sensitization to mold Mx2 was identified in 4% (28/677) of this cohort. Mold Mx2 positive sera were then tested for specific IgE to a panel of potential farm-specific mold allergens using the commercially available Phadia ImmunoCap test. Allergic sensitization in the atopic farmer population (n=144) was greatest to common fungal contaminants including A. alternata (10%), P. betae (9%), E. purpurascens (9%), C. albicans (9%), P. chrysogenum (8%), and A. fumigatus (8%), and not to common crop contaminating species. Because of NTP interest in additional fungal contaminants, all 677 sera were screened for deoxynivalenol (DON) levels in the serum. DON levels in the serum were evaluated using a modified commercially competitive ELISA produced by NEOGEN with a quantification range between 0.6-10 ng/mL. No DON has been found in the farmers serum.