Fungi Are Among the Most Common Foreign Aerobiological Particles That We Inhale

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Chapter 1∗

1. Introduction Fungi are among the most common foreign aerobiological particles that we inhale. Personal exposure to airborne microorganisms, particularly fungi both in indoor, outdoor, residential and occupational environments, is widely recognized as exacerbating agents of non-infectious respiratory diseases such as allergic rhinitis, asthma, bronchitis, organic dust toxic syndrome and hypersensitivity pneumonitis. However, the role of fungi in mediating allergic reactions remains unclear. Although many species are known to produce allergens inducing an Immunoglobulin E (IgE) and IgG response, the importance of many other additional species remains unrecognised. This is partly because of the vast number of fungi that occur naturally and the diversity of allergens within different species, which is further confounded by the poor quality of commercial extracts available for diagnosis and the methods of analysis used to quantify airborne counts. In addition, the interaction of fungi with the airways is complex and involves a number of innate and acquired mechanisms, including allergic responses. This introduction will discuss the current understanding of allergy, the biology of fungi, the nature and location where people may come into contact with fungal allergens and review the methods available to measure exposure to airborne fungi and diagnose fungal allergic sensitisation.

1.1 Respiratory allergy – a brief history of the problem

The first documented report of an allergic reaction occurred to King Menes of Egypt around 2, 600 B.C., who supposedly died from anaphylaxis following a wasp sting.

∗ Chapter 1, Section 1.6 was published as “Measuring environmental fungal exposure” in Medical Mycology in 2005.

Other reports from ancient history include, Britannicus, the son of the Roman emperor Claudius, who was described to develop swelling around his eyes following horse riding and King Richard III of England, who was reported to get urticaria from strawberries (Cohen and Evans, 1991). Adverse health effects related to microbial growth in the indoor environment were documented as far back as Leviticus, in the 3rd book of the Old Testament, in which home occupants were warned against the growth and spread of red or green-like depressions on the walls, which was most likely mould growth (Schoental, 1980; Leviticus, 14th Chapter, versus 34-45). The most comprehensive early clinical description of hay-fever was published by Dr. Charles Blackley in 1873, who observed that the inhalation of grass pollen during winter induced hay-fever (Blackley, 1873). The term allergy (from the Greek word “allos” meaning changed or altered state and “ergon” meaning reaction or reactivity) was later coined by the Venetian paediatrician, Clemons von Pirquet in 1906 (von Pirquet, 1907). Further development of the field occurred during 1911, when Noon and Freeman injected allergen solutions into subjects to build up immunotolerance, in addition to performing provocation tests by putting drops of allergen extracts in the eyes of subjects (Noon, 1911). In 1918, Robert Crooke opened the first clinic in a New York hospital to treat allergic symptoms and along with colleague Arthur Cocoa, were the first to use the term “atopy” and the serum factors called “reagins”. These were further characterised throughout the 1960’s and were later termed “IgE antibodies” by Ishizaka and colleagues (Roitt et al., 1998). This breakthrough enabled further progress into the understanding of the mechanisms of atopy and sensitisation to allergen sources (Bennich et al., 1968). Allergy is currently defined as the hypersensitive response of the immune system to ingested or inhaled foreign proteins and allergens (Bierman and Van Arsdel, 1999; Blumenthal and Rosenberg, 1999; Chiu and Fink, 2002). The clinical manifestation of symptoms, including wheeze, coughing, shortness of breath, sneezing, nasal discharge, urticaria, angioedema and anaphylaxis are conditions associated with allergic rhinitis, conjunctivitis and bronchial asthma. Environmental exposure to allergens is an important step in the aetiology and exacerbation of allergic conditions, in

particular allergic rhinitis, conjunctivitis and bronchial asthma (Blumenthal and Rosenberg, 1999). However, even after extensive environmental exposure to certain aeroallergen sources, it has been shown that not all individuals develop allergic disease, which is strong evidence for genetic predisposition (Marsh et al., 1981; Howard et al., 1997; Cookson, 1999; Holloway et al., 1999; Ong and Hirsch, 1999).

1.2 Prevalence of allergic respiratory disease

The prevalence of “allergy” affects approximately 20-30% of the population, although this figure has been shown to be highly variable between countries in the recent International Study of Asthma and Allergies in Childhood (ISAAC, 1998). In the questionnaire-based survey, which included 155 centres in 56 countries, the highest prevalence (>20%) of allergic rhinitis and asthma among children aged 13-14 years were found in Australia, New Zealand, The United Kingdom and Ireland. Other studies have identified similar prevalence rates (~10-20%) (Hagy and Settipane, 1969; Gergen et al., 1987; Wuthrich, 1989; Ogino et al., 1990), although recent evidence suggests that allergic diseases may be increasing for reasons that remain to be identified. Epidemiologic studies have hypothesised that the rise in allergic diseases, particularly in industrialised countries, can be attributed to a number of societal and environmental variables. Increasing levels of atmospheric pollutants, including cigarette smoke (Wuthrich, 1989; Davies et al., 1998), suspended particulate matter (Takafuji and Nakagawa, 2000), diesel exhaust particulate (D'Amato et al., 2000), ozone and sulphur dioxide (D'Amato, 2000) have been shown to damage the mucosal membrane and promote IgE mediated immune responses. However, these factors alone may not necessarily account for the increased prevalence of disease. Societal changes, including shifts towards single parent families, childhood vaccinations, parental control, children spending more time indoors and cleaner home environments are factors believed to have changed the incidence of common childhood diseases, which have resulted in differences of allergen exposure (Erwin and Platts-Mills, 2005). In addition, factors associated with the lifestyle of populations or families, such as socio-economic status,

sibship size, early childhood infection, dietary habits, growing up in anthroposophic families or a farming environment are important parameters that are often neglected in epidemiologic studies and might prove to be of greater relevance in the study of allergic disease (Kitch et al., 2000; von Mutius, 2000). Thus, anthropogenic change has lead to the creation of what is known as the “Hygiene hypothesis”. This is currently the subject of much research. The Hygiene hypothesis refers to a paradox, whereby the greater hygiene of modern lifestyles reduces microbial exposure, but also modulates immune responses, such that the likelihood of developing allergic diseases is increased. Within the atopic population, the prevalence of mould allergy has been estimated to range from 2 to 90% (Hasnain et al., 1985; Santilli et al., 1985; Lehrer et al., 1986; Portnoy et al., 1987; Sprenger et al., 1988; Brunekreef et al., 1989; Santilli et al., 1990; O'Hollaren et al., 1991; Szantho et al., 1992; Helbling et al., 1994; Horner et al., 1995; Vijay et al., 1998; Black et al., 2000; Zureik et al., 2002). The heterogeneity between the prevalence of reactivity can be accounted for by variations in airborne fungal concentrations in different sampling environments, the selection criteria for test subjects and the source and batch of commercial fungal extracts. Previous studies have shown that reactions to commercial extracts vary anywhere from 6-70% in the same population (Aas et al., 1980; Portnoy et al., 1987). Using recently available assays for individual fungal allergens, the allergenic content of commercial extracts of Aspergillus fumigatus may vary by up to 400 fold (Vailes et al., 2001). Given the ubiquitous nature of fungi, the number and diversity of species, their composition and the vast numbers of tiny, buoyant spores they frequently produce, it is not surprising that variations in allergen extracts exist and thus the true prevalence of fungal allergy will remain unknown until standardized extracts are made available and tested on well-defined populations.

1.3 Innate and adaptive immunity

The host response against fungal pathogens, foreign bodies and infection requires an integrated response between innate immunity, adaptive immunity and fungal virulence

factors. Innate immunity is the first line of defence against pathogen invasion until adaptive immunity develops and is driven by the mediation of specific cell types, including macrophages, eosinophils, basophils and T-lymphocytes, such as γδ T-cells or natural killer cells (Nicod and Spiteri, 2001). This arm of immunity initially senses the incoming pathogens via complex interactions between secreted pathogenic macromolecules and a family of host Toll-like receptors (TLRs), which then initiate a range of host defensive mechanisms. To date, at least 10 members of TLRs and their associated ligands including lipopolysaccharides, lipoproteins, bacterial peptidoglycans and bacterial deoxyribonucleic acid have been identified (Krutzik et al., 2001; Nicod and Spiteri, 2001; Takeda and Kaisho, 2003). Further recent findings have also suggest the signalling of an innate immune response due to interactions with fungal cell wall beta-glucans through the newly discovered receptor, dectin-1 (Brown and Siamon, 2003; Hauswirth and Sundy, 2004), as well as with heat shock protein (60) and polysaccharides through TLR2 and TLR4 (Sieling and Modlin, 2002). Mast cells, eosinophils and T-cells can be activated by their own specific receptors during the innate immune response and under the appropriate conditions these cells can release a wide range of biologically active mediators that control the invading pathogen and chemokines to attract mono- or polymorphonuclear cells. These cells are also able to release danger signals, such as interleukin (IL)-1, IL-12 and IL-18, tumour necrosis factor (TNF)-α and interferon (INF) to activate dendritic cells (Nicod et al., 2000; Nicod and Spiteri, 2001). In contrast, these various modifying cells can also down regulate the innate response by mediating the release of IL-4, IL-10 and transforming growth factor (Herring and Huffnagle, 2001; Huffnagle and Deepe, 2003). Dendritic cells, which are localised around the airways and blood vessels, are a family of leukocytes that are derived from lymphoid or myeloid lineages and have evolved specifically to translate innate recognition into adaptive immunity (Huffnagle and Deepe, 2003; Moll, 2003; Reis e Sousa, 2004). Upon activation by TLR ligands (Kaisho and Akira, 2003), the dendritic cell can detect and engulf pathogens and neighbouring epithelial cells invaded by viruses by phagocytosis (Albert et al., 1998; Nicod et al., 2000), produce a variety of cytokines, in addition to orchestrating the

activation of the adaptive immune response by migrating to lymphoid organs and presenting the pathogen-derived peptides to T cells, thereby inducing T cell activation and differentiation (Moll, 2003; Pope, 2003; Steinman, 2003). Recent studies have demonstrated that activated dendritic cells may take up to 6-24 hours to travel to nearby lymph nodes and a further 3-4 days to trigger and drive T-cells to replicate before the migration to the peripheral lymphoid organs and tissues in the form of memory cells (Masopust et al., 2001; Vermaelen et al., 2001). Moreover, it is now clear that the activation of the innate immune system through dendritic cells and TLRs is a critical event that shapes the emerging response, thereby controlling the course of infection and thus may influence allergic diseases such as asthma (Heine and Lien, 2003). The regulation of opportunistic fungal pathogens by the innate immune system and dendritic cells is well recognized, although the signalling processes required to discriminate and mount an appropriate response to the vast array of fungal pathogens has been speculated and is currently the focus of many research groups. Signalling from different groups of microbes can be mediated via the TLRs, which lead to the activation of conserved host defence signalling pathways that control the expression of a variety of immune response genes. Different dendritic cells express various recognition molecules, which indicate that they are more or less efficient when responding to certain pathogens (Buentke and Scheynius, 2003). Results of Bozza et al. (2002) and d’Ostiani et al. (2000) demonstrate that fungi or possibly fungal derived products provide a powerful activation stimulus to dendritic cells, which results in dendritic cells having functional plasticity and the capacity to distinguish between different forms of a fungal species prior to differentiating into IL-12 or IL-4/ IL-10 producing cells that support distinct T- cell responses. However, the ability of fungi to produce a number of fungal virulence factors during germination and bioactive lipids, such as PG2, that modulate dendritic cell function and TLR4-mediated proinflammatory signals, in addition to down regulating the maturation of dendritic cells have been recently explored and demonstrated in Schistosome and Aspergillus species (Angeli et al., 2001; Netea et al., 2003). Thus, these findings provide a new paradigm for fungal and parasite

pathogenesis and demonstrate the ability of certain fungal genera to evade immune recognition. If the innate immune response does not rapidly eliminate a pathogen, adaptive immune responses are activated. To initiate adaptive immunity, soluble antigen and antigen-presenting cells (APCs), macrophages and dendritic cells containing antigen are carried in the lymph via circulation to nearby lymphoid organs as aforementioned. There, antigen binds B cells and APCs present antigen peptides on their membrane to major histocompatibility complex II (MHC) to activate antigen-specific T cells. T helper (Th2) cells activate antigen-binding B cells to produce antibody and become effector plasma cells. In allergic diseases, the sensitisation to allergens in atopic individuals involves adaptive immunity, which includes both inflammatory and immunological responses involving T-cell-mediated and B-cell or antibody mediated responses. Although IgE antibody is believed to have a functional role in allergic disease, eosinophils, in addition to T cell immune and inflammatory pathways have also been identified to be important in allergy and asthma symptomology. T cells regulate or organize most types of immune responses to foreign proteins by secreting cytokines such as IL or IFN, which can be categorised into phenotypes (Th0, Th1, Th2) on the basis of their products. Th 1 and Th 2 dependent adaptive immune responses underlie some of the most common and important of lung diseases, including tuberculosis and asthma, respectively. In allergy, Th2 cells initiate the immediate allergic response by releasing proinflammatory cytokines such as IL-4, IL-5, IL-10 and IL-13. The interaction between the cytokines IL-4 and IL-13 and their receptors on B-cells can promote antibody class switching, IgE synthesis, induction of tissue eosinophilia and promote the growth of mucosal mast cells (Bacharier et al., 1998; Kurup et al., 2000; Prussin and Metcalfe, 2003). To initiate IgE synthesis, a B-cell requires two signals that are delivered by T-cells through a complex series of interactions. The process is initiated when an allergen is captured on the surface of the B-cell by an allergen specific IgM molecule, internalised and processed into polypeptides before being presented on the B- cell surface in association with MHC molecules (Vercelli, 2002; Prussin and Metcalfe,

2003). The recognition of the antigen-MHC class II molecule by T-cell receptors leads to two critical events for antibody class switching: (1) the immediate secretion of lymphokines (IL-4, IL-13) and (2) the ligation of CD40 ligand (CD40L) on B-cells, which activates T-cells and promotes the switch recombination of the B-cell to IgE and the further amplification of IgE synthesis (Renshaw et al., 1994; Vercelli, 1997; Oettgen and Geha, 2001; Vercelli, 2002; Prussin and Metcalfe, 2003). By contrast, Th1 cells are primarily involved in delayed hypersensitivity and have been proposed to inhibit the Th2 driven processes.

Several transcription factors (STAT 4, STAT 6, NF-Kβ and BSAP) have also been identified and shown to bind to genes associated with inflammatory and allergic responses (Del Prete et al., 1994; Abbas et al., 1996; Bacharier et al., 1998). STAT 4 is essential for IFN-γ production and the up regulation of the Th 1 response (Del Prete et al., 1994), while STAT 6 is associated with the IL-4 response and the up regulation of the Th 2 pathway (Prussin and Metcalfe, 2003). Other transcription factors, including

NF-Kβ and BSAP are known to mediate both IL-4 and CD-40 delivered signals (Mosmann and Coffman, 1989; Del Prete et al., 1994; Renshaw et al., 1994; Abbas et al., 1996; Bacharier et al., 1998). The immune reactions that drive allergic disease and asthma can be considered a combination of Gell and Coombs type I and type IV hypersensitivity reactions (Cohen, 1988; Horner et al., 1995; Menz et al., 1998). It is known that personal exposure to aeroallergens leads to sensitisation and immunorecognition, and that successive exposures result in the interaction of allergens with receptor bound IgE antibodies on various cells. Previous studies have characterised two different types of affinity receptors, (1) high affinity receptors (FCεRI) that are bound to basophils, mast cells and antigen-presenting cells, and (2) low-affinity receptors (FCεRII/CD23) that are expressed by B cells, monocytes, macrophages, dendritic cells and eosinophils (Vercelli, 1997; Prussin and Metcalfe, 2003). The interaction of allergens with IgE bound to low affinity receptors on APCs results in the mediation of endocytosis, phagocytosis and pinocytosis before being processed and presented to antigen specific T-cells. Conversely, the interaction of allergens with IgE-sensitised mast cells

characterises classical type I hypersensitivity, which results in the release of pharmacological mediators. This produces an acute early phase inflammatory reaction within minutes. A late phase reaction or “delayed hypersensitivity” develops 3-4 hours after exposure and is due to cellular infiltrates as a response to the mediators released in the early phase reaction. The manifestation of clinical symptoms induced by the immediate response include sneezing, wheezing and urticaria, in part due to the release of histamine from mast cells, while chronic symptoms such as bronchial hyper reactivity may be explained by eosinophil-mediated tissue damage induced by various proinflammatory mediators and proteins. In contrast, eosinophils have also been identified to have a critical role in the aetiology and pathogenesis of allergy and asthma (Gleich, 2000; Leckie et al., 2000; Prussin and Metcalfe, 2003). Eosinophils are granulocytes that are characterised by two phenotypes, specific and primary granules. The outer surface of the eosinophil contains many different types of surface cell molecules, including cytokine, chemokine and complement receptors, in addition to adhesion molecules and CD69 that act as eosinophil up regulation activation markers and are localized in regions of inflammation (Rothernberg, 1998; Bochner, 2000; Bochner and Schleimer, 2001; Prussin and Metcalfe, 2003). The development of eosinophils occurs in bone marrow, however the differentiation and proliferation of eosinophils is controlled by the cytokine, IL-5 (Kita et al., 1998; Gleich, 2000; Prussin and Metcalfe, 2003). Bronchial provocation with allergen extracts has been shown previously to induce the release of mature eosinophils from the bone marrow, upon the distal mediation of IL-5 at sites of allergic inflammation (Denburg et al., 2000). Although the proliferation of eosinophils from the bone marrow to the sites of development are well recognized (Bochner and Schleimer, 2001), the physiological mechanisms involved in their activation remain unclear. Moreover, in allergic disease eosinophils release a number of proinflammatory mediators such as major basic protein at sites of inflammation, which have been shown to directly cause bronchial hyper responsiveness and bronchoconstriction in asthma (Gleich, 2000; Prussin and Metcalfe, 2003).

Genetic predisposition or host specific factors are also recognized to contribute to inflammatory and allergic responses (Marsh et al., 1981). Studies demonstrating the genetic control of specific IgE production have shown that IgE production to some purified allergens was strongly linked to the HLA-DR allele, although immunoblotting studies with Alternaria extracts using allergic monozygotic twins, show only moderate concordance between the different allergens (Karihaloo et al., 2002). The future development of molecular biological techniques will enable the further identification and characterisation of genes linked to the exacerbation of allergy and asthma.

1.4 Allergens and sensitisation

Many allergens can be defined as soluble proteins or glycoprotein molecules of molecular weight 5-100kDa that can induce allergic respiratory disease via a number of immunological pathways. The proteins that make up allergens are heterogeneous and have many biochemical functions that may include proteolytic enzymes, recognition molecules and transport molecules, yet a large proportion has still not had their function characterised. While the nature of an allergen is generally defined in terms of the induction of an IgE response, the molecules stimulate a much broader immune response, including via receptors on T-cells, other antibody isotypes (IgG, IgA) and through innate mechanisms (Wang et al., 2001). All of the latter occur whether people are allergic or not and are probably definitive in regulating the overall response. Allergy may be induced through numerous exposure routes, including inhalation, ingestion, injection, dermal and indirectly via the placenta or breast milk. Which allergies are common in a population are probably determined by the molecules that are common in the environment, so factors including geography, season, dwellings, occupation, lifestyle and social settings are important determinants. Within a community, at the level of mapping individual responses to different allergenic molecules, almost everyone has a different fingerprint. Immunoblotting studies of identical twins show only moderate concordance, suggesting the major influence of

genetics on allergy, concerns the ability to regulate total IgE production and not the response to individual allergens (Sluyter et al., 1998; Tovey et al., 1998). Sensitisation to allergens from various synthetic and environmental sources is the general precursor to the development of respiratory disease, including allergy and asthma in genetically predisposed individuals. Synthetic allergens are derived from processed commercial materials and synthesised organic compounds, such as plastics, food dyes and preservatives that are included in packaged foods and building materials. Environmental sources of allergen on the other hand are heterogeneous, consist of proteins and glycoproteins, are generally of biological origin and have either perennial or seasonal distributions. Perennial allergen sources are present throughout the year and include proteins from cockroach, house-dust mite, cat, dog and rat allergen sources. Seasonal allergens typically include airborne pollen and fungi, which are dynamic aeroallergen sources that are reliant on many meteorological parameters that aid in their development and distribution. The most widely recognized sensitising sources that have been implicated to have a causal role in the development of sensitisation include house-dust mite (Sporik et al., 1990; Peat et al., 1996), animal dander (Ingram et al., 1995; Sporik et al., 1995), cockroach (Rosenstreich et al., 1997), pollen (Pollart et al., 1988; Cakmak et al., 2002) and mould (Halonen et al., 1997) allergens. However, due to the heterogeneity between mould species and the variations between allergen extracts and the methods of analysis, associations between mould sensitisation and the severity and incidence of respiratory disease have often been difficult to interpret and is poorly understood (Aas et al., 1980; Helbling et al., 1994; Bush and Portnoy, 2001; Hyvarinen et al., 2001). This is further confounded by the subjective methods available to quantify predicting parameters, which include spore counts, surveys and indoor assessments of mould growth that are commonly used in statistical analyses to determine the risk factors for mould induced respiratory allergic disease. Epidemiologic studies that have established associations between mould and respiratory symptoms, usually implicate indoor and outdoor spore counts (Klabuschnigg et al., 1981; Delfino et al., 1997; Downs et al., 2001; Dales et al., 2003), skin prick test reactions (Halonen et al., 1997; Bibi et al., 2002), damp buildings

(Brunekreef et al., 1989; Koskinen et al., 1999; Chapman, 2003), and bronchial provocation (Licorish et al., 1985) as important predicting parameters, in addition to demonstrating the significance of fungal exposure. Outdoor studies have primarily focused on the allergenic species of Alternaria, Cladosporium and Basidiomycetes as these have been suggested to be a risk factor for allergic rhinitis (Li and Kendrick, 1995a; Andersson et al., 2003), asthma (Santilli et al., 1985; Ward et al., 1989; Abramson et al., 1996; Helbling et al., 1999; Fung et al., 2000; Reese et al., 2000; Stieb et al., 2000; Downs et al., 2001; Zock et al., 2002; Zureik et al., 2002; Dales et al., 2003) and even death (Salvaggio et al., 1971; O'Hollaren et al., 1991; Targonski et al., 1995; Black et al., 2000; Newson et al., 2000). Sensitisation to the outdoor mould spores of Alternaria alternata and Basidomycetes have been implicated as a major risk factor for fatal asthma attacks in Chicago and New Orleans, USA, where deaths from asthma at ages 5-34 years were twice as common on days with a high total mould spore count (> 1000 spores/m3) compared to days with lower spore counts (Salvaggio et al., 1971; Newson et al., 2000). Previous studies have identified links between hospital admissions and outdoor spore counts, where asthma admissions were highest on days with concentrated total spore counts (Salvaggio et al., 1971; Dales et al., 2000; Newson et al., 2000). Other investigations have also linked additional species whose clinical significance is relatively unknown, in particular Trichophyton, Leptosphaeria, Wallemia and Basidiomycete species to induce respiratory disease (Sakamoto et al., 1989; Ward et al., 1989; Hasnain, 1993; Rosas et al., 1998; Helbling et al., 1999). Thus, sensitisation to moulds is a powerful risk factor for severe asthma and is common among asthmatic subjects, which has recently been demonstrated in several multi-centre studies (Zureik et al., 2002). The influence of other fungal propagules that are more difficult to identify, such as hyphae and fungal fragments, as well as other uncharacterised genera is unknown and remains to be investigated (Gorny et al., 2002; Gorny, 2004).

1.5 Fungal respiratory allergy

Fungi are a diverse lineage of ubiquitous eukaryotic microorganisms. They grow as saprophytes on non-living organic matter or function as invasive or symbiotic organisms in living tissue where their thread like-hyphae form mycelial networks. Approximately 69,000 species have been identified out of an estimated 1.5 million species (Levetin, 1995) and in some cases over 100 different individual stains of a species of agricultural, food or medical interest have been recognized (Swardnordmo et al., 1984; Steringer et al., 1987; Portnoy et al., 1993a). The principal dispersal vectors of fungi are sexual spores or asexual conidia. These vary in size (2–70 microns), shape (spheres, rods, chains, etc) and depending on the species are phenotypes that can be used to differentiate between fungal genera (Flannigan, 1997; Park et al., 2004). Aerosolised viable hyphae may also disperse the organisms, although they lack the morphological characteristics to be directly identified (Menetrez et al., 2001; Gorny et al., 2002; Gorny, 2004). The prevalence of fungal spores in the environment follows seasonal patterns that are influenced specifically by temporal (Li and Kendrick, 1995b) and spatial variables (Chao et al., 2002), geographic location (Rantio-Lehtimaki et al., 1991; Muilenberg et al., 1997; Mitakakis and McGee, 2000; Bergamini et al., 2004) and meteorological parameters (Levetin, 1995; Frenz, 2000). Although seasonal fungal spore counts are not as distinctive as those of pollen varieties, studies assessing outdoor fungal spore concentrations have shown that they are present throughout the entire year with the highest numbers occurring during the spring and summer months. Conversely, indoor spore counts are generally five fold lower than prevailing outdoor spore concentrations (Gots et al., 2003). The most common fungi isolated from indoor environments include Aspergillus, Penicillium and Cladosporium species, although this is dependent on the levels of internal moisture. Thus, personal exposure to moulds, both indoors and outdoors, is inevitable and airborne concentrations in the range of 101–105 spores per cubic meter of air are common (Gots et al., 2003).

Fungi capable of releasing allergens and exacerbating fungal allergic diseases currently comprise more than 80 species and belong to three distinct taxonomic groups; the Ascomycota, Basidiomycota and the Anamorphic fungi (Horner et al., 1995; Bush and Portnoy, 2001; Esch, 2004). A list of fungal genera most frequently associated with IgE mediated allergy is presented in Table 1.1. The most widely recognized allergenic fungi include Alternaria, Aspergillus, Penicillium and Cladosporium species (Cruz et al., 1997) for reasons that relate to their airborne abundance in many geographic locations and morphologically recognizable phenotypic features. This is particularly the case for Alternaria, which has been widely studied in epidemiologic investigations, due to the ease of identifying its large and visibly distinctive spores that have been shown in many environments to exacerbate rhinitis (Andersson et al., 2003) and asthma (Licorish et al., 1985; O'Hollaren et al., 1991; Halonen et al., 1997; Neukirch et al., 1999; Fung et al., 2000; Negrini et al., 2000; Downs et al., 2001). In contrast, the biology of fungi has slowed the characterization and standardisation of fungal allergen extracts compared to other aeroallergen sources. This has restricted the numbers of fungal extracts available for in vivo and in vitro diagnostic techniques and molecular probes to quantify airborne fungi. As a result of these factors, the interpretation of personal exposure to fungal allergens has been restricted to the inhalation of fungal spores from a small and select number of species. Thus, the influence of previously unrecognised fungal genera, in addition to other fungal propagules to fungal allergy currently remains unknown and relates to the inadequacies and availability of current methods of analysis. Fungal allergic disease has also been implicated with a number of other medical disorders, including cerebral phaeohyphomycoses (Osiyemi et al., 2001), eczema (Koskinen et al., 1999; Helbling, 2003), fungal sinusitis (Ponikau et al., 1999; Karpovich-Tate et al., 2000; Braun et al., 2003; Shin et al., 2004) and allergic bronchopulmonary aspergillosis (Geller et al., 1999; Skov et al., 2000). Although many studies have described the epidemiology of these diseases, the biological and environmental conditions required to exacerbate clinical symptomology of many of these conditions is often not discussed nor studied. Mould and their associated allergens are unlike any other bioaerosol, in that they are biologically dynamic particles that are

heterogeneous between species and are able to actively secrete molecules that have a variety of pathogenic, inflammatory and allergenic functions. A thorough understanding of the processes involved with the growth, development, reproduction, classification and dispersal of fungi is required to further the understanding of fungal allergic disease.

1.5.1 Fungal biology

The Mycenaean civilisation, the greatest civilization ever developed according to historians, may have been named after a mushroom. From the Greek word mykes (mushroom) and logos (discourse) is derived the word mycology, meaning the study of mushrooms. Mycology as a science is only 250 years old and was founded by Pies Antonio Micheli, an Italian botanist who published Nov Planatarum Genera in 1729, however the uses of this group extend back for many thousands of years. The Egyptians considered biological fermentation a gift of the gods, while the Romans attributed the appearance of mushrooms to lightning that had been hurled by Jupiter. Nevertheless, fungi in some way on a daily basis affect each and every one of us whether the effects are beneficial, negligible or harmful. Fungi are widely recognised as a causal agent of many known plant, animal and human diseases, however the group has many holistic applications that extend into medicine, particularly the commercial production of medical drugs, including ergometrine, cortisone, vitamin preparations and a number of antibiotics (penicillin and griseofulvin) (Burge, 1992). Today fungi are also important to cytologists, geneticists and biochemists that have identified fungi as an important tool in understanding and studying biological processes. The requirements of less space, rapidity of fungal growth cycles and the shortness of generation times compared to many other plant and animal species, in addition to being an easier organism to work with has meant that fungi are a good model organism. The unique ability of certain species of fungi to exploit the toughest environments has led to the classification of certain fungi as business-related and industrial pests. These fungi often conflict with human interests and breakdown commonly used materials that contain cellulose, such as cloth, paint, cartons, leather,

waxes, jet fuel, petroleum, wood, paper, insulation cables, wires, photographic equipment and food (Raven et al., 1999). It has also been shown that a number of fungal species grow under a wide range of conditions, in particular Cladosporium herbarum, which contaminates meat in cold storage and can grow at temperatures as low as –6oC. On the other hand, other species such as Chaetomium can survive and grow at temperatures exceeding 50oC (Ingold and Hudson, 1993). The rare nature of these organisms to exploit extreme conditions can also be transformed for commercial purposes, in the instance of yeast, which is used in baking, brewing and wine production (Marfenina et al., 1994; Raven et al., 1999). Moulds are a heterogeneous group of non-photosynthetic organisms that are ubiquitous in nature and because of the presence of a cell wall have been grouped in the plant kingdom, although some authorities now place them in a separate lineage because of their ability to synthesise lysine (Whittaker, 1969; Marguilis and Schwartz, 1982; Burge, 1992; Ingold and Hudson, 1993; Alexopoulos et al., 1996). They are 80-90% polysaccharide in composition and enjoy a relative humidity from 75% to 95% (Dhillon, 1991). Fungi occur naturally throughout the environment and facilitate the aerobic decay of nonliving organic material (Burge, 1992). Their activities degrade plant and animal material into carbon dioxide, which is released into the atmosphere in addition to returning nitrogenous compounds back to the soil, where plants and animals can utilize them later.

1.5.1.1 Morphology

The body of the fungus (soma) is made up of a number of filaments that elongate by apical growth into a substrate that gathers nutrients for growth. “Hyphae” is the term given to individual filaments (Figure 1.1) and these consist of a tubular wall filled with a layer of protoplasm of varying thickness (Alexopoulos et al., 1996). In some fungi, the protoplasm lining the cell wall is often interrupted at regular intervals by cross walls that divide hyphae into compartments termed “septa”, which are formed by centripetal growth from the hyphal wall inward (Cooper, 1985). Hyphae are composed of a firm aqueous gel (Figure 1.1) with a complex fine structure that is multilaminate with

lamellae consisting of various oriented microfibrils (Ingold and Hudson, 1993; Alexopoulos et al., 1996). The fungal cell wall is recognized as a non-living membrane, which surrounds and protects the protoplasm. The cell wall is composed of a range of polymers, including glucans, chitin, proteins (Ingold and Hudson, 1993) and the sterol, ergosterol (Burge, 1992). Throughout the wall there is a matrix of glucans, which create a web of long interwoven microfibrils that maintain the form of the hyphae and are often composed of chitin (Cooper, 1985). Proteins localised in the cell wall occur as glycoproteins, which are proteins combined with a carbohydrate. The membrane is made up of two layers of lipid molecules coated with proteins and is approximately 8nm thick (Ingold and Hudson, 1993). More recently, a diverse group of related proteins that are found widely in fungi and are excreted from the tip of the hyphae during growth have been recently described and are collectively termed hydrophobins (Wessels, 1996; Wessels, 1999). These consist of a hydrophobic and hydrophilic domain, which provide an extraordinary array of functions. When the hyphae are in solution the hydrophobins pass into solution, however if the hyphae emerge from the solution, the polypeptide then polymerises on the surface resulting in the formation of parallel rodlets. The hydrophobin protein is attached to the wall by the hydrophilic end and the hydrophobic domain remains exposed, which reduces the movement of water through the wall of the hyphae protecting it from desiccation and provides extra strength for the cell wall (Wessels, 1996). In addition, the exposed hydrophobic domain enables the attachment of the hyphae to other hydrophobic surfaces, including other hyphae and waxy plant surfaces. Hydrophobins have also been identified to have important immunologic functions. The rodlets have been shown to contain the highest concentrations of conidial allergen in Cladosporium species and these function to regulate the permeability of many cytoplasmic components including antigen-presenting macromolecules (Bouziane et al., 1989). A mass of hyphal filaments is collectively termed the mycelium. The mycelium often forms thick strands and usually grows apically on the surface (or penetrates cells

in the case of parasitic fungi) of nutrient rich substratum, which leads to the degradation of organic material and the direct diffusion of nutrients through the hyphal cell walls (Cooper, 1985). Throughout the lifecycle of the mycelium, a number of organisational events take place, where the mycelium arranges into loosely or compacted woven tissue as distinguished from the loose hyphae ordinarily composing a thallus. Plectinchyma designates all fungal tissues and two general types are recognized. Loosely woven tissues where the component hyphae are parallel to one another are regarded as prosenchyma, while closely packed oval cells that resemble the parenchyma cells of vascular plants are referred to as pseudoparenchyma. Towards the end of the life cycle of the mycelium, older sections of hyphae form vacuoles in a process termed vacuolation. These sections of hyphae eventually die and can be aerosolised as fragments into the atmosphere or reabsorbed back into the biosphere (Paul et al., 1994; Papagianni et al., 1999). Since the development of the scanning electron microscope (SEM), the ultra structure of the hyphae and mycelium has been able to be studied. Fungal protoplasts have the same general structure as eukaryotic protoplasts. They contain a nucleus, which is bound by a nuclear envelope consisting of two membranes with pores. Within the cytoplasm, the usual eukaryotic organelles are found, including mitochondria, vacuoles, vesicles, endoplasmic reticulum, ribosomes, microbodies and microtubules (Alexopoulos et al., 1996). However, membranous structures lying between the plasma membrane and the cell wall, called “lomasomes”, are more common in fungi than in other organisms, while Golgi bodies are not present in fungi.

1.5.1.2 Growth and nutrition

The temperature parameters required for fungal growth can vary between species and range from anywhere as low as -10 oC to as high as 50oC (Horner et al., 1995). Optimum growth conditions for most fungal species range between 18-32 oC (Bush and Portnoy, 2001). There are a number of species termed thermophiles that can tolerate extreme conditions throughout the environment, including warm prevailing conditions greater than 50 oC. Conversely, there are species that have adapted to both dry

(xerophilic) and wet (hydrophilic) conditions (Bush and Portnoy, 2001). Fungi also have the ability to withstand freezing temperatures by shifting into a dormant state, which facilitates the storage of fungal cultures for prolonged periods of time. In contrast, the optimum pH is a difficult variable to control in culture conditions compared to temperature due to the chemical and biochemical considerations of the growing mycelium (Ingold and Hudson, 1993). Optimum pH conditions have been identified (Ingold and Hudson, 1993; Alexopoulos et al., 1996) and are often broad for fungi, generally within the region of pH 6. Few fungi grow below pH 3 or above pH 9 (Ingold and Hudson, 1993). Oxygen is another crucial prerequisite for fungal growth. In the presence of oxygen, aerobic respiration enables normal metabolic function, however when oxygen is limited fungal growth is often inhibited. Water is also critical to the survival of the fungus. A number of xerophylic species have adapted to environments where the water potential is extremely low. The reproductive spores of most fungal groups can remain dormant and survive for decades without water but once the right conditions arise, metabolic and physiological activity can resume. Light, although it is not required for growth for some species, is necessary for the development of fungal spores and the process of sporulation (Leach, 1965). The physiological process in which light triggers sporulation, however remains unclear, although it is thought that many fungi are positively phototropic and release their spores towards the light (Leach, 1965; Leach, 1971; Rotem and Cohen, 1978; Honda and Nemoto, 1984). Under favourable conditions fungal hyphae are capable of constant growth and in nature they grow apically over or through solid substrates or are submerged in water (Ingold and Hudson, 1993; Alexopoulos et al., 1996). Although it is virtually impossible to study the dynamics of fungal growth in nature, vegetative growth can be studied in the lab in either liquid culture or solid nutrient agar. Upon inoculation with spores, there is generally a lag phase where the synthetic and metabolic processes are revitalised. The emergence of hyphae occurs upon the germination of the spore, where a germ tube is developed. The germ tube grows by the incorporation of material at the tip in a region called the extension zone (Ingold and Hudson, 1993; Raven et al., 1999). The area of

the extension zone, which is the only growing point of the fungus (Alexopoulos et al., 1996), is often soft and elastic to allow for growth, differentiation and diffusion. The initial extension of the germ tube from the spore is at an accelerated rate as the spore reserves are utilized for growth and establishment. Once the reserves have been exhausted, a section known as the peripheral growth zone that is constantly distal to the extension zone, develops to support prolonged hyphal expansion. In this zone, the compartments of cell wall synthesis, precursors and enzymes are made and then released to form new wall (Ingold and Hudson, 1993; Alexopoulos et al., 1996). After the initial burst of growth, hyphae extend at a constant rate, which is determined by the quantity of nutrients, space and competition. The section of hyphae immediately behind the extension and peripheral growth zones remains metabolically active and constantly absorbs nutrients, but does not increase in size. The absorbed nutrients are utilized in the development of lateral branches. Branch development is the formation of a new tip, where localized softening and the partial dissolution of the rigid cell wall occurs (Ingold and Hudson, 1993). Like the main extension zone, a cluster of vesicles develops in the newly formed branched tip that is associated with an accelerated rate of growth. This is followed by constant growth by its own peripheral zone. More branches form with time and eventually leads to the formation of a young spherical mycelium (Ingold and Hudson, 1993). Fungi attain their nutrients by attacking and infecting living organisms (parasites) or by decomposing dead organic material/detritus (Alexopoulos et al., 1996). Most fungi can synthesise their own proteins when they acquire carbohydrates (glucose and maltose) by utilizing organic and inorganic sources of nitrogen, which are essential for growth. Various other mineral elements are vital and include phosphorous, potassium, magnesium, silicon, bromide, manganese, copper, iron and zinc (Ingold and Hudson, 1993; Alexopoulos et al., 1996).

1.5.1.3 Culture of fungi

Saprophytic fungi can be isolated from many environments, commercial products or clinical specimens and inoculated on culture plates with nutrient media as a means to

recover the fungi of interest. A variety of media are available for the primary inoculation and recovery of fungi, however no one specific nutrient or combination of nutrients is adequate for all species. The most commonly used nutrient media consists of a mixture of fruit and vegetable material with subsequent additions of various sugars and agar, which are then added to Petri dishes and allowed to set. The organic and mineral fractions are designed to supplement nutrients that are similar to those found in the environment. Several common media varieties include soil, potato dextrose, vegetable juice, malt extract and dung agar. The isolation of fungi from indoor and outdoor environments requires a versatile nutrient media that provides suitable nutrients to facilitate the heterogeneous growth requirements of a wide range of species that are present in the atmosphere. This is particularly important, as many small-spored forms are unable to be subjectively identified by light microscopy following volumetric air sampling and require culture to differentiate them (Burge et al., 1977). Previous studies that have evaluated environmental survey culture media have established that the total recovery of fungal isolates was highest for Sabouraud’s dextrose, malt extract, vegetable juice and potato dextrose agar varieties (Burge et al., 1977; Meletiadis et al., 2001). In particular, malt extract agar recovers a broad spectrum of fungi and has been recommended by the American Conference of Governmental Industrial Hygienist, Committee on Bioaerosols for the detection and enumeration of fungi in indoor environments (Burge et al., 1987). However, the collection of large concentrations of airborne microbes may alone present numerous problems for enumerating discrete colonies after seven days of colony growth. The addition of high NaCl concentrations to nutrient media, in addition to the use of DG18 media has been shown in several studies to restrict the apical growth of colonies, although identification may be more difficult as sporulation is often inhibited (Swaebly, 1950; Rogerson, 1958; Burge et al., 1977; Wu et al., 2000). The isolation of clinical mould specimens from blood cultures or from other areas of the body may require more specific nutrient media, which target individual fungal species and enable diagnosis, intervention and treatment. This is well recognized for the recovery of many Aspergillus species from patients with invasive aspergillosis.

The genus Aspergillus requires specific nutrient regimes and the use of selective media enables the differentiation of species based on the morphological characteristics of the colony (Procop et al., 2000). In contrast, the growth of bacteria on the nutrient media can be suppressed by adding antibiotic supplements to the agar. Common antibiotic supplements include, Penicillin, Streptomycin, Tetracycline or Chloramphenicol. Although the addition of many of these antibiotic agents restricts the growth of bacteria, the growth of fungal colonies can also be inhibited as shown by Burge and co-workers (1977), who demonstrated that Rose Bengal containing media with streptomycin recovered significantly fewer fungal colonies than other popular nutrient medias that contained no antibiotics.

1.5.1.4 Reproduction

Traditionally, fungal life cycles have been divided into teleomorph (sexual) and anamorph and/or imperfect (asexual) states, which is dependant on the spore type produced. Asexual reproduction, often called somatic or vegetative reproduction, does not involve the union of two nuclei, sex cells or sex organs. Asexual reproduction is important for the production of numerous spores and is a simple division of unicellular organisms into daughter cells or of a multi cellular thallus into a number of fragments, which is repeated several times during one season. There are four varieties of asexual reproduction. (1) Soma fragmentation - each fragment grows into new individuals, (2) Fission - of somatic cells into daughter cells, (3) Budding - of somatic cells and spores, each bud produces a new individual and (4) Sporulation - the production of spores, where each spore produces a germ tube that develops into a mycelium. The asexual reproductive lifecycle of a fungus is depicted in Figure 1.2. The most common form of fungal asexual reproduction is sporulation. Spores vary in colour, some being hyaline (clear) whilst others are pigmented (green, yellow, orange, red, brown and black), thick walled and hydrophobic, which favours long-range transport through dry air (Burge, 1992). The size and shapes of spores varies from small 3µm microconidia (Aspergillus sp.) to large 50µm macroconidia (Exserohilum sp.);

globose to oval, oblong and needle shaped to helical. Spores also vary in the number of cells from unicellular to multicellular, in addition to the arrangement and birth of those cells (Figure 1.3). Some fungi produce only one type of spore while others can produce as many as four different types of spores. Fungal spores are produced in two different types of asexual bearing structures, sporangia and conidia. A sporangium is a sack like structure whose contents cleave to form spores (sporangiospores) that may be motile (simpler fungi) or non motile. Fungi may also bear asexual structures on the tips of hyphae, whose spores are referred to as conidia. These fungi rely on a high rate of somatic mutation and the production of heterokaryons by hyphal fusion to produce new gene combinations that allow them to adapt to variations in the environment (Burge, 1992). Fungi can also reproduce sexually by means of the fusion of two compatible nuclei. Sexual reproduction consists of three distinct phases; plasmogamy, karyogamy and meiosis. Plasmogamy is the process of bringing two haploid nuclei together in one cell. The fusion of the two nuclei brought together in plasmogamy is known as karyogamy, which occurs initially after the first stage. Following the fusion of the nuclei, meiosis usually follows and reduces the number of chromosomes to the haploid condition with four daughter nuclei resulting from it. The growth state induced by sexual recombination is referred to as the teleomorph or perfect state and provides the basis for classifying fungi belonging to the Zygomycota, Ascomycota or Basidiomycota (Cooper, 1985). The sexual organs of fungi vary across species with some producing distinguishable male and female (hermaphroditic) organs on each thallus while others produce only either male or female organs on each thallus (dioecious). Fungal sexual organs are referred to as gametangia and they may contain one or more gamete nuclei or differentiated gametes. The male gametangium is referred to as the antheridium while the female gametangium is called the oogonium. There are five different ways in which fungi bring compatible nuclei together to fuse, these include; plasogametic copulation, gametangial contact, gametangial copulation, spermatization and somatogamy.

1.5.1.4 Classification and taxonomy

Fungal classification is an extremely complex and subjective field with countless difficulties. On the simplest level, two of the most common fungi associated with allergic disease can be differentiated into two types based on the macroscopic appearance of the colonies. Those that produce opaque, creamy or pasty colonies are known as yeasts, whereas those that produce cottony, woolly, fluffy or powdery aerial growths above the culture medium are called moulds. Further macroscopic and microscopic identification can be accomplished by differentiating between the colour and gross morphology of the colonies, however specific identification requires the induction of the fungus to display its characteristic conidia, which may require specific nutrient media or culture growth conditions (Cooper, 1985). To date, many taxonomic issues have still not been resolved, which has lead to differences in opinion amongst mycologists. Unlike plants and animals, fungi have a limited fossil record, which makes it difficult to determine the relationships between specific families and genera. Recent paleontological evidence suggests that fungi arose during the Pre-Cambrian period, approximately one billion years ago, however most of the present day fungal families are represented in the stratigraphic record by the end of the Palaeozoic, some 400 million years ago. Furthermore, the advent of molecular techniques, in particular polymerase chain reaction (PCR), is replacing subjective taxonomic techniques and has enabled the investigation of evolutionary and phylogenetic relationships to further differentiate fungal groups. At present, 69,000 species of fungi have been identified and 1.5 million species are estimated to exist. According to Ainsworth (2001), these species belong to five separate divisions termed the Ascomycota, Basidiomycota, Chytridiomycota, Zygomycota and the Anamorphic fungi or Deuteromycota (Table 1.2). The division that has attracted the most amount of review with reference to respiratory tract disorders are the anamorphic fungi, which release a large quantity of spores into the atmosphere (Table 1.1). The Zygomycota is one of the oldest lineages of inconspicuous fungi to evolve with the Ascomycota, however the Basidiomycota diverged much later (Cooper, 1985;

Ingold and Hudson, 1993). This group is distinguished from others by coenocytic multi nucleate vegetative hyphae, sexual zygospores and asexual spores that are developed inside a sporangium. There are approximately 1000 named species within the Zygomycota (Figure 1.4) and include fast growing saprophytic moulds that are the first visible colonizers of decomposing organic material, insect pathogens, parasitic, biological control agents and are the fungal partners of arbuscular mycorrhiza (Ingold and Hudson, 1993). The largest division of fungi, the Ascomycota (Figure 1.4), are the most important degraders of cellulose in the ecosystem and are also important plant and animal pathogens. Its members share saprobic, symbiotic or parasitic characteristics have septate hyphae, produce eight sexual ascospores (meispores) inside a sac-like ascus and produce asexual conidia that are borne directly on hyphae. The vegetative mycelium is haploid, multinucleate and heterokaryotic and has septa with simple spores. The phylum Basidiomycota includes 29,000 known species (Figure 1.4) and comprises the mushrooms, puffballs, bracket fungi, rusts and smuts. These are saprobic, symbiotic or parasitic fungi that reproduce by forming Basidiospores, which are generated on a club-like cell known as the basidium (Ingold and Hudson, 1993). Nuclear fusion takes place inside the basidium, which is formed from the dikaryotic mycelium and may be surrounded by a large basidiocarp such as a mushroom or toadstool. The vegetative mycelium is dikaryotic and consists of divided septate hyphae. The Basidiomycota often have complex lifecycles with more than one type of asexual spore. The Anamorphic fungi or the Deuteromycota comprise 16,000 species (Figure 1.4) and are saprobic, symbiotic or parasitic. Anamorphic fungi are a polyphyletic group in which many species are related to other members in the Ascomycota and Basidiomycota. The term “anamorphic” is given to these fungi because they have lost the ability to reproduce sexually or the sexual stage has not yet been discovered. Most reproduce asexually by the dissemination of airborne conidia, although a few are purely mycelial and develop no spores (Ingold and Hudson, 1993). Anamorphic fungal classification is based upon the morphological characteristics of the asexual spores.

More typically, these are unicellular with septate hyphae and produce conidia from various types of conidiogenous cells (Alexopoulos et al., 1996). Genetic diversity is achieved through the parasexual cycle and is harboured in heterokaryons (Ingold and Hudson, 1993; Alexopoulos et al., 1996).

1.5.1.5 Fungal aerospora and dispersal

Particulate matter, bacteria, plant pollen and fungal spores are the major types of aerospora that can be found throughout the atmosphere. Assemblages of microorganisms suspended in the atmosphere are often dominated by one type with lesser numbers of other types (Gregory, 1973). The diversity of the aerospora within the atmosphere at any specified time is influenced by environmental factors such as rainfall, humidity and temperature. The interests in airborne microbes accelerated with the inception of kites and aircraft, which made it possible to explore the lower few kilometres of the atmosphere and gave rise to the field of Bioaerosol Science or otherwise known as Aerobiology (Gregory, 1973). Aerobiology is defined as the scientific discipline focused on the transport of organisms and biologically significant materials through the atmosphere (Gregory, 1973). The study of atmospheric bioaerosols has enhanced the understanding of the atmospheric environment and has generated information pertaining to the distribution, deposition, adhesion and release of bioaerosols. A bioaerosol is an aerosol whose components contain or have attached to them, one or more microorganisms, their components or effluents (Lighthart and Mohr, 1994). Microbial bioaerosols include organisms, such as algae, bacteria, particulate and mineral matter, soot, plant debris, pollen and fungi. Loading of bioaerosols into the atmosphere originates from natural and anthropogenic sources (Lighthart and Mohr, 1994). Natural loading is generated by winds that are later transported to the atmosphere and include wave spray, dust and soil. Anthropogenic bioaerosols are divided into two separate categories, extramural and intramural. Extramural bioaerosols are produced as sprays from agricultural sprinklers,

waste management plants and vehicular activity, while intramural bioaerosols are generated while walking, sneezing, coughing and talking. The atmosphere, weather and climate are factors that impact on the dispersion and survival of bioaerosols. Pollen, fungi and other particulate matter are transported away from the area of generation, commonly by the wind. The distance of travel relies on the size, mass, volume and aerodynamic nature of the bioaerosol. Throughout their journey, individual aerosols are constantly removed from the atmosphere by gravity, scavengers or when they adhere to either rainwater, snow droplets, plants, water or when they hit the ground. However, some aerosols are so small in structure that they adjust to the nature and movement of the air (Lighthart and Mohr, 1994). The atmosphere is composed of four distinct layers, the troposphere, stratosphere, mesosphere and thermosphere (Gregory, 1973; Lighthart and Mohr, 1994), whose boundaries are referred to as the tropopause, stratopause and mesopause. The earth’s atmosphere is stably stratified with warm light air located above cold dense air, therefore the vertical movement of air is retarded by stratification. Aerosols are mainly confined within the troposphere and its boundary with the stratosphere, which extends up to 10 kilometres. This region is characterized by a temperature lapse or a decrease of temperature with height. There are five transitional layers confined to the troposphere that are influential to aerosol distribution and dispersion (Gregory, 1973); the convective layer closest to the tropopause, the outer frictional turbulence layer, the turbulent boundary layer, local eddy layer, the laminar boundary layer and the layer closest to the ground comprises the surface boundary layer. Aerosols are initially disseminated in close proximity to the ground within the surface boundary layer (Lighthart and Mohr, 1994). Spore particles were thought to remain in small clouds at the turn of the century, although later it was proposed that microbes were in a state of randomness. It is now accepted that once released into the atmosphere, the concentration of spores per unit volume of air decreases with distance (Gregory, 1973). The early pioneers of Aerobiology recognized that fungal rust, which devastated rye plots was dispersed and patients suffering from hay-fever were advised to avoid grass pastures during the

flowering season (Gregory, 1973). These early investigations laid the foundations to a series of approaches used to understand spore dispersal. More recently, evidence suggests that fungal spores and associated pathogenic particles can be dispersed not just on a regional scale, but also on global and continental scales. In a review by Brown and co workers (2002), plant diseases were shown to be aerially dispersed over long distances and implicated in outbreaks of plant disease in other geographic regions (Brown and Hovmeller, 2002). Well-documented examples include the aerial transport of sugar-cane rust (Puccinia melanocephala) from Cameroon in West Africa to the Dominican Republic by cyclonic winds in 1978 and the outbreak of coffee leaf rust (Hemileia vastatrix) from Angola to Brazil in 1970 (Brown and Hovmeller, 2002). Fungi disperse their spores from the sporophore (spore holding structure) by a variety of active or passive means that appear in many cases to be adaptations to specific habitats. Active release mechanisms include fungi that disseminate spores either by ballistospore release or by spore discharge. Passive spore release involves the dissemination of spores in dry conditions by exposure to the wind or by the release of spores in mucilage in association with a vector. Researchers studying spore dispersal concentrate primarily on geometrical, empirical and meteorological approaches (Gregory, 1973). The geometrical approach, takes into account the laws of radiation, due to their relevance to the dispersion of spore clouds. The empirical method has been based on field records of dispersal gradients, such as the scatter of seedlings on the ground. The data accumulated is manipulated statistically to find an empirical formula for the line of best fit. The empirical method is advantageous but it is difficult to compare results from different sources. The atmospheric approach proved to be the area most likely to unveil the mystery behind spore dispersal. The atmosphere is a dynamic environment that varies constantly. Wind direction and eddies are processes that can fluctuate and generate irregular or regular spore dispersal patterns. These findings have led the way to further research of eddies and atmospheric processes to determine larger and smaller scale dispersal mechanisms.

1.5.1.6 Fungal spore deposition

The word deposition is given to all processes in which airborne particulates are transferred from the atmosphere to the surface (Gregory, 1973). Spores and other materials suspended throughout the atmosphere are deposited in a number of ways. Particle deposition occurs either as gravitational settling, molecular diffusion, impaction onto the surface, or in the precipitation process, through rainout and washout. In addition, microbiological particles vary widely in size, shape, density and aerodynamics, which all effect the processes of deposition. The speed at which particles are removed from the atmosphere is dependent on the settling velocity. The calculation of the settling velocity is widely recognized to be complex and relates to the numerous aforementioned variables, however for fungal particles of unit density and diameter range 1.6-70µm, Stokes’ law can be used to determine the settling velocity. Larger spores, such as those belonging to Alternaria species, settle more quickly than smaller spores belonging to Aspergillus species, however these smaller fungal particles may also be influenced by electrostatic forces and Brownian motion and thus may keep a particle aerosolised. The study of aerosol impaction has important implications in rain scavenging, dry deposition and aerosol sampling. Little was known about the deposition process until the development of the wind tunnel. Originally built for aerodynamic purposes (Gregory, 1973), the device was efficient in studying the behaviour of spores in controlled wind environments. Wind tunnel experiments have shown that impaction is dependent on the orientation and type of surface; the size, shape and density of the particle as well as the air temperature and pressure. It has been shown that small spores that approach large objects at low wind speeds follow the air trajectory and impact inefficiently, while large objects that approach small objects at high speeds impact efficiently (Gregory, 1973). These findings have been incorporated into the design and development of a number of aerosol impaction devices. In addition, other parameters that may affect the speed at which microbiological particles are removed from the air include, the characteristics of surrounding vegetation and the degree of turbulence in the lower atmosphere.

1.5.1.7 Medical disorders associated with fungi

Fungi are also recognized to cause a number of infectious diseases that range from cutaneous infections to fatal systemic mycoses, in addition to producing potent toxins (Horner et al., 1995; Bhatnager et al., 2002). Fungi that affect humans can be divided primarily into two groups; pathogenic fungi and opportunistic fungi. The major difference between these two divisions is that opportunistic fungi are incapable of infecting healthy hosts, whereas pathogenic forms can be recovered from both immunocompromised and non-immunocompromised patients (Guppy et al., 1998). To date, the incidence of medical disorders associated with fungi has risen dramatically for reasons that relate to the exponential intake of antibiotics, which suppress the natural bacterial competitors of fungi, the incidence of immunocompromising disease states, the intake of pharmacological mediators following tissue transplants and infectious organisms that suppress normal immune responses, such as human immunodeficiency virus and hepatitis (Guppy et al., 1998). The clinical manifestations of fungal disease can be categorised into three different groups and include; (1) cutaneous infections of the outer layers of the skin, (2) subcutaneous infections, where fungi have penetrated through wounds or have been injected into the tissue; and (3) systemic infections, where the fungus becomes established in the host (Kendrick, 1992). Well characterised fungal infections that occur frequently throughout the literature include fungal sinusitis, fungal pneumonia, invasive aspergillosis, lung abscesses, fungal infections of the spine, cerebral fungal infections, sinus mycetoma, nasal polyposis, rhinosinusitis and eczema (Osiyemi et al., 2001).

1.5.1.7.1 Cutaneous and subcutaneous infections

Many of us have been subjected to a cutaneous fungal infection at some point, which is caused by a group of fungi known as the dermatophytes. These fungi are able to degrade keratin and have been established to belong to Trichophyton, Epidermophyton and Microsporum species. Each of these fungi have specific micro environmental conditions that are required to facilitate growth and colonisation, however they generally become a problem for a host when the skin is either dead or remains warm and moist for

prolonged periods of time. The dermatophyte can be transmitted freely between hosts without the need for any dispersal vectors and examples of these diseases include tinea and ringworm (Kendrick, 1992). At the site of infection the fungi also releases a number of macromolecules, enzymes and metabolites, which have been shown to induce allergic or immunological reactions in many susceptible individuals (Kanny et al., 1996; Savolainen et al., 2001). Subcutaneous infections involve the entry, colonisation and future infection by a fungus through an opening in the skin or wound caused by either passive or active trauma. Fungi belonging to the genus Sporothrix cause the most common subcutaneous fungal infections. These fungi can enter the body in a hyphal form and cause localised infection before converting to a yeast, which can then spread throughout the lymphatic system, joints, bones and internal organs (Kendrick, 1992).

1.5.1.7.2 Systemic mycoses

Systemic mycoses are classified as fungal infections that are systemic and borne within the body (Kendrick, 1992). The most frequent point of entry for infection vectors includes the inhalation of viable spores or hyphae into the lung or through wounds following surgery. Systemic disease may also follow ingestion of mould contaminated food and water, which are regarded as additional transmission vectors (Bouakline et al., 2000). The most described fungal infections include phaeohyphomycoses, spine infections, invasive aspergillosis and fungal sinusitis. Phaeohyphomycosis is a histopathological term coined to encompass cutaneous, subcutaneous and systemic infections caused by byphaeroid (dark-coloured), hyaline, septate and filamentous moulds that develop as dematiaceous septate hyphae in host tissue in affected patients (Odell et al., 2000; Osiyemi et al., 2001). Phaeohyphomycosis is a rare disease, which affects immunocompromised hosts and is caused by various bacterial and fungal species including, Cladophialophora bantiana, C. trichoides, Exophiala dermatitidis, Ochroconis gallopavum, Chaetomium atrobrunneum, Aspergillus species, Blastomyces dermatididis, Candida albicans, Coccidioides immitis, Cryptococcus neoformans, Histoplasma capsulatum and Ramichloridium mackenziei

(Guppy et al., 1998; Odell et al., 2000; Osiyemi et al., 2001). The fungal species, Cladophialophora bantiana, is the most common fungal type to be isolated from brain tissue of patients and it has been shown to negatively influence the central nervous system and the integument (Osiyemi et al., 2001). According to Osiyemi al. (2001), phaeohyphomycoses due to C. bantiana, mainly affect males within the age range of 6 to 76 years. Cerebral abscesses have been reported to occur in healthy hosts, mainly in the Middle East and are caused by the fungal species, Ramichloridium mackenziei (Osiyemi et al., 2001). Conversely, Guppy et al (1998) established that out of 58 patients who underwent bone marrow transplants, approximately 92% were diagnosed with a cerebral fungal infection. Thus, the majority of these fungal species have a predilection for brain tissue, are highly neurotropic and cause disease in vertebrates and humans with an immune deficiency. Common clinical symptomology associated with cerebral phaeohyphomycoses include acute headaches, paresis and somnolence. In contrast, fungal spine infections are noncaseating, acid-fast-negative infections that occur primarily as opportunistic infections in immunocompromised patients (Frazier et al., 2001). Since the original article published by Keating in 1932, there has been an expanding body of scientific knowledge concerning the diagnosis and treatment of this unusual disease (Keating, 1932). A recent study by Frazier et al (2001), established that spinal infection sources typically arise from implantation from trauma, hematogenous spread and elective spine surgery. After being infected paralysis developed in eight of eleven patients, two patients died and the average delay in diagnosis was ninety-nine days, which demonstrates the difficulties involved with the diagnosis of the disease (Frazier et al., 2001). Fungal spine infection is a rare systemic mycosis, however fungi that characterise this disease include Candida albicans and Petriellidium boydii that are generally slow growing and are difficult to identify by culture.

1.5.1.7.3 Fungal sinusitis

Sinusitis is defined as the inflammation of one or more of the paranasal sinuses, which are air-filled cavities in the facial bones that are lined with pseudostratified ciliated

columnar epithelium and mucous goblet cells (Dykewicz, 2003). Recent estimates suggest that the overall United States of America health care expenditure attributable to sinusitis was approximately $5.8 billion dollars in 1996 (Ray et al., 1999) and with chronic conditions presenting symptomology that might extend for more than 12 weeks, demonstrates the significance of the disease. Fungi have been increasingly recognized as pathogenic agents involved in the exacerbation of sinusitis, particularly when histological similarities have been made between inspissated sinus material from patients with chronic sinusitis and bronchial mucus plugs from patients with allergic bronchopulmonary aspergillosis (Millar et al., 1981; Katzenstein et al., 1983a; Manning et al., 1997). This was further supported when clinical and pathogenic observations suggested that fungi could contribute to inflammatory disorders of the nose and paranasal sinuses in the absence of tissue invasion. However, whether or not fungi can exist in sinus mucous without causing disease remains unclear (deShazo, 1998) and a contentious issue (Ponikau et al., 1999). Fungal sinusitis or otherwise known as “aspergillus sinusitis” can be accurately diagnosed by the visualization of fungal elements by histopathologic examination of material or tissue removed from a sinus (deShazo, 1998). Even though fungi can be cultured from nasal secretions of healthy individuals this does not represent a diagnostic criteria for fungal sinusitis, especially as fungi are often difficult to culture from infected sinuses. The pathogenesis of fungal sinusitis is characterised by two different disease states, which include non-invasive and invasive forms (deShazo et al., 1997; deShazo, 1998; Asero and Bottazzi, 2000). The key feature in distinguishing invasive from non-invasive forms is by the breaching of the sinus mucosa by fungal elements with fungal growth into the submucosa and underlying bone, in addition to tissue necrosis (deShazo et al., 1997; deShazo, 1998). Non-invasive fungal sinusitis can be differentiated into two separate forms, allergic fungal sinusitis (AFS) and sinus mycetoma. AFS is a newly characterized disease that has generated a great deal of interest over the last decade and was first described in the 1980’s by Miller et al. (1981) in Great Britain and Katzenstein et al. (1983a) in the United States. Katzenstein et al. (1983a) described the disease as an

allergic response to fungi in the paranasal sinuses similar to that of allergic bronchopulmonary aspergillosis, however it became apparent that Aspergillus species were not the only organisms to be associated with the symptomology (Michaels et al., 2000). Anamorphic fungi, including Alternaria (Bartynski et al., 1990), Exserohilum (Friedman et al., 1991), Curvularia (Brummond et al., 1986; Bartynski et al., 1990) and Bipolaris (Gourley et al., 1990) species, as well as non-dermataceous fungi, including Fusarium (Peltroche-Llacsahuanga et al., 2001), Chrysosporium (Scheer and Schultz, 1992), Rhizopus and Mucor (Goldstein et al., 1992) species have also been identified to be causative organisms. Patients with AFS are generally immunocompetent and have a history of allergic rhinitis and asthma. These patients may also have a history of chronic sinusitis and have often undergone multiple sinus surgeries prior to diagnosis (Katzenstein et al., 1983b; Bent and Khun, 1994; deShazo and Swain, 1995). The clinical manifestations include the incidence of fungal elements in mucous material and within sinus contents, which generate an immune response and result in mucous exudate, nasal polyps and bone erosion (Michaels et al., 2000). The contents of the sinuses include eosinophil rich material called allergic mucin, which has a consistency similar to that of peanut butter or cottage cheese and may be foul smelling (deShazo et al., 1997). The nasal polyposis associated with this condition may form an expansive mass that causes bony necrosis. According to Michaels (2000), bone erosion occurs in 19%-80% of patients and in some cases patients might present with a complete breach of the bony sinus wall, which is the result of the secretion of substances from the vascular inflammatory tissue (Michaels et al., 2000). Nevertheless, very little is actually known about the inflammatory and pathogenic mechanisms that give rise to AFS, although it is known that dematiaceous and non-dematiaceous fungi are present within the lumina of seromucinous glands, ducts within the nasal cavity and the parasinal sinuses (Michaels et al., 2000). Under certain circumstances, when conditions supporting the colonisation and proliferation of fungi are present in the nose and paranasal sinuses, another form of non- invasive fungal sinusitis may develop known as sinus mycetoma or fungus ball. In these cases, unlike AFS, fungal hyphae grow throughout any available spaces in the nose and

paranasal sinuses in the absence of inflammation (Marple and Mabry, 2002). Patients who present with symptoms of chronic sinusitis, might complain of blowing out gravel- like material from their noses or be found to have opacifications on sinus radiographs, in addition to having facial pain or seizures (deShazo, 1998). The management of such infections include the clinical removal of the mycetoma (Ferguson, 2000), however it has been shown that exposure to small mycetomas in certain fungal atopic individuals can lead to a cascade of inflammatory responses (Marple and Mabry, 2002).

1.5.1.7.4 Invasive aspergillosis

The potential for mycotic disease of the lower respiratory tract has been known for the better part of a century. Invasive aspergillosis is an opportunistic infection that is a life threatening disease predominantly in immunocompromised patients (Ruchel and Reichard, 1999; Reboux et al., 2001). The fungal species A. fumigatus is widely recognised as the second most frequent opportunistic fungal infection and the species most frequently isolated in invasive aspergillosis. This can be accounted for by its virulence, wide ranging distribution and the increasing prevalence of immunocompromised patients (Beck-Sague and Jarvis, 1993). Its importance in aspergillosis also relates to the species pathogenic virtues, which include its omnipresence, its small size permitting penetration into the airways and its readiness to transform into tissue-invasive hyphae under host conditions. However, other fungal species have additionally been implicated in the pathogenesis of aspergillosis and include A. flavus, A. niger, A. terreus, A. versicolor, Bipolaris hawaiiensis, and Curvularia lunata (Lake et al., 1991; Ruchel and Reichard, 1999). The pathogenesis of aspergillosis follows the inhalation of spores that release various extracellular proteins, including lytic enzymes or toxins on contact with the lining of the respiratory mucosa. In the airways, the defence against inhaled Aspergillus involves the clearance of spores and other viable propagules by ciliary transport and phagocytosis by alveolar macrophages. If these mechanisms are inhibited by the secreted enzymes and toxins, chronic colonization and invasive infection take place as happens in patients with cystic fibrosis (Wojnarowski et al., 1997). Although the exact

molecular mechanisms employed by Aspergillus to evade the immune system are not known, it has been recently shown that the in vivo germination of A. fumigatus conidia promotes the synthesis of macromolecules that function to evade TLRs, which are important in the recognition of pathogens. Furthermore, the dosage of viable conidia inhaled and the relationship to impairment of the respiratory defence are critical as demonstrated by the role of house plant sources of Aspergillus near neutropenic patients (Summerbell et al., 1989; Staib, 1992) and the potential risk associated with infested dump sites (Kramer et al., 1989).

1.5.1.7.5 Mycotoxicosis

Many fungi produce toxic secondary metabolites that are either beneficial or detrimental to human health. To date, approximately 400 known mycotoxins exist and may have developed to function as a chemical defence system against insects, other microorganisms, nematodes, grazing animals and humans (Bhatnager et al., 2002). Most fungi release the secondary metabolites during the digestion process. Mycotoxins do not appear to have any physiologic function other than to reduce competition from other fungal colonies. Mycotoxins are characterised by a low molecular weight and can elicit acute toxic, mutagenic, teratogenic and carcinogenic effects (Burge, 2001; Bhatnager et al., 2002). The toxic effects of mycotoxins are termed “mycotoxicosis”, which is characterised by damage to the cells of most major organs, endocrine and immune systems (Bhatnager et al., 2002). A number of mycotoxins have been identified and the most frequently encountered of human importance include ergot alkaloids, aflatoxin and to some extent trichothecenes. The health effects caused by exposure to these mycotoxins are well documented and wide-ranging, but are primarily derived from human ingestion data. The ergot alkaloids and aflatoxin are probably the most widely recognized mycotoxins and were the first to be recognized to have health implications, which date back to the 9th century (Etzel, 2002). Persons who ingested ergot alkaloid contaminated mouldy rye grains were exposed to vasoconstrictive properties of the ergot alkaloids, which led to ergotism or otherwise known as St. Anthony’s fire (Lewis, 1977). Aflatoxin is primarily

produced by Aspergillus species and is a common contaminant of peanuts, soybeans and cassava roots in tropical environments. The ingestion of aflatoxin B1 has been shown in epidemiologic studies to increase the risk of contracting hepatocellular cancer in humans (Van Rensburg, 1977) and has been hypothesised to induce allergic sensitisation (Kocaba and Sekerel, 2003). Exposure to aflatoxin has also been shown to induce chronic effects termed “acute aflatoxicosis” that include abdominal pain, vomiting, hepatitis and even death (Krishuamachari et al., 1975; Etzel, 2002). However, the link between human respiratory exposures to aflatoxin and disease remains unclear (Baxter et al., 1981; Burg et al., 1981; Burge, 2001). The fungal genera Fusarium and Stachybotrys have been shown to produce mycotoxins called trichothecenes. Personal exposure to trichothecenes, following ingestion of contaminated wheat and corn, has been shown to produce alimentary toxic aleukia, which was responsible for the deaths of more than 100, 000 people in Russia between 1942-1948 (Etzel, 2002). The clinical symptomology of alimentary toxic aleukia include the development of necrotic ulcers in the nose, mouth, throat, stomach and intestines, in addition to haemorrhage from the nose, mouth, gastrointestinal tract and kidneys (Etzel, 2002). Acute toxicosis following the inhalation of Stachybotrys mycotoxins was also described in the early forties by Russian scientists (Drobotko, 1945). Presenting symptoms include a sore throat, bloody discharge from the nose, dyspnea, cough, low- grade fever and chest tightness (Etzel, 2002). However, recent studies focussing on the production of saratoxin from Stachybotrys chartarum growing in indoor moist environments have confirmed and extended these findings. Etzel and co workers (1998) found that 10 infants with life-threatening acute pulmonary bleeding were more likely to live in homes with S. chartarum and other moulds than a matched group of 30 comparison infants (Etzel et al., 1998). Other studies have also shown an association between S. chartarum exposure and acute pulmonary haemorrhage in infants located in Kansas City, Missouri (Flappan et al., 1999) and Houston, Texas (Elidemir et al., 1999). However, these reported findings linking S. chartarum exposure to infant pulmonary

haemorrhage remains contentious and requires further investigation (Etzel, 2002; CDC, 2000). To date, a vast array of other fungi produce toxins, however their identity, human benefits and health effects have not yet been identified. Furthermore, the quantity of toxins required to elicit health effects remain unknown and difficult to determine due to the complexities of isolating airborne mycotoxins. Preliminary data using animal models suggests that very large doses are required to be inhaled before acute toxic effects are exacerbated (Nikulin et al., 1996; Nikulin et al., 1997; Reijula et al., 1997; Burge, 2001).

1.5.2 Airborne distribution

The frequency and distribution of aerosolised fungal spores in indoor and outdoor environments is dependent on a number of prevailing seasonal and meteorological parameters, in addition to temporal, spatial and geographic variables. Depending on the environmental conditions that are required for fungal growth, reproduction and spore dissemination, spore counts have been shown to be present in outdoor air throughout the year and frequently exceed pollen counts by 100-1000 fold. Indoor fungi are also heterogeneous in composition, although these are characterized by a mixture of spore types that have entered from the outside, in addition to those that grow and multiply due to damp conditions indoors. These counts are further influenced by a number of other variables that relate to intramural and extramural disturbances as well as air sampling parameters. The most common fungi identified in indoor and outdoor environments include Aspergillus, Penicillium, Cladosporium, Aureobasidium and Basidiomycete species and these have seasonal spore releasing patterns (Bush and Portnoy, 2001). During the last quarter of a century, the emerging interest in Allergology together with the inception of volumetric and personal air sampling techniques has witnessed the exponential increase in the number of studies that have assessed the relative abundance of fungal spores in both indoor and outdoor environments. The majority of these studies have occurred throughout Europe and have provided valuable information on the seasonal distribution of airborne fungal counts and the types of fungi

that were prevalent and important in the exacerbation of allergic rhinitis and asthma. These studies have shown that the most common spores belong to Cladosporium, Botrytis, Ustilago, Alternaria, Epicoccum, Erysiphe, Entomophthora, Torula, Stemphylium and Polythrincium species and peak spore counts range anywhere between 1 000 – 10 000 000 spores per m-3 (Nikkels et al., 1996). The presence of individual genera has also been shown in most cases to fluctuate throughout the year. The most dominant fungus identified with the highest airborne concentrations in the majority of these studies include Cladosporium species during the spring and summer months (Comtois and Mandrioli, 1996; Nikkels et al., 1996; Pelizzari, 1996), however during the winter months Penicillium and Aspergillus species were often predominant indoors (Cosentino and Palmas, 1996; Meriggi et al., 1996; Pasanen et al., 1997; Katz et al., 1999). Furthermore, a number of these genera, in particular Cladosporium, Penicillium and Alternaria have also been shown by a number of investigators to settle in high concentrations in mattresses, carpet, the bedroom, and living areas of indoor environments (Benguin, 1995; Benguin and Nolard, 1996; Cosentino and Palmas, 1996; Pasanen et al., 1997). In other regions of the world, such as in North America, the research into airborne fungal spores has primarily addressed the problems associated with indoor fungal exposure, particularly in the homes of low-income urban communities (Rogers, 2003; O'Connor et al., 2004). These aerobiological studies have shown that the concentrations and distribution of fungal spores varies significantly to those reported in European studies. These differences may relate to variations in vegetation, climate and sampling methodologies (Rogers, 2003). In the northern regions of the United States, spore concentrations have been shown to be highest during the spring and summer months, however in the southern areas of the country, fungal spores are present throughout the entire year with peak spore counts occurring in late summer and early autumn months (Bush and Portnoy, 2001). The most abundant fungi that are reflected in spore counts include Cladosporium, Penicillium, Aspergillus, Paecilomyces, Alternaria, Trichoderma, Ulocladium, Stachybotrys, Fusarium, Aureobasidium, Phialophora, Wallemia, Acremonium and Rhodotorula species (Levetin et al., 1995; Cole et al., 1999;

Wedner et al., 1999; Ren et al., 2001). However, numerous other fungal spore types, such as those belonging to Basidiomycetes are also abundant (Kramer et al., 1959), particularly in Kansas and New Orleans and have been implicated as a causal factor for asthma exacerbations in these regions (Salvaggio et al., 1971). These studies have also shown a wide range of indoor exposures, which range from less than 100 colony forming units (CFUs) per m-3 for viable fungi and 100 spores per m-3 to concentrations over 1000 CFUs per m-3 for viable fungi and 15 000 spores per m-3 for total fungi (Levetin et al., 1995; Ren et al., 2001; O'Connor et al., 2004). In contrast, spore surveys conducted in equatorial Asian countries have established that fungal spores are abundant throughout the entire year and these do not follow any distinctive seasonal periodicity. In a survey of airborne fungal spores at Dehra Dun, India, Singh and co-workers (1987) demonstrated that the most prevalent fungi belong to Cladosporium, Alternaria, Curvularia, Aspergillus, Penicillium, Dreschera, Chaetomium and Epicoccum species with July through to October identified as the period of greatest spore concentrations. However, in Taiwan and Japan, the predominant fungal genera are restricted to only a handful of fungal spore types including Cladosporium, Aspergillus, Penicillium and Alternaria species (Su et al., 2001; Ara et al., 2004). In Australia and New Zealand, only a handful of studies have assessed the incidence of airborne fungal spores in indoor and outdoor environments. Of these, spores belonging to the genus Cladosporium have been established to be the most predominant outdoor airborne fungal spore type in Australia and accounts for over 40% of the total fungal spore count in Melbourne and Brisbane (Rutherford et al., 1997; Mitakakis and Guest, 2001). However, in Auckland, New Zealand, Ascospores belonging to Leptosphaeria species were the most abundant (Hasnain, 1993). Other common outdoor genera that have been identified include Alternaria, Ustilago, Epicoccum, and Botrytis species (Hasnain et al., 1985; Bass and Morgan, 1997; Mitakakis et al., 1997; Rutherford et al., 1997; Mitakakis and Guest, 2001). Seasonally, spore levels peak through spring and summer and begin to decline in total concentrations towards the end of autumn (Mitakakis and Guest, 2001). Several

residential studies have also assessed the relative distribution of fungal spores in indoor residential environments and identified Cladosporium and Penicillium species to be the most prevalent indoors, while Aspergillus, Cephalosporium and Gliocladium spores were also abundant but restricted to the winter months (Garrett et al., 1996; Dharmage et al., 1999). Furthermore, it has also been shown by Dharmage and co-workers (1999), that approximately 55% of all residential houses have viable counts exceeding 550 CFUs per m-3 and that these higher indoor concentrations were associated with infrequent ventilation, vacuuming, presence of pets and old carpets. The contribution of other fungal propagules, including fungal fragments and airborne hyphae to the total airborne fungal count is not currently known and is a parameter that is largely dismissed in many aerobiological and epidemiologic studies. The reasons for this relate to the difficulties involved with resolving and subjectively differentiating between the morphologically indiscernible particles. However, recent studies suggest that airborne hyphal fragments are an abundant component of the airborne mycoflora indoors (Li and Kendrick, 1995b) and in some cases these fragments outnumber spores of individual allergenic genera (Gorny et al., 2002; Gorny, 2004). Li and Kendrick (1995a) observed that the highest concentrations of hyphal fragments were localized in the living room of residential environments and often outnumbered Alternaria, Epicoccum and Leptosphaeria spore counts (Li and Kendrick, 1995b). Culturally derived studies have shown that the aerosolization of fungal fragments associate with the fungal species, air velocity, the substrate surface and intensity of vibration (Gorny, 2002). These fragments can be released up to 320 times higher than spore counts, however the two measurements are not correlated (Gorny et al., 2002; Gorny, 2004). In addition, the incorporation of hyphal counts with spore counts has also been shown to greatly enhance the associations with asthma exacerbations (Delfino et al., 1997). As a result, future investigations should incorporate hyphal fragment counts to further characterize the distribution of these particles in indoor and outdoor environments and to determine their effect on the aetiology of fungal allergic disease. Airborne fungal spore counts may be affected by a number of weather, disturbance and methodological variables. According to Bush et al. (2001), fungal spore

numbers can be influenced by meteorological parameters, including temperature, rainfall and relative humidity. For instance, fungal spores belonging to Fusarium, Phoma, Ascomycete and Basidiomycete species predominate during periods of precipitation, while spores that belong to Cladosporium, Alternaria, Epicoccum, Helminthosporium and Dreschera species predominate during dry weather (Mitakakis et al., 1997; Nayak et al., 1998; Bush and Portnoy, 2001; Stennett and Beggs, 2004). Personal disturbance, which includes daily activities such as walking and mowing the lawn, in addition to exercise was shown by Mitakakis and co-workers (2001b) to aerosolise greater quantities of Alternaria spores, which significantly increased personal exposure. Analogous findings were made by Buttner and co-workers (1993, 2002), who demonstrated that walking significantly increased the proportion of spores that were aerosolised from loop pile carpets. Cropping activities in rural environments also provides a much larger disturbance activity, which has been shown to significantly increase the concentration of spores aerosolised from wheat crops in Western New South Wales, Australia (Mitakakis et al., 2001b). Alternatively, variations in the methods of air sampling and analysis have been shown to obtain dramatically varying results from the same geographic environment. The height at which air samplers operate effectively has been a widely debated subject with numerous studies demonstrating that spore numbers decrease in concentration with increasing height (Rantio-Lehtimaki et al., 1991; Alcazar et al., 1999; Fiorina et al., 1999; Bergamini et al., 2004). Variations in methods of counting are also known to further confound the interpretation of fungal exposure measurements, which makes it difficult to compare between studies (Sterling et al., 1999). Thus, to date, there are no standardized methods for measuring fungal exposure and with recent estimates of more than 50% of fungi misidentified in aerobiological studies (Flannigan, 1997), demonstrates the need to implement standardized and objective methods of analysis.

1.5.3 Allergenic fungal extracts

Progress in understanding fungal allergic diseases has been obstructed by the complexity and ubiquity of fungal allergens and species, the instability and variability

of their allergens, the disagreement on current nomenclature and the selection of adequate source material for the preparation of extracts. For these reasons the characterization and standardization of fungal allergen extracts has lagged behind that of other aeroallergen sources. The most commonly studied and characterized allergens of the anamorphic fungi belong to A. alternata, A. fumigatus, and C. herbarum, which relate to their airborne abundance in many geographic environments. Photomicrographs of the colony features and reproductive structures of these species are shown in Figure 1.5. However, the importance of other fungal species, particularly Exserohilum rostratum, Epicoccum nigrum and Botrytis cinerea is becoming increasingly recognized (Karlsson-Borga et al., 1989). Fungi produce a number of proteins, glycoproteins and carbohydrates that are capable of stimulating the immune system and bind specifically to IgE antibody produced in atopic individuals in response to stimulation (Esch, 2004). Fungal allergens range in molecular weight from roughly 10 to >100 kDa and any species may contribute as few as 4 to more than 30 fungal allergens (Horner et al., 1995; Rogers, 2003) (Table 1.3). Although most of the IgE binding potential resides in the protein components of the fungal extracts (Doukes et al., 1993), it has been shown that glycoproteins comprise the greatest proportion of several fungal allergens, particularly for C. herbarum and A. alternata that consist of up to 80% carbohydrate. Experimental studies show that when this fraction is removed, IgE binding is significantly reduced (Portnoy et al., 1991; Einarsson and Aukrust, 1992). Thus the challenge for many researches is to remove and extract the allergens from the fungus in significant quantities without modifying or altering the allergenicity or potency of the macromolecules. This is particularly important as the diagnosis of fungal allergic disease by in vivo and in vitro tests is principally based on the availability of well-characterized allergen preparations. Fungal allergen extracts used for skin prick and provocation tests are typically crude, unpurified, highly variable and are usually prepared using culture media. Fungal growth of most species is dependent on specific temperature, nutrient, moisture and light regimes. When these conditions vary slightly or are limited, the expression of many proteins and enzyme systems decreases and to adapt to these altered conditions

the fungus responds by synthesizing a different assortment of macromolecules. Recent studies with E. nigrum and C. lunata have demonstrated that the most biologically potent extracts were cultured on semi-synthetic Sabouraud’s broth after 11-13 days and these had the highest antigenic and allergenic reactivity compared to natural media selections (Gupta et al., 1999; Bisht et al., 2000). These findings further confirmed earlier studies that showed that semi-synthetic nutrient media yielded the most allergenically potent extracts that approached the activity of a commercially prepared extract (Vijay et al., 1985). Allergenic fungal extracts derived from synthetic media demonstrate less variability and provide a rich source of culture filtrate in two to three weeks, while reliable mycelial antigens can be obtained from short-term fungal growth of aerated culture. The methods and time of extraction may also result in variations between fungal allergen extracts. Bouziane et al. (1989) demonstrated that the amount of products released by C. cladosporioides during the extraction procedure depended on the composition of the solution used and the concentration of conidia. Other researches have extended on this concept and included methods of disruption, in addition to the time of extraction as important parameters for extraction. Paris and co-workers (1990b) showed that greater amounts of allergen were extracted from Alternaria following active spore disruption, while passive methods generally yielded lower quantities (Paris et al., 1990b). The amount of protein and carbohydrate released from Alternaria was also shown to be dependent on extraction time with the majority of allergens extracted within 1 hour (Portnoy et al., 1993a). In contrast, some allergens have been shown to be more prevalent after a 24-hour extraction (Portnoy et al., 1993a), although the explanations for this are debated. The pattern of allergen release also varies according to the spore wall structure (Horner et al., 1993). Spores with thinner walls, including Pleurotus ostreatus and Lentinus edodus release the majority of allergen within four hours, whereas thicker spore walls of Psilocybe cubensis and Calvatia cyathiformis can take up to 24 hours (Horner et al., 1993). It has been suggested that thicker spore walls contain less protein and unlike the hydrophobic rodlet layer present in Cladosporium species, the wall

functions to reduce cytoplasmic leakage and absorbs allergen. However, longer incubation periods decrease the total allergen content in most fungal extracts, due to the activity of extracellular serine proteases. After 24 hour extractions, it has been shown that between 0-25% of the maximal protein concentration remains (Bouziane et al., 1989; D'Rosario et al., 1997). Proteolysis can be blocked by protease inhibitors, such as phenyl-methylsulfonyl fluoride or by using either a 5-50mM Tris buffer pH 9.0 or a variety of commercially available protease inhibitor cocktails. Such proteolytic degradation may have a deleterious effect on the potency of the extract and may explain why some individual allergens can vary from 1000% to 5% of a standard reference (Salvaggio and Aukrust, 1981). To date, kinetic studies have demonstrated that maximal sugar and protein release occurs 3-4 hours after the beginning of incubation (Bouziane et al., 1989; Horner et al., 1993; D'Rosario et al., 1997). The source material for many fungal extracts previously consisted of fungal spores as it was presumed throughout the literature that personal exposure occurred principally to these propagules. However, studies exploring the antigenic and allergenic composition of spores and mycelium have shown that various spore and mycelium specific allergens exist (Hoffman et al., 1981; Einarsson and Aukrust, 1992). In Alternaria, spore extracts contained eight-spore specific antigens not detected in the mycelium extract. On the other hand, Lehrer et al. (1986) compared several Basidiomycete mycelia and spore allergen extracts by skin prick testing patients. Of the patients, 32% had positive skin prick test responses to the mycelial extracts compared to 27% that showed positive reactions to the basidiospore extracts (Lehrer et al., 1986). Although these studies demonstrate that spore preparations contain a more diverse variety of allergens, allergenic extracts generally contain additional mycelia components as these have the same allergenic molecules but are often in higher concentrations (Fadel et al., 1992). Biochemical variability within a fungal species has been documented for several allergenically important fungi, including A. alternata (Schumacher and Jeffery, 1976; Vijay et al., 1984), A. fumigatus (Wallenbeck et al., 1984) and C. herbarum (Vijay et al., 1991). Extracts from different strains of these species vary greatly in their

composition and in some cases it has not been possible to grow two consecutive cultures with similar antigenic profiles (Schumacher and Jeffery, 1976). A number of researchers and commercial suppliers try to overcome issues of isolate variability by culturing several isolates and growing different batches of the specific fungus at the one time and then pooling the extracted material from each culture. The stability of allergenic extracts depends on a number of variables that relate to the storage temperature, the nature and structure of the allergen, the quality of the allergen extract and the presence of preservatives and proteolytic inhibitors in the mixture. Various studies have reported that lyophilization is the best method to preserve and maintain the allergenic potency of fungal extracts, however this process may permanently alter and inactivate some allergens (Kurup et al., 2000). Several studies have shown that Alternaria extracts lyophilised and stored at various temperatures below 4oC were stable for up to 21 months (Vijay et al., 1987; Helm et al., 1988). Vijay and co-workers (1987) also concluded that aqueous Alternaria extracts, stored under analogous conditions, remained stable for similar periods of time. Similarly, other studies evaluating extract stability have shown that Cladosporium extracts remained unaltered for at least five years when stored at –20oC (Sward-Nordmo et al., 1988), although this was not the case for A. fumigatus extracts, which only remained stable for less than 21 days (Hansen et al., 1994). Moreover, the addition of certain preservatives to allergen extracts such as phenol are recognized to have a destructive effect, but this can be prevented by adding a non-toxic stabilizer such as human serum albumin or glycerol (Niemeijer et al., 1996).

1.5.4 Diagnosis of fungal allergic disease

Fungal allergic disease can be diagnosed by in vivo skin prick test (SPT), in vitro detection of allergen specific serum IgE antibodies (RAST, Pharmacia CAP System™) and in some cases by provocative challenge tests (Licorish et al., 1985). The most frequently used diagnostic test is the SPT, which uses a commercial fungal extract (Malling, 1992; Esch, 2004). The assessment of allergen-specific sensitisation in the SPT consists of the development of a wheal or erythematic reaction, in which the

diameter of the reaction is measured after 15 minutes. Patients are regarded as sensitised to an allergen if the mean wheal diameter is 3mm greater than the negative control. Alternatively, in vitro tests such as ELISA, RAST and Western blots detect circulating antibodies against specific allergens in sera from atopic patients. The advantages of these in vitro techniques include the ability to quantify the potency of allergenic extracts, test against multiple fungal species at once, in addition to examining patients with skin diseases and patients under medication, which may interfere with SPT and in some cases avoid the possibility of anaphylaxis (Kurup et al., 2000). Although the SPT is recognized as an inexpensive diagnostic alternative to other commercially available tests, the allergen content in many mould extracts is questionable due to extract variations as aforementioned (Esch, 2004). Recent studies have demonstrated that commercial allergenic products, where a single allergen has been assayed, may vary up to more than 400 fold for A. fumigatus extracts (Vailes et al., 2001) and up to 3000 fold for A. alternata extracts (Yunginger et al., 1976). Furthermore, the lack of concordance between fungal in vivo and in vitro diagnostic tests has added to the clinical uncertainty about the accuracy and effectiveness of current fungal allergen diagnostic systems (Corsico et al., 1998; Mabry et al., 1999; Negrini et al., 2000). It is also recognized that fungal extracts contain a larger proportion of carbohydrate-associated allergens (Swardnordmo et al., 1984), which makes them less likely to be covalently coupled to the solid-phase used in CAP assays (Kozak and Hoffman, 1984). Within the community, the median wheal size for fungal allergy lies close to the cut-off value conventionally used to discriminate between positive and negative responses (Nordvall et al., 1990). Thus, it is likely that the variability of existing extracts, the use of a few selected extracts to represent a broader range of fungi and the uncertainty of an appropriate cut-off for a positive response have all contributed to the under-diagnosis of the prevalence of fungal allergy.

1.5.5 Cross-reactivity

Cross-reactivity or the sharing of epitopes between fungal genera is widely recognized. The term cross-reactivity refers to the antigenic similarities of various fungal species

and their associated allergens. According to Horner (1995), cross reactivity is a function of the shared epitopes and should be distinguished from parallel, independent sensitisation. The best method to detect shared epitopes is by inhibition immunoassays. A number of shared epitopes have been described for phylogenetically related species. Using RAST inhibition, Agarwal et al. (1982) identified some degree of cross- reactivity between Alternaria and Stemphylium extracts with multiple shared allergenic determinants. In fact, double-antibody radioimmunoassay studies for Alt a 1 demonstrated higher Alt a 1 activity in Stemphylium than in Alternaria. Significant IgE cross-reactivity has been detected between Alternaria and other fungal genera, including Epicoccum (Dixit and D'Rosario, 1997; Bisht et al., 2002), Cladosporium (Tee et al., 1987), Spondylocladium (Hoffman and Kozak, 1979), Dreschera (Hoffman and Kozak, 1979) and Curvularia species (Gupta et al., 2002). Tee et al. (1987) studied cross reactivity between different fungal genera using RAST-inhibition and identified considerable cross-reactivity between Alternaria and Cladosporium. More recent studies have shown the allergenic cross-reactivity of both C. lunata and E. nigrum to other airborne species, particularly A. alternata and to a lesser extent C. herbarum and A. fumigatus (Bisht et al., 2002; Gupta et al., 2002). Different species within a genus have also been shown to share similar epitopes, particularly for Aspergillus (Kim and Chaparas, 1979) and Alternaria (Vijay et al., 1979). Thus, these findings suggest a high degree of homology between allergens belonging to various fungal species (Achatz et al., 1995), which may in the future restrict the characterization and standardization of fungal allergen extracts to only a fraction of the current total now recognized from different species. Whether this is the case remains to be determined, nevertheless such findings would have significant implications for the development of immunotherapies and the characterization of health affects due to fungal aerosols.

1.5.6 Fungal spore germination and allergen release in vivo

Fungal spores possess a number of mechanisms, which serve to help them evade the defence system, which would normally remove them. Previous studies have shown that spores belonging to A. fumigatus produce a number of fungal virulence factors that

inhibit cilia beat frequency (Tomee and Kauffman, 2000), phagocytosis and macrophage adherence (Arruda et al., 1992; Amitani et al., 1995; Tomee and Kauffman, 2000), which promotes spore germination and further colonization within the respiratory tract and bronchioles. Recent research has indicated that germinated A. fumigatus spores express greater amounts of allergen compared to ungerminated spores (Reijula et al., 1991; Sporik et al., 1993). Mitakakis and co-workers (2001a) later confirmed these results by demonstrating that the main fungal allergen, Alt a 1, is expressed in greater quantities following spore germination (Mitakakis et al., 2001a). These studies have lead to the hypothesis that fungal spores may be capable of germination in the human respiratory tract following inhalation and may express greater concentrations of allergen than previously thought. Fungal spores are small enough to be inhaled and trapped within the bronchioles for periods extending to 24 hours before mucocillary clearance (Salvaggio, 1994). This provides enough time for spores to germinate. In fact under appropriate conditions, Alternaria will germinate in less than 6 hours (Rotem and Aust, 1991). This hypothesis is further supported by studies that have isolated a wide-range of pathogenic and virulent fungal species from the upper respiratory tract (Katzenstein et al., 1983b; Gourley et al., 1990; Cosentino and Palmas, 1996; Ponikau et al., 1999; Karpovich-Tate et al., 2000). In addition, the variable temperature regimes that are associated with nasal anatomy (Keck et al., 2000) may provide conditions that are optimal for site-specific fungal spore germination for some fungi, such as thermotolerant species including A. fumigatus, that may be localized deeper within the respiratory tract. However, fungal genera that are sensitive to temperature variations, such as Alternaria and Epicoccum species, may be restricted to the nasal cavity and turbinates. Thus, the influence of germination on the expression of allergen for other fungal species and whether germinated fungal spores are present within the nasal cavity remains unknown and requires further investigation.

1.6 Measuring environmental fungal exposure

Airborne fungi are ubiquitous in the environment and human exposure is inevitable. Such fungi differ greatly in their taxonomic, physical, ecological and pathogenic characteristics. Many strategies have evolved to sample, identify and interpret fungal exposure and their choice is determined by the hypotheses involved. While fungi and associated macromolecules can be sampled directly from surfaces and house dust, results do not generally reflect human exposure (Chew et al., 2001; Chew et al., 2003). For this reason, airborne spores are commonly sampled, by either filtration or impaction, using volumetric air samplers. Identification is commonly performed by either culture on nutrient medium or by light microscopy using morphological criteria, although new techniques using DNA probes or characteristic antigens or toxins continue to be developed. Interpretation of such exposure data is both complex and contentious, but while there are numerous recommendations there is no consensus on exposure thresholds. A better understanding of the complex pathogenic roles of fungi and susceptibilities of their hosts will enable refinement of techniques for sampling and interpretation. Although many studies have described environmental conditions that lead to fungal growth and distribution, there is no consensus on the most clinically relevant methods for measuring fungal exposure. Methods applied to estimate exposure to fungi must balance the level of information required to answer specific questions within the numerous practical and technical limitations of the techniques that have been developed. There are three phases to measuring environmental exposure: sampling, identification and interpretation.

1.6.1 Sampling

For simple measurement of mould presence, mycelia can be directly sampled from surfaces by swabbing or by lifting with an adhesive tape. Such samples are generally then subjected to either culture or direct microscopy (Macher, 1989; Flannigan, 1997; Rogers, 2003). The principle advantages are speed, simplicity and the high recovery of organisms available for analysis. The main disadvantage is the lack of quantitative

correlation of these samples with both the number and identity of airborne organisms a person may be exposed to and thus any interpretation in terms of relevance to disease pathology may be limited (Macher, 1989; Rogers, 2003). The measurement of ambient mould spores requires their recovery and isolation from a volume of air. Not only is this more representative of personal exposure but in many circumstances there are no appropriate surfaces available to represent multiple and distant sources. Many different designs of the devices used to collect air samples have evolved to match the different characteristics of the organisms and the forms of analysis to be applied. These devices allow various sized particles to be settled, impacted, filtered or impinged onto collection agents such as porous filters, agar media, adhesive films or into nutrient liquids. The collection efficiency for the various sizes and shapes of spores differs widely, due to the interaction between the characteristics of particles (particularly drag and inertia) and the sampler’s design and use (inlet shape, face velocity, and the orientation relative to air currents etc) (Gregory, 1973; Lighthart and Mohr, 1994). In addition, some spores behave less predictably, due to the effects of their non-spherical shape and surface ornamentation. Such mechanical sampling devices partly imitate the complex series of events occurring during human exposure to spores. Here, inhalation via the nose occurs through two small downward-facing orifices with oscillating airflow and the spores are collected at different sites within the airways by a combination of impingement, adhesion and impaction. Sampling strategies need to reflect the many different ecological variables involved in exposure, where spore concentrations differ in space, time and in the mixture of species present. At one end of the spectrum are immobile (static) samplers used in a fixed location to represent ambient concentration over an area (Figures 1.6 and 1.7), while at the other end are small personal samplers, worn on the body to represent an individual’s local exposure (Figure 1.8). The ambient concentration of spores can fluctuate by several magnitudes over a period of time, including minutes, hours or days, which is dependant on the situation. The time used for collection also varies, from ‘grab’ samples collected over seconds or minutes (Anderson-type; Figure 1.6) to samplers operating independently for up to a week (Hirst-type; Figure 1.7). The choice

of samplers reflects both the limitations of the analysis (e.g., long sampling times could overload culture plates) as well as the need for temporal information. Thus, any sample only provides an approximation of the exposure an individual or a community may receive over time in that location. Such a strategy may need to perform repeated sampling over the course of days and use multiple samplers. Our group has developed small filters termed Intra-nasal air samplers (INAS) that are worn inside the nostrils to collect inhaled particles (Figures 1.9-1.11). This air sampling methodology provides a unique insight into personal exposure to airborne fungal propagules and has been used extensively in personal exposure studies. There are several limitations, which include the particle capture efficiency for particles less than 3 microns in diameter and the impact of mouth breathing, however the latter is a negligible source of error as recruited subjects receive sampling instructions prior to use (Graham et al., 2000; O'Meara et al., 2005).

1.6.2 Identification

Numerous methods have been developed to identify the collected fungi. The two most common methods, which require specialist identification skills, are direct histochemical staining followed by microscopic visualization of spore morphology, and secondly, culture of viable spores on specific nutrient media. More refined techniques, commonly used to differentiate between strains of a species, involve the production of characteristic proteins or toxins, fine surface morphology resolved by SEM or the development of specific molecular probes. While culture may provide great discrimination, it has disadvantages: it is slow, it can be critically influenced by the nutrient medium chosen, non-viable species are not detected and competition and inhibition from other colonies may inhibit growth. Fungi may also be quantified by analysis of specific component molecules, for example, by RT-PCR, providing suitably characterized primers are available. Extracted antigens, proteins or toxins that are characteristic of species or genera may be immunoassayed, providing suitable antiserum is available. The advantages of such molecular techniques to monitor specific spores are

that they can be performed on a large number of samples, often quickly and without a strong technical background in mycological techniques. In many cases the production of suitable biomarkers by the affected host is used to detect previous exposures of pathological importance. The most common biomarkers are human IgG (of different subclasses) and IgE. The former can be used both as a function of airborne exposure and is used as a component of diagnosis for a range of fungal diseases (e.g., fungal allergy and hypersensitivity pneumonitis) (Lappalainen et al., 1998; Eduard et al., 1993; Eduard et al., 1992). Immune responses to specific A. fumigatus antigens can be used diagnostically (e.g., for allergic bronchopulmonary aspergillosis in cystic fibrosis), although in this case, the antibodies are the consequence of colonization of the airways and not aerosol exposure. Fungal-specific IgE is the hallmark of allergic sensitisation and in some communities such allergy is the strongest risk factor for asthma (Zureik et al., 2002). Our group has developed the Halogen Immunoassay (HIA, Appendix 1.1 and 1.2), which enables the co-visualization of individual spores together with their expressed allergens, which are immunostained with IgE or IgG (Mitakakis et al., 2001a). The method enables an environmental sample to be directly analysed to determine which type of spores a patient is allergic to (Figure 1.12). Biotrophs (which cannot be cultured and therefore extracts cannot be prepared for conventional allergy diagnosis) have also been identified as allergen sources by this method. Further, dual staining of Alternaria and Aspergillus spore-allergens with both human IgE and species- specific monoclonal antibodies is possible, which enables the identification of spores that are the source of allergens in situations where morphological criteria are not sufficient for their identification. The HIA also powerfully demonstrates the importance of germination in achieving allergen expression (Mitakakis et al., 2001a). It has been speculated that intra-nasal germination, rather than inhalation of numbers of spores per se, may form a critical event in providing ‘exposure’ to fungi. The wide diversity of fungi present in environmental samples and their possession of common pathogenic components, such as cell wall extracellular polysaccharides, has led to the use of general proxies for their biomass as markers of

overall fungal exposure. These markers include ergosterol and beta (1-->3)-glucans (Douwes et al., 1997; Chew et al., 2001). While such markers may lack the ability to discriminate between species, they do integrate the diversity of exposure to a variety of viable and non-viable components, the pathogenic role of these as inducers of innate mechanisms and the differences in biomass between spores of different species, which is about 200 fold between Alternaria and Aspergillus spores, for example.

1.6.3 Interpretation

Interpretation of sampling data continues to provide a challenge, particularly in a litigious society, balancing health risks with enterprises. Risk depends on the context; what may be acceptable for a dwelling or occupational setting, may be a serious risk for immunocompromised patients. Concern about the health effects of domestic airborne exposure to specific fungal mycotoxins is not widely substantiated, although fungal contamination provides a significant impact on both the structural integrity and aesthetics of buildings and may affect the health of occupants. There are currently no defined exposure thresholds, although a figure of 500 spores per m3, combined with higher exposure indoors than outdoors, would appear to provide a simple benchmark for indoor sites (Gots et al., 2003). A recent meta-analysis for asthma risk, linked disease activity to indices of fungal exposure (Zock et al., 2002). This is substantiated by studies showing increases in asthma symptoms and airways hyper-responsiveness with airborne spore levels (Downs et al., 2001) and experimental challenge studies that have produced symptoms with levels of spores that occur in high-exposure dwellings (Licorish et al., 1985).

1.7 Conclusion

After more than a century where the basic collection and analytical techniques have changed little, it is likely that the need to rapidly sample and detect airborne allergenic fungal propagules and toxins will contribute new methods to measure fungal exposure. Such methods range from rapid and miniature assay systems using specific antibodies,

protein fingerprints or DNA that can be applied to collected samples, to spectral UV or IR backscatter systems that might detect airborne organisms at a distance. The technical challenge remains formidable, characteristic macromolecules can be difficult to extract and some are only expressed under specific conditions, while the organisms of interest occur among a rich and variable background mixture of other materials that can obscure detection. However, the legacy of this will be more rapid and accurate systems to identify both the fungal organisms and their molecules involved in human disease.

1.8 Aims of the thesis

This thesis aims to examine the HIA as a new technique to diagnose fungal allergic sensitisation, to investigate the distribution, sources and factors influencing fungal allergens and to understand what is actually inhaled in exposure settings.

The aims will be addressed by investigating:

• the relation of HIAs to conventional immunodiagnostic techniques

• the distribution and sources of airborne fungal allergens in indoor environments

• the relation of airborne spore concentrations measured by traditional techniques to individual inhalation of spores

• how new diagnostic methods improve the characterization of personal exposure to fungal aeroallergens

• the factors that influence the amount of allergen released from spores

• and the patterns of fungal spore germination in the nasal cavity

Table 1.1: Fungal genera frequently associated with IgE mediated (atopic) allergya

Agaricus Curvularia Phoma

Alternaria Epicoccum Pisolithus

Amanita Eurotium Psilocybe

Aspergillus Exserohilum Rhizopus

Aureobasidium Fusarium Scleroderma

Bipolaris Ganoderma Stachybotrys

Botrytis Leptosphaeria Stemphylium

Calvatia Leptosphaerulina Trichoderma

Candida Monilia Trichophyton

Chaetomium Mucor Ulocladium

Cladosporium Paecilomyces Ustilago

Corprinus Penicillium Xylaria

a Adapted from Kurup (2002) and Bush (2001)

Table 1.2: Fungal nomenclaturea

Hyphochytriomycota

Labyrinthulomycota Chromista Oomycota

Ascomycota • Ascomycetes • Neolectomycetes • Pneumocystidomycetes • Saccharomycetes • Taphrinomycetes

Basidiomycota • Basidiomycetes Fungi • Urediniomycetes • Ustilaginomycetes

Chytridiomycota

Zygomycota • Trichomycetes • Zygomycetes

Anamorphic fungi

Acrasiomycota

Myxomycota Protozoa • Dictyosteliomycetes • Myxomycetes • Protosteliomycetes Plasmodiophoromycota

a According to the systematic arrangement presented by Ainsworth and Bisby (2001).

Table 1.3: Relevant characterised allergens from fungi and their function approved by the Allergen Nomenclature Committee a

Allergen Molecular size Reactivity with patient Accession Fungal species Function designation kD sera No. Alternaria alternata Alt a 1 28 IgE binding - U82633 Alt a 2 25 IgE binding - U62442 Alt a 3 - IgE binding Heat shock protein 70 U87807 Alt a 4 57 - Protein disulfidisomerase X84217 Alt a 6 11 IgE binding Ribosomal protein P2 X-78222 Alt a 7 22 IgE binding YCP4 protein X-28225 Alt a 10 53 IgE binding Aldehyde dehydrogenase X-78277 Alt a 11 45 - Enolase U82437 Acid ribosomal protein Alt a 12 11 - X84216 P1 Aspergillus fumigatus Asp f 1 18 IgE and IgG binding Ribonuclease M-83781 Asp f 2 37 IgE binding Laminin binding U-56938 Asp f 3 19 - Peroxisomal protein U20722 Asp f 4 30 - - AJ001732 Asp f 5 42 - Metalloproteinase Z-30424 Asp f 6 26.5 IgE binding Mn superoxide dismutase U53561 Asp f 7 12 - - AJ-223315 Asp f 8 11 - Ribosomal protein P2 AJ224333 Asp f 9 34 - - AJ223327 Asp f 10 34 - Aspartic proteinase X85092 Asp f 11 24 - Peptidyl-prolyl isomerase * Asp f 12 90 IgE and IgG binding Heat shock protein P90 * Alkaline serine Asp f 13 34 - * proteinase

Asp f 15 16 - - AJ002026 Asp f 16 43 - - g3643813 Asp f 17 - - - AJ224865 Vacuolar serine Asp f 18 34 - * proteinase Asp f 22 22 - Enolase AF284645 Cladosporium herbarum Cla h 1 13 IgE binding - * Cla h 2 23 IgE binding - * Cla h 3 53 IgE binding Aldehyde dehydrogenase X-78228 Acid ribosomal protein Cla h 4 11 - X-78223 P2 Cla h 5 22 - YCP4 protein X-78224 Cla h 6 46 - Enolase X-78226 Acid ribosomal protein Cla h 12 11 - X85180 P1

a Adapted from Bush (2001) and Kurup (2002). * http://www. Allergen.org (IUIS Allergen List)

Figure 1.1. Transverse sectional view of fungal hyphae, organelles and associated structures.

Figure 1.2. Life cycle of Anamorphic fungi.

Figure 1.3. Morphological characteristics of asexual Anamorphic conidia and associated reproductive structures

Figure 1.4. Number of fungal species represented in each fungal division.

Figure 1.5. Photomicrographs of fungal cultures in nutrient media and microscopic morphology of the spores. (A-B) A. alternata conidia arranged in chains on hyphae, (C- D) individual A. alternata conidia, (E-G) colony features of A. fumigatus, (H) A. fumigatus conidial head and attached chain of conidia, (I-J) unicellular A. fumigatus conidia, (K-N) colony features of C. herbarum and (O-R) C. herbarum conidia.

Figure 1.6. Hirst-type volumetric spore trap.

Figure 1.7. Anderson sampler

Figure 1.8. IOM personal air sampler

Figure 1.9. The Intra-nasal air sampler. The disassembled components for the nasal sampler – a soft silicon strap spans the septum of the nose and connects the two silicon frames and that house the collection cups.

Figure 1.10. The fully assembled Intra-nasal air sampler worn by a subject.

Figure 1.11. Cross sectional view of a nose with the Intra-nasal air sampler in place.

Figure 1.12. Halogen Immunoassay system for fungi. (a) This assay is a membrane based immunoblotting technique. The particles carrying the allergen are laminated permanently between a protein binding membrane and the adhesive, which they were collected on. This permanent laminate is wetted and the allergens elute from the particles and are bound around the particles by the protein binding membrane. The allergens are then probed with the patient’s sera and stained with a precipitating substrate. (b) The particles, in this case a germinated Epicoccum spore, and their halo of allergen can be visualised through the transparent adhesive tape layer. Each particle the patient has inhaled and their IgE reacts with will appear with a halo of immunostained allergen surrounding it.

Chapter 2∗

2. A new immunodiagnostic technique

More than 80 species of fungi are suspected of inducing IgE mediated hypersensitivity (Horner et al., 1995). The most commonly studied fungal species are A. alternata, A. fumigatus, C. herbarum and E. purpurascens, which are prevalent aeroallergen sources throughout the world and allergy to them is a risk factor for allergic rhinitis (Li and Kendrick, 1995a), asthma (Zureik et al., 2002) and even death (O'Hollaren et al., 1991). Exposure to mould occurs both indoors and outdoors and can vary between individuals, geographical location, time and airflow (Rogers, 2003). The incidence of respiratory allergy to fungi varies geographically, although it is estimated that 20-30% of atopic individuals and up to 6% of the general population are atopic to fungi (Kurup et al., 2002). Fungal allergic disease can be diagnosed by in vivo SPT, in vitro detection of allergen specific serum IgE antibodies (RAST, Pharmacia CAP System™) and in some cases by provocative challenge tests (Licorish et al., 1985). The most frequently used diagnostic test is the SPT, which uses a commercial fungal extract (Malling, 1992; Esch, 2004). It is well recognized that such extracts are heterogeneous in allergen content and vary between manufacturers, batches and strains. This has contributed to the slower rate of allergen standardization, compared to other aeroallergen sources (Malling, 1992; Esch, 2004). Furthermore, the lack of concordance between fungal in vivo and in vitro diagnostic tests has added to the clinical uncertainty about the accuracy and effectiveness of current fungal allergen diagnostic systems (Corsico et al., 1998;

∗ This chapter has been accepted for publication as “A new method (Halogen Immunoassay) for the detection of sensitization to fungal allergens; comparisons with conventional techniques” in Allergology International in 2005.

Mabry et al., 1999; Negrini et al., 2000). Within the community, the median wheal size for fungal allergy lies close to the cut-off value (3mm) conventionally used to discriminate between positive and negative responses (Nordvall et al., 1990). It is likely that the variability of existing extracts, the use of a few selected extracts to represent a broader range of fungi and the uncertainty of an appropriate cut-off for a positive response have all contributed to the under-diagnosis of the prevalence of fungal allergy. Until now, the collection of wild-type fungi from a patient’s environment and directly demonstrating allergic sensitisation to the same fungal particles has been a technically formidable task (Popp et al., 1988). The recently developed HIA enables the simultaneous visualization of individual particles collected by volumetric air sampling, together with their expressed antigens following their immunostaining with human IgE (Tovey et al., 2000). Originally developed as a novel analytical technique for a range of common aeroallergen sources, including house dust mite (Poulos et al., 1999), cat (O’Meara et al., 1998), cockroach (De Lucca et al., 1999b) and pollen (Razmovski et al., 2000), the HIA has recently been successfully adapted to the detection of allergens expressed by germinated fungal conidia (Mitakakis et al., 2001a; Green et al., 2003). These preliminary studies have demonstrated that the HIA provides a mechanism to test for reactivity to the allergens that are expressed by germinating fungi and thus simulates natural exposure to conidia. In this Chapter we compare two groups of sera from subjects classified as being either SPT positive or negative to two common fungi (A. alternata and A. fumigatus). The analysis of allergy to these species, in addition to two more (C. herbarum and E. purpurascens) by the HIA, using fungi derived from culture, were compared to the conventional diagnostic systems of SPT and the Pharmacia UniCap® assay (CAP).

2.1 Materials and Methods

2.1.1 Study population

Atopic serum was sourced from a previous study of adult respiratory diseases in different Australian communities (Duffy et al., 1990). Ages of the subjects ranged from

17 to 50 years, however as samples were de-identified the calculation of a median age was not possible. The subjects were classified as having allergy-associated disease if they previously had a history of wheeze, asthma or hay-fever (Tovey et al., 1998). The Human Ethics Committee of the Northern Rivers Area Health Service approved the study protocol and the subjects gave written informed consent following a full explanation of the study.

2.1.2 Skin testing procedures

SPT was performed with a panel of eleven common standardized aeroallergen and mould extracts using a standardized procedure and lancets from Hollister-Stier Laboratories (Hollister-Stier, Spokane, WA, USA) (Sluyter et al., 1998). The mean of the longest and perpendicular wheal size was recorded after 15 minutes and expressed as mean wheal mm. Sensitisation to mould was defined as having a minimum wheal diameter of 3mm or greater, 15 minutes after the testing procedure to one of either A. alternata or A. fumigatus SPT extracts (Karihaloo et al., 2002).

2.1.3 Measurement of total and specific IgE

Following the SPT, sera was obtained from consenting subjects for in vitro diagnostic testing and stored in aliquots at –70oC until required. Sera from 60 atopic subjects were selected to be tested; 30 were sensitised to at least one of A. alternata or A. fumigatus extracts and the other 30 subjects were SPT negative to these fungi, but sensitised to other non-fungal allergens. Undiluted sera were serologically tested at the Clinical Immunology Laboratory (Central Sydney Laboratory Service, NSW) for specific IgE antibodies to A. alternata (m6), A. fumigatus (m3), C. herbarum (m2) and E. purpurascens (m14) using the CAP assay according to manufacturers instructions (Pharmacia CAP System™, Pharmacia and Upjohn Diagnostics AB, Uppsala, Sweden).

The positive cut-off for the specific IgE assays was 0.35kUA/L and was calibrated against the World Health Organization standard 75/502 for IgE in the range 0.35-

100kUA/L.

2.2.3 Fungal isolates

Fungal isolates of A. alternata (28008), A. fumigatus (28004), C. herbarum (28006) and E. purpurascens (28007) were supplied by the Queensland Department of Primary Industries (Brisbane, Australia). Isolates of each species were subcultured from stock sources and grown for 10 days at 24.9oC on vegetable juice nutrient media until sufficient sporulation occurred.

2.2.4 Cultured spore sampling

Conidia were aerosolised from culture plates using an air jet and then collected by suction onto a polyvinylidene difluoride protein-binding membrane (PBM; 0.45µm pore size) that had been pre-incubated with 30% sucrose and then dried as described previously (Green et al., 2003). After collection, the membrane was kept in a humid box overnight at room temperature to allow the germination of conidia, which has been shown previously to significantly increase the expression of allergen from fungi (Green et al., 2003). Germinated conidia samples were permanently laminated to the membrane by overlaying it with a clear water-based adhesive film (Woolcock Institute of Medical Research, Sydney, Australia).

2.2.6 Halogen immunostaining of cultured spore samples

Briefly in the HIA (Appendix 2.2), previously laminated PBMs with germinated conidia were immersed in 80% methanol until wet, rinsed in deionized water and incubated in borate buffer (pH 8.2) for four hours to enable allergens to elute and bind to the PBM in close proximity to the germinated conidia. Membranes were blocked in 5% skim milk (SM) in phosphate buffered saline (PBS, pH 7.4) for 45 minutes and then incubated overnight at 4oC with individual patient sera (dilution 1:3, vol/vol) in 2% SM-PBS- 0.05% Tween 20. After the primary antibody incubation, the membranes were rinsed three times in PBS-Tween 20 and incubated for 1.5 hours with biotinylated goat antihuman IgE (Kirkegaard & Perry Laboratories, Gaithersburg, Md) diluted 1:500 in 2% SM-PBS-Tween 20. This was followed by an incubation for 1.5 hours with

ExtrAvidin alkaline phosphatase conjugate (Sigma Chemical Co, St Louis, Mo), diluted 1:1000 in 2% SM-PBS-Tween 20. For immunostaining, all samples were incubated with the alkaline phosphatase substrate NBT/BCIP (Pierce Chemical Co, Rockford, Ill) and then staining was monitored for 20 minutes periodically until an optimum purple precipitate was achieved. Positively immunostained germinated conidia have visible immunostaining of the allergen that has been bound to the PBM in close proximity to the conidia or hyphae. Entire PBMs were examined at a magnification of 200x by standard light microscopy. Three replicates were evaluated and the experiment was repeated.

2.2.5 Halogen immunoassay rank score

Two independent observers using a subjective ordinal ranking system quantified the intensity, occurrence and localization of the resultant IgE immunostained haloes around germinated conidia. The intensity of the precipitate staining around hyphae and germinated conidia was estimated as 3+ for strong immunostaining (dark purple), 2+ for moderate immunostaining (medium purple), 1+ for weak immunostaining (light purple) and 0 for no immunostaining reaction. For each species, a series of photomicrographs showing different intensities of immunostaining were used as references for the classification of ordinal rank scores.

2.2.6 Statistical analysis

The statistical agreement between the ordinal rank scores that were assigned by each independent observer was calculated using Cohen’s kappa statistic (Cohen, 1960) using Analyse-It software (Analyse-It for Microsoft Excel, Version 1.68, Analyse-It Software Ltd., Leeds, United Kingdom). In the Kappa test for agreement, the resultant co- efficient (k) gives values between –1 and 1. When there is exact agreement for the two measures of all subjects the kappa statistic equals 1. A value less than 0.6, represents a non-significant agreement between two variables. The kappa statistic was interpreted as

follows: 1.00 – 0.81 = very good agreement; 0.80 – 0.61 = good agreement; 0.60 – 0.41 = moderate agreement; 0.40 – 0.21 = poor agreement; and 0.20 – 0.00 = no agreement. The associations between the HIA, CAP and SPT were analysed for significance using the non-parametric Spearman correlation analysis (Analyse-It for Microsoft Excel, Version 1.68, Analyse-It Software Ltd., Leeds, United Kingdom). The criterion for significance for all analyses was a P value of less than .05. Except otherwise noted, data are expressed as Spearman coefficients (rs) and P values.

2.3 Results

2.3.1 Localization of immunostaining

The fungi showed differing patterns of allergen expression from both the hyphae and the site of conidial germination following HIA analysis, which is consistent with earlier observations (Mitakakis et al., 2001a; Green et al., 2003). Resultant immunostaining for high positive A. alternata sera was heterogeneous and confined to hyphal septal junctions and at the tips of hyphae often in high concentrations (Figure 2.1A). For medium and low positive ordinal rank scores, the intensity of resultant immunostaining qualitatively weakened and antigen expression was restricted to along the entire hyphae and the sites of conidial germination (Figure 2.1B and 2.1C). For germinated conidia belonging to A. fumigatus (Figure 2.2), E. purpurascens (Figure 2.3) and C. herbarum (data not shown) resultant immunostaining was homogeneous. The expression of allergen for each of these respective species was localized around the perimeter of the hyphae, especially at the site of conidial germination. The concentration, intensity and the localization of resultant immunostaining qualitatively decreased with each respective ordinal rank score. Although conidia and hyphae themselves showed some staining within the negative control serum, this was confined to the organelles themselves and there was no immunostaining of the PBM. Such staining is due to the NBT functioning as a vital stain for fungi and is not a consequence of immunostaining IgE.

2.3.2 Inter-observer agreement

The intensity and occurrence of the resultant IgE immunostaining around germinated conidia and hyphae were quantified by two independent observers using a predefined ordinal rank scale. The agreement between the HIA rank scores assigned to the samples by each observer is demonstrated in Table 2.1. Overall, the agreement between the observers for the ranking of A. alternata, C. herbarum and E. purpurascens was very good (k ≥ 0.8). However, the agreement between A. fumigatus rank scores was slightly lower than that found for the other species and indicated good agreement (k = 0.66).

2.3.3 Correlations between immunodiagnostic techniques

The results in Table 2.2 demonstrate the proportion of sera samples that were identified in each of the immunodiagnostic techniques to be either fungal positive or negative. For fungal SPT negative sera, 7% (n=2) of serum samples were identified to be positive to A. alternata in the HIA, whereas 10% (n=3) were identified in CAP. Similarly, 3% (n=1) and 7% (n=2) of the fungal negative sera were identified to be positive to A. fumigatus in the HIA and CAP respectively, whereas only one negative serum sample was established by CAP alone to be positive to E. purpurascens (Table 2.2). For the fungal positive SPT sera samples, the resultant proportion of positive and negative sera within this group was heterogeneous for each of the species (Table 2.2). There were greater numbers of sera identified to be positive in the HIA to A. alternata, E. purpurascens and C. herbarum compared to in CAP and SPT, respectively (Table 2.2). Furthermore, only 30 and 40% of fungal positive sera was established to be positive to A. fumigatus in the HIA and CAP respectively, compared to SPT in which 60% of the sera was positive to A. fumigatus. The correlations between HIA, SPT and CAP are shown in Table 2.3. Significant correlatations were found between HIA and CAP for each of the four fungal species studied, in particular A. alternata (rs = 0.79, P<0.0001), A. fumigatus (rs = 0.67,

P<0.0001) and C. herbarum (rs = 0.66, P<0.0001). However for E. purpurascens, the

association between HIA and CAP was weaker (rs = 0.52, P<0.0035) compared to the other species (Table 2.3). The associations between HIA and SPT for A. alternata and A. fumigatus were less strong and consistent compared to the CAP correlations. For A. alternata, there was a significant but weak correlation between the two techniques (rs = 0.44, P<0.0153), while there was no correlation between HIA and SPT for A. fumigatus (Table 2.3). Similarly, there was a significant but weak correlation between CAP and SPT for A. alternata (rs = 0.43, P<0.0182) and again no correlation for A. fumigatus (Table 2.3).

2.4 Discussion

This study showed that as a diagnostic technique, HIA correlated well with CAP and to a lesser extent with SPT. This finding represents a new approach to the diagnosis of fungal allergic diseases and the technique may have significant implications for our understanding of diagnosing fungal allergy. Sera previously categorized as either SPT negative or positive to at least one of two fungal extracts were found to show only a moderate correlation on retesting by HIA and CAP to those two fungi, while the HIA and CAP showed good overall correlation with each other as in vitro assays using four fungal species. The novelty of the HIA is that it uses freshly germinated spores as the source of fungal allergens. This potentially avoids many of the confounding factors that limit both SPT and the in vitro tests, which depend on retaining the stability of allergens over prolonged periods of storage. Measurement of the subject’s allergic response to an allergen, whether in terms of the quantity of specific IgE or SPT wheal diameter, involves introducing categories into what occurs naturally as a continuous variable. Defining a boundary for a clinically relevant skin test or in vitro response is arbitrary and is only loosely linked to the probability of any clinical outcome. Conventionally with skin tests, a wheal size of

≥3mm is often taken as this cut-off point (Crobach et al., 1998), although rarely ≥2mm or even ≥1mm are used (Green et al., 2002). In a large population study close to 50% of fungal allergic subjects had skin tests close to the 3 mm cut-off point (Maccario et al.,

2003), whereas for mite allergens, larger wheals were proportionately more common. In this study, we introduced three categories of classification for the HIA that were based on an observed intensity of immunostaining. All three categories showed some IgE binding of extracted allergen on the PBM and could be presumed to reflect a corresponding immune recognition had the spores been inhaled. The lower limit of detection in the HIA is also subject to variability, however this is not different than interpreting CAP or SPT scores, where lower levels of response (e.g 2mm SPT) are thought to be artifacts of a non-specific nature or clinically less relevant responses. While more objective measures of IgE binding in the HIA could presumably be made using a measurable label, such as fluorescent anti-IgE, this is technically complex in terms of the required image software and processing. Such improvements may not be required as visual scoring alone may be adequate for most purposes, as it is in the case for quantifying SPT results. It is well recognized that the expression of allergens by a fungal species in culture is highly dependent on the growth media (Gupta et al., 1999; Bisht et al., 2000), fungal strain (Swardnordmo et al., 1984; Steringer et al., 1987; Portnoy et al., 1993b), proteolytic enzyme content (Nordvall et al., 1990; Malling, 1992) and other variables (Esch, 2004). Those strains chosen for preparation of commercial SPT and CAP extracts are usually done so on the basis of high yields under specific culture conditions (Esch, 2004). While the concordance between in vitro and in vivo tests for common allergens such as mites, pollens and animals is usually around 90% (Williams et al., 2001), for fungi the concordance is lower, varying from 39 to 57% (Corsico et al., 1998; Mabry et al., 1999; Negrini et al., 2000). In most studies where such concordance has been compared, the allergens are sourced from different manufacturers, further adding to the variables. As a diagnostic system, skin tests are popular as they are less expensive, more convenient and are generally regarded as being more sensitive. However, they are also influenced by a range of biological and reading variables, which confound the data (Gordon, 1998). These include an individual’s tissue histamine responsiveness, diurnal variation, site along the arm, operator variation, reading error and non-allergic

contaminants in the extracts. At least hypothetically, skin tests provide a more indicative biological response to an allergen. There is limited data comparing SPT to CAP using single purified allergens. These show that IgE binding by an allergen is only weakly associated with its ability to generate skin test wheals (Witteman et al., 1996) and some allergens, for unclear reasons, seem to be biologically more potent than others (Niederberger et al., 2001). It is also recognized that fungal extracts contain a larger proportion of carbohydrate-associated allergens (Swardnordmo et al., 1984) than do other allergen sources. While the importance of carbohydrates as allergens is debated (Hansen et al., 1994), such allergens are less likely to be covalently coupled to the solid- phase used in CAP assays (Kozak and Hoffman, 1984) than to the polyvinylidene difluoride PBM used in the HIA. It has previously been shown that carbohydrate allergens, such as mannan from Candida albicans, will bind to nitrocellulose membranes (Nermes et al., 1994). Currently, to demonstrate allergy to individual fungal bioaerosols within an environment to which a subject is exposed, would require separate collections, culture and preparation of extracts for each species present. The HIA can provide the diagnosis of IgE mediated hypersensitivity to not only wild-type fungal conidia, but also airborne fungal hyphae, fragmented conidia and fungal genera not previously considered as allergen sources (Green et al., 2005). The advantage of the HIA assay is that fungal species from a wide range of sources can be used. While in this study spores from culture were used, the technique lends itself to the use of wild-type fungi that the subject is exposed to. In addition, the conidia in the HIA are maintained for a short period at high humidity and naturally express their allergens, whereas those used for commercial extracts are cultured on artificial media, which could influence the expression of allergens. The significance of this technique derives from its unique ability to provide allergen sources that are actively secreted by the germinating spores and hyphae. This may reflect the allergens to which exposure occurs naturally after conidia are deposited on the mucous membrane. Thus the assay uses the un-degraded allergens that are the natural agent of exposure to fungi and as such, are most likely to be relevant to clinical disease.

Several experimental studies have shown that the germination of fungal conidia is an important process in the release of fungal allergens. The detection of Asp f 1 and Alt a 1, in addition to the immunostaining of IgE of a number of other fungal species significantly increases upon conidial germination in both culture (Mitakakis et al., 2001a; Green et al., 2003) and house dust (Sporik et al., 1993). Germinated conidia consist of growing hyphae, in which the hyphal tips are metabolically active and functionally differentiating regions that have been shown in cross sectional studies to bind considerably greater amounts of Asp f 1 and human IgE compared to conidia (Reijula et al., 1992). Other fungal extract studies have also demonstrated that mycelium extracts bind significantly higher concentrations of IgE compared to spore extracts (Fadel et al., 1992). In the present study, germination increased the detectable thresholds of the assay and confirms earlier studies that ungerminated Aspergillus conidia cannot be accurately enumerated using the HIA. Such unicellular Aspergillus conidia share small dimensions (<3µm) and compared to germinated spores contain only negligible quantities of antigen-bearing macromolecules. The amounts of antigen per conidia (probably in the low pg quantity per conidial) impose a lower limit of detection on the assay making ungerminated Aspergillus conidia difficult to detect. Even though the detectable thresholds of the assay had been improved upon conidial germination, the between-observer agreement for A. fumigatus was still slightly lower compared to the other fungal species. The between-observer variation can be accounted for by the difficulty differentiating the intensity of immunostaining between lower ordinal rank scores, in addition to visualizing germinated and ungerminated A. fumigatus conidia on the PBM. Future studies should explore ways to improve the detection thresholds. This would further clarify wild-type fungal conidia as well as enable the resultant immunostaining to be semi-quantitatively analysed. However to date, this has not been possible using current materials as preliminary tests reveal that a combination of available fluorophores and precipitating substrates were less sensitive and confounded by quenching (unpublished data). These results extend and are consistent with the findings of previous studies that the patterns of IgE responses to fungal allergens are heterogeneous amongst individuals

(Karihaloo et al., 2002). The variable immunostaining patterns observed may be determined by the specificity of the sera for different individual allergens in the extract. This in turn is dependent on the immunodominance to individual fungal allergens and is controlled by either environmental or genetic factors (Karihaloo et al., 2002). Studies demonstrating the genetic control of specific IgE production have shown that the IgE production to some purified allergens was strongly linked to the HLA-DR allele, although immunoblotting studies with Alternaria extracts using allergic monozygotic twins, show only moderate concordance for the different allergens (Karihaloo et al., 2002). The resultant individual immunostaining patterns from germinated conidia may also be a function of whether the antigen is expressed during conidial germination. In a separate study, an Alt a 1 homolog was differentially expressed during hyphal growth (Cramer and Lawrence, 2003), whereas Mitakakis et al. (2001a) demonstrated that a greater proportion of germinated conidia were immunostained with Alt a 1 specific monoclonal antibodies compared to ungerminated conidia. Thus, the visualization of resultant immunostaining on an individual basis using the HIA is dependent on conidial germination and provides novel insight into the localization and variability of human antibody immunostaining of germinated fungal conidia. Future studies can apply this technique to further investigate the influence of genetic factors on IgE responses to fungal allergens.

2.5 Conclusions

The results of this study demonstrate that the use of freshly germinated spores in the HIA correlated well with conventional in vitro techniques and less well with SPT. The results also indicated a good agreement between the independent observer ordinal rank scores for each fungal species, which enabled the semi-quantification of the HIA. These outcomes form a background against which to determine the role of fungal exposure and fungal allergens in asthma, with the eventual aim of reducing its burden through better diagnosis, management and prevention.

Table 2.1: Inter-observer agreement between the HIA ordinal rank scores that were assigned by each independent observer.

HIA ordinal rank score a Fungal species 3+ 2+ 1+ 0 Agreement b

T1 T2 T1 T2 T1 T2 T1 T2

Alternaria alternata 2 (3) 3 (5) 6 (10) 6 (10) 20 (33) 20 (33) 32 (53) 31 (52) 0.81 Aspergillus fumigatus 2 (3) 2 (3) 3 (5) 3 (5) 11 (18) 7 (12) 44 (73) 48 (80) 0.66 Cladosporium herbarum 1 (2) 1 (2) 2 (3) 1 (2) 19 (32) 15 (25) 38 (63) 43 (72) 0.79 Epicoccum purpurascens 2 (3) 2 (3) 4 (7) 3 (5) 19 (32) 21 (35) 35 (58) 34 (57) 0.82

a Values presented represent the total number of serum samples assigned to each HIA ordinal rank score by the observer (T1 or T2), with the percentage frequency given in parentheses. b Agreement identifies the inter-observer agreement between HIA ordinal rank scores as indicated by the kappa statistic (k) for each fungal species.

Table 2.2: The number of serum samples identified by each immunodiagnostic technique to be either sensitive to fungi (+ve) or not sensitive to fungi (-ve).

Immunodiagnostic techniquea Fungal species HIA CAP SPT

+ ve - ve + ve - ve + ve - ve Fungal –ve sera (n=30) Alternaria alternata 2 (7) 28 (93) 3 (10) 27 (90) 0 (0) 30 (100) Aspergillus fumigatus 1 (3) 29 (97) 2 (7) 28 (93) 0 (0) 30 (100) Epicoccum purpurascens 0 (0) 30 (100) 1 (3) 29 (97) NDb ND Cladosporium herbarum 0 (0) 30 (100) 0 (0) 30 (100) ND ND

Fungal +ve sera (n=30) Alternaria alternata 24 (80) 6 (20) 20 (66) 10 (33) 19 (63) 11 (37) Aspergillus fumigatus 9 (30) 21 (70) 12 (40) 18 (60) 18 (60) 12 (40) Epicoccum purpurascens 23 (77) 7 (23) 15 (50) 15 (50) ND ND Cladosporium herbarum 17 (57) 13 (43) 14 (47) 16 (53) ND ND

a Values presented represent the total number of serum samples identified by each immunodiagnostic technique to be either fungal positive or negative with the percentage frequency given in parentheses. b ND = not detected

Table 2.3: Spearman correlation results for the SPT fungal positive sera between the three-immunodiagnostic techniques.

Immunodiagnostic techniquea Fungal species HIA Rank Score SPT CAP Alternaria alternata HIA Rank Score 1 0.44 (0.0153) b 0.79 (0.0001) SPT (mm) 1 0.43 (0.0182) CAP (kUA/L) 1 Aspergillus fumigatus HIA Rank Score 1 - 0.04 (0.8095) 0.67 (0.0001) SPT (mm) 1 -0.02 (0.9249) CAP (kUA/L) 1 Cladosporium herbarum HIA Rank Score 1 - 0.66 (0.0001) SPT (mm) - - CAP (kUA/L) 1 Epicoccum purpurascens HIA Rank Score 1 - 0.52 (0.0035) SPT (mm) - - CAP (kUA/L) 1

a Data show the non-parametric Spearman rs co-efficient and resultant P value. b Bold type identifies the Spearman correlation coefficients, which were significant (P<0.05).

Figure 2.1. Germinated Alternaria alternata showing different intensities of immunostaining of conidia (arrow a) and hyphae (arrow b) using the Halogen immunoassay to detect the binding of human IgE (arrow c) to expressed allergens. Rank scoring is as follows; A. high positive (3+). B. medium positive (2+). C. low positive (1+) and D. negative (0). Scale Bar – 50 µm.

Figure 2.2. Germinated Aspergillus fumigatus showing different intensities of immunostaining of conidia (arrow a) and hyphae (arrow b) using the Halogen immunoassay to detect the binding of human IgE (arrow c) to expressed allergens. Rank scoring is as follows; A. high positive (3+). B. medium positive (2+). C. low positive (1+) and D. negative (0). Scale Bar – 20 µm.

Figure 2.3. Germinated Epicoccum purpurascens showing different intensities of immunostaining of conidia (arrow a) and hyphae (arrow b) using the Halogen immunoassay to detect the binding of human IgE (arrow c) to expressed allergens. Rank scoring is as follows; A. high positive (3+). B. medium positive (2+). C. low positive (1+) and D. negative (0). Scale Bar – 50 µm.

Chapter 3∗

3. Fungal fragments as new aeroallergen sources

Fungi are ubiquitous throughout the environment and commonly grow as saprophytes on non-living organic material or as invasive pathogens in living tissue. They are principally dispersed as sexual spores or asexual conidia, which are common components of the atmospheric aerospora. These agents each have distinctive morphological features that facilitate the recognition of the genera or species. Fungal hyphae are also aerosolised in large numbers but lack sufficient morphological characteristics to be taxonomically identified. Counts of the airborne conidia fluctuate widely in indoor and outdoor environments, with time and between different geographical regions and climatic conditions. The concentrations can range from 0 to more than 100,000 CFUs per cubic meter of air. The most frequent taxa are Cladosporium, Penicillium, Aspergillus, Alternaria and Aureobasidium species (Burge et al., 2000; Gots et al., 2003). Horner et al. (1995) estimated that of the 69 000 fungal species described to date (Levetin, 1995), more than 80 species have been identified as sources of allergens associated with allergic respiratory diseases mediated by IgE hypersensitivity. Studies of the aerobiology of allergenic fungi usually enumerate 10 to 20 of the more common genera, whereas the diagnosis of fungal allergy is usually made on the basis of responses to 3 or 4 species. This pragmatic approach reflects both the enormous diversity of fungi and the confounders of the diagnostic processes resulting from the lack of standardization, low stability of extracts and the variability of source materials.

∗ This Chapter was published as “Fungal fragments and undocumented conidia function as new aeroallergen sources” in The Journal of Allergy and Clinical Immunology in 2005 and was selected as one of the Editors Choice articles for the issue.

The association between personal exposure to airborne fungi and the manifestation of respiratory disease is complex. In epidemiologic studies, exposure to airborne fungal conidia has been linked to the symptoms of seasonal rhinitis (Li and Kendrick, 1995a; Andersson et al., 2003), asthma (Downs et al., 2001; Zureik et al., 2002) and even death (O'Hollaren et al., 1991) in subjects with fungal allergy. This paradigm has been supported by bronchial provocation (Licorish et al., 1985) and longitudinal community (Downs et al., 2001) studies. However, these investigations have seldom included the measurement of other fungal propagules, including airborne hyphae that might additionally function as aeroallergen sources. Fungal fragments, including airborne hyphae, have been shown to become airborne at significantly higher concentrations than conidia in simulated aerosolization experiments (Gorny et al., 1999; Gorny et al., 2002; Gorny, 2004) and the incorporation of hyphal counts with those of conidia in epidemiologic investigations improved the association with asthma severity (Delfino et al., 1997). To determine the extent to which airborne fungal hyphal fragments and other fungal species function as aeroallergen sources, we collected air samples from a well-ventilated indoor environment and detected the different sources of environmental airborne fungal antigens by using the recently described HIA (Mitakakis et al., 2001a; Green et al., 2003).

3.1 Materials and Methods

3.1.1 Personal air sampling

Personal volumetric air samplers (PASs), which are extensively used in occupational health settings, were used for the current study. The PASs consisted of an Institute of Occupational Medicine (IOM) sampling head (SKC Ltd, Dorset, United Kingdom) (Mark and Vincent, 1986) connected to a diaphragm pump providing a constant 2.0 L min -1 air flow through a mixed cellulose ester protein binding membrane (MPBM). The IOM sampling head was sterilised and fitted with a 0.8 µm pore size MPBM (Millipore Corp., Bedford, Mass) for use in the HIA (Woolcock Institute of Medical Research,

Australia). An indoor residential environment located in Sydney, Australia (34o0′S

151o0’E) was sampled daily over a 21-day period during spring (August-September) for a period of 150 minutes on each day. The IOM sampling heads were placed with the MPBM face on a vertical plane in a location with ample natural ventilation. The collection of air samples coincided with a daily mean temperature of 18.5oC and a mean relative humidity of 46% (Bureau of Meteorology, NSW, Australia). Sampling was not conducted if there had been rainfall in the previous 24 hours. Before and following collection, the flow rate of the PAS was remeasured to ensure that a constant 2.0 L min - 1 had been maintained.

3.1.2 Human serum samples

Human sera from 30 subjects highly allergic to Alternaria species and other fungi were collected and pooled. The diagnosis was based on a documented positive clinical history of asthma or allergy specifically caused by mould, which was determined on the basis of a positive epicutaneous SPT with a wheal diameter of 3mm or greater. In addition, specific IgE was detected with Pharmacia UniCAP (Pharmacia, Uppsala, Sweden) to a panel of fungal allergens. After collection, all samples were stored in aliquots for future use at –70oC. Pooled serum IgE from 10 subjects with negative SPT responses to fungi but sensitised to other non-fungal allergens was included in the study and used as a negative control.

3.1.3 Immunostaining of environmental samples

The MPBM was removed from the IOM sampling head, placed in a humid chamber overnight to enable conidia germination and immunostained using the HIA as described previously (Mitakakis et al., 2001a; Green et al., 2003). Briefly, in the HIA MPBMs were laminated with an adhesive coverslip and immersed in borate buffer (pH 8.2) for 4 hours to enable allergens and other macromolecules to elute and bind to the MPBM in close proximity to the conidia and hyphae. Membranes were blocked in 1% bovine serum albumin (BSA) in PBS and 0.05% Tween 20 (BSA-PBS-Tween 20) for 45 minutes and then incubated overnight at 4oC with pooled human Alternaria species-

positive sera diluted 1:3 in BSA-PBS-Tween 20. After the primary antibody incubation, the membranes were washed and incubated for 1.5 hours with biotinylated goat antihuman IgE (Kirkegaard & Perry Laboratories, Gaithersburg, Md) diluted 1:500 in BSA-PBS-Tween 20; this was followed by incubation for 1.5 hours with ExtrAvidin alkaline phosphatase conjugate (Sigma Chemical Co, St Louis, Mo) diluted 1:1000 in BSA-PBS-Tween 20 and developed with NBT/BCIP substrate (Pierce Chemical Co, Rockford, Ill), as described previously (Mitakakis et al., 2001a; Green et al., 2003). Samples were examined at a magnification of ×200 by using standard light microscopy. Positively immunostained particles displayed visible purple immunostaining only if the sera used contained IgE antibodies specific to the proteins associated with the particles. The number of non-immunostained and immunostained hyphae, conidia and fragmented conidia was counted and taxonomically identified to genus level. Negative controls consisted of similar environmental samples collected on MPBMs and were probed with either: (1) non-atopic human fetal chord sera or (2) pooled adult human sera from 10 subjects with negative SPT responses to fungi but sensitised to other non-fungal allergens in place of the pooled human Alternaria species-positive sera.

3.1.4 Statistical analysis

Differences between the proportion of immunostained and non-immunostained hyphae, in addition to the total fungal conidia and fungal hyphal numbers, were analysed for significance using the nonparametric Mann-Whitney U Test (Analyse-It for Microsoft Excel, Version 1.68, Analyse-It Software Ltd, Leeds, United Kingdom). The criterion for significance for all analyses was a P value of less than .05. Except otherwise noted, all data are expressed as medians and 25th and 75th percentiles.

3.2 Results

Airborne fungal spores, conidia and hyphae expressed detectable levels of antigen in all personal air samples. Collected fungal hyphae varied markedly in size (5-100µm), shape, colour, and hyphal septation (Figure 3.1). Resultant immunostaining was

heterogeneous and localized primarily to the outer margins of hyphal tips (Figure 3.1A, B, D and F), the septal junctions (Figure 3.1C), and around the entire fragment (Figure 3.1E), and restricted to the site of conidial fragmentation (Figure 3.1G-H). The proportion of fungal hyphae demonstrating immunostaining is presented in Figure 3.2. Approximately 25% of all hyphae collected on the MPBM demonstrated resultant immunostaining, which was significantly lower (P<0.05) than the proportion of non- stained hyphae (Figure 3.2). Similarly, the total number of conidia and hyphae collected in all personal air samples (Figure 3.3) showed that fungal hyphae were significantly higher in airborne concentration than the conidia counts belonging to Alternaria species (P<0.05), Aspergillus-Penicillium species (P<0.05) and Cladosporium species (P<0.05). The expression of allergen and subsequent immunostaining of conidia from well-documented allergenic genera, including Alternaria, Aspergillus-Penicillium, Cladosporium, Exserohilum, Curvularia and Pithomyces (Figure 3.4), was similar to the patterns of IgE staining described in previous culturally derived studies (Green et al., 2003). Resultant immunostaining of wild-type conidia was primarily restricted to the germinated hyphal tips, septal junctions, basal regions and the outer periphery of the conidia of Alternaria (Figure 3.4A), Exserohilum (Figure 3.4E), Curvularia (Figure 3.4D) and Pithomyces (Figure 3.4F) species, whereas for unicellular conidia belonging to Aspergillus-Penicillium (Figure 3.4B) and Cladosporium (Figure 3.4C) species, immunostaining was localized around the entire conidia. In addition, the conidia of fungal genera, previously uncharacterised as allergen sources, were demonstrated for the first time to function as sources of aeroallergen (Figure 3.5). The localization of IgE binding to expressed antigen was concentrated around the entire spore for Amphisphaeria species (Figure 3.5A), Myxomycetes (Figure 3.5E), Spegazzinia species (Figure 3. 5G), and Ascomycete cleistothecium of the Erysiphales (Figure 3.5I); basal regions of the conidia for Arthrinium species (Figure 3.5B), Leptosphaerulina species (Figure 3.5D) and Sporidesmium species (Figure 3.5H); and around the germinated hyphal tips of Leptosphaeria species (Figure 3.5C), Pleospora species (Figure 3.5F) and germinated Ascospores belonging to the Xylariaceae (Figure 3.5J). The total airborne

concentrations for each of these previously undocumented allergenic genera fluctuated widely between air samples and accounted for approximately 8% of the total conidia collected (data not shown).

3.3 Discussion

This study conclusively demonstrates that airborne fungal hyphae commonly function as sources of aeroallergen because positively immunostained hyphae were frequently observed on all indoor air samples. The resultant staining was heterogeneous and primarily localized around the entire length of the hyphae, outer margins of the tips and septal junctions. It is suspected that allergen expression in the vicinity of these sites is attributable to the processes of separation and shear by environmental factors in addition to the cell walls of the hyphae being significantly thinner in regions of septation and cross walls. Several in situ factors, including the age of the mycelium and nutrient limitation, have previously been shown to fragment hyphae by initiating hyphal vacuolation, which weakens the tensile strength and rigidity of the hyphae (Paul et al., 1994; Li et al., 2002). Fragmentation, however, was not only restricted to hyphae, and several examples of fragmented conidia were observed. All fragmented conidia were restricted to the genera Curvularia and Exserohilum and shared similar morphological characteristics including conidial septation. The manifestation of allergen was confined to the area surrounding the site of conidial fragmentation, often in higher concentrations compared with that seen in intact conidia belonging to the same genera. It is unknown what environmental processes are involved in causing fragmentation, however it can be expected that these fragmented conidia that would function as allergen sources were non-viable. Our findings demonstrate that the proportion of non-stained hyphae was approximately 4 times greater than positively immunostained hyphae. The interpretation of this variation is relatively unclear, although the amount of allergen released from a hyphal fragment might be a function of the critical fragment size, which is the minimum size to which a fungal fragment remains viable. Cenocytic or non-septate hyphae have

been shown to have the largest critical fragment size, whereas dematiaceous septate hyphal varieties, including Alternaria and Penicillium species, have significantly smaller critical fragment sizes (Marfenina et al., 1994). This suggests that smaller-sized dematiaceous fragments may be more capable of releasing greater quantities of allergen than cenocytic varieties. The extent to which viability influences the release of allergen from fragments of hyphae has not been studied in detail. Environmental variables, including the ambient temperature, humidity, solar radiation and duration that a particle remains airborne might account for this variation in immunostaining by either denaturing or solubilizing antigens, however this remains anecdotal and requires further investigation. The proportion of hyphae demonstrating immunostaining would also be a function of the specificity of the sera used. In this case a pool of serum from subjects selected for Alternaria species skin test sensitivity was used and individual serum or different serum pools could be expected to have a different spectrum of detection. Collected airborne hyphae could be morphologically characterized by a number of discernable features, including hyphal septation and the presence of melanin, however further identification on the basis of taxonomic criteria involves uncertainty. The concentration of total airborne hyphae was significantly higher than the conidia of a number of common and well-characterized individual genera, including Alternaria, Aspergillus, Penicillium and Cladosporium. Aerosolization studies using culturally derived fungal sources also showed fungal fragments outnumbering conidia (Gorny et al., 2002; Gorny, 2004). Particles smaller than 2.5µm collected on the MPBM were unable to be accurately identified by means of standard light microscopy and it is possible that smaller hyphal and conidial fragments containing allergens were undetected. Personal exposure to airborne fungi in indoor and outdoor environments is heterogeneous and has been shown to differ between individuals in experimental studies (Mitakakis et al., 2000). The aerosolization of fungal hyphae and conidia is dependent on a number of location-specific atmospheric parameters, including wind velocity, wind direction, and substrate disturbance (Gorny et al., 2002; Gorny, 2004). The release of fungal hyphae is additionally dependant on the morphological characteristics of the

species and of the mycelium, as shown in the case of Aspergillus versicolor and Penicillium melinii (Gorny et al., 2002). It could be anticipated that personal exposure to a range of fungal conidia and hyphae would fluctuate independently, as well as widely, between seasons and study locations. In addition to well-recognized allergen sources, such as Alternaria, Aspergillus- Penicillium, Cladosporium, Exserohilum, Curvularia and Pithomyces species, a number of other fungal genera (approximately 8% of the total conidia collected) were demonstrated as sources of allergen, as determined by IgE binding to expressed antigen in the HIA. These included conidia and spores belonging to Ascomycetes, Deuteromycetes and Myxomycetes, all of which had characteristic localization of allergen concentrated around the basal regions of the spores, conidia, cleistothecia and growing tips of the germinating hyphae. Our findings suggest a much more diverse range of fungi function as sources of allergen. This provides a new paradigm of natural exposure, in which a substantial proportion of the airborne fungal biomass rather than a limited group of genera contributes to the aeroallergen load. Although these experiments were performed with a pooled serum and thus might over-represent the diversity for an individual, the same experiments could be repeated with individual serum providing a view of individual responses to the environmental fungal load. It is plausible that individuals will recognize allergens from the common species as frequently as they did the less common species because all genera showed a similar strength of immunostaining.

3.4 Conclusions

The interpretation of personal exposure and sensitization to fungal allergens has, to date, remained restricted to the inhalation of fungal conidia belonging to a select number of species that have been established in bioaerosol studies to be abundant in most geographical locations. Our results independently demonstrate, using a novel immunodiagnostic technique, that airborne fungal hyphae and fragmented conidia, as

well as fungal genera not previously considered as allergen sources, express detectable levels of allergen and contribute to personal exposure to fungal allergens.

Figure 3.1. Resultant immunostaining of airborne hyphal fragments confined to the hyphal tips (A-B, D, F; arrow a), regions of septation or cross wall linkages (C; arrow b) and around the entire fragment (E; arrow c). Fragmented conidia (G and H) expressed antigen localized at the site of fragmentation (arrow d). Scale Bar, 10 µm (E and F) and 20 µm (A-D, G and H).

Figure 3.2. The proportion of airborne hyphal fragments not expressing detectable levels of fungal allergen was significantly higher than positively immunostained fragments (P<0.05). Results are presented as medians and 25th and 75th percentiles (n=21). Significant differences between the numbers of positively and negatively immunostained airborne hyphal fragments were indicated as follows * P<0.05.

Figure 3.3. Total fungal counts demonstrated that airborne hyphal fragments were significantly higher in concentration compared to other fungal genera. Results are presented as medians and 25th and 75th percentiles (n=21). Significant differences between fungal fragment counts and other genera were indicated as follows * P<0.05.

Figure 3.4. Deuteromycete conidia (arrow a) and germinated conidia (arrow b) represented by Alternaria (A), Aspergillus-Penicillium (B), Cladosporium (C), Curvularia (D), Exserohilum (E) and Pithomyces species (F) demonstrated IgE binding to expressed antigens. Scale Bar, 10 µm (B) and 20 µm (A and C- F).

Figure 3.5. Undocumented fungal genera demonstrated IgE binding to expressed antigens, including Amphisphaeria species (A), Arthrinium species (B), Leptosphaeria species (C), Leptosphaerulina species (D), Myxomycete spores (E), Pleospora species (F), Spegazzinia species (G), Sporidesmium species (H), Ascomycete cleistothecium of the Erysiphales (I), and germinated ascospores belonging to the Xylariaceae (J). Scale Bar, 10 µm (A and B), 20 µm (C-H and J) and 50 µm (I).

Chapter 4

4. Individual exposure to airborne fungi

Fungi are a diverse lineage of spore-bearing microorganisms that are ubiquitous bioaerosols in indoor and outdoor environments. Currently, 69 000 species have been identified out of 1.5 million estimated to exist (Levetin, 1995). To date, more than 80 fungal species have been identified to induce IgE mediated hypersensitivity (Horner et al., 1995; Esch, 2004). The airborne spores, hyphae and fragments of these fungi are the principal dispersal agents, many of which are small enough to be inhaled into the lower airways (Mitakakis et al., 2000; Gorny et al., 2002; Green et al., 2005). Personal exposure to these fungal propagules, in particular Alternaria spores, are widely recognized to be risk factors for allergic rhinitis (Li and Kendrick, 1995a; Andersson et al., 2003), asthma (Downs et al., 2001; Zureik et al., 2002) and even respiratory arrest (O'Hollaren et al., 1991; Targonski et al., 1995). However, the numbers of ambient spores that are required to elicit an adverse health effect is contentious and varies on an individual basis (Licorish et al., 1985). Industrial hygiene guidelines developed by the United States Occupational Safety and Health Administration have set a value of 1000 CFUs per m-3 or greater as unacceptable levels of fungal contamination (OSHA, 1992). Nonetheless, the enumeration of airborne fungi is highly dependent on the methods of sampling and analysis applied and such counts are difficult to standardise and interpret. Additional variations arise from temporal, spatial and geographic variables (Flannigan, 1997; Rogers, 2003). The benchmark for outdoor aerobiological sampling is the Hirst (Burkard) volumetric spore trap (Hirst, 1952). This is usually used while located in a fixed position, such as on the roof of a building. The trap operates by drawing in ambient air through an orifice at a constant rate (10 l/min) and deposits the airborne particles onto an adhesive tape, which is fixed to a rotating drum. The particles are then resolved by

light microscopy and subjectively identified. The counts are used to estimate the level of exposure to airborne fungal spores in the region surrounding the spore trap. Similar devices, which correlate with Burkard counts that are based on similar air sampling principles have been developed and utilized in a number of outdoor exposure investigations; these include the Rotorod (Aylor, 1993; Frenz, 1999) and liquid impinger (Barnes et al., 2000) air samplers. More recently, the development of personal air samplers has enabled the measurement of personal exposure to fungal aerosols in occupational environments. Such personal samplers consist of a small filter membrane held in a sampling head and connected to a portable air pump. The filter head is generally worn on the lapel of an individual, so that it samples from the ‘breathing zone’. An example is the IOM personal aerosol sampler (SKC Inc, USA). The pump draws in the surrounding air at 2 l/min and has been shown in field trials to represent the inhalable fraction (Mark and Vincent, 1986). Alternate personal air sampling devices have also been developed and evaluated against benchmark aerobiological air sampling techniques (Aizenberg et al., 2000; Adhikari et al., 2003; Adhikari et al., 2004). However, variations between collection efficiencies of these mechanical samplers and differences in sampling strategies make it difficult to interpret personal exposure to airborne fungi as well as compare between studies. To date, there is no validated and accurate ‘gold standard’ of personal exposure, although measurements made with IOM samplers are often seen as the closest approximation to this. Intra-nasal air samplers (INASs) are worn in the nose and use impaction to collect inhaled particles (Graham et al., 2000). The INASs have high collection efficiency for larger particles (>5µm in diameter), low air flow resistance and do not clog (Graham et al., 2000). The INASs have been used to accurately measure individual personal exposure to a range of aeroallergen sources, including house dust mite (Poulos et al., 1999), cat (De Lucca et al., 2000), cockroach (De Lucca et al., 1999b), latex (Poulos et al., 2002), rat (Renstrom et al., 2002) pollen and larger mould spores (Mitakakis et al., 2000). These samplers provide novel insight into the quantity and distribution of inhaled particles as well as the variation in exposure between individuals. The aim of this Chapter was to simultaneously measure exposure to fungal spores in an

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outdoor community setting with a Burkard spore trap, IOM personal aerosol samplers and INASs to qualitatively compare between the three different sampling methods. Variations between individual exposure patterns in the same location and at the same time were additionally investigated.

4.1 Materials and Methods

4.1.1 Study location

The study was carried out in a public park in the town of Casino (Figure 4.1) in northern

New South Wales, Australia (28o52′S 153o03’E). The climate of Casino is classified as subtropical with an average yearly mean temperature of 19ºC and an average yearly rainfall of 110cm (Bureau of Meteorology, NSW, Australia). The major vegetation formation of the trapping site consisted of several acres of open Eucalyptus woodland with an understorey of grass and herbaceous species, including Paspalum notatum, Cynodon dactylon and Ambrosia artemisiifolia (Figure 4.1). Surrounding creeks and rivers are vegetated by riparian communities and residential and industrial areas have numerous representatives of exotic taxa (Bass et al., 2000).

4.1.2 Subjects

The samples in this Chapter were collected as part of a study where the INASs were being assessed as a personal nasal filter and information pertaining to the recruitment, age and clinical symptomology of the subjects has been published elsewhere (O'Meara et al., 2005). The Human Ethics Committee of the Northern Rivers Area Health Service gave approval for the study and written informed consent was obtained from all subjects.

4.1.3 Study design

The study occurred on two consecutive Sundays (7th and 14th April, 2002) during autumn and subjects were asked to attend on both sampling days (SaD). On each SaD, subjects assembled at the study site at approximately 10am and were randomly

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allocated into groups of 8-10. Random allocation to each group was based on arrival time at the study site. Each group was then randomly allocated to receive active or placebo INASs after baseline measurements for a concurrent study (O'Meara et al., 2005). The subjects then placed the INAS into their nostrils. Subjects were asked to breathe through the nose for the two-hour duration of the study, while remaining in their groups and engaging in only mild activity (sitting, walking, eating) in a central location in the park (Figure 4.1). On each SaD the meteorological parameters characterizing the study site consisted of warm and dry conditions with no wind, however precipitation was recorded throughout the week prior to SaD 2.

4.1.4 Fungal exposure measurements

Ambient fungal spore levels were measured by three varying methods of analysis. A Burkard 7-day volumetric spore trap (Burkard Manufacturing Co., Rickmansworth, United Kingdom), located 3.5m above the ground on the roof of a sporting pavilion, operated continuously at the study site from the 14th of March to the 25th of May. The sampler was located less than 100m away from where people were performing the measurements of personal exposure. The trap was calibrated to sample air at 10 L/min and the atmospheric particulate matter was deposited onto Melanex tapes coated with a thin film of Vaseline/paraffin/toluene mixture. The 7-day tapes were prepared as twenty-four hour sections and were then mounted onto microscope slides and stained with Carberla’s solution (Appendix 4.2) to enumerate pollen and fungal spores (Schumacher et al., 1988). Data from the portion of tape corresponding to 10am to 12 noon was used. The fungal conidia and hyphae were counted using light microscopy at a magnification of 200X and expressed as the number of spores per 2 hours. On each SaD, 4 and 6 subjects (day 1 and 2, respectively) concurrently wore IOM personal samplers (SKC Inc, USA). These were run at 2 L/min and IOM sampling heads (Institute of Occupational Medicine, Edinburgh, UK) contained a Millipore BVXA (Millipore, Bedford, MA) membrane. The filters were removed from the heads and the collected fungal propagules were resolved using light microscopy as described above. The total fungal count was expressed as the number of spores per 2 hours.

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Inhaled fungal exposure was also measured for each SaD using the INAS. For counting, the adhesive core of the active filter was removed and Carberla’s stain was placed directly onto the adhesive core and examined as described above (Appendix 4.1). The fungal conidia and hyphae were counted using light microscopy at a magnification of 200X and the left and right INAS adhesive cores were added and the total fungal counts were expressed as the number of spores per 2 hours.

4.1.5 Intra-nasal air samplers

The INASs, which were also evaluated as prophylactic filters in a concurrent study, are shown in Figure 4.2. The outer surface is made of a soft medical grade silicone and the inner polypropylene core was coated with Vaseline (Lever Rexona, North Rocks, Australia). The airflow through the device is non-linear and inhaled particles tend to remain in a linear trajectory and impact onto the adhesive surface. To accommodate different sized noses, two sizes of the INAS were used, with the appropriate size being allocated on the basis of external nasal appearance. INASs were kept in a sealed container until the time of their issue to study participants. Capture efficiency of the INASs was established in an airflow rig. Rye grass (Lolium perenne), Ragweed (Ambrosia artemisiifolia), Bermuda (Cynodon dactylon) and Bahia (Paspalum notatum) grass pollen (Greer Laboratories Inc., Lenoir, NC) were aerosolised using a medical powder blower (Professional Medical Products, Greenwood, SC). Aerosol laden air was drawn in a steady flow from a plenum through the nasal filter and a downstream Millipore RAWG filter at flow rates spanning the normal human inspiratory flow range (4.6, 10.3, 21.7 and 32.5 L/min). Pollen grains were counted using an Olympus BX60 fluorescent microscope and capture efficiency was calculated as the number of particles collected by the nasal filter divided by the sum of particles collected by the nasal filter and the downstream membrane filter. Airflow resistance of the INASs was 4.1 cm H20/L/sec and was measured as previously described (Graham et al., 2000).

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4.1.6 Statistical analysis

All data are reported as total fungal counts per two hour period and the results are expressed as medians and 25th and 75th percentiles, which were analysed using Graphpad prism software (Prism 4, Graphpad, San Diego, CA). Since values were non- normally distributed the values for total fungal counts were log10 transformed for all individual genera counts and all absolute counts were log10 transformed plus 0.5, to account for the high number of zero values. Overall differences between the total counts of individual fungal genera for each SaD and the total left and right nostril fungal counts were examined by one-way analysis of variance (ANOVA). The association between the INAS and IOM was calculated using the Pearson correlation co-efficient. Statistical significance was defined for all tests as P < 0.05.

4.2 Results

The median and 25th and 75th percentile values of airborne fungi per two hours measured by each of the three sampling methodologies are presented in Table 4.1. The static Burkard spore trap measurements consisted of only one observation and there were a total of 25 genera collected on both SaD 1 and SaD 2 (Figure 4.3). The most frequent fungi collected on SaD 1 included Cladosporium, Aspergillus-Penicillium, Leptosphaeria, and Xylariaceae species with smaller concentrations of Coprinus, Curvularia and hyphal fragments encountered. IOM and INAS devices showed a similar, although smaller spectrum of airborne fungal genera compared to the Burkard spore trap (Figure 4.3). The total spore numbers per two hours for the Burkard spore trap were higher than IOM and INAS for several smaller-spored species, including Aspergillus-Penicillium, Cladosporium Sporomiella, Coprinus and Leptosphaerulina. However, a number of other species were less frequently collected on the Burkard compared to IOM and INAS, for example, Alternaria, Curvularia, Exserohilum, Spegazzinia, Arthrinium, Xylariaceae, in addition to hyphal fragments (Table 4.1). Most of these are larger-spored species.

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The spectrum of fungal genera and the total counts collected by each method of analysis on SaD 2, qualitatively differed compared to SaD 1. The Burkard spore trap collected similar quantities of Cladosporium, Curvularia and Xylariaceae ascospores on both days, however greater total numbers of Ascobolus, Coprinus, Leptosphaerulina, Pithomyces and Sporomiella species were collected on SaD 2. The number of genera collected on the IOMs was less on SaD 2, while the numbers of genera collected by INAS remained unchanged (Figure 4.3). As in SaD 1, greater airborne counts of fungal spores >10µm in size were collected by both IOMs and INASs compared to Burkard measurements. The most frequent taxa were represented by Cladosporium, Curvularia, Bipolaris, Epicoccum, Spegazzinia and Xylariaceae species, in addition to hyphal fragments. Other genera collected by IOM and INAS and not by the Burkard spore trap included Basidiomycete, Fusariella, Fusarium and Tetraploa species (Table 4.1). For both SaDs, the mean proportion of unknown mould spores accounted for 6.5% of the total count for the Burkard spore trap, 0.4% for IOM and 2.5% for INAS. The total number of fungi collected by each sampling method varied between each SaD (Table 4.1) with INAS spore counts significantly higher on SaD 2 compared to SaD 1 (P<0.05). On an individual genera basis, the overall variations between INAS genera counts for each SaD were heterogeneous (Table 4.1). Spore counts measured by INASs were significantly higher on SaD 1 (P<0.05) for Tetraploa, Pithomyces, Arthrinium, Stemphylium, Xylariaceae, Torula, Beltrania, Cladosporium, Leptosphaeria and Delitschia species and significantly higher on SaD 2 for Alternaria, Bipolaris, Fusariella, Epicoccum, Spondylocladiella and Fusarium species (P<0.05). No significant differences were found between spore counts for SaDs 1 and 2 for species belonging to Curvularia, Exserohilum, Acrodictys, Isthmospora, Smut, Rust, Basidiomycete, Ulocladium and Leptosphaerulina, in addition to hyphal fragments (Table 4.1). The total number of spores belonging to the five most frequent taxa that were inhaled by each subject on each SaD is presented in Figure 4.4. These results showed that inhaled exposure in most people varied within a 2-fold range with 10-fold outliers. Photomicrographs of the most common taxa collected by INASs are also presented in

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Figure 4.5. Furthermore, the comparison between the numbers of spores belonging to the nine most frequently recorded genera that were captured on left and right nostril INAS collection cups is shown in Figure 4.6. No significant difference between left and right nostrils was observed for any of the fungal genera (P>0.05). Pearson correlation co-efficients between fungal genera counts for IOM and INAS samplers are presented in Figure 4.7. The number of inhaled airborne spores and hyphae collected by INAS was highly associated with those counts collected on IOM air samples (r=0.74, P<0.0001).

4.3 Discussion

This is the first study to quantify both the numbers and genera of fungi that are actually inhaled during normal outdoor activities. Furthermore, this study provides novel insight into the variations between traditional measurement techniques and the number and types of fungi inhaled between subjects in the same location. Overall we showed that there was a wide (~10x) fold range in the total number of particles, although the overall differences within the group were not large with only a 2-fold difference between the 25th and 75th percentile for total counts. The variability between subjects probably reflects combined differences in the distribution of fungi within the local microenvironments, plus variations in activity, affecting the disturbance of spores by individuals as well as respiratory patterns. Previous studies investigating the inhalation of Alternaria and Cladosporium spores alone using INASs, have shown that variations in spore levels within families of the same household were largely associated with the type and intensity of personal activity (Mitakakis et al., 2000). Similarly, the dispersal of fungal spores from various flooring materials has been shown to be greatest following disturbances either by walking or vacuuming (Buttner and Stetzenbach, 1993). Personal activity in this study was restricted to mild daily events including sitting on the ground and walking within a 100x100 m area, however it has not been explored what differences on exposure such events might have.

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The requirements for fungal growth and reproduction include the breakdown of living or decaying plant material, which restrict many genera to the uppermost soil horizon, the organic layer. Microscopic examination of soil has shown that fungi are present both as spores and hyphae in the O, A, B and C soil horizons with the highest concentrations restricted to highly organic layers (Vardavakis, 1990). The fungal genera most commonly isolated from the soil include Alternaria, Aspergillus, Cladosporium, Curvularia, Epicoccum and Xylariaceae species (Vardavakis, 1990; Guiraud et al., 1995; Arif and Hashem, 1998; San Martin and Lavin, 1999; Stchigel et al., 2000). It has been proposed that disturbances to the surface O Horizon result in the detachment of spores and fragmentation of hyphae from the fungal colony and with the aid of wind and other atmospheric vectors; these fungal propagules may aerosolize directly into the inhalable zone of the subject (Mitakakis et al., 2000). This is well illustrated on an environmentally larger disturbance scale, where cropping activities of wheat have been shown to significantly increase the airborne concentration of Alternaria in rural environments (Mitakakis et al., 2001b). Thus, the collection of larger concentrations of fungi associated with soil by INAS, in addition to high concentrations of hyphal fragments might have resulted from the mild personal activity engaged by the subjects. This chapter demonstrates the detection of variability between collected spore numbers on different air sampling days was dependant on both the sampling methods and genera counted. Airborne fungal counts are well recognized to fluctuate between spatial, temporal and geographic parameters as well as between the methods of air sampling (Flannigan, 1997; Rogers, 2003). Regional airborne counts are largely dependent on the prevailing weather conditions, including temperature, humidity and wind direction of the surrounding environment, in addition to the pre-existing substratum nutrient conditions, which facilitate in the production and release of fungal spores. Disturbing this subtle balance alters the growth, reproduction and aerosolization of fungal propagules from fungal colonies, thus modifying daily concentrations. Previous studies have demonstrated that the spores of specific fungal genera in some cases, require moisture in order to disseminate the spores, whereas other fungi require dry conditions (Bush and Portnoy, 2001). This is particularly evident on the first SaD

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when the study location was characterized by dry environmental conditions, which facilitated higher airborne spore counts of Arthrinium, Spegazzinia and Xylariaceae species. However, pre-sampling precipitation on SaD 2 favoured the release of Epicoccum, Bipolaris and Curvularia spores. The variations observed in the number and spectrum of fungal spores collected by each method of analysis can be attributed to a number of parameters that are associated with the operation, collection and quantification of each air-sampling device. For instance, the Burkard volumetric spore trap is a static air-sampling device that is generally placed at ground level or on top of a building. The local topography, environment and orientation of the air sampler has been shown in numerous studies to yield a wide spectrum of fungi, in addition to total fungal concentrations (Li and Kendrick, 1995b; Rutherford et al., 1997; Bush and Portnoy, 2001). The collection of airborne fungal particles by a spore trap is influenced by a combination of localized and more distant sources and may not accurately represent the distribution of particles in a neighbouring geographic locale (Mitakakis and McGee, 2000). The height, at which a sampler is placed, is likely to also influence the final outcomes of a study. It has been shown that fungal spore concentrations decrease with increasing height as demonstrated for Alternaria and other spore types (Rantio-Lehtimaki et al., 1991; Hart et al., 1994; Bergamini et al., 2004). Thus, spore concentrations are generally greatest at ground level and this was particularly reflected in the INAS and IOM spore counts reported in the present study. To date, the INAS method is the closest air sampling technique available for a true assessment of personal exposure over short temporal intervals. The sampler has been shown to efficiently collect most spherical latex particles above 5µm (Graham et al., 2000), however for smaller particles (<5µm), including unicellular fungal conidia and hyphal fragments the collection efficiency decreases. This is particularly evident when comparing between Burkard, IOM and INAS samples. Our findings show that relatively low numbers of small fungal particles (<5µm), in particular Aspergillus- Penicillium and Cladosporium spores were collected on the INASs compared to IOM and Burkard counts. In some cases the total numbers of fungi collected by IOM

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personal aerosol samplers were approximately 4-fold greater than that collected by INAS. These variations can be accounted for by limitations in the INAS collection efficiencies for smaller particles and by difficulties visualizing and subjectively identifying smaller spores collected on the opaque INAS impaction surface. Conversely, the Burkard spore trap has a much broader collection efficiency for airborne particles compared to the other devices, which is reflected by the diversity and size ranges of airborne fungal spores collected in the present study. In addition, smaller spores belonging to Aspergillus-Penicillium and Cladosporium species were much more easily resolved on the stained Burkard tape than the other methods and this is probably why smaller fungal spores, as well as some larger opaque spores; for example Leptosphaerulina and Aspergillus-Penicillium were only observed on the Burkard impaction surface. Thus, to accurately monitor inhaled airborne exposure to fungi, it will be necessary to continue to develop INAS to collect particles down to 2µm in diameter. The differences between airborne fungal levels obtained in the stationary and personal exposure measurements confirm previous findings using various personal exposure monitoring devices, including the volumetric Button sampler (Aizenberg et al., 2000; Riediker et al., 2000; Toivola et al., 2002). However, several other studies have demonstrated similarities between airborne counts collected by the two different measurement techniques. Adhikari et al. (2004) used multiple stationary samplers and found similarities in airborne fungal levels between stationary and personal exposure measurements. In addition, the differences in airborne fungal levels in the present study, may be accounted for by other variations in the sampling techniques and the ability to resolve particles as demonstrated in various other studies (Sterling et al., 1999; Irdi et al., 2002). The results of this study also demonstrate for the first time the collection of inhaled fungal fragments and hyphae in an outdoor environment. These morphologically indiscernible particles have recently been shown to express detectable allergen in environmental samples detected with the HIA (Green et al., 2005). The proportion of inhaled hyphae confirms previous investigations that airborne fragments

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are often higher in concentration than the numbers of spores of a single allergenic genus (Gorny et al., 2002; Gorny, 2004; Green et al., 2005). The importance of these airborne particles, often ignored in previous airborne studies, is slowly being recognized and the results of this study further demonstrate the significant concentration in which these particles are inhaled. Furthermore, the genera collected by the INASs provide an entirely different spectrum of fungal exposure compared to those reported in previous studies. Although dependent on the geographic locale and efficiency of the sampler, the dominant inhaled fungi included Arthrinium, Curvularia, Epicoccum, Pithomyces, Spegazzinia, Xylariaceae and Bipolaris species, which have all been recently shown to express detectable allergen (Green et al., 2005). These findings further support a new paradigm, where a substantial proportion of the airborne fungal biomass rather than a limited group of genera contributes to the aeroallergen load.

4.4 Conclusions

The measurement of personal exposure to airborne fungi using INASs is a sensitive and unique technique compared to pre-existing methods of analysis. The collected fungal particles represent are a true reflection of what is actually being inhaled, although this is confounded by low collection efficiency for smaller particles. The results of this study showed that inhaled fungal exposure, in most people in the same location, varied within a 2-fold range with 10-fold outliers. Comparatively, the INASs and IOM personal aerosol samplers were associated more to each other than to Burkard spore traps. The variations between each of the air sampling techniques investigated can be attributed to variables associated with the operation, collection and quantification of each air- sampling device. Our results further demonstrate the significance of recently identified fungal allergen sources, including airborne hyphae and numerous other allergenic genera to personal exposure. These findings advance our understanding of personal exposure to airborne fungi, however future investigations are required to improve diagnostic techniques, in addition to understanding the role of fungal allergens in allergic rhinitis and asthma.

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Table 4.1. Median, 25th and 75th percentile values of the total number of airborne fungi measured by three different air sampling methods in an outdoor community setting.

Method of Analysis Method of Analysis Fungal genera Burkarda IOMb INASc Burkard IOM INAS Sampling Day 1 Sampling Day 2 Fungal spores ≥ 10µm Acrodictys 3 0 (0-0) 0 (0-0) 20 0 (0-0) 0 (0-0) Alternaria 18 64 (42-80) 34 (23-87) 8 48 (21-80) 128 (76-228)† Ascobolus 9 0 (0-0) 0 (0-0) 100 0 (0-0) 0 (0-0) Beltrania 27 0 (0-4) 0 (0-1) 16 0 (0-0) 0 (0-0)† Bipolaris 12 16 (12-18) 5 (1-13) 4 200 (0-369) 108 (56-246)† Cerabella 0 0 (0-0) 0 (0-0) 0 0 (0-0) 0 (0-0) Curvularia 45 176 (172-228) 160 (116-224) 48 304 (296-352) 196 (96-266) Dictyosporium 0 0 (0-0) 0 (0-0) 12 0 (0-0) 0 (0-0) Epicoccum 18 8 (8-14) 47 (25-64) 88 56 (16-80) 204 (102-290)† Exserohilum 6 32 (20-36) 20 (9-35) 12 32 (5-56) 20 (16-30) Fusariella 1 8 (8-8) 6 (4-10) 0 32 (24-32) 16 (8-28) Fusarium 0 8 (8-12) 0 (0-3) 0 0 (0-8) 12 (5-38)† Hyphal fragments 30 656 (624-692) 250 (170-357) 8 944 (656-1024) 232 (142-356) Isthmospora 0 0 (0-0) 0 (0-0) 0 0 (0-0) 0 (0-0) Leptosphaeria 108 8 (8-8) 8 (5-12) 4 0 (0-0) 4 (0-8)† Leptosphaerulina 27 0 (0-0) 0 (0-0) 140 0 (0-0) 0 (0-0) Pithomyces 2 64 (42-116) 99 (49-148) 20 32 (24-40) 52 (32-72)† Pleospora 1 0 (0-0) 0 (0-0) 0 0 (0-0) 0 (0-4) Rust 0 0 (0-0) 0 (0-0) 0 0 (0-0) 0 (0-0) Spondylocladiella 27 56 (43-64) 4 (0-13) 24 28 (24-40) 44 (24-62)† Sporidesmium 0 8 (4-8) 0 (0-0) 0 0 (0-0) 0 (0-0) Stemphylium 0 0 (0-0) 0 (0-0) 0 0 (0-0) 0 (0-0)† Spegazzinia 6 96 (80-104) 47 (31-79) 8 16 (16-21) 16 (12-34)† Tetraploa 2 8 (8-12) 16 (10-31) 0 0 (0-8) 16 (4-24)† Ulocladium 0 0 (0-0) 0 (0-1) 0 0 (0-0) 0 (0-4) Xylariaceae 195 1056 (720-1080) 697 (402-1093) 112 968 (688-984) 372 (274-552)† Fungal spores 3-10µm Amphisphaeria 0 0 (0-0) 0 (0-0) 0 0 (0-0) 0 (0-0) Arthrinium 20 1312 (1004-5724) 410 (194-781) 8 120 (104-128) 108 (62-166)† Basidiomycete 3 8 (5-14) 11 (1-16) 0 8 (0-8) 8 (4-18) Botrytis 0 0 (0-0) 0 (0-0) 16 0 (0-0) 0 (0-0) Cladosporium 3495 1048 (968-1256) 42 (32-85) 3528 4970 (4578-5180) 32 (8-106)† Coprinus 57 0 (0-0) 0 (0-0) 440 0 (0-0) 0 (0-0) Delitschia 0 0 (0-0) 0 (0-0) 0 0 (0-0) 0 (0-0)† Myxomycete 0 0 (0-0) 0 (0-0) 28 0 (0-0) 0 (0-0) Smut 0 0 (0-0) 0 (0-0) 0 0 (0-0) 0 (0-0) Sporomiella 21 0 (0-0) 0 (0-0) 118 0 (0-0) 0 (0-0) Torula 0 0 (0-0) 2 (0-5) 0 0 (0-0) 0 (0-0)† Fungal spores ≤ 3µm Aspergillus-Penicillium 528 0 (0-0) 0 (0-0) 96 0 (0-0) 0 (0-0)

Unknown 591 16 (12-18) 78 (36-138) 356 40 (24-40) 36 (20-66) Total 5202 4794 (4718-8642) 2593 (1749-3663) 5214 7522 (7521-8300) 1808 (1284-2358)†

a. Burkard counts are representative of only one observation (n=1). b. IOM spore counts include Median, 25th and 75th percentiles (n=4 SaD 1 and n=6 SaD 2) c. INAS spore counts represent the median (25th and 75th percentiles) number of spores inhaled by each subject (n=34 SaD1 and n=31). The total spore count for each genus is derived from the addition of left and right nostrils. † Denotes the level of significance of ANOVA statistical analyses between SaDs 1 and 2 INAS spore counts, which were log transformed to normalize the data. † P<0.05.

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Figure 4.1. The sampling site and surrounding vegetation with subjects participating in the study on SaD 1 with and inset of the map of Australia depicting the geographical position of Casino, northern New South Wales and the () sampling region.

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Figure 4.2. (A) The intra-nasal air sampler. The disassembled components for the nasal sampler – a soft silicon strap spans the septum of the nose and connects the two silicon frames and that house the collection cups. (B) The fully assembled intra-nasal air sampler worn by a subject.

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5 Total number of genera collected / 2 hours

0 Burkard SaD1 Burkard SaD2 PAS SaD1 PAS SaD2 INAS SaD1 INAS SaD2

Figure 4.3. The total number of fungal genera collected by each air sampling technique on each sampling day.

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20000 Xylariaceae 17500 A Hyphal fragments Arthrinium Curvularia 15000 Epicoccum Alternaria 12500

10000

7500

5000

2500

0 1 2 3 4 5 6 7 8 9 10 21 22 23 24 25 26 27 28 29 30 41 42 43 44 45 46 47 48 49 50 61 62 63 64 6000 Xylariaceae B Hyphal fragments 5000 Arthrinium Curvularia Epicoccum 4000 Alternaria

Total number of spores collected / 2 hours 3000

2000

1000

0 81 82 83 84 85 86 87 88 97 98 99 100 101 102 103 104 113 114 115 116 117 118 119 120 129 130 131 132 133 134 135

INAS Subject Identification Number

Figure 4.4. The relative abundance of fungal spores belonging to the five most frequent genera inhaled by each subject. A. Sampling Day 1. B. Sampling Day 2.

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Figure 4.5. Photomicrographs of collected fungal conidia and hyphal fragments. A. Ascospores belonging to the Xylariaceae (arrow a) and hyphal fragments (arrow b) and B. fungal spores of Arthrinium (arrow c) and Curvularia (arrow d) species. Scale bar, 20µm.

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Xylariaceae INAS Hyphal fragments INAS Arthrinium spp. INAS 700 1500 10000 600 1250 7000 500 4000 1000 400 1000 300 750 300 200 500 200 100 250 100 Spores collected / 2 hours / Spores collected Spores collected / 2 hours / Spores collected Spores collected / 2 hours / collected Spores 0 0 0 Left Nostril Right Nostril Left Nostril Right Nostril Left Nostril Right Nostril

Curvularia spp. INAS Epicoccum spp. INAS Alternaria spp. INAS

400 600 1100 350 500 900 300 400 700 250 300 500 150 150 200 150 100 100 100 50 50 Spores collected / 2 hours / Spores collected

50 2 hours / Spores collected Spores collected / 2 hours / Spores collected 0 0 0 Left Nostril Right Nostril Left Nostril Right Nostril Left Nostril Right Nostril

Pithomyces spp. INAS Cladosporium spp. INAS Spegazzinia spp. INAS

200 500 125 400 100 150 300 200 75 100 100 75 50

50 50 25 25 Spores collected / 2 hours / Spores collected Spores collected / 2 hours / Spores collected Spores collected per 2 hours per 2 collected Spores 0 0 0 Left Nostril Right Nostril Left Nostril Right Nostril Left Nostril Right Nostril

Figure 4.6. Comparison between the relative abundance of total fungal spores collected by left and right nostril INAS collection cups for the nine most frequent genera. The box plot denotes minimum, 25th percentile, median, 75th percentile and maximum values. There were no significant differences observed between left and right nostrils for any of the genera.

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5 r = 0.74 P < 0.0001 4

1 Log INAS Counts INAS Log

-1 -1 0 1 2 3 4 5 Log IOM Counts

Figure 4.7. Correlation between IOM and INAS total fungal counts. In the figure: r = Pearson rank correlation co-efficient. Correlations are significant at the 0.05 level.

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Chapter 5∗

5. Part A. HIA double immunostaining

Personal exposure to airborne fungi is recognized to be a risk factor for seasonal rhinitis (Li and Kendrick, 1995a; Andersson et al., 2003), asthma (Downs et al., 2001; Zureik et al., 2002) and even death (O'Hollaren et al., 1991; Black et al., 2000). The collection and enumeration of airborne fungal conidia, hyphae and more recently fungal fragments in bioaerosol and occupational investigations is complex. Various viable and non-viable sampling techniques are available but are often confounded by a lack of specificity, long incubation times and subjective identification methods. These limitations are in part due to the viability of conidia, lack of suitable molecular probes and the inability to speciate small conidia and morphologically indiscernible fragments (Rogers, 2003; Schmechel et al., 2003a). Furthermore, current in vitro methods to diagnose allergy to fungi are restricted by the availability and variability of allergen extracts (Esch, 2004). It is likely that fungal allergy is both under diagnosed and airborne fungi are incorrectly identified as causes of allergic symptoms. Not since the integration of direct microscopy and immunohistochemistry has it been possible to collect and enumerate airborne wild-type fungal particles and concurrently demonstrate antigen-antibody interactions (Popp et al., 1988). Such immunohistochemical techniques, however, are confined to only recognizing surface antigens fixed in or on the particles themselves. The development of the HIA has enabled the co-visualization of individual fungal conidia and hyphae collected by

∗ Chapter 5, Part A has been accepted for publication as “Enumeration and detection of aerosolized Aspergillus fumigatus and Penicillium chrysogenum conidia and hyphae using a novel double immunostaining technique” in the Journal of Immunological Methods in 2005. Chapter 5, Part B was published as “Detection of aerosolized Alternaria alternata conidia, hyphae and fragments using a novel double immunostaining technique” in Clinical and Diagnostic Laboratory Immunology” in 2005.

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volumetric air sampling together with their expressed antigens immunostained around the particle as a halo with human IgE (Tovey et al., 2000). However, the identification of the small (2-3µm) and unicellular Aspergillus and Penicillium conidia and hyphae collected onto PBMs has previously been based entirely on conidial morphological criteria and to date remains subjective (Green et al., 2005). In this proof of principle study, we describe a novel double immunostaining technique using the HIA, that enables the enumeration and identification of culturally derived unicellular A. fumigatus and P. chrysogenum conidia and hyphae with monoclonal antibodies (mAbs) and the concurrent immunostaining of allergens with human IgE to the same fungal propagules.

5.1 Materials and Methods

5.1.1 Cultivation, aerosolization and collection of fungal conidia

Fungal isolates of A. fumigatus (28004) and P. chrysogenum (28002) were supplied by the Queensland Department of Primary Industries (Brisbane, Australia). The isolates were sub-cultured from stock sources and grown for 10 days on vegetable juice nutrient agar at 24.9oC. Conidia were aerosolised from sporulating cultures by use of an air jet and then collected by suction onto a MPBM (0.8 µm pore size; Millipore Corporation, Bridgewater, MA) as described previously (Green et al., 2003). To germinate conidia, the membrane was moistened in deionized water and placed in a humid box for 12 hours at 24.9oC to allow germination. Both germinated and ungerminated conidia samples were permanently laminated to the MPBM by overlaying it with a glass coverslip that had been pre-coated with a film of optically clear adhesive (Woolcock Institute of Medical Research, Sydney, Australia).

5.1.2 Human serum samples

Human sera from 30 subjects with asthma who were allergic to Alternaria and other fungal genera was collected and pooled. The diagnosis was based upon a documented clinical history of asthma, and allergy was determined by a positive SPT with a wheal diameter of 3mm or greater. Specific IgE towards a panel of fungal allergens was detected in the pool by Pharmacia UniCAP (Pharmacia, Uppsala, Sweden) (Pharmacia

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CAP score; specific IgE to A. alternata = 60.7 kUA/L). All samples were stored in aliquots for future use at –70oC. Pooled serum IgE from 10 subjects, SPT negative to fungi but sensitized to other non-fungal allergens (Pharmacia CAP score; specific IgE to

A. alternata < 0.35 kUA/L), in addition to an in house rabbit polyclonal antibody raised against a crude Lolium perenne pollen extract were used as a negative control (Green et al., 2003). The local research ethics committee approved the study protocol and the subjects gave written informed consent following a full explanation of the study.

5.1.3 Monoclonal antibody production

Briefly, mice were immunized with extracts of P. chrysogenum conidia as described previously for Aspergillus versicolor (Schmechel et al., 2003b). The mAb 18G2 (IgG1) was found to extensively cross-react with the mycelium or spores of a number of fungal species commonly identified in indoor environments, including A. fumigatus.

5.1.4 Immunostaining

Previously laminated ungerminated or germinated conidia (from 4.2.1 above) were immersed in borate buffer (pH 8.2) for four hours to enable antigens and other macromolecules to elute and bind in close proximity to the conidia and hyphae on the MPBM. Membranes were blocked in 1% BSA in PBS for 45 minutes and then incubated overnight with a positive IgE serum pool diluted 1:3 in 1% BSA-PBS-0.05% Tween 20. After the primary antibody incubation, the membranes were rinsed three times in PBS-0.05% Tween 20 and incubated for 1.5 hours with biotinylated goat antihuman IgE (Kirkegaard & Perry Laboratories, Gaithersburg, MD) diluted 1:500 in 1% BSA-PBS-0.05% Tween 20. This was followed by an incubation for 1.5 hours with a mouse mAb 18G2 raised against P. chrysogenum (Health Effects Laboratory Division, Centres for Disease Control and Prevention, National Institute of Occupational Safety and Health, Morgantown, WV), which was diluted 1:5 in 1% BSA-PBS-0.05% Tween 20. Following the mAb incubation, the membranes were rinsed and then incubated for 1.5 hours with an anti-mouse IgG horseradish peroxidase (HRP) conjugate (Sigma Chemical Co, St Louis, Mo). Finally, biotinylated anti-human IgE antibodies were

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labelled with an ExtrAvidin alkaline phosphatase conjugate (Sigma Chemical Co, St Louis, Mo) diluted 1:1000 in 1% BSA-PBS-0.05% Tween 20 and incubated for 1.5 hours. The membranes were then rinsed three times in PBS-0.05% Tween 20 before being developed. For immunostaining, Vector NovaRED substrate for HRP (Vector Laboratories, Burlingame, CA) was first prepared as per manufacturers instructions and samples were incubated with the substrate for approximately one hour to allow adequate development of the red precipitate. The membrane was then rinsed three times in PBS-0.05% Tween 20 and transferred to a separate staining well containing the alkaline phosphatase NBT/ BCIP substrate (Pierce Chemical Co, Rockford, Il). Staining was then monitored periodically for approximately 20 minutes until an optimum dark blue precipitate was achieved. Entire membranes were then examined at a magnification of 200X by using standard light microscopy as described previously (Green et al., 2003) and double immunostained conidia and hyphae were counted. Three replicates were evaluated and the experiment was repeated with appropriate controls, including the rabbit polyclonal antibody raised against a crude Lolium perenne pollen extract and the pooled adult human sera SPT negative to fungi but sensitized to other non-fungal allergens.

5.1.5 Statistical analysis

Differences between mean values for double immunostained subsets among ungerminated and germinated groups were analysed for significance using the Independent samples t-test (Analyse-It for Microsoft Excel, Version 1.68, Analyse-It Software Ltd., Leeds, United Kingdom). The criterion for significance for all analyses was P<0.0001. Except otherwise noted, all data are expressed as mean and ranges.

5.2 Results

The observed antigen immunostaining as detected by the mAb and the human IgE varied amongst each of the species and the state of germination (Figure 5.1 and 5.2). For ungerminated conidia belonging to A. fumigatus, resultant mAb immunostaining was primarily localized as a red halo around the outer extremities and on either margin of the conidia, whereas resultant IgE immunostaining was in close proximity around the

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entire conidia and appeared as a dark purple halo (Figure 5.1A). In addition, significant concentrations of antigen were expressed in regions of clustered A. fumigatus conidia (Figure 5.1B). For P. chrysogenum, the mAb and IgE immunostaining was localized around the perimeter of the conidia and staining by both antibodies was homogeneously distributed (Figure 5.2A and 5.2B), often in greater concentrations compared to A. fumigatus. Upon germination, greater concentrations of both mAb and IgE immunostaining were observed with both species, when compared to the ungerminated conidia. Similar patterns of antigen immunostaining were evident for A. fumigatus (Figure 5.1C and 5.1D) and P. chrysogenum (Figure 5.2C and 5.2D), however a number of subtle variations existed between the genera. For A. fumigatus the resultant mAb and IgE immunostaining was localized in the same regions around the growing hyphal tips of germinated conidia (Figure 5.1C and 5.1D). However, for P. chrysogenum resultant mAb and IgE immunostaining was heterogeneous and primarily localized in the vicinity of growing hyphal tips (Figure 5.2C and 5.2D), in addition to around the area of conidial germination (Figure 5.2C and 5.2D). For A. fumigatus, there was greater staining of the hyphal structure itself by the mAb (Figure 5.1C and 5.1D), whereas with P. chrysogenum, the hyphae showed greater direct staining with IgE (Figure 5.2C and 5.2D). In addition, negative controls showed no immunostaining around the outer edges of the ungerminated (Figure 5.1E, Figure 5.2E and Table 5.1) and germinated conidia or hyphae (Figure 5.1G, Figure 5.2G and Table 5.2). In the controls there was some purple staining of the interior of the conidia but not the hyphae, which is consistent with the NBT functioning as a vital stain of viable spores. The proportion of ungerminated and germinated conidia demonstrating double immunostaining for each of the species is presented in Table 5.1. Approximately 31% of A. fumigatus and 14% of P. chrysogenum ungerminated conidia expressed dual mAb and human IgE immunostaining (Table 5.1). Upon conidial germination however, the percentage showing double immunostaining (Table 5.1) rose to 42% and 80%, respectively (P<0.0001).

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5.3 Discussion

Traditional methods of sampling airborne fungi have been confounded by a number of limitations, which has made data interpretation and comparisons between studies difficult. Viable culture detects only those fungi that are capable of growth on the selected nutrient medium, while other factors including, environmental conditions, sampling practices, transport of materials and culture conditions also influence the final outcome (Stetzenbach et al., 2004). Alternatively, non-viable methodologies are often subjective and imprecise with recent estimates of up to 50% of airborne fungi in aerobiological investigations misclassified (Flannigan, 1997). The recent development of more reliable non-viable methods have corresponded with the advances in molecular biological techniques, which have seen the development of PCR probes (Portnoy et al., 1993c) and recombinant mAbs specific to individual fungal allergens, the most common including Alt a 1 and Asp f 1 (Vailes et al., 2001). These mAbs have enabled the quantification of specific fungal allergens in environmental air samples using immunoassays, as well as demonstrated the localization of allergens on the surface of conidia and hyphae in immunohistochemical studies (Reijula et al., 1992). However, even with these molecular advances, the enumeration and identification of collected wild-type unicellular fungal conidia and hyphae continues to be taxonomically challenging. Currently, there are no techniques, which allow the identification and simultaneous demonstration of allergic sensitisation to the same collected airborne wild- type fungal propagule. While immunoassay techniques for the quantification of antigens and the identification of cells have found wide applications with many areas of biology, so far its contribution to mycology has been less productive. The available immunohistochemical techniques based on the staining of surface structures (Popp et al., 1988; Reijula et al., 1992) are not practical for general use, while immunoassays of some soluble allergens from conidia are confounded by the production of such allergens by different fungal genera (Bisht et al., 2000). HIAs, particularly when used with this double immunostaining technique, provide novel insight. They allow the application of

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traditional morphological techniques to identify structures and combine this with the ability of monoclonal or polyclonal antibodies to further identify conidia along with the use of a second human antibody to diagnose whether an individual is allergic to that particular species. Our proof of principle study has demonstrated that the localization of the antigens detected by both primary antibodies was heterogeneous between the two fungal genera and varied between the states of germination. Although conidial structure and allergen function were not investigated in the present study, the heterogeneous patterns of antigen expression for A. fumigatus and P. chrysogenum suggest that morphological characteristics of the conidial walls are important features in determining the resultant double immunostaining of expressed antigen in the HIA. The expression of fungal antigens from intact conidia and hyphae has been poorly investigated, however several studies using thin sectioning and immunoelectron microscopy, have demonstrated the location of allergens belonging to C. cladosporioides (Bouziane et al., 1989) and A. fumigatus (Reijula et al., 1991). Here, the cell wall, cell membrane (Bouziane et al., 1989) and to a lesser extent, the cytoplasm (Reijula et al., 1991) are the principle locations in which allergens are expressed in the conidia. In addition, the conidia of a number of airborne fungi are covered by an interwoven fibrous network of rodlets, which have been identified by immunoassays to contain the highest concentrations of conidial allergen and function to regulate the permeability of many cytoplasmic components including antigen-presenting macromolecules (Bouziane et al., 1989). Several experimental studies have also demonstrated that the germination of Aspergillus (Sporik et al., 1993), Alternaria (Mitakakis et al., 2001a) and a number of other fungal genera (Green et al., 2003) increases the amount of detectable allergen. Newly emerged hyphal tips from germinated conidia are functionally differentiating and metabolically active regions of the hyphae, which have been shown to bind considerably greater amounts of antibody compared to conidia (Reijula et al., 1991). In the current study, fewer ungerminated A. fumigatus and P. chrysogenum conidia were detected than germinated conidia. Furthermore, the germination of conidia increased the amount of expressed antigen and when compared to ungerminated conidia, provided a

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greater concentration of double immunostaining. Unicellular Aspergillus and Penicillium conidia share small dimensions (<3µm) and compared to hyphae contain only negligible quantities of antigen-bearing macromolecules. These amounts of antigen (probably in the low pg quantity per conidial) pose a lower limit of detection on the assay. Future studies should explore ways to improve the detection thresholds. This would further clarify wild-type fungal conidia as well as enable the resultant immunostaining to be semi-quantitatively analysed. However, to date, this has not been possible using current materials as preliminary tests reveal that a combination of available fluorophores and precipitating substrates were less sensitive and confounded by quenching (unpublished data). In addition to identifying fungal conidia, this methodology may also be applied to the identification of airborne hyphae and morphologically indiscernible fungal fragments. Aerosolization studies have demonstrated that fungal fragments and hyphae are significantly higher in concentration than airborne conidia of any single species (Gorny et al., 2002; Gorny, 2004; Green et al., 2005) and that these fragments released detectable quantities of allergen (Green et al., 2005). However, the identification and quantification of such particles using non-viable methods has not been possible to date, using available mAbs and subjective identification techniques. The speciation of fungal hyphae and fragments requires species-specific mAbs, which up till now, have not been available for the majority of medically important fungi, other than for Stachybotrys chartarum (Schmechel et al., 2003c). Although our study was based on a cross-reactive mAb, it clearly demonstrates the potential and practicality of the HIA in combination with double immunostaining. As further species-specific mAbs become available, our approach will enable the differentiation of wild-type fungi in air samples, in addition to providing informative patient-specific exposure profiles, which may eventually lead to instructive environment-specific exposure-disease patterns in epidemiologic studies.

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5.4 Conclusions

Understanding the clinical significance of exposure to airborne fungi is well recognized, however progress has been limited by problems associated with present methodologies (Rogers, 2003). Currently, allergen extracts used for in vivo and in vitro diagnostics lack standardization, are restricted to only a few major fungal species and research based on them are often difficult to reproduce. Furthermore, extensive cross reactivity exists between fungal genera (Horner et al., 1995; Esch, 2004), leading to uncertainties in the identification of species to which exposure and hypersensitivity is occurring. Double immunostaining based on the HIA has the potential to identify fungal species in personal air samples without the need for any fungal extracts, in addition to simultaneously demonstrating allergic sensitisation to individual fungi for each patient. It thus combines environmental with serological monitoring on a patient-specific basis and may well become a significant immunodiagnostic tool ultimately contributing to better patient management and the characterization of adverse health effects due to fungal aerosols.

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Table 5.1: Summary of the proportion of ungerminated and germinated A. fumigatus and P. chrysogenum conidia treatments demonstrating double immunostaining with mAb and human IgE.

Mean proportion of fungal conidia (range)a State of fungal germination No immunostaining mAb + IgE immunostaining Aspergillus fumigatusc Ungerminated conidia 68.8 (60.4-73.3) 31.2 (26.7-39.5) Germinated conidia 58.3 (44.5-67.1) 41.7 (32.9-55.5)b

Penicillium chrysogenumd Ungerminated conidia 86.1 (82.9-13.9) 13.9 (18.6-17.1) Germinated conidia 19.5 (11.8-31.4) 80.4 (68.6-88.2)b

a Values presented represent the mean determined from counting the number of ungerminated and germinated conidia expressing double immunostaining from a total count of all conidia present. In some instances, under development of the precipitating stains may have under represented the proportion of conidia demonstrating double immunostaining. b Bold type identifies the germinated conidia double immunostained values, which are significantly higher than those double immunostained in the ungerminated conidia treatment (P<0.0001). c All A. fumigatus negative control treatments showed no immunostaining around the ungerminated conidia and germinated conidia and hyphae. d All P. chrysogenum negative control treatments showed no immunostaining around the ungerminated conidia and germinated conidia and hyphae.

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Figure 5.1. Dual immunostaining of culture derived A. fumigatus conidia (A-B) and germinated conidia (C-D) using mAb 18G2 (arrow a; red precipitate) and human serum IgE (arrow b; purple precipitate). Immunostaining was confined to (A) around the entire conidia (arrow c), (B) regions of clustered conidia (arrow c) and (C- D) around the hyphae and hyphal tips (arrow d and e) of germinated conidia (arrow c). Scale bar, 20 µm. (E-F). Negative controls using a pool of sera from atopic but Alternaria negative subjects (Pharmacia CAP score; specific IgE to A. alternata <0.35 kUA/L) and an in house rabbit pAb raised against a crude Lolium perenne pollen extract showed no localized immunostaining around the conidia (arrow c) and hyphae (arrow d). Scale bar, 20 µm.

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Figure 5.2. Dual immunostaining of culture derived P. chrysogenum conidia (A-B) and germinated conidia (C-D) using mAb 18G2 (arrow a; red precipitate) and human serum IgE (arrow b; purple precipitate). Immunostaining was confined to (A-B) around the entire conidia and (C) around the periphery of the hyphal tips (arrow d and e), as well as (D) in close proximity to the site of conidial germination (arrow c). (E-F) Negative controls using a pool of sera from atopic but Alternaria negative subjects (Pharmacia CAP score; specific IgE to A. alternata <0.35 kUA/L) and an in house rabbit pAb raised against a crude Lolium perenne pollen extract showed no localized immunostaining around the conidia (arrow c) and hyphae (arrow d). Scale bar, 20 µm.

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5. Part B. Alternaria double HIA immunostaining

The fungus A. alternata, is an extensively distributed plant pathogen and allergy to it is a risk factor for asthma severity (Downs et al., 2001). The reproductive structures, which characterize this species and aid in the organism’s dispersal, include multicellular conidia and septate hyphae. Airborne A. alternata conidia have been established in numerous studies to be prevalent in both indoor and outdoor environments (Burge et al., 2000), although the role of aerosolised hyphae in exacerbating asthma and allergy has not been established. Recent preliminary investigations have demonstrated that airborne wild-type fungal hyphae express detectable quantities of allergen (Green et al., 2005) and can be significantly greater in concentration than conidia of any single species in indoor residential environments (Gorny et al., 2002; Gorny, 2004; Green et al., 2005). Until now, the enumeration and identification of airborne hyphae, using non-viable methods remains subjective and imprecise and improved objective detection methods for fungal bioaerosol monitoring are required. Numerous attempts have been made to detect and identify specific fungal components that serve as allergen sources. One such technique has been the direct immunostaining using human IgE against insoluble fungal antigens on the surface of wild-type fungal conidia (Popp et al., 1988). However, this did not permit speciation or detect hyphae. The recent development of monoclonal antibodies specific for some soluble fungal allergens does not provide a basis for speciation either, as these allergens are often shared between fungal species (Schmechel et al., 2003a). Up until now there has not been a method that can potentially differentiate between different species of aerosolised hyphae collected during volumetric air sampling that function as allergen sources. In this study, we describe for the first time proof-of-principle of a novel double immunostaining methodology based on the HIA, which enables (1) the identification of A. alternata conidia, hyphae and fragments by immunostaining eluted antigens with a polyclonal antibody (pAb) and (2) concurrently demonstrates allergy to the same fungal particles by immunostaining with human IgE.

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5.5 Materials and methods

The method is briefly described as follows. Conidia and hyphae belonging to A. alternata were double immunostained using the HIA with an anti-Alternaria pAb (Woolcock Institute of Medical Research, Sydney, Australia) and a serum IgE pool from Alternaria-sensitive subjects (Pharmacia CAP score; specific IgE to A. alternata =

60.7 kUA/L). The rabbit antiserum was raised against a crude in-house Alternaria extract and was shown in these studies to have a broad specificity for different Alternaria components. ELISA analysis using crude fungal extracts showed the sera to have strong binding to this species and weak binding to several other species tested (data not presented). Negative controls consisted of a pool of sera from atopic, but Alternaria negative subjects (Pharmacia CAP score; specific IgE to A. alternata < 0.35 kUA/L) and rabbit pAb raised against a crude Lolium perenne pollen extract. For the HIA, components of A. alternata were aerosolised from culture plates using an air jet and then collected by vacuum onto a MPBM (0.8 µm pore size; Millipore Corporation, Bridgewater, MA) as previously described (Green et al., 2003). The collected fungal particles were either germinated under humid conditions for 12 hours or were not germinated, before being permanently laminated to the MPBM by overlaying them with a glass coverslip that had been pre-coated with a thin film of optically clear adhesive (Green et al., 2003). The laminated samples were immersed in borate buffer (pH 8.2) for four hours to enable antigens and other macromolecules to elute from the conidia and hyphae and bind to the membrane. Membranes were blocked in 1% BSA in PBS for 45 minutes and then incubated overnight with the positive IgE serum pool diluted 1:3 in 1% BSA-PBS-0.05% Tween 20. After the primary antibody incubation, the MPBMs were rinsed three times in BSA-PBS-0.05% Tween 20 and incubated for 1.5 hours with biotinylated goat antihuman IgE (Kirkegaard & Perry Laboratories, Gaithersburg, MD) diluted 1:500. This was followed by an incubation for 1.5 hours with the pAb, diluted 1:100. The membranes were rinsed and then incubated for 1.5 hours with an anti-rabbit IgG HRP conjugate (Sigma Chemical Co, St Louis, Mo) and an ExtrAvidin alkaline phosphatase conjugate (Sigma Chemical Co, St Louis,

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Mo), each at a titre of 1:1000 and then rinsed three times. For immunostaining, Vector NovaRED substrate for HRP was prepared as per manufacturers instructions (Vector Laboratories, Burlingame, CA) and added to the MPBM for approximately one hour to allow adequate development of the red precipitate. The membrane was then rinsed three times and transferred to a separate staining well containing the alkaline phosphatase substrate NBT/ BCIP (Pierce Chemical Co, Rockford, Il). Staining was then monitored periodically for up to 20 minutes until optimum dark blue precipitate development was achieved. Samples were examined at a magnification of 200X by using standard light microscopy and double immunostained fungal particles were considered positive and counted. A Student’s t-test was performed with Analyse-It software (Analyse-It Software Ltd, Leeds, United Kingdom) to compare mean values for double immunostained subsets among ungerminated and germinated groups. A difference was considered significant when P was <0.0001.

5.6 Results and Discussion

As shown in Table 5.2, results of double immunostaining demonstrated that 48% of ungerminated conidia expressed detectable pAb and IgE immunostaining, however upon germination this rose to 94% (P<0.0001). Negative controls showed no immunostaining around the outer edges of the ungerminated and germinated conidia or hyphae (Figure 5.3G, Table 5.2). In the controls there was some purple staining of the interior of the conidia but not the hyphae, which is consistent with the NBT functioning as a vital stain of viable spores. These findings are consistent with previous studies, which have demonstrated that the germination of conidia belonging to Aspergillus fumigatus and other fungal genera increases the proportion of conidia releasing detectable allergen (Sporik et al., 1993; Mitakakis et al., 2001a; Green et al., 2003), which are detected in the periphery of the components. Conidial germination is an important process in this immunoassay as it significantly increases the number of conidia being detected, in addition to improving the detectable thresholds of the immunoassay.

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Results from this study also indicate that the localization of immunostaining was heterogeneous between both conidia and the state of germination. The sites of immunostaining associated with ungerminated conidia varied from being restricted to the basal regions (Figure 5.3A) and septal junctions (Figure 5.3C) to around the entire conidia (Figure 5.3B). It is unclear whether this represents different sites of antigen or allergen release or is an artifactual effect of the assay, as the antigens have to diffuse from the particle until bound to the membrane. Upon conidial germination, where long hyphae are evident, greater concentrations of double immunostaining were observed. In the germinated conidia, antigen staining by the pAb was associated with the entire hyphae, whereas the IgE immunostaining was confined to the hyphal tips and the sites of conidial germination (Figure 5.3E and 5.3F). The heterogeneous localization of resultant immunostaining between conidia and the states of germination confirm our earlier reports investigating other fungal genera (Mitakakis et al., 2001a; Green et al., 2003). Improvements in technical aspects of the HIA allow improved resolution of the expression of allergens by the conidia and other fungal components, not previously observed (Mitakakis et al., 2001a) and demonstrate for the first time, the localized expression of soluble antigens released from A. alternata conidia. Previous studies have shown that the release of proteins, glycoproteins and carbohydrates may be determined by the conidial wall structure (Bouziane et al., 1989; Horner et al., 1993). Thin walled conidia have been shown to release allergen quickly, whereas those with thicker walls have been found to contain a rodlet layer (Bouziane et al., 1989), which functions to reduce cytoplasmic leakage. These results suggest that the localization of A. alternata conidial antigen could be due to variations in the thickness of the conidial wall, especially as immunostaining was localized around thin walled septal junctions. The development of the described double immunostaining technique was initiated as a result of recent observations, which showed that the airborne concentration of fungal hyphae and fragments was higher than conidia of any single species (Gorny et al., 2002; Gorny, 2004; Green et al., 2005) and that these fragments released detectable quantities of allergen (Green et al., 2005). Fungal fragments are heterogeneous particles,

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which are unable to be speciated using non-viable morphological characteristics nor distinguished from conidia as antigen sources using current techniques. Our findings demonstrate that morphologically indiscernible fragments and hyphae were double immunostained with pAb and IgE (Figure 5.3D). This proof of principle finding is important as it enables for the first time, the identification of whether the fragment is of fungal origin by detecting expressed fungal antigens, in addition to demonstrating IgE binding to allergens eluted from these fragments. The determination of individual fungal species by immunostaining techniques is dependant on the specificity of the antibodies available. Although several fungal allergen-specific monoclonal antibodies have been produced, these allergens are produced by multiple fungal species and are not restricted to a single species (Schmechel et al., 2003a). To our knowledge only one species-specific antibody, against Stachybotrys chartarum, has been described (Schmechel et al., 2003c). The HIA enables visualization of the morphology of particulates as well as staining of eluted antigens and so speciation on the basis of immunostaining combined with morphology is feasible for conidia, but without species-specific sera cannot currently be used to speciate hyphae. This paper provides a proof of concept how this could proceed and the future availability of different species-specific antibodies will allow this technique to be applied to environmental samples to further differentiate the airborne fragments and provide a comprehensive immunodiagnostic and environmental monitoring technique based not only on conidia but also on hyphae and conidial fragments. This would not be possible by any other current technique.

5.7 Conclusions

In conclusion, this Chapter has demonstrated a new approach to the identification of aerosolized fungal particles and demonstrating allergy to them. It combines environmental with serological monitoring on a patient specific basis and has the potential to be a significant immunodiagnostic tool and contribute to improved characterization of adverse health effects due to fungal aerosols.

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Table 5.2: Summary of the proportion of ungerminated and germinated A. alternata conidia treatments demonstrating double immunostaining with pAb and human IgE.

Mean proportion of fungal conidia (range)a State of fungal germination No immunostaining pAb + IgE immunostaining Ungerminated conidiac 51.7 (48.7-54.7) 48.3 (45.2-51.3) Germinated conidiac 5.8 (2.7-10.6) 94.2 (89.3-97.2)b

a Values presented represent the mean determined from counting the number of ungerminated and germinated conidia expressing double immunostaining from a total count of all conidia present. In some instances, under development of the precipitating stains may have under represented the proportion of conidia demonstrating double immunostaining. b Bold type identifies the germinated conidia double immunostained values, which are significantly higher than those double immunostained in the ungerminated conidia treatment (P<0.0001). c All A. alternata negative control treatments showed no immunostaining around the ungerminated conidia and germinated conidia and hyphae.

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Figure 5.3. Resultant double immunostaining of culture derived A. alternata conidia (A-C), A. alternata hyphal fragment (D) and germinated A. alternata conidia (E-F) using a pAb raised against a crude Alternaria extract (arrow a, red precipitate) and human serum IgE (arrow b, purple precipitate). Immunostaining was confined to (A) basal regions of the conidia (arrow c), (B) around the entire conidia (arrow c), (C) septal junctions of the conidia (arrow c), (D) around the entire fungal hyphal fragment (arrow d) and (E-F) around the hyphae (arrow e) and hyphal tips (arrow f) of germinated conidia (arrow c). (G) Negative controls using a pool of sera from atopic but Alternaria negative subjects (Pharmacia CAP score; specific IgE to A. alternata <0.35 kUA/L) and an in house rabbit pAb raised against a crude Lolium perenne pollen extract showed no localized immunostaining around the conidia (arrow c), hyphal fragments (arrow d) and hyphae (arrow e). The diffuse homogeneous background staining observed in the positive samples (A-F) was absent in the negative controls (G). Scale bar, 25 µm

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Chapter 6∗

6. Part A. Spore germination and allergen release

Sensitisation to fungi has been shown to associate with both asthma severity (Neukirch et al., 1999; Black et al., 2000) and mortality (Targonski et al., 1995). Although allergic sensitisation has been demonstrated to more than 80 species of fungi (Horner et al., 1995; Bush and Portnoy, 2001), only a few have been studied in any detail. This may partly be attributed to the difficulty of detecting allergen when few spores are present. Sporik and colleagues (1993) used an ELISA assay for the major allergen from A. fumigatus (Asp f I), to show the importance of incubating dust in a humid environment to be able to detect the presence of this allergen, suggesting that germination may be a lag factor in allergen release or production. Using techniques that enabled individual spores to be observed, we previously demonstrated that when Alternaria spores were germinated, the number of spores eluting allergen increased substantially and that allergen was released along the entire length of the growing hyphal tubes (Mitakakis et al., 2001a). While the genus Alternaria has been strongly associated with asthma (O'Hollaren et al., 1991; Downs et al., 2001), other types of fungi are emerging as clinically relevant (Karlsson-Borga et al., 1989). It is unknown what proportion of individual spores from different fungal genera other than Alternaria release allergen, nor how the process of germination affects the release of their allergens. In this Chapter, we aimed to characterize allergen elution from a range of fungi having different sized spores, that are known to be allergenic, in order to further understand the delivery of fungal allergens to the respiratory tract.

∗ Chapter 6, Part A was published as “Allergen detection from 11 fungal species before and after germination” in The Journal of Allergy and Clinical Immunology in 2003.

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6.1 Materials and Methods

6.1.1 Fungal isolates

The isolates studied were A. alternata (28008), A. fumigatus (28004), C. herbarum (28006), B. spicifera (11583) Botrytis cinerea (28005), C. lunata (14192), E. nigrum (28007), Exserohilum rostratum (28001), P. chrysogenum (28002), Stemphylium botryosum (27560) and Trichoderma viride (15719) (Queensland Department of Primary Industries, Brisbane, Australia). The isolates were sub-cultured from stock sources and grown for 10 days on vegetable juice nutrient agar at 24.9oC.

6.1.2 Cultured spore sampling

Spores were aerosolised from culture plates using an air jet and then collected by suction onto a polyvinylidene difluoride PBM (0.45µm pore size). If the spores were to be germinated, the membrane was pre-incubated with 30% sucrose and then dried prior to spore collection. After collection, the PBM was kept in a humid box for 48 hours at room temperature to allow germination. A plain membrane was used for spores that were not germinated. Both germinated and ungerminated spore samples were fixed to the membrane by overlaying it with a clear adhesive film (Woolcock Institute of Medical Research, Australia).

6.1.3 Immunostaining of cultured spore samples

Allergen was detected using the HIA using the same method and Alternaria sensitive serum IgE pool as described previously (Mitakakis et al., 2001a). Pooled serum (n = 10) from atopic subjects not sensitised to Alternaria was used as a negative control. In the HIA, previously laminated spores and germinated spores were briefly immersed in 80% methanol until wet, rinsed in deionized water and incubated in borate buffer (ph 8.2) for four hours to enable allergens to elute and bind to the PBM. Membranes were blocked in 5% SM in PBS for 45 minutes and then incubated overnight with positive IgE serum pool diluted 1:3 in 2% SM-PBS-0.05% Tween 20. After the primary antibody incubation, the PBMs were washed and incubated for 1.5 hours with biotinylated goat

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antihuman IgE (Kirkegaard & Perry Laboratories, Gaithersburg, Md) diluted 1:500, followed by incubation for 1.5 hours with ExtrAvidin alkaline phosphatase conjugate (Sigma Chemical Co, St Louis, Mo), diluted 1:1000 and developed with BCIP/NBT substrate (Pierce Chemical Co, Rockford, Ill). Positively immunostained spores have visible immunostaining of the allergen that has been bound to the membrane in close proximity to the spore or hyphae. Samples were examined at a magnification of 200X by light microscopy. The number of spores and germinated spores exhibiting haloes were expressed as percentages of total spore counts. A minimum of 100 spores was counted per sample. Three replicates were evaluated and the experiment was repeated.

6.1.4 Statistical analysis

The effect of germination was determined using single factor analysis of variance (ANOVA) by use of Analyse-It for Microsoft Excel (Analyse-It Software Ltd, Leeds, UK).

6.2 Results

The proportion of ungerminated spores that released allergen varied markedly among the species (Table 6.1). Of the 11 species, 9 released allergen, with proportions ranging between 5.7 - 92% of spores. Epicoccum, Stemphylium and Alternaria released allergen from more than 70% of spores, whereas Cladosporium, Exserohilum, Aspergillus, Bipolaris, Botrytis and Curvularia released allergen from 5 - 40% of spores. Penicillium and Trichoderma did not release detectable allergen. No allergen was detected from the ungerminated spores stained with negative control serum. The percentage of spores that germinated varied across the species, such that Alternaria, Exserohilum, Epicoccum, Stemphylium and Bipolaris recorded the greatest number of germinated spores (> 85%) whereas fewer spores of the remaining six species germinated (14.8 - 60%) (Table 6.1). Of the spores that germinated, all spores (100%) from all genera released allergen, even those of Penicillium and Trichoderma

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where no allergen release could be detected prior to germination (Table 6.1). No allergen was detected from the germinated spores stained with negative control serum. To identify whether the total number of spores eluting allergen increased after germination, we assumed that the subset of spores that eluted allergen before germination would also elute allergen after germination. Given that all germinated spores eluted allergen, the percentages of spores eluting allergen prior to germination were compared to the percentages of spores that germinated. A significant increase was observed for all species except Epicoccum, Cladosporium and Aspergillus (Table 6.1). Alternaria, Exserohilum, Bipolaris, Botrytis and Curvularia recorded substantial increases in the percentage of spores eluting allergen (25.6 - 83.5%). The fungi showed differing patterns of allergen elution from their hyphae (Figure 6.1-6.8). The pattern of elution for Alternaria was similar to our earlier observations (Mitakakis et al., 2001a). Allergen elution from Exserohilum (Figure 6.1), Bipolaris (Figure 6.2) and Stemphylium (data not shown) was primarily localised to hyphal tips, extension zones and septal junctions. All other genera eluted allergen from the entire length of their hyphae (Figure 6.3-6.8). Of the three fungi observed to produce allergen in a localized pattern around the hyphal tips, extension zones and septal junctions, it can be seen that if the hyphal tube had not grown far, the intensity of allergen deposition was similar to the other fungi that elute allergen from the entire hyphal length. For example, Exserohilum compared to Epicoccum (Figure 6.1 and 6.4).

6.3 Discussion

Germination of Aspergillus and Alternaria has been shown in earlier studies to increase the amount of detectable allergen (Sporik et al., 1993; Mitakakis et al., 2001a). In this Chapter, these findings were confirmed and extended to demonstrate that several other common fungal species also release more allergen upon germination. Indeed, allergen release occurred from all germinated spores. By visual examination of the germinated spores, the sites of allergen release were observed to vary in localisation along the hyphae.

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On the basis of observed amounts of immunostaining, the total amount of allergen post-germination was greater than before germination and was contributed mainly by the germinated spores, although with several species, many of the ungerminated spores also released allergen, but only small amounts compared to after germination. Viability is not a condition for allergen release as we have previously shown that even autoclaved spores can release some allergen (Mitakakis et al., 2003). For some of the species the percentage releasing allergen may have been underestimated by the technique used. Fungi with small, clear unicellular spores were difficult to resolve through the adhesive film used in the HIA and this may have confounded the counting of both the spores and their haloes. In particular this applied to individual, ungerminated fungal spores < 5µm in diameter, particularly those of Penicillium, Aspergillus and Trichoderma. We cannot exclude the possibility that unstained spores released small amounts of allergen below the detectable thresholds. Indeed, no allergen was detected from Penicillium and Trichoderma prior to germination, but was readily detected post-germination. These experiments provide only a limited model of the behaviour of wild-type spores in contact with the human respiratory mucosa. These spores were germinated using only sucrose as the nutrient and at one temperature, whereas in culture the success of germination is known to vary according to the nutrient media and temperature. The sucrose solution media used in this case was selected due to its similarities with Sabouraud’s agar, although peptone was omitted to avoid blocking the protein binding sites on the PBM. Sabouraud’s agar has been found to give a greater recovery of Aspergillus species (Noble and Clayton, 1963; Noble, 1967; Einarsson and Aukrust, 1992) as well as yield allergenically more potent extracts of C. lunata and E. nigrum (Gupta et al., 1999; Bisht et al., 2000) than either the common Czapek-Dox agar or Malt agars. Selective species also have varying temperature requirements that enable germination. According to Noble (1963; 1967), thermotolerant species including A fumigatus and C. albicans can be selected by incubating the spores at 37oC, however temperatures between 24 - 25oC were chosen by us for culture as they are considered optimal for culturing most species of Anamorphic fungi. To our knowledge there has

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been no experiments comparing human mucous as a culture medium to common synthetic media. It is unknown whether the allergens detected from the ungerminated spores are the same as those released from germinated spores. In our earlier study, we compared detection of Alternaria allergens recognised by the pooled IgE and two monoclonal antibodies directed against the major allergen, Alt a 1 (Mitakakis et al., 2001a). Germination significantly increased the proportion that released allergen and suggested Alt a 1 was a minor component of total allergen released prior to germination. Studies using SDS-PAGE and protein blotting of several spore and hyphal extracts, show they contain differing proportions and numbers of individual allergens (Hoffman et al., 1981; Aukrust et al., 1985; Paris et al., 1990a; Einarsson and Aukrust, 1992). This could be further tested in a HIA with monoclonal antibodies or allergen-specific IgE prepared from blots (Tovey et al., 1989). The determinants of release of different allergens during the early phase of germination presumably relate to processes of host colonisation. So far the majority of allergenic proteins characterized are considered household cytoplasmic proteins (Achatz et al., 1996) that are required for amino acid translation, protein folding and basic metabolism (Arruda et al., 1990; Zhang et al., 1995; De Vouge et al., 1996; Shen et al., 2001). A few of the many proteins associated with germination have been identified and have had their functions identified or proposed. A recent summary of 25 functions for 34 allergens from A fumigatus, C. herbarum and A alternata can be found in the literature (Breitenbach and Simon-Nobbe, 2002; Crameri, 2002). The different patterns of allergen release from fungi, involving tips, septal junctions or along the entire length of the hyphae may affect subsequent mucosal responses. If fungi elute allergen along the length of their hyphae as opposed to from spores only or intermittently at septal junctions, the potential surface area of the respiratory mucosa that allergens contact and potentially irritate may be larger. Allergen release from fungal hyphal tips has also been observed using transmission electron microscopy and gold labelling with A fumigatus (Reijula et al., 1991) and along the entire length of the hyphal tube in A alternata (Mitakakis et al., 2001a).

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The clinical relevance of allergens associated with fungal germination remains to be resolved. Ungerminated spores may enter the lung or nasal cavity, in which they are deposited in the favourable environment of warmth and moisture and have been speculated to subsequently germinate and act as an additional source of allergen (Sporik et al., 1993). While the ciliary transport and immune mechanisms may largely prevent such invasion in normal subjects, several dermataceous fungi, including Bipolaris, Curvularia, Penicillium, Stemphylium, Alternaria, Aspergillus and Exserohilum species are capable of invasive forms of fungal disease, particularly allergic bronchopulmonary disease and fungal sinusitis (Lake et al., 1991; Cody et al., 1997; Yang et al., 2000) in immunocompromised people. Not all regions of the nasal cavity are cleared efficiently and spores deposited in less well cleared areas may initiate germination prior to removal and while not causing infection, they may release allergen during this process. A number of pathogenic mechanisms have been described for the mould A fumigatus that may inhibit its clearance from the respiratory tract. These include the production of conidial inhibitory factors (Tomee and Kauffman, 2000), low molecular weight toxic metabolites that inhibit phagocytosis and macrophage adherence (Arruda et al., 1992; Amitani et al., 1995; Tomee and Kauffman, 2000) and factors that enhance spore binding in epithelial cells (Yang et al., 2000). A fumigatus may also have receptor binding sites for epithelial components (Tronchin et al., 1997). Of additional interest are the recent descriptions of TLRs for fungal hyphae on human monocytes (Wang et al., 2001) and mast cells (Supajatura et al., 2002), indicating innate immune mechanisms as well as those associated with humoral immunity.

6.4 Conclusions

In conclusion, we have used a new technique to investigate the release of allergen from individual fungal spores before and after germination. We characterized allergen elution from a range of fungi with different sized spores and found that not all ungerminated spores released allergen, whereas all spores that germinated released allergen from their hyphae. This suggests that a critical event determining exposure to allergen may be the

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opportunity for spores to germinate following inhalation. To date there has been no systematic attempt to identify spores able to germinate in the respiratory tract or to characterize the spore and mycelial allergens of a number of these species. Further examination of these important species is required to understand the delivery of fungal allergens to the respiratory tract.

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Table 6.1: Percentage of ungerminated spores and germinated spores with hyphae (± SE) eluting allergen, detected by the Halogen immunoassay. * P<0.05, ** P<0.0001 calculated by ANOVA between percentage of spores with haloes before germination against percentage of spores that germinated.

% Ungerminated % Spores Fungal species spores with haloes germinated

Alternaria alternata 67.7 ± 2.3 96.4 ± 0.7** Aspergillus fumigatus 11.6 ± 2.7 27.2 ± 7.6** Bipolaris spicifera 9.7 ± 1.7 93.2 ± 2.8** Botrytis cinerea 5.7 ± 1.5 33.9 ± 6.8** Cladosporium herbarum 40.8 ± 7.4 43.9 ± 5.0** Curvularia lunata 23.8 ± 0.8 49.4 ± 9.1** Epicoccum nigrum 92.0 ± 0.6 89.4 ± 1.9** Exserohilum rostratum 13.3 ± 2.4 89.9 ± 2.7** Penicillium chrysogenum 0 ± 0 60.0 ± 3.8** Stemphylium botryosum 87.3 ± 2.4 97.8 ± 0.1** Trichoderma viride 0 ± 0 14.8 ± 2.1**

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Figure 6.1. Individual (A) ungerminated and (B) germinated Exserohilum rostratum spores immunostained by the Halogen immunoassay with human serum IgE. (Scale bar = 50 µm.)

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Figure 6.2. Individual (A) ungerminated and (B) germinated Bipolaris spicifera spores immunostained by the Halogen immunoassay with human serum IgE. (Scale bar = 50µm.)

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Figure 6.3. Individual (A) ungerminated and (B) germinated Curvularia lunata spores immunostained by the Halogen immunoassay with human serum IgE. (Scale bar = 50µm.)

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Figure 6.4. Individual (A) ungerminated and (B) germinated Epicoccum nigrum spores immunostained by the Halogen immunoassay with human serum IgE. (Scale bar = 50µm.)

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Figure 6.5. Individual (A) ungerminated and (B) germinated Trichoderma viride spores immunostained by the Halogen immunoassay with human serum IgE. (Scale bar = 50 µm.)

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Figure 6.6. Individual (A) ungerminated and (B) germinated Botrytis cinerea spores immunostained by the Halogen immunoassay with human serum IgE. (Scale bar = 20 µm.)

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Figure 6.7. Individual (A) ungerminated and (B) germinated Aspergillus fumigatus spores immunostained by the Halogen immunoassay with human serum IgE. (Scale bar = 50 µm.)

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Figure 6.8. Individual (A) ungerminated and (B) germinated Cladosporium herbarum spores immunostained by the Halogen immunoassay with human serum IgE. (Scale bar = 50 µm.)

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6. Part B. The relationship between inhaled fungi and germination

Airborne fungal conidia, hyphae and associated fragments are abundant in most indoor and outdoor environments (Burge et al., 2000). Concentrations of these vary widely, depending on the location, season, time of day, and prevailing weather conditions (Chew et al., 2003). The inhalation of fungi has been recognized to exacerbate allergic rhinitis (Andersson et al., 2003) and asthma (Downs et al., 2001), however the importance of exposure to fungal conidia and other fungal components in the progression of these diseases remains unclear. Fungi can be cultured from the nasal airways of most people, including those with no respiratory disease, as well as those with chronic rhinosinusitis (Ponikau et al., 1999). Inhaled fungi are mostly deposited in the nasal cavity, due to the efficiency of the nose in removing particles greater than 10µm from the inhaled air (Knutsen et al., 2002). Although the presence of fungi in the airways does not necessarily indicate a pathological state, it may lead to symptoms of allergic or invasive disease in susceptible individuals (Braun et al., 2003). The hypothesis that colonization by fungi plays a major role in the development of chronic fungal sinusitis has recently drawn attention to their potential role in the pathology of this common condition (Braun et al., 2003). Experiments have shown that the release of allergens from fungal conidia significantly increases as a consequence of germination (Sporik et al., 1993; Mitakakis et al., 2001a). In contrast to other allergens, which are carried on non-viable particles, fungal allergens are synthesized and actively secreted at the sites of conidial deposition in the respiratory tract. Furthermore, germinating conidia may also release proteolytic enzymes that induce epithelial damage (Robinson et al., 1990), inflammation (Reed and Kita, 2004) and lead to enhanced antigen presentation to the immune system (Kauffman, 2003). Hence, the germination state of fungal conidia either before or shortly after deposition on the nasal mucosa may be important in determining their pathogenic potential.

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Although conidia have been recovered from the nasal cavity (Karpovich-Tate et al., 2000) and hyphae and conidia isolated from mucin originating from inflamed nasal sinuses (Ponikau et al., 1999), no studies to date, have investigated the state of germination of inhaled fungal conidia following normal environmental exposure. Using an anterior nasal lavage technique (Greiff et al., 1990), we demonstrated the relative numbers of ungerminated and germinated conidia of a range of fungal genera recovered from the human nasal cavity following exposure to indoor and outdoor environments.

6.5 Materials and methods

6.5.1 Subjects

The study population consisted of 20 adults (13 male) including 11 atopic and 9 nonatopic subjects, ranging in age from 20 to 53 years, with a mean of 30.5 years. No subject reported any recognized nasal disease. The University of Sydney Human Research Ethics Committee approved the experimental protocol, and all participants gave their informed consent in writing.

6.5.2 Environmental exposure

The anterior nasal lavage procedure was performed at 3 consecutive intervals: (1) at the start of the experiment to quantify and remove the bulk of fungi inhaled prior to controlled exposure, (2) after 1 hour in an indoor environment, and (3) after 1 hour outdoors adjacent to a grassed sports field. No delays were allowed between exposures and lavages. IOM personal aerosol samplers (SKC Inc., Eighty Four, PA.), worn by the subjects, were run concurrently during the indoor and outdoor exposures to monitor the subjects’ exposure to airborne fungi. The study was performed on consecutive days, around midday, during Spring (August) in Sydney, Australia. The study coincided with mean daily maximum and overnight minimum temperatures of 21ºC and 6ºC, respectively (Bureau of Meteorology, NSW, Australia). No testing was performed within 2 days of any rainfall.

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6.5.3 Nasal lavage procedure

The nasal pool device (Figure 6.9) (Greiff et al., 1990) was used to instil 10 ml of warmed (approx 32ºC) sterile normal saline into one nostril at a time and was administered by the subject themselves with the assistance of the technician. The liquid was held in each nostril for 1 minute, with the head tilted 30 degrees forward and then re-drawn into the nasal pool device. This was immediately repeated in the same nostril using the same liquid for another 1 minute. The liquid was then expelled from the nasal pool device into a sterile tube. The other nostril was lavaged in an identical manner, using a separate 10 ml of sterile saline. Nasal lavages from both left and right nostrils were combined and the volume of liquid recovered was recorded. In this procedure, on average, 14.4 ml of the 20 ml instilled into the nose was recovered. The nasal lavage liquid was immediately divided into 2 equal portions, A and B. In preliminary experiments, where 5 identical nasal lavage procedures were performed consecutively, the first lavage was found to remove an average of 93% of the total culturable fungi recovered (data not shown).

6.5.4 PAS staining

Portion A was treated immediately with Kathon CG (Rohm and Haas Ltd., UK) (0.5% v/v) to prevent the germination of conidia subsequent to their recovery from the nasal cavity. Trial experiments confirmed that Kathon CG treatment immediately prevented conidial germination (data not shown). These samples were then treated with Sputolysin (Calbiochem, CA) sufficient to achieve a final concentration of 0.528 mg/ml Dithiothreitol and incubated for 30 minutes at room temperature in order to dissolve nasal mucous. Aliquots were impacted by Cytospin centrifugation onto silanased microscope slides, then fixed in 100% ethanol and air-dried. Periodic Acid Schiff (PAS) staining was performed as described previously (McManus, 1946) and preparations examined by light microscopy at 200x magnification. PAS stains fungi, mucous and glycogen crimson, allowing the enumeration and identification of conidia, hyphae and fragments.

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6.5.5 Nasal lavage culture

Portion B was treated with Sputolysin, as described above, in order to dissolve nasal mucous. An aliquot of 7 ml was then concentrated by centrifugation at 1250g for 10 minutes, resuspended in sterile deionized water and plated onto 20% vegetable juice (V8) and Rose-Bengal Chloramphenicol (RB) (Oxoid Ltd., UK) nutrient media for culture-based identification of viable fungi. Our preliminary experiments indicated that treatment with Sputolysin did not affect the viability of fungal conidia (data not shown), which confirms previous findings (Ponikau et al., 1999). Culture plates were incubated at 25ºC and inspected daily for 20 days until sufficient sporulation had occurred. Fungal colonies were taxonomically identified to genus level by macroscopic and microscopic morphological features, by 2 observers.

6.5.6 IOM personal aerosol sampler counts

Fungal conidia were collected concurrently during each of the indoor and outdoor environmental exposures by IOM personal aerosol samplers, which were fitted with 1.0µm polyvinylidene difluoride PBMs (Millipore, Bedford, MA). The collected fungal propagules were identified morphologically and counted at a magnification of 200x using light microscopy.

6.5.7 Statistical analysis

Associations between the numbers of fungi quantified by each method of analysis were expressed as Spearman correlation coefficients. Differences between the numbers of viable fungi isolated on culture plates following indoor and outdoor exposure and differences in the numbers of fungal conidia collected by IOM personal air samplers in each exposure setting were tested using a paired t-test on log normalized values. Statistical calculations were performed using Analyse-it for Microsoft Excel (Analyse-it Software Ltd., United Kingdom). The criterion for significance for all analyses was P<0.05.

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6.6 Results

PAS staining of Cytospin preparations of nasal lavage fluid allowed the identification and quantification of fungal propagules in 95% of the 60 samples processed. Figure 6.10 shows germinated and ungerminated fungal conidia, in addition to hyphal fragments recovered from nasal lavages and stained using the PAS technique. Both germinated and ungerminated Aspergillus and Penicillium conidia (Figure 6.10) were by far the most frequent genera observed in the PAS stained samples (Table 6.2). However, it was necessary to analyse Aspergillus and Penicillium conidia as a combined group in the PAS results, since it was not possible to discriminate between these conidia morphologically. Other species, such as Curvularia (Figure 6.10) were also identified, along with isolated hyphal fragments, discernible by their septal junctions (Figure 6.10). Table 6.2 shows the median, maximum and minimum numbers of conidia in the nasal lavage samples from each of the 20 subjects. The most numerous fungal conidia that were observed in a state of germination belonged to the Aspergillus/Penicillium group. Due to the comparatively low numbers of other species that were found germinated, a meaningful analysis of the percentage of conidia germinated was only possible for the Aspergillus/Penicillium group. The mean percentages of germinated Aspergillus/Penicillium conidia were 32% for the start condition, 36% after 1 hour indoors and 33% after 1 hour outdoors. There were no significant differences (P>0.05) between the percentage germination of Aspergillus/Penicillium observed following each exposure condition. The percentage germination of Aspergillus/Penicillium conidia observed in each individual subject, by environmental exposure condition is shown in Table 6.3. Viable fungi were recovered from the nasal lavage fluids of 95% of subjects participating in the study (Table 6.2). The most common fungi cultured from the nasal lavages were Alternaria, Aspergillus, Cladosporium, Epicoccum, Penicillium and yeast species. The diversity of fungal genera and the total numbers of fungi recovered varied between subjects and exposure conditions. The total number of all viable filamentous

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fungi recovered after 1 hour of outdoor exposure was significantly greater than after 1 hour of indoor exposure on both V8 (P=0.0024) and RB (P =0.0019) nutrient media. The number of all filamentous fungi combined, isolated on V8 media, correlated significantly to that isolated on RB nutrient media (rs=0.96, P <0.001). The total number of fungal conidia collected by the IOM samplers was highly variable between individuals and there was a significant difference between the IOM counts obtained from the two environmental exposure settings (P <0.0001). In addition, there was a significant association between the numbers of fungi collected on the IOM personal air sampler and the culture colony counts on V8 (rs=0.73, P <0.001) and on RB

(rs=0.58, P <0.001) nutrient media. Speciation of the conidia captured on the IOM samplers and the determination of their germination state, was not possible due to the opaque nature of the filter membrane.

6.7 Discussion

Fungal propagules, including germinated conidia, ungerminated conidia and hyphal fragments were recovered from the nasal cavity of 95% of subjects following various environmental exposures in typical everyday settings. For the Aspergillus and Penicillium group, over 30% of conidia recovered from nasal lavage samples were in a state of germination. Numbers of viable conidia estimated by culture on both V8 and RB nutrient media correlated significantly (P<0.001) to the corresponding IOM personal air sampler measurements of each subject’s environmental fungal exposure. The results of this Chapter demonstrate for the first time that fungal conidia inhaled, as part of ordinary environmental exposure, can exist in the nasal cavity in a germinated state. These findings implicate the germination of conidia as another major event leading to natural exposure to fungal allergens. We found that the numbers of fungi recovered from the nasal lavages was heterogeneous between subjects. This is likely to reflect their various exposures during the study period, as demonstrated by the significant correlations between nasal lavage culture counts and the IOM personal air sampler counts collected concurrently. Other

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sources of variation include the subject’s individual breathing patterns, nasal anatomy, and nasal lavage technique. Overall, the numbers of many fungal genera recovered were low in a number of samples, which is consistent with previous studies (Braun et al., 2003) that describe the difficulties encountered in recovering fungi from nasal mucous. Our preliminary experiments also demonstrated that the nasal lavage procedure recovered a mean of 93% of recoverable fungi in the first lavage alone. Thus, it is not known whether there is a subset of fungi that remain localised in the nasal cavity and have a greater level of adhesion to the mucosa than those that can be collected by lavage. Such fungi, due to their virulent adhesive properties and difficulties of removal by normal mucocillary clearance would most likely provide an even greater allergen challenge than transient and less virulent fungal species. The results of the study show that in a number of samples, the conidia of up to 5 of the 7 genera identified in the PAS stained specimens were in a state of germination. Conidial germination has been linked to a substantial increase in the expression of allergen following in vitro experiments using monoclonal antibodies (Sporik et al., 1993; Mitakakis et al., 2001a) and fungal-specific human IgE from individuals sensitised to fungi (Green et al., 2003). Conidial germination is also associated with protein synthesis, protein release and enzymatic activity at the hyphal tips and sites of germination. Previous studies have shown that upon germination of Alternaria conidia, an Alt a 1 homologue was differentially expressed during hyphal growth (Cramer and Lawrence, 2003), while it was also demonstrated that a greater amount of Alt a 1 could be detected by immunostaining with Alt a 1 specific monoclonal antibodies following conidial germination (Mitakakis et al., 2001a). Although conidial germination is a natural part of the life cycle of the fungus, it has also been shown to lead to the expression of specific pathogenic enzymes that have been shown to decrease the clearance of conidia from the respiratory mucosa (Robinson et al., 1990), promote adhesion to the respiratory epithelium (Yang et al., 2000) and inhibit macrophage function (Kauffman et al., 2000; Yang et al., 2000). When these processes occur in contact with the nasal epithelium of a sensitised individual, an IgE mediated allergic reaction is likely. In the present study, we have shown that substantial numbers of

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germinated fungal conidia exist in contact with the nasal epithelium following normal respiration in indoor and outdoor settings. The requirements for conidial germination include the availability of nutrients as well as specific temperature and humidity profiles. The site of deposition of fungal propagules in the nasal cavity is dependant upon their aerodynamic characteristics as part of the inhaled air stream (Proctor et al., 1973). Within the nasal cavity there are various temperature zones, which relate to regions of nasal anatomy (Keck et al., 2000) and might function as a selective parameter in the determination of conidial germination, especially for thermotolerant species (Lake et al., 1991). Although it is not known whether the recovered conidia in the present study were in a state of germination prior to inhalation, we could speculate that following inhalation, the temporal interval between exposures might be sufficient to allow in vivo conidial germination for a number of fungal genera (data not shown). The relationship between the fungi in the nasal sinuses and those in the nasal cavity in both healthy subjects and subjects with nasal disease is unclear, but it is likely that the content of the sinuses is normally more stable than that of the nasal cavity. It is known that the sinus cavities are not sterile, even in disease-free states (Su et al., 1983) and that infants within 4 months of birth display similar concentrations of fungi in nasal mucous samples as adults do (Lackner et al., 2004). The interaction of viable fungi with the mucosa of the nasal sinuses has been shown to induce specific inflammatory responses that have been proposed to be the root cause of the hyper-immune disorder, chronic fungal sinusitis (CFS) (Schubert et al., 2004). Clinical interventions that reduce the amount of viable fungi in the nasal cavities of CFS patients, such as daily lavage with anti-fungal drugs, have been tested on the premise that these treatments will also have some effect on the sinuses. Such treatments have been identified as being effective in some studies (Ponikau et al., 2002; Dennis, 2003) and not effective in others (Weschta et al., 2004). Our results confirm that viable fungi are common in the nasal cavity, and suggest that any anti-fungal treatment applied nasally would have to cope with the constant influx of newly inhaled fungi.

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Our findings demonstrate that not only are fungal conidia present in the nasal cavities of normal individuals, reflecting recent environmental exposure, but also that these conidia are in a state of germination. These findings are in agreement with Braun et al. (2003) who identified viable fungi in the nasal mucous of 91.3% of normal controls. These authors suggested that positive nasal fungal cultures should be considered a normal finding, but that chronic rhinosinusitis (a more broad definition of CFS) can be identified by fungal elements and clumps of eosinophils present in the mucous. In this Chapter we used two types of nutrient agar to allow the recovery of viable fungi from the nasal lavage samples. Our choice of V8 and Rose Bengal Chloramphenicol nutrient media was based on the findings of previous studies, which reported that at least two types of media are necessary to recover the full spectrum of environmental fungi, whilst protecting against overgrowth, which could cause errors in counting. The choice of one type of non-selective and selective nutrient media, is often recommended for environmental fungal air sampling (Burge et al., 1977). Our observation in the current study that the numbers of fungal colonies isolated using each type of media correlate not only to each other but to the numbers of conidia collected using the IOM personal air samplers (a non-viable method) indicates that no sampling bias was introduced by the culture methods. We used the PAS staining technique on fixed Cytospin preparations of nasal lavage liquid. Limitations of this technique include the often-difficult identification of unicellular conidia, hyphae and fragments in samples containing small numbers of fungal propagules. In this study, it was not possible to differentiate between Aspergillus and Penicillium conidia, however culture based methods reported more Penicillium colonies than that of any other unicellular spore bearing species. Furthermore, the clinical significance of differentiating between Penicillium and Aspergillus exposure in regard to their effect as aeroallergen sources remains unknown, and it is the fact that unicellular conidia of these fungal varieties were often seen to be germinating that is of interest.

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We have demonstrated that fungal conidia in the state of germination are commonly present in the nasal cavity of normal humans. Future studies investigating the in vivo consequences of the inhalation of viable fungal propagules will be required to confirm the existence of an increased allergenic effect due to germinating conidia.

6.8 Conclusions

We have demonstrated that germinated conidia, ungerminated conidia and hyphal fragments are commonly present in the human nasal cavity following natural environmental exposure. The numbers of these fungi present also relate to the nature of exposure. This has profound implications for the study of allergic rhinitis, asthma and fungal sinusitis.

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Table 6.2: Numbers of fungal colonies recovered by culture and the numbers of germinated and ungerminated fungal conidia recovered by PAS staining from nasal lavage samples.

Recovery Methoda Fungal species V8 media culture RB media culture PAS ungerminated conidia PAS germinated conidia Start Indoor Outdoor Start Indoor Outdoor Start Indoor Outdoor Start Indoor Outdoor Aspergillus 0 (0-9) 0 (0-9) 0 (0-43) 0 (0-41) 0 (0-8) 0 (0-39) ------Penicillium 0 (0-94) 0 (0-15) 0 (0-9) 0 (0-69) 0 (0-9) 0 (0-8) ------Asp-Pen combined ------543 (0-5976) 208 (0-3981) 157 (11-1382) 172 (0-24336) 44 (0-8240) 61 (0-3224) Alternaria 0 (0-26) 0 (0-15) 0 (0-18) 0 (0-17) 0 (0-0) 0 (0-15) 0 (0-10) 0 (0-0) 0 (0-25) 0 (0-0) 0 (0-0) 0 (0-25) Bipolaris 0 (0-0) 0 (0-0) 0 (0-9) 0 (0-41) 0 (0-17) 0 (0-35) 0 (0-0) 0 (0-0) 0 (0-620) 0 (0-13) 0 (0-0) 0 (0-12) Cladosporium 0 (0-174) 0 (0-9) 8 (0-4003) 0 (0-273) 0 (0-8) 9 (0-4606) 0 (0-157) 0 (0-24) 0 (0-1538) 0 (0-0) 0 (0-0) 0 (0-471) Curvularia 0 (0-8) 0 (0-0) 0 (0-97) 0 (0-0) 0 (0-0) 0 (0-71) 0 (0-36) 0 (0-0) 0 (0-12) 0 (0-0) 0 (0-0) 0 (0-0) Epicoccum 0 (0-9) 0 (0-17) 0 (0-673) 0 (0-9) 0 (0-9) 0 (0-416) 0 (0-0) 0 (0-0) 0 (0-13) 0 (0-0) 0 (0-0) 0 (0-12) Exserohilum 0 (0-0) 0 (0-0) 0 (0-0) 0 (0-0) 0 (0-0) 0 (0-0) 0 (0-0) 0 (0-0) 0 (0-25) 0 (0-0) 0 (0-0) 0 (0-0) Mucor 0 (0-0) 0 (0-0) 0 (0-9) 0 (0-0) 0 (0-0) 0 (0-0) 0 (0-0) 0 (0-0) 0 (0-0) 0 (0-0) 0 (0-0) 0 (0-0) Mycelia Sterilia 0 (0-33) 0 (0-0) 0 (0-248) 0 (0-41) 0 (0-17) 0 (0-35) ------Hyphae ------0 (0-0) 0 (0-0) 0 (0-51) Pollen ------0 (0-0) 0 (0-26) 0 (0-12) - - -

aAll counts given as Median (Min-Max) for n=20 subjects.

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Table 6.3: Percentage of germinated Aspergillus and Penicillium conidia recovered from nasal lavage samples, grouped by experimental subject and environmental exposure.

Percentage of germinated Aspergillus/Penicillium conidia, for each subject (%).

Subject number Start Indoor Outdoor 1 81.4 71.2 75.6 2 0.0 16.7 0.0 3 27.8 37.5 47.1 4 22.2 28.9 23.5 5 31.3 0.0 22.2 6 27.3 0.0 25.9 7 69.9 66.4 58.0 8 34.5 0.0 40.0 9 31.9 31.3 34.4 10 13.1 28.2 16.2 11 31.2 44.1 58.6 12 63.9 74.3 73.6 13 10.0 6.7 0.0 14 7.5 2.9 0.0 15 71.4 100.0 50.0 16 0.0 0.0 0.0 17 26.3 18.2 23.1 18 92.3 88.2 52.0 19 0.0 5.9 66.7 20 0.0 100.0 0.0

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Figure 6.9: The nasal pool device used for nasal lavage.

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Figure 6.10: Photomicrographs of ungerminated and germinated fungal conidia and hyphae recovered from nasal lavage samples and stained with PAS. A. Ungerminated Alternaria conidia (arrow a).B. Germinated Alternaria conidia (arrow a) and hyphae (arrow b). C. Ungerminated Cladosporium conidia (arrow a). D. Germinated Cladosporium conidia (arrow a) and hyphae (arrow b). E. Ungerminated Epicoccum conidia (arrow a). F. Germinated Epicoccum conidia (arrow a) and hyphae (arrow b). G. Ungerminated (arrow a) and germinated (arrow b) Aspergillus-Penicillium conidia. H. Ungerminated Curvularia conidia (arrow a). I, J. Fungal hyphae (arrow c) with septal junctions (arrow d). Scale Bar = 30 µm.

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Chapter 7

7. General discussion and conclusions

The main aim of this thesis was to investigate both the sources of personal exposure to fungal allergens in indoor and outdoor environments and the factors influencing their release, utilizing a novel immunodiagnostic technique termed the HIA. The current understanding of personal exposure and sensitisation to fungal allergens is restricted to the inhalation of conidia belonging to a small and select number of species. The contribution of other genera and smaller fungal propagules is poorly understood. This relates to the inadequacies of current immunodiagnostic techniques and methods of analysis, in addition to the heterogeneity, diversity and abundance of airborne fungi in the environment. It was hypothesised that three major factors appear to determine personal exposure to airborne fungal allergens and the immunodiagnosis of allergic disease; (1) the contribution of airborne hyphae and other previously unrecognised genera to airborne counts and as allergen sources, (2) the expression and release of allergens from germinating fungal spores and (3) the methods available for immunodiagnosis. Methods to diagnose fungal allergic disease are confounded by a number of variables that relate to the biology of the fungus, in addition to the method of analysis (Malling, 1992; Esch, 2004). The most popular and traditional techniques that are used by clinicians to diagnose fungal allergy are in vivo SPT, in vitro CAP assays and in rare cases by bronchial provocation (Licorish et al., 1985), all of which require fungal extracts of the particular fungus in question (Esch, 2004). A separate examination of an individual’s environment, where exposure takes place can also be tested and viable fungi cultured, quantified by molecular probes or subjectively identified to determine the most prevalent airborne fungal sources (Rogers, 2003).

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For pragmatic reasons, fungal extracts are restricted to a handful of species to represent a much broader spectrum of fungi and these few have been shown to vary between the methods of extraction, extraction time and between manufactures, batches and strains (Malling, 1992; Esch, 2004). The low yield of allergens extracted from fungi for use in commercial extracts and the lack of concordance between immunodiagnostic tests have also been hypothesised to account for the uncertainties of appropriate cut-off values for a positive response (Nordvall et al., 1990). All of these factors can account for the slower rate of standardization compared to other common aeroallergen sources and might contribute to the under-diagnosis of the prevalence of fungal allergic disease within the community. The technological advances in molecular biological techniques over the last decade have enabled the development of other methods and molecular probes to quantify airborne fungal concentrations and to diagnose fungal allergic disease. The most widely used of these techniques include enzyme linked immunoabsorbant assays, polymerase chain reaction and immunoblotting, however each of these methods are restricted by the availability of fungal extracts and species-specific molecular probes. The development of direct immunohistochemistry enabled for the first time, the immunostaining of collected wild-type fungal spores to diagnose fungal allergic disease from the environment to which exposure occurred (Popp et al., 1988). Although this technique provided novel insight into personal exposure to fungal allergens in occupational settings, it was confounded by the solubilization of allergens from the outer walls of the fungal spore, which significantly reduced the detectable thresholds of the assay. Thus, understanding the clinical significance of exposure to airborne fungi has been limited by problems associated with present methodologies and the lack of standardization of fungal allergen extracts, which has made it difficult to reproduce research based on these techniques. To date, it has been a technically formidable task to concurrently collect airborne fungal particles representing personal exposure and demonstrate patient- specific allergy to the same particles. The development of the HIA has enabled for the first time, the simultaneous visualization of individual airborne particles and the

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immunodiagnosis of allergic disease (Tovey et al., 2000). The HIA has been tested successfully against a range of common aeroallergen sources in personal exposure studies (O’Meara et al., 1998; De Lucca et al., 1999a; De Lucca et al., 1999b; Poulos et al., 1999; Poulos et al., 2002) and has recently been adapted to detect allergens expressed by the fungus Alternaria (Mitakakis et al., 2001a). However, the ability of the technique as a test for reactivity to the allergens expressed by other fungi and its performance as an immunodiagnostic technique compared to conventional tests was not known. The application of the HIA to other culturally derived fungal sources including A. fumigatus, C. herbarum and E. purpurascens that vary in size, shape and allergen content was confirmed in Chapter 2. The detection of expressed allergen with subjects human serum IgE of varying IgE titres was shown in the HIA and was found to strongly correlate with the in vitro CAP assay. Weaker associations were identified between HIA and SPT, which confirm previous studies. These variations can be predicated by the variability of fungal extracts, individual SPT responses and between examiner variations of the HIA. Compared to in vitro CAP assays, where the specific IgE titre is able to be determined, the HIA was restricted to a semi-quantitative and subjective method of analysis. Although these methodological considerations did not hinder the performance of the HIA in the identification of fungal positive serum samples, further methods development would enable the measurement of specific IgE. This may provide a very useful and objective immunodiagnostic technique. To date, the development of more objective quantification using a fluorescent anti-IgE label has not been achieved by our group. However, such improvements may not be required, as visual scoring alone in the HIA may be adequate, as it is in the case of quantifying SPT results. The significance of the HIA derives from its unique ability to provide allergen sources that are actively secreted by the collected fungal spore and hyphae. The assay uses undegraded allergens from fungal particles that are the natural agent of exposure and has the capacity to combine environmental with serological monitoring on a patient specific basis. Thus, the HIA may become an important immunodiagnostic technique in

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the future, which will ultimately contribute to better patient management and characterisation of fungal allergic diseases. Personal exposure to airborne allergenic fungi was previously assumed to be mainly restricted to the spores of a handful of genera, including Alternaria, Aspergillus, Cladosporium and Penicillium species. Using the HIA, it was hypothesised in Chapter 3 that personal exposure to airborne fungi in indoor environments was not only to previously characterised allergenic genera but also to a much wider spectrum of fungal sources. This hypothesis was based on previous studies that had shown that aerosolised fungal fragments were significantly higher in concentration than conidia counts (Gorny et al., 2002; Gorny, 2004). Fungal fragments, largely ignored in many epidemiologic studies, have also been shown to associate with exacerbations of asthma when included with conidia counts (Li and Kendrick, 1995a). Our hypothesis was largely supported and it is now clear that aerosolised fungal hyphae release allergens and such particles are significantly higher in airborne concentration than the spores of many well- characterized allergenic genera. It can also be expected that smaller fragmented hyphae, based on their size range (<3µm), are more likely to enter the lung than conidia and as conidia and hyphae often occur independently, the hyphae may be an unrecognised source of asthma exacerbations. Furthermore, a number of conidia that were not previously known to be allergenic were demonstrated by the HIA for the first time to express detectable allergen and accounted for approximately 8% of the total fungal count. These findings suggest that natural fungal exposure is not only restricted to prevalent airborne fungal genera, but implicates a much wider spectrum of fungal sources, in particular airborne hyphae to contribute to personal exposure. The results of Chapter 3 would also be expected to vary with the geographical location and between indoor and outdoor environments. In Australia, residential premises are characterized by open plan living, which is often well ventilated with high air exchange rates, whereas those in the northern hemisphere, in particular North America, have closed ventilation to retain the warmth in winter and circulation is based on internal air conditioning. These factors alone may have influenced the findings of the study. It is likely that other residential environments with lower exchange rates will

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have limited numbers of airborne genera and this might reflect an entirely different spectrum of airborne fungi compared to those collected in Chapter 3. The various measurement techniques used to quantify airborne fungi are known to be confounded by a variety of factors and provide only a proxy measure of personal exposure. Sampling methods, such as static volumetric air sampling and personal air sampling, collect only a sub-fraction of the particles that are in the general region and not what an individual or group of individuals inhales. The development of the INAS has allowed for the first time, the collection of inhaled particles, which is a true reflection of personal exposure to airborne fungi. The use of INASs in outdoor environments provides novel insight into the variations between conventional air sampling techniques, the number of fungi inhaled by individuals and the types of fungal propagules actually inhaled. It was hypothesised in Chapter 4 that the inhalation of airborne fungi was associated with the local microenvironment and that the relative numbers of fungi inhaled between individuals was heterogeneous. Although concurrent HIAs were not undertaken in this study, allergenic genera identified in Chapter 3 by HIA in indoor environments were present and in some cases, were just as frequent in outdoor environments. It is now known that the spectrum of fungal genera inhaled is heterogeneous and does not necessarily reflect the counts of static volumetric air sampling devices. Although previous studies have demonstrated that Alternaria and Cladosporium are the most frequently inhaled spore types (Mitakakis et al., 2000), the results of Chapter 4 extend these findings and implicate other genera including Arthrinium, Xylariaceae and even hyphal fragments as the most frequently inhaled fungi. Many of these genera are currently not included in airborne counts for epidemiologic assessments, although their allergenic potential was described for the first time in Chapter 3. The genera most frequently collected on INASs are sourced from either the O Horizon layer or decaying vegetation and upon disturbance, these fungi are locally aerosolised into the inhalable zone (Mitakakis et al., 2000; Buttner et al., 2002). This results in an individual inhaling larger concentrations of these spores compared to those at higher altitudes, as sampled with a Burkard. In addition, these disturbance activities

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may also explain the higher concentration of hyphal fragments collected compared to static Burkard counts. Conversely, other disturbance parameters that liberate spores on a much larger geographical scale, including wind direction, wind speed, temperature and the mowing of lawns, parks and paddocks can lead to significantly higher concentrations of fungi in a geographic location (Mitakakis et al., 2001b). Thus, while disturbance activities have been shown to disperse substantial quantities of spores, personal exposure to airborne fungi is heterogeneous between individuals and the types of fungi inhaled does not necessarily relate to static measurements. These findings also demonstrate that any intervention to reduce the contact with fungal allergens must consider all fungal sources and not specific fungal types. Individuals can reduce their exposure to fungal allergens by limiting activities on soil and vegetated areas as well as avoiding paddocks, farms, lawns and sporting fields during disturbance activities. The quantification of airborne fungi is restricted by the method of analysis. As discussed briefly in previous chapters, the availability of molecular probes and the type of measurement technique used determine the detection of allergenic fungi in air samples. Until now, it has not been possible to enumerate and identify airborne spores of a particular genus and concurrently demonstrate allergy to the same particles. This is particularly the case for those particles with indiscernible morphological features, such as hyphae and unicellular conidia. In previous studies A. alternata, A. fumigatus and P. chrysogenum have been quantified using ELISA’s (Schmechel et al., 2003a), whereas airborne counts based on the identification of non-conidial fungal particles have posed a taxonomically difficult task (Flannigan, 1997). Although the amount of allergen that is sourced from an environment may be of more relevance in certain studies, it remains unknown as to what role small fungal fragments function in the exacerbation of allergic rhinitis and asthma. These fragments are smaller than conidia, which are able to be inhaled and penetrate further into the respiratory tract and are often higher in airborne concentration than conidia. The emerging clinical significance of aerosolised hyphae has recently been confounded by the inability to accurately identify environmental unicellular conidia and hyphae (Gorny, 2004).

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A novel modification of the HIA was developed in Chapter 5 to enable for the first time, the dual identification and diagnosis of allergy to morphologically indiscernible particles by double immunostaining culturally derived conidia, hyphae and fungal fragments with monoclonal and human IgE antibodies. This technique combines serological with environmental monitoring and can be applied as a tool to detect and accurately identify such fragments on a patient-specific basis, which has not been possible before. However, for many medically important fungi species-specific monoclonal antibodies are not currently available, except in the case of Stachybotrys chartarum (Schmechel et al., 2003c) and thus limit the technique, for the moment, to the detection and differentiation of fungi to the genus level. Further development of mAbs in the future will provide a much more sensitive immunodiagnostic tool that will ultimately contribute to better patient management and the characterization of adverse health affects due to fungal aerosols. The release of allergens from individual fungal spores and the biological factors affecting their release were not known for most fungal species other than A. alternata (Mitakakis et al., 2001a) and A. fumigatus (Sporik et al., 1993). It was hypothesised in Chapter 6 that the germination of culturally derived fungal spores belonging to other fungal genera was associated with an increased release of allergen. The hypothesis was predicated on the results of previous studies, which had shown that the majority of A. alternata fungal spores expressed greater amounts of allergen upon germination from hyphae (Mitakakis et al., 2001a). The hypothesis was largely supported and it is now clear that the germination of spores belonging to many other fungal species increased both the proportion of spores and the amount of allergen released. Furthermore, the findings of the study also demonstrate the heterogeneity between the proportions of spores expressing allergen within species. Unlike Alternaria, it cannot be expected that the majority of wild-type spores belonging to other fungi, including Aspergillus, Penicillium and Trichoderma species release allergen immediately on deposition in the respiratory tract. Although, factors such as assay sensitivity, culture conditions, spore viability and fungal strain may have influenced these findings, the process of germination seems to be an important step in

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the expression and release of fungal allergens in many fungal species, particularly in those species, which are characterised by small unicellular conidia. Furthermore, the germination of fungal spores collected onto PBMs significantly increased the detectable thresholds of the HIA and enabled the visualization of both smaller Aspergillus and Penicillium unicellular conidia and the immunostained antigen. This method of analysis has not been possible to date and allows a more accurate tool for the identification and differentiation of these fungal propagules. Our observations of the generation of allergens by fungi raise questions about the process of allergen production following deposition of conidia in the respiratory tract. Although there are efficient removal mechanisms for deposited particles in the upper respiratory tract, including via mucocillary transport, fungi also possess functions that inhibit these processes in various ways. These include the inhibition of cilia beat frequency and reactive oxygen species (Cody et al., 1997; Tomee and Kauffman, 2000), the degradation of the respiratory epithelium and cell tight junctions (Tronchin et al., 1993) and cellular adhesive properties (Yang et al., 2000). At present, the process involved in the delivery of allergen from the deposited spore to effector sites in the epithelium is unclear, although the results from this thesis provide new insight and techniques to elucidate these processes. The recovery of germinated wild-type fungal spores, in particular Aspergillus and Penicillium unicellular conidia, from the nasal cavities of both normal and atopic individuals provides for the first time, novel insight into the state of germination of fungal conidia and the delivery of fungal allergens in vivo. It is proposed the viability and in vivo conditions are important parameters, which influence the germination of fungal conidia. Thus, germination may play a crucial role in the pathogenesis of fungal allergic disease but also other fungal induced diseases, however this remains speculative and requires further investigation. This thesis points to numerous new lines of research. Further research is required to clarify the factors affecting personal exposure to airborne fungi, the species to which exposure occurs most frequently and the nature of allergen release from fungi. The first direction of research would be a detailed evaluation of personal exposure to

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airborne fungi on an individual basis using the HIA. The development of the HIA for fungi has enabled for the first time, the combination of environmental with serological monitoring on a patient specific basis to demonstrate fungal allergic disease to aerosolised fungal particles. The findings from the current project have shown that personal exposure to fungi is heterogeneous and that the number of genera capable of mediating IgE hypersensitivity covers a much wider spectrum than previously thought. However, the relationship between airborne concentrations of these newly identified species, prevalence of sensitisation and exacerbations of respiratory symptoms remains unclear. In addition, the research should also focus on the clinical consequences of different sized fungal particles, the influence of dust raising activities and the influence of various temporal, spatial and geographic variables on the distribution of airborne allergenic fungi. Each of these areas of research can be uniquely explored by the techniques developed in this thesis. Another direction of research is to further improve the detectable thresholds of the HIA. The novelty of the technique derives from the unique ability to immunostain naturally secreted allergen from spores or hyphae that reflect actual exposure, however the classification of immunostaining intensity is currently limited to a subjective ordinal rank score. This was highlighted in Chapter 2, particularly in the case of classifying positive sera with low IgE titres. Future studies should also focus on the sensitivity of the assay with the aim of incorporating new objective measures of IgE binding in the HIA. Such measurement labels include fluorescent anti-IgE, however preliminary studies (data not shown) have demonstrated that this is technically complex using current materials and methods and further development of image processing software is required to enable accurate quantification. The success of the experiments in this thesis has encouraged others to also try to refine them. A senior researcher from the National Institute of Occupational Health in Norway is spending their 2006 sabbatical in Sydney with a view to modifying the technique for use with SEM. The clinical significance of fungal fragments including aerosolised hyphae and fragmented conidia as aeroallergen sources is becoming increasingly recognized. In the present study, the size of these fragments varied from 5-100µm, however Gorny et al.

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(2004) demonstrated using SEM that fragments also included particles less than 2µm in size, which were often higher in concentration than conidia counts. The application of HIA, utilizing the aforementioned objective measurement labels and SEM, would allow the further quantification of morphologically indiscernible particles. Thus, it would be of interest to apply the described double immunostaining technique to differentiate between fungal fragments. Such a study would provide further novel insight into the interpretation of personal exposure, the inhalation of respirable particles and the sensitisation of fungal allergens. The results of this thesis have shown that not all spores are equal in the amount of allergen that is released. The HIA provides a novel way to compare the dose of allergen delivered by different species. Currently, species are simply enumerated but large spores (>10µm), such as those belonging to Alternaria, Bipolaris, Curvularia and Exserohilum species, proportionally produce many times more allergen than unicellular Aspergillus and Penicillium conidia. This has never been explored and the development of this would greatly enhance models of fungal allergen exposure. Until now, natural fungal exposure and sensitisation has been restricted to the inhalation of fungal conidia belonging to a select number of fungi, generally Alternaria, Aspergillus, Penicillium and Cladosporium species. Each of these has been shown to cross react with other species including C. lunata and E. nigrum (Bisht et al., 2002; Gupta et al., 2002), however it is not known whether these allergens are homologous with the newly identified species in Chapter 3. It can be hypothesised that if these allergens are homologous between species, a much larger proportion of airborne fungi might contribute to the overall aeroallergen load. Indeed, if cross reactivity is common amongst most fungi, more significant efforts to prepare standardized allergen extracts could be undertaken, which could have many benefits extending into the immunodiagnosis of fungal allergic disease and immunotherapy. The expression and function of many allergens from germinating fungal spores and hyphae remains unclear. Although the processes of in vitro germination was shown to significantly increase the amount of allergen expressed compared to ungerminated spores, it was not within the scope of this thesis to conduct protein expression studies

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due to the numerous confounding variables, which relate to the biology of the fungus. Previous studies have shown that variations between fungal strains, nutrient media and incubation parameters alter the allergen content of a number of allergenic species (Vijay et al., 1984). In Chapter 6, fungal spore germination and the measurement of hyphal growth was to be investigated at various time and temperature intervals to characterize the allergens expressed during growth. Although this would have provided novel insight into the nature of allergen production, it became apparent from preliminary experiments using fungi sourced from the Queensland Department of Primary Industries that the results would not be able to be repeated using other fungal isolates of the same species (data not shown), due primarily to inter-isolate variation. Future studies should utilize a specific isolate of a fungal species that has been catalogued with the American Type Culture Collection to enable the replication of similar studies with the same fungal source. Another direction would be to examine the influence of synthetic nutrient media, temperature, relative humidity, atmospheric pressure, temporal intervals, and the variations between wild-type and culturally derived conidia on the synthesis and release of fungal allergens. The spectrum of allergens expressed in wild-type fungi may be vastly different compared to culturally derived sources, due to variations in available nutrients, dehydration of the spore in the environment and exposure to ultra violet radiation, however this remains anecdotal and requires further investigation. The nature by which allergens are released and delivered into the respiratory tract remains to be established. The fact that germinated fungal spores were recovered from the nasal cavity suggests that some spores may have the ability to resist immediate removal by mucocillary mechanisms, germinate and release greater quantities of allergen, thus increasing personal exposure to fungal allergens. Previous studies have shown that A. fumigatus possess innate colonizing mechanisms, such as mucocillary inhibition, adhesive properties and the ability to break down the respiratory mucosa and inhibit macrophages, however the extent to which other species have these features remains to be determined. Understanding the interaction between the spore and respiratory tract upon deposition may assist in the interpretation of the potency of some fungal aeroallergens sources compared to others, such as in the case of Alternaria. It

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might also aid in the treatment of other respiratory diseases such as CFS and Aspergillosis. Investigating the in vivo germination of fungal spores in the respiratory tract may also be important in understanding the conditions and time required by some fungi to germinate and release allergen. In addition, questions related to the fate of inhaled fungal fragments, the processes involved with spore removal and whether ingested fungi release allergens requires further investigation. This thesis established a new technique for the immunodiagnosis of fungal allergic disease. The HIA method can be used to demonstrate IgE binding to fungal particles, including spores, hyphae and fragments in environments where exposure actually occurs on a patient specific basis. This method of analysis also avoids many of the problems associated with extract variability based on the performance of current diagnostic techniques for fungal allergens. Using this technique, it has been possible for the first time, to immunostain wild-type fungi with patient sera and determine, which fungi and types of fungal propagules were sources of airborne allergens. The findings demonstrated that the interpretation of personal exposure and sensitisation to fungal allergens was not just restricted to fungal conidia belonging to a select number of species, but implicated airborne hyphae and fungal fragments and the conidia of genera previously not recognized as aeroallergen sources. The significance of these findings represents a new paradigm of natural fungal exposure, which was later confirmed in Chapter 4 using INASs in an outdoor environment. The inhalation of airborne fungi is also heterogeneous between individuals and may be determined by the release of spores from dust raising activities, in particular personal disturbances of local vegetation. Additionally, fungal spores that are inhaled are capable of germinating, which potentially increases personal exposure to fungal allergens. The HIA technique for fungi has extensive applications in many fields of exposure analysis, including personal or occupational related exposure settings and can be used as an alternate immunodiagnostic method in epidemiologic and clinical investigations. These findings will assist in the reduction of symptoms suffered by sensitised individuals.

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Appendices

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Appendix 1.1 Original Halogen immunoassay protocol.

Halogen Immunoassay Method

Description of assay

The HALOgenTM IgE immunoassay has been developed by Woolcock Institute of Medical Research, for the measurement of patient’s IgE reactions to the particles they inhale. Allergen carrying particles that the patient inhales may include: cat, house dust mite (Dermatophagoides pteronyssinus and D. farinae), cockroach (Blatella and Periplanta species), dog, pollen grains and fungal spores. By performing this assay you are helping patients identify what allergens they are exposed to, where their exposure is occurring and measure their level of allergen exposure.

The samplers that you have received have been worn for ~20 minutes by a patient with an allergic disease such as asthma or rhinitis. The sampler collects particles the patient inhales that contain allergen. The inhaled particles are collected onto an adhesive strip inside the sampler. The sampling sheets that you have been sent with the samplers describe what allergen the patient would like the samplers tested for. Some patients will require samples tested for IgE reactivity, cat allergen and house dust mite allergen, while others will want them tested for the one allergen only.

The type and amount of allergen the patient has inhaled is determined using the HALOgenTM immunoassay. This assay is a membrane based immunoblotting technique. The particles carrying the allergen are laminated permanently between a protein binding membrane and the adhesive, which they were collected on. This permanent laminate is wetted and the allergens elute from the particles and are bound around the particles by the protein binding membrane. The allergens are then probed with the patient’s sera and stained with a precipitating substrate. The particles and their halo of allergen can be visualised through the transparent adhesive tape layer. Each particle the patient has inhaled and their IgE reacts with will appear with a halo of immunostained allergen surrounding it.

By accurately counting the particles containing allergen you are providing the patient with valuable information about their level of exposure. By examining each particle with a halo you may be able to identify the source of the allergens, for example the number of grass pollen grains. You may also mount the stained samples and scan them using the HALOgenTM reader, which will generate a picture of the inhaled allergen. One copy of this picture should be sent to the patient and the other to the physician.

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Assay Protocol 4.1. Laminating the inhaled allergens with a protein binding membrane (Figure 1).

adhesive film with particles removed from sampler

pair of strips are placed in the same well for immunostaining

adhesive film laminated with protein-binding and cross-section, showing ends trimmed particles between adhesive & membrane

PLACE LAMINATE WITH THE TAPE SIDE DOWN IN WELLS

1. Slowly remove the silicon seals and tape from the nasal samplers, ensuring that the sticky collection surface is not touched.

2. Remove the roll of protein binding membrane from the cassette. The membrane is 11mm wide and will fit the adhesive strip placed across it. Gloves should be worn at all times when handling the membrane.

3. Holding the tape by one end, place the tape, adhesive side down, onto the protein binding membrane with the tabbed tape ends sticking over the edge as shown in the diagrams above. Thus, the collected particles are “laminated” between the protein binding membrane and the adhesive tape.

4. Repeat step 3 for the other sampler in the pair, ensuring that the collection tapes from the same pair of samplers are placed on the same piece of protein binding membrane. Tapes placed up to 4mm apart will still fit into the assay plate.

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5. Place the protein binding membrane /adhesive laminate tape side down, on a clean flat surface and apply gentle and even pressure to ensure the adhesive tape and protein binding membranes are laminated. Cut the laminate from the roll of membrane and trim the laminate so that the overhanging ends are removed.

6. Place the laminate with the membrane side facing upwards, in the well of the 24 well plate. Sample identification details eg: subject, location, length of sampling time and activities performed while sampling can be noted on the lid of the 24 well plate.

7. Repeat steps 1 to 6 for each pair of nasal samplers.

Remove adhesive strip from sampler and laminate

Elute allergens 4h-overnight

Blocking 45 mins at room temp

Incubate with serum for 4h – O/N at room temp

wash Second antibody (biotinylated anti-human IgE) 1.5 hours at room temp wash Conjugate (streptavidin alkaline-phos) 1.5 hours at room temp wash Soak overnight at room temp

Substrate (BCIP/NBT) 10 mins -1 hour at room temp

Stop and mount

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Probing of Samples with Patient’s serum

1. Each protein binding membrane /adhesive laminate is placed in a well of the 24 well plate with the membrane side of the laminate facing upwards. At least one positive control, which contains laminated allergen carrying particles, should also be placed in a separate well of the assay plate.

2. The membranes used are extremely hydrophobic and need to be wet in wetting solution (80% methanol in DDW). Add 400µl of wetting solution to each well and soak the membranes for 20-60 seconds (or until membrane is evenly opaque). Prolonged soaking will lead to the separation of the membrane/adhesive laminate.

3. Remove the wetting solution and rinse three times each with approximately 500µl of deionised water. Once the membranes have been wet it is imperative that they are not allowed to dry at any stage during the assay.

4. Add 600µl of elution buffer (0.2M Borate Buffer pH 8.2, 6.18g of Boric acid in 500mls DDW plus NaOH) to all membranes, elution can be varied from 2h to overnight. The optimum elution time is overnight. Some gentle agitation is desirable prior to refrigeration and again after removal the following morning. This can be done on a plate shaker. Air bubbles should not be trapped underneath the membrane during incubations.

5. Following elution, remove the elution buffer and add 400µl of blocking solution (Bottle 3). Block the membrane-adhesive sandwich for approximately 45 minutes.

6. Following blocking, remove the Blocking agent (5% skim milk or 3% BSA in PBS or TBS or PBS Tween) and add 400µl of the patient’s serum. Incubation can be from 2h to overnight at room temperature on the plate shaker. The optimum serum incubation time depends on the level of total IgE in the patient’s serum. For patients with high total IgE, such as RAST 4, 2 hours is sufficient, while for patients with low IgE, overnight may be preferable. For most sera an incubation time of 4 hours is optimum.

7. Wash for 3 x 10 minutes in 500 µl PBS/Tween (0.05%).

8. Add 400µl of second antibody (Bottle 4 (your preferred conjugate) for example) to each well. Incubate for 1.5 hours at room temperature with agitation on the plate shaker.

9. Wash for 3 x 10 minutes in 500µl of PBS/Tween (0.05%).

10. Mix the 11 µl of concentrated streptavidin alkaline phosphatase conjugate (vial) with the 11mls of conjugate dilution buffer (Use your preferred enzyme conjugate and dilutent, such as 2% skim milk or 1% BSA and PBS Tween or other buffer.). Add 400µl of diluted conjugate to each well. Incubate for 1.5 hours at room temperature with agitation on the plate shaker.

11. Wash for 3 x 10 minutes in 500µl of PBS/Tween (0.05%). The soak overnight.

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12. Develop membranes with 400µl BCIP/NBT substrate (Bottle 6 = BCIP/NBT substrate). The substrate reaction is stopped after membranes have been stained purple. For IgE staining this should be monitored every 10 minutes. Staining should be stopped at a time where the particle is still clearly visible and halo a purple halo surrounding it. The staining is stopped by rinsing three times in deionised water. Stained samples can be stored in deionised water in a refrigerator for up to 3 days before analysis.

Probing of Specific Allergens with Monoclonal Antibodies

1. As per IgE Protocol

2. As per IgE Protocol

3. As per IgE Protocol

4. As per IgE Protocol

5. As per IgE Protocol

6. After blocking remove the blocking solution and add 400µl of anti-allergen monoclonal antibody at the same concentration you would use in Western Blotting. Incubation can be from 1 h to overnight at room temperature, depending on the concentration and avidity of the monoclonal, this incubation should be on the plate shaker. The optimum monoclonal incubation time is 3 hours.

7. Wash for 3 x 10 minutes in 500 µl PBS/Tween (0.05%).

8. Add 400µl of diluted anti-mouse conjugate to each well. Incubate for 1.5 hours at room temperature with agitation on the plate shaker.

9. Wash for 3 x 10 minutes in 500µl of PBS/Tween (0.05%).

10. Develop membranes with 400µl BCIP/NBT substrate. The substrate reaction is stopped after membranes have been stained purple (approximately 1 hour) by rinsing three times in deionised water. Stained samples can be stored in deionised water in a refrigerator for up to 3 days before analysis.

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Remove adhesive strip and laminate

Elute allergens 4h-overnight

Blocking 45 mins at room temp

Monoclonal antibody 1-4 hours at room t wash Conjugate (anti-mouse alk-phos) 1 hour at room temp

wash Substrate (BCIP/NBT) 10 mins- 1 hour at room temp

Stop and mount

Scan or count

Viewing under a microscope

Upon completion of the assay, laminates can be removed from the assay plate and placed on a microscope slide, adhesive side facing up for viewing under a microscope.

The laminate should be kept wet with deionised water. A temporary mountant is provided with the kit (Bottle 7 = is not necessary), which also serves to clear the membrane and enhance visibility.

Under 200X magnification you should be able to clearly see the particles and particles with allergen halos (Figure 2). Any particle with a halo is a particle carrying allergen to which the patient has IgE. Many of the particles will have no distinct shape; these are most likely house dust mite or cat allergen. Patient’s exposure to these allergens can also be tested using the HALOgen assay.

Particles with a distinct shape may be identified as pollens grains or fungal spores. Fungal spores generally release only small amount of allergens.

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Notes: Samples can be viewed up to 3 days following completion of immunostaining, if kept wet and stored at 4oC. If the laminates need to be preserved they can be mounted in aqueous mounting media.

Trouble shooting

If you have any problems running this assay then please contact us. Some trouble shooting tips are provided below:

Problem: No particles have been collected on the samples. Solution: The patient has not worn the samplers correctly. Contact the patient

Problem: The adhesive protein binding laminate separates. Solution: Prolonged soaking in the wetting buffer will lead to the separation of the membrane/adhesive laminate. It is very important that the wetting steps only occurs for 60 seconds. Other steps in the process do not influence delamination of the sample.

Problem: Particles are present on the samples, but no allergen halos are seen on the samples or on the positive controls. Solution: This suggests that the assay has not worked. Check all reagents to make sure that each reagent was added in the correct order. Repeat the assay using the supplied positive controls.

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Appendix 1.2 Initial allergen detection protocol developed for spores of Alternaria alternata developed by Teresa Mitakakis.

Halogen immunoassay - fungal spore protocol

Day 1:

1. Grow Fungal Culture, aged between 10-20 days old, grown on V8 agar. 2. Sample spores onto the 0.45 um PVDF membrane for thirty seconds. 3. Apply Inhalix Adhesive, binding it firmly with roller, removing air bubbles. 4. Cut out the pieces for 3 replicates per variable. 5. Wet in 80% methanol for 1 minute, 400 µl. 6. Wash in dH2O 3 times, 400 µl. 7. Soak in PBS for five minutes on rocker, 400 µl. 8. Transfer to micro titre tray with Borate buffer – elute for 4 hours on shaker, 800 µl. 9. Wash in PBS/Tween 0.05% 3 times, 400 µl. 10. Block in 5% skim milk in PB/Tween 0.05% on shaker for 45 minutes, 800 µl. 11. Wash in PBS/Tween 0.05% 3 times, 400µl. 12. Incubate samples with 350 µl (24 well plate) anti-Fungal human sera diluted 1:3 in 2% skim milk/PBS/Tween 0.05% (Pool B), leave on shaker or rocker overnight at room temperature.

Day 2:

13. Wash in PBS/Tween 0.05% 3 times, 400 µl. 14. Incubate samples with 350 µl (24 well plate) biotinylated goat anti human IgE (1:500 diluted with 2% skim milk/PBS/Tween 0.05%) for 90 minutes on shaker. 15. Wash in PBS/Tween 0.05% 3 times, 400 µl. 16. Incubate samples with 350 µl (24 well plate) ExtrAvidin alkaline phosphatase (EAP) (1:1000 diluted with 2% skim milk/PBS/Tween 0.05%) for 90 minutes on shaker. 17. Wash in PBS/Tween 0.05% 2 times and leave over night in fridge, 400 µl. 18. Wash in PBS/Tween 0.05% 1 time, 400 µl. 19. Incubate in BCIP/NBT checking for stain at 30 minutes, 400 µl. 20. Stop by rinsing in d H2O 3 times, leaving sample in dH2O, 800µl. 21. Clear samples in 9:1 ethylene glycol/glycerol (app 1/5.1) 22. Count percentage haloes, calculate the average for three replicates

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Appendix 2.1 Recipes for agars and buffers

V8 juice agar PBS (pH 7.4)

200ml V8 juice 8g NaCl

800ml dH2O 0.2g KH2PO4

20g agar 1.15g Na2HPO4 mix together, adjust to pH 6.0 and 0.2g KCl autoclave 1000ml deionised water

adjust pH to 7.4

Rose Bengal Chloramphenicol agar

16g Rose Bengal Chloramphenicol agar PBS/0.05% Tween

500ml dH2O To 1 litre of PBS (pH 7.4)

1 vial Chloramphenicol selective Add 0.5ml of Tween 20 supplement SR78 mix together, adjust to pH 6.0 and 1% BSA autoclave To 100ml of dH2O

Add 1g of bovine serum albumen

Borate buffer (0.2M, pH 8.2)

12.366g of boric acid 5% Skim milk

1000ml deionised water To 100ml of dH2O adjust pH to 8.2 Add 5g of skim milk powder

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Appendix 2.2 Fungal germination Halogen immunoassay procedure

Halogen immunoassay - 2-day fungal germination protocol

Day 1:

1. Grow Fungal Culture, aged between 10-20 days old, grown on V8 agar. 2. Quickly wet in 80% methanol the 0.45µm PVDF membranes. 3. Soak 0.45µm PVDF membranes in a 30% sucrose solution, for 30 minutes. 4. Allow the membranes to dry. 5. Sample spores onto the 0.45 µm PVDF membrane for thirty seconds. 6. Incubate PVDF membranes in a humid chamber at 25oC overnight to enable spore germination. 7. Apply Inhalix Adhesive, binding it firmly with roller, removing air bubbles. 8. Cut out the pieces for 3 replicates per variable. 9. Wet in 80% methanol for 1 minute, 400 µl. 10. Wash in dH2O 3 times, 400 µl. 11. Soak in PBS for five minutes on rocker, 400 µl. 12. Transfer to micro titre tray with Borate buffer – elute for 4 hours on shaker, 800 µl. 13. Wash in PBS/Tween 0.05% 3 times, 400 µl. 14. Block in 5% skim milk in PB/Tween 0.05% on shaker for 45 minutes, 800 µl. 15. Wash in PBS/Tween 0.05% 3 times, 400µl. 16. Incubate samples with 350 µl (24 well plate) anti-Fungal human sera diluted 1:3 in 2% skim milk/PBS/Tween 0.05% (Pool B), leave on shaker or rocker overnight at room temperature.

Day 2:

16. Wash in PBS/Tween 0.05% 3 times, 400 µl. 17. Incubate samples with 350 µl (24 well plate) biotinylated goat anti human IgE (1:500 diluted with 2% skim milk/PBS/Tween 0.05%) for 90 minutes on shaker. 18. Wash in PBS/Tween 0.05% 3 times, 400 µl. 19. Incubate samples with 350 µl (24 well plate) ExtrAvidin alkaline phosphatase (EAP) (1:1000 diluted with 2% skim milk/PBS/Tween 0.05%) for 90 minutes on shaker. 20. Wash in PBS/Tween 0.05% 3 times, 400 µl. 21. Incubate in BCIP/NBT checking for stain at 30 minutes, 400 µl. 22. Stop by rinsing in d H2O 3 times, leaving sample in dH2O, 800µl. 23. Clear samples in 9:1 ethylene glycol/glycerol (app 1/5.1) 24. Count percentage haloes, calculate the average for three replicates

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Appendix 4.1 Coating Intra nasal air samplers with adhesive.

Requirements:

Intra nasal air samplers Adhesive mix (available from The Woolcock Allergen Lab) Aluminium foil Cotton swabs Dust free environment in which to work

Instructions:

Make up 1:2 v/v Woolcock Adhesive / acetone mix.

Have clean, dry Intra nasal air samplers ready in a dust-free environment.

Aliquot some of the diluted adhesive into a little “boat” made of aluminium foil (it is very difficult to wash off beakers).

Dip a fresh cotton bud in the adhesive mix, holding cup in gloved hand paint liberally onto inside of cup. The adhesive is self-levelling as the acetone evaporates. Ensure the bottom of the cup has a complete coat.

Re-wet the cotton bud with the adhesive and coat the insides of the silicone outer part of the Maggie. Swirl the bud around to get a thin, even coat on the entire inside including where the cup will contact the silicone.

After about 10 mins, assemble cups and silicone outer parts of the Intra nasal air sampler.

Leave covered to exclude dust but not airtight for >1 hr to allow all acetone to evaporate.

Package the prepared devices in small sealable plastic containers. Clean specimen jars are ideal.

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Appendix 4.2 Calberla's stain for pollen grains.

1. Make saturated solution of Basic Fuchsin by mixing 3mg in 1mL of distilled water.

CAUTION: FUCHSIN IS TOXIC BY SKIN CONTACT AND INHALATION OF THE POWDER. - Wear protective gloves and mask when handling the powder.

2. Mix 5mL glycerol, 10mL 95% ethanol, and 10 drops of the Basic Fuchsin solution made in (1) and mix.

3. Calberla's stain can be stored (away from light) for up to 1 month.

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