Final Screening Assessment for Aspergillus oryzae ATCC 11866

Environment and Climate Change

Canada Health Canada

January 2017

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Table of Contents

List of Tables

List of Figures


Pursuant to paragraph 74(b) of the Canadian Environmental Protection Act, 1999 (CEPA), the Minister of the Environment and Climate Change and the Minister of Health have conducted a screening assessment on A. oryzae strain ATCC 11866.

A. oryzae ATCC 11866 is a fungus that is a member of the Aspergillus flavus group and has characteristics in common with two members of that group, A. oryzae and A. flavus.

A. oryzae occurs where it is used in food fermentation facilities, primarily in Japan and China, but it may also be found sporadically in soil or on decaying plant materials. A. oryzae is considered by some taxonomists as a group of domesticated strains of A. flavus that have lost the ability to produce aflatoxins, exhibit sparse sporulation and have floccose aerial mycelia. Although genetically almost identical to A. flavus, A. oryzae is classified as a separate species to indicate strains that are suitable for food production. There is no evidence in the scientific literature indicating that A. oryzae is a plant or animal pathogen, although a few cases of infection in animals with predisposing conditions have been reported. Under normal circumstances, it is unlikely to be a serious hazard to healthy livestock or to other organisms in the environment. Information from the scientific literature indicates that A. oryzae is unlikely to cause infection in healthy or debilitated humans.

A. flavus is generally considered to be ubiquitous in nature, and able to thrive in a variety of terrestrial and aquatic habitats. A. flavus is a plant pathogen and is known to cause disease in maize, cottonseed, tree nuts, and oilseed crops. A. flavus has also been reported as an opportunistic animal pathogen, causing mycosis (i.e., infection) mostly in birds and mycotoxicosis (i.e., disease from ingestion of toxin-contaminated feed), which triggers a range of symptoms that can debilitate the host. A. flavus can cause sinus and eye infections in otherwise-healthy humans, and potentially fatal lung disease and systemic infections in susceptible groups (i.e., infants and the elderly, the immunocompromised and individuals with debilitating comorbidities).

Both species have been reported infrequently to cause allergic reactions in humans and animals, including hypersensitivity in susceptible individuals.

A. oryzae ATCC 11866 was originally selected for high protease production. This property makes it of possible commercial and industrial interest for fermentation, enzyme production, chemical production, as a livestock probiotic, for bioremediation, biodegradation, industrial effluent treatment, municipal wastewater treatment (particularly grease traps and sewer lines), organic waste treatment, biosorption of environmental contaminants and use as a host organism for recombinant protein and enzyme production.

The morphological characteristics of A. oryzae ATCC 11866, particularly small conidial size, which is associated with virulence, more closely resemble those of A. flavus; however, A. oryzae ATCC 11866 is not known to produce aflatoxin, and it is susceptible to the major clinical antifungals used to treat aspergillosis. These are mitigating factors in this risk assessment.

This assessment considers the aforementioned characteristics of A. oryzae ATCC 11866 with respect to human health and environmental effects associated with product use and industrial processes subject to CEPA, including releases to the environment through waste streams and incidental human exposure through environmental media. A conclusion under CEPA on this living organism is not relevant to, nor does it preclude, assessment of products produced by or containing A. oryzae ATCC 11866as prescribed under the purview of the Food and Drugs Act. To update information about current uses, the Government launched a mandatory information-gathering survey under section 71 of CEPA as published in the Canada Gazette, Part I, on October 3, 2009 (section 71 Notice). Information submitted in response to the section 71 Notice indicates that A. oryzae ATCC 11866 was not imported into or manufactured in Canada in 2008.  Based on the information available, A. oryzae ATCC 11866 is not currently in commerce in Canada.

Based on the information available, it is concluded that A. oryzae ATCC 11866 does not meet the criteria under paragraph 64(a) or (b) of CEPA as it is not entering the environment in a quantity or concentration or under conditions that have or may have an immediate or long-term harmful effect on the environment or its biological diversity or that constitute or may constitute a danger to the environment on which life depends. It is also concluded that A. oryzae ATCC 11866 does not meet the criteria under paragraph 64(c) of CEPA as it is not entering the environment in a quantity or concentration or under conditions that constitute or may constitute a danger in Canada to human life or health.

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Pursuant to paragraph 74(b) of the Canadian Environmental Protection Act, 1999 (CEPA), the Minister of the Environment and Climate Change and the Minister of Health are required to conduct screening assessments of living organisms added to the Domestic Substances List (DSL) by virtue of section 105 of the Act to determine whether they present or may present a risk to the environment or human health (according to criteria as set out in section 64 of CEPA)Footnote 1. A. oryzae ATCC 11866 was added to the DSL under subsection 25(1) of CEPA 1988 and to the DSL under subsection 105(1) of CEPA  because it was manufactured in or imported into Canada between January 1, 1984 and December 31, 1986.

This screening assessment considers hazard information obtained from the public domain and from unpublished research data generated by Health CanadaFootnote 2, as well as comments from scientific peer reviewers. Exposure information was obtained from the public domain and from a mandatory CEPA  section 71 Notice published in the Canada Gazette, Part I, on October 3, 2009. Further details on the risk assessment methodology used are available in the Risk Assessment Framework document entitled "Framework on the Science-Based Risk Assessment of Micro-organisms under the Canadian Environmental Protection Act, 1999" (Environment Canada and Health Canada 2011).

In this report, data that are specific to the DSL-listed Aspergillus oryzae ATCC 11866 are identified as such. Strain-specific data are limited and are from five sources: the Nominator, the American Type Culture Collection (ATCC), unpublished data generated by Health CanadaFootnote 3, and two published papers [i.e., Baens-Arcega et al. (1956) and Wei and Jong (1986)]. Where strain-specific data were not available, surrogate information from literature searches was used. Surrogate information was considered in this report relating to other strains of the species A. oryzae, as well as information on the species A. flavus, given that the morphological characteristics of A. oryzae ATCC 11866 may make its behaviour more similar to A. flavus than to other A. oryzae strains. When applicable, literature searches conducted on the organism included synonyms, and common and superseded names. Surrogate organisms are identified in each case to the taxonomic level provided by the source. Literature searches were conducted using scientific literature databases (i.e., SCOPUS, CAB Abstracts, Google Scholar and NCBI PubMed), web searches, and key search terms for the identification of human health and environmental hazards. Information identified up to May 2014 was considered for inclusion in this screening assessment.

Decisions from Domestic and International Jurisdictions


A. oryzae ATCC 11866 is a Risk Group 1 pathogen for humans and terrestrial animals (low individual risk, low community risk) according to the Public Health Agency of Canada (PHAC) (personal communication, PHAC 2015). Itis not considered to be a regulated plant pest in Canada by the Canadian Food Inspection Agency (CFIA) (personal communication, CFIA 2013).

A. oryzae, as a taxonomical synonym for Aspergillus flavus var. oryzae, is listed in the Natural Health Products Ingredients Database (NHPID) with a medicinal role and as a source material for alpha-amylase, catalase, cellulase, fungal protease, glucoamylase, hemicellulase, lactase, leucyl aminopeptidase, malt diastase, oligopeptidase B, polygalacturonase, protease, and lipase (NHPID 2015).  Although A. oryzae is found in the Licensed Natural Health Products Ingredients Database as being present as medicinal ingredient in currently licensed natural health products (NHPs), these products are not expected to contain or provide live A. oryzae, as consistent with the Food Chemicals Codex and the information found in the NHPID. A. oryzae (strain unknown) is used as the production organism for the various enzymes currently found in licensed NHPs (LNHPD 2015; FCC 2012).


A. oryzae and the enzymes it can produce are accepted as constituents of food (Barbesgaard et al. 1992). The Organisation for Economic Co-operation and Development (OECD) has assigned A. oryzae a Good Industrial Large-Scale Practice (GILSP) host status (OECD 1992), and the United States Food and Drug Administration (U.S. FDA) has accepted A. oryzae as a safe recipient organism for the purposes of enzyme production (Olempska-Beer et al. 2006). A risk assessment of A. oryzae was conducted under the Toxic Substances Control Act (TSCA) by the biotechnology program of the United States Environmental Protection Agency (U.S. EPA), and this species is included as a recipient micro-organism at õ 725.420 (recipient organism) for the tiered exemption (U.S. EPA 1997a; U.S. EPA 1997b), allowing it to be used in containment in the U.S. for production of enzymes and organic acids without notifying the U.S. EPA.

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1. Hazard Assessment

1.1 Characterization of Aspergillus oryzae

1.1.1 Taxonomic identification and strain history

Binomial name: Aspergillus oryzae

Taxonomic designation:
Synonyms and common names:

The most common synonyms associated with the species A. oryzae include: A. flavus Link 1809 (Roskov et al. 2014); A. flavus var. oryzae (Kurtzman et al. 1986); and Eurotium oryzae (Machida et al. 2008). A more exhaustive list of synonyms is available in the Catalogue of Life (Roskov et al. 2014).

The species A. oryzae can be described by the common name of koji mould (Machida et al. 2008). A. oryzae ATCC 11866 is also referred to as the Philippine strain of A. oryzae (Baens-Arcega et al. 1956).

Strain history: A. oryzae ATCC 11866 was originally isolated as a contaminant of indoor air in the Institute of Science and Technology in Manila, Philippines and was deposited to the ATCC by L. Baens-Arcega (Baens-Arcega et al. 1956). The strain was of interest for its high protease activity and for possible use in the production of soy sauce from copra meal (a coconut by-product). The isolate was identified as a typical strain of A. oryzae by Drs. D. I. Fennell and C.W. Hesseltine at the Agricultural Research Service, U.S. Department of Agriculture, Peoria, Illinois after the isolate was sent to ATCC for identification. No information on the basis of their identification of the isolate as A. oryzae was provided. Phenotypic and molecular characteristics

A. oryzae is a filamentous, aerobic fungus belonging to the Aspergillus Section Flavi, which is commonly referred to as the A. flavus group. This group also includes Aspergillus sojae, Aspergillus tamarii, Aspergillus flavus, Aspergillus parasiticus and Aspergillus nomius. The original A. flavus group comprised nine species and two varieties distinguished by the colour and ornamentation of the conidial heads (Raper and Fennell 1965). Currently, the A. flavus group contains 18 accepted species (reviewed in Samson et al. 2006).

A. oryzae is the name given to the domesticated strains of A. flavus used as koji moulds in Japan and China for the fermentation of rice and soy products (Barbesgaard et al. 1992). The species A. oryzae may have emerged as a natural variant of A. flavus through long-term successive cultivation on Oryza sativa (rice) (Wicklow 1984a). Domestication is thought to have reduced the selective pressure required by A. flavus to maintain genes associated with spore formation and dispersal, and those associated with the production of aflatoxins (Jorgensen 2007) resulting in sparse sporulation, floccose aerial mycelia and production of few or no scleriotia (Chang and Ehrlich 2010). Furthermore, the loss or inactivation of these genes through domestication created morphological differences between the domesticated and wild type species, namely: differences in colony texture, conidiophore length and texture, conidia texture, and in the arrangement of sterigmata, as well as the loss of some metabolic capabilities, such as the formation of aflatoxins (Barbesgaard et al. 1992; Kurtzman et al. 1986; Wicklow 1984a). Nevertheless, the lack of production of aflatoxins alone is not a criterion to differentiate A. oryzae from A. flavus since some strains of A. flavus do not produce aflatoxins (Jorgensen 2007).

Because they are almost genetically identical, it was once proposed to reclassify A. oryzae as a variant of A. flavus (reviewed in Blumenthal 2004).The two species remain difficult to differentiate based on morphological, physiological or genotypic characteristics (Jorgensen 2007). According to Ehrlich (2008), it is becoming clear that A. oryzae is not a separate species, but rather a group of non-aflatoxigenic variants of A. flavus. However, A. oryzae continues to be classified as a different species for economic and food safety reasons (Chang and Ehrlich 2010).

Since A. oryzae and A. flavus are so closely related, information on both species is presented in this report.


As for many fungi, the taxonomy of Aspergillus species is primarily based on morphological features. The conidiophore morphology and associated terminology used for the identification of Aspergillus species are illustrated in Figure 1-1.
In spite of subtle morphological differences, A. oryzaeand A. flavus have a very broad range of overlapping characteristics. As such, several characteristics must be used simultaneously to distinguish them accurately (Barbesgaard et al. 1992; Klich and Pitt 1988). Morphologically:

The morphological characteristics of the DSL strain, ATCC 11866, are consistent with those of the group of yellow-green moulds belonging to the A. flavus group. Using Raper and Fennell's (1965) identification keys, the identifying characteristics of the A. flavus group include: biseriate sterigmata in most species; non clavate vesicles; bright yellow-green conidial heads in young cultures, becoming brown with time; loosely radiate conidial heads; spore chains usually separate, sometimes forming poorly defined columns; and conidiophores usually presenting as roughened and colourless. At the time of its isolation, Baens-Arcega et al. (1956) could not determine whether the DSL strain was A. flavus or A. oryzae based on its morphology (Table 1-1). Based on the length and diameter of the conidiophores, the size and form of the vesicles, the manner of colouring the agar, and the appearance of the conidial heads and conidia, it appeared closer to A. flavus than to A. oryzae; however, it could not be identified as A. flavus with certainty because the colour of the colony was not as deep a green as the colour of the control A. flavus culture used. The basis of the identification of the isolate as A. oryzae by Drs. D.I. Fennell and C.W. Hesseltine was not provided by Baens-Arcega et al. (1956).

Figure 1-1: Terminology used to describe conidiophore morphology for the identification of Aspergillus species: biseriate, metula, phialides or sterigmata, conidia, vesicle, uniseriate and stipe
Terminology used to describe conidiophore morphology for the identification of Aspergillus species: biseriate, metula, phialides or sterigmata, conidia, vesicle, uniseriate and stipe (See long description below)
Long description for figure 1-1

The figure shows the top part of a conidiophore (the foot cell is not part of the figure) which includes the stipe terminating into a vesicle. On the vesicle, there are flask-shaped structures known as phialides or sterigmata which can be either uniseriate or biseriate. In the uniseriate form, phialides are directly attached to the vesicle; in the biseriate form, phialides are attached to the vesicle via a structure called a metula. Conidia form chains on top of the phialides.

Table 1-1 Comparison of morphological characteristics of A. oryzae ATCC 11866 with the type strain for A. oryzae and the type strain for the closest pathogenic strain, A. flavus.
CharacteristicA. oryzae
ATCC 1011
(type strain)a
A. flavus
ATCC 16883
(type strain)
A. oryzae
ATCC 11866
(DSL strain)c
A. oryzae
ATCC 11866
(DSL strain)d
Growth temperatureOptimal at 34°COptimal at 37°CbNot reported by the source15-42°C (not tested below 15°C)
Colony colorYoung colonies yellow, maturation giving yellow green, green/olive; brown in aged culturesYellow-green to leaf greenb
Green colonies and white or lighter green edges and black specks at the point of the colony radiating away from the center (7 days growth on CYA at 25°C)d
Colony turning yellow, then russet green as it matures; surface of the colony becoming radiantly wrinkledWhite colonies with yellowish centres (7 days growth on CYA at 25°C)
Conidial headGlobose, radiate or columnarShape is radiate to loosely columnar; aged heads split into columnsbSubglobose or nearly globose, radiateNA
Conidial head size (µm)Not reported by the source60d30 to 50 (horizontal diameter)72.2 +/- 8.6
Conidiophore/stipeColorless and roughenedNo coloration, nearly smooth to echinulatebColourless conidiophores with wart-roughened wallsNA
Conidiophore (µm)1000 and longer in lengthNot reported by the source350-750 (length) 5.0-8.0 (diameter)NA
VesicleSubglobose to clavateElongate, subglobose to globosebSubglobose vesiclesglobose
Vesicle diameter (µm)Not reported by the sourceNot reported by the source10.0-20.0NA
Sterigmata/phialides20% biseriate sterigmata, mostly uniseriatePhialides biseriate (20-80%); uniseriate when on small vesiclesbArranged in single series, greenish and closely packed over one-half to two-thirds of the vesicular surfaceNA
Sterigmata size (µm)Not reported by the sourceNot reported by the source6.4-9.6 (length) 3.2-4.8 (diameter)NA
ConidiaOften globose or subglobose, or elliptical to pyriform. Slightly roughSubglobose to globose; smooth to definitely echinulatebIn chains, greenish, mostly globose, wall roughened with spinules or wartsNA
Conidia diameter (µm)5-6 and larger3-7.5b
4.0-4.83.9 +/- 0.8
Presence of sclerotiaRare; globose, purple-brown or black when matureNANo sclerotiaNA

a Data for A. oryzae ATCC 1101 are from Murakami (1971)

b Data for A. flavus ATCC 16883 are from Christensen (1981)

c Data for A. oryzae ATCC 11866 are from Baens-Arcega et al. (1956)

d Data generated by Health Canada's Environmental Health Science and Research Bureau

NA = Not available


The close evolutionary proximity of the species A. flavus and A. oryzae is reflected in their genomes, which are nearly identical in size and gene content (Payne et al. 2006). Comparison of A. oryzae and A. flavus 18S rRNA and 26S rRNA gene sequences and ITS regions, and DNA/DNA hybridization studies, show the species to be almost identical (Nakamura et al. 2011; Kurtzman et al. 1986). Amplified fragment length polymorphism (AFLP) cannot differentiate A. oryzaefrom A. flavus (Montiel et al. 2003). Nonetheless, A. oryzae and A. flavus can be distinguished using SmaI digestion of total DNA and multilocus sequence analysis (MLSA) using four aflatoxin biosynthetic gene loci (aflR, aflT, norA, and vbs) (Klich and Mullaney 1987; Nakamura et al. 2011).

The genome of A. oryzae RIB40 has been sequenced, and the strain has a high gene content related to metabolism and transport (Machida et al. 2005). A. oryzae strain RIB40 is considered to be representative of the species (but is not a type strain), as its characteristics are typical of industrial strains used for sake brewing, including morphology, growth, amylase production, and potent protease activity, which are important properties for soy sauce fermentation (Abe et al. 2006). The DSL strain ATCC 11866 shares the property of potent protease activity with strain RIB40, but it is not known to what degree further properties of the DSL strain are common to RIB40 (Baens-Arcega et al. 1956). The genome of A. oryzae ATCC 11866 has not been sequenced.

1.1.2 Biological and ecological properties

Other than Baens-Arcega et al. (1956) and Wei and Jong (1986), there are no additional publications in the literature on the DSL strain ATCC 11866. Therefore, this section relies on surrogate information relating to other members of the species A. oryzae. This section also includes surrogate information on A. flavus, given that A. flavus is considered to be the wild type of domesticated A. oryzae (Blumenthal 2004; Kurtzman et al. 1986); the behaviour of A. oryzae in the environment (i.e., outside a fermenter) is not well understood, and its potential to revert to the wild type phenotype is unclear; morphological characteristics of the DSL strain (e.g., small conidia) may make its behaviour more similar to A. flavusthan to other A. oryzae strains, as described below. Natural occurrence

A. oryzae is mainly found in Japan and China where it has been intensively used in food fermentation. Outside those countries, the fungus may be found sporadically in soil or on decaying plant materials (Barbesgaard et al. 1992).
Researchers have also reported isolating this fungus from:

Nevertheless, some specialists argue that there are no wild strains of A. oryzae and that these environmental isolates are, in fact, A. flavus. A. flavus is distributed worldwide in soil and litter (Horn 2003; Wicklow 1984a). It is most commonly found at tropical and subtropical latitudes, and is uncommon above 45 degrees latitude (Klich 2007; Klich 2002). Soils containing high levels of organic matter, and abundant nitrate, phosphate, and potassium are linked to denser populations of A. flavus (Zablotowicz et al. 2007). A. flavus is also common in marine environments (Zuluaga-Montero et al. 2010).

A. flavus has been isolated from a diversity of terrestrial, aquatic, and marine habitats. Terrestrial sources of isolation include cotton (Klich 1986), corn and maize (Shearer et al. 1992; Wicklow et al. 1998), plants and leguminous trees (Boyd and Cotty 2001), peanuts (Pinto et al. 2001), fresh date fruits (Shenasi et al. 2002), bee pollen (Gonzalez et al. 2005), and from bird and mammal tissues (Gallagher et al. 1978). In a survey on the overall diversity of the Mahanadi River in India, A. flavus comprised 2.5% of the fungal population; more dominant species were A. niger (15%) and A. vesicolor(9.5%). It was also found to be among the dominant fungi associated with common house mosquito (Culex pipiens) larvae, pupae, and adult life stages collected from a fresh water pond in Egypt (Parveen et al. 2011; Badran and Aly 1995). In marine environments, A. flavus has been isolated from sponges, sea fans, and corals (Zuluaga-Montero et al. 2010). A. flavus has also been isolated in hospitals together with A. fumigatus, A. oryzae, and A. niger (Allo et al. 1987; Arnow et al. 1991; Buffington et al. 1994; Grossman et al. 1985; Hahn et al. 2002; Humphreys et al. 1991; Iwen et al. 1994; Lai 2001; Leenders et al. 1996; Loo et al. 1996; Lutz et al. 2003; Opal et al. 1986; Pegues et al. 2002; Rotstein et al. 1985; Sherertz et al. 1987; Singer et al. 1998; Thio et al. 2000). Growth Conditions

Under laboratory conditions, optimal growth of A. oryzae occurs within a temperature range of 32°C to 36°C and a pH range between 2 and 8, and it requires ions of the trace elements Fe and Zn (Domsch et al. 1980) for growth. The optimum growth temperature of A. flavus is 37°C, within a growth temperature range of 12°C to 48°C (Hedayati et al. 2007). The culture method recommended for A. oryzae ATCC 11866 is on potato dextrose agar at 24°C (ATCC 2014).

Characterization of the growth kinetics of A. oryzae ATCC 11866 was performed at Health Canada laboratories at different temperatures in liquid media (Appendix A, Table A-1), and growth on a variety of solid media at 28°C and 37°C was also tested (Appendix A, Table A-2). A. oryzae ATCC 11866 can grow at 37°C, but grows poorly at 42°C. Life cycle, persistence and survival

A sexual life cycle has not been observed in A. oryzae(Abe et al. 2006; Jorgensen 2007).However, the presence of mating loci, recombination and meiosis-associated genes in A. oryzae all indicate that a yet to be identified heterosexual generation exists (Goffeau 2005; Rokas 2009); the sexual stage of A. flavus is Petromyces flavus (Horn et al. 2009).

In the asexual life cycle, asexual spores (conidia) are readily formed by A. oryzae and released into the air (Barbesgaard et al. 1992; Klich and Pitt 1988). Although A. oryzaesporulation is sparser, its conidia typically germinate 3 hours earlier than those of A. flavus, which could be a competitive advantage in some environments such as fermentation processes, which may favour selection of conidia that germinate rapidly (Wicklow 1984b). A. oryzae conidia are also typically larger. Larger conidia contain greater endogenous spore reserves, which could increase the species' colonization capacity and have a positive impact on its competitive ability; however, greater size might also be a disadvantage for dispersal in nature, and this may be the more important selective factor for wild-living A. flavus (Wicklow 1984b).

The DSL strain, ATCC 11866, has conidia that are within the size range described for A. flavus, and smaller in size than the range described for A. oryzae; thus, this strain might be expected to behave more like A. flavus in the environment.

A wide range of factors, including temperature, pH, water activity, presence of preservatives, and gaseous atmosphere may affect sporulation and spore germination in the fungal life cycle. In A. flavus, sporulation is optimal at an atmospheric water activity (a­­w) range between 0.86 and 0.96 (Vujanovic et al. 2001). When compared to some mycotoxin-producing Aspergillus, Cladosporium, and Penicilliumspecies, A. flavus may require more time to germinate at a lower aw (Vujanovic et al. 2001). In addition, A. flavusspore germination declines by 45% with an increase in temperature from 37°C to 41°C (Pasqualotto 2009). No germination of A. flavus spores is observed at 18°C or 12°C (Vujanovic et al. 2001).

All tested strains of A. flavus produce sclerotia, which are hardened, thick-walled spherical structures formed for survival under adverse conditions (Vujanovic et al. 2001). The majority of A. flavus sclerotia buried in sandy soils in the U.S. survived at least 36 months burial, while buried A. flavus conidia survived for a maximum of 24 months (Wicklow et al. 1993). Although most A. oryzae strains, including the DSL strain ATCC 11866, have lost the ability to produce sclerotia (reviewed in Wada et al. 2014), some strains such as A. oryzae RIB40 produce large and abundant sclerotia (Rank et al. 2012). In a study testing the persistence of A. oryzaeCCFC008014 live cells inoculated into intact soil microcosms, a reduced quantity of A. oryzae DNA within the soil was seen by day 46 post-inoculation, and by day 126, the concentration of DNA had declined approximately 32 fold relative to the concentration on day 2. Both qualitative and quantitative PCR analyses indicated that A. oryzae declined in abundance initially, but survived for the full test period of 126 days (Hynes et al. 2006). A. oryzae can survive in soil for several months – including under cold conditions – which indicates that A. oryzae used in industrial applications could survive in highly competitive soil environments, and if released into the Canadian environment, are likely to persist for at least one growing season (Hynes et al. 2006). However, information that A. flavus and A. oryzae are uncommon above 45 degrees latitude suggests that introduced populations are unlikely to overwinter successfully enough to be maintained in many parts of Canada. In addition, maintenance of high numbers of introduced micro-organisms beyond background levels is unlikely due to natural competition with naturally occurring micro-organisms in the environment (Leung et al. 1995). Antifungal susceptibility

Overall, antifungal resistance in cases of human mycoses is increasing (Hedayati et al. 2007). Until recently, aspergilloses have mainly been treated with itraconazole or amphotericin B, but now, other antifungal drugs such as voriconazole, posaconazole and caspofungin have been approved to treat aspergillosis (Hedayati et al. 2007). The antifungal susceptibility profile of A. oryzae ATCC 11866 generated by Health Canada scientists (Table 1-2) shows that the DSL strain is susceptible to amphotericin B, itraconazole and voriconazole, which is consistent with susceptibility profiles reported for clinical isolates of A. flavus. Voriconazole is considered the most effective antifungal agent against A. flavus (Misra et al. 2011), and caspofungin (Denning 2006; Maertens et al. 2004) and terbinafine (Li et al. 2008) have been proposed for use when other drugs fail to control infection. Futhermore, the DSL strain is resistant to anidulafungin, caspofungin, fluconazole and miconazole for which resistance has been reported in isolates of A. flavus (Eschertzhuber et al. 2008; Fattahi et al. 2012; Li et al. 2008).

Table 1-2: Minimal Inhibitory Concentration (MIC) for A. oryzae ATCC 11866a
Antifungal DrugMIC after 48h (µg/mL)Breakpointb
Amphotericin B0.5A. flavus IE+; A. fumigatus S less than or equal to 1 R greater than 2
Anidulafungingreater than 8IE
Caspofungingreater than 8IE
5-Flucytosinegreater than 64NA
Itraconazole0.12S less than or equal to 1 R greater than 2
Micafungingreater than 8IE
Posaconazole0.06A. flavus IE+; A. fumigatus S less than or equal to 0.12 R greater than 0.25
Voriconazole0.5A. flavus IE+; A. fumigatus S less than or equal to 1 R greater than 2

IE: insufficient evidence that A. flavus is a good target for therapy with the drug.
IE+: insufficient evidence that A. flavus is a good target for therapy with the drug and the epidemiological cut-off values for A. flavus are in general one step higher than for A. fumigatus.
-: susceptibility testing is not recommended as the species is a poor target for therapy with the drug
S; Susceptible, R: Resistant, NA: Not Available

a Data generated by Health Canada's Environmental Health Science and Research Bureau. Work conducted by using Sensititre Assay Kit (TREK Diagnostic Systems) and by following manufacturer's protocol. The MIC was interpreted as the lowest concentration corresponding to a blue-coloured well, indicating no growth.

b Breakpoint for interpretations of Minimal inhibitory concentration from EUCAST 2014.

The fungal cell targets of antifungal drugs such as polyenes, nystatin and azole are structurally analogous to some components of human cells (reviewed in Lamb et al. 2000); and as a result, some antifungal drugs are relatively toxic to humans. However, antifungals such as echinicandins (which include caspofungin, micafungin and anidulafungin) target fungal cell wall components that are lacking in humans; and therefore, have few side effects (S. Perkhofer, personal communication). In lieu of antifungal therapy, less commonly employed treatments for fungal infection include surgical debridement of the infected tissue and the use of steroid sprays. In refractory cases, the use of a combination of these treatments with antifungal therapy may be required; however, these procedures are also damaging. Pathogenesis

In Aspergillus species, small conidial size is one of the most significant factors affecting pathogenicity in humans (Denning 1998; Speth and Rambach,2012) and presumably also in dogs, horses and in particular birds, as in all, the most common route of infection is through the inhalation of conidia (Denning 1998; Speth and Rambach 2012). It is generally recognized that particles between 0.01 and 5 microns in diameter can be deeply inhaled into the pulmonary alveoli (Witschi et al. 2008). Pathogenic Aspergillus species have smaller conidia compared to non-pathogenic species; the conidial size of A. flavusranges from 3 to 7.5 microns in diameter (Christensen 1981; Larone 2011), while the conidia of A. oryzae are typically larger, ranging from 5 to over 6 microns in diameter. However, the DSL strain, ATCC 11866, has conidia between 4.0 and 4.8 microns in diameter, which are within the size range that can be deeply inhaled into the pulmonary alveoli.

After conidia have gained access to the pulmonary alveoli within the lungs, they germinate and grow hyphae (Denning 1998). The hyphal tip secretes extracellular enzymes into the pulmonary tissue, including aspartyl proteinase, serine proteinase, metalloproteinase, alkaline proteinase and lipases. These enzymes act as virulence factors, by breaking down complex host protein and lipid molecules, the subunits of which are used as fungal nutrients. This process also digests the extracellular matrix of the host to facilitate tissue penetration (Krishnan et al. 2009). Other hydrolases, such as α-amylases, pectinases, and other proteases and lipases are also involved in fungal virulence (reviewed in Amaike and Keller 2011). The DSL strain ATCC 11866 was originally selected for its high proteolytic activity, but it is unclear whether this affects its virulence.

Pectinases produced by plant pathogens macerate plant tissues thereby facilitating plant invasion and capture of nutrients (reviewed in Mellon et al. 2007). Polygalacturonase, P2c, is a major player in the maceration of host tissues in A. flavus and is correlated with virulence to plants (reviewed in Mellon et al. 2007). Other hydrolases such as amylase can breakdown the plant storage polysaccharides, creating intermediates such as glucose, maltose and maltotriose that have a role in the induction of aflatoxins biosynthesis (reviewed in Mellon et al. 2007). It is not known whether the DSL strain of A. oryzae, ATCC 11866, produces these hydrolases.

Elastase produced by A. flavus is also considered to play a role in the invasion of host tissues because elastin is a major structural component of the lung. Of 23 A. flavusstrains isolated from human patients suffering from invasive aspergillosis, 19 had elastase activity (Alp and Arikan 2008). It is not known whether the DSL strain of A. oryzae, ATCC 11866, produces elastase.

A. oryzae has haemolysin (Nayak et al. 2013) and haemolysin-like (Bando et al. 2011) coding genes, with a promoter for one of these genes possessing high activity. Haemolysins are potential virulence factors produced by A. terreus and A. fumigatus (Nayak et al. 2011; Wartenberg et al. 2011) that lyse erythrocytes and other cells (Nayak et al. 2011). Hemolysis was not observed when the DSL strain, ATCC 11866, was grown on sheep blood agar (Appendix A, Table A-2).

A. flavus can synthesize and release a complement inhibitor (CI) molecule within the human host, which inhibits both the activation of the complement cascade and the resulting fungal opsonisation. CI prevents activation of the alternative pathway, and interferes with complement protein C3b-induced phagocytosis and fungal cell killing (reviewed in Speth and Rambach 2012). It is not known whether the DSL strain ATCC 11866 produces CI.

Various Aspergillus species, including A. flavus, produce dihydroxynaphthalene (DHN) – melanin (Thywißen et al. 2011), a pigment that covers the conidia and protects them from ingestion by macrophages, as well as heat, ultraviolet radiation, and extremes of pH (Krishnan et al. 2009). There is limited information on DHN-melanin in A. oryzae. Genes for DHN-melanin synthesis are present in the genome of A. oryzae RIB 40 but gene expression has not been shown (Baker 2008). DHN-melanin has not been reported for the DSL strain ATCC 11866.

In vitro tests conducted by Health Canada scientists to evaluate the potential of A. oryzae ATCC 11866 cells to cause cytotoxicity and adverse immune effects demonstrated that ATCC 11866 was non-cytotoxic to moderately cytotoxic towards human colonic epithelial cells (HT29) and macrophage cells (J774A.1) after 4- and 24-hour exposuresFootnote 4. Toxigenesis

Species in the A. flavus group are known to produce secondary metabolites, including mycotoxins. Secondary metabolites are compounds produced by an organism that are not required for a physiological function (i.e., growth, development or reproduction of the organism), some of which are presented here because they have been reported to have negative effects. Mycotoxins, a subset of these, are small organic molecules produced by filamentous fungi that can cause disease and death in humans and animals through a natural exposure route (Bennett 1987). Mycotoxins enter the human food chain when the fungus grows and produces the toxin in foods such as vegetables or grains or when food animals ingest the toxins in contaminated animal feed. Toxins may also be inhaled along with spores when handling infected material. Mycotoxins and some secondary metabolites produced by A. oryzae and A. flavus are listed in Appendix B (Table B-1). For A. oryzae, these include: cyclopiazonic acid, kojic acid, maltoryzine and 3-nitropropionic acid (Blumenthal 2004; Samson et al. 2006). It is unclear whether the DSL strain, ATCC 11866, produces these or other mycotoxins associated with the A. flavus group, including: aflatrem, aspergillic acid, aspertoxin, gliotoxin, or sterigmatocystin (Appendix B, Table B-1).

Some strains of A. flavus produce the potent, carcinogenic mycotoxins, aflatoxins, which is regulated in numerous countries (reviewed in Klich 2007). The DSL strain ATCC 11866 did not produce aflatoxins when tested on rice, peanut or YES medium after incubation at 25 °C for 8-10 days, whereas strains of A. flavus, the wild type counterpart of A. oryzae, produced aflatoxins under the same conditions (Wei and Jong 1986). A. oryzae is considered by some taxonomists as a group of domesticated strains of A. flavus that have lost the ability to produce aflatoxins (Jorgensen 2007). A full range of intraspecific variation exists within the A. oryzaeaflatoxins biosynthetic pathway, from deletion of 0.8 kb to 1.5 kb of the aflatoxins gene cluster to no deletions (reviewed in Jorgensen 2007). In many strains of A. oryzae, all the genes necessary for aflatoxins biosynthesis are present, yet inactive (Chang et al. 1995; Klich et al. 1995; Kusumoto et al. 1998; Lee et al. 2006; Nakamura et al. 2011; Watson et al. 1999). No data are available on the completeness of the aflatoxins synthesis gene cluster in the DSL strain of A. oryzae, ATCC 11866.

The production of secondary metabolites (including toxins) in A. oryzae is strain-specific and environment dependent (Park et al. 2008). Environmental factors which are known to have an impact on toxin production by strains of A. oryzaeinclude: growth temperature, where optimal toxin production is achieved in the range of 25-35°C; the growth medium or substrate composition; and incubation time, where toxin production is favoured by longer incubation periods (Adebajo 1992; Blumenthal 2004; Jorgensen 2007). In the practice of koji fermentation, the normal fermentation time period is short (2 to 3 days), whereas secondary metabolites are produced by A. oryzae typically after 3 days of incubation. This may explain, in part, why mycotoxins are not detected in the final fermented products. Some secondary metabolites, including kojic acid and cyclopiazonic acid, could also be degraded during the fermentation process (reviewed in Jorgensen 2007).

It is reasonable to assume that if the DSL strain, ATCC 11866, encounters suitable conditions in environments to which it is released, it may produce any of the above-mentioned mycotoxins that it is able to produce.

1.1.3 Effects Environment

Since A. oryzae and A. flavus are essentially indistinguishable by most molecular techniques and the DSL strain is morphologically similar to A. flavus, environmental effects caused by both A. oryzae and A. flavushave been considered in this assessment. Available information on A. oryzae is presented first, since few cases are reported in the literature, followed by information on A. flavus.

A) A. oryzae

Enzymes secreted by A. oryzae (e.g., xylanase, polygalacturonase, cellulase and α-amylase) can degrade plant cell walls (reviewed in Al-Hindi et al. 2011; Chang et al. 2012b; Um and Walsum 2010), and may allow it to act as a saprophyte on decaying plant material; however, there are no reports of negative effects on living terrestrial or aquatic plants in the scientific literature.

Although A. oryzae is not considered to be an animal pathogen, a few cases of infection have been reported. A. oryzae caused one case of spontaneous keratomycosis of a previously injured cornea in a horse (Marolt et al. 1984). Several cases of spontaneous fatal aspergillosis in various species of parrot were associated with A. oryzae, confirmed through morphological and histopathological examination (Kaplan et al. 1975). All the parrots were predisposed to infection because of stress associated with capture and shipment to the U.S., co-infection with psittacosis, and treatment with either antibiotics or corticosteroids. Histologic investigations identified hyphal structures and conidia in the pulmonary tissues (Kaplan et al. 1975). Possible allergic effects in animals have also been reported (Lugauskas et al. 2004).

A. oryzae was also reported as a parasite of the Beet Armyworm (Spodoptera exigua) (Kenneth and Olmet 1975), and in pathogenicity tests (inoculation of food, spraying of spore suspensions and dusting of spores), A. oryzae caused mortality in all life stages of the Maize Stem Borer (Chilo partellus) and was pathogenic to the Codling Moth larvae (Cydia pomonella) (Gardezi 2006).

No reports were found in the scientific literature of A. oryzae having negative effects on aquatic vertebrate or invertebrate species.

Given that it is difficult to differentiate A. oryzaefrom A. flavus, it is possible that the causative agent in some of the cases described above was A. flavus, erroneously identified as A. oryzae.

B) A. flavus

A. flavus has been widely reported in the literature to infect many terrestrial and aquatic species of organisms:


As a known plant pathogen, A. flavus commonly affects maize, cottonseed, tree nuts, oilseed crops, and peanut plants (reviewed in Klich 2007). Insect damage to plant tissues increases the susceptibility of plants to fungal infection (reviewed in Klich 2007). In maize, A. flavus infection causes ear rot, while in peanuts, infection causes a disease termed yellow mould of seedlings (also known as aflaroot), which manifests as chlorosis and necrotic lesions of the plant's upper tissues, rot of mature peanuts, and developmental failure of secondary roots (reviewed in Klich 2007). In cotton plants, A. flavus infection can lead to boll rot and infection of the fibre, causing yellow spot disease, although infection is most likely in bolls under water stress during maturation (reviewed in Klich 2007).

Plant embryos can be damaged by mycotoxins in A. flavus-contaminated grain crops, resulting in germination failure or reduced fitness of the seedling (Hasan 1999). Experimental challenges reported in the literature indicate that A. flavus infection is associated with lower seed viability of cottonseed plants (Klich 2007), peanut plants (Pitt et al. 1991), and Himalayan Cedar (Cedrus deodara) (Mittal 1983), and that viability further decreased in the presence of aflatoxins (Klich 2007).

No reports of negative effects on aquatic plants from A. flavus were found in the scientific literature.


A. flavus is the second most common cause of avian aspergillosis after A. fumigatus, and frequently kills 5-10% of birds in domestic poultry flocks (reviewed in Richard et al. 1984). The primary route of A. flavus infection in birds is inhalation of large amounts of spores (Richard et al. 1984). Immunocompromised birds are generally at the greatest risk of infection and turkeys are reported to be more susceptible than chickens (Richard et al. 1984). Young waterfowl (ducks and geese) are often affected by A. flavus after exposure to high levels of spores in a hatchery environment (Morishita 2004).
Fatal outbreaks of aspergillosis caused by A. flavuswithin commercial poultry houses have been reported:

A. flavus is known to cause keratomycosis in horses. In one case, A. flavus was recovered from the affected cornea of a horse in Japan. Fungal hyphae were observed in the corneal scrapings (Wada et al. 2013). Aspergillus species (A. niger, A. flavus, and unidentified species) were the most common isolates (64%) in equine keratomycosis in California (Reed et al. 2013).

Additional cases have been reported involving the successful treatment of A. flavus infection in animals using antifungal therapy:

There are a few reports of experimental infections induced using A. flavus in terrestrial vertebrate species:

A. flavus is ubiquitous in marine environments; however, little is known about the behavior of fungi in marine ecosystems (Zuluaga-Montero et al. 2010). An outbreak of aspergillosis caused by A. flavus and A. nigerwas reported among tilapia fish (Sarotherodon species) in a Kenyan fish farm (Olufemi et al. 1983).


A. flavus is the most common Aspergillusspecies to infect insects (reviewed in Foley et al. 2014). It is associated with different stages of a variety of insects and is very potent in decomposing chitin (Badran and Aly 1995; reviewed in Ismail and Abdel-Sater 1993). It is a rare facultative pathogen of honey bees (Apis mellifera), in which it is the primary cause of stonebrood disease, causing mummification of larvae and infecting adult bees in colonies weakened by predisposing factors (Vojvodic et al. 2011). A. flavus infection in the bee species Tetralonia lanuginose can cause up to 50% decay of pupae and nymphs (Mohamed and El-Khadem 1976, referred to in the abstract of a German-language journal article). A. flavuswas found at an incidence of 10% in a population of grasshopper (Zonocerus variegatus) in Nigeria with a 76% total fungal incidence (Balogun and Fagade 2004), and was found to cause natural infection in 6% of a Southern Cattle Tick (Rhipicephalus {Boophilus} microplus) population surveyed in Mexico (Miranda-Miranda et al. 2012).

Experimental infections with A. flavus in terrestrial invertebrate species at unspecified challenge doses had the following effects:

Effects of experimental infections with specified doses of A. flavus were reported as follows:

A. flavus is also known to affect marine invertebrates, as a common isolate from the tissue of diseased sea fans (Gorgonia ventalina), indicating its potential role in sea fan aspergillosis (reviewed in Zuluaga-Montero et al. 2010). No reports were found in the scientific literature of A. flavus having negative effects on fresh water invertebrate species.

C) Mycotoxicosis

Consumption of food or feed that is contaminated with mycotoxins may cause a variety of symptoms, depending on the type of mycotoxin, quantity and duration of exposure (Kanora et al. 2009), animal species, its age, and nutritional and health status at the time of exposure to contaminated feed (Prelusky et al. 1994). Mycotoxicosis can affect a wide range of susceptible animal species, including livestock, poultry and fish (Marasas and Nelson 1987; Moss 1996). Grains, cereals or products made from such grains are common sources of mycotoxin exposure (Binder 2007; Richard 2007; Sweeney and Dobson 1998). The signs elicited by mycotoxin consumption vary with the amount ingested. Mycotoxin concentrations occurring under field conditions lead to reduced animal performance or immune suppression without causing overt clinical signs (Oswald and Comera 1998; Marquardt 1996). Clinically overt disease results from ingestion of higher concentrations. Signs of mycotoxin intoxication include diarrhea, liver and kidney damage, pulmonary edema, vomiting, hemorrhaging and tumours (Binder 2007; Bryden 2012). The following are examples of the case reports related to mycotoxins produced by A. oryzae and A. flavusfound in the literature: Human health


In spite of the long-term traditional use of A. oryzaein fermentation, no reports of infection are associated with occupational or household exposure to A. oryzaefermentation strains. However, members of the species A. oryzae have caused a small number of infections in immunocompromised, debilitated, or injured patients:

Given that it is difficult to differentiate A. oryzaefrom A. flavus, it is possible that the causative agent in some of the cases described above was A. flavus, erroneously identified as A. oryzae. A. flavus, which is considered to be the wild type of A. oryzae, is a human opportunistic pathogen, and systemic infection is often fatal in affected immunocompromised individuals (reviewed in Amaike and Keller 2011). A. flavus is the second most common cause of aspergillosis after A. fumigatus (Guarro et al. 2010).

In the following cases, A. flavus was identified as a cause of infection in patients with predisposing health conditions including nephritis, uremia, hepatitis, cholecystitis, tuberculosis, haemoptysis, diabetes, heart disease, hypertension, leukemia, lung cancer, and neuroblastoma. Many of these patients had undergone chemotherapy, surgery, or organ transplants as treatments for these diseases, and were thereby immunocompromised, debilitated, or were exposed to A. flavus through significant breaches in normal barriers to infection:

Hospital-acquired infections associating A. flavus have also been reported:

There are a few reports of A. flavus infections in otherwise-healthy individuals:


There have been multiple reports in the literature of allergy to alpha–amylase produced by A. oryzae, which are often linked to breadmaking as baker's asthma. Hypersensitive pneumonitis in brewers exposed to A. oryzae has also been reported (Tsuchiya et al. 1993).

Allergic bronchopulmonary aspergillosis (ABPA) has been associated with A. oryzae, which is a form of aspergillosis related to the inhalation of Aspergillusspores. Five similar cases of A. oryzae ABPA were reported in Japan related to the production of soybean products (Akiyama et al. 1987), and a case of ABPA was reported in which immunological tests indicated that both A. fumigatus and A. oryzae were involved, but fungal material was not isolated from the patient (Kurosawa et al. 1990). In Japan, it is estimated that more than 30,000 individuals are exposed to high concentrations of A. oryzae spores from the fermentation of soy products in small family workshops (Akiyama et al. 1987); given this context, Kurosawa et al. (1990) stressed the fact that surprisingly only a few cases of ABPA due to A. oryzaehave been reported.

A. flavus caused allergic fungal sinusitis (Chhabra et al. 1996) and allergic Aspergillus pneumonitis (Bakri et al. 2010) in two patients with either a history of asthma, or a weak immune system resulting from leukemia. A. flavus also caused allergic reactions or hypersensitivities among people in India with no predisposing conditions, including allergic fungal rhinosinusitis (Taj-Aldeen et al. 2004; Taj-Aldeen et al. 2003).

In vivo testing of A. oryzae ATCC 11866 using BALB/c mice as a model for human infection were done at Health CanadaFootnote 5. Four replicate BALB/c mice per time point exposed to 1 x 106 conidia/25 μL by endotracheal instillation for 2 hours to 1 week showed that there were no changes in behaviour or physical appearance. In addition, significantly elevated levels of proinflammatory cytokines, IL-1beta, IL-6, and TNF-alpha and KC in the lungs were observed, maximally between 4 and 48 hours post-exposure, declining to control levels by 4 days to 1 week post-exposure. These observations were accompanied by an elevation in levels of pulmonary granulocytes between 4 and 48 hours post-inoculation. These results are indicative of transient local inflammation resolving within one week; furthermore, there was clearance of A. oryzae ATCC 11866 from the lungs by 4 days post-exposure, with the lungs remaining clear at one week.

1.2 Hazard Severity

A. oryzae strains have a long history of use in the production of enzymes for brewing and baking, and as "koji moulds" for the fermentation of soy sauce, miso and sake (Barbesgaard et al. 1992; Blumenthal 2004; Jorgensen 2007). Until 1965, cultivation of A. oryzae in Europe took place in open trays, and massive amounts of conidia were released into the environment (Barbesgaard et al. 1992). Despite its long history of use in food production, there have been no reported safety concerns associated with exposure to A. oryzae.

A. oryzae is considered to be a domesticated strain of the wild type species A. flavus, and is thought, through domestication, to have lost, inactivated or modified some genes related to spore formation and dispersal, sclerotium formation and aflatoxins production (Jorgensen 2007; Kurtzman et al. 1986; Wicklow 1984a).These changes also appear to make A. oryzaeless hazardous to environmental species and humans than A. flavus; however, the DSL strain ATCC 11866 has characteristics more typical of A. flavus.This is relevant especially with respect to conidial size, which in the DSL strain could favour infectivity in organisms in which inhalation is the principal route of infection. Futhermore, as with A. flavus, the DSL strain can grow at 37°C. Nevertheless, the DSL strain, ATCC 11866, differs from A. flavus in ways that reduce the estimation of hazard: ATCC 11866 is non-hemolytic and non-aflatoxigenic.

Sources of uncertainty in the assessment of hazard include a lack of information on the ability of A. oryzae ATCC 11866 to produce mycotoxins other than aflatoxins, a lack of information on the potential for A. oryzae strains to regain characteristics favouring fitness and infectivity under selective pressures encountered in the environment, and an absence of a history of safe use of A. oryzae ATCC 11866.

To help prevent the formation and consumption of mycotoxins, the livestock feed industry has established internal monitoring methods. Similarly, government regulatory agencies, including the CFIA, regulate mycotoxin levels in livestock feeds; non compliance with the CFIA Feeds Regulations is subject to the compliance and enforcement policies of that agency (Bennett and Klich 2003; CFIA 2014).

1.2.1 Environment

A. oryzae is implicated in a small number of animal infections, whereas some A. flavus isolates are pathogenic to plants, birds, and insects, and to a lesser extent, mammals. Most mammals and birds that were reported to be naturally infected by A. oryzae and A. flavus had predisposing health conditions and were exposed to high concentrations of spores. No negative effects were reported when A. oryzaewas used as a probiotic in mammals and birds; therefore, ingestion of A. oryzae would not appear to be of concern. In the event of an infection, antifungal treatments are available to mammals. However, terrestrial invertebrates seem to be highly susceptible to A. oryzae and A. flavuspathogenicity.

Given that the DSL strain, ATCC 11866, shares characteristics with A. flavus that could favour its infectivity (i.e., small conidia, growth at 37°C), but lacks other determinants of pathogenicity and toxicity, and taking uncertainties into account, its hazard severity is estimated to be intermediate between that of A. flavus and 'typical' A. oryzae. Based on surrogate information relating to the species A. oryzaeand A. flavus, the DSL strain ATCC 11866 may pose a potential hazard to plants, birds, insects, and mammals, especially those that are predisposed to infection.

Thus, based on these considerations, and on the uncertainties described above, the environmental hazard severity for A. oryzae ATCC 11866 is estimated to be medium.

1.2.2 Human Health

Hazards related to micro-organisms used in the workplace should be classified accordingly under the Workplace Hazardous Materials Information System (WHMIS)Footnote 6.

In spite of a long history of safe use of A. oryzae in food production (both commercially and domestically), there are very few case reports of A. oryzae infection in humans. In addition, the human body temperature (37°C) is optimal for the growth of A. flavus, but A. oryzae grows optimally at a lower temperature (34°C). The DSL strain can grow at normal, but not at febrile (42°C) human body temperature. The conidial size of the DSL strain could favour infectivity, making its hazard potential closer to that of A. flavus for pulmonary infections. Since there are no reports of gastrointestinal illness caused by A. oryzae or A. flavus, ingestion of A. oryzae ATCC 11866 is not of concern.

The vast majority of A. flavus-related diseases in healthy humans are mild, self-resolving and usually treatable, but there have been mortalities in immunocompromised individuals.

A lower estimation of hazard is supported by experimental testing conducted by Health Canada in a mouse model of human infection, in which no adverse effects were observed following endotracheal exposure to A. oryzae ATCC 11866. Although there was an inflammatory cytokine response, it was transient, and A. oryzae was cleared from the lungs within one week.

Based on these considerations, and taking into account the uncertainties described above, the human health hazard severity for A. oryzae ATCC 11866 is estimated to be low for healthy individuals and medium for immunocompromised individuals.

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2. Exposure Assessment

2.1 Sources of exposure

The focus of this assessment is to characterize exposure to A. oryzae ATCC 11866 from its deliberate addition to consumer or commercial products or its use in industrial processes.

A. oryzae ATCC 11866 was nominated to the DSL in 1997 for its use in consumer and commercial products. In 2007, a voluntary questionnaire was sent to a subset of key biotechnology companies. These results, combined with information obtained from other federal government regulatory and non-regulatory programs, indicate that 10,000 to 100,000 kg of products potentially containing A. oryzae ATCC 11866 (formulation and concentration unknown) were imported into or manufactured in Canada in 2006.

To update information about current uses, the Government of Canada conducted a mandatory information-gathering survey under section 71 of CEPA, as published in the Canada Gazette, Part I, on October 3, 2009 (hereafter referred to as the section 71 Notice). The section 71 Notice applied to any persons who, during the 2008 calendar year, manufactured or imported A. oryzae ATCC 11866, whether alone, in a mixture, or in a product. No commercial or consumer activities using A. oryzae ATCC 11866 were reported in response to the section 71 Notice.

The 2007 and 2009 surveys differed significantly in target and scope. In this assessment, results from the 2009 survey were used to estimate exposure from current uses because it required that requested information on uses of the micro-organism strain that is listed on the DSL be provided, whereas the 2007 survey asked about uses of the products that had been associated with the micro-organism at the time it was nominated to the DSL. Because product formulations may have changed, information from the 2009 mandatory survey may more accurately represent current uses. Uses reported in the 2007 voluntary survey were also considered in the assessment of potential uses.

The DSL strain ATCC 11866 was originally selected for its high protease activity. This characteristic could be applied for preparing bates for the tanning industry and for hydrolyzing fish, animal, and vegetable proteins into food (Baens-Arcega et al. 1956). A search of the public domain (MSDS, literature and patents) revealed the following consumer, commercial and industrial applications for A. oryzae ATCC 11866:

A. oryzae is metabolically diverse, which makes it of commercial interest in a variety of industries, particularly in enzyme production and food fermentation. Although no uses were reported for ATCC 11866 during the mandatory survey, it is available for purchase from the ATCC. As it is on the DSL, and so can be used in Canada without prior notification, it could be an attractive choice for commercialization.

A search of the public domain (MSDS, literature and patents) revealed the following consumer, commercial and industrial applications of other strains of A. oryzae. These represent possible uses of the DSL strain, as strain ATCC 11866 is likely to share the characteristics (modes of action) with other commercialized A. oryzae strains:

These uses are largely industrial, but its ability to degrade organic wastes makes it of possible interest for use in certain consumer products, including septic tank treatments, drain cleaners and degreasers.

2.2 Exposure characterization

2.2.1 Environment

The environmental exposure for A. oryzae ATCC 11866 is estimated to be low based on the absence of responses to the 2009 section 71 Notice, suggesting that this strain is no longer used in consumer or commercial products or for industrial processes in Canada.

Nevertheless, environmental exposure scenarios, in the event that consumer, commercial or industrial activities with A. oryzae ATCC 11866 resume, have been considered along with the persistence and survival properties of this micro-organism. Due to the expanding commercialization of microbial-based products, some potentially containing A. oryzae ATCC 11866, there is a likelihood of an increase in the use and release of this micro-organism in the environment (Chatzipavlidis et al. 2013).

Former and possible future uses of the DSL strain are described in Section 2.1. These would likely introduce A. oryzae ATCC 11866 into both aquatic and terrestrial ecosystems. For example, uses in bioremediation or biodegradation would involve direct application to soils, and subsequent rainfall events could introduce A. oryzae ATCC 11866 into waterways. In addition, the potential uses of A. oryzae ATCC 11866 in waste water treatment facilities and for the production of biofuels, organic acids or enzymes could lead to direct input into waterways in wastewater effluent.

The magnitude of plant and animal exposure to A. oryzae ATCC 11866 will depend on the nature of the use and on its persistence and survival in the environments to which it is released. Hynes et al. (2006) demonstrated that A. oryzaecan persist 4 months after inoculation into soil microcosms; the ability of A. oryzae to survive wastewater treatment from industrial discharges, probably due to its resistant conidia, suggests that it is likely to persist after introduction into aquatic environments (U.S. EPA 1997a).

On the basis of its larger conidial size, A. oryzae is generally considered less fit to disperse in nature than A. flavus (Horn 2003; Wicklow 1984a); however, the conidia of A. oryzae germinate earlier and produce a larger mycelium than wild relatives. In an environment where it is in competition with its wild-type counterparts, it could provide a competitive advantage over its wild-type counterparts in accessing available substrates at a particular site (Wicklow 1984b). The DSL strain,ATCC 11866, shares morphological characteristics with A. flavus, such as smaller conidia, that could favour its dispersal in the environment, but reduce its competitiveness. In addition, the DSL strain does not produce sclerotia, and this limits its ability to persist under unfavourable conditions in soil relative to A. flavus. Considering that A. oryzaeand A. flavus are uncommon above 45 degrees latitude, it is unlikely that introduced populations of these species can overwinter successfully enough to be maintained in many parts of Canada.

2.2.2 Human

Human exposure to A. oryzae ATCC 11866 is estimated to be low based on responses to the 2009 section 71 Notice, which indicated that this strain was no longer used in consumer or commercial products or for industrial processes in Canada in 2008.

Nevertheless, human exposure scenarios in the event that consumer, commercial or industrial activities with A. oryzae ATCC 11866 resume have been considered. These are based on former and probable future uses as described in Section 2.1. Should potential uses of A. oryzae ATCC 11866 be realized, human exposure would be expected primarily through direct contact with consumer products containing A. oryzae ATCC 11866. The handling and application of such products would be expected to result in direct exposure to the skin, as well as exposure through inhalation of aerosolized droplets or dusts containing A. oryzae ATCC 11866. Secondary to product application, residual A. oryzae ATCC 11866 on surfaces or in reservoirs such as treated drains could result in dermal exposure; secondary exposure through inadvertent ingestion could occur where the organism persists on food preparation surfaces; inhalation may occur where aerosols are generated (e.g., from kitchen garbage disposal units), or where spores are formed in reservoirs such as treated drains. If custodial products containing A. oryzae ATCC 11866 were used in hospitals, clinics, or long-term care facilities, there would be an increased risk that susceptible individuals could be exposed. Since A. oryzae ATCC 11866 is expected to persist in organic-rich sites following application, such exposures may be temporally distant from the time of application.

The general population could be exposed to A. oryzae ATCC 11866 as bystanders during the application of commercial products containing this strain. The route and extent of exposure will depend on the nature of the product, the application method, the concentration of A. oryzae ATCC 11866 in the product, the amount of product applied, and the proximity of the bystander to the site of application, but in general is expected to be moderate to low. The general population could also come into contact with residual A. oryzae ATCC 11866 on surfaces treated with commercial products.

Indirect exposure to A. oryzae ATCC 11866 in the environment subsequent to its use in bioremediation and biodegradation, bioleaching, textile processing, municipal or industrial wastewater treatment or in disposal of waste from its use in the production of enzymes and fermentation extracts is likely to occur. Certain uses in waste and wastewater treatment or in industrial processes may introduce A. oryzae ATCC 11866 into bodies of water. Human exposure to this strain through recreational activities is expected to be low. Drinking water treatment processes might not eliminate this micro-organism (Sisti et al. 2012); however, ingestion of this micro-organism is not of concern. The micro-organism could possibly be inhaled from water droplets (e.g., while showering), but this would be expected to occur only in minute quantities due to dilution and because growth conditions for A. oryzae are not optimal in drinking water.

The exposure route of primary concern for human infection with A. oryzae, as for A. flavus, is through the inhalation of conidia; of secondary concern is infection through contact with broken skin or wounds, and the contamination of intravenous solutions and wound dressings (Kagen et al. 1983). Inhalation of fungal spores through smoking contaminated tobacco or marijuana has been reported (reviewed in Amaike and Keller 2011), but is an unlikely route of exposure from foreseeable future uses of A. oryzae ATCC 11866.

In the event that consumer, commercial or industrial activities resume, the human exposure to A. oryzae ATCC 11866 could change based on the exposure scenarios described above.

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3. Risk Characterisation

In this assessment, risk is characterized according to a paradigm whereby a hazard and exposure to that hazard are both required for there to be a risk. The risk assessment conclusion is based on the hazard, and on what is known about exposure from current uses.

Hazard has been estimated for A. oryzae ATCC 11866 to be medium for the environment, and for human health, low for healthy individuals, and medium for immunocompromised individuals. Environmental and human exposure to A. oryzae ATCC 11866 from its deliberate use in industrial processes or consumer or commercial products in Canada is not currently expected, so the risk associated with current uses is estimated to be low for both the environment and human health.

The determination of risk from current uses is followed by consideration of the estimated hazard in relation to foreseeable future exposures.

Risks to the environment from foreseeable future uses:

This assessment concludes that the hazard associated with A. oryzae ATCC 11866 is likely to be intermediate between the hazard typically associated with A. oryzae and that associated with A. flavus.

A. flavus is a plant pathogen commonly affecting maize, cottonseed, tree nuts, oilseed crops, and peanut plants (Klich 2007). It is also an opportunistic animal pathogen, causing mycosis, particularly in poultry, but also affecting insects, including bees and grasshoppers, and in marine environments, sea fans.

Wild plants and animals (including birds and insects) could be exposed to elevated concentrations of A. oryzae ATCC 11866 if it were used in bioremediation, biotransformation or biodegradation in contaminated sites; however, such releases would be areas already affected by contamination with toxic compounds such as textile dyes, phenols and chromium, which are also likely to affect plants and animals, and these sites are expected to be small relative to the total Canadian land mass. Crops and domestic animals, especially poultry, could be affected if such sites were adjacent to farms or pastures, but this is expected to be an exceedingly rare occurrence.

Aquatic or marine animals could inadvertently be exposed to A. oryzae ATCC 11866 through its use in waste water treatment, through runoff from terrestrial applications or release in industrial effluents. A. oryzae could persist and survive in the aquatic environment due to its conidia; however, no adverse effects have been found in the literature regarding freshwater species. In the marine environment, few effects were found in sea fans and sea farm fishes; even these effects are unlikely to be realized from commercial uses of A. oryzaeproducts because of the expected moderate extent of their use, and because of conidial dilution in receiving waters. Furthermore, the Canadian climate would limit survival of the conidia in the receiving water.

Therefore, the use of A. oryzae ATCC 11866 in bioremediation, biodegradation, and waste water treatment or in industrial processes is unlikely to have a serious impact on terrestrial or aquatic populations and trends over an entire ecosystem or an ecozone, and the risk from foreseeable future uses remains low.

Risks to human health from foreseeable future uses:

A. flavus is a human opportunistic pathogen leading to a wide array of infections including lung, sinus, skin, bone, eyes, ear, heart and systemic infections.

Industrial uses are not of significant concern from a human health perspective; however, its ability to degrade organic wastes makes A. oryzae ATCC 11866 of possible interest for use in certain consumer products, including septic tank treatments, drain cleaners and degreasers. Susceptible individuals could be exposed to A. oryzae ATCC 11866 during the application of consumer products containing A. oryzae ATCC 11866, or from contaminated surfaces or reservoirs such as drains. If custodial products containing A. oryzae ATCC 11866 were used in hospitals, clinics, or long-term care facilities, there would be an increased risk that susceptible individuals could be exposed and that medical devices could become contaminated. These risks are expected to increase with growth in the market for "greener" microbial-based cleaning products (Spök and Klade 2009; Vandini et al. 2014)), and so the risk from foreseeable future uses of A. oryzae ATCC 11866 is considered to be medium for human health.

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4. Conclusion

Based on the information presented in this screening assessment, it is concluded that A. oryzae ATCC 11866 is not entering the environment in a quantity or concentration or under conditions that:

Therefore, it is concluded that A. oryzae ATCC 11866 does not meet the criteria as set out in section 64 of the CEPA.

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5. References

Abd El-Moneim, O.M., Abd El-Rahim, A.H., Farag, I.M., and Fadel, M. (2013). Bioremediation of contaminated diets with industrial wastewater - phenol compounds using marine and earth fungi: DNA fragmentation, cytogenetic and sperm studies in mice. World Appl. Sci. J. 27, 302-310.

Abe, K., Gomi, K., Hasegawa, F., and Machida, M. (2006). Impact of Aspergillus oryzae genomics on industrial production of metabolites. Mycopathologia 162, 143-153.

Adebajo, L.O. (1992). Spoilage moulds and aflatoxins from poultry feeds. Nahrung 36, 523-529.

Akiyama, K., Takizawa, H., and Suzuki, M. (1987). Allergic bronchopulmonary aspergillosis due to Aspergillus oryzae. Chest 91, 285-286.

Alastruey-Izquierdo, A., Mellado, E., Peláez, T., Pemán, J., Zapico, S., Alvarez, M., Rodríguez-Tudela, J.L., and Cuenca-Estrella, M. (2013). Population-based survey of filamentous fungi and antifungal resistance in Spain (FILPOP study). Antimicrob Agents Chemother. 57, 3380-3387.

Al-Hindi, R.R., Al-Najada, A.R. and Mohamed, S.A. (2011). Isolation and identification of some fruit spoilage fungi: screening of plant cell wall degrading enzymes. Afr.J.l Microbiol. Res. 5, 443-448.

Allo, M.D., Miller, J., Townsend, T., and Tan, C. (1987). Primary cutaneous aspergillosis associated with Hickman intravenous catheters. N. Engl. J. Med. 317, 1105-1108.

Alp, S., and Arikan, S. (2008). Investigation of extracellular elastase, acid proteinase and phospholipase activities as putative virulence factors in clinical isolates of Aspergillusspecies. J. Basic Microbiol. 48, 331-337.

Amadi, J.E., and Adeniyi, D.O. (2009). Mycotoxin production by fungi isolated from stored grains. Afr. J. Biotechnol. 8, 1219-1221.

Amaike, S., and Keller, N.P. (2011). Aspergillus flavus. Annu. Rev. Phytopathol. 49, 107-133.

Antinori, S., Corbellino, M., Meroni, L., Resta, F., Sollima, S., Tonolini, M., Tortorano, A.M., Milazzo, L., Bello, L., Furfaro, E., Galli, M., and Viscoli, C. (2013). Aspergillusmeningitis: A rare clinical manifestation of central nervous system aspergillosis. Case report and review of 92 cases. J. Infect. 66, 218-238.

Arnow, P.M., Sadigh, M., Costas, C., Weil, D., and Chudy, R. (1991). Endemic and epidemic aspergillosis associated with in-hospital replication of Aspergillus organisms. J. Infect. Dis. 164, 998-1002.

Atalla, M.M., Hassanein, N.M., El-Beih, A.A., and Youssef, Y.A. (2003). Mycotoxin production in wheat grains by different Aspergilli in relation to different relative humidities and storage periods. Nahrung 47, 6-10.

ATCC. (2014). Product Description of Aspergillus oryzae ATCC 11866 (viewed June 2014).

Aziz, N.H., Youssef, Y.A., El-Fouly, M.Z., and Moussa, L.A. (1998). Contamination of some common medicinal plant samples and spices by fungi and their mycotoxins. Bot. Bull. Acad. Sin. 39, 279-285.

Baens-Arcega, L., Maranon, J., and Palo, M.A. (1956). Proteolytic enzyme from Philippine strain of Aspergillus oryzae (Ahlburg) Cohn. Philipp. J. Sci. 85, 189-201.

Baker, S.E. (2008) Aspergillus genomics and DHN-melanin conidial pigmentation. In Aspergillus in the genomic era, Varga, J., and Samson, R. A. eds., (The Netherlands: Wageningen Academic Publishers) pp. 77-85.

Bakri, S.J., Sears, J.E., Procop, G.W., Shrestha, R., and Gordon, S.M. (2010). Excellent visual outcome following Aspergillus flavus endogenous endophthalmitis-farmer's lung disease. Int. Ophthalmol. 30, 99-101.

Balogun, S.A., and Fagade, O.E. (2004). Entomopathogenic fungi in population of Zonocerus variegatus (l) in Ibadan, Southwest, Nigeria. Afr. J. Biotechnol. 3, 382-386.

Bando, H., Hisada, H., Ishida, H., Hata, Y., Katakura, Y., and Kondo, A. (2011). Isolation of a novel promoter for efficient protein expression by Aspergillus oryzae in solid-state culture. Appl. Microbiol. Biotechnol. 92, 561-569.

Barbesgaard, P., Heldt-Hansen, H.P., and Diderichsen, B. (1992). On the safety of Aspergillus oryzae: A review. Appl. Microbiol. Biotechnol. 36, 569-572.

Barton, J.T., Daft, B.M., Read, D.H., Kinde, H., and Bickford, A.A. (1992). Tracheal aspergillosis in 6 1/2-week-old chickens caused by Aspergillus flavus. Avian Dis. 36, 1081-1085.

Badran, R.A.M., and Aly, M.Z.Y. (1995). Studies on the mycotic inhabitants of Culex pipiens collected from fresh water ponds in Egypt. Mycopathologia 132, 105-110.

Behera, B. C., Mishra, R. R. and Thatoi, H. N. (2012). Diversity of soil fungi from mangroves of Mahanadi delta, Orissa, India. J. Microbiol. Biotechnol. Res. 2, 375-378.

Behnke, C.N., and Yendol, W.G. (1969). Pathogenesis of an Aspergillus flavus infection of Galleria mellonella eggs. Entomophaga 14, 177-184.

Belli, D.C.A., Eigenmann, P., Kahn, J.-M. and Polla, B. (1994) Organic Compounds. Patent CA 2125793 A1.

Beluffi, G., Bernardo, M.E., Meloni, G., Spinazzola, A., and Locatelli, F. (2008). Spinal osteomyelitis due to Aspergillus flavus in a child: A rare complication after haematopoietic stem cell transplantation. Pediatr. Radiol. 38, 709-712.

Benkhemmar O., Gaudemer F., Bouvier-Fourcade I. (1985). Heterokaryosis between Aspergillus oryzae cyclopiazonic acid-defective strains: method for estimating the risk of inducing toxin production among cyclopiazonic acid-defective industrial strains. Appl. Environ. Microbiol. 50,1087-93.

Bennett, J.W. (1987). Mycotoxins, mycotoxicoses, mycotoxicology and Mycopathologia. Mycopathologia 100, 3-5.

Binder, E.M. (2007). Managing the risk of mycotoxins in modern feed production. Anim Feed Sci. Tech. 133, 149-166.

Bhargava, S., Frisner, H., Bisgard-Frantzen, H. and Tams, J.W. (2005) A process of producing a fermentation product. Patent CA 2566252.

Blumenthal, C.Z. (2004). Production of toxic metabolites in Aspergillus niger, Aspergillus oryzae, and Trichoderma reesei: Justification of mycotoxin testing in food grade enzyme preparations derived from the three fungi. Regul. Toxicol. Pharmacol. 39, 214-228.

Boel, E., Tove, C. and Woldike, H. F. (2009) Process for the production of protein products in Aspergillus oryzae and a promoter for the use in aspergilllis. Patent CA 1341593.

Boyd, M.L., and Cotty, P.J. (2001). Aspergillus flavusand aflatoxin contamination of leguminous trees of the Sonoran Desert in Arizona. Phytopathology 91, 913-919.

Bryden, W.L. (2012). Mycotoxin contamination of the feed supply chain: implications for animal productivity and feed security. Anim.Feed Sci. Technol. 173, 134-158.

Buffington, J., Reporter, R., Lasker, B.A., McNeil, M.M., Lanson, J.M., Ross, L.A., Mascola, L., and Jarvis, W.R. (1994). Investigation of an epidemic of invasive aspergillosis: Utility of molecular typing with the use of random amplified polymorphic DNA probes. Pediatr. Infect. Dis. J. 13, 386-393.

Burdock, G.A., Soni, M.G., and Carabin, I.G. (2001). Evaluation of health aspects of kojic acid in food. Regul. Toxicol. Pharmacol. 33, 80-101.

Burwen, D.R., Lasker, B.A., Rao, N., Durry, E., Padhye, A.A., and Jarvis, W.R. (2001). Invasive aspergillosis outbreak on a hematology-oncology ward. Infect. Control Hosp. Epidemiol. 22, 45-48.

Byard, R.W., Bonin, R.A., and Haq, A.U. (1986). Invasion of paranasal sinuses by Aspergillus oryzae. Mycopathologia 96, 41-43.

Cafarchia, C., Paradies, R., Figueredo, L.A., Padalino, B., Greco, M.F., Greco, G., and Otranto, D. (2012). A Case of Equine Aspergillosis: A Novel Sampling Procedure for Diagnosis. J. Equine Vet. Sci. 32, 634-637.

CBSG (2013). Canadian biosafety standards and guideline for facilities handling human and terrestrial animal pathogens, prions and biological toxins. First Edition. Public Health Agengy of Canada, Ottawa, Canada.

Chang, H.M., Yu, H.H., Yang, Y.H., Lee, W.I., Lee, J.H., Wang, L.C., Lin, Y.T., and Chiang, B.L. (2012a). Successful treatment of Aspergillus flavus spondylodiscitis with epidural abscess in a patient with chronic granulomatous disease. Pediatr. Infect. Dis. J. 31, 100-101.

Chang, J., Cheng, W., Yin, Q.Q., Zuo, R.Y., Song, A.D., Zheng, Q.H., Wang, P., Wang, X. and Liu, J.X. (2012b). Effect of steam explosion and microbial fermentation on cellulose and lignin degradation of corn stover. Biores. Technol. 104, 587-592.

Chang, P.K., Bhatnagar, D., Cleveland, T.E., and Bennett, J.W. (1995). Sequence variability in homologs of the aflatoxin pathway gene aflR distinguishes species in Aspergillus section Flavi. Appl. Environ. Microbiol. 61, 40-43.

Chang, P.K. and Ehrlich, K.C. (2010). What does genetic diversity of Aspergillus flavus tell us about Aspergillus oryzae? Int. J. Food Microbiol. 138, 189-199.

Chang, P.K., and Ehrlich, K.C. (2011). Cyclopiazonic acid biosynthesis by Aspergillus flavus. Toxin Rev. 30, 79-89.

Chang, P.K., Ehrlich, K.C., and Fujii, I. (2009). Cyclopiazonic acid biosynthesis of Aspergillus flavus and Aspergillus oryzae. Toxins (Basel) 1, 74-99.

Chattopadhyay, S.K., Parihar, N.S., and Sikdar, A. (1996). Experimental infection with Aspergillus flavus in goats. Indian J. Anim. Sci. 66, 1003-1009.

Chatzipavlidis, I., Kefalogianni, I., Venieraki, A., and Holzapfel, W. (2013). Status and trends of the conservation and sustainable use of micro-organisms in agroindustrial processes. Commission on Genetic Resources for Food and Agriculture, Food and Agriculture Organization of the United Nations.

Chavan, M.N., Dandi, N.D., Kulkarni, M.V., and Chaudhari, A.B. (2013). Biotreatment of melanoidin-containing distillery spent wash effluent by free and immobilized Aspergillus oryzae MTCC 7691. Water Air Soil Pollut. 224, 1755.

Chaves, F.C., Gianfagna, T.J., Aneja, M., Posada, F., Peterson, S.W. and Vega, F.E. (2012) Aspergillus oryzae NRRL 35191 from coffee, a non-toxigenic endophyte with the ability to synthesize kojic acid. Mycol. Progress 11, 263-267.

Chhabra, A., Handa, K.K., Chakrabarti, A., Mann, S.B., and Panda, N. (1996). Allergic fungal sinusitis: clinicopathological characteristics. Mycoses 39, 437-441.

Chi, C.Y., Fung, C.P., and Liu, C.Y. (2003). Aspergillus flavus epidural abscess and osteomyelitis in a diabetic patient. J. Microbiol. Immunol. Infect. 36, 145-148.

Chiquette, J. (2009). Evaluation of the protective effect of probiotics fed to dairy cows during a subacute ruminal acidosis challenge. Anim.Feed Sci. Technol.153, 278–291.

Choi, J. Y., Shinde, P. L., Ingale, S. L., Kim, J. S., Kim, Y. W., Kim, K. H., Kwon, I. K. and Chae, B. J. (2011). Evaluation of multi-microbe probiotics prepared by submerged liquid or solid substrate fermentation and antibiotics in weaning pigs. Livestock Sci. 138,144-151.

Christensen, M. (1981). A synoptic key and evaluation of species in the Aspergillus flavus group. Mycologia 73, 1056-1084.

Ciegler, A., and Vesonder, R.F. (1983). Microbial food and feed toxicants: fungal toxins. In CRC handbook of foodborne diseases of biological origin, Rechcigl, M. ed., (Boca Raton, Florida: CRC Press) pp. 57-166.

CFIA. (2014). Canadian Food Inspection Agency. Feeds Regulations 1983 (SORS/83-539). (viewed November 2014).

Corso, C.R., Almeida, E.J.R., Santos, G.C., Morão, L.G., Fabris, G.S.L., and Mitter, E.K. (2012). Bioremediation of direct dyes in simulated textile effluents by a paramorphogenic form of Aspergillus oryzae. Water Sci. Technol. 65, 1490-1495.

Corso, C.R., and Maganha De Almeida, A.C. (2009). Bioremediation of dyes in textile effluents by Aspergillus oryzae. Microb. Ecol. 57, 384-390.

Denning, D.W. (1998). Invasive aspergillosis. Clin. Infect. Dis. 26, 781-805.

Denning, D.W. (2006). Aspergillus and aspergillosis - Progress on many fronts. Med. Mycol. 44, 1.

Domsch, K.H., Gams, W., and Anderson, T.H. (1980). Compendium of soil fungi (London, UK: Academic Press).

Doxtader, K.G., and Alexander, M. (1966). Role of 3-Nitropropanoic acid in nitrate formation by Aspergillus flavus. J. Bacteriol. 91, 1186-1191.

Ehrlich, K.C. (2008). Genetic diversity in Aspergillus flavus and its implications for agriculture. In Aspergillus in the genomic era, Varga, J., and Samson, R. A. eds., (The Netherlands: Wageningen Academic Publishers) pp. 233-244.

El-Kassas, H. Y. and El-Taher, E. M. (2010). Mycoremediation of monocyclic aromatic hydrocarbons by a local marine Aspergillus oryzae (statistical analysis of the main and substrate interaction effects). Egypt.J. Microbiol 45,15-29.

El-Nagerabi, S.A.F., and Abdalla, R.M.O. (2004). Note: Survey of seedborne fungi of Sudanese cultivars of onion, with new records. Phytoparasitica 32, 413-416.

Eschertzhuber, S., Velik-Salchner, C., Hoermann, C., Hoefer, D., and Lass-Florl, C. (2008). Caspofungin-resistant Aspergillus flavus after heart transplantation and mechanical circulatory support: A case report. Transpl. Infect. Dis. 10, 190-192.

EUCAST (2014). The European Committee on Antimicrobial Susceptibility Testing: Antifungal Agents Breakpoint tables for interpretation of MICs, Version 7.0. (viewed November 2014).

Fattahi, A., Zaini, F., Kordbacheh, P., Hashemi, S.J., Mahmoudi, M., and Safara, M. (2012). In vitro susceptibility of aflatoxigenic and non-aflatoxigenic Aspergillus flavus strains to conventional antifungal agents. Acta Med. Iran. 50, 798-804.

FCC (2012).  Food Chemicals Codex Eighth Edition.  The United States Pharmacopeial Convention.

Finer, G., Greenberg, D., Leibovitz, E., Leiberman, A., Shelef, I., and Kapelushnik, J. (2002). Conservative treatment of malignant (invasive) external otitis caused by Aspergillus flavuswith oral itraconazole solution in a neutropenic patient. Scand. J. Infect. Dis. 34, 227-229.

Florio, M., Marroni, M., Morosi, S., and Stagni, G. (2004). Nosocomial Aspergillus flavus wound infections following cardiac surgery. Infez. Med. 12, 270-273.

Foley, K., Fazio, G., Jensen, A.B., and Hughes, W.O.H. (2014). The distribution of Aspergillus spp. opportunistic parasites in hives and their pathogenicity to honey bees. Vet. Microbiol. 169, 203-210.

Foley, P. R. and Jones, L. A. (2000) Detergent Compositions. Patent CA 2183745.

Frank, K.A., Merz, W.G., and Hutchins, G.M. (1988). Sclerotium formation in an Aspergillus flavus wound infection. Mycopathologia 102, 185-188.

Galimberti, R., Kowalczuk, A., Hidalgo Parra, I., Gonzalez Ramos, M., and Flores, V. (1998). Cutaneous aspergillosis: A report of six cases. Br. J. Dermatol. 139, 522-526.

Gallagher, R.T., Richard, J.L., Stahr, H.M. and Cole, R.J. (1978). Cyclopiazonic acid production by aflatoxigenic and non-aflatoxigenic strains of Aspergillus flavus. Mycopathologia. 66, 31-36.

Gardezi, S.R.A. (2006). Studies on the application of fungi and bacteria controlling insect pests in Azad Jammu and Kashmir, Pakistan. Arch. Phytopathol. Plant Prot. 39, 49-67.

Gayatri, D. and Hasmukh, M. (2013). Phytase producing microbial species associated with rhizosphere of mangroves in an Arid Coastal Region of Dholara. Acad. J. Biotechnol. 1, 027-035.

Glare, T.R., Harris, R.J., and Donovan, B.J. (1996). Aspergillus flavus as a pathogen of wasps, Vespula spp., in New Zealand. N. Z. J. Zool. 23, 339-344.

Goffeau, A. (2005). Genomics : Multiple moulds. Nature 438, 1092-1093.

Gomez-Lopez, A., Garcia-Effron, G., Mellado, E., Monzon, A., Rodriguez-Tudela, J.L., and Cuenca-Estrella, M. (2003). In vitro activities of three licensed antifungal agents against Spanish clinical isolates of Aspergillus spp. Antimicrob. Agents Chemother. 47, 3085-3088.

Gonzalez, G., Hinojo, M.J., Mateo, R., Medina, A. and Jiménez, M. (2005) Occurrence of mycotoxin producing fungi in bee pollen. Int. J. Food Microbiol. 105, 1-9.

Gordon, M.A., Holzman, R.S., Senter, H., Lapa, E.W., and Kupersmith, M.J. (1976). Aspergillus oryzae meningitis. JAMA 235, 2122-2123.

Gornatti Churria, C.D., Reynaldi, F.J., Origlia, J.A., Marcantoni, H.A., Píscopo, M.V., Herrero Loyola, M.A., Reinoso, E.H., and Petruccelli, M.A. (2012). Pulmonary aspergillosis due to Aspergillus flavus Infection in a captive Eclectus parrot (Eclectus roratus). Braz. J. Vet. Pathol. 5, 4-6.

Grossman, M.E., Fithian, E.C., Behrens, C., Bissinger, J., Fracaro, M., and Neu, H.C. (1985). Primary cutaneous aspergillosis in six leukemic children. J. Am. Acad. Dermatol. 12, 313-318.

Guarro, J., Xavier, M.O., and Severo, L.C. (2010). Differences and similarities amongst pathogenic Aspergillus species. In Aspergillosis: from diagnosis to prevention, Comarú Pasqualotto, Alessandro ed., (The Netherlands: Springer) pp. 7-32.

Hahn, T., Cummings, K.M., Michalek, A.M., Lipman, B.J., Segal, B.H., and McCarthy Jr., P.L. (2002). Efficacy of high-efficiency particulate air filtration in preventing aspergillosis in immunocompromised patients with hematologic malignancies. Infect. Control Hosp. Epidemiol. 23, 525-531.

Hasan, H.A.H. (1999). Phytotoxicity of pathogenic fungi and their mycotoxins to cereal seedling viability. Mycopathologia 148, 149-155.

Hattori, A., Uchida, N. and Kitaoka, M. (2002) Phospholipase A1, processes for its preparation and the use thereof. Patent CA 2098421.

Hedayati, M.T., Pasqualotto, A.C., Warn, P.A., Bowyer, P., and Denning, D.W. (2007). Aspergillus flavus: Human pathogen, allergen and mycotoxin producer. Microbiology 153, 1677-1692.

Heinemann, S., Symoens, F., Gordts, B., Jannes, H., and Nolard, N. (2004). Environmental investigations and molecular typing of Aspergillus flavus during an outbreak of postoperative infections. J. Hosp. Infect. 57, 149-155.

Higa, T. (1997) Microbiological method for disposing of organic waste materials. Patent CA 2098969.

Horn, B.W. (2003). Ecology and population biology of aflatoxigenic fungi in soil. Toxin Rev. 22, 351-379.

Horn, B.W., Moore, G.G., and Carbone, I. (2009). Sexual reproduction in Aspergillus flavus. Mycologia 101, 423-429.

Howell, A., Midturi, J., Sierra-Hoffman, M., Carpenter, J., Hurley, D., and Winn, R. (2005). Aspergillus flavusscleritis: Successful treatment with voriconazole and caspofungin. Med. Mycol. 43, 651-655.

Hsueh, P.R., Lau, Y.J., Chuang, Y.C., Wan, J.H., Huang, W.K., Shyr, J.M., Yan, J.J., Yu, K.W., Wu, J.J., Ko, W.C., et al. (2005). Antifungal susceptibilities of clinical isolates of Candida species, Cryptococcus neoformans, and Aspergillus species from Taiwan: surveillance of multicenter antimicrobial resistance in Taiwan program data from 2003. Antimicrob. Agents Chemother. 49, 512-517.

Huang, C. and Huang, C.P. (1996) Application of Aspergillus oryzae and Rhizopus oryzae for Cu (II) removal. Wat. Res. 30, 1985-1990.

Huffman, G.A. (2001) Regulatory element conferring tapetum specificity. Patent CA 2188280.

Humphreys, H., Johnson, E.M., Warnock, D.W., Willatts, S.M., Winter, R.J., and Speller, D.C.E. (1991). An outbreak of aspergillosis in a general ITU. J. Hosp. Infect. 18, 167-177.

Hynes, S.S., Chaudhry, O., Providenti, M.A., and Smith, M.L. (2006). Development of AFLP-derived, functionally specific markers for environmental persistence studies of fungal strains. Can. J. Microbiol. 52, 451-461.

IARC. (1976). IARC (International Agency for Research on Cancer), Some Naturally Occurring Substances. Monographs, vol. 10, Lyon, France.

Iizuka, H., and Iida, M. (1962). Maltoryzine, a new toxic metabolite produced by a strain of Aspergillus oryzae var. microsporus isolated from the poisonous malt sprout. Nature 196, 681-682.

Ismail, M.A. and Abdel-Sater, M.A. (1993). Fungi associated with the Egyptian cotton leafworm Spodoptera littoralisBoisdoval. Mycopathologia. 124, 79-86.

Iwen, P.C., Davis, J.C., Reed, E.C., Winfield, B.A., and Hinrichs, S.H. (1994). Airborne fungal spore monitoring in a protective environment during hospital construction, and correlation with an outbreak of invasive aspergillosis. Infect. Control Hosp. Epidemiol. 15, 303-306.

Jaime-Garcia, R., and Cotty, P.J. (2004). Aspergillus flavus in soils and corncobs in South Texas: Implications for management of aflatoxins in corn-cotton rotations. Plant Dis. 88, 1366-1371.

Johnson, J.R., Bruce, W.F., and Dutcher, J.D. (1943). Gliotoxin, the antibiotic principle of Gliocladium fimbriatum. I. Production, physical and biological properties. J. Am. Chem. Soc. 65, 2005-2009.

Jorgensen, T.R. (2007). Identification and toxigenic potential of the industrially important fungi, Aspergillus oryzaeand Aspergillus sojae. J. Food Prot. 70, 2916-2934.

Jouany, J.P., Mathieu, F., Senaud, J., Bohatier, J., Bertin, G and Mercier, M. (1998). Effect of Saccharomyces cerevisiaeand Aspergillus oryzae on the digestion of nitrogen in the rumen of defaunated and refaunated sheep. Anim. Feed Sci, Technol. 75, 1–13.

Kagen, S.L., Kurup, V.P., Sohnle, P.G., and Fink, J.N. (1983). Marijuana smoking and fungal sensitization. J. Allergy Clin. Immunol. 71, 389-393.

Kanora, A., and Maes, D. (2009). The role of mycotoxins in pig reproduction: A review. Vet.Med., 54, 565-576.

Kaplan, W., Arnstein, P., Ajello, L., Chandler, F., Watts, J., and Hicklin, M. (1975). Fatal aspergillosis in imported parrots. Mycopathologia 56, 25-29.

Kenneth, R., and Olmert, I. (1975). Entomopathogenic fungi and their insect hosts in Israel: Additions. Israel J. Entomol. X, 105-112.

Khan, Z.U., Gopalakrishnan, G., Al-Awadi, K., Gupta, R.K., Moussa, S.A., Chugh, T.D., and Krajci, D. (1995). Renal aspergilloma due to Aspergillus flavus. Clin. Infect. Dis. 21, 210-212.

Khan, Z.U., Sanyal, S.C., Mokaddas, E., Vislocky, I., Anim, J.T., Salama, A.L., and Shuhaiber, H. (1997). Endocarditis due to Aspergillus flavus. Mycoses 40, 213-217.

Kharchenko, S.N. and Yatsyshin, A.I. (1984). Species of Aspergillus mich. molds producing kojic acid on mixed feed and the role of this mycotoxin in animal pathology. Mikrobiol. Zh. 46, 41-47.

Kim, G.W., and Chan, P.H. (2002). Involvement of superoxide in excitotoxicity and DNA fragmentation in striatal vulnerability in mice after treatment with the mitochondrial toxin, 3-nitropropionic acid. J. Cereb. Blood Flow Metab. 22, 798-809.

Kiuchi, A. and Tanaka, Y. (1977). Beta.-galactosidase production by Aspergillus oryzae. Patent CA 1016092.

Klich, M.A., and Pitt, J.I. (1988). A laboratory guide to common Aspergillus species and their teleomorphs. CSIRO Division of Food Processing ,North Ryde, Australia.

Klich, M.A. (2007). Aspergillus flavus: The major producer of aflatoxin. Mol. Plant Pathol. 8, 713-722.

Klich, M.A. (1986). Presence of Aspergillus flavus in developing cotton bolls and its relation to contamination of mature seeds. Appl. Environ. Microbiol. 52, 963-965.

Klich, M.A., and Mullaney, E.J. (1987). DNA restriction enzyme fragment polymorphism as a tool for rapid differentiation of Aspergillus flavus from Aspergillus oryzae. Exp. Mycol. 11, 170-175.

Klich, M.A., Yu, J., Chang, P.K., Mullaney, E.J., Bhatnagar, D., and Cleveland, T.E. (1995). Hybridization of genes involved in aflatoxin biosynthesis to DNA of aflatoxigenic and non-aflatoxigenic aspergilli. Appl. Microbiol. Biotechnol. 44, 439-443.

Klich, M.A. (2002). Biogeography of Aspergillus species in soil and litter. Mycologia 94, 21-27.

Koss, T., Bagheri, B., Zeana, C., Romagnoli, M.F., and Grossman, M.E. (2002). Amphotericin B-resistant Aspergillus flavusinfection successfully treated with caspofungin, a novel antifungal agent. J. Am. Acad. Dermatol. 46, 945-947.

Krishnan, S., Manavathu, E.K., and Chandrasekar, P.H. (2009). Aspergillus flavus: An emerging non-fumigatus Aspergillus species of significance. Mycoses 52, 206-222.

Kriván, G., Sinkó, J., Nagy, I.Z., Goda, V., Reményi, P., Bátai, Á, Lueff, S., Kapás, B., Réti, M., Tremmel, A., and Masszi, T. (2006). Successful combined antifungal salvage therapy with liposomal amphothericin B and caspofungin for invasive Aspergillus flavus infection in a child following allogeneic bone marrow transplantation. Acta Biomed. 77, 17-21.

Kulshrestha, V., and Pathak, S.C. (1997). Aspergillosis in German cockroach Blattella germanica (L.) (Blattoidea: Blattellidae). Mycopathologia 139, 75-78.

Kumar, M.A., Krishnagopal, S., Manivannan, D., and Ramraj, V. (2013). Invasive orbital aspergillosis in an immunocompetent individual after functional endoscopic sinus surgery. Ann. Trop. Med. Public Health 6, 254-255.

Kumar, V., Singh, G.P., and Babu, A.M. (2004). Surface ultrastructural studies on the germination, penetration and conidial development of Aspergillus flavus Link : Fries infecting silkworm, Bombyx mori Linn. Mycopathologia 157, 127-135.

Kumeda, Y., and Asao, T. (2001). Heteroduplex panel analysis, a novel method for genetic identification of Aspergillussection Flavi strains. Appl. Environ. Microbiol. 67, 4084-4090.

Kurita, H., Ichikawa, T., Oyama, K., Mori, K., Sato, M., Isowa, Y. and Ohmori, M. (1979) Process for producing a peptide. Patent CA 1059051.

Kurosawa, M., Kobayashi, S., Yanagihara, Y., and Shida, T. (1990). A case of occupational allergic bronchopulmonary aspergillosis unique to Japan. Br. J. Clin. Pract. 44, 482-489.

Kurtzman, C.P., Smiley, M.J., Robnett, C.J., and Wicklow, D.T. (1986). DNA relatedness among wild and domesticated species in the Aspergillus flavus group. Mycologia 78, 955-959.

Kusumoto, K., Yabe, K., Nogata, Y., and Ohta, H. (1998). Transcript of a homolog of aflR, a regulatory gene for aflatoxin synthesis in Aspergillus parasiticus, was not detected in Aspergillus oryzae strains. FEMS Microbiol. Lett. 169, 303-307.

Labelle, A.L., Hamor, R.E., Barger, A.M., Maddox, C.W., and Breaux, C.B. (2009). Aspergillus flavus keratomycosis in a cat treated with topical 1% voriconazole solution. Vet. Ophthalmol. 12, 48-52.

Lai, K.K. (2001). A cluster of invasive aspergillosis in a bone marrow transplant unit related to construction and the utility of air sampling. Am. J. Infect. Control 29, 333-337.

Lamb, D.C., Kelly, D.E., Baldwin, B.C., and Kelly, S.L. (2000). Differential inhibition of human CYP3A4 and Candida albicans CYP51 with azole antifungal agents. Chem. Biol. Interact. 125, 165-175.

Larone, D.H. (2011). Medically important fungi: A guide to identification. ASM Press, Washington, DC.

Lee, C.Z., Liou, G.Y., and Yuan, G.F. (2006). Comparison of the aflR gene sequences of strains in Aspergillus section Flavi. Microbiology 152, 161-170.

Leenders, A., Van Belkum, A., Janssen, S., De Marie, S., Kluytmans, J., Wielenga, J., Löwenberg, B., and Verbrugh, H. (1996). Molecular epidemiology of apparent outbreak of invasive aspergillosis in a hematology ward. J. Clin. Microbiol. 34, 345-351.

Leung, K., Trevors, J.T., and Lee, H. (1995). Survival of and lacZ expression recombinant Pseudomonas strains introduced into river water microcosms. Can. J. Microbiol. 41,461-469.

Li, D.M., Xiu, D.R., Li, R.Y., Samson, R.A., De Hoog, G.S., and Wang, D.L. (2008). Aspergillus flavus myositis in a patient after liver transplantation. Clin. Transplant. 22, 508-511.

Liao, W., Shao, J., Li, S., Wan, G., Da, Z., Sun, Y., Qi, Z., Chen, Q., and Sun, Z. (1988). Mycological identification of pulmonary aspergilloma caused by Aspergillus oryzae with proliferating heads. Chin. Med. J. 101, 601-604.

LNHPD (2015). Licensed Natural Health Products Database [database on the internet]. Health Canada, Government of Canada; [Accessed January 2015].

Lomax, L.G., Cole, R. J. and Dorner, J.W. (1984). The toxicity of cyclopiazonic acid in weaned pigs. Vet. Pathol. 21, 418-424.

Loo, V.G., Bertrand, C., Dixon, C., Vityé, D., DeSalis, B., McLean, A.P.H., Brox, A., and Robson, H.G. (1996). Control of construction-associated nosocomial aspergillosis in an antiquated hematology unit. Infect. Control Hosp. Epidemiol. 17, 360-364.

Lugauskas, A., Krikstaponis, A., and Sveistyte, L. (2004). Airborne fungi in industrial environments - Potential agents of respiratory diseases. Ann. Agric. Environ. Med. 11, 19-25.

Luh, B.S. (1995). Industrial production of soy sauce. J. Ind. Microbiol. 14, 467-471.

Lutz, B.D., Jin, J., Rinaldi, M.G., Wickes, B.L., and Huycke, M.M. (2003). Outbreak of invasive Aspergillus infection in surgical patients, associated with a contaminated air-handling system. Clin. Infect. Dis. 37, 786-793.

MacDonald, J.C. (1973). Toxicity, analysis, and production of aspergillic acid and its analogues. Can. J. Biochem. 51, 1311-1315.

Machida, M., Asai, K., Sano, M., Tanaka, T., Kumagai, T., Terai, G., Kusumoto, K., Arima, T., Akita, O., Kashiwagi, Y., et al. (2005). Genome sequencing and analysis of Aspergillus oryzae. Nature 438, 1157-1161.

Machida, M., Yamada, O., and Gomi, K. (2008). Genomics of Aspergillus oryzae: learning from the history of koji mold and exploration of its future. DNA Res. 15, 173-183.

Maertens, J., Raad, I., Petrikkos, G., Boogaerts, M., Selleslag, D., Petersen, F.B., Sable, C.A., Kartsonis, N.A., Ngai, A., Taylor, A., et al. (2004). Efficacy and safety of caspofungin for treatment of invasive aspergillosis in patients refractory to or intolerant of conventional antifungal therapy. Clin. Infect. Dis. 39, 1563-1571.

Mahajan, V.M. (1988). Experimental orbital mycosis. Mycoses 31, 11-16.

Mahajan, V.M., Dayal, Y., Patra, C.P., and Bhatia, I.M. (1978). Experimental aspergillosis in monkeys. Sabouraudia 16, 199-201.

Mahalingam, V., and Muralirangan, M.C. (1996). Haemocytic responses of Atractomorpha crenulata (Fabricius) (Orthoptera: Pyrgomorphidae) to fungal (Aspergillus flavus) infection. Entomologist 115, 97-101.

Manabe, M., Tanaka, K., Goto, T., and Matsuura, S. (1984). Producing capacity of kojic acid and aflatoxin by koji mold. In Toxigenic Fungi - their toxins and health hazard, Proceedings of the mycotoxin symposia held at the Third International Mycological Congress, Tokyo, 1983, Kurata, H., and Ueno, Y. eds., (Tokyo: Elsevier) pp. 4-14.

Marasas, W. F. O., and Nelson, P. E. (1987). Mycotoxicology. The Pennsylvania State University Press, University Park, Pa.

Marolt, J., Naglic, T., and Hajsig, D. (1984). Aspergillus oryzae as a cause of keratomycosis in the horse. Tierarztl. Prax. 12, 489-492.

Marquardt, R.R. (1996). Effects of molds and their toxins on livestock performance: a western Canadian perspective. Anim. Feed Sci.Technol. 58, 77-89.

Martin, M.P., Bouck, K.P., Helm, J., Dykstra, M.J., Wages, D.P., and Barnes, H.J. (2007). Disseminated Aspergillus flavusinfection in broiler breeder pullets. Avian Dis. 51, 626-631.

Masaki, M., Chigira, Y., and Ohta, M. (1966). Total syntheses of racemic aspergillic acid and neoaspergillic acid. J. Org. Chem. 31, 4143-4146.

Matsudo, T. and Sasaki, M. (1995). Simple determination of cyclopiazonic acid. Biosci. Biotech. Biochem. 59, 355-357.

Mellon, J.E., Cotty, P.J. and Dowd, M.K. (2007). Aspergillus flavus hydrolysases: their roles in pathogenesis and substrate utilization. Appl. Microbiol. Biotechnol. 77,497-504.

Miranda, R. L. A., Mendoza, M. G. D., Barcena-Gama, J. R.,Gonzalez, M. S. S., Ferrara, R., Ortega, C. M. E. and Cobos, P. M. A. (1996). Effect of Saccharomyces cerevisiae or Aspergillus oryzae cultures and NDF level on parameters of ruminal fermentation. Anim. Feed Sci. Technol. 63, 289-296.

Miranda-Miranda, E., Cossio-Bayugar, R., Martínez-Ibañez, F., Casasanero-Orduña, R., and Folch-Mallol, J. (2012). Natural occurrence of lethal aspergillosis in the cattle tick Rhipicephalus (Boophilus) microplus(Acari:Ixodidae). Parasitology 139, 259-263.

Misra, R., Malik, A., and Singhal, S. (2011). Comparison of the activities of amphotericin B, itraconazole, and voriconazole against clinical and environmental isolates of Aspergillusspecies. Indian J. Pathol. Microbiol. 54, 112-116.

Mittal, R.K. (1983). Studies on the mycoflora and its control on the seeds of some forest trees. I. Cedrus deodara. Can. J. Bot. 61, 197-201.

Mohamed, M.I., and El-Khadem, M. (1976). Aspergillus flavus Link -- a new parasite on Tetralonia lanuginosa Klug. Zentralbl Bakteriol Parasitenkd Infektionskr Hyg. 131, 569-570.

Montiel, D., Dickinson, M.J., Lee, H.A., Dyer, P.S., Jeenes, D.J., Roberts, I.N., James, S., Fuller, L.J., Matsuchima, K., and Archer, D.B. (2003). Genetic differentiation of the Aspergillus section Flavi complex using AFLP fingerprints. Mycol. Res. 107, 1427-1434.

Morishita, T.Y. (2004). Common diseases in backyard ducks and geese. Seminars in Avian and Exotic Pet Medicine 13, 191-196.

Moss, M.O., (1996). Centenary review. Mycotoxins. Mycol. Res. 100, 513-523.

Mothapo, N. V.; Chen, H. H.; Cubeta, M. A.; Shi, W. (2013). Nitrous oxide producing activity of diverse fungi from distinct agroecosystems. Soil Biol. Biochem. 66, 94-101.

Mullbacher, A., Waring, P., and Eichner, R.D. (1985). Identification of an agent in cultures of Aspergillusfumigatus displaying anti-phagocytic and immunomodulating activity in vitro. J. Gen. Microbiol. 131, 1251-1258.

Muniraj, I.K., Xiao, L., Hu, Z., Zhan, X., and Shi, J. (2013). Microbial lipid production from potato processing wastewater using oleaginous filamentous fungi Aspergillus oryzae. Water Res. 47, 3477-3483.

Murakami, H. (1971). Classification of the koji mold. J. Gen. Appl. Microbiol. 17, 281-309.

Myoken, Y., Sugata, T., Fujita, Y., Kyo, T., Fujihara, M., Kohara, T., Katsu, M., and Mikami, Y. (2003). Molecular epidemiology of invasive stomatitis due to Aspergillus flavus in patients with acute leukemia. J. Oral Pathol. Med. 32, 215-218.

Nakamura, H., Narihiro, T., Tsuruoka, N., Mochimaru, H., Matsumoto, R., Tanabe, Y., Hagiya, K., Ikeba, K., Maruyama, A., and Hanada, S. (2011). Evaluation of the aflatoxin biosynthetic genes for identification of the Aspergillus section Flavi. Microbes Environ. 26, 367-369.

Nayak, A.P., Blachere, F.M., Hettick, J.M., Lukomski, S., Schmechel, D., and Beezhold, D.H. (2011). Characterization of recombinant terrelysin, a hemolysin of Aspergillusterreus. Mycopathologia 171, 23-34.

Nayak, A.P., Green, B.J., and Beezhold, D.H. (2013). Fungal hemolysins. Med. Mycol. 51, 1-16.

NHPID (2015). Natural Health Products Ingredients Database [database on the internet]. Health Canada, Government of Canada; [Accessed January 2015].

Nicholson, M.J., Koulman, A., Monahan, B.J., Pritchard, B.L., Payne, G.A., and Scott, B. (2009). Identification of two aflatrem biosynthesis gene loci in Aspergillus flavus and metabolic engineering of Penicillium paxilli to elucidate their function. Appl. Environ. Microbiol. 75, 7469-7481.

Nicolle, A., De La Blanchardière, A., Bonhomme, J., Hamon, M., Leclercq, R., and Hitier, M. (2013). Aspergillus vertebral osteomyelitis in immunocompetent subjects: Case report and review of the literature. Infection 41, 833-840.

Noda, F. (1982). Process for producing solid koji. Patent US 4329370 A.

Noda, F. (1983). Process for producing soy sauce or Miso. Patent US 4382964 A.

OECD. (1992). Safety Considerations for Biotechnology. Organisation for Economic Co-operation and Development, Paris, France.

Offner, F., Krcmery, V., Boogaerts, M., Doyen, C., Engelhard, D., Ribaud, P., Cordonnier, C., De Pauw, B., Durrant, S., Marie, J.P., et al. (2004). Liposomal nystatin in patients with invasive aspergillosis refractory to or intolerant of amphotericin B. Antimicrob. Agents Chemother. 48, 4808-4812.

Okoye, J.O.A., Gugnani, H.C., and Okeke, C.N. (1989). Pulmonary infections due to Aspergillus flavus in turkey poults and goslings. Mycoses 32, 336-339.

Olempska-Beer, Z.S., Merker, R.I., Ditto, M.D., and DiNovi, M.J. (2006). Food-processing enzymes from recombinant micro-organisms--a review. Regul. Toxicol. Pharmacol. 45, 144-158.

Olufemi, B.E., Agius, C., and Roberts, R.J. (1983). Aspergillomycosis in intensively cultured tilapia from Kenya. Vet. Rec. 112, 203-204.

Opal, S.M., Asp, A.A., Cannady Jr., P.B., Morse, P.L., Burton, L.J., and Hammer II, P.G. (1986). Efficacy of infection control measures during a nosocomial outbreak of disseminated aspergillosis associated with hospital construction. J. Infect. Dis. 153, 634-637.

Orth, R. (1977). Mycotoxins of Aspergillus oryzaestrains for use in the food industry as starters and enzyme producing molds. Ann. Nutr. Aliment. 31, 617-624.

Oswald, I.P. and Comera,C. (1998). Immunotoxicity of mycotoxins. Rev. Med. Vet. 149, 585-590.

Panda, N.K., and Reddy, C.E.E. (2005). Primary frontal sinus aspergillosis: An uncommon occurrence. Mycoses 48, 235-237.

Pandita, A., Sadana, J.R., and Asrani, R.K. (1991). Studies on clinical signs and haematological alterations in pneumonic aspergillosis due to Aspergillus flavus in Japanese quail. Mycopathologia 116, 119-123.

Pardo, J., Urban, C., Galvez, E.M., Ekert, P.G., Müller, U., Kwon-Chung, J., Lobigs, M., Müllbacher, A., Wallich, R., Borner, C., and Simon, M.M. (2006). The mitochondrial protein Bak is pivotal for gliotoxin-induced apoptosis and a critical host factor of Aspergillus fumigatus virulence in mice. J. Cell Biol. 174, 509-519.

Park, S.C., Yoo, N.C., Kim, J.Y., Park, H.K., Chae, B.J., Shin, S.Y., Cheong, H., Park, Y., and Hahm, K.S. (2008). Isolation and characterization of an extracellular antimicrobial protein from Aspergillus oryzae. J. Agric. Food Chem. 56, 9647-9652.

Parveen, S., Lanjewar, S., Sharma, K., and Kutti, U. (2011). Isolation of fungi from the surface water of river. J. Exp. Sci. 2, 58-59.

Pasqualotto, A.C. (2009). Differences in pathogenicity and clinical syndromes due to Aspergillus fumigatus and Aspergillus flavus. Med. Mycol. 47, S261-S270.

Pasqualotto, A.C., and Denning, D.W. (2008). An aspergilloma caused by Aspergillus flavus. Med. Mycol. 46, 275-278.

Payne, G.A., Nierman, W.C., Wortman, J.R., Pritchard, B.L., Brown, D., Dean, R.A., Bhatnagar, D., Cleveland, T.E., Machida, M., and Yu, J. (2006). Whole genome comparison of Aspergillus flavus and A. oryzae. Med. Mycol. 44, 9-11.

Pegues, D.A., Lasker, B.A., McNeil, M.M., Hamm, P.M., Lundal, J.L., and Kubak, B.M. (2002). Cluster of cases of invasive aspergillosis in a transplant intensive care unit: Evidence of person-to-person airborne transmission. Clin. Infect. Dis. 34, 412-416.

Pinto, V.F., Patriarca, A., Locani, O., and Vaamonde, G. (2001). Natural co-occurrence of aflatoxin and cyclopiazonic acid in peanuts grown in Argentina. Food Addit. Contam. 18, 1017-1020.

Pitt, J.I., Dyer, S.K., and McCammon, S. (1991). Systemic invasion of developing peanut plants by Aspergillus flavus. Lett. Appl. Microbiol. 13, 16-20.

Prelusky, D., Rotter, B., and Rotter, R. (1994).Toxicology of Mycotoxins. In: Mycotoxins in Grain: Compounds other than Aflatoxin, Miller, J. and Trenholm, R. (Eds.). Eagan Press, St. Paul, pp: 359-403.

Product Sheet A (2014) Sigma-Aldrich (viewed September 2014).

Product Sheet B (2014) TCI America (viewed September 2014).

Product Sheet C (2014) BIO-CAT Microbials (viewed September 2014).

Purchase, I.F.H., and Van der Watt, J.J. (1969). Acute toxicity of sterigmatocystin to rats. Food Cosmet. Toxicol. 7, 135-139.

Qiao, M.F., Ji, N.Y., Liu, X.H., Li, K., Zhu, Q.M. and Xue, Q.Z. (2010). Indoloditerpenes from an algicolous isolate of Aspergillus oryzae. Bioorg. Med. Chem. Lett. 20, 5677-5680.

Rank, C., Klejnstrip, M. L., Petersen, L. M., Kildgaard, S., Frisvad, J. C., Gotfredsen, C. H. and Larsen, T. O. (2012). Comparative chemistry of Aspergillus oryzae (RIB40) and A. flavus (NRRL 3357). Metabolites 2, 39-56.

Rao, K., and Saha, V. (2000). Medical management of Aspergillus flavus endocarditis. Pediatr. Hematol. Oncol. 17, 425-427.

Raper, K.B., and Fennell, D.I. (1965). The Genus Aspergillus. Williams & Wilkins, Baltimore, MD.

Reddy, R.V., and Sharma, R.P. (1989). Effects of aflatoxin B1 on murine lymphocytic functions. Toxicology 54, 31-44.

Reed, Z., Thomasy, S.M., Good, K.L., Maggs, D.J., Magdesian, K.G., Pusterla, N., and Hollingsworth, S.R. (2013). Equine keratomycoses in California from 1987 to 2010 (47 cases). Equine Vet. J. 45, 361-366.

Richard,J.L. (2007). Some major mycotoxins and their mycotoxicoses-An overview. Int J Food Microbiol. 119, 3-10.

Richard, J.L., Thurston, J.R., Peden, W.M., and Pinello, C. (1984). Recent studies on aspergillosis in turkey poults. Mycopathologia 87, 3-11.

Richards, C.S., Hill, M.P., and Dames, J.F. (2005). The susceptibility of small hive beetle (Aethina tumida Murray) pupae to Aspergillus niger (van Tieghem) and A. flavus (Link:Grey). Am. Bee J. 145, 748-751.

Riou, C. and Gunata, Z. (1999) Cloning and expressing the Aspergillus oryzae BGII beta-glucosidase gene. Patent CA 2326496.

Rodricks, J.V., Lustig, E., Campbell, A.D., Stoloff, L., and Henery-Logan, K.R. (1968). Aspertoxin, a hydroxy derivative of o-methylsterigmatocystin from aflatoxin-producing cultures of Aspergillus flavus. Tetrahedron Lett. 9, 2975-2978.

Rokas, A. (2009). The effect of domestication on the fungal proteome. Trends in Genetics 25, 60-63.

Roskov, Y., Kunze T., Orrell T., Abucay L., Culham A., Bailly N., Kirk P., Bourgoin T., Baillargeon G., Decock W., De Wever A., eds. (2014). Species 2000 & ITIS Catalogue of Life, 30th May 2014. Digital resource at Species 2000: Naturalis, Leiden, the Netherlands.

Rotstein, C., Cummings, M., Tidings, J., Killion, K., Powell, E., Gustafson, T.L., and Higby, D. (1985). An outbreak of invasive aspergillosis among allogeneic bone marrow transplants: A case-control study. Infect. Control 6, 347-355.

Rupa, V., and Thomas, M. (2013). Different types of fungal sinusitis occurring concurrently: Implications for therapy. Eur. Arch. Otorhinolaryngol. 270, 603-608.

Russo, A., Falcone, M., Vena, A., Venditti, C., Mancini, C., Morelli, A., and Venditti, M. (2011). Invasive pulmonary aspergillosis in non-neutropenic patients: Analysis of a 14-month prospective clinical experience. J. Chemother. 23, 290-294.

Samson, R.A., Hong, S.B., and Frisvad, J.C. (2006). Old and new concepts of species differentiation in Aspergillus. Med. Mycol. 44, 133-148.

Sannasi, A. (1968). Apparent infection of queens and drones of the mound-building termite Odontotermes obesus by Aspergillus flavus. J. Invertebr. Pathol. 10, 434-435.

Sannasi, A., and Amirthavalli, S. (1970). Infection of the velvet mite, Trombidium gigas by Aspergillus flavus. J. Invertebr. Pathol. 16, 54-56.

Sarubbi Jr., F.A., Kopf, H.B., Wilson, M.B., McGinnis, M.R., and Rutala, W.A. (1982). Increased recovery of Aspergillus flavus from respiratory specimens during hospital construction. Am. Rev. Respir. Dis. 125, 33-38.

Sawasaki, H., Horie, K., Yamada, M., Tajima, G., Katsura, S., Naito, Y., Watabe, S., Murabayashi, A., Kukidome, S., and Jo, K. (1969). Bronchial stump aspergillosis. Experimental and clinical study. J. Thorac. Cardiovasc. Surg. 58, 198-208.

Schwetz, I., Horina, J., Buzina, W., Roob, J., Olschewski, H., and Krause, R. (2007). Aspergillus oryzae peritonitis in CAPD: Case report and review of the literature. Am. J. Kidney Dis. 49, 701-704.

Sepehr, M.N., Nasseri, S., Mazaheri Assadi, M., and Yaghmaian, K. (2005). Chromium Bioremoval from Tannery Industries Effluent by Aspergillus oryzae. Iran. J. Environ. Health. Sci. Eng. 2, 273-279.

Shearer, J.F., Sweets, L.E., Baker, N.K., and Tiffany, L.H. (1992). A study of Aspergillus flavus/parasiticus in Iowa crop fields: 1988-1990. Plant Dis. 76, 19-22.

Shenasi, M., Aidoo, K.E., and Candlish, A.A.G. (2002). Microflora of date fruits and production of aflatoxins at various stages of maturation. Int. J. Food Microbiol. 79, 113-119.

Sherertz, R.J., Belani, A., Kramer, B.S., Elfenbein, G.J., Weiner, R.S., Sullivan, M.L., Thomas, R.G., and Samsa, G.P. (1987). Impact of air filtration on nosocomial Aspergillusinfections. Unique risk of bone marrow transplant recipients. Am. J. Med. 83, 709-718.

Shim, Y. H., Shinde, P. L., Choi, J. Y., Kim, J. S., Seo, D. K., Pak, J. I., Chae, B. J. and Kwon, I. K. (2010). Evaluation of multi-microbial probiotics produced by submerged liquid and solid substrate fermentation methods in broilers. Asian Austral.J. Anim.Sci. 23, 521-529.

Shoar, M.G., Zomorodian, K., Saadat, F., Hashemi, M.J., and Tarazoei, B. (2004). Fatal endocarditis due to Aspergillus flavus in Iran. J. Pak. Med. Assoc. 54, 485-486.

Singer, S., Singer, D., Rüchel, R., Mergeryan, H., Schmidt, U., and Harms, K. (1998). Outbreak of systemic aspergillosis in a neonatal intensive care unit. Mycoses 41, 223-227.

Sisti, M., Brandi, G., De Santi, M., Rinaldi, L., and Schiavano, G.F. (2012). Disinfection efficacy of chlorine and peracetic acid alone or in combination against Aspergillus spp. and Candida albicans in drinking water. J. Water Health 10, 11-19.

Speth, C., and Rambach, G. (2012). Complement attack against Aspergillus and corresponding evasion mechanisms. Interdiscip. Perspect. Infect. Dis. 2012, 463794.

Spök, A., and Klade, M. (2009). Environmental, Health and Legal Aspects of Cleaners Containing Living Microbes as Active Ingredients. IFZ 1-17.

Stenson, S., Brookner, A., and Rosenthal, S. (1982). Bilateral endogenous necrotizing scleritis due to Aspergillus oryzae. Ann. Ophthalmol. 14, 67-72.

Stodulski, D., Kowalska, B., and Stankiewicz, C. (2006). Otogenic skull base osteomyelitis caused by invasive fungal infection: Case report and literature review. Eur. Arch. Otorhinolaryngol. 263, 1070-1076.

Sukumaran, K. (1991). Ulcerative keratomycosis--case reports on three different species of fungi. Med. J. Malaysia 46, 388-391.

Sutton, P., Newcombe, N.R., Waring, P., and Mullbacher, A. (1994). In vivo immunosuppressive activity of gliotoxin, a metabolite produced by human pathogenic fungi. Infect. Immun. 62, 1192-1198.

Sweeney, M.J. and Dobson, A.D.W. (1998). Mycotoxin production by Aspergillus, Fusarium and Penicilliumspecies. Int. J. Food Microbiol. 43, 141-158.

Taj-Aldeen, S.J., Hilal, A.A., and Schell, W.A. (2004). Allergic fungal rhinosinusitis: a report of 8 cases. Am. J. Otolaryngol. 25, 213-218.

Taj-Aldeen, S.J., Hilal, A.A., and Chong-Lopez, A. (2003). Allergic Aspergillus flavus rhinosinusitis: A case report from Qatar. Eur. Arch. Otorhinolaryngol. 260, 331-335.

Tanaka, K., Goto, T., Manabe, M., and Matsuura, S. (2002). Traditional Japanese fermented foods free from mycotoxin contamination. Jpn. Agric. Res. Q. 36, 45-50.

Tendolkar, U., Sharma, A., Mathur, M., Ranadive, N., and Sachdev, M. (2005). Epidural mass due to Aspergillus flavus causing spinal cord compression - A case report and brief update. Indian J. Med. Microbiol. 23, 200-203.

Thio, C.L., Smith, D., Merz, W.G., Streifel, A.J., Bova, G., Gay, L., Miller, C.B., and Perl, T.M. (2000). Refinements of environmental assessment during an outbreak investigation of invasive aspergillosis in a leukemia and bone marrow transplant unit. Infect. Control Hosp. Epidemiol. 21, 18-23.

Thywißen, A., Heinekamp, T., Dahse, H.M., Schmaler-Ripcke, J., Nietzsche, S., Zipfel, P.F., and Brakhage, A.A. (2011). Conidial dihydroxynaphthalene melanin of the human pathogenic fungus Aspergillus fumigatus interferes with the host endocytosis pathway. Front. Microbiol. 2, 96.

Tokuoka, M., Seshime, Y., Fujii, I., Kitamoto, K., Takahashi, T. and Koyama, Y. (2008). Identification of a novel polyketide synthase-nonribosomal peptide synthetase (PKS-NRPS) gene required for the biosynthesis of cyclopiazonic acid in Aspergillus oryzae. Fungal Genet. Biol. 45, 1608-1615.

Tsuchiya, Y., Shimokata, K., Ohara, H., Nishiwaki, K., and Kino, T. (1993). Hypersensitivity pneumonitis in a soy sauce brewer caused by Aspergillus oryzae. J. Allergy Clin. Immunol. 91, 688-689.

Um, B. H. and Walsum, G.P. van. (2010). Evaluation of enzyme mixtures in releasing fermentable sugars from pre-pulping extracts of mixed northeast hardwoods. Appl.Biochem. Biotechnol. 161, 432-447.

Upadhyay, M.P., West, E.P., and Sharma, A.P. (1980). Keratitis due to Aspergillus flavus successfully treated with thiabendazole. Br. J. Ophthalmol. 64, 30-32.

U.S. EPA. (1997a). Aspergillus oryzae Final Risk Assessment.

U.S. EPA. (1997b). Aspergillus oryzae TSCA Section 5(h)(4) Exemption: Final Decision.

Van Den Broek, P. and Affolter, M. (1999) Enhanced expression of proteolytic enzymes in koji mold. Patent CA 2263947.

Vandini, A., Temmerman, R., Frabetti, A., Caselli, E., Antonioli, P., Balboni, P.G., Platano, D., Branchini, A. and Mazzaxane, S. (2014). Hard surface biocontrol in hospitals using microbial-based cleaning products. PLOS One, 9, e108598.

Vega, F.E., Simpkins, A., Aime, M.C., Posada, F., Peterson, S.W., Rehner, S.A., Infante, F., Castillo, A., and Arnold, A.E. (2010). Fungal endophyte diversity in coffee plants from Colombia, Hawai'i, Mexico and Puerto Rico. Fungal Ecol. 3, 122-138.

Verghese, S., Chellamma, T., and Cherian, K.M. (2009). Osteomyelitis of the rib caused by Aspergillus flavusfollowing cardiac surgery. Mycoses 52, 91-93.

Verschraegen, C.F., Van Besien, K.W., Dignani, C., Hester, J.P., Andersson, B.S., and Anaissie, E. (1997). Invasive Aspergillus sinusitis during bone marrow transplantation. Scand. J. Infect. Dis. 29, 436-438.

Vigushin, D., Mirsaidi, N., Brooke, G., Sun, C., Pace, P., Inman, L., Moody, C., and Coombes, R. (2004). Gliotoxin is a dual inhibitor of farnesyltransferase and geranylgeranyltransferase I with antitumor activity against breast cancer in vivo. Med. Oncol. 21, 21-30.

Vojvodic, S., Jensen, A.B., James, R.R., Boomsma, J.J., and Eilenberg, J. (2011). Temperature dependent virulence of obligate and facultative fungal pathogens of honeybee brood. Vet. Microbiol. 149, 200-205.

Vujanovic, V., Smoragiewicz, W., and Krzysztyniak, K. (2001). Airborne fungal ecological niche determination as one of the possibilities for indirect mycotoxin risk assessment in indoor air. Environ. Toxicol. 16, 1-8.

Wada, R., Jin, F.J., Koyama, Y., Maruyama, J., and Kitamoto, K. (2014). Efficient formation of heterokaryotic sclerotia in the filamentous fungus Aspergillus oryzae. Appl. Microbiol. Biotechnol. 98, 325-334.

Wada, S., Hobo, S., Ode, H., Niwa, H., and Moriyama, H. (2013). Equine keratomycosis in Japan. Vet. Ophthalmol. 16, 1-9.

Wartenberg, D., Lapp, K., Jacobsen, I.D., Dahse, H.M., Kniemeyer, O., Heinekamp, T., and Brakhage, A.A. (2011). Secretome analysis of Aspergillus fumigatus reveals Asp-hemolysin as a major secreted protein. Int. J. Med. Microbiol. 301, 602-611.

Watson, A.J., Fuller, L.J., Jeenes, D.J., and Archer, D.B. (1999). Homologs of aflatoxin biosynthesis genes and sequence of aflR in Aspergillus oryzae and Aspergillussojae. Appl. Environ. Microbiol. 65, 307-310.

Wei, D.L., and Jong, S.C. (1986). Production of aflatoxins by strains of the Aspergillus flavus group maintained in ATCC. Mycopathologia 93, 19-24.

Wicklow, D.T. (1984a). Adaptation in wild and domesticated yellow-green aspergilli. In Toxigenic fungi: their toxins and health hazards, Kurata, H., and Ueno, Y. eds., (Amsterdam, The Netherlands.: Elsevier) pp. 78-86.

Wicklow, D.T. (1984b). Conidium germination rate in wild and domesticated yellow-green aspergilli. Appl. Environ. Microbiol. 47, 299-300.

Wicklow, D.T., McAlpin, C.E., and Platis, C.E. (1998). Characterization of the Aspergillus flavus population within an Illinois maize field. Mycol. Res. 102, 263-268.

Wicklow, D.T., Wilson, D.M., and Nelsen, T.C. (1993). Survival of Aspergillus flavus sclerotia and conidia buried in soil in Illinois or Georgia. Phytopathology 83, 1141-1147.

Wilson, B.J., and Wilson, C.H. (1964). Toxin from Aspergillus flavus: Production on Food Materials of a Substance Causing Tremors in Mice. Science 144, 177-178.

Witschi, H.R., Pinkerton, K.E., Van Winkle, L.S., and Last, J.A. (2008). Toxic Responses of the Respiratory System. In Casarett & Doull's Toxicology: the Basic Science of Poisons Seventh Edition, Klaassen, C. D. ed., (United States of America: McGraw Hill) pp. 609-630.

Wu, N., Huang, Y., Li, Q., Bai, C., Huang, H.D., and Yao, X.P. (2010). Isolated invasive Aspergillus tracheobronchitis: A clinical study of 19 cases. Clin. Microbiol. Infect.16, 689-695.

Xavier, M.O., De Oliveira, F.M., De Almeida, V., Prolla, G., and Severo, L.C. (2009). Invasive Aspergillus flavussinusitis: Case report in a patient with biphenotypic acute leukemia. Rev. Inst. Med. Trop. Sao Paulo 51, 57-58.

Yoon, I. K. and Stern, M. D. (1995). Influence of direct-fed microbials on ruminal microbial fermentation and performance of ruminants: A review. Asian Austral. J. Anim. Sci. 8, 533-555.

Yu, O.H.Y., Keet, A.W.W., Sheppard, D.C., and Brewer, T. (2010). Articular aspergillosis: Case report and review of the literature. Int. J. Infect. Dis. 14, e433-e435.

Zablotowicz, R.M., Abbas, H.K., and Locke, M.A. (2007). Population ecology of Aspergillus flavus associated with Mississippi Delta soils. Food Addit. Contam. 24, 1102-1108.

Zhang, X., Li, B., Wang, Y., Guo, Q., Lu, X., Li, S., and Ma, P. (2013). Lipopeptides, a novel protein, and volatile compounds contribute to the antifungal activity of the biocontrol agent Bacillus atrophaeus CAB-1. Appl. Microbiol. Biotechnol. 97, 9525-9534.

Zhirong, Y., Wanqing, L., and Weihua, P. (1999). Case reports. Invasive pulmonary aspergillosis in non-neutropenic patients treated with liposomal amphotericin B. Mycoses 42, 679-682.

Zhu, L.P., Chen, X.S., Wu, J.Q., Yang, F.F., and Weng, X.H. (2011). Aspergillus vertebral osteomyelitis and ureteral obstruction after liver transplantation. Transpl. Infect. Dis. 13, 192-199.

Ziskind, J., Pizzolato, P., and Buff, E. (1958). Aspergilloma of the brain: report of a case. Am. J. Clin. Pathol. 29, 554-559.

Zuluaga-Montero, A., Ramírez-Camejo, L., Rauscher, J., and Bayman, P. (2010). Marine isolates of Aspergillus flavus: Denizens of the deep or lost at sea? Fungal Ecol. 3, 386-391.

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Appendix A: Growth of Aspergillus oryzae ATCC 11866 in various media

Table A-1 Growth of A. oryzae ATCC 11866 in liquid media at various temperaturesa
Sabouraud Liquid Medium+++~
100% Fetal Bovine Serum~++
Dulbecco's Modified Eagles Medium (mammalian cell culture)(+)(+)(+)
10 % Sheep Blood Serum~~~

– no growth, + growth, ~ low level growth, (+) delayed growth (after 15h)

a Data generated by Health Canada's Environmental Health Science and Research Bureau. Growth of Aspergillus oryzae ATCC 11866 in broth culture, as measured by increase in absorbance at 500 nm, in four different growth media and over a range of temperatures: Measurements were taken every 15 minutes over a 24-hour period with a multi-well spectrophotometer.

Table A-2 Growth Characteristics of A. oryzae ATCC 11866 on Solid Media at various temperaturesa
Blood Agarb growth++
Blood Agarb hemolysis--
Czapek Agarc++
Dermatophyte test agard--
Mycosel Agare--
Potato Dextrose Agarf++
Sabouraud Dextrose Agarg++
Staib's Hivey Bird Seed Agarh++
Yeast Mould Agari++

(+) Positive for growth (-) Negative for growth

a Data generated by Heath Canada's Environmental Health Science and Research Bureau

b Sheep Blood Agar. Used to detect lysis of blood cells (hemolysis).

c Czapek-Dox Agar is recommended in Standard Methods for the Examination of Water and Wastewater for the isolation of Aspergillus, Penicillium, Paecilomycesand other types of fungi with similar physiological requirements (Hardy Diagnostics).

d Dermatophyte test agar. Selective medium used for the isolation of pathogenic fungi from cutaneous specimens.

e Mycosel Agar. For the isolation of pathogenic fungi from materials having a large flora of other fungi and bacteria.

f Potato Dextrose Agar. Used for cultivation and isolation of yeasts and molds

g Sabouraud Dextrose Agar (SAB). A standard medium for the isolation and maintenance of a wide variety of fungi commonly encountered in a clinical setting.

h Staib's Hivey Bird Seed Agar. For the selective isolation of Crytococcus neoformans from other yeasts.

i Yeast Mould Agar. Cultivation of yeasts, molds and other aciduric micro-organism

Appendix B: Secondary metabolites produced by Aspergillus oryzae and Aspergillus flavus

Table B-1 List of toxins and secondary metabolites produced by Aspergillus oryzae/flavus
  • Produced by some strains of A. flavus (Klich 2007).
  • Multiple forms: B1, B2, G1, G2, M1 and M2 which differ slightly in structure (reviewed in Amaike and Keller 2011).
  • Link to crop and feed contamination (Adebajo 1992; Jaime-Garcia and Cotty 2004).
  • In mice, aflatoxin B1 decreases DNA synthesis in lymphocyte cultures, peripheral leukocyte counts, DNA, protein and RNA synthesis in splenic lymphocytes, and concanavalin A-induced suppressor cells, suggesting a direct and complex effect on lymphocytes (Reddy and Sharma 1989).
  • Daily intake of low dose of aflatoxins can cause chronic aflatoxin poisoning resulting in anorexia, stunted growth, immune suppression and possible liver cancer development (reviewed in Amaike and Keller 2011).
  • Acute aflatoxin poisoning with high doses can kill humans and animals (reviewed in Amaike and Keller 2011).
  • A. oryzae ATCC 11866 does not produce aflatoxin (Wei and Jong 1986).
  • Produced by some strains of A. flavus (Nicholson et al. 2009).
  • Potent tremorgenic toxin, part of a group of fungal secondary metabolites known as indole-diterpenes (Nicholson et al. 2009).
  • In mice, gavage doses between 0.5 g to 1.0 mg of the partially purified toxin induced tremors, and when the dose was increased to 2 mg, tremors were followed by convulsions. Lower doses produced either no effect or mild tremors for a short period (Wilson and Wilson 1964).
  • No LD50 available.
  • No indication if A. oryzae ATCC 11866 produces this mycotoxin.
Aspergillic acid
  • Produced by some strains of A. flavus (Masaki et al. 1966).
  • A hydroxypyrazine derivative secondary metabolite with bactericidal properties (Masaki et al. 1966).
  • Induces acute severe toxicity in mice at 150 mg/kg (intraperitoneal), however no chronic toxicity is indicated at sublethal doses, unlike aflatoxins (MacDonald 1973).
  • No LD50 available.
  • No indication if A. oryzae ATCC 11866 produces aspergillic acid.
  • Produced by some strains of A. flavus (Rodricks et al. 1968).
  • Hydroxy derivative of another metabolite produced by A. flavus, O-methylsterigmatocystin.
  • The structure resembles aflatoxin M1, aflatoxin B1 and sterigmatocystin (Rodricks et al. 1968).
  • No LD50 or information on toxicity available.
  • No indication if A. oryzae ATCC 11866 or any A. oryzae strains produce aspertoxin.
Cyclopiazonic Acid (CPA)
  • Produced by some strains of A. flavus and A. oryzae (Benkhemmar et al. 1985; Chang et al. 2009; Matsudo and Sasaki 1995; Tokuoka et al. 2008).
  • Associated with contamination of crop and animal foods (Chang et al. 2009; Gallagher et al. 1978).
  • An indole-tetramic acid neurotoxin (Chang et al. 2009).
  • Causes the inhibition of Ca2+-ATPase which in turn causes the sarcoplasmic reticulum (Smooth ER) to lose function (reviewed in Blumenthal 2004).
  • LD50: 2 mg/kg in rat (intraperitoneal) 13 and 64 mg/kg in mouse (intraperitoneal and oral) and 12 mg/kg in chickens (oral) (reviewed in Blumenthal 2004).
  • Caused leukocytosis and lesions in gastrointestinal tract, liver and kidney of pigs (NOEL between 0.1 and 0.01 mg/kg). Altered renal or hepatic function was not determined (Lomax et al. 1984).
  • Some A. oryzae strains used in food fermentation are capable of CPA production (Chang and Ehrlich 2011; Orth 1977), but in most, domestication has likely resulted in a loss of biosynthetic pathways for CPA because it is of no adaptive value to the fungus under conditions of domestication (Wicklow 1984a).
  • No indication if A. oryzae ATCC 11866 produces cyclopiazonic acid.
  • Produced by some strains of A. flavus (reviewed in Hedayati et al. 2007).
  • An immunosuppressive toxin (Mullbacher et al. 1985; Sutton et al. 1994).
  • Inhibits macrophage and polymorphonuclear cell function and generation of alloreactive cytotoxic T cells; inhibits the transcription factor of activated B cells; toxic to mitochondria by reducing the production of ATP, which leads to apoptosis (Pardo et al. 2006).
  • LD50 (intraperitoneal): 25mg/kg in mice (Johnson et al. 1943; Vigushin et al. 2004).
  • No indication if A. oryzae ATCC 11866 or any other A. oryzae strains produce gliotoxin.
Kojic Acid (KA)
  • Produced by some A. oryzae and A. flavusstrains (Blumenthal 2004; Manabe et al. 1984).
  • Inhibits melanosis by hindering the uptake of oxygen required for enzymatic browning (reviewed in Burdock et al. 2001).
  • Inhibits the oxidation of D-amino acids, xanthine, L-phenylalanine, and L-methionine in the liver of rats in vitroand inhibits 4-nitroanisole O-demethylation in vitro and in vivoin Helicoverpa zea (moth) and Spodoptera frugiperda(Army worm) (reviewed in Burdock et al. 2001).
  • Insecticidal, antibacterial and antifungal properties (Chaves et al. 2012).
  • Can cause toxicity in mice, rats, dogs, and poultry (Burdock et al. 2001).
  • v (intraperitoneal): 250 mg/mouse (Blumenthal 2004).
  • Considered a contaminant and is present at negligible levels in fermented foods. The incubation period for sake, shoyu and miso is about two days and no kojic acid is found at that time (Tanaka et al. 2002).
  • No indication if A. oryzae ATCC 11866 produces kojic acid.
  • Produced by A. oryzae var. microsporus and possibly by very few other strains of A. oryzae (Ciegler and Vesonder 1983).
  • Highly toxic to dairy cows (Iizuka and Iida 1962).
  • LD50 (mouse): 3mg/kg and causes muscular narcotism (Iizuka and Iida 1962).
  • No indication if A. oryzae ATCC 11866 produces maltoryzine.
3-Nitropropionic Acid (NPA)
  • Produced by some strains of A. oryzae (reviewed in Blumenthal 2004) and A. flavus (Doxtader and Alexander 1966).
  • Considered neurotoxic by irreversibly inhibiting succinate dehydrogenase (reviewed in Blumenthal 2004).
  • Can cause energy impairment, excitotoxicity (apoptotic neuronal death) and oxidative stress in rats and mice (Doxtader and Alexander 1966; Kim and Chan 2002).
  • LD50: 67 mg/kg in rat (intraperitoneal), 22 mg/kg in rat (subcutaneous) 140 and 50 mg/kg in mouse (intraperitoneal), 68.1 mg/kg in mouse (oral) and 25.1mg/kg in chickens (oral) (reviewed in Blumenthal 2004).
  • No indication if A. oryzae ATCC 11866 produces 3-Nitropropionic acid.
  • Produced by some strains of A. flavus (reviewed in Hedayati et al. 2007).
  • An intermediate in aflatoxin synthesis (Blumenthal 2004).
  • Caused lung tumours in mice, liver tumours in rats after oral administration, and skin and liver tumours in rats after application to the skin, and is therefore considered carcinogenic in mice and rats (reviewed in IARC 1976).
  • LD50 in mice (oral) 166mg/kg in males (dimethylformamide as vehicle) and 120 mg/kg in females (wheat-germ oil as vehicle) and LD 50 (intraperitoneal) in males were 60 and 65 mg/kg using respectively dimethylformamide and wheat-germ oil as vehicle (Purchase and Van der Watt 1969).
  • No indication if A. oryzae ATCC 11866 produces sterigmatocystin.

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