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Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
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Appendix A

Contributed Manuscripts

A1

THE EMERGENCE OF CRYPTOCOCCUS GATTII IN BRITISH COLUMBIA AND THE PACIFIC NORTHWEST1

Karen H. Bartlett, Sarah E. Kidd, and James W. Kronstad2

An unprecedented emergence of cryptococcal infections in animals and otherwise healthy humans was recognized in 1999 on the east coast of Vancouver Island, British Columbia. Unexpectedly, these infections were caused by Cryptococcus gattii, a species closely related to the AIDS-associated fungal pathogen Cryptococcus neoformans. Human cases have continued over the past 8 years and now total approximately 170 with eight deaths. Extensive environmental

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1 Reprinted with kind permission from Springer Science+Business Media: Current Infectious Diseases Reports, The emergence of Cryptococcus gattii in British Columbia and the Pacific Northwest, 10, 2008, p. 108–115, Karen H. Bartlett, Sarah E. Kidd, and James W. Kronstad.

Current Infectious Disease Reports 2008, 10:58-65
Current Medicine Group LLC ISSN 1523-3847
Copyright © 2008 by Current Medicine Group LLC

Papers of particular interest, published recently, have been highlighted as:
+ Of importance
++ Of major importance

2Karen H. Bartlett, PhD, Sarah E. Kidd, PhD, and James W. Kronstad, PhD. Corresponding author: James W. Kronstad, PhD, The Michael Smith Laboratories, University of British Columbia, 2185 East Mall, Vancouver, BC, V6T 1Z4, Canada. Email: kronstad@interchange.ubc.ca.

Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
×

sampling, coupled with detailed molecular typing of isolates, revealed areas of permanent and transient colonization with primarily three genotypes of the fungus. C. gattii was found in air, soil, water, and in association with numerous tree species. Importantly, there is solid evidence for human-mediated dispersal of the pathogen, and C. gattii has now been detected in the environment on the mainland of British Columbia and in the Pacific Northwest. Associated animal and human cases are now being reported and further spread of the pathogen may be inevitable.

Introduction

The basidiomycetous yeast Cryptococcus neoformans has a global distribution and has achieved prominence in recent decades because of its propensity to infect immunocompromised people (Casadevall and Perfect, 1998). In fact, cryptococcosis is recognized as an AIDS-defining illness, and in the absence of highly active antiretroviral therapy, the disease is a significant cause of death in individuals with HIV infection (Bicanic and Harrison, 2005; Bicanic et al., 2005). People and animals acquire the fungus via the inhalation of desiccated yeast cells or basidiospores from environmental sources such as avian guano, soil, and trees. Pulmonary infection often results in dissemination to the central nervous system and C. neoformans is the leading cause of fungal meningitis (Casadevall and Perfect, 1998).

Isolates of C. neoformans have previously been divided into three varieties known as grubii, neoformans, and gattii and into serotypes (A–D and hybrids such as AD) defined by antigenic differences in the capsular polysaccharide that is the major virulence factor (Casadevall and Perfect, 1998). The gattii variety is now recognized as a separate species based on phenotypic and molecular traits, and mating (Kwon-Chung et al., 2002). Thus the current view is that the species C. neoformans (var grubii and neoformans) contains strains of serotypes A, D, and AD, and the distinct species C. gattii contains isolates of the B and C serotypes (Kwon-Chung and Varma, 2006). An excellent review of the differences between C. gattii and C. neoformans has been published by Sorrell (Sorrell, 2001).

Extensive surveys have been performed over the past 10 years to characterize the genotypes and distribution of C. neoformans and C. gattii isolates (Barreto de Oliveira et al., 2004; Boekhout et al., 2001; Boukhout et al., 1997; Fraser et al., 2005+; Kidd, 2003; Kidd et al., 2004 ++; Kidd et al., 2005+; Meyer et al., 1999; Meyer et al., 2003). These surveys used a variety of DNA-based typing methods to provide detailed classifications of isolates into molecular types. Thus, isolates of C. neoformans var grubii (serotype A) are represented by the VNI, VNII, and VNB (Litvintseva et al., 2006) molecular types, var neoformans (serotype D) is represented by the VNIV type, and isolates of the AD hybrid serotype are the VNIII type. Four molecular types are recognized for C. gattii isolates (designated VGI–VGIV) and further divisions within the molecular types have been identified

Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
×

img-103

Figure A1-1 Map of the forecasted ecologic niche and region of emergence of C. gattii in British Columbia (BC). The optimal, potential, and unsuitable ecologic niches of C. gattii in BC are indicated based on biogeoclimatic data for the region (Mak, 2007). Note that the distribution of human and animal cases and the locations of positive environmental samples coincide primarily with the optimal ecologic niche. The information on human and animal cases, and environmental sampling, from Washington (WA) is not included.

(Fraser et al., 2005+; Kidd et al., 2005+; Kidd et al., 2007++). For example, VGII strains can be further classified into VGIIa and VGIIb subtypes, as well as other less-well characterized subtypes (Kidd et al., 2004; MacDougall et al., 2007++).

There is currently an intense focus on C. gattii due to the unprecedented emergence of the VGI, VGIIa, and VGIIb molecular types as primary pathogens of humans and animals on Vancouver Island in British Columbia (BC) (Kidd et al., 2004; MacDougall et al., 2007++) (Fig. A1-1). Remarkably, the majority of human cases have occurred in people without recognized immunologic defects, thus highlighting the unusual pathogenicity of C. gattii relative to C. neoformans. The purpose of this review is to summarize recent progress in the investigation of this fascinating emergence with regard to human and animal exposure, environmental colonization, isolate characterization, and the potential for further dispersal.

Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
×

Overview of Veterinary and Clinical Aspects of the Emergence of C. gattii in BC

Animal sentinels played a key role in the study of the emergence of C. gattii in BC and in particular contributed to our understanding of the range of environmental niches for the pathogen. A single veterinary pathology laboratory handled clinical specimens from the majority of southern BC veterinary practices, and this allowed early detection and monitoring of C. gattii in the animal population. In addition, the BC Provincial Animal Health Branch Laboratory was able to perform necropsies on porpoises that were found stranded and dead on Vancouver Island and nearby islands, and these became index cases (Stephen et al., 2002). Beginning prior to the first documented human case in 1999 and continuing to the present, veterinary cases have been diagnosed two to three times more frequently than human cases (Lester et al., 2004); this disparity is likely an underestimate given that only those animals seen by a veterinarian are diagnosed and that infections in wildlife are not considered. The diagnosed cases have primarily been in companion animals (dogs, cats, and ferrets) but also include other domesticated species such as llamas, horses, mink, and psittacine birds (Duncan et al., 2006b; Lester et al., 2004; Stephen et al., 2002). Sampling in the environs of these animal cases has been particularly productive for identifying sources of C. gattii (Kidd et al., 2007a++; MacDougal et al., 2007++).

Unlike the colonized koalas of Australia (Krockenberger et al., 2002), no significant wild animal host or reservoir has been identified in BC. Limited surveys of wild animals were performed between 2003 and 2007 with the examination of necropsy samples of nares, lung, anus or cloacae, and brain for C. gattii. In two surveys, all fatally injured animals turned into rescue facilities were studied. In the first study, 91 animals (14 species) were examined, and only two eastern gray squirrels were positive (Duncan et al., 2006a). In the second study, only one great blue heron was found to have a pulmonary C. gattii infection of 226 animals necropsied (Bartlett, unpublished data). Additionally, 18 river otters were trapped in early spring 2007, but none showed signs of disease or colonization with C. gattii (Bartlett and Balke, unpublished data). Duncan et al. (2005b) established sentinel veterinary practices in areas known to have exposure to airborne C. gattii and found positive C. gattii cultures from nasal swabs of asymptomatic animals in 4.3% of 94 cats, 1.1% of 280 dogs, and 1.5% of 351 horses. Additionally, six cats and two dogs were found to have cryptococcal antigen titers of greater than 1:2. Of seven cats and five dogs that were selected from the asymptomatic but culture- or antigen-positive cohorts and followed over 27 months, only two cats progressed to clinical disease, suggesting that the majority of animals exposed to C. gattii may naturally clear the organism (Duncan et al., 2005a).

In the first years of recognition of both the emergence of C. gattii disease and the stability of the pathogen’s environmental niche, it appeared that all human and animal cases had some contact with Vancouver Island. MacDougall and Fyfe (MacDougall and Fyfe, 2006) were able to identify human cases of disease

Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
×

with historic travel to Vancouver Island and to determine a likely incubation period (median 6–7 months) based on isolated exposure. In addition, Hoang et al. (Hoang et al., 2004) performed a retrospective chart review examining all cases of cryptococcosis identified between 1997 and 2002 at the largest teaching hospital located on the BC mainland. They discovered that there had been a sudden increase in cryptococcal cases of all origins (C. neoformans var grubii, C. n. var neoformans, C. gattii, and C. laurentii), but all C. gattii cases (3/26 charts) reported travel history to Vancouver Island (Hoang et al., 2004). The first cases of mainland-acquired C. gattii infection were identified in animals (ferret, llama, and cats) in 2003, and three cases in cats in Washington were reported in 2005. Eight off-island human cases with no travel history to an endemic area were documented (five in BC and two in Oregon) in 2004 to 2005 (MacDougall et al., 2007++). Upton et al. (Upton et al., 2007+) recently reported the first confirmed human case in Washington presenting in 2006, and the Whatcom County Public Health Department has now identified four additional cases diagnosed in 2007 (Stern, personal communication). Unlike in BC, cryptococcosis is not yet a reportable disease in Washington, although public health officials are actively soliciting case studies. The VGIIa genotype accounted for 78% of the examined veterinary cases and 87% of the human cases; all off-island veterinary cases to date had the VGIIa genotype (Bartlett, unpublished data) (MacDougall et al., 2007++).

Environmental and Dispersal Studies on Vancouver Island

Competing theories have been proposed regarding the origin of C. gattii on Vancouver Island (eg, recent introduction, long-term colonization, specific imported vectors). Suffice it to say, the colonization pattern and dispersal of the organism argues against a one-time introduction to Vancouver Island, particularly if the timeline extends only to the first animal and human cases (1998–1999). The first systematic sampling performed on Vancouver Island in 2002 mapped the colonization of C. gattii along a 200 km north-south and a 40 km east-west corridor. This study revealed that C. gattii is not homogeneously spread in the environment, with central Vancouver Island having a higher percentage of colonized trees and higher concentration of the organism in soil. The heterogeneous pockets of colonization could explain why limited-sampling strategies may miss the organism. Additionally, even though C. gattii has been found to be permanently colonized in some areas, it appears to be transiently colonized in others. The permanently colonized sites have yielded C. gattii repeatedly over the last 5 years, although transiently positive results may be due to limits of detection or failure of the organism to establish true colonization (Kidd et al., 2007a++). As well, sites that initially appeared to be negative for C. gattii have more recently yielded positive environmental samples (Bartlett, unpublished data). It has been shown that in addition to the airborne spread of propagules, wood products,

Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
×

soil, water, vehicles, and shoes can act as dispersal mechanisms for the organism (Kidd et al., 2007a++). These mechanisms are consistent with the findings of a veterinary case-control study, where statistically significant risk factors for disease in cats and dogs related to soil disturbance within 10 km of cases, logging within 10 km, travel to Vancouver Island, or owner hiking within 6 months of diagnosis (Duncan et al., 2006c). Although limited environmental sampling in the San Juan Islands, Olympic Peninsula, and Oregon has not yielded C. gattii (Fraser et al., 2006; Kidd et al., 2007b++; Upton et al., 2007+). Kidd et al. (2007a++,2007b++) reported finding positive environmental samples from islands in the Georgia Strait and in northern Washington.

A rather surprising finding was that co-isolated C. gattii strains are heterogeneous. The first isolates distributed to the research community were mostly from one sampling site (central Vancouver Island) and may have unduly influenced our thinking about the composition of the BC outbreak strains (Kidd et al., 2004++; Fraser et al., 2005+; Fraser et al., 2003). In the initial analysis of the C. gattii isolates from this site, Kidd et al. (Kidd et al., 2004++) used polymerase chain reaction (PCR)-fingerprinting to demonstrate that 5% represented the VGI molecular type and 95% belonged to VGII (90% of these were VGIIa and 10% were VGIIb based upon a one polymorphic band in the PCR-fingerprint profiles). Subsequent work revealed that the composition of the C. gattii population varies in different regions where detailed molecular subtyping of isolates has been undertaken. In the southern extreme of Vancouver Island, VGIIa accounts for 91% of the isolates and the remainder are VGIIb, whereas at another site VGIIa accounts for only 66% of the isolates, with VGIIb and VGI at 19% and 15%, respectively (Bartlett and Kidd, unpublished data). Of course, the genotype frequencies are likely to be dynamic, and repeated sampling is important. Also, additional diagnostic tools sensitive enough to detect and differentiate isolates directly in environmental samples (eg, PCR on soil samples) would facilitate a better understanding of the population structure and mechanisms of spread of the organism. Already heightened awareness of changing ecologic niches has resulted in an expansion of knowledge of the environmental origins of other cryptococcal species (Filion et al., 2006).

Molecular Characterization of Isolates from BC and the Pacific Northwest

Following the initial analyses of genotype frequency described above, Kidd et al. (2005+) used multilocus sequence typing (MSLT) and gene genealogy analyses with four genes to examine patterns of molecular variation as well as population structure of the isolates from Vancouver Island compared with a worldwide sample of C. gattii strains. This work demonstrated that the VGIIa and VGIIb genotypes originally established by PCR-fingerprinting (Kidd et al., 2004++) corresponded to specific MLST profiles. Similar MLST results with additional genes were obtained by Fraser et al. (Fraser et al., 2005+). Of specific

Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
×

interest from these studies was the identification of isolates from other areas of the world with identical or similar genotypes to the VGIIa (as represented by isolate A1MR265) and VGIIb (represented by isolate A1MR272) strains from Vancouver Island. For example, the VGIIa genotype was also shared by the NIH444 strain (from a patient in Seattle, ca 1971), CBS7750 (from a Eucalyptus tree in San Francisco, ca 1990) and with isolates from other parts of North America (KB10455 and KB9944) (Fraser et al., 2005+; Kidd et al., 2005+). A Brazilian clinical isolate, ICB107, differed from the VGIIa genotype at only one of 22 loci (Fraser et al., 2005+). The VGIIb genotype was also observed among environmental isolates from Australia (eg, Ram002, Ram005, WM1008), clinical isolates from Australia (eg, NT-6, NT-13), as well as a clinical isolate from Thailand (MC-S-115) (Fraser et al., 2005+; Kidd et al., 2005+). A Caribbean strain 99/473 of the VGIIb type was also found to differ at only one of 22 loci (Fraser et al., 2005+). Intriguingly, two isolates from human cases in Oregon (2004) were recently found to represent subtypes within the VGII genotype that have not identified among any other strains to date (MacDougall et al., 2007++).

The VGIIa and VGIIb isolates from Vancouver Island have been obtained from both clinical and environmental sources. However, the situation is more complex for strains of the VGI genotype from clinical and environmental sources. Specifically, Kidd et al. (2005+) characterized six VGI isolates from Vancouver Island and identified four different genotypes by MLST analysis. Two of these were environmental isolates with a different genotype from the clinical isolates. Thus, in contrast to the VGII types, it was not possible to establish an epidemiologic link between environmental and clinical isolates of the VGI type. However, recent analysis of further environmental VGI isolates from Vancouver Island indicated that they were highly similar to a porpoise isolate (A1MF2863), being identical at four MLST loci (Kidd and Bartlett, unpublished data). It is possible that the clinical isolates of the VGI type represent strains acquired during travel outside of Vancouver Island.

Overall, Kidd et al. (2005+) found that the Vancouver Island isolates were part of a predominately clonal population with little evidence of sexual recombination occurring between them. Fraser et al. (2005+) also presented evidence that the VGIIa and VGIIb strains from Vancouver Island were related in that they shared 14 identical loci out of the 30 examined and proposed that the genotypes represent either siblings arising from a past mating event, or that one may be the parent of the other, perhaps as the result of same-sex mating between MATα parents. Selected isolates from Vancouver Island and other parts of the world have been tested for mating competence. These studies revealed that the VGII isolates are generally fertile whereas VGI strains are not (Campbell et al., 2005; Fraser et al., 2003; Kidd et al., 2004++). In general, the ability of C. gattii isolates to mate has implications for recombination events that might generate strains with different virulence properties and environmental adaptability.

Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
×

The Global Distribution of C. gattii

Prior to the emergence of C. gattii on Vancouver Island, it was commonly accepted that this species was restricted to tropical and subtropical regions of the world, and that infection was associated with exposure to Eucalyptus trees (Ellis and Pfeiffer, 1990; Kwon-Chung and Bennett, 1984; Sorrell et al., 1996). The idea of a limited geographic distribution came from a study that surveyed a worldwide collection of clinical isolates (Kwon-Chung and Bennett, 1984). This survey revealed that C. gattii was prevalent only in regions with tropical and subtropical climates (22%–50% of isolates) relative to C. neoformans (50%–71% of isolates). However, this study also reported that 13% of the strains from North America, and 3.3% of the strains from Europe were C. gattii (without reference to travel histories). More recent surveys have focused on identifying the molecular types of C. gattii found in collections from various regions. In this regard, VGI appears to be the most widely distributed type worldwide (Kidd, 2003; Meyer et al., 2003), and this type is also found most frequently among clinical and environmental isolates in Australia (Campbell et al., 2005). Strains of the VGII type are also found in parts of Australia as well as in North and South America (Fraser et al., 2005+; Kidd, 2003; Kidd et al., 2004++; Kidd et al., 2005+; Meyer et al., 2003). In a recent, large-scale study of IberoAmerican isolates, VGIII predominated, and this type has also been found in India and the United States (Kidd, 2003; Meyer et al., 2003). The VGIV type has been found in Central America and South Africa (Kidd, 2003; Meyer et al., 2003). Notably, the VGIII and VGIV types were not found in the collections from Vancouver Island suggesting that these genotypes may have a more limited distribution.

More recently, Meyer et al. (2007) have surveyed 160 VGII strains recovered globally since 1986 using PCR-fingerprinting, amplified fragment length polymorphism analysis and MLST with eight loci. This work revealed that the VGIIa genotype from Vancouver Island is also found among Brazilian isolates and that Colombian isolates are closely related. Interestingly, the majority of the latter isolates are mating type a in contrast to mating type α for the Vancouver Island strains (Escandon et al., 2006), and mating was demonstrated between the Colombian MATa strains and VGIIa MATα strains from Brazil and Vancouver Island. This work suggests that the VGIIa genotype was present in South America as early as 1986 and it sheds additional light on the potential mating interactions for VGII types of C. gattii that may be relevant for the situation on Vancouver Island.

Overall, these surveys provide an interesting view that the genotypes of C. gattii (at least for VGI and VGII) are likely to have a worldwide distribution and the concomitant potential for permanent colonization of suitable environments. This view highlights the need for more extensive environmental sampling globally to generate a detailed picture of genotype frequency over time and location. The most extensive view is now available from the work on Vancouver Island and the lessons learned from this work can be applied in other locations (Kidd

Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
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et al., 2007a++), especially with regard to the need for extensive multisource sampling over many years. The wide distribution of C. gattii genotypes should also be considered in light of recent reports that infections with this species are occurring in patients with AIDS (South Africa [Morgan et al., 2006], Southern California [Chaturvedi et al., 2005a]). Therefore, it will be important to identify the endemic areas for specific C. gattii genotypes in order to monitor human and animal disease.

Origin of the C. gattii in BC and the Pacific Northwest:
Aboriginal Species or Landed Immigrant?

It is fun to speculate about the origin of the genotypes on Vancouver Island, and this activity has consumed much energy in the research community. However, the extent of global strain dispersal has been demonstrated to be significant (Kidd et al., 2005+, Xu et al., 2000), making it difficult to accurately determine a specific origin of any given genotype. It is possible that the species has been a long-term resident of BC and that changing conditions (eg, climate or land use) or improved surveillance are responsible for the current level of awareness. Alternatively, it has been suggested that the emergence is due to the recent introduction of a particularly virulent genotype that may be well adapted to the local conditions such that large numbers of infectious cells are propagated (Fraser et al., 2005+). Although it may be difficult to garner strong evidence for a given theory, it is clear that much more information is needed about the C. gattii genotypes on Vancouver Island and worldwide and about the disease caused by C. gattii in immunocompetent hosts. Below, we discuss some of the studies that are needed to generate a more detailed view of C. gattii that may help in infection control.

Ecologic adaptability, colonization, and dispersal

The environmental sampling revealed a high level of soil colonization on Vancouver Island, and it would be interesting to examine soil persistence and competition in laboratory and field settings. These types of experiments may be relevant to addressing how the fungus becomes aerosolized and the nature of the infectious particle. An investigation of conditions required for the propagation of the infectious particles in soil/trees would also be highly relevant to understanding the factors that influence exposure of humans/animals.

It is likely that no one factor can explain the dramatic emergence of C. gattii on Vancouver Island, and there may be interplay between soil conditions, temperature, and moisture. Current weather station data are insufficient to adequately describe the microclimates in areas colonized by the pathogen. Climate oscillations driven by alternating El Niño and La Niña currents have produced both drier and wetter than normal summer conditions in BC over the last few decades. Outbreaks of another fungal disease, coccidioidomycosis, have been

Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
×

shown to follow soil disruption in California (Zender and Talamantes, 2006). Data gathered from the BC environment conclusively show that C. gattii is well adapted to survive in dry, low nutrient soil and is more likely to be airborne during dry summer weather (Kidd et al., 2007a++). The stability of the colonization of soil and trees at permanently colonized sites suggests that the pathogen can effectively compete with resident soil microflora. Longer cycles of meteorology patterns and finer tools of climate measurement will be needed to understand the complex relationship of microbe, climate, and ecologic niche.

Additional sampling around the world is needed to investigate predicted favorable climate/soil/water conditions that might allow colonization by C. gattii. Mak (2007) has recently developed ecologic niche models that predict the probable extent of environmental colonization of C. gattii based on human, animal, and environmental data and climate projections for the Pacific Northwest (Fig. A1-1). Areas that may eventually be impacted include the Lower Mainland of BC with a population base of approximately 2 million people. These projections could be used by public health officials on both sides of the US-Canada border to plan strategies for risk communication and anticipated morbidity and mortality (Mak, 2007).

Clinical considerations

Perhaps the most relevant topics regarding the emergence of C. gattii have to do with identifying risk factors for people, designing ways to limit exposure, and developing effective methods to treat the infections that do occur. It is common to see statements in the literature that C. gattii is a primary pathogen that infects immunocompetent people, and that C. neoformans is an opportunistic pathogen that infects immunocompromised people. The distinction may be less clear given that C. gattii is now being found in AIDS patients and C. neoformans can infect seemingly immunocompetent people (Chaturvedi et al., 2005a; Morgan et al., 2006; Speed and Dunt, 1995). There is clearly a need for retrospective studies of patients to determine host risk factors as well as prospective case studies to determine efficacy of treatments. The number of cases continuing to occur on Vancouver Island (and among tourists [Lindberg et al., 2007]) would allow this type of investigation.

An interesting consideration in terms of exploring possible virulence differences for C. gattii versus C. neoformans is whether mouse virulence studies have relevance for human disease. For example, the strains with the VGIIa and VGIIb genotypes from Vancouver Island both cause disease in humans, but laboratory studies revealed virulence differences between the two strains tested (Fraser et al., 2005+). The more virulent strain, A1MR265, of the VGIIa genotype showed equal virulence in the mouse model to strain H99 that is representative of the most common VNI type of C. neoformans (var grubii). It is possible that these results reflect the fact that only one isolate of each genotype from Vancouver Island

Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
×

was tested and the isolates selected may not be representative. It is clear, however, that strains of C. gattii show virulence differences (Kronstad, unpublished data) (Chaturvedi et al., 2005b; Fraser et al., 2005+) and that multiple isolates from Vancouver Island and worldwide collections need to be tested. The same is true for C. neoformans as demonstrated by the range of virulence detected by Clancy et al. (2006). Thus, we need to develop better models to assess differences in virulence and to explore possible differences that may be relevant to infection of immunocompetent versus immunocompromised hosts.

Applications of genomic approaches to develop a detailed understanding of C. gattii

The emergence of C. gattii provided the impetus to sequence the genomes of isolates representing the VGI (WM276) and VGIIa (A1MR265) genotypes (Michael Smith Genome Sciences Center, 2007++; The Broad Institute, 2007++). These are important resources for the next steps in characterizing the virulence of C. gattii, the genetic diversity of the species and the interactions of the fungus with the environment. One can imagine, for example, using the genomes for transcriptome and proteome studies to identify differences in expression for C. gattii relative to C. neoformans. Some of these differences may reveal factors that contribute to the primary pathogenesis of C. gattii relative to C. neoformans. The two C. gattii genomes also provide a platform for more detailed analyses of genotypes and comparative studies of genome variability. In the latter case, comparative hybridization or genome resequencing approaches can be used to study the microevolution of genomes in strains in the environment and clinical strains during passage through human and animal hosts (eg, during relapse or drug therapy). Comparative genome hybridization experiments with the VGI and VGIIa genomes have been initiated to identify genomic changes in mutants that have lost virulence and to examine genome variation in strains representing the VGI, VGIIa, and VGIIb genotypes (Kronstad, unpublished data). The declining cost of sequencing will also allow further genome-sequencing projects to provide a deeper view of genome content and variability. The more detailed information may eventually lead to the separation of the molecular types of C. gattii into distinct varieties or species.

Media Coverage of the Emergence of C. gattii

Any emerging infectious disease represents a challenge to the public health system. The system must respond to educate caregivers about appropriate interventions while balancing the message to allow the public to make informed choices. For example, the lay press recently reported concern by members of the public in Alabama where experimental plots of genetically engineered Eucalyptus trees will be grown; the fear being that C. gattii will be imported into the environment

Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
×

through the Eucalypts (United Press International, 2007), even though no link to Eucalyptus was shown in the BC experience (Kidd et al., 2007a++). In an examination of press coverage of C. gattii as an emerging infectious disease agent, researchers at the University of BC Centre for Health and Environment Research found that during the period 2001 to 2006, BC newspapers carried 422 articles warning the public about West Nile Virus (although no West Nile Virus cases have been reported in BC) compared with 79 articles about C. gattii (170 human cases, eight deaths) (Nicol et al., unpublished data). The research group concluded that because West Nile Virus is a public health risk with identifiable precautionary actions in central Canada, newspapers were more likely to print stock West Nile Virus stories. C. gattii was seen to be a local phenomenon with no identifiable risk aversion strategies and to have potential economic repercussions to the areas affected and so was less reported. There also seemed to be confusion by news writers about the biology of Cryptococcus because the term “virus” seems to be better understood as a pathogen compared to “yeast” (Nicol et al., unpublished data). Similarly, some news items labeled C. gattii as an “Australian” fungus despite the body of literature cited above on the global distribution of the pathogen. Overall, these observations demonstrate that effective education of the media and the public is a critical component of the management of an emerging infectious disease.

Conclusions

A great deal has been learned about the emergence of C. gattii in BC over the past 8 years. We now have a clear picture of the environmental sources of the pathogen and mechanisms of dispersal, we have an understanding of the genotypes that are causing disease in humans and animals, and we have some information about clinical presentation and treatment. Certainly, there is a great deal more to investigate in terms of risk factors for the human population and treatment outcomes. In this regard, the situation on Vancouver Island presents an opportunity to develop a detailed view of an emerging infectious disease with regard to environmental exposure, the role of sentinel animals in monitoring risk, and the underlying factors that influence human susceptibility. This information may prove useful for other emerging diseases and provide methods to manage both the ongoing situation in BC and the apparent emergence of the disease in the Pacific Northwest.

Acknowledgments

The authors thank the members of the BC Cryptococcal Working Group (http://www.cher.ubc.ca/cryptococcus/) and the BC Centre for Disease Control (http://www.bccdc.org/) for helpful discussions and Sunny Mak for the preparation of Figure A1-1. The authors are supported in part by grants from the US

Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
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National Institute of Allergy and Infectious Disease (Dr. Kronstad, award RO1AI-053721), the Canadian Institutes of Health Research (Drs. Kronstad and Bartlett), British Columbia Lung Association (Dr. Bartlett), and WorkSafe BC (Dr. Bartlett). Dr. Kronstad is a Burroughs Wellcome Fund Scholar in Molecular Pathogenic Mycology, and Dr. Bartlett is a Michael Smith Foundation for Health Research Scholar.

References and Recommended Reading

Barreto de Oliveira MT, Boekhout T, Theelen B, et al.: Cryptococcus neoformans shows a remarkable genotypic diversity in Brazil. J Clin Microbiol 2004, 42:1356–1359.

Bicanic T, Harrison TS: Cryptococcal meningitis. Br Med Bull 2005, 72:99–118.

Bicanic T, Wood R, Bekker LG, et al.: Antiretroviral roll-out, antifungal roll-back: access to treatment for cryptococcal meningitis. Lancet Infect Dis 2005, 5:530–531.

Boekhout T, Theelen B, Diaz M, et al.: Hybrid genotypes in the pathogenic yeast Cryptococcus neoformans. Microbiol 2001, 147:891–907.

Boekhout T, van Belkum A, Leenders ACAP, et al.: Molecular typing of Cryptococcus neoformans: taxonomic and epidemiological aspects. Int J Sys Bacteriol 1997, 47:432–442.

Campbell LT, Fraser JA, Nichols CB, et al.: Clinical and environmental isolates of Cryptococcus gattii from Australia that retain sexual fecundity. Eukaryot Cell 2005, 4:1410–1419.

Casadevall A, Perfect JR: Cryptococcus neoformans. Washington, DC: American Society for Microbiology Press; 1998.

Chaturvedi S, Dyavaiah M, Larsen RA, Chaturvedi V: Cryptococcus gattii in AIDS patients, southern California. Emerg Infect Dis 2005a, 11:1686–1692.

Chaturvedi S, Ren P, Narasipura SD, Chaturvedi V: Selection of optimal host strain for molecular pathogenesis studies on Cryptococcus gattii. Mycopath 2005b, 160:207–215.

Clancy CJ, Nguyen MH, Alandoerffer R, et al.: Cryptococcus neoformans var. grubii isolates recovered from persons with AIDS demonstrate a wide range of virulence during murine meningoencephalitis that correlates with the expression of certain virulence factors. Microbiol 2006, 152:2247–2255.

Duncan C, Schwantje H, Stephen C, et al.: Cryptococcus gattii in wildlife of Vancouver Island, British Columbia, Canada. J Wildl Dis 2006a, 42:175–178.

Duncan C, Stephen C, Campbell J: Clinical characteristics and predictors of mortality for Cryptococcus gattii infection in dogs and cats of southwestern British Columbia. Can Vet J 2006b, 47:993–998.

Duncan C, Stephen C, Lester S, Bartlett KH: Follow-up study of dogs and cats with asymptomatic Cryptococcus gattii infection or nasal colonization. Med Mycol 2005a, 43:663–666.

Duncan C, Stephen C, Lester S, Bartlett KH: Sub-clinical infection and asymptomatic carriage of Cryptococcus gattii in dogs and cats during an outbreak of cryptococcosis. Med Mycol 2005b, 43:511–516.

Duncan CG, Stephen C, Campbell J: Evaluation of risk factors for Cryptococcus gattii infection in dogs and cats. J Am Vet Med Assoc 2006c, 228:377–382.

Ellis DH, Pfeiffer TJ: Natural habitat of Cryptococcus neoformans var gattii. J Clin Microbiol 1990, 28:1642–1644.

Escandon P, Sanchez A, Martinez M, et al.: Molecular epidemiology of clinical and environmental isolates of the Cryptococcus neoformans species complex reveals a high genetic diversity and the presence of the molecular type VGII mating type a in Colombia. FEMS Yeast Res 2006, 6:625–635.

Filion T, Kidd S, Aguirre K: Isolation of Cryptococcus laurentii from Canada goose guano in rural upstate New York. Mycopathologia 2006, 162:363–368.

Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
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+ Fraser JA, Giles SS, Wenink EC, et al.: Same-sex mating and the origin of the Vancouver Island Cryptococcus gattii outbreak. Nature 2005, 437:1360–1364.
An extensive MLST analysis of C. gattii isolates from Vancouver Island and from around the world. The authors found shared genotypes between the VGIIa and VGIIb strains from BC and strains of these molecular types from other parts of the world. This study presents interesting hypotheses about the origin of the VGIIa genotype in BC and reports the first virulence tests of VGIIa and VGIIb strains from Vancouver Island.

Fraser JA, Lim SM, Diezmann S, et al.: Yeast diversity sampling on the San Juan Islands reveals no evidence for the spread of the Vancouver Island Cryptococcus gattii outbreak to this locale. FEMS Yeast Res 2006, 6:620–624.

Fraser JA, Subaran RL, Nichols CB, Heitman J: Recapitulation of the sexual cycle of the primary fungal pathogen Cryptococcus neoformans var. gattii: implications for an outbreak on Vancouver Island, Canada. Eukaryot Cell 2003, 2:1036–1045.

Hoang LM, Maguire JA, Doyle P, et al.: Cryptococcus neoformans infections at Vancouver Hospital and Health Sciences Centre (1997–2002): epidemiology, microbiology and histopathology. J Med Microbiol 2004, 53:935–940.

Kidd SE: Molecular epidemiology and characterization of genetic structure to assess speciation within the Cryptococcus neoformans complex [PhD thesis]. Sydney: University of Sydney; 2003.

++ Kidd SE, Chow Y, Mak S, et al.: Characterization of environmental sources of the human and animal pathogen Cryptococcus gattii in British Columbia, Canada, and the Pacific Northwest of the United States. Appl Environ Microbiol 2007a, 73:1433–1443.

This important study describes a systematic and thorough investigation of the environmental colonization of C. gattii on Vancouver Island and the Pacific Northwest. Key findings include the isolation of the pathogen from air, trees, soil, freshwater, and seawater, and the identification of colonization hotspots. Additionally, this study identified characteristics of soil that may favor C. gattii colonization.

++ Kidd SE, Bach PJ, Hingston AO, et al.: Cryptococcus gattii dispersal mechanisms, British Columbia, Canada. Emerg Infect Dis 2007b, 13:51–57.

This study employed systematic environmental sampling strategies to document patterns of C. gattii colonization on Vancouver Island and to obtain evidence for human-mediated dispersal of the fungus.

+ Kidd SE, Guo H, Bartlett KH, et al.: Comparative gene genealogies indicate that two clonal lineages of Cryptococcus gattii in British Columbia resemble strains from other geographical areas. Eukaryot Cell 2005, 4:1629–1638.

This study employed MLST analysis and gene genealogy to reveal a predominantly clonal population among the Vancouver Island isolates and to demonstrate that the genotypes of isolates from BC resembled those of strains from other parts of the world.

++ Kidd SE, Hagen F, Tscharke RL, et al.: A rare genotype of Cryptococcus gattii caused the cryptococcosis outbreak on Vancouver Island (British Columbia, Canada). Proc Natl Acad Sci USA 2004, 101:17258–17263.

This paper describes the results of the first marshaling of the expertise of the international research community to tackle the analysis of the emergence of C. gattii in BC. The investigators described initial studies on the environmental source of the pathogen and identified the molecular types of C. gattii that were responsible for the human and animal cases.

Krockenberger MB, Canfield PJ, Malik R: Cryptococcus neoformans in the koala (Phascolarctos cinereus): colonization by C n var gattii and investigation of environmental sources. Med Mycol 2002, 40:263–272.

Kwon-Chung KJ, Bennett JE: Epidemiologic differences between the two varieties of Cryptococcus neoformans. Am J Epidemiol 1984, 120:123–130.

Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
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Kwon-Chung KJ, Boekhout T, Fell JW, Diaz M: (1557) Proposal to conserve the name Cryptococcus gattii against C. hondurianus and C. bacillisporus (Basidiomycota, Hymenomycetes, Tremellomycetidae). Taxon 2002, 51:804–806.

Kwon-Chung KJ, Varma A: Do major species concepts support one, two or more species within Cryptococcus neoformans? FEMS Yeast Res 2006, 6:574–587.

Lester SJ, Kowalewich NJ, Bartlett KH, et al.: Clinicopathologic features of an unusual outbreak of cryptococcosis in dogs, cats, ferrets, and a bird: 38 cases (January to July 2003). J Am Vet Med Assoc 2004, 225:1716–1722.

Lindberg J, Hagen F, Laursen A, et al.: Cryptococcus gattii risk for tourists visiting Vancouver Island, Canada. Emerg Infect Dis 2007, 13:178–179.

Litvintseva AP, Thakur R, Vilgalys R, Mitchell TG: Multilocus sequence typing reveals three genetic subpopulations of Cryptococcus neoformans var grubii (serotype A) including a unique population in Botswana. Genetics 2006, 172:2223–2238.

MacDougall L, Fyfe M: Emergence of Cryptococcus gattii in a novel environment provides clues to its incubation period. J Clin Microbiol 2006, 44:1851–1852.

++ MacDougall L, Kidd SE, Galanis E, et al.: Spread of Cryptococcus gattii in British Columbia, Canada, and detection in the Pacific Northwest, USA. Emerg Infect Dis 2007, 13:42–50.

This paper describes the detection of C. gattii in three people and eight animals without a travel history to Vancouver Island, and the detection of the pathogen in air, soil, water and on trees from sites off the island. The study also reported locally acquired C. gattii infections in three cats in Washington and two people in Oregon; interestingly, the genotypes of the strains from the Oregon cases were VGIIa- and VGIIb-like, but MLST results indicated differences from the isolates of the corresponding subtypes from Vancouver Island.

Mak S: Ecological niche modeling of Cryptococcus gattii in British Columbia [MSc thesis]. Vancouver: University of British Columbia; 2007.

Meyer W, Castaneda A, Jackson S, et al.: Molecular typing of IberoAmerican Cryptococcus neoformans isolates. Emerg Infect Dis 2003, 9:189–195.

Meyer W, Kaocharoen S, Trills L, et al.: Global molecular epidemiology of Cryptococcus gattii VGII isolates traces the origin of the Vancouver Island outbreak to Latin America [abstract]. Presented at the 24th Fungal Genetics Conference. Pacific Grove, CA; March 20–25, 2007.

Meyer W, Marszewska K, Amirmostofian M, et al.: Molecular typing of global isolates of Cryptococcus neoformans var neoformans by PCR-fingerprinting and RAPD—a pilot study to standardize techniques on which to base a detailed epidemiological survey. Electrophoresis 1999, 20:1790–1799.

++ Michael Smith Genome Sciences Center: Cryptococcus Neoformans Summary. http://www.bcgsc.ca/project/cryptococcus/summary/. Accessed July 9, 2007.

The sequences of the genomes of VGI and VGIIa strains are exceptional resources for detailed investigations of the virulence properties of C. gattii. In addition, the sequences allow genome-wide comparative studies with the genomes of C. neoformans var neoformans strains and a var grubii strain.

Morgan J, McCarthy KM, Gould S, et al.: Cryptococcus gattii infection: characteristics and epidemiology of cases identified in a South African province with high HIV seroprevalence, 2002–2004. Clin Infect Dis 2006, 43:1077–1080.

Sorrell TC, Brownlee AG, Ruma P, et al.: Natural environmental sources of Cryptococcus neoformans var gattii. J Clin Microbiol 1996, 34:1261–1263.

Sorrell TC: Cryptococcus neoformans variety gattii. Med Mycol 2001, 39:155–168.

Speed B, Dunt D: Clinical and host differences between infections with the two varieties of Cryptococcus neoformans. Clin Infect Dis 1995, 21:28–34.

Stephen C, Lester S, Black W, et al.: Multispecies outbreak of cryptococcosis on southern Vancouver Island, British Columbia. Can Vet J 2002, 43:792–794.

++ The Broad Institute: Cryptococcus neoformans Serotype B Database. http://www.broad.mit.edu/annotation/genome/cryptococcus_neoformans_b. Accessed July 9, 2007.

Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
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The sequences of the genomes of VGI and VGIIa strains are exceptional resources for detailed investigations of the virulence properties of C. gattii. The genome sequence of a C. neoformans var grubii strain is also available at the Broad Institute.United Press International: GE eucalyptus tree investigation urged. http://www.sciencedaily.com/upi/index.php?feed=Science&article=UPI-1-20070614-13565200-bc-us-eucalyptus.xml. Accessed June 17, 2007.

+ Upton A, Fraser JA, Kidd SE, et al.: First contemporary case of human infection with Cryptococcus gattii in Puget Sound: evidence for spread of the Vancouver Island outbreak. J Clin Microbiol 2007, In press.
This report and MacDougall et al. (2007) document the recent emergence of C. gattii outside of BC.

Xu J, Vilgalys R, Mitchell TG: Multiple gene genealogies reveal dispersion and hybridization in the human pathogenic fungus Cryptococcus neoformans. Mol Ecol 2000, 9:1471–1481.

Zender CS, Talamantes J: Climate controls on Valley Fever incidence in Kern County, California. Int J Biometeorol 2006, 50:174–182.

A2

THE GOOD, THE BAD, AND THE UGLY:
FUNGI MOLD YOUR WORLD

Meredith Blackwell3

Fungi4 are important members of many ecosystems. As heterotrophs they are involved in nutrient cycles, especially of carbon, nitrogen, and phosphorus. The effects of fungi were observed in prehistoric times, and their part in causing plant disease was understood before the germ theory was advanced. Today fungi are featured in the popular press and science Internet postings, indicating that they are of increasing interest and importance. Molecular methods have helped to popularize fungi by bringing rapid progress to fungal classification and discovery and have enhanced understanding of their biology. Fungi are associates of all major groups of organisms and are especially well known for their interactions with plants and insects. Fungi also are economically important and provide drugs, foods, and fermented beverages. The value of fungal activities and products far exceeds the costs of the diseases they cause.

Introduction

Human beings were aware of fungal fruiting bodies in prehistoric times, and the sudden appearance of mushrooms after rain awed those who did not comprehend the fungus lifecycle. Lowy (1974) wrote that the sudden appearance of mushrooms of Amanita muscaria was believed to have been caused by thunder-

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3 Louisiana State University.

4 In addition to members of Kingdom Fungi, several other organisms of the fungus-like group Oomycota (Phytophthora) are included.

Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
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bolts as they struck the ground, a belief held independently in Roman, Hindu, and Mayan cultures. Humans endowed mushrooms with magical properties (Wasson, 1968), and evidence of early fungal use exists in many parts of the world. Grave guardians, masks, clothing ornaments, and other artifacts were made from the fruiting bodies of wood-decaying basidiomycetes such as Fomitopsis officinalis and Haploporus odorus (Blanchette, 1997; Blanchette et al., 1992a, 2002). A surviving mushroom culture centered on magic mushrooms existed in Oaxaca for many years, and the celebrated curandera, Maria Sabina, was visited by a number of prominent individuals and notable musicians who sought her spiritual guidance (Wasson, 1957, 1976). Although yeasts themselves were not known, evidence of their activity comes from residues in nine millennia-old Neolithic vessels (Vouillamoz et al., 2006).

Plant pathogenic fungi also were known in ancient times. Three centuries BC, Theophrastus recognized fungi as the cause of certain diseases of crops, but by the first century, the knowledge had been lost, and Pliny attributed lost yields to the gods or stars (Carefoot and Sprott, 1969). Fungal effects such as disease were not understood by many until the observations and experiments of Miles Joseph Berkeley and Anton de Bary around the time of the Irish potato famine of 1845–1846. This work actually came before the general acceptance of the germ theory. The contribution of de Bary also argued strongly against a lingering belief in spontaneous generation (Matta, 2010). Fungi continue to appear suddenly as they invade natural landscapes to cause diseases of plants and animals. The invading organisms often are not noticed until they encounter naïve hosts in new regions where they cause devastating diseases. Earlier invasions included the chestnut blight fungus and several waves of Dutch elm disease fungi (Alexopoulos et al., 1996). The papers in this volume, Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health (IOM, 2011), cover the newest waves of invasive fungal diseases and their attack on naïve hosts.

More important, however, is the realization that fungi are essential for life on Earth. Fungi are decomposers that destroy plant and animal bodies and return carbon, nitrogen, phosphorus, and other minerals to nutrient cycles. Compatible with their primary role in decomposition, fungi interact with other living organisms in nutritional relationships, and their secondary metabolites and enzymes supply medicines, food and drink, and industrial products for profitable enterprises. Fungi appear regularly in newspapers and magazines. Over the past year, the New York Times featured fungi prominently. Articles have included reports of the identification of a microsporidian fungus partly responsible for colony collapse disorder of bees, a chytrid responsible for global amphibian decline, Geomyces destructans of bats, and pathogens of home garden vegetables. Ecological topics included interactions between bark beetles and fungal symbionts, mycorrhizal associations, sexual reproduction in truffles, a fungus that exerts selective pressure on rotifers, and fungal function in the environment. Fungi also have been covered in the Wall Street Journal as food items, inhabitants of saunas, and the “Torula

Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
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of Cognac,” Baudoinia compniacensis, the fungus that grows on walls of wine cellars in mists of alcoholic vapors. One fungus was reported widely because it prompted a murder investigation in a German forest when its sulfurous odor of decay was mistaken for that of a dead body (Anonymous, 2005). Coverage of a broad range of fungal topics also can be found in science blogs and Internet postings with reports of jet lag expressed in circadian rhythms of fungi; wood decay; the evolutionary arms race between a smut fungus and maize; a new species of introduced, beetle-associated fungus that kills plants in the Lauraceae; and yeast genome sequencing leading to improved bioethanol production. National Geographic News also reviews interesting fungal topics, including stories on endophyte biology and “bringing order to the fungus among us,” describing the Assembling the Fungal Tree of Life project (see below). Only 2 days before this meeting (December 12, 2010), USA Today published an article by Elizabeth Weise, “Why it’s cool to have a fungus among us.” The informative article could have been the basis for this talk—if only it had appeared earlier. The range of examples cited indicates a growing interest in and knowledge of fungi.

Fungi influence our daily lives in ways we seldom appreciate. Several entrenched fungal-influenced cultural practices are the result of fungal plant diseases. These include tea drinking in the United Kingdom, a switch imposed by devastation of coffee plants in Ceylon (present-day Sri Lanka) by the coffee rust fungus (Hemileia vastatrix) in the late 1800s (Horsfall and Cowling, 1978); consumption of cornbread as a staple in the southern United States colonies was imposed because the wheat rust fungus prevented wheat cultivation in the humid South (Horsfall, 1958); and the enjoyment of gingerbread comes from the time when the effects of stinking smut of wheat were masked by molasses and ginger (Carefoot and Sprott, 1969). We rely on fungi for clothing fads such as use of cellulase enzymes of species of Trichoderma to speed the “stone washing” of our blue jeans (Bhat, 2000). Perhaps, fungi may make us more beautiful when certain “integrative approaches to better skin”5 are followed using a blend of fungi that includes Cordyceps, reishi mushrooms, and other ingredients.

The Classification and Discovery of New Species of Fungi

Early phylogenetic studies based on DNA sequences defined a monophyletic6 group of Fungi. Oomycota and relatives, various slime mold clades, and several other groups previously considered as zygomycetes have been excluded from the monophyletic Fungi. Asexual and sexual fungi could be combined on the basis of their genetic relationships, and asexual groupings of asexual fungi were abandoned (Alexopoulos et al., 1996). More recently, mycologists have increased

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5 Several brands of skin creams include a variety of basidiomycete fruiting bodies as ingredients that are said to provide for skin relief and other effects (e.g., Dr. Weil’s Mega-Mushroom lotions, cleansers, and serums).

6 A group of taxa containing an ancestor and all its descendants.

Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
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the number of DNA markers and taxa in diverse clades to produce increasingly well-resolved phylogenies,7 the basis of predictive classifications (Figure A2-1) (Hibbett et al., 2007; James et al., 2006; White et al., 2006). An issue of the journal Mycologia (98:829–1103, 2006) was devoted to the phylogenetics of many major groups of fungi. Recent phylogenetic studies have provided new insights into fungal relationships and show that the earliest diverging lineages of zoosporic8 and zygosporic9 groups are not monophyletic as previously assumed on the basis of morphological characters and that they are more diverse than previously understood. Other findings provide data to include microsporidia within or very near the fungi (Lee et al., 2010). The new phylogenetic studies are largely the result of several National Science Foundation projects (Research Coordination Networks: A Phylogeny for Kingdom Fungi [Deep Hypha] and Assembling the Fungal Tree of Life 1 and 2) that involved more than 100 biologists from about 20 countries (Blackwell et al., 2006; Hibbett et al., 2007). Current projects under way include adding taxa to expand the fungal tree of life and pursuing an increasing number of genomics projects.

About 100,000 species of fungi have been described, but a conservative estimate suggests that there are 1.5 million fungi on Earth (Hawksworth, 1991, 2001). The estimate has spurred exploration for the million fungi that remain undiscovered (Figures A2-2A through A2-2D).

More recently the 1.5 million estimate was surpassed by a higher estimate of 3.1 to 5.1 million species based on the use of molecular methods, including highthroughput sequencing (O’Brien et al., 2005). Because of the great discrepancy between known and estimated fungal species numbers, mycologists have a renewed interest in fungal discovery. Many have wondered, where are the missing fungi (Hyde, 2001)? If the higher estimates are realistic, the number of fungi is equal to the number of animal species and may exceed the number of plants by 10:1. Abundant evidence shows that many tropical fungi remain to be discovered based on species accumulation curves of fungi collected in plots (Aime et al., 2010). Other habitats reporting large numbers of fungi include living leaves of tropical trees (Arnold, 2007), soil fungi (O’Brien et al., 2005; Taylor et al., 2010), and even the fungi in the buildings in which we spend most of our time (Amend et al., 2010). Many fungi, however, remain to be discovered in northern temperate regions, including far northeastern Asia (Petersen and Hughes, 2007). We do not have to look for undescribed fungi in completely new places or tropical regions, however, because they may be in our backyards. My colleagues and I look for new species among the yeasts and other microscopic fungi that are difficult to see

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7 A phylogeny is an inferred history of evolutionary relationships of organisms; often depicted in a tree diagram.

8 Zoospores are flagellated cells of certain fungi (see Figure A2-1) that are produced in sporangia in asexual reproduction.

9 Zygospores are thick-walled spores produced in some fungi (see Figure A2-1) resulting from the fusion of like gametes.

Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
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img-120

Figure A2-1 Diagrammatic representation of relationships of fungal taxa, examples (ex.), and approximate number of species in each group. Zoosporic and zygosporic fungi are more diverse than previously recognized on the basis of morphological traits, and they are not monophyletic. Two flagellated taxa, Rozella and Olpidium, are of uncertain taxonomic placement. Evidence from multilocus sequencing and genomics reveals that microsporidians branch within or near fungi. Ascomycota and Basidiomycota, the most speciose phyla, are each divided into three subphyla. The largest number of fungal species are classified in the subphyla Pezizomycotina and Agaricomycotina.

Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
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img-121

Figure A2-2 Images of representative fungal groups. (A) Hyphae of the blastocladialean fungus, Allomyces sp. Note a terminal zoosporangium (ZS) containing zoospores. The spiny, dark, thick-walled resting spores (RS) within the hyphae are those of a zoosporic fungal parasite, Rozella allomycis, of uncertain taxonomic placement. Bar = 10 µm. (B) Lobosporangium transversale. The zygosporic fungus in the Mortierellales has unusual spiny lobed sporangia (Benny and Blackwell, 2004). Bar = 50 µm. (C) Sarcoscypha coccinea. The several cm diameter fruiting body of the scarlet cup ascomycete, Sarcoscypha coccinea. Ascospores are formed within asci on the inner surface of the cup. (D) Mutinus sp. A stinkhorn similar to one mentioned in the text that caused a search for a dead body (Anonymous, 2005). Stinkhorns produce noxious compounds that attract insect spore dispersers. The dark slimy mass of spores has been partially removed by flies.
SOURCES: (A) photo courtesy of Timothy Y. James, provided by Meredith Blackwell (2009). (B) micrograph courtesy of Kerry O’Donnell, provided by Meredith Blackwell (2004). (C) photo courtesy of eriotropus/coqui, provided by Meredith Blackwell (2002). (D) photo courtesy of Nhu H. Nguyen, provided by Meredith Blackwell (2005).

Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
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with the unaided eye (Boekhout, 2005; Suh et al., 2005), and members of early diverging lineages that often are difficult to isolate and culture. Ascomycetes and basidiomycetes are expected to provide the greatest diversity of additional taxa based on numbers of currently known fungi, but certainly the developing methods using high-throughput sequencing of DNA will lead to the discovery of more of the early diverging groups (Figure A2-1) (Kirk et al., 2008).

Examples of large numbers of species isolated into culture from certain substrates include the finding of 418 unique morphotypes of endophytic fungi from 83 leaves in Panama (Arnold, 2007), 257 fungal endophyte genotypes in coffee plants (Vega et al., 2010), and 650 yeast isolates representing 290 genotypes of nearly 200 undescribed taxa from the gut of beetles (Suh et al., 2005). Acquiring cultures and specimens will remain important in cases when fungi and cultures are needed for certain purposes, including population studies, environmental remediation, and secondary metabolites. Taylor and his colleagues (2010) used high-throughput sequencing to estimate the presence of more than 200 taxa in a 0.25 g soil sample with only 14 percent overlap in taxa in a sample taken a meter away. If we are to determine the number of fungi on Earth, environmental sequencing will be necessary to speed fungal exploration and discovery. In addition to new species, entire lineages, some probably at the level of subphylum, may be recognized by DNA sequences such as Soil Clone Group 1 (Porter et al., 2008; Rosling et al., 2010). More work will be needed to determine geographical and substrate ranges in order to obtain more accurate estimates of species numbers.

Species discovery is relevant to the topic of this workshop because previously unknown plant and animal pathogenic fungi have been introduced into the United States many times. These fungi probably caused few symptoms and went unnoticed in their native hosts. Devastation of naïve hosts, however, led to their recognition and subsequent description as new species. This scenario certainly is repeated by the fungi discussed in this meeting, including Batrachochytrium dendrobatidis, the pathogenic chytrid of amphibians spread around the world; Geomyces destructans, the pathogen of bats in North America; and Phytophthora ramorum, causing declines of certain plants in North America and Europe. Prior invasions have included several fungal agents of Dutch elm disease; the chestnut blight fungi; the newly arrived agent of the laurel wilt delivered within the mycangia10 of its ambrosia beetle vector; and Discula destructans, a pathogen of North American dogwoods (Alexopoulos et al., 1996; Harrington and Fraedrich, 2010; Zhang and Blackwell, 2001). Recently, a new approach to discovering the native ranges of certain fungi has been profitable. Ning Zhang (Personal communication, Rutgers University, December 10, 2010) designed an efficient assay method using specific primers to detect the dogwood pathogenic fungus in herbarium specimens. The method promises to greatly reduce the time involved in determining geographical and host ranges and is ideal for working with col-

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10 Mycangia are pouch-like invaginations in the cuticle of certain insects used to transport cells and spores of symbiotic fungi, found especially in some species of bark and ambrosia beetles as well as a few other groups of insects.

Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
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laborators at herbaria throughout the world. Because patterns of introduction of pathogens may exist, determination of native ranges is essential in combating invasive organisms.

Supermodels

Fungi are important as model systems in research. Saccharomyces cerevisiae (Figure A2-3) is a supermodel known for its baking and brewing prowess and as the first eukaryote to have its entire genome sequenced.

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Figure A2-3 Saccharomyces cerevisiae (Y-2235), baker’s yeast and model organism. Note the many budding cells in the stained preparation. Bar = 5 µm.
SOURCE: Photo courtesy of Cletus P. Kurtzman, provided by Meredith Blackwell (2008).

Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
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In addition to S. cerevisiae, three other fungi that have been important in research and were the subjects of Nobel Prize–winning research are Schizosaccharomyces pombii, another fast-growing organism with a yeast growth form; Penicillium crysosporium, producer of the first effective antibiotic; and Neurospora crassa. In his Nobel Prize acceptance speech, Tatum (1958) acknowledged, among others, “B.O. Dodge for his establishment of this Ascomycete as a most suitable organism for genetic studies.” Beadle (1958) also spoke of Neurospora crassa and pointed out that “Dodge was an enthusiastic supporter of Neurospora as an organism for genetic work. ‘It’s even better than Drosophila,’ he insisted to Thomas Hunt Morgan, whose laboratory he often visited. He finally persuaded Morgan to take a collection of Neurospora cultures with him from Columbia University to the new Biology Division of the California Institute of Technology, which he established in 1928.” This was the beginning of the development of Neurospora crassa in genetics research.

As mentioned above, S. cerevisiae was the first eukaryotic organism to have its entire genome sequenced. This yeast and other species in the Saccharomycotina have relatively small genomes that make them economical candidates for sequencing (Mewes et al., 1997). In addition, yeasts and other model fungi are easy to grow and complete their lifecycles in culture in a few days; because they are haploid throughout most of their lifecycle, induced mutations are expressed rapidly. Many fungi, including some yeasts, also have a sexual state from which all products of meiosis can be isolated in addition to asexual spores and somatic cells from which uniform populations can be established. They also are excellent organisms for population studies (Anderson et al., 2010). Some fungi, including S. cerevisiae, have morphological cues that indicate the occurrence of certain cell cycle events, and a large body of background information is available for previously established model fungi studies. Improvements in genome sequencing have made it possible to develop many new “models,” including plant and animal pathogens and their hosts. For example, yeasts from the gut of wood-feeding beetles have been of particular interest because many of them ferment xylose, a requirement for efficient digestion of lignocellulose in biofuel production. These species have undergone biochemical and metabolic engineering to obtain more information on xylose fermentation pathways, and genome sequencing is important toward this end (Jeffries et al., 2007; Joint Genome Institute, 2007; Van Vleet and Jeffries, 2009).

Fungi Make Money: Useful Fungal Products

Humans have used a variety of fungal products for different purposes, including cures. In fact some of the magical fungi mentioned above also have been used for their medicinal properties, which may have been known since prehistoric times. Evidence exists for the use of fungi by early humans. Ötzi the Iceman lived about 5,300 years ago, and his mummified body was discovered in 1991

Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
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on the border of Italy and Austria. He carried pieces of the fruiting bodies from two species of wood-rotting basidiomycetes, Piptoporus betulinus and Fomes fomentarius, perhaps for medicinal uses (Peinter et al., 1998). Other writers have suggested that one of the fruiting bodies was used as a strop for sharpening knives and tools, but whatever their use, fungi appear to have been important to Copper Age Europeans.

Some basidiomycetes have been used medicinally in more recent times. Extracts of Inonotus obliquus was used in Europe as a treatment for cancer, and the fruiting bodies of Fomitopsis officinalis (the quinine conk), mentioned earlier as grave guardians in the Pacific Northwest, were also harvested for medicinal properties. A different kind of medicinal use by foresters was the application of sheets of mycelium on ax injuries to stop bleeding (Gilbertson, 1980). The spore masses of giant puffballs that were discovered stockpiled along Hadrian’s Wall (in Northern England) also have been used as a styptic (Personal communication, Roy Watling, former Head of Mycology and Plant Pathology, Royal Botanic Garden Edinburgh, August 27, 1977), and spores of unspecified puffballs also were widely used as a styptic by natives of North America as well (Blackwell, 2004).

Certain ascomycete fungi, previously known as species of Cordyceps, have been used in Asian traditional medicine for several centuries (Spatafora et al., 2007). One of these fungi, a parasite of caterpillars, known as Cordyceps sinensis since 1878, now is Ophiocordyceps sinensis based on a phylogenetic study (Sung et al., 2007). Recent interest in the fungus has provided evidence that it may be effective in the treatment of certain tumors (Spatafora et al., 2007). The revision of the entire group of insect–pathogenic fungi previously placed in the genus Cordyceps has resulted in the placement of species in three different families (Sung et al., 2007). This is an important development because phylogenies are predictive of traits common to closely related fungi, and other Ophiocordyceps species may be targeted for the mining of metabolites. The efforts to develop penicillin for the treatment of bacterial infections at the beginning of World War II resulted in the discovery of a long-sought magic bullet and hastened the rise of the modern pharmaceutical industry. In addition to the fungus-derived drug penicillin, three statin drugs for lowering cholesterol levels (e.g., Lipitor®) and the immune suppressant cyclosporine each have earned more than a billion dollars annually. Cyclosporine, once critical to transplant surgery, is today used to treat dry eye as well as more serious conditions (Blackwell, 2011).

Fungi also are big business in the food and beverage industries. In addition to the usual fresh fruiting bodies of basidiomycetes (mushrooms) and a few highly favored ascomycetes (truffles and morels), other fungi, such as cuitlacoche (corn smut) and rice smut, are eaten in Mexico and Asia, respectively. Processed foods also are made from fungi. These include yeast extract spreads such as marmite and vegemite and the meat substitute, Quorn™, a product of hyphae of an ascomycete, a species of Fusarium. Several species of Aspergillus are used in the processing of soy sauce, and fungi play a part in the flavoring process of cheeses.

Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
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Throughout the world many fermented foods rely on fungi at least in part to increase nutritional value, improve texture and flavor, and preserve the foodstuff. In one short street block in Brussels, I examined shop windows to count the many products that had been touched by fungi: coffee, certain teas, chocolate, cheeses, bread, salami and dry-cured hams, and numerous fermented beverages (Tamang and Fleet, 2009). Many African and Asian foods, including miso, ontjom, and tempeh, are the products of fermentation (Nout, 2009; Rodríguez Couto and Sanromán, 2006).

As in the case of other fungal products, the making of alcoholic beverages almost certainly was discovered millennia ago, found accidently in prehistoric times when wild yeasts settled into a sugary beverage. Yeasts are essential to the multibillion-dollar alcoholic beverage industry. In the United States, sales of beer, spirits, and wine were $116 billion in 2003 (Library Index, 2011). The yeasts involved in brewing were first isolated into pure culture by Emil Hansen at the Carlsberg Brewery in Copenhagen, and the brewery lab became an important site of classic yeast genetics and biotechnology research (Hansen and Kielland-Brandt, 2003). Pretorius (2000) suggested that many additional yeast species might be used in winemaking. In this context my colleagues and I have discovered nearly 300 previously unknown yeasts, many of which have the ability to ferment a variety of sugars, yet are untried for making beverages (Suh et al., 2005; Urbina and Blackwell, unpublished). In addition to its significance in brewing and bread making, S. cerevisiae, of course, has been extremely important in industrial biotechnology because of the development of efficient transformation methods and specialized expression vectors, and for a variety of other genetics tools (Nevoigt, 2008).

Fungi Interact with Other Organisms

Fungi interact with all major groups of organisms. Specific interactions with photosynthetic organisms are generally well known (Table A2-1). About 80 percent of all plant species and 92 percent of plant families form close associations with fungi known as mycorrhizae (Smith and Read, 2008; Trappe, 1987). Fungi and plant roots or underground stems form several kinds of mycorrhizae that are classified by the morphology of the interacting fungus in relation to the root. The associations are important for carbon, mineral, and water exchange, with carbon generally transferred from the plant to the fungus.

Arbuscular mycorrhizal (AM) fungi are known from the 400 million-year-old Rhynie chert. The fungi penetrate the plant cell wall and form a highly branched arbuscule that invaginates the plasma membrane of the root cortex cells. The 200 members of the asexually reproducing phylum Glomeromycota are obligate fungal partners of about 60 percent of all plant species. Hosts include a variety of crop and forage plants such as maize, rice, alfalfa, and citrus, as well as many non-cultivated plants. Molecular methods have detected previously unknown host

Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
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TABLE A2-1 Examples of Fungal Associations with Plants

Association Plants Fungus Reference
AM Mycorrhizae 60% of all species Glomeromycota Selosse et al. (2006)
Ectomycorrhizae 2,000 species ~5,000 species of basidiomycetes, ascomycetes, Endogenales Smith and Read (2008)
Endophytes 95% of all plants Many groups of ascomycetes and some basidiomycetes Rodriguez et al. (2009)
Lichens ~100 species of photobionts (green algae, blue/green bacteria) ~32,000 ascomycetes (Leotiales, Dothideales, and Pezizales), a few basidiomycetes Schoch et al. (2009)

specificity in some cases (Selosse et al., 2006). Ectomycorrhizal fungi (FIGURE A2-4) are associated with fewer hosts, including certain dominant forest trees such as birch, dipterocarp, eucalyptus, oak, and pine. Greater ectomyccorhizal fungal diversity is evident, and basidiomycetes, ascomycetes, and a few zygomycetes are involved in these associations. Many of the fungi are generalists, but more specificity occurs than among AM associates. The fungi produce an external mantle over young roots and often cause dramatic shortening and dichotomous branching of the mycorrhizal root (Smith and Read, 2008).

Endophytes are fungi that usually grow within above-ground plant parts without causing disease symptoms in about 95 percent of all plants examined (Arnold, 2007). The fungi that form the associations have been placed in four groups, depending on host specificity, tissues colonized, and amount of colonization within the plant (Rodriguez et al., 2009). Hypocrealean endophytes of grasses and sedges produce alkaloids that have been suggested to deter feeding by insects and vertebrates. Endophyte-infected grasses have enhanced growth and drought resistance (Rodriguez et al., 2009). A different group of endophytes is more taxonomically diverse and has broad plant host range with restricted growth within the plant, often occupying only a single cell. Some of these horizontally transmitted endophytes convey protection from plant pathogens (Arnold et al., 2003; Rodriguez et al., 2009). An endophyte was reported to convey heat tolerance to its grass host near a hot springs in Yellowstone National Park, but additional research has shown that only virus-infected endophytes convey thermal tolerance, a sign of the complexity of such associations (Márquez et al., 2007).

About half of the estimated 64,000 ascomycetes (e.g., Leotiales, Dothideales, and Pezizales) and a few basidiomycetes are the fungal associates (mycobionts) of about 100 species of photosynthetic organisms (photobionts) to form lichens

Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
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Figure A2-4 Anaptychia ciliaris. Small colonies of the lichen-forming fungus on agar medium after 3 months of growth. Bar = 200 µm.
SOURCE: Photo courtesy of Ning Zhang, provided by Meredith Blackwell (2010).

(Schoch et al., 2009). Lichens have been used as indicators of pollution. In addition to the photosynthetic partner, usually a green alga, a photosynthetic, nitrogen-fixing blue/green bacterium also may occur in a tripartite association in the lichen. Although the fungal associate can be grown on artificial media, they usually grow very slowly (Figure A2-5). Lichens are hosts for pathogenic fungi as well as endolichenic fungi, the lichen equivalent of endophytes. Each partner in the lichen has a scientific name, but the name of the lichen as a whole is that of the fungus (Ahmadjian, 1993; Nash, 2008).

Wood-Decaying Fungi

Fungi are heterotrophic and their ability to degrade organic materials and return them to nutrient cycles is an essential activity in almost all ecosystems. The ability of a fungus to degrade specific substrates depends on the enzymes it produces, and certain fungi are especially important in forest ecosystems where they are the primary decomposers of wood. Basidiomycetes and some ascomycetes are the primary decomposers of plant cell wall carbohydrates (cellulose and

Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
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Figure A2-5 Ectomycorrhizal root. The hyphae of Rhizopogon rubescens enveloping the young roots of a Virginia pine seedling. The mycelium extends from the roots into the surrounding environment.
SOURCE: Photo by J. B. Anderson, provided by Meredith Blackwell (1996).

hemicellulose) and lignin polymers (Gilbertson, 1980). Some wood-decaying fungi invade living trees and attack non-functional tissues, especially heartwood, the non-conducting vascular tissue in the center of a cross section of the trunk. Few wood-decaying fungi actually cause diseases and most of the damage comes from the weakening of tree trunks so that they fall in wind or ice storms. The loss of weakened trees is a natural process that culls branches and entire trees to create clearings in older forests (Gilbertson, 1980). Aldo Leopold recognized the value of wood decay for wildlife in the chapter “November” of A Sand County Almanac and Sketches Here and There. He referred to his woodlot as “a mighty fortress that fell heir to all the diseases of plants” known to humankind. The importance of wood-decaying fungi in the formation of nesting holes for wildlife is well known (Gilbertson, 1980). The red-cockaded woodpecker prefers to nest in mature pines about 60 years old that have been rotted by the basidiomycete Phellinus pini. Old pine stands are a diminishing habitat in regions where pines are grown in plantations on a 15-year rotation or less for commercial use. The ivory-billed woodpecker may be extinct because the extensive old-growth, bottomland hardwood forests the species inhabited have been lost (Gilbertson, 1980).

A less significant but interesting use of wood decay is the creation of wooden objects that have been modified by wood-decaying fungi. Spalted wood is distinguished by zone lines, the dark lines formed by oxidation at the points of contact between closely related fungal colonies. The patterned wood is often favored by

Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
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collectors and increases the cost of hand-turned bowls at craft fairs. These fungal effects include the deep blue/green stain of an ascomycete fungus that remains green in intarsia of fine Italian furniture and the inlay of Tunbridge Ware objects (Blanchette et al., 1992b). Even Stradivarius violins may have been made more resonant by the partial decay of the wood (Schwarze et al., 2008).

Insects Associated with Fungi and Vertebrates

The importance of many insects in the ecosystem is overlooked, but many of them are important in degradation of course particles, dispersal of bacteria and fungi, and, as is well known, as agents of fungal fertilization. Fungi clearly provide benefits for insects, although the exact advantages to the fungi beyond providing habitat and a means of dispersal often are not clear (Buchner, 1965; Gilbertson, 1984; Mueller et al., 2005). Few animals have the enzymes necessary to digest refractory plant cell wall materials or to synthesize vitamins. Fungi also may detoxify plant toxins and produce pheromones for insects (Table A2-2) (Dowd, 1991; Vega and Dowd, 2005; Wheeler and Blackwell, 1984; Wilding et al., 1989). The best known fungus–insect associations include the farming interactions of basidiomycetes with Old World termites (Macrotermitinae) (Aanen

TABLE A2-2 Examples of Fungal Associations with Insects

Insect Group Fungi Reference
Macrotermitinae Termitomyces spp. Aanen et al. (2002)
Formicidae: Attini (derived clades) Leucocoprinus spp. Mueller et al. (2005)
Formicidae: Attini (most in Apterostigma pilosum clade) Pterulaceae spp. Munkacsi et al. (2004)
Scolytinae and Platypodinae Ophiostomatoid ascomycetes Farrell et al. (2001);
(Bark and ambrosia beetles) Harrington (2005)
Siricidae (wood wasps) Certain species of Amylostereum, Stereum, and Daedalea Martin (1992)
Passalidae (bess beetles) Several clades of xylose-fermenting yeasts Suh et al. (2003)
Mushroom-feeding beetles Candida tanzawaensis clade yeasts Suh et al. (2005)
Drosophila in cacti Various yeasts Starmer et al. (2006)
Nectar feeding beetles Various yeasts Lachance et al. (2001)
Coccidae Septobasidium Henk and Vilgalys (2007)
Certain insects, especially aquatic larvae Harpellales, Asellariales Lichtwardt et al. (2001)
Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
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et al., 2002) and attine ants (Figure A2-6) (Formicidae: Attini) (Mueller et al., 2005) and of ascomycetes by bark and ambrosia beetles (Scolytinae and Platypodinae) (Harrington, 2005). The females of another insect group, siricid wood wasps (Siricidae), are less well studied, but they have been considered by some to form farming interactions with fungi (see Gilbertson, 1984). The interaction, however, does not meet all the criteria established for what has been defined as “agriculture” (Mueller et al., 2005).

The farming association of the basidiomycete Termitomyces with Old World macrotermitine termites arose once in Africa. Since that event no additional fungal lineages have been domesticated and no reversals of the fungus to a free-living state have been found. Repeated host switching, however, has occurred within termite clades as reflected in the phylogenetic trees of termites and associated fungi (Aanen et al., 2002). Nest initiation by both males and females of certain species has been suggested to have influenced the mode of transmission of the fungus, usually acquired from the environment or some source other than a parent (horizontal transmission) (Aanen et al., 2002). In the New World it is not termites, but attine ants that are involved with basidiomycetes in farming interactions, and Aanen and his colleagues (2002) compared the associations. The attines have become associated with several clades of fungi, and in contrast to termite transmission, transmission of the fungi is usually directly from parent to offspring (vertical) except in the early diverging ant lineages. Another important difference is that the ant-associated fungi apparently do not reproduce sexually. The work on the fungus–attine ant associations have revealed that ants have evolved with several groups of fungi on several different occasions. Although the best-known fungal mutalists are species of Leucocoprinus, other fungal groups, including certain species of Pterulaceae, have an association with ants in the Apterostigma pilosum clade (Munkacsi et al., 2004). The intensive studies of the fungi and attine ant associations have led to the discovery of other organisms that participate in the complex interactions. Species of hypocrealean ascomycetes in the genus Escovopsis are parasites of the cultivated fungus. Actinomycete associates of the ants produce antibiotics that have been reported to be specific in inhibiting Escovopsis (Currie et al., 1999), but more recently Sen et al. (2009) found that the bacteria they isolated had more generalized antibiotic activity, including activity against the cultivated fungus. The association of a fourth component of the association is black yeasts that apparently reduce the efficiency of the antibiotics (Little and Currie, 2008). This attine and—cultivated fungus—Escovopsis parasite associations provide the best example of coevolution, in this case tripartite association, among fungi and associates (Currie et al., 2003).

Unlike the termite and ant interactions, fungus-beetle associations have arisen multiple times. Some bark and ambrosia beetles have mycangia already mentioned above in which they carry inoculum of certain fungi (Malloch and Blackwell, 1993). The fungi, often Ceratocystis and Ophiostoma or relatives, may be the agents of plant diseases, and some of the fungi have been introduced

Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
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Figure A2-6 Excavation of deeply entrenched nest of the ant Atta texana requires heavy equipment or, alternatively, ground-penetrating radar to map such nests. The ant is native to adjacent parts of Texas and Louisiana, and the nests are said to be able to contain a three-story house. Visual materials based on a ground-penetrating radar nest model are available on the Internet from Carol LaFayette, Department of Visualization, Texas A&M. http://www.viz.tamu.edu/faculty/lurleen/main/attatunnel/tunnel.html.
SOURCE: Photo courtesy of John Moser, provided by Meredith Blackwell (2009).

Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
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with the beetles as in the case of Raffaelea laurelensis, the agent of laurel wilt disease (Harrington, 2005; Harrington and Fraedrich, 2010). Ophiostoma ulmi and similar fungi have been introduced into the United States, where they are virulent pathogens of trees, including American elms. The most efficient dispersers of some of these fungi actually were introduced before the fungus, Ophiostoma ulmi (Alexopoulos et al., 1996). In this discussion of beneficial fungi, these interactions benefit the insects and call attention to potential devastating effects of efficient insect dispersal in the context of emerging plant diseases.

Other beneficial fungal associates of insects involve siricid wood wasps and wood-decaying basidiomycetes, species of Amylostereum, Stereum, and Daedalea. The wasps lay their eggs through long ovipositors, tube-shaped organs at the posterior of the abdomen, and the larvae probably rely on fungal enzymes to decompose and detoxify the wood they ingest (Gilbertson, 1984; Martin, 1992). Many more fungi are associated with insects as necrotrophic parasites (FIGURE A2-7), and some of these deadly fungi have potential for development as biological control agents (Vega et al., 2009). In addition, many of about 1,000 described yeast species have close associations with insects (Table A2-2), and the yeasts provide important services to the insects (Vega and Dowd, 2005). Cer-

img-133

Figure A2-7 Hirsutella citriformis (Ophiostomataceae) on a delphacid planthopper. The asexual fruiting structure of this fungus erupted through the cuticle of the parasitized insect soon after its death.
SOURCE: Photo courtesy of Jennifer Luangsa-ard, provided by Meredith Blackwell (2010).

Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
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tain clades of gut yeasts appear to have diversified with insect hosts into certain habitats, and the yeasts provide basic resources for the insects to survive when subjected to new nutritional situations (Suh et al., 2003, 2006). About 200 species of Septobasidium in the Septobasidiales are known as associates of scale insects; only a few related species of Pachnocybe grow on wood (Henk and Vilgalys, 2007). The use of insect hosts is unusual for fungi that are related to the plant pathogenic rust fungi. The fungi are parasites of a few of the scale individuals, but in general benefit the entire insect colony by providing a protective covering against parasitic wasps (Henk and Vilgalys, 2007). Two orders of zygomycetes, Harpellales and Asellariales, were previously placed in a polyphyletic group known as Trichomycetes. The results of several studies indicate that these gut fungi produce vitamins and perhaps other benefits for their aquatic insect hosts (Lichtwardt et al., 2001). One species is known to parasitize simulid black flies (Lichtwardt et al., 2001), potentially a benefit to those who engage in outdoor activities.

Another nutritional interaction between fungi and animals is only briefly noted here, but is extremely important. An early diverging lineage of obligately anaerobic multiflagellated fungi, the Neocallimastigomycota, and vertebrate herbivores are closely associated (Griffith et al., 2010). The fungi reside in the host rumen or another anaerobic part of the gut, where they are important in supplying cellulases and other enzymes for the degradation of the large quantities of cellulose ingested by the herbivore (James et al., 2006).

Conclusion

Many fungi are obligate, beneficial associates of other groups of organisms. These are the “good fungi” of this article, and we often fail to appreciate their value because the fungi usually are unseen within their substrates unless they form macroscopic fruiting bodies. More often it is the effects of the fungi that we observe when they ferment fruit juice, or fitting to this volume, cause dramatic new outbreaks of disease. The Robert Frost poem quoted in the prologue of this publication describes the costs of the introduction of the disease caused by the chestnut blight fungus, Cryphonectria parasitica. The poem predicts that the disease will ravage until a new pathogen comes to kill the fungus, and in fact a virus did appear to suppress the fungus. In 1974, however, yet another pathogen, the oriental chestnut gall wasp, was introduced to attack the trees, an additional turn not predicted by the verse.

Today, as one out of every six or seven humans on Earth is reported to be malnourished or hungry (FAO, 2010), the war against pathogenic diseases of plants and animals is as important as ever. An earlier writer, Jonathan Swift (1667–1745) addressed the topic of hunger with his essay, A Modest Proposal, written to bring attention to the starvation of Irish tenant farmers during the potato famine. In Gulliver’s Travels he wrote directly of the importance of increasing agriculture yields:

Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
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And he gave it for his opinion, “that whoever could make two ears of corn, or two blades of grass, to grow upon a spot of ground where only one grew before, would deserve better of mankind, and do more essential service to his country, than the whole race of politicians put together.”

—Jonathan Swift, Gulliver’s Travels, Part II, Voyage to Brobdingnag,
first published in 1726–1727.

This volume, Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health, provides a discussion of new fungal diseases of plants and the animals that we strive to overcome at a time when introduced diseases contribute to hunger.

Acknowledgments

I am grateful to Dr. Fernando Vega, who improved the original manuscript through his careful editing. Several colleagues provided images, and Dr. Matthew Brown kindly prepared the plate. I acknowledge support from the National Science Foundation (NSF-0732671 and DEB-0417180) and the Louisiana State University Boyd Professor support fund.

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Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
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Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
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Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
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Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
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A3

THE FUNGI: 1, 2, 3 … 5.1 MILLION SPECIES?11,12,13

Meredith Blackwell14,15

Premise of the Study

Fungi are major decomposers in certain ecosystems and essential associates of many organisms. They provide enzymes and drugs and serve as experimental organisms. In 1991, a landmark paper estimated that there are 1.5 million fungi on the Earth. Because only 70000 fungi had been described at that time, the estimate has been the impetus to search for previously unknown fungi. Fungal habitats include soil, water, and organisms that may harbor large numbers of understudied fungi, estimated to outnumber plants by at least 6 to 1. More recent estimates based on high-throughput sequencing methods suggest that as many as 5.1 million fungal species exist.

Methods

Technological advances make it possible to apply molecular methods to develop a stable classification and to discover and identify fungal taxa.

_____________________

11 Reprinted with kind permission from the Botanical Society of America, www.amjbot.org.

12 Manuscript received 10 August 2010; revision accepted 19 January 2011.

13 Key words: biodiversity; fungal habitats; fungal phylogeny; fungi; molecular methods; numbers of fungi.

14 Department of Biological Sciences; Louisiana State University; Baton Rouge, Louisiana 70803 USA.

15 Author for correspondence (e-mail: mblackwell@lsu.edu) doi:10.3732/ajb.1000298.

Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
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Key Results

Molecular methods have dramatically increased our knowledge of Fungi in less than 20 years, revealing a monophyletic kingdom and increased diversity among early-diverging lineages. Mycologists are making significant advances in species discovery, but many fungi remain to be discovered.

Conclusions

Fungi are essential to the survival of many groups of organisms with which they form associations. They also attract attention as predators of invertebrate animals, pathogens of potatoes and rice and humans and bats, killers of frogs and crayfish, producers of secondary metabolites to lower cholesterol, and subjects of prize winning research. Molecular tools in use and under development can be used to discover the world’s unknown fungi in less than 1000 years predicted at current new species acquisition rates.

What are Fungi?

Fungal biologists debated for more than 200 years about which organisms should be counted as Fungi. In less than 5 years, DNA sequencing provided a multitude of new characters for analysis and identified about 10 phyla as members of the monophyletic kingdom Fungi (Fig. A3-1). Mycologists benefited from early developments applied directly to fungi. The “universal primers,” so popular in the early 1990s for the polymerase chain reaction (PCR), actually were designed for fungi (Innis et al., 1990; White et al., 1990). Use of the PCR was a monumental advance for those who studied minute, often unculturable, organisms. Problems of too few morphological characters (e.g., yeasts), noncorresponding characters among taxa (e.g., asexual and sexual states), and convergent morphologies (e.g., long-necked perithecia producing sticky ascospores selected for insect dispersal) were suddenly overcome. Rather than producing totally new hypotheses of relationships, however, it is interesting to note that many of the new findings supported previous, competing hypotheses that had been based on morphological evidence (Alexopoulos et al., 1996; Stajich et al., 2009). Sequences and phylogenetic analyses were used not only to hypothesize relationships, but also to identify taxa rapidly (Kurtzman and Robnett, 1998; Brock et al., 2009; Begerow et al., 2010).

Most fungi lack flagella and have filamentous bodies with distinctive cell wall carbohydrates and haploid thalli as a result of zygotic meiosis. They interact with all major groups of organisms. By their descent from an ancestor shared with animals about a billion years ago plus or minus 500 million years (Berbee and Taylor, 2010), the Fungi constitute a major eukaryotic lineage equal in numbers to animals and exceeding plants (Figs. A3-2–10). The group includes molds, yeasts, mushrooms, polypores, plant parasitic rusts and smuts, and Penicillium chrysoge-

Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
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Figure A3-1 Fungal phyla and approximate number of species in each group (Kirk et al., 2008). Evidence from gene order conversion and multilocus sequencing indicates that microsporidians are Fungi (see below; Lee et al., 2010). Note also that zoosporic and zygosporic fungal groups are not supported as monophyletic. Tree based on Hibbett et al. (2007), White et al. (2006), and James et al. (2006).

num, Neurospora crassa, Saccharomyces cerevisiae, and Schizosaccharomyces pombe, the important model organisms studied by Nobel laureates.

Phylogenetic studies provided evidence that nucleriid protists are the sister group of Fungi (Medina et al., 2003), nonphotosynthetic heterokont flagellates are placed among brown algae and other stramenopiles, and slime mold groups are excluded from Fungi (Alexopoulos et al., 1996). Current phylogenetic evidence suggests that the flagellum may have been lost several times among the early-diverging fungi and that there is more diversity among early diverging

Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
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zoosporic and zygosporic lineages than previously realized (Bowman et al., 1992; Blackwell et al., 2006; Hibbett et al., 2007; Stajich et al., 2009).

Sequences of one or several genes are no longer evidence enough in phylogenetic research. A much-cited example of the kind of problem that may occur when single genes with different rates of change are used in analyses involves Microsporidia. These organisms were misinterpreted as early-diverging eukaryotes in the tree of life based on their apparent reduced morphology (Cavalier-Smith, 1983). Subsequently, phylogenetic analyses using small subunit ribosomal RNA genes wrongly supported a microsporidian divergence before the origin of mitochondria in eukaryotic organisms (Vossbrinck et al., 1987). More recent morphological and physiological studies have not upheld this placement, and analyses of additional sequences, including those of protein-coding genes, support the view that these obligate intracellular parasites of insect and vertebrate hosts are members of the Fungi (Keeling, 2009; Corradi and Keeling, 2009). Additional evidence from genome structure as well as phylogenetic analyses, supports the inclusion of microsporidians within the Fungi and indicates that comparison of whole genomes contributes to the solution of challenging phylogenetic problems (Lee et al., 2010).

The level of resolution and sophistication of systematics studies made possible by molecular markers and phylogenetic analyses put mycologists on equal footing with other biologists for competitive funding, and they joined in several community-wide efforts to organize fungal diversity within a phylogenetic classification. Three projects funded by the National Science Foundation were initiated, including the Research Coordination Network: A Phylogeny for Kingdom Fungi (Deep Hypha) and successive Tree of Life projects, Assembling the Fungal Tree of Life (AFTOL-1) and a second ongoing project (AFTOL-2) (Blackwell et al., 2006). A major product of the Deep Hypha project was the publication of 24 papers on fungal phylogeny in a single journal issue (Mycologia 98: 829–1103). The papers included an introduction to progress in fungal phylogeny, a paper on dating the origin of Fungi, one on the evolution of morphological traits, and 21 articles with multilocus phylogenies of most major groups. Participants included 156 authors with some involved in more than one paper; only 72 of the authors were originally from North America. The multi-investigator AFTOL-1 publication (Hibbett et al., 2007) included a widely used and often cited phylogenetic classification to the level of order (e.g., Kirk et al., 2008; The NCBI Entrez Taxonomy Home-page, http://www.ncbi.nlm.nih.gov/taxonomy; Science Watch, http://sciencewatch.com/dr/nhp/2009/09jannhp/09jannhpHibb). The paper included 68 authors from more than 20 countries.

It is important to note that there was broad participation and, essentially, global involvement on these projects, emphasizing that studies of biodiversity are indeed global endeavors. Additional pages were contributed to the Tree of Life web project (http://www.tolweb.org/Fungi/2377) to make information on fungi more accessible to students and the general public. Two objectives of the ongoing AFTOL-2 project include increased taxon sampling of fungi for molecular

Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
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Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
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FIGURES A3-2–10 Examples of fungal diversity. 2. Lemonniera sp. Tetraradiate conidia developed on a submerged leaf in a well-aerated freshwater stream surrounded by lush vegetation. This type of aquatic species, an Ingoldian ascomycete, is named for C. T. Ingold, who pioneered the study of these fungi, that are characterized by highly branched conidia. Photo courtesy of H. Raja. 3. The aero-aquatic ascomycete Helicoon gigantisporum produces distinctive tightly coiled conidia. As the spore develops air is trapped in the coil and causes it to be buoyant. This feature is an adaptation for the polyphyletic aero-aquatic fungi that grow on leaves in slow-moving or stagnant freshwater. Photo courtesy of H. Raja. 4. The smut Testicularia sp. develops in the ovary of grasses and (as shown here) sedges. The spores mature sequentially, with the dark spores being more mature. A plant taxonomy student once thought he had discovered a new species of Leersia, distinguished by large ovaries of ca. 1 cm, only to be disappointed that the enlargement was caused by a fungus. It is helpful to mycologists when plant taxonomists collect and accession fungal diversity by selecting some diseased plant specimens, an activity that should be encouraged. 5. Perithecia of Pyxidiophora sp. (Laboulbeniomycetes) developed in moist chamber on moose dung from Meredith Station, New Brunswick, Canada. The 150 µm long ascospores are seen at the tip of the perithecium neck in the center. Spores adhere to phoretic mites that are carried by dung beetles to fresh dung piles. Some fungi have complex animal dispersal systems. Pyxidiophora species are usually mycoparasites that grow on fungi in dung or other substrates including wrack washed up on beaches. The genus is a “missing link” and provided clues to confirm that Laboulbeniomycetes are ascomycetes and not other kinds of fungi or floridian red algae. 6. The ca. 8 cm wide basidiomata of Pycnoporus sp., a wide-ranging, brightly colored, wood-decaying polypore, photographed at Barro Colorado Island, Panama. Some collectors have referred to basidiomycetes that produce colorful basidiomata as charismatic megamycota of the fungus world. 7. Peniophorella baculorubrensis, a bark-decaying basidiomycete common on and restricted to living live oak (Quercus virginiana), decays the bark and changes its water-holding capacity. The effect of decay on bryophyte communties by this fungus was first studied by ecologists (Penfound and Mackaness, 1940) more than 70 yr ago but was not described until a specialist on wood-decaying fungi happened to notice it on the Louisiana State University campus, Baton Rouge (Gilbertson and Blackwell, 1984). The inconspicuous basidiomata are shown growing on the lower side of a 7 cm long bark segment aimed downward for basidiospore discharge in response to gravity. 8. Basidiomata of Perenniporia phloiophila on the bark of living Quercus virginiana. Although the basidiomata are obvious against the darker bark, this species was not described until it was discovered at the same time and often on the same trees as Peniophorella baculorubrensis. Although the fungus usually rots only the outer bark, it will invade and decay wood whenever the vascular cambium is broached by a bird or insect. In addition to the two species on live oak, six other species have been described from the campus, illustrating the need for specialists to study noncharismatic fungi. 9. A basidioma (8 cm diameter) of the wood-decaying fungus, Favolus tenuiculus, a favorite food of several species of mushroom-feeding beetles (see Fig. A3-10). Photo courtesy of N. H. Nguyen. 10. The small (>10 mm long) brightly colored beetle, Mycotretus sp. (Erotylidae), was collected at Barro Colorado Island, Panama. Many erotylid beetles have specialized yeast-packed pouches at the anterior end of the midgut. More than 200 novel yeasts have been isolated from the gut of ca. 15 families of mushroom-feeding beetles (Suh et al., 2005). Photo courtesy of James A. Robertson.

Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
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data and the discovery of correlated morphological and biochemical characters (AFTOL Structural and Biochemical Database, https://aftol.umn.edu; Celio et al., 2006).

Known Fungal Species

The Dictionary of Fungi (Kirk et al., 2008) reported 97330 species of described fungi at the “numbers of fungi” entry. The addition of 1300 microsporidians brings the total of all described fungi to about 99000 species (Fig. A3-1). The Dictionary’s estimate of known species has almost tripled in the period between the first edition in 1943 (38000 described species) and now, amounting to an increase of more than 60000 described species over the 65-yr period (Fig. A3-11). Factors such as difficulty of isolation and failure to apply molecular methods may contribute to lower numbers of species in certain groups, but there cannot be any doubt that ascomycetes and basidiomycetes comprise the vast majority of fungal diversity (Fig. A3-1).

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Figure A3-11 Numbers of known fungi from the Dictionary of the Fungi (editions 1–10, 1950–2008). Authors state that the large increase in species numbers in the 10th edition may be inflated because asexual and sexual forms were counted separately and molecular techniques that distinguish close taxa have been used.

Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
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Estimated total fungal numbers

In 1991, a landmark paper provided several qualified estimates of the number of fungi on the Earth based on ratios of known fungi to plant species in regions where fungi were considered to be well-studied (Hawksworth, 1991). “Estimate G” of 1.5 million species was accepted as a reasonable working hypothesis based on a fungus to plant ratio of 6:1, in contrast to the much lower 50–60-yr-old estimates by Bisby and Ainsworth (1943) of 100000 fungal species and by Martin (1951) of 250000 species based on one fungus for every phanerogam known at the time. A more recent estimate of the total number of fungi, 720 256 (Schmit and Mueller, 2007), is also low compared to present estimates that include environmental samples.

Hawksworth’s (1991) estimate now is considered to be conservative by many, including Hawksworth (Hawksworth and Rossman, 1997), because numerous potential fungal habitats and localities remain understudied (Hawksworth, 2001). Furthermore, the use of molecular methods had not yet been considered as a means of species discovery. For example, analysis of environmental DNA samples from a soil community revealed a high rate of new species accumulation at the site, and these data supported an estimate of 3.5 to 5.1 million species (O’Brien et al., 2005). Using the present discovery rate of about 1200 fungal species per year based on the last 10 years, Hibbett and his colleagues (in press) estimated that it would take 1170 years to describe 1.4 million fungi (based on Estimate G of Hawksworth [1991]) and 2840 to 4170 yr to describe 3.5 to 5.1 million (based on O’Brien et al., 2005).

Using present higher estimates of land plant numbers as somewhat under 400000 species (Paton et al., 2008; Joppa et al., 2010) fungal species numbers now are expected to outnumber land plants by as much as 10.6:1 based on O’Brien et al. (2005). Even higher ratios have been predicted using data from highthroughput sequencing of clone libraries, although individual ecosystems will vary (L. Taylor, University of Alaska, Fairbanks, personal communication, January 2011). The large gap between known and estimated species numbers has led to a series of papers and symposia (e.g., Hawksworth and Rossman, 1997; Hawksworth, 2001; Hyde, 2001; Mueller and Schmit, 2007) attempting to answer the question “Where are the missing fungi?”

How to Discover New Fungi

Collecting and culturing fungi from the environment will remain important because of the need to identify specimens, revise taxonomy, assess the roles in the environment, and provide strains for biological control, environmental remediation, and industrial processes. A physical specimen, including an inert culture, is still required as a type specimen (but see Conclusions later), and vouchers of known fungi are used for documenting DNA sequences deposited in some databases (Nilsson et al., 2006). For example, the current AFTOL project has

Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
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a requirement that each sequence deposited as part of the project be linked to a specimen, including a culture.

All taxa biological inventories (ATBIs) attempt to survey organisms within particular geographical regions by collection of specimens and culture of substrates. One of these, Discover Life in America, All Taxa Biological Inventory, seeks to survey an estimated 50000 to 100000 species of organisms in the Great Smoky Mountains National Park. Karen Hughes and Ronald Petersen have been successful in collecting more than 3000 species of fungi, mostly agarics housed in the University of Tennessee Fungal Herbarium (http://tenn.bio.utk.edu/fungus/database/fungus-browse-results.asp?GSMNP=GSMNP), out of about 17000 species of all taxa that have been collected by others in the park (Biodiversity Surveys and Inventories: Agaric Diversity in the Great Smoky Mountains National Park, NSF DEB 0338699). All fungal specimens have been identified, and the agarics have been studied to the extent that a culture, ITS barcode sequence, and genetic analysis are available for many species. This successful project has required hours of time over a number of years and costly resources for studying the material, but it serves as an example of the commitment needed to acquire specimen-based information on fungi.

DNA methodology makes it possible to use independent sampling methods to discover the presence of organisms without ever seeing a culture or a specimen. Several new methods significantly outperform previous automated sequencing methods (e.g., Jumpponen and Jones, 2009; Metzker, 2010). Although there may be certain limitations and biases for the different methods (Amend et al., 2010a; Tedersoo et al., 2010), mycologists have been quick to embrace them in ecological and biodiversity studies. O’Brien and colleagues (2005) pointed out that collection and culture methods revealed numbers of fungi similar to those acquired by sampling environmental DNA. Hibbett et al. (in press), however, used data from GenBank to show that by 2008 and 2009 the number of environmental samples, excluding overwhelming numbers of sequences discovered by pyrosequencing, exceeded the accessions of specimen-based sequences. The rapid development of automated, high-throughput methods also has made it possible to acquire whole genome sequences for population level studies (Liti et al., 2009; Neafsey et al., 2010).

Which Regions of the Earth Harbor Fungal Diversity?

Fungi grow in almost all habitats on Earth, surpassed only by bacteria in their ability to withstand extremes in temperature, water activity, and carbon source (Raspor and Zupan, 2006). Tropical regions of the world are considered to have the highest diversity for most groups of organisms (Pianka, 1966; Hillebrand, 2004), and this is generally true for fungi as well (Arnold and Lutzoni, 2007).

A group of researchers are studying the diversity of the Guyana Shield. For the last 11 years, Terry Henkel and Cathie Aime and their colleagues have studied the fungi in six 1-km2 plots—three in a Dicymbe corymbosa-dominated

Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
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forest and three in a mixed tropical forest. Their current collections contain 1200 morphospecies, primarily basidiomycetes. Approximately 260 species were collected repeatedly only in the Dicymbe plots. Thus far, two new genera and ca. 50 new species have been described. On the basis of groups already studied, Aime estimated that ca. 120 new ectomycorrhizal taxa have been discovered. Including novel saprobes as well as ectomycorrhizal fungi, ca. 500 new species are expected among the 1200 taxa collected. It is clear, however, that these are not simply high numbers of new taxa, but biologically interesting fungi as well (Aime et al., 2010). One species is so unusual, that a reviewer of the original report called it “the find of the century” (Redhead, 2002). As Aime has quipped “if one were to compare the ratio of fungi to plants in the Dicymbe plots as did Hawksworth (1991), the ratio would be 260 to 1, obviously an overestimate but also a cautionary exercise in basing any estimate on a single ecotype” (M. C. Aime, Louisiana State University, personal communication, August 2010).

Many fungi have in fact come from temperate regions, and some studies report a high diversity of fungi. For example, in a study of indoor air from buildings using culture-independent sampling methods, diversity was found to be significantly higher in temperate sites independent of building design or use. The authors also alluded to the possibility that previous studies of certain mycorrhizal fungi showed similar trends (Amend et al., 2010b). More investigation in this area is needed, but it is clear that many undescribed fungi are present in temperate regions. Popular literature often rationalizes the need to save the rainforests, not because of their intrinsic value, but because of the potential drug-producing organisms that may be found there. Many of the commercially most successful fungal drugs, however, come from temperate fungi. Penicillium chrysogenum, producer of penicillin, was found in a northern temperate city. Another remarkable fungus, Tolypocladium infl atum from Norwegian soil, synthesizes cyclosporine, an immune-suppressant drug that revolutionized organ transplants (Borel, 2002); the sexual state of this fungus was collected in New York, USA (Hodge et al., 1996). Today the drug is commonly used to treat dry eye (Perry et al., 2008), as well as many serious conditions. Statins produced by fungi such as Aspergillus terreus from temperate regions, combat high cholesterol levels, as well as providing other benefits (Vaughan et al., 1996; Askenazi et al., 2003; Baigent et al., 2005).

In temperate deserts, mycorrhizal boletes, agarics, and rust and smut fungi, are common. A surprising number of wood-decaying basidiomycetes have been discovered on living and dead desert plants, including cacti and are in the University of Arizona, Robert L. Gilbertson Mycological Herbarium (http://ag.arizona.edu/mycoherb/herbholdings). When a noted mycologist moved to Arizona early in his career, he became excited about the new and unreported fungal diversity found in the desert. His proposed study of the wood-decaying fungi of the Sonoran Desert was poorly received with a comment that wood-decaying fungi were not present in the desert (R. L. Gilbertson, University of Arizona, personal communication, August 1979). The Sonoran Desert, however, has many plants

Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
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(e.g., cacti, ocotillo, and mesquite and other desert legumes) that are substrates for polypores and resupinate basidiomycetes (e.g., Gilbertson and Ryvarden, 1986, 1987).

Fungi also grow at low temperatures. An example involves fungal deterioration of historic huts built between 1901 and 1911 for use by Antarctic explorers including Robert Scott and Ernest Shackleton, and although there are not large species numbers, it is important not to overlook this fungal habitat in diversity studies (Held et al., 2005). Lichens have often been reported to be common in Arctic and Antarctic regions (Wirtz et al., 2008), and yeasts are active under frozen conditions in the Antarctic (Vishniac, 2006; Amato et al., 2009). In some cases, a yeast isolated from the Antarctic (based on 28S rDNA barcoding) also has been reported from varied habitats, including human infections, the gut of insects, deep seas, and hydro-carbon seeps (Kurtzman and Fell, 1998; Bass et al., 2007; personal observation). Although some fungi are specialized for cold regions, others simply occupy a wide variety of environmental conditions.

Many regions and habitats of the world need to be included in fungal discovery. In general, microscopic fungi and those that cannot be cultured are very poorly known. Parts of Africa remain to be collected for many, although not all, fungal groups (Crous et al., 2006). Fungi are important as symbionts, and they are associated with every major group of organisms, bacteria, plants and green algae, and animals including insects. Because certain under-studied symbiotic associations are known to include large numbers of fungi, these are a good place to search for new taxa. The associated organisms also allow for resampling, a quick way to obtain data about host specificity. Targeting hosts also is a productive method for discovering fungal fossils, such as those associated with plants of the Rhynie Chert (Taylor et al., 2004). Examples of diversity in particular fungal habitats are reviewed in the following sections.

Fungi and Plant Roots

Mycorrhizal plants and their fungal partners have been studied by a number of mycologists (Trappe, 1987; Smith and Read, 2008). The fungi often are essential to their plant hosts because they take up water, nitrogen, phosphorus, and other nutrients from the soil and transfer them to the plant roots. Some of these fungi may not prosper or even grow without the host. In addition to flowering plants and conifers, many bryophytes and ferns are mycorrhizal (Pressel et al., 2010). Certain mycorrhizal fungi specialize on orchids and ericoid plants, and some are known to have invaded new habitats with successful invasive plants (Pringle et al., 2009).

There are two main types of mycorrhizal fungi, arbuscular mycorrhizae (AM) and ectomycorrhizae. AM associations are more common and occur with up to 80% of all plant species and 92% of plant families. AM fungi are all members of the phylum Glomeromycota, a less diverse group than ectomycorrhizal

Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
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fungi with about 250 described species in a variety of taxa (Gerdemann, 1968; Schüssler and Walker, 2011; Wang and Qiu, 2006). Evidence from recent molecular studies, however, indicates that cryptic species with higher levels of host specificity than previously realized will increase the number of known AM fungi (Selosse et al., 2006; Smith and Read, 2008). More than 6000 species, mostly of mushroom-forming basidiomycetes, form ectomycorrhizae with about 10% of all plant families. Greater host specificity usually occurs in the ectomycorrhizal fungus–plant associations than in AM associations (Smith and Read, 2008). Vast parts of the world remain to be sampled (Mueller et al., 2007), and it is expected that barriers to inter-breeding have led to high genetic diversity among these fungi (Petersen and Hughes, 2007).

Inside Plant Leaves and Stems

Almost all plants on Earth are infected with endophytes, fungi that do not cause disease symptoms (Saikkonen et al., 1998). Endophytes occur between the cells, usually of above ground plant parts, and represent a broad array of taxonomic groups (Arnold, 2007; Rodriguez et al., 2009). The earliest studies of endophytes were of those associated with grasses (Diehl, 1950). Some grass endophytes are specialized members of the Clavicipitaceae, relatives of insect and fungal parasites in the Hypocreales, and many species produce alkaloid toxins effective against insects, other invertebrate animals, and vertebrates (Clay et al., 1993). Some grass endophytes are transmitted to the host offspring in seeds, and others inhibit sexual reproduction in the host and are dispersed within plant parts such as leaf fragments. For grass endophytes that reproduce sexually, fertilization may occur by insect dispersal. Water intake is increased in infected hosts, and these plants often grow taller than uninfected hosts.

A much more diverse group of endophytic fungi are associated with plants in addition to grasses, including a variety of dicots and conifers (Carroll, 1988; Rodriguez et al., 2009). In some tropical forests considered to be diversity hotspots for endophytes, there are extremely large numbers of the fungi, sometimes with hundreds reported from a single tree species, judged by both cultural and molecular methods of discovery and identification (Arnold et al., 2001; Arnold and Lutzoni, 2007; Pinruan et al., 2007; Rodriguez et al., 2009). In one study, more than 400 unique morphotypes were isolated from 83 leaves of two species of tropical trees. A subset of the fungi was distributed among at least seven orders of ascomycetes (Arnold et al., 2000). Leaves usually acquired multiple infections as they matured, and there was strong evidence that the endophytes protected leaves of plants, such as Theobroma cacao, from infection when they were challenged with pathogens (Arnold et al., 2003). Vega and colleagues (2010) also found high diversity of endophytes in cultivated coffee plants. Interestingly, some of these were insect pathogens and experiments are being conducted to develop endophytes as biological control agents of insect pests.

Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
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Plant Pathogens

Plant pathogens differ from endophytes in that they cause disease symptoms. Although some zoosporic and zygosporic fungi are plant pathogens, most plant pathogens are ascomycetes and basidiomycetes. A large number of ascomycetes and ca. 8000 species of basidiomycetes are plant pathogens. In addition to crop pathogens, it is important to remember that many pathogens are numerous and important in natural ecosystems (Farr et al., 1989; Burdon, 1993). Nonpathogenic phylloplane yeasts occupy leaf surfaces of many plants and are increasingly recognized for their control of potential leaf pathogens (Fonseca and Inácio, 2006). In addition to the thousands of native fungi that parasitize plants in the United States, pathologists are constantly on the lookout for introduced pathogens that often are undescribed when they arrive to decimate naïve native plant populations. For example, invasive fungi such as those grouped as Dutch elm disease fungi, chestnut blight fungus, dogwood anthracnose fungus, and redbay wilt fungus, were all unknown until they were observed soon after their introduction (Alexopoulos et al., 1996; Zhang and Blackwell, 2001; Harrington et al., 2008). Exotic localities will need to be searched for undescribed fungi that probably go largely unnoticed on their native hosts. It is important to note that although fungi may cause only minor symptoms to hosts in their native habitats, one of these may have the potential to be the next destructive disease after introduction to a new region.

Molecular methods have helped to clarify limits of closely related species and to establish host ranges (e.g., Crous et al., 2008). In a study of 26 leaf spot fungi in Australia, three genera of Myrtaceae, including Eucalyptus, were hosts for three new genera and 20 new species (Cheewangkoon et al., 2009). Although the authors acknowledged the high level of new taxa discovered, they pointed out that the potential for host shifts within plantations might lower estimates of fungal species numbers worldwide. Host or substrate specificity is a concept that can be applied to fungal groups that are closely associated with hosts such as endophytes, pathogens, and mycorrhizal fungi but not usually for saprobic species (Zhou and Hyde, 2001). In the past species of plant pathogens often were based on host identity, a practice that is not always effective because some groups are host-specific while others are not.

Lichens and Lichenicolus Fungi

About 20% of all fungi and 40% of the ascomycetes (13500 species) are lichen-forming fungi (Lutzoni and Miadlikowska, 2009). Lichenicolous fungi, parasites, and other associates of lichens are not well collected, but an estimate for the combined lichens and lichenicolous fungi is about 20000 species (Feuerer and Hawksworth, 2007). Lichens and lichenicolous fungi are polyphyletic, and several different groups of ascomycetes and a few species of basidiomycetes have become associated with green algae and cyanobacteria (Lutzoni and

Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
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Miadlikowska, 2009). Feuerer (2010) can be consulted for information on lichen diversity worldwide. This checklist also highlights the absence of collections in certain regions.

Deserts are rich in lichens. Of 1971 lichen species and associated fungi reported from the Sonoran Desert, about 25% studied since 1990 are new. Three volumes on lichens of the greater Sonoran Desert region have been published (Nash et al., 2002, 2004). Other habitats of high lichen diversity are Arctic and Antarctic regions (Feuerer, 2010).

Fungi From Arthropod and Invertebrate Animals

There is a need for more information on arthropod- and insect-associated fungi. As was mentioned earlier, estimates of global fungal diversity usually omit insect-associated species because they are so poorly known (Hawksworth, 1991; Rossman, 1994; Mueller and Schmit, 2007; Schmit and Mueller, 2007). Several post-1991 estimates of insect-associated fungi suggested that 20 000–50 000 species exist (Rossman, 1994; Weir and Hammond 1997a, b; Schmit and Mueller, 2007). Some parasites are biotrophic, associated with living insects, and many do not grow in culture. These also usually require special methods for removal and mounting, and few mycologists or entomologists have ever seen members of the Laboulbeniomycetes or the fungal trichomycetes, Asellariales and Harpellales (Lichtwardt et al., 2001; Cafaro, 2005). Laboulbeniomycetes are seta-sized, ectoparasitic ascomycetes of insects, mites, and millipedes (Weir and Blackwell, 2005). All 2000 known species have distinctive life cycles with determinate thalli arising from two-celled ascospores. About 90% of the species have been found on adult beetles (12 of 24 superfamilies) or on flies. New arthropod hosts at the level of family are still being discovered (Weir and Hammond, 1997a, b; Rossi and Weir, 2007), and there is an indication that there is some degree of host specificity (De Kesel, 1996). In the future, increased use of molecular methods will make it possible to determine the degree of species level host specificity, but the information is not available now. Septobasidiales, relatives of the basidiomycete rust fungi are associated with scale insects, and their felty basidiomata presumably protect the insects from parasitoid wasps. Many microsporidians also are parasites of a broad group of host insects.

Necrotrophic parasites of insects include some members of Chytridiomycota, Blastocladiales (Coelomomyces), Entomophthorales, and Tubeufiaceae (Podonectria) (Benjamin et al., 2004). About 5000 members of three families of Hypocreales are necrotrophic parasites of arthropods (Spatafora et al., 2007, 2010). These species show an evolutionary pattern of host shifting among plants, fungi, and insects in addition to displaying a high level of host specificity.

Fungi also occur in ancient, obligate gardening associations with bark and ambrosia beetles, attine ants, and Old World termites, and new species are still being discovered in these groups (Benjamin et al., 2004; Little and Currie, 2007;

Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
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Harrington et al., 2008; Aanen et al., 2009). Many yeasts are associated with insects, particularly insects that feed on nectar (Lachance, 2006; Robert et al., 2006).

Other insects contain gut yeasts, a habitat where few have looked for them. Isolations from the gut of mushroom-feeding beetles yielded up to 200 new species of yeasts (Suh et al., 2004, 2005; see also Lachance et al., 2010). Because only about 1500 ascomycete yeasts (Saccharomycotina) have been described, the gut yeasts represent a dramatic increase in diversity from a limited geographical range (Boekhout, 2005; C. Kurtzman, USDA-ARS, personal communication, July 2010). In fact, the estimated total number of yeast species worldwide could be increased by as much as 50% by simply recollecting in previously collected sites from the study (Suh et al., 2005). As Lachance (2006) pointed out, based on predictions of yeast numbers using data from species in slime fluxes and in associations with flower-visiting insects, it is necessary to obtain more information on specificity and geographical ranges before better estimates can be made. Although not all insects harbor large numbers of yeasts in their guts, those with restricted diets in all life history stages such as mushrooms or wood are often associated with yeasts. Host insects may acquire digestive enzymes or vitamins from the yeasts. This contention is supported by the fact that unrelated insects feeding on mushrooms (e.g., beetles in different lineages, lepidopteran larvae) all have gut yeasts with similar assimilative capabilities and vitamin production. The high rate of discovery of yeasts in under-collected habitats and localities suggests that far more taxa await discovery (Suh et al., 2005), and the gut habitat has been considered a yeast diversity hotspot (Boekhout, 2005).

Insects may be food for fungi, especially in low nitrogen environments. The mycelium of Pleurotus ostreatus, a favorite edible species for humans, secretes toxic droplets that kill nematodes. A study involving the mushroom-producing, ectomycorrhizal basidiomycete, Laccaria bicolor, was designed to determine the amount of predation by springtails on the fungal mycelium. The study led to the surprise discovery that the fungus was not insect food, but rather, it, and indirectly, the host tree benefited by obtaining substantial amounts of nitrogen from the insects (Klironomos and Hart, 2001). The predatory habit has arisen independently on several occasions in at least four phyla of fungi and oomycetes. Predaceous fungi such as species of Arthrobotrys and Dactylella lure, then trap, snare, or grip nematodes and other small invertebrate animals in soils and in wood (Barron, 1977).

Ødegaard (2000) revised global estimates of arthropods downward from 30 million to 5–10 million. Not all insects and arthropods are tightly associated with fungi, but even the revised species estimates indicate that the numbers of insect-associated fungi will be very high.

Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
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Soil Fungi

Soil is a habitat of high fungal diversity (Waksman, 1922; Gilman, 1957; Kirk et al., 2004; Domsch et al., 2007). Soil fungi and bacteria are important in biogeochemical cycles (Vandenkoornhuyse et al., 2002), and the diversity of soil fungi is highest near organic material such as roots and root exudates. Per volume, large numbers of microscopic fungi occur in pure soil, and these are largely asexual ascomycetes and some zygomycetes, including animal-associated Zoopagales. Gams (2006) estimated that 3150 species of soil fungi are known, and ca. 70% are available in culture. There presently is a high rate of new species acquisition, and the group appears to be better known than most ecologically defined groups. Molecular studies, however, are predicted to increase the total number (Bills et al., 2004). In fact a study of soil communities in several forest types at the Bonanza Creek Long Term Ecological Research site, Fairbanks, Alaska, United States, revealed not only seasonal changes in community composition but also in dominance of fungi over bacteria. The data acquired by several molecular methods including high-throughput sequencing greatly increased the total number of fungal sequences in GenBank at the time (Taylor et al., 2010). Taylor and his colleagues found more than 200 operational taxonomic units in a 0.25 g soil sample with only 14% overlap in a sample taken a meter away. This study is not directly comparable with the soil fungi reported by Gams (2006) because Gams’ figures excluded fungi such as mycorrhizal species.

Another study of soil fungi based on environmental DNA sequences showed an unexpected distribution of a group of zoosporic fungi, Chytridiomycota. The chytrids, were found to be the predominate group of fungi in nonvegetated, high-elevation soils at sites in Nepal and in the United States in Colorado, where more than 60% of the clone libraries obtained were from chytrids. A phylogenetic analysis of the sequences compared with those of a broad selection of known chytrids, indicated that a diverse group of Chytridiomycota representing three orders was present (Freeman et al., 2009).

Most major fungal lineages are known from cultures and specimens, but there have been a few surprises even in well-sampled habitats such as soil. Soil clone group I (SCGI) represents a major lineage of fungi that occurs in temperate and tropical soils on three continents, but no one has ever seen or isolated any of the species into culture (Schadt et al., 2003; Porter et al., 2008).

The phylogenetic position of this lineage, perhaps a new phylum, appeared as a sister group to the clade of Pezizomycotina–Saccharomycotina (Porter et al., 2008). Other unexpected higher taxonomic level fungal clades have been detected from environmental DNA sequences (Vandenkoornhuyse et al., 2002; Jumpponen and Johnson, 2005; Porter et al., 2008). Another lineage detected by environmental sequences was subjected to fluorescent in situ hybridization (FISH). The outline of a single-celled, flagellated organism was detected (Jones and Richards, 2009), but apparently none of these fungi has been cultured either. Higher-level

Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
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bacterial taxa have been discovered by environmental sampling, but this is a far less common occurrence for fungi (Porter et al., 2008).

Fungi form crusts that stabilize desert soils. Crusts usually are made up of darkly pigmented ascomycetes, lichens, and nitrogen-fixing cyanobacteria (States and Christensen, 2001). Rock-inhabiting fungi occur in the surface and subsurface layers of desert rocks. These darkly pigmented ascomycetes are members of the classes Dothideomycetes and Arthoniomycetes, but basidiomycetes and bacteria may occur in the associations (Kuhlman et al., 2006; Ruibal et al., 2009). Easily cultured asexual ascomycetes and other fungi also occur in desert soils, and these include an unusual zygomycete, Lobosporangium transversale (Ranzoni, 1968), known only from three isolations including Sonoran Desert soil. Yeasts are well known from American deserts in association with cacti and flies where they detoxify plant metabolites (Starmer et al., 2006).

Freshwater Fungi

Certain fungi are adapted for life in fresh water. More than 3000 species of ascomycetes are specialized for a saprobic life style in freshwater habitats where they have enhanced growth and sporulation (Shearer et al., 2007; Kirk et al., 2008; Shearer and Raja, 2010). The asci are evanescent, and ascospores have appendages and sticky spore sheaths, that anchor the spores to potential substrates in the aquatic environment. Conidia have several dispersal strategies, and these are designated as Ingoldian (Fig. A3-2) and aero-aquatic (Fig. A3-3) conidia. Ingoldian conidia are sigmoidal, branched, or tetraradiate and attach to plants and other material in the water. The conidia float on foam that accumulates at the banks of streams, especially during heavy runoff, and when the bubbles burst, the spores may be dispersed for great distances from the water and into trees, where they can be isolated from water-filled tree holes (Bandoni, 1981; Descals and Moralejo, 2001; Gönczöl and Révay, 2003). Aero-aquatic fungi have multicellular, often tightly helical conidia with air spaces to make them buoyant on the surface of slower-moving waters (Fisher, 1977).

Other, less obviously modified fungi are present in water, and some of these are active in degrading leaves in streams after the heavy autumn leaf fall. A few specialized freshwater basidiomycetes also are known, and several have branched conidia similar to those of the Ingoldian ascomycetes. Flagellated fungi occur in aquatic habitats, including Chytridiomycota, Blastocladiomycota, and Monoblepharomycota (James et al., 2006). Batrachochytrium dendrobatidis, the recently described amphibian killer, is an aquatic chytrid (Longcore et al., 1999). Members of Neocallimastigomycota also live in a specialized largely aquatic environment, the gut of vertebrate herbivores, where they are essential for digestion of cellulosic substrates.

Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
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Marine Fungi

Marine waters provide a habitat for certain specialized fungi (Kohlmeyer and Volkmann-Kohlmeyer, 1991), and Hyde et al. (1998) estimated that more than 1500 species of marine fungi occur in a broad array of taxonomic groups. Many of these fungi are distinct from freshwater aquatic species, and they may be saprobic on aquatic plant substrates. Some species have characters such as sticky spore appendages, indicators of specialization for the marine habitat (Kohlmeyer et al., 2000).

It is interesting that few fungi from early-diverging lineages have been reported from marine environments, perhaps in part because mycologists studying these groups sampled more often from fresh water habitats. More recently, an investigation of deep-sea hydrothermal ecosystems revealed not only novel species of ascomycetes and basidiomycetes, but also what may be a previously unknown lineage of chytrids (Le Calvez et al., 2009).

Most marine fungi are ascomycetes and basidiomycetes, and these include ascomycete and basidiomycete yeasts (Nagahama, 2006). Some of the yeasts degrade hydrocarbon compounds present in natural underwater seeps and spills (Davies and Westlake, 1979). Certain ascomycetes are specialists on calcareous substrates including mollusk shells and cnidarian reefs. Even a few mushroom-forming basidiomycetes are restricted to marine waters (Binder et al., 2006). Some fungi use other marine invertebrates as hosts (Kim and Harvell, 2004), including antibiotic producers that live in sponges (Bhadury et al., 2006; Pivkin et al., 2006; Wang et al., 2008). A wide variety of fungi considered to be terrestrial also are found in marine environments. Basidiomycete (i.e., Lacazia loboi) and ascomycete yeasts, and other fungi including Basidiobolus ranarum, may occur in marine waters where they infect porpoises and other vertebrates (Kurtzman and Fell, 1998; Murdoch et al., 2008; Morris et al., 2010).

Fungal Species

Currently, molecular methods provide large numbers of characters for use in phylogenetic species discrimination (e.g., Kohn, 2005; Giraud et al., 2008). In the past, biologists relied primarily on phenotype for species delimitation, and most of the formally described species known today were based on morphology. In addition, mating tests have been used to distinguish so-called biological species, especially among heterothallic basidiomycetes (Anderson and Ullrich, 1979; Petersen, 1995). The ability to mate, however, may be an ancestral character. For example, Turner et al. (2010) found evidence that fungi have evolved strong barriers to mating when they have sympatric rather than allopatric distributions. Distant populations would not have had strong selective pressure against hybridization, thereby avoiding production of progeny less fit than conspecific progeny (e.g., Garbelotto et al., 2007; Stireman et al., 2010). This phenomenon, known as reinforcement, helps to explain how fungi from different continents can mate

Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
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in the laboratory but never in nature and is an argument in favor of recognizing species by phylogenetics. A number of researchers have recognized species using “phylogenetic species recognition” criteria (Taylor et al., 2000). The operational phylogenetic method is based on a “concordance of multiple gene genealogies,” and in addition to discriminating species, the method indicates whether fungal populations actually exchange genes in nature (Taylor et al., 2000; Fisher et al., 2002; Dettman et al., 2006; Jacobson et al., 2006).

The use of phylogenetic species criteria results in recognition of more species than those delimited by morphological characters. For example, work on Neurospora species resulted in the discovery of 15 species within five previously recognized species (Dettman et al., 2006; Villalta et al., 2009). There are many such examples among other groups of fungi, and eventually these may be a significant source of new species discovery in the effort to discover 5 million fungi. Fungal species recognized in this way may be described without a phenotypic diagnosis, but it is not uncommon for distinguishing characters to be found with guidance from the phylogenetics study (e.g., Otrosina and Garbelotto, 2010).

Conclusions

Until recently, estimates of numbers of fungi did not include results from large-scale environmental sequencing methods. Newer estimates based on data acquired from several molecular methods, however, have predicted as many as 5.1 million species of fungi (O’Brien et al., 2005; Taylor et al., 2010). Mycologists also are beginning to use high-throughput methods to gain insight into questions including geographical ranges and host and substrate specificity, topics that have direct bearing on species numbers (Lumbsch et al., 2008). For example, high-throughput methods have been used to determine the amount of overlap between species within a given region by comparing soil samples a meter apart to find only 14% species overlap (Taylor et al., 2010).

A better estimate of fungal numbers also can be speeded by enlisting more biologists to accomplish the goal. When amphibian populations first were observed to be dwindling and some species were determined to have disappeared almost 20 yr earlier, a number of causes, all nonfungal, were suggested as the explanation. The revelation that a chytrid was involved brought to mind that there were probably fewer than 10 mycologists in the world who could collect, isolate, culture, and identify the novel flagellated fungus, Batrachochytrium dendrobatidis (Longcore et al., 1999). Since that time interest in and publications on chytrids have increased dramatically (e.g., Freeman et al., 2009; LeCalvez et al., 2009). The interest in amphibian disease was in part the impetus for a large number of recent publications on amphibian decline, but amphibian decline also justified other projects, including training new chytrid systematists in monographic work. This effort has resulted in the discovery of many new chytrid species and the description of five new orders between 2008 and 2010. The rise of AIDS and the

Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
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accompanying large number of fungal infections brought about a similar interest in medical mycology several decades ago.

In addition to any sudden influx of biologists to obtain better estimates of fungal numbers, a new approach clearly is needed. In a thoughtful paper, Hibbett and colleagues (in press) called for obtaining clusters of similar sequences and assigning Latin binomials to these molecular operational taxonomic units (MOTUs). The names would allow the sequences to be integrated into a specimen-based taxonomic data stream. They considered inclusion of the sequence-based taxa among all taxa to be a better alternative than the candidate taxon status used by bacteriologists. Changes in the International Code of Botanical Nomenclature would be needed if sequence-based materials were to be allowed as nomenclatorial types. This proposal seems to be a practical approach to handling the overwhelming fungal diversity being discovered.

Recent experience in working as a broadly inclusive group to plan and produce a phylogenetic classification, the development of freely accessible databases, and the use of new tools to survey fungi in ecological studies has prepared the mycological community to accomplish a number of new goals, including the discovery of millions of fungi.

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Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
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A4

BAT WHITE-NOSE SYNDROME IN NORTH AMERICA16

David S. Blehert, Jeffrey M. Lorch, Anne E. Ballmann, Paul M. Cryan, and
Carol U. Meteyer
17

Since 2007, infections by a previously unrecognized, perhaps imported fungus killed an estimated 1 million bats in North America.

Summary

  • The newly described fungus, Geomyces destructans, causes an invasive skin infection in bats and is the likely agent of white-nose syndrome (WNS).

_____________________

16 Reprinted with permission from the American Society for Microbiology (Microbe, June 2011, pp. 267–273).

17 David S. Blehert is the head of the diagnostic microbiology laboratory at the U.S. Geological Survey (USGS)–National Wildlife Health Center, Madison, Wis. (dblehert@usgs.gov), Jeffrey M. Lorch is a graduate student with the Molecular and Environmental Toxicology Center, University of Wisconsin–Madison, Medical Sciences Center, Madison, Wisconsin (jmlorch@wisc.edu), Anne E. Ballmann is a wildlife disease specialist at the USGS–National Wildlife Health Center, Madison, Wis. (aballmann@usgs.gov), Paul M. Cryan is a bat ecologist at the USGS–Fort Collins Science Center, Fort Collins, Colo. (cryanp@usgs.gov), and Carol U. Meteyer is a wildlife pathologist at the USGS–National Wildlife Health Center, Madison, Wis. (cmeteyer@usgs.gov).

Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
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  • With immune system functions and body temperatures reduced during hibernation, bats may be unusually susceptible to a pathogenic fungus such as G. destructans.
  • WNS was first observed in a popular show cave near Albany, New York, leading some investigators to suspect that a visitor inadvertently introduced G. destructans at this site, triggering a wider WNS outbreak in North America.
  • Biologists trying to manage WNS within North American bat populations face major challenges, including the variety of susceptible host species, incredible dispersal capabilities of bats, difficulties in treating such populations, and persistence of the pathogen in their vulnerable underground habitats.

In 2007 bats in eastern North America began dying in unprecedented numbers from a previously undocumented disease, now called white-nose syndrome (WNS). Although the ecological and economic impacts of this disease are not fully elucidated, this severe loss of insectivorous bats threatens decreased crop yields, forest defoliation, and a rise in insect-borne diseases. The recent emergence of WNS in bats of eastern North America, its rapid spread, and the severity of the outbreak highlight the importance of wildlife disease as an integral component of ecosystem health.

Biologists with the New York State Department of Environmental Conservation first recognized WNS as a problem in late winter 2007 at five hibernation sites near Albany, N.Y. Subsequently, a recreational caver furnished a photograph from February 2006 in nearby Howes Cave depicting bats with clinical signs of WNS, implicating this location as the likely index site and suggesting disease emergence the winter before New York state biologists drew public attention to the disease. By 2011 WNS had spread south along the Appalachian Mountains into eastern Tennessee, as far west as southern Indiana and western Kentucky, and north into the Canadian provinces of Quebec, Ontario, and New Brunswick (Figure A4-1). Experts estimate that more than 1 million bats have died from WNS thus far. Modeling studies show that, if such mortality trends continue, one of the most abundant bat species in eastern North America, the little brown bat (Myotis lucifugus), could disappear from this region within 16 years. Sustained killing of this magnitude from an infectious disease is unprecedented among the approximately 1,100 species of bats known worldwide.

The Host, Pathogen, and Environment

The likely agent of WNS is a newly described fungus, Geomyces destructans, which causes an invasive skin infection that is the hallmark of this disease (Figure A4-2). G. destructans belongs to the order Helotiales within the phylum Ascomycota. Characteristics that distinguish it from other Geomyces spp. include

Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
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img-169

Figure A4-1 Occurrence of white-nose syndrome and/or Geomyces destructans in the United States (by county) and Canada (by county or district) from winter 2005–2006 through April 2011.

curved conidia (Figure A4-2), slow growth on laboratory medium, cold adaptation, and pathogenicity to bats. Species of Geomyces exist in soils worldwide, especially in colder regions.

Any infectious disease involves interactions among a susceptible host, pathogen, and the environment. To comprehend the ecology of WNS, we must consider the physiological and behavioral aspects of bats that make them susceptible to the disease, the characteristics of the fungus that allow it to act as a pathogen, and the role of underground sites (hibernacula) such as caves and mines in providing conditions conducive to maintaining this pathogen and enabling it to infect these hosts.

WNS appears to occur only in bats, suggesting they possess unique traits that make them a suitable host. Bats are nocturnal and the only mammals capable of powered flight. Their forelimbs are highly modified, consisting of elongated phalanges connected by a thin layer of skin to form wings. This body plan provides bats with selective advantages that allow them to dominate the night skies, making them the second most diverse group of mammals, accounting for approximately 1,100 of 5,400 mammalian species. Of 45 bat species in the United

Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
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img-170

Figure A4-2 Micrograph of Geomyces destructans showing distinctive asymmetrically curved conidia either free or borne singly at the tips and sides of branched conidiophores (bar, 10 µm).

States, at least 6 of the approximately 25 that hibernate have been documented with WNS, including the little brown bat, the northern long-eared bat (M. septentrionalis), the eastern small-footed bat (M. leibii), the endangered Indiana bat (M. sodalis), the tricolored bat (Perimyotis subflavus), and the big brown bat (Eptesicus fuscus). All six of those species are insectivorous and cope with winter food shortages by hibernating in cold and humid, thermally stable caves and mines. When hibernating, the animals typically congregate in large numbers, dramatically reduce metabolic functions, and assume a body temperature close to that of their surroundings (2–7°C). These physiological adaptations and behaviors likely predispose bats to infection by G. destructans and consequent development of WNS. Because approximately half the bat species of the United States are obligate hibernators, another 19 species are at risk for infection by G. destructans if it spreads beyond its current range.

G. destructans colonizes the skin of bat muzzles, wings, and ears, then erodes the epidermis and invades the underlying skin and connective tissues. This pattern is distinctive and is more severe than that caused by typical transmissible dermatophytes. Although the disease was named for the characteristic white growth visible around an infected animal’s nose, the primary site of infection is

Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
×

the wing (Figure A4-3A). Gross damage to wing membranes such as depigmentation, holes, and tears are suggestive of WNS, but these lesions are nonspecific, and histopathologic examination is necessary to diagnose the disease.

Specifically, fungal invasion of wing membranes ranges from characteristic cup-like epidermal erosions filled with fungal hyphae to ulceration and invasion of underlying connective tissue, with fungal invasion sometimes spanning the full thickness of the wing membrane (Figure A4-3B). Fungal hyphae can also fill hair follicles and destroy skin glands and local connective tissue. Bat wings play an important role in the pathogenesis of WNS by providing a large surface area for the fungus to colonize. Once infected, the thin layer of skin that composes the bat wing is vulnerable to damage that may catastrophically disrupt homeostasis during hibernation.

In North America, bat hibernacula range in temperature from approximately 2–14°C, temperatures all permissive to growth of G. destructans. Within this temperature range, G. destructans exhibits increasing growth rates with increasing temperature (Figure A4-4), but the fungus does not grow at temperatures of approximately 20°C or higher. This temperature sensitivity helps to explain why WNS is observed only among hibernating or recently emerged bats and why the disease is not diagnosed in bats during their active season when body temperatures are consistently elevated above those permissive to growth of G. destructans.

Looking for Other Host and Environmental Susceptibility Factors

Hosts with impaired immune functions tend to be susceptible to opportunistic fungi in their environments. Guided by this concept, some investigators suspected that insults such as exposure to environmental contaminants or infections by viral pathogens compromised bat immunity and made them vulnerable to G. destructans. However, neither contaminant exposure nor viral coinfections can be consistently identified in bats infected with that fungus.

Hibernating bats with WNS generally do not exhibit signs of an inflammatory response. However, severe inflammation typifies fungal skin infections of bats aroused from hibernation, providing evidence that such animals are not immunocompromised. Although studies of bat immune functions are in their infancy, studies of other mammalian species indicate that their immune functions are naturally suppressed during hibernation. Thus, rather than suggesting immune-function impairment, the lack of inflammatory response to fungal infection by hibernating bats may reflect an immune suppression that is part of hibernation physiology.

In addition, the body temperature of hibernating bats drops dramatically, providing another vulnerability to infection by G. destructans. Fatal fungal diseases are relatively rare among endothermic, or warm-blooded, animals because their tissues are too warm to support the growth of most fungal species. However,

Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
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img-172

Figure A4-3 (A) Three little brown bats (Myotis lucifugus) photographed by Alan Hicks (New York State Department of Environmental Conservation) in Graphite Mine, New York, in November, 2008. Note the white fungus colonizing the muzzles and nostrils of all three bats. Also note the extensive fungal colonization of the skin of the ears and wings of the bat pictured on the right; (B) Periodic acid-Schiff (PAS) stained microscopic section of wing membrane from a little brown bat with white-nose syndrome collected in Pennsylvania in February, 2009. Dense colonies of fungal hyphae erode skin and fill the cup-shaped depressions (arrow). Ulceration of epidermis with penetration and replacement of subcutaneous tissue (arrow heads) dramatically alters the integrity of wing membrane. Bar = 25 µm.

Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
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img-173

FIGURE A4-4 Colony expansion rates of Geomyces destructans when grown on cornmeal agar at 3, 7, 14, and 20°C. The trend line estimates colony expansion rates at temperatures ranging from 3–20°C.

fungi are more apt to cause fatal diseases in ectothermic, or cold-blooded, organisms such as insects, fish, amphibians, and plants. Bats and other mammals that hibernate are unique in that they are warm-blooded when metabolically active, but cold-blooded during hibernation—a period when their metabolism and body temperatures are dramatically suppressed. Although lowered body temperatures may predispose torpid bats to infection by G. destructans, the mechanism enabling this specific fungus to be a pathogen for bats while other cave-associated fungi remain innocuous is not known.

How G. destructans kills bats is under active investigation. One possibility is that fungal infection disrupts how bats behave while hibernating, leading to more frequent or longer arousals from torpor and thus accelerating usage of fat reserves. However, fat depletion is not consistently observed among all bats with WNS. Infected bats also may exhibit other aberrant behaviors midway through the hibernation season, such as shifting from thermally stable roost sites deep within hibernacula to areas with more variable temperatures near entrances.

Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
×

Sometimes, they depart early from hibernacula. Thus, exposure to cold could account for some WNS-associated mortality.

Further, fungal damage to wing membranes, which can account for more than 85% of the total surface area of a bat, may increase fatality rates. In addition to the key role that wings play in flight, wing membrane integrity is essential for maintaining water balance, temperature, blood circulation, and cutaneous respiration. Disrupting any of these functions could increase WNS mortality rates.

As with so many other diseases, the environment affects the progress and transmission of WNS. Some pathogenic fungi such as Histoplasma capsulatum, Cryptococcus spp., and Batrachochytrium dendrobatidis can persist in the environment without an animal host for survival. This independence contrasts with host-requiring viruses or other pathogens for which transmission dynamics tend to moderate as infected hosts are removed from a population. G. destructans likely does not require bat hosts to survive and can persist in caves by exploiting other nutrients.

The cool and humid conditions of underground hibernacula provide ideal environmental conditions for G. destructans or other fungal growth. While most G. destructans isolates were cultured from skin or fur of bats collected in or near underground hibernacula during winter, DNA from the same fungus is found in soil samples from several hibernacula that harbor WNS-infected bats in the northeastern US. Also, G. destructans has been cultured from soil samples from hibernacula in three states where WNS occurs, supporting the hypothesis that bat hibernacula are reservoirs for this pathogen and that bats, humans, or fomites may transport G. destructans between hibernacula. How temperature and humidity differences among hibernacula influence G. destructans and WNS is not known.

Uncertainties about WNS Emergence

What caused WNS to emerge in a North American cave during the winter of 2005 to 2006? Bats with clinical signs consistent with WNS were first observed in Howes Cave, a hibernaculum connected to a popular North American show cave. Because of its high human traffic, a tourist might have inadvertently introduced G. destructans at this site.

Europe might be the source for the fungus causing WNS. Reports dating back several decades describe hibernating bats in Germany with white muzzles resembling bats with WNS in North America. Recent culture and PCR surveys indicate that G. destructans is widespread in Europe, including among hibernating bats in hibernacula in the Czech Republic, France, Germany, Hungary, Slovakia, and Switzerland. Unlike in North America, however, mortality rates and population declines remain normal among European bat species. This sharp contrast between disease manifestation among bats in Europe and North America provides an opportunity to investigate how bat species may differ in terms of their susceptibilities to fungal infection, continental variability among fungal strains, and the influence of environmental conditions and bat behavior on this fungal disease.

Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
×

Challenges in Managing WNS, Conserving Bat Populations

Bat conservation efforts have historically focused mainly on reducing human causes of bat mortality, including habitat destruction, detrimental intrusions into roosts, and intentional extermination of colonies. Bat census figures prior to the emergence of WNS in North America indicate many populations of cave-hibernating bats were stable or increasing. However, the current WNS outbreak brings an even more serious threat to bat populations of North America, confronting biologists with a new set of conservation and management challenges.

Mitigating diseases in free-ranging wildlife populations requires very different approaches from those applied in agriculture for domestic animals. Once established, diseases in free-ranging wildlife are rarely, if ever, eradicated. Biologists trying to manage WNS within bat populations face multiple challenges, including the need to deal with numerous host species, long-distance migrations of infected hosts, poor access to some host populations, impracticalities associated with treating individual wild animals, infected hosts that are sensitive to being disturbed and that inhabit fragile ecosystems, and environmental persistence of the pathogen.

The guiding principle for physicians and veterinarians, “first, do no harm,” will help to prevent WNS management efforts from having unintended adverse consequences. For example: depopulating an infected colony would not be effective unless all infectious animals are removed and all hibernacula used by the population are decontaminated—conditions unlikely to be achieved among free-ranging wildlife; using disinfectants to decontaminate hibernacula could have toxic effects on other organisms reliant on those environments; treating individual bats with antifungal agents is labor intensive, is not self-sustaining, and could be toxic for treated animals or their symbionts; and careless intervention could disrupt natural selective processes that might yield behaviorally or immunologically resistant bats.

However, “first, do no harm” does not mean “do nothing.” State and federal agencies already are taking measures to combat WNS, including closing caves and mandating decontamination procedures. Such steps are intended to prevent people from disturbing hibernating bats and to reduce the chance that intruding humans will transfer G. destructans from one hibernaculum to another. For example, taking a proactive approach prior to the appearance of WNS, state wildlife officials in Wisconsin conferred threatened status on four cave bat species that hibernate within its borders and designated G. destructans a prohibited invasive species providing state resource managers with legal authorities to take disease management actions.

Since the first description of G. destructans in 2008, its genome has been sequenced, and WNS pathology has been more fully defined. Additionally, hibernacula are being surveyed internationally, and ongoing analyses are revealing much about the biodiversity of fungi associated with bat hibernacula. With these and other advances in understanding WNS, opportunities will arise to better

Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
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manage the disease cycle. The sudden and unexpected emergence of WNS exemplifies the importance of monitoring, investigating, and responding to emerging wildlife diseases and the ecological and societal threats that they present.

SUGGESTED READING

Blehert, D. S., A. C. Hicks, M. Behr, C. U. Meteyer, B. M. Berlowski-Zier, E. L. Buckles, J. T. H. Coleman, S. R. Darling, A. Gargas, R. Niver, J. C. Okoniewski, R. J. Rudd, and W. B. Stone. 2009. Bat white-nose syndrome: an emerging fungal pathogen? Science 323:227.

Casadevall, A. 2005. Fungal virulence, vertebrate endothermy, and dinosaur extinction: Is there a connection? Fungal Genet. Biol. 42:98–106.

Cryan, P. M., C. U. Meteyer, D. S. Blehert, and J. G. Boyles. 2010. Wing pathology of white-nose syndrome in bats suggests life-threatening disruption of physiology. BMC Biol. 8:135.

Desprez-Loustau, M-L., C. Robin, M. Buée, R. Courtecuisse, J. Garbaye, F. Suffert, I. Sache, and D. M. Rizzo. 2007. The fungal dimension of biological invasions. Trends Ecol. Evol. 22:472–480.

Frick, W. F., J. F. Pollock, A. C. Hicks, K. E. Langwig, D. S. Reynolds, G. G. Turner, C. M. Butchkoski, and T. H. Kunz. 2010. An emerging disease causes regional population collapse of a common North American bat species. Science 329:679–682.

Gargas, A., M. T. Trest, M. Christensen, T. J. Volk, and D. S. Blehert. 2009. Geomyces destructans sp. nov. associated with bat white-nose syndrome. Mycotaxon 108:147–154.

Kunz, T. H. and M. B. Fenton (ed.). 2003. Bat ecology. University of Chicago Press, Chicago.

Lindner, D. L., A. Gargas, J. M. Lorch, M. T. Banik, J. Glaeser, T. H. Kunz, and D. S. Blehert. 2010. DNA-based detection of the fungal pathogen Geomyces destructans in soil from bat hibernation sites. Mycologia 103:241–246.

Meteyer, C. U., E. L. Buckles, D. S. Blehert, A. C Hicks, D. E. Green, V. Shearn-Bochsler, N. J. Thomas, A. Gargas, and M. J. Behr. 2009. Pathology criteria for confirming white-nose syndrome in bats. J. Vet. Diag. Invest. 21:411–414.

Wibbelt, G., A. Kurth, D. Hellmann, M. Weishaar, A. Barlow, M. Veith, J. Prüger, T. Görföl, T. Grosche, F. Bontadina, U. Zöphel, H.-P. Seidl, P. M. Cryan, and D. S. Blehert. 2010. White-nose syndrome fungus (Geomyces destructans) in bats, Europe. Emerg. Infect. Dis. 16:1237–1242.

Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
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A5

MAMMALIAN ENDOTHERMY OPTIMALLY RESTRICTS FUNGI AND METABOLIC COSTS18,19

Aviv Bergman20and Arturo Casadevall21

Abstract

Endothermy and homeothermy are mammalian characteristics whose evolutionary origins are poorly understood. Given that fungal species rapidly lose their capacity for growth above ambient temperatures, we have proposed that mammalian endothermy enhances fitness by creating exclusionary thermal zones that protect against fungal disease. According to this view, the relative paucity of invasive fungal diseases in immunologically intact mammals relative to other infectious diseases would reflect an inability of most fungal species to establish themselves in a mammalian host. In this study, that hypothesis was tested by modeling the fitness increase with temperature versus its metabolic costs. We analyzed the tradeoff involved between the costs of the excess metabolic rates required to maintain a body temperature and the benefit gained by creating a thermal exclusion zone that protects against environmental microbes such as fungi. The result yields an optimum at 36.7°C, which closely approximates mammalian body temperatures. This calculation is consistent with and supportive of the notion that an intrinsic thermally based resistance against fungal diseases could have contributed to the success of mammals in the Tertiary relative to that of other vertebrates.

Importance

Mammals are characterized by both maintaining and closely regulating high body temperatures, processes that are known as endothermy and homeothermy,

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18 Originally published as: Bergman, A. and A. Casadevall. 2010. Mammalian Endothermy Optimally Restricts Fungi and Metabolic Costs. mBio 1(5): e00212-10. doi:10.1128/mBio.00212-10.

19Received 17 August 2010 Accepted 11 October 2010 Published 9 November 2010 Citation Bergman, A., and A. Casadevall. 2010. Mammalian endothermy optimally restricts fungi and metabolic costs. mBio 1(5):e00212-10. doi: 10.1128/mBio.00212-10. Editor Françoise Dromer, Institut Pasteur Copyright © 2010 Bergman and Casadevall. This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-Share Alike 3.0 Unported License, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited. Address correspondence to Arturo Casadevall, arturo.casadevall@einstein.yu.edu.

20 Department of Systems and Computational Biology, Albert Einstein College of Medicine, Bronx, New York, USA.

21 Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, New York, USA.

Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
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respectively. The mammalian lifestyle is energy intensive and costly. The evolutionary mechanisms responsible for the emergence and success of these mammalian characteristics are not understood. This work suggests that high mammalian temperatures represent optima in the tradeoff between metabolic costs and the increased fitness that comes with resistance to fungal diseases.

Endothermy and homeothermy are fundamental aspects of mammalian physiology whose evolutionary origin remains poorly understood. Although many explanations have been suggested for the origins of endothermy and homeothermy, none are fully satisfactory given their high metabolic costs (Kemp, 2008; Ruben, 1995). Furthermore, the factors responsible for the mammalian set point remain unknown, posing the additional question of why mammals are so hot. Recently, the observation that fungal diseases are common in plants and insects but rare in mammals, combined with the thermal susceptibility of fungi, led to the proposal that mammalian endothermy and homeothermy create a thermal exclusionary zone that protects mammals against mycoses (Robert and Casadevall, 2009). Endothermy was also suggested to have provided a fitness advantage in the fungal bloom that followed the end of the Cretaceous such that it could have contributed to the success of mammals in the Tertiary (Casadevall, 2005; Robert and Casadevall, 2009).

Assuming that a relationship exists between endothermy and reduced susceptibility to certain classes of microbes, we hypothesized a tradeoff relationship whereby the high costs of endothermy were mitigated by protection against infectious diseases. In other words, we posited that increases in body temperature would protect against microbes by creating a thermal exclusionary zone but that such increases would be increasingly costly with regard to metabolic rates as the host body temperature diverged from ambient temperatures. Given that there is robust information on fungal thermal tolerances (Robert and Casadevall, 2009), we decided to test this hypothesis by attempting to identify body temperatures that confer maximal fitness for certain metabolic rates.

To address this question, we propose a first-order model wherein a tradeoff exists between the excess metabolic rates required to maintain a body temperature, T, and the benefit gained by protection against deleterious microbes because of the creation of a thermal exclusion zone. Metabolism, the exchange of energy between the organism and its environment, as well as the transformation of that energy to material within an organism, is affected by two main factors, body mass, M, and body temperature, T. Due to the fractal nature of transport networks, that is, vessel architecture and branching (Gillooly et al., 2001; Savage et al., 2008), over ontogeny, the resting metabolic rate, Brest, scales with body mass, m, as Brest = B0m3/4, where B0 is a normalization constant for a given taxon. Also, the normalization coefficient, B0, exponentially increases with body temperature B0 ~ e–E0/KT, where E0 is the average activation energy for the rate-limiting enzyme-catalyzed biochemical reactions of metabolism (ca. 0.65 eV), K is Boltzmann’s constant (8.62 × 10–5eV/K), and T is body temperature (Brown et al., 2004;

Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
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Gillooly et al., 2001). The scaling relationship between resting metabolic rate and body mass, ∝m3/4, has been predicted from allometric theories and supported by data on a diverse set of organisms, including mammals, birds, fish, and mollusks (Brody, 1964; Moses et al., 2008; Savage et al., 2004; West et al., 1997). As can be seen from the formulas above, body temperature affects the metabolic rate through its effects on rates of biochemical reaction kinetics according to Boltzmann’s factor, e–Ei/kT, where T is measured in kelvins (absolute temperature). The resting metabolic rate, Brest, is proportional to the product of these two effects and again has been shown to be well approximated, within a biologically relevant temperature range (0°C to 40°C), as B(T) ∝e–Ei/kTm3/4 (Gillooly, 2001). The first part of our analysis examined the excess cost for an organism of body mass m to maintain a body temperature T (assuming no dependence of body mass on temperature).

In the second part of our analysis, the benefit, noted here as F(T), is calculated as the reduction in the number of fungal species capable of infecting a host; this number is reduced approximately by s ≈ 6% for every degree Celsius in the temperature range of 27°C to 40°C (Robert and Casadevall, 2009). The increased benefit of the successive elimination of fungal species can thus be expressed as F(T) ∝F0[1 – (1 – s)T], where F0 is a constant scaling factor. The quantity W(T) = F(T)/B(T) can represent the balance between cost and benefit; thus, W(T) can be viewed as the total fitness of an organism as a function of its body temperature. Within the biologically relevant temperature range, the proposed fitness measure reaches its maximum at approximately 37°C (Fig. A5-1). Note that in this formulation, the optimal body temperature, where W(T) attains its maximum value, does not depend on the organism’s body mass. Furthermore, the one parameter that is determined from biological observation is the reduction in the number of fungal species capable of infecting a host; thus, to determine our model’s dependence on this parameter, we calculated the optimal temperature over a wide range of possible reduction percentages, i.e., 4% to 8%. In this range, the optimal temperature was found to remain in a tight range of less than 2°C, from 37.7°C to 35.9°C, respectively, which is still within the biologically relevant range of mammalian body temperatures. The insensitivity of the model to its only parameter further strengthens our hypothesis.

In summary, we present a minimal, parsimonious model to account for the cost of maintaining a high body temperature in mammalian organisms. A body temperature of 36.7°C maximizes fitness by restricting the growth of most fungal species relative to its metabolic cost. Our model suggests that no additional elaborations are required to explain the evolution of endothermy other than the tradeoff between protection against environmentally acquired microbial diseases and the cost of metabolism. Although we cannot rule out the possibility that this body temperature optimum arose by some remarkable coincidence, we think this highly unlikely because it emerges from considering two unrelated processes, fungal thermal tolerance and mammalian metabolic costs. Nonethe-

Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
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img-180

Figure A5-1 Organism fitness as a function of body temperature. We normalized fitness, W(T), to attain a maximum value of 1 and plotted body temperature in degrees Celsius over a range of 27°C to 60°C. Fitness reaches a maximum value at WMAX(T) ≈ 36.7°C.

less, we acknowledge that similar temperature optima might emerge from other considerations. For example, the specific heat capacity of water has a minimum at 36°C, and if the efficiency of metabolic processes is related to heat capacity, then using this parameter as the optimality criterion may result in a similar range of solutions. Nevertheless, we note the internal consistency in the theme that fungal diseases are rare in immunologically intact mammals and the tradeoff between increased fitness and metabolic costs closely approximates mammalian body temperatures.

Acknowledgements

Aviv Bergman is supported by 5P01AG027734-04 and 5R01AG028872-04. Arturo Casadevall is supported by AI33774-11, HL59842-07, AI33142-11, AI52733-02, and U54-AI057158-Lipkin.

References

Brody, S. 1964. Bioenergetics and growth. Hafner, New York, NY.

Brown, J. H., J. F. Gillooly, A. P. Allen, V. M. Savage, and G. B. West. 2004. Toward a metabolic theory of ecology. Ecology 85:1771–1789.

Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
×

Casadevall, A. 2005. Fungal virulence, vertebrate endothermy, and dinosaur extinction: is there a connection? Fungal Genet. Biol. 42:98–106.

Gillooly, J. F., J. H. Brown, G. B. West, V. M. Savage, and E. L. Charnov. 2001. Effects of size and temperature on metabolic rate. Science 293:2248–2251.

Kemp, T. S. 2008. The origin of mammalian endothermy: a paradigm for the evolution of complex biological structure. Zool. J. Linn. Soc. 147:473–488.

Moses, M. E., C. Hou, W. H. Woodruff, G. B. West, J. C. Nekola, W. Zuo, and J. H. Brown. 2008. Revisiting a model of ontogenetic growth: estimating model parameters from theory and data. Am. Nat. 171:632–645.

Robert, V. A., and A. Casadevall. 2009. Vertebrate endothermy restricts most fungi as potential pathogens. J. Infect. Dis. 200:1623–1626.

Ruben, J. 1995. The evolution of endothermy in mammals and birds: from physiology to fossils. Annu. Rev. Physiol. 57:69–95.

Savage, V. M., E. J. Deeds, and W. Fontana. 2008. Sizing up allometric scaling theory. PLoS Comput. Biol. 4:e1000171.

Savage, V. M., J. F. Gillooly, W. H. Woodruff, G. B. West, A. P. Allen, B. J. Enquist, and J. H. Brown. 2004. The predominance of quarter-power scaling in biology. Funct. Ecol. 18:257–282.

West, G. B., J. H. Brown, and B. J. Enquist. 1997. A general model for the origin of allometric scaling laws in biology. Science 276:122–126.

A6

VERTEBRATE ENDOTHERMY RESTRICTS MOST FUNGI AS POTENTIAL PATHOGENS22,23

Vincent A. Robert24and Arturo Casadevall25

The paucity of fungal diseases in mammals relative to insects, amphibians, and plants is puzzling. We analyzed the thermal tolerance of 4802 fungal strains from 144 genera and found that most cannot grow at mammalian temperatures. Fungi from insects and mammals had greater thermal tolerances than did isolates from soils and plants. Every 1°C increase in the 30°C–40°C range excluded an

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22 Vincent A. Robert and Arturo Casadevall, “A Vertebrate Endothermy Restricts Most Fungi as Potential Pathogens”, Journal of Infectious Diseases, 2009, Vol. 200, Iss. 10, pp. 1623–1626. Reprinted by permission of Oxford University Press.

23 Received 23 May 2009; accepted 18 June 2009; electronically published 14 October 2009. Potential conflicts of interest: none reported. Financial support: National Institutes of Health (awards 5R01AI033774, 5R01HL059842, and 2U54AI057158). Reprints or Correspondence: Dr Arturo Casadevall, Department of Medicine, Albert Einstein College of Medicine, Yeshiva University, 1300 Morris Park Ave, Bronx, NY 10461 (arturo.casadevall@einstein.yu.edu).

The Journal of Infectious Diseases 2009;200:000–000

© 2009 by the Infectious Diseases Society of America. All rights reserved. 0022-1899/2009/20010-00XX$15.00

DOI: 10.1086/644642

24 Centraalbureau voor Schimmelcultures, Utrecht, the Netherlands.

25 Department of Microbiology and Immunology and Division of Infectious Diseases, Department of Medicine, Albert Einstein College of Medicine, Yeshiva University, Bronx, New York.

Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
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additional 6% of fungal isolates, implying that fever could significantly increase the thermal exclusion zone. Mammalian endothermy and homeothermy are potent nonspecific defenses against most fungi that could have provided a strong evolutionary survival advantage against fungal diseases.

Of the 1.5 million fungal species, only a few hundred are pathogenic to mammals (Kwon-Chung and Bennett, 1992). Fungal diseases in mammals often reflect impaired immune function, and fungi did not emerge as major pathogens for humans until the late 20th century. For example, candidiasis was uncommon until the 1950s, when thrush was associated with the introduction of antibiotics that disrupted bacterial flora. Similarly, diseases such as cryptococcosis, aspergillosis, and histoplasmosis were rare until recently, when their prevalence increased with the human immunodeficiency virus epidemic and the development of immunosuppressive therapies. In contrast, the number of fungal species pathogenic to plants and insects is estimated to be 270,000 and 50,000, respectively (Hawksworth and Rossman, 1997). Amphibians are particularly vulnerable to certain fungal infections, as evidenced by the current catastrophic epidemic of chytridiomycosis in frogs.

The resistance of mammals with intact immune systems to systemic fungal diseases, coupled with their endothermic and homeothermic lifestyles, suggested that these costly physiological adaptations were evolutionarily selected because they conferred a survival advantage by protecting against environmental pathogens (Casadevall, 2005). However, testing this hypothesis was difficult because knowledge of fungal thermal tolerance is largely anecdotal. Consequently, we evaluated the thermal growth tolerances of fungal species in a reference collection and compared them to mammalian temperatures.

Methods

A total of 4802 fungal strains belonging to 144 genera in the Centraalbureau voor Schimmelcultures (Utrecht) collection were tested for growth at 4°C, 12°C, 15°C, 18°C, 21°C, 25°C, 30°C, 35°C, 37°C, 40°C, 42°C, and 45°C. Strains were grown for times ranging from a few days to a few weeks on the most suitable medium, generally glucose–peptone–yeast extract agar, potato-dextrose agar, or yeast extract–malt extract agar. Growth was considered positive when a colony was visible without magnification. The strain set included Ascomycetes and Basidiomycetes but excluded Zygomycetes, which is not in the yeast database.

The culture deposit records were reviewed to identify the isolation source. Fungi isolated from flowers, grains, and herbal exudates were grouped under plant isolates. Animal isolates were classified depending on whether they originated from endothermic (mammals and birds) or ectothermic (insects, nematodes, fishes, and crustaceans) species. Another group comprised isolates from nonliving environmental sources, which included predominantly soils; this group is referred to as soil isolates. These groups were compared for thermal tolerance at 2 tem-

Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
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peratures, 25°C and 37°C, which reflect ambient and mammalian temperatures, respectively.

To test the significance of the difference in growth patterns between fungal strains isolated from different groups, we calculated the test statistics

img-183

where p1 and p2 are the observed sample proportions for each group of fungal strains at a given temperature, n1 and n2 are the size of the 2 groups under comparison, and

P = (p1 × n1 + p2 × n2)/(n1 + n2).

The statistic z was assumed to be distributed normally. The 2-tailed probability from the absolute z score to infinity on both tails of the distribution was calculated (http://www.danielsoper.com/statcalc/calc21.aspx) and confirmed using the NORMSDIST function in Excel (Microsoft) to assess the significance of differences in growth between groups at different temperatures.

Results and Discussion

Knowledge of fungal thermal tolerance is limited to a few species because the subject has not been systematically studied. In fact, such studies may be very difficult to do, and a comprehensive prospective study of fungal thermal tolerance would require a gargantuan effort. However, culture collections provide an attractive alternative for initial explorations of this subject. Culture collections store and maintain fungal strains and record basic nutritional needs and temperature tolerances. This information, when accessed and analyzed with bioinformatics tools, provides a useful starting point for the analysis of fungal thermal tolerances.

Our results show that most strains grew well in the 12°C–30°C range, but there was a rapid decline in thermal tolerance at temperatures >35°C (Figure A6-1). A plot of the fraction of fungal strain that grew versus temperature in the 30°C–42°C range revealed a linear relationship with an equation of y = -0.0166x × 2.7911, such that for every 1° increase in temperature >30°C, ~6% fewer strains could grow.

For 3020 strains, there was information on both source isolation and temperature tolerance. This group included isolates from the environment (primarily soils), plants, ectothermic animals, and endothermic animals. The majority of these isolates grew at 25°C regardless of their source (Table A6-1). Nevertheless, the proportion growing at 25°C was significantly greater for isolates recovered from living hosts than from soils, irrespective of whether the hosts were ectothermic plants and animals or endothermic animals. At 37°C, the proportion of

Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
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img-184

Figure A6-1 Frequency histogram of thermal growth tolerance for 4802 fungal strains (bars). Lines connect percentages for 49 mammalian (blue) and 12 bird (red) species core temperatures. Obtained from McNab (1970).

Table A6-1 Growth Tolerances for Fungi from Soils, Animals, and Plants at 2 Temperatures

Origin, host type Isolate Growth Total P valuesb
Yes No Unknowna P1 P2 P3
at 25°C
Soils, NA 657 42 7 706
Plant, ectotherm 1108 30 5 1143 <.001
Animal
   Ectotherm 490 0 6 496 <.001 .29
   Endotherm 661 5 9 675 <.001 .214 .263
at 37°C
Soils, NA 146 535 15 706
Plant, ectotherm 304 871 22 1143 .292
Animal
   Ectotherm 193 284 19 496 <.001 .004
   Endotherm 466 202 7 605 <.001 <.001 <.001

NOTE. NA, not applicable

aRefers to a small no. of isolates for which the temperature growth data was not complete.

bP1 refers to the comparison of isolates from soils, P2 refers to the comparison versus plant isolates, and P3 refers to the comparison between isolates from ectothermic and endothermic animals.

Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
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fungi that grew was much higher for isolates from endothermic animals than from ectothermic animals. The proportions of Ascomycetes and Basidiomycetes fungi in each group were comparable, except for ectothermic hosts, which yielded predominantly Ascomycetes fungi.

Isolates from ectothermic hosts (such as plants and insects) were significantly more thermotolerant than isolates from soils. A significantly greater percentage of fungal strains from insects grew at 37°C relative to those recovered from plants, possibly reflecting the fact that insects can increase their temperature through behavioral fevers that increase survival after fungal infection (Thomas and Blanford, 2003). However, this explanation is unlikely to apply to plants, which have much lower metabolic rates. Since thermal tolerance must be associated with numerous metabolic changes that mitigate fungal damage, the association between greater thermotolerance and plant pathogenicity could mirror adaptation to survival in a host with potent antifungal defenses, raising the tantalizing possibility that selection pressures by virulence may contribute to thermal stability and vice versa. In this regard, we note that Hsp90 orchestrates morphogenesis in Candida albicans (Shapiro et al., 2009), thus providing a molecular association for heat shock and a virulence-related phenotype that may be conserved in other fungi.

A survey of the fungal genera represented in our sample collection revealed differences in the percentage of isolates capable of growth at 37°C. All genera studied included some thermotolerant species, as defined by their ability to grow at 37°C, but there were large differences in the percentage of species within each genera. Thermotolerant genera included those from both Ascomycetes and Basidiomycetes, but basidiomycetous genera were disproportionately more common among the thermotolerant genera (P < .001, Fisher exact test). The strains grouped within the sexually related basidiomycetous genera Filobasidiella (a telemorph of Cryptococcus) and Cryptococcus (an anamorph of Filobasidiella) included comparable numbers of thermotolerant species (61% among 116 strains and 53% among 287 strains, respectively). These data suggest an association between phylogeny and thermotolerance.

The capacity for thermotolerance was interspersed among Ascomycetes and Basidiomycetes, suggesting that it may have emerged independently several times in evolution. Alternatively, thermotolerance may be an ancient fungal trait that was lost by those species that cannot grow at 37°C. In this regard, we note that the climate for much of Earth’s history was much warmer than in recent geologic epochs, having cooled by ~5°C during the Eocene-Oligocene transition ~34 million years ago (Liu et al., 2009). The fact that thermotolerance is a complex trait that can be lost by a single mutation, as demonstrated by laboratory-generated temperature-sensitive mutants, makes the explanation of a retained phenotype attractive.

Our results may be relevant to the ongoing debate on the origin and function of endothermy, homeothermy, and fever, each a major unsolved problem in vertebrate physiology (Kemp, 2008; Ruben, 1995). There is no consensus as to

Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
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why mammals have adopted such an energetically costly lifestyle. Endothermy is associated with certain metabolic benefits and thermodynamic efficiency, but these benefits come at a high cost since endothermic vertebrates require ~10 times more oxygen to support metabolism than do ectothermic vertebrates (Ruben, 1995). Our analysis suggests that part of the cost is mitigated by the creation of a thermal exclusionary zone that can protect against environmental microbes. Given the high metabolic cost of endothermy, the core temperatures of individual mammal and bird species are likely to be a compromise between its benefits and costs. If endothermy was selected for protection against infectious disease, then a case could be made that endothermy preceded homeothermy. Similarly, if one considers fever as a mechanism to extend the thermal exclusionary zone against environmental microbes such as fungi, increases in temperature of only 1°–3° can significantly reduce the proportion of such microbes that can inhabit the host.

The benefits of endothermy and homeothermy in protection against microbes do not appear to have been previously considered as mechanisms for evolutionary selection, possibly because most of the viral and bacterial diseases that currently plague animals are often acquired from other warm hosts, and these necessarily involve thermotolerant microbes. However, the perspective is very different when one focuses on environmentally acquired microbes and the fungi in particular. Pathogenic microbes are a very small subset of the total terrestrial microbial flora, and these can be divided as to whether they are acquired from other hosts or directly from the environment (Casadevall and Pirofski, 2007). For mammals, pathogenic microbes acquired from other hosts are usually adapted to mammalian temperatures, but microbes acquired directly from the environment would not be subject to such selection pressures. Hence, the potential benefit of endothermy and homeothermy to host defense may become apparent only when one considers the entire microbiota and that subset of pathogenic microbes that is acquired directly from the environment. In support of this notion, we note that bats become susceptible to a cold-loving fungus when hibernation greatly reduces their body temperatures (Blehert et al., 2009) and that primitive mammals (such as the egg-laying platypus, which has a body temperature of 32°C) are susceptible to fungal diseases (Obendorf et al., 1993). An epidemiological observation consistent with the protective function of endothermy comes from the observation that serotype D Cryptococcus neoformans are less thermotolerant (Martinez et al., 2001) than other varieties and are associated with cutaneous cryptococcosis (Dromer et al., 1996). Experimental support for the notion that endothermy restricts fungal infection comes from the observation that rabbits, which have core temperatures of 38°C–39°C, are notoriously resistant to cryptococcosis, and infection can be induced only in cooler organs, such as testes (Perfect et al., 1980). However, the same system also shows that mammalian immune systems also make a decisive contribution to host defense against fungi since systemic cryptococcosis can be induced in rabbits after corticosteroid administration (Perfect et al., 1980).

This study reflects the power of a bioinformatics analysis of archival data

Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
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from culture collection, which allows comparison of temperature growth data on thousands of isolates. However, there are certain limitations that should be considered in evaluating the data. Strains from plants and insects were disproportionately represented in the collection, and this may introduce certain biases. The relative paucity or absence of strains from certain sources and taxonomic groups could contribute to bias in the statistical analysis. For example, there were relatively few isolates from birds and ectothermic animals other than insects, no Zygomycetes fungi, and only a few filamentous fungi. Furthermore, the catalogued information was insufficiently detailed to distinguish between skin and systemic isolates from endotherms, which could differ in thermotolerance.

The discovery of fossilized fungal proliferation at the Cretaceous-Tertiary boundary was proposed to contribute to extinction events at the end of the Cretaceous epoch that replaced reptiles with mammals as the dominant large animals (Casadevall, 2005). Thermal tolerance is a necessary, but not sufficient, characteristic of microbes being capable of causing invasive disease in mammals. Since thermal tolerance almost certainly involves many genes and biochemical processes, it is unlikely that this trait can be rapidly acquired by any one microbial species. Consequently, new human pathogenic fungi are likely to emerge from genera that are already tolerant to higher temperatures; such species may warrant special attention given likely climatic changes in the years ahead that could alter patterns of fungal prevalence.

References

Blehert DS, Hicks AC, Behr M, et al. Bat white-nose syndrome: an emerging fungal pathogen? Science 2009; 323:227.

Casadevall A. Fungal virulence, vertebrate endothermy, and dinosaur extinction: is there a connection? Fungal Genet Biol 2005; 42:98–106.

Casadevall A, Pirofski LA. Accidental virulence, cryptic pathogenesis, martians, lost hosts, and the pathogenicity of environmental microbes. Eukaryot Cell 2007; 6:2169–74.

Dromer F, Mathoulin S, Dupont B, Letenneur L, Ronin O. Individual and environmental factors associated with infection due to Cryptococcus neoformans serotype D. Clin Infect Dis 1996; 23:91–6.

Hawksworth DL, Rossman AY. Where are all the undescribed fungi? Phytopathology 1997; 87:888–91.

Kemp TS. The origin of mammalian endothermy: a paradigm for the evolution of complex biological structure. Zool J Linn Soc 2008; 147:473–88.

Kwon-Chung KJ, Bennett JE. Medical mycology. Philadelphia: Lea & Febiger, 1992.

Liu Z, Pagani M, Zinniker D, et al. Global cooling during the eocene-oligocene climate transition. Science 2009; 323:1187–90.

Martinez LR, Garcia-Rivera J, Casadevall A. Cryptococcus neoformans var. neoformans (serotype D) strains are more susceptible to heat than C. neoformans var. grubii (serotype A) strains. J Clin Microbiol 2001; 39:3365–7.

McNab BK. Body weight and the energetics of temperature regulation. J Exp Biol 1970; 53:329–48.

Obendorf DL, Peel BF, Munday BL. Mucor amphibiorum infection in platypus (Ornithorhynchus anatinus) from Tasmania. J Wildl Dis 1993; 29:485–7.

Perfect JR, Lang SDR, Durack DT. Chronic cryptococcal meningitis. Am J Path 1980; 101:177–93.

Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
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Ruben J. The evolution of endothermy in mammals and birds: from physiology to fossils. Annu Rev Physiol 1995; 57:69–95.

Shapiro RS, Uppuluri P, Zaas AK, et al. Hsp90 orchestrates temperature-dependent Candida albicans morphogenesis via Ras1-PKA signaling. Curr Biol 2009; 19:621–9.

Thomas MB, Blanford S. Thermal biology in insect-pathogen interactions. Trends Ecol Evol 2003; 18:344–50.

A7

SURVEILLANCE FOR EMERGING DISEASES IN WILDLIFE

Peter Daszak, Carlos Zambrana-Torrelio, and Tiffany Bogich26

Impact of Fungal Diseases on Wildlife

At this meeting, a number of presenters discussed the role of specific fungal diseases in rapid declines and even extinctions of wildlife. Getting an accurate measure of the impact of a pathogen or group of pathogens on wildlife is notoriously difficult. First, wildlife populations undergo often dramatic shifts that are difficult to distinguish from the effects of an outbreak. These may be seasonal or interannual, and occur in response to long-term climatic fluctuations, variation in predator–prey cycles, or a range of other difficult-to-measure factors (Daszak et al., 2005; Pechmann et al., 1991). Second, outbreaks of disease in wildlife may cause significant mortality, but these events may be difficult to detect due to rapid scavenging or decay of carcasses. Even when carcasses are found, they may be too decayed to conduct proper pathological investigations. Third, despite a range of infectious agents linked to recent declines, these are relatively new to ecologists and wildlife managers, so that die-offs are often attributed to other factors, and diseases may not be examined. Despite these and other issues, emerging diseases—indeed, emerging fungal diseases—have been shown to cause significant population declines and even extinctions in wildlife (Daszak and Cunningham, 1999; Frick et al., 2010; Schloegel et al., 2006).

The emerging fungal disease chytridiomycosis is a good example of this trend. As discussed elsewhere in this report, it is caused by the fungal pathogen Batrachochytrium dendrobatidis, which infects the keratin-rich cells on the skin of adult amphibians (Kilpatrick et al., 2010). This disease was discovered in the 1990s and associated with significant population declines in Australia and Central America (Berger et al., 1998). Substantial support from other studies shows it is the major cause of global amphibian declines (Crawford et al., 2010; Lips et al., 2006; Skerratt et al., 2007). However, when this disease was first reported, a debate took place in the literature over whether amphibians were undergoing

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26 EcoHealth Alliance, 340 West 34th Street, New York, NY 10001.

Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
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population declines, or simple fluctuations (Blaustein, 1994). Debate continued on the importance of chytridiomycosis and other factors. The ecological community took about a decade to accept the role of disease in these wild animals as important enough to call for large-scale global action to prevent further spread (Mendelson et al., 2006).

Despite these issues, reports of emerging diseases affecting wildlife populations have grown rapidly (Aguirre and Tabor, 2008; Cunningham, 2005; Daszak et al., 2000; Deem et al., 2001; Nettles, 1996; Wildlife and emerging disease, 2009; Williams et al., 2002). These include diseases that have caused a number of high-profile declines, such as mycoplasmal conjunctivitis of house finches in the United States (Fischer et al., 1997); trichomonosis in declining birds in the United Kingdom (Robinson et al., 2010); chronic wasting disease of cervids in the United States (Miller and Williams, 2004; Sigurdson, 2008); and white-nose syndrome (Blehert et al., 2009) or geomycocis (Chaturvedi and Chaturvedi, 2011) of bats. Among these are a surprising number of fungal diseases, which often have a high impact. In humans, fungal pathogens do not represent a major cause of emerging diseases.

Analysis of a global database of emerging pathogens (Jones et al., 2008) suggests that fungi are responsible for only 5.9 percent of the emerging infectious diseases of people in the past four decades (Figure A7-1). Yet in wildlife, fungi have been implicated in global declines of amphibians, leading to extinction of species (Schloegel et al., 2006), multistate declines of bat populations (Frick et al., 2010), the near extinction of the Florida Torreya tree (Schwartz et al., 1995, 2000), and the collapse of eel grass beds, leading to global extinction of the eel grass limpet Lottia albicans (Carlton, 1993; Carlton et al., 1991).

Challenges in the Surveillance of Wildlife for Fungal and Other Pathogens

The emergence of so many high-impact diseases in wildlife and the role of wildlife as reservoirs for human emerging infectious diseases, or EIDs (Mahy and Brown, 2000; Taylor et al., 2001), have led to expanding efforts in surveillance of wildlife populations, both for new pathogens of human significance and for potential EIDs affecting wildlife. However, a number of important factors hinder this strategy. Here, we highlight the two most important challenges to effective global surveillance in wildlife, as well as review some approaches to address these challenges; and put them into context for emerging pathogens of humans and wildlife, and for fungal diseases in particular.

Jurisdictional Problems in the Surveillance of Wildlife

The agencies, funding bodies, non-governmental organizations (NGOs), and researchers that normally work with wildlife populations are usually distinct from those involved in public health and agricultural diseases. For example, in

Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
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img-190

Figure A7-1 Proportion of emerging infectious diseases caused by different taxonomic groups of pathogens. Fungi are responsible for only 5.9 percent of emerging human disease, yet have a disproportionate impact on wildlife, with responsibility for a series of major population declines and extinctions. The data are taken from the database published in a recent global analysis of emerging infectious diseases of humans over the last four decades.
SOURCE: Adapted from Jones et al. (2008).

the United States, the U.S. Fish and Wildlife Service is the agency responsible for protecting wildlife, but if the threat is an emerging pathogen, this agency has little capacity for outbreak investigation and control. Similarly, the national agency responsible for funding ecological research in the United States is the National Science Foundation (NSF). The National Institutes of Health (NIH) oversees a broad range of issues, from infections to organ dysfunction to mental health and other areas. The U.S. Department of Agriculture oversees agricultural health. At the international scale, the intergovernmental agency to protect wildlife is the International Union for the Conservation of Nature (IUCN), whereas the global health agenda falls under the World Health Organization (WHO), agricultural health under the Food and Agriculture Organization (FAO), and trade-related disease issues under the World Organisation for Animal Health (OIE). This siloed approach is followed by many countries globally; they tend to have separate ministries for health, agriculture, trade, and environment/forestry/wildlife. These approaches work well until the threats to human health cross these jurisdictional boundaries. With emerging diseases, they have done so repeatedly. For example, the emergence of severe acute respiratory syndrome involved wildlife reservoir

Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
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species (Li et al., 2005), the national and international trade in hunted and farmed wildlife and livestock (Xu et al., 2004), and international travel and migration (Anderson et al., 2004). Likewise, the global emergence of amphibian chytridiomycosis has been linked to trade and climate change (Lips et al., 2008), and involves the medical industry (Weldon et al., 2004), the production of amphibians for food (Schloegel et al., 2009), and introduced or invasive species (Kilpatrick et al., 2010).

One simple approach to overcoming these challenges is to encourage cross-disciplinary, cross-agency collaboration. This approach to research and policy has been led by the fields of “conservation medicine” (Daszak et al., 2004), One Health (Karesh and Cook, 2005), and EcoHealth (Daszak, 2009; Wilcox and Daszak, 2006). In the United States, some efforts have successfully bridged the funding gap between NIH and NSF, notably the Ecology of Infectious Diseases program launched jointly by the NSF and the NIH John E. Fogarty International Center in 2000 (Scheiner and Rosenthal, 2006). Likewise, there is a unique U.S. federal agency with a specific remit to address with wildlife diseases, the National Wildlife Health Center (NWHC) (Fleischli et al., 2004; Skerratt et al., 2005). The NWHC has been conducting surveillance, monitoring, investigation, research, and response on wildlife diseases for 35 years, and is registered with the Centers for Disease Control and Prevention Select Agent Program, marking it as a laboratory of significant relevance to human and livestock as well as wildlife health. It has a sophisticated network of laboratories, including Biosecurity Level 3 biocontainment labs, necropsy suites, and isolation rooms. In addition, it publishes quarterly reports of mortality investigations, and acts as a national focal point for similar activities in universities and NGOs. At the intergovernmental scale, there has been a recent flurry of activity to bring together agencies around the One Health agenda, including formal links among the OIE, FAO, and WHO, which originated from their collaborative efforts to tackle avian influenza (Anderson et al., 2010). Additionally, wildlife health has two significant nuclei within the United Nations system.

First, in the IUCN, there is the Species Survival Commission Wildlife Health Specialist Group (http://www.iucn.org/about/work/programmes/species/about_ssc/specialist_groups/specialist_group_pprofiles/veterinary_sg_profile/), which has a network of more than 400 wildlife veterinarians and researchers globally. Second, the OIE has a Working Group on Wildlife Diseases (http://web.oie.int/wildlife/eng/en_wildlife.htm), which has operated for more than 15 years and advises the OIE on wildlife health issues.

These initiatives have begun to bring diverse disciplines together to understand the drivers and impacts of wildlife EIDs, and to conduct effective surveillance and control of wildlife as reservoirs for human EIDs. However, they could be improved significantly with some simple approaches. First, within the United States, the government has the capacity to form interagency task forces for specific issues that cross agency mandates. In a previous administration, the complex issue of amphibian declines was addressed with the formation of the Interagency

Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
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Taskforce on Amphibian Declines and Deformities. Similar task forces are likely to be useful to address the need for better surveillance of the wildlife trade for pathogens (Smith et al., 2009a), or the threat of white-nose syndrome in bats. Second, at a global scale, strengthening of laboratory capacity and personnel for wildlife diseases, and support for One Health approaches from the development community, could be extremely useful in fostering linkages among disparate ministries, universities, NGOs, and others. Recently, the U.S. Agency for International Development launched an Emerging Pandemic Threats program that specifically adopted a One Health approach to build capacity in the regions where emerging zoonoses most commonly originate (http://www.usaid.gov/our_work/global_health/home/News/ai_docs/ept_brochure.pdf) (Daszak, 2009). This includes specific collaboration among human and veterinary medical scientists; ministries of health, agriculture, and environment; and OIE, FAO, and WHO.

Limited Resources for Surveillance and Lack of Predictive Capacity

During the past decade, significant economic investment has been made in global surveillance for EIDs. Funding was provided by intergovernmental agencies, and national governments in particular, to counter the specific threat of H5N1 avian influenza. Although much of the surveillance and control efforts focused on Southeast Asia, the recent emergence of an H1N1 triple reassortant (Neumann et al., 2009; Smith et al., 2009b; WHO gets mixed reviews for H1N1 response, 2011) from a different region may have changed the public’s perception of pandemic risk, and a shift of funding priorities. At the same time, the prominence of H5N1 in the media over the past decade and its inability so far to mount a pandemic heightens the perception of emerging diseases as difficult to predict or forecast. With limited global resources for pandemic prevention or control, predictive efforts that can help target resources are greatly needed.

Recent work has shown that analyzing past trends in disease emergence and correcting data for surveillance biases allows us a strategy to identify the regions most likely to propagate a new emerging zoonosis (Jones et al., 2008; King et al., 2006; Taylor et al., 2001; Woolhouse and Gowtage-Sequeria, 2005). These approaches may provide a way to geographically target surveillance efforts. They suggest that surveillance programs should target regions with high biodiversity and dense human populations, essentially in tropical and subtropical regions (Jones et al., 2008). This approach essentially analyzes the underlying drivers of emerging diseases, which are usually socioeconomic factors (e.g., travel, trade, agricultural changes) or environmental factors (e.g., climate, land use change). If we adapt this to fungal pathogens, we see that two drivers in particular are important—climate and trade. For human EIDs, analysis of the database in Jones et al. (2008) suggests that the majority of emerging fungal diseases were associated with HIV/AIDS or antimicrobial use. For plant EIDs, analyses suggest that agricultural trade and climate are the most important drivers of new emerging diseases (Anderson et al., 2004). With the limited work that has been conducted

Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
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on emerging fungal pathogens of wildlife, trade appears to be an important driver of emergence. For example, significant evidence points to the global amphibian trade for food as a major driver of the spread of the amphibian fungal disease chytridiomycosis (Schloegel et al., 2010). The similarity of U.S. isolates of Geomyces destructans (the causative agent of bat white-nose syndrome) with those from Europe and its continued spread as if from a focal point at a tourist cave in New York state strongly suggest anthropogenic introduction.

Two important recommendations emerge: First, targeting of wildlife trade routes, wet markets,27 and international ports of entry for surveillance of wildlife may be particularly fruitful in identifying novel EIDs (Karesh et al., 2005; Pavlin et al., 2009; Smith et al., 2009a). Second, use of novel modeling approaches to geographically targeting wildlife for surveillance may be particularly useful for identifying novel pathogens. This “smart surveillance” approach (see Daszak, 2009) would help identify a pool of microbes harbored by a reservoir, some of which ultimately may become zoonotic under the right circumstances. Clearly, issues remain in that many microbes will be unable make the species jump to humans, so that distinguishing the truly potential zoonoses from these is not a simple task. However, conducting PCR-based surveillance in wildlife using conserved (or “degenerate”) primers for known pathogen groups would allow the targeting of novel agents related to known pathogens. This would enable a greater understanding of the risk of new zoonoses from each wildlife species examined, and would be a simple, cost-effective way to better evaluate the diversity of likely zoonoses. Diagnostic assays could then be developed for these pathogens, and used to screen people who live in close contact with the wildlife reservoirs.

References

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Anderson, P. K., A. A. Cunnigham, N. G. Patel, F. J. Morales, P. R. Epstein, and P. Daszak. 2004. Emerging infectious diseases of plants: Crop homogeneity, pathogen pollution and climate change drivers. Trends in Ecology and Evolution 19(10):535–544.

Anderson, R. M., C. Fraser, A. C. Ghani, C. A. Donnelly, S. Riley, N. M. Ferguson, G. M. Leung, T. H. Lam, and A. J. Hedley. 2004. Epidemiology, transmission dynamics and control of SARS: The 2002–2003 epidemic. Philosophical Transactions of the Royal Society of London Series B: Biological Sciences 359:1091–1105.

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27 Markets which sell live wildlife (often mixed with livestock) are called “Wetmarkets,” particularly with reference to Asia.

Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
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Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
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Li, W. D., Z. Shi, M. Yu, W. Ren, C. Smith, J. H. Epstein, H. Wang, G. Crameri, Z. Hu, H. Zhang, J. Zhang, J. McEachern, H. Field, P. Daszak, B. T. Eaton, S. Zhang, and L. F. Wang. 2005. Bats are natural reservoirs of SARS-like coronaviruses. Science 310:676–679.

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Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
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A8

GEOGRAPHY, CLIMATE, DUST, AND DISEASE: EPIDEMIOLOGY OF VALLEY FEVER (COCCIDIOIDOMYCOSIS) AND WAYS IT MIGHT BE CONTROLLED

John N. Galgiani28,29

Introduction

Human disease resulting from infection by Coccidioides spp. was first recognized late in the 19th century. Since then, with more information and changing demographics, our understanding of this problem and our perception of its importance has evolved in many ways (Galgiani, 2007). First thought of as a rare

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28 Valley Fever Center for Excellence, University of Arizona College of Medicine.

29 Correspondence and current address: John N. Galgiani, M.D.; Professor, University of Arizona College of Medicine; Director, Valley Fever Center for Excellence; P.O. Box 245215, Tucson, AZ 85724; Tel.: 520-626-4968; Fax: 520-626-4971; e-mail: spherule@u.arizona.edu.

Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
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and always fatal illness, coccidioidomycosis later was appreciated as a common and frequently self-limited illness known as valley fever, in which only a small percentage of those affected suffered serious complications (Smith, 1940).

With U.S. military training within the endemic regions of California and Arizona during World War II, the potential of coccidioidomycosis as a significant problem affecting military readiness quickly became apparent, a problem that persists for the military into the present (Crum-Cianflone, 2007; Smith, 1958). With the rapid and extensive population expansion within south-central Arizona over the past decades, the impact of coccidioidomycosis has emerged from a rural to a much larger public health problem. With continued population growth within the central valley of California, in Mexico along the U.S. border, and in other endemic regions throughout the Western Hemisphere, it is expected that the numbers of infected persons will continue to increase. In addition to the medical problem for humans, a variety of other species, especially dogs, are susceptible to coccidioidomycosis and suffer considerable morbidity and mortality as a result (Shubitz, 2007).

A comprehensive assessment of coccidioidomycosis should address the fungus as it exists in the environment as well as how it creates medical problems. In both arenas, there are opportunities for better understanding which in turn could lead to improvements in public health. Hopefully, this article will be useful to call attention to where our knowledge is limited and how advances in those areas could benefit prevention and management of coccidioidal disease.

Coccidioides spp. in the Environment

Our primary source for our understanding of the relative endemnicity for regions within the United States is derived from studies conducted in the 1950s of coccidioidin skin-test prevalence of naval recruits from across the country (Palmer et al., 1957). These primarily include extensive portions of southern California, the lower deserts of Arizona, west Texas, and smaller areas of Utah, Nevada, and New Mexico. More recent information from California and Arizona (Hector et al., 2011), where coccidioidomycosis is a reportable infectious disease, is consistent with the earlier estimate. Coccidioidal infections are found in numerous other countries within the Western Hemisphere. What is known about these areas also suggests that the endemic distribution has been stable over the past several decades (Laniado-Laborin, 2007). On the other hand, the endemic regions in a longer time scale may not be constant. Recent archeological findings in Nebraska identified a Holocene bison dating back 8,500 years with coccidioidomycosis in a bone (Morrow, 2006). Also, models of wind pattern trends with global warming would predict increased westerly currents from the Coccidioides-endemic regions of west Texas toward the more populated portions of the state (Reheis and Rademaekers, 1997). Thus, with climate change we might see the endemic region expand.

Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
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Recent population studies of fungal isolates have identified genetic differences in isolates from patients in California (now classified as Coccidioides immitis) as compared to isolates from elsewhere (now classified as Coccidioides posadasii) (Burt et al., 1997; Fisher et al., 2000, 2001). This geographic separation is surprising, given the likelihood that coccidioidal spores should travel freely in and out of California, either by wind currents or as contaminants of trains, planes, or road vehicles. One possibility is that C. immitis is much better suited to its California niche than is C. posadasii and vice versa. Another explanation might relate to the biology of how soil becomes infected and endemic regions become established. For example, simply inoculating new soil may not be sufficient to create a new coccidioidal colony. Instead, a more complex interaction with rodents, plants, or other factors in the environment may be needed. Reinforcing this speculation is the sparse distribution of Coccidioides spp. within the environment. For example, in one systematic study in the central valley of California, only four genetically distinct isolates were found from 720 soil samples (Greene et al., 2000). Soil sampling in southern Arizona has also found isolates in a very small proportion of samples tested (Barker et al., 2010).

Researchers have known for some time that Coccidioides spp. are more prevalent in specimens from rodent burrows than from random subsurface soil (Elconin et al., 1957), although the reasons for this association remain unclear (Barker et al., 2010). In contrast, repeated sampling from a site known to be positive often yields positive results. One site identified as containing Coccidioides spp. in the 1960s (Converse and Reed, 1966) has again yielded C. posadasii half a century later (Personal communication, January 2011, M. J. Orbach). The sparse and stable nature of coccidioidal residence within the endemic regions affords an implication as to what factors are more likely to be associated with risk of exposure. Although there have been well-documented outbreaks associated with archeological and other dirt-disrupting sites (Pappagianis, 1983; Werner et al., 1972), in general occupational exposure does not appear to be a major risk factor (Kim et al., 2009). What might appear to be a paradox is actually consistent with the sparse prevalence of the fungus within the soil of even its most endemic regions, in which case much of the soil-disrupting activities occur at sites where the fungus is not present. In contrast, climate and especially wind patterns have been shown to affect seasonal incidence of infection (Comrie and Glueck, 2007; Hugenholtz, 1957). Taken together, the evidence would suggest that simply length of endemic exposure rather than a specific activity constitutes the more dominant effect for risk of infection.

With our current understanding of Coccidioides spp. residing in the environment, it is not possible to predict whether it exists in a specific location with any degree of precision from a physical or chemical analysis of the soil. Furthermore, high-throughput methods do not exist for microbial detection of Coccidioides spp. in soil samples. These missing tools prevent us from identifying specific locations which, if disrupted, would likely create a release of fungal spores. If

Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
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methods were available to do this, methods for treating the soil exist that would minimize or prevent this exposure from happening. Advances in this area would have practical public health benefits.

Coccidioidomycosis: The Scope of the Problem

Statistics about newly diagnosed coccidioidomycosis from California and Arizona were expected to total more than 16,000 new infections in 2010 (Figure A8-1). The sharp increase in reported cases in Arizona in 2009 is the result of an administrative change by a single large clinical laboratory to report a more sensitive coccidioidal serologic test as indicative of a new infection when positive. However, for both states there has been increased disease activity in the last half of 2010 that is unexplained.

In Arizona, the Department of Health Services conducted a telephone questionnaire of 10 percent of newly identified patients in 2007. It provides a better understanding of the impact of this disease (Tsang et al., 2010). According to the survey, illness lasted for an average of 6 months; three quarters of employed

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Figure A8-1 Annual cases of coccidioidomycosis.
SOURCE: Figure courtesy of John N. Galgiani (data from the California Department of Health and Arizona Department of Health Services).

Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
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patients lost more than a month of work; a quarter of patients needed 10 or more physician visits; and 40 percent of patients required hospitalization for their illness (Tsang et al., 2010). The same report shows Arizona hospital costs for coccidioidomycosis were more than $86 million. Extrapolating from these costs, estimates that include all outpatient medical care, often lasting for years if not entire lives, could easily reach a quarter of a billion dollars.

As significant as these findings are, other projections suggest that the actual number of persons seeking medical attention for coccidioidal infection is several times greater than those diagnosed and included in state public health statistics. One study found that only 3–13 percent of patients with pneumonia in Phoenix were tested for coccidioidomycosis (Chang et al., 2008). By contrast, a prospective study in Tucson in which patients with community-acquired pneumonia were tested for coccidioidomycosis demonstrated that nearly a third of these subjects had a coccidioidal infection (Valdivia et al., 2006).

State statistics show case rates for college-age persons in Pima County to be from 34 to 48 cases per 100,000 annually. However, recent surveillance of scholarship athletes at the University of Arizona, which is in Pima County, indicated 374 cases per 100,000 (Stern and Galgiani, 2010). Further analysis suggested that the most important reason for this much higher case rate was that the athletes received many more serologic tests for coccidioidomycosis. Evidently, more patients would be accurately identified as to the true cause of their illness if patients with endemic exposure to Coccidioides spp. were tested more routinely for this possibility. This is now the recommendation of the Arizona Department of Health Services and a growing number of Arizona state medical specialty societies and other professional organizations (Tsang et al., 2010).

With the commercial and recreational growth of the southwestern United States, coccidioidomycosis has become an increasing problem for the rest of the country as well. For example, persons who develop a respiratory illness within a month after returning from vacation or business conferences in south-central Arizona would have the same risk (approximately 30 percent) that their illness is due to Coccidioides spp. as would residents of the endemic regions. Using Arizona Department of Tourism statistics for 2008, the chance of an individual visitor developing any clinical illness would be expected to be small (approximately 1 in 17,000). However, because more than 22 million persons visit Arizona for an average of 4 to 5 days, the total number of illnesses occurring after leaving Arizona would add up to more than 1,300 per year. Evidence suggests that most of these illnesses would be diagnosed incorrectly in the course of routine medical care (Standaert et al., 1995).

Even if physicians obtain appropriate testing, establishing a diagnosis of early coccidioidal infection is often difficult. Coccidioidal serology is very specific when results are positive. Moreover, in progressive forms of infection, serology is very likely to be diagnostic (Fish et al., 1990; Pappagianis and Zimmer, 1990). However, these tests are not nearly as sensitive early in the course of the

Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
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acute pneumonia syndrome, the most common manifestation of a coccidioidal infection. In one study, depending on the method of analysis, false-negative results were estimated to occur from one third to two thirds of the time on first testing (Wieden et al., 1996). Although improved methods for detecting Coccidioidesspecific antibodies may improve the sensitivity, approaches such as detection of coccidioidal DNA by polymerase chain reaction (Clark and McAllister, 1996) or detection of coccidioidal antigens by enzyme-linked immunosorbent assay (ELISA) (Durkin et al., 2008; Helfrich et al., 2011) offer theoretical advantages to earlier detection. Improvements in early diagnosis would be very useful to clinicians trying to manage their patients.

Therapy for Coccidioidomycosis and Its Current Limitations

The most serious complications of coccidioidomycosis include progressive chronic pneumonia and hematogenous spread of infection to parts of the body outside of the chest. Patients with these problems have benefited greatly from the advent of orally absorbed azole antifungal agents (Galgiani et al., 2005). Fluconazole and itraconazole are commonly used for these complications (Galgiani et al., 1993, 2000; Tucker et al., 1990). The more recently available azoles, voriconazole and posaconazole, offer additional options (Catanzaro et al., 2007; Prabhu et al., 2004; Proia and Tenorio, 2004; Stevens et al., 2007). This class of antifungal drugs has been found to be relatively safe and generally well tolerated for extended courses of administration. However, approximately a quarter of patients with such infections do not respond adequately to these drugs. Even in patients who appear to respond to azole treatment, relapses occur in approximately a third of those when treatment is discontinued. Thus, for some patients, including all patients with coccidioidal meningitis, treatment is recommended to be lifelong (Dewsnup et al., 1996). The most currently available drugs offer a suppressive effect; they do not eradicate infections or cure patients.

Surprisingly, there is virtually no published experience on the use of any antifungal drug for the most common manifestations of coccidioidal infection, that of the early respiratory syndrome. In a recent prospective observational study (Ampel et al., 2009), patients treated with oral azoles (usually fluconazole) appeared neither to improve at a faster rate nor subsequently to avoid progressive complication than did patients who did not receive antifungal treatment. Although not a randomized controlled trial, this study presents the only information available and provides little encouragement for the value of early treatment.

Prospects for new drugs to treat coccidioidomycosis are limited (Ostrosky-Zeichner et al., 2010). This is due in part to the general contraction in anti-infective drug development in general (Talbot et al., 2006), but is especially problematic for coccidioidomycosis because of its status as an orphan disease, defined by the Food and Drug Administration (FDA) as having a U.S. prevalence of less than 200,000. Even for new antifungals, such as voriconazole, posacon-

Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
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and caspofungin (Gonzalez et al., 2001, 2007), which already have FDA approval for other fungal diseases, there are very limited or no controlled clinical trials conducted in patients with any form of coccidioidomycosis.

One exception to this pattern has been the persistent interest in bringing nikkomycin Z into clinical trials. Nikkomycin Z is a competitive inhibitor of chitin synthase, first discovered by German scientists in the 1970s. It was part of a fungicide discovery program at the Bayer Company (Fiedler, 1988). Its potential as a therapeutic for coccidioidomycosis was identified in the 1980s (Hector et al., 1990). In mice, nikkomycin Z treatment produced sterile lungs under conditions in which the lungs of untreated mice yielded several million viable fungal colonies. This observation raises the possibility that nikkomycin Z might offer a curative treatment for coccidioidomycosis. If so, this would provide even more incentive to diagnose coccidioidomycosis early in order to eradicate it and thereby prevent later and serious complications. Clinical development of nikkomycin Z was begun in the 1990s by Shaman Pharmaceuticals (Galgiani, 2007). However, the program became inactive when Shaman ceased to exist in 2000, and for several years nikkomycin Z development remained dormant.

In 2005, the University of Arizona acquired the program along with several kilograms of bulk nikkomycin Z that remained from Shaman’s program. Since then faculty at the University of Arizona and a small start-up company, Valley Fever Solutions, have successfully competed for research awards and small business grants from the National Institutes of Health (NIH) and the FDA Office of Orphan Products Development. With these funds as well as philanthropic support, clinical trials with nikkomycin Z were resumed with a 2-week multidose safety trial that was completed in 2009. This support is also being used to develop a more efficient manufacturing process. Supplies of nikkomycin Z made by this new process are planned to be available to begin a Phase II clinical trial in 2011 or 2012. It is hoped that this progress will advance the program sufficiently to attract pharmaceutical or investment interest to complete the commercialization process.

Prospects for a Preventative Vaccine

A large majority of the estimated 150,000 U.S. coccidioidal infections occurring annually resolve with or without symptoms and whether or not specific antifungal treatment is instituted (Galgiani et al., 2005). Healing occurs as a result of the patient’s cellular immunity, which is remarkably durable, usually lasting a lifetime (Galgiani et al., 2010). The fact that natural infection so often produces resistance to second infections has attracted a long-time interest in engendering this immunity through active immunization, leading to a clinical trial of a whole-cell killed vaccine (Pappagianis and Valley Fever Vaccine Study Group, 1993). This preparation did not produce protection. In retrospect it was surmised that the irritation at the injection site of the fungal cell wall polysaccharides prevented sufficient doses of protective immunogens to be delivered during vaccination. For

Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
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the past 15 years, a collaboration of several research groups has yielded a number of immunogenic coccidioidal proteins and vaccines prepared from recombinant proteins with adjuvants, some of which have shown excellent protection in mice and efficacy in primates (Cole et al., 2004; Cox and Magee, 2004; Herr et al., 2007; Johnson et al., 2007; Shubitz et al., 2006; Tarcha et al., 2006).

The next step for existing recombinant vaccine candidates would be for them to be moved into clinical trials. However, these candidates have met with major challenges including developing a suitable manufacturing process; identifying a suitable and available adjuvant; and compounding a suitable formulation appropriate for human experimentation (Galgiani, 2008). None of these challenges are insurmountable, but all require significant development investment. The overall impact of coccidioidomycosis within the endemic region is not so dissimilar to that caused by polio in the United States before a polio vaccine was available (approximately 10 per 100,000 population). However, the impact of coccidioidomycosis involves a much smaller population at risk as compared to the worldwide distribution of polio. This difference in market size makes it unlikely that a commercial vaccine manufacturer will invest in developing a coccidioidal vaccine even though such a vaccine, once developed, could arguably be profitable to manufacture and distribute (Barnato et al., 2001). Moving a coccidioidal vaccine into clinical trials probably requires the discovery of new, more easily formulated protective antigens; a breakthrough in vaccine technology that greatly reduces the cost of development; or a growing public health imperative to underwrite the costs needed for vaccine development.

Summary

Coccidioidomycosis is a major public health problem for a major, growing segment of the U.S. population as well as other endemic regions throughout the Western Hemisphere. A more complete understanding of its biology and ecology where it exists in the endemic environment could lead to risk abatement strategies not currently available. Improved recognition by healthcare professionals of coccidioidomycosis as a cause of community-acquired pneumonia when it occurs in their patients could improve management. This could be assisted further by developing more sensitive and clinically available diagnostic tests based on biosignatures such as DNA or proteins from the fungus itself. Curative therapies are also needed, but none exist today. Finally, eliminating problems caused by Coccidioides spp. might be possible if a preventive vaccine were developed. Even though coccidioidomycosis is an orphan disease, pursuit of these objectives is more than justified by the potential public health benefit and the reduced medical costs to society that their achievement would provide.

Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
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Acknowledgments

This presentation was supported in part by Award Number U54AI065359 from the National Institute of Allergy and Infectious Diseases (NIAID). The content is the sole responsibility of the authors and does not necessarily represent the official views of the NIAID or NIH.

Disclosure

Dr. Galgiani is chief medical officer, chair of the board, and a significant stock holder in Valley Fever Solutions, Inc.

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Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
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Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
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Helfrich, F. S. E., L. F. Shubitz, T. Peng, K. S. Knox, N. M. Ampel, J. N. Galgiani, and V. H. Wysocki. 2011. Proteomic identification of coccidioidal antigens from lung fluid of infected mice: MRM analysis to confirm presence in biological fluids. Proceedings of the Third Annual Meeting of the Association for Mass Spectrometry Applications to the Clinical Lab, San Diego, CA, February 2011.

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Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
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A9

CRYPTOCOCCUS GATTII: AN EMERGING PATHOGEN IN THE UNITED STATES

Julie R. Harris30

The genus Cryptococcus comprises 37 different species, of which only 2 are relevant for clinical infection (C. gattii and C. neoformans). Cryptococcus spores are inhaled from the environment, causing a primary lung infection that may or may not be symptomatic. Disseminated disease may result in meningitis and death (Li and Mody, 2010). Intricately linked with severe immunosuppression, C. neoformans was rarely reported before the 1950s, when cancer treatments and organ transplants—conditions that often require immunosuppressive treatments—began occurring with increasing frequency (Perfect and Casadevall, 2011). The era of AIDS led to an exponential increase in the

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30 Centers for Disease Control and Prevention.

Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
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numbers of immunosuppressed persons, and a corresponding massive increase in C. neoformans infections worldwide (Mitchell and Perfect, 1995; Perfect and Casadevall, 2011). Today, cryptococcal infections due to C. neoformans are among the most common AIDS-defining infections (Park et al., 2009; Warkentien and Crum-Cianflone, 2010). In contrast, infections caused by C. gattii are reported much less frequently. Gatti and Eeckels produced the first report of C. gattii infection in 1970, from a 7-year-old boy in Congo in 1966 (Gatti and Eeckels, 1970). The boy was found to have a cryptococcal infection clinically similar to C. neoformans, but with a different pathogen morphology (Gatti and Eeckels, 1970). The newly observed pathogen was deemed to be a variant of C. neoformans called C. neoformans var. gattii.

Today, C. gattii is considered its own species (Kwong-Chung et al., 2002). During the mid-1980s, studies of geographic sources of C. gattii and C. neoformans isolates demonstrated that although C. neoformans was found from all areas of the world, C. gattii was found only in tropical and subtropical climatic zones (Kwon-Chung and Bennett, 1984a,b). The authors concluded that C. gattii was likely to be restricted to locations where the minimum winter temperatures typically remained above freezing (Kwon-Chung and Bennett, 1984a). During the next decade, limited information became available about the epidemiology of C. gattii, much of it from papers describing infections in endemic Australia and Papua New Guinea (Ellis, 1987; Mitchell et al., 1995; Seaton et al., 1996b, 1997; Speed and Dunt, 1995). These studies demonstrated that C. gattii, unlike C. neoformans, was found almost exclusively in immunocompetent persons. In agreement with the findings of Kwon-Chung and Bennett (1984a), C. gattii infections were still seen only in patients living in tropical and subtropical climates (Chen et al., 2000; Lalloo et al., 1994; Laurenson et al., 1993, 1996, 1997; Mitchell et al., 1995; Seaton et al., 1996a, 1997; Slobodniuk and Naraqi, 1980; Speed and Dunt, 1995).

Beginning in 1999, the rate of cryptococcal infections among HIV-uninfected persons living on temperate Vancouver Island, British Columbia (B.C.), Canada, began increasing rapidly (Hoang et al., 2004; Fyfe et al., 2002). Early investigations demonstrated that most of these infections were caused by C. gattii, rarely reported before from Canada (Kwon-Chung and Bennett, 1984a). Veterinarians, too, noted that a wide range of animals were becoming infected with C. gattii where they had not previously been found with non-neoformans cryptococcosis (Duncan et al., 2006). During the following years, the disease continued spreading in B.C., infecting humans and animals on the nearby mainland who had no history of travel to Vancouver Island (MacDougall et al., 2007), and by 2007, 218 human infections had been reported from B.C. Although four genetic groups of C. gattii have been identified (VGI, VGII, VGIII, and VGIV), most infections in British Columbia were caused by the relatively uncommon VGII genotype. Due to the high numbers of isolates available from the outbreak, further genetic subdivision of outbreak-associated isolates was performed, demonstrating that

Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
×

approximately 90 percent of isolates in B.C. were of a “major strain” genetic subtype VGIIa, with a smaller number of “minor strain” subtype VGIIb isolates (Galanis and MacDougall, 2010).

Clinicians began noting C. gattii infections among patients in the Pacific Northwestern (PNW) states of Washington and Oregon in 2004 and 2005, respectively (MacDougall et al., 2007). In addition to the VGIIa and VGIIb infections, a new genotype of infection was noted in the United States, called VGIIc (Byrnes et al., 2010a). Beginning in October 2009, the Pacific Northwest Cryptococcus gattii Public Health Working Group, comprising the U.S. Centers for Disease Control and Prevention (CDC) and state health departments and laboratories in the Pacific Northwest, began retrospectively and prospectively collecting standardized information on C. gattii infections in the United States. As national awareness about the outbreak began to grow, other states also reported rare infections or submitted isolates for speciation and genotyping at the CDC.

Outbreak of C. gattii in the United States

To date, nearly 80 laboratory-confirmed human cases have been reported in the United States (Harris et al., 2010), most from Washington and Oregon (Figure A9-1). As awareness of the outbreak in the PNW has spread throughout the United States, infections from other states have also been reported. Case counts have increased each year, from a single case reported during 2004 to 24 cases reported during 2010 (Figure A9-2). The increase in reported cases is likely to be a result of both improved surveillance, as indicated by the recent increase in cases being reported from areas outside the PNW (Figure A9-2), and actual increases in case occurrences.

Among human patients in the United States identified through surveillance as having laboratory-confirmed C. gattii infection, approximately half are male, with a median age of 56 (range, 15–95). Patients aged 30 and older comprise the majority of cases (Table A9-1). The most commonly reported symptoms are headache, nausea, and cough, affecting more than half of all patients; more than half present with pneumonia and approximately half have meningitis. Most (73 percent) patients have an underlying immunosuppressive condition, including (but not limited to) a recent history of oral steroid use; lung, heart, kidney, or liver disease; a history of cancer; or a solid organ transplant (Table A9-2). HIV infection was more frequent among C. gattii patients than is found in the general U.S. population (5.9 percent vs. 0.6 percent) (CIA World Factbook, 2010), but was still the least commonly reported underlying condition. Of 59 patients with data, 17 (29 percent) had no detectable underlying immunocompromising condition. Cryptococcomas, or fungal masses, were found in the lungs and/or brains of substantial proportions of patients who received the corresponding scans or x-rays. Nearly all patients were hospitalized, and nearly one third with follow-up information died with or from their infections (Table A9-1).

Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
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img-210

Figure A9-1 Human infections with C. gattii, United States, December 2004–January 2011 (n = 79).
NOTE: * indicates patients that reported extensive travel to Washington and/or Oregon during the year before their illness onsets.

Outbreak-Strain vs. Non-Outbreak-Strain C. gattii Infections in the United States

Currently, 77 of 79 reported U.S. infections have been genotyped. Of these 77, 38 (49 percent) were VGIIa; 19 (25 percent) were VGIIc; 6 (8 percent) were VGIIb; and 14 (18 percent) were other genotypes (VGI, VGIII, and unrelated VGII).

Clear delineations exist between C. gattii infections in Oregon and Washington (“PNW-associated infections”) and those occurring in other parts of the United States (Figure A9-2). In particular, most PNW-associated infections are genotype VGIIa, VGIIb, or VGIIc (“outbreak-strain” genotypes). Outbreak-strain infections have also been reported from humans living in states outside of Washington and Oregon; those that have been reported were linked to these states by residential or travel history (Harris et al., 2010). One case in Idaho (subtype VGIIc) (CDC, 2010; Iqbal et al., 2010) and one case from Alaska (subtype VGIIa) (Harris et al., 2010) are considered linked to the outbreak, with both patients reporting extensive travel throughout Oregon, Washington, and/or B.C. during the year before their illness onsets (CDC, 2010).

Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
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img-211

Figure A9-2 U.S. human cases of C. gattii, by year of illness onset (n = 71*).
* NOTE: Onset year is reported for 62 patients and is estimated by initial report year for 9 patients for whom onset date was not available. 2010 data are current as of January 2011; complete case data typically lag illness onset by several months. PNW = Pacific Northwest.

While a small number of non-outbreak-strain infections have occurred in the PNW, most have been reported from states outside the PNW. A patient from North Carolina was diagnosed with C. gattii (genotype VGI) in 2007 following travel to San Francisco (Byrnes et al., 2009), and two cases of C. gattii (genotypes VGI and VGIII) were reported in 2010 from patients in New Mexico who did not have any recent history of travel outside of the state (Harris et al., 2010). In 2010, a patient was reported from Hawaii with a novel subtype of infection belonging to the genotype VGII (Harris et al., 2010). Since 2004, two patients have been reported from California and one from Michigan with VGIII-type infections. Thus, at least one focus of infection involving genotypes novel in the United States appears to be ongoing in the PNW; it also appears that sporadic C. gattii infection is occurring elsewhere, with genotypes distinct from those found in the PNW.

The collection of standardized data on U.S. outbreak-strain and non-outbreak-strain infections has provided an opportunity to examine clinical

Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
×

Table A9-1 Characteristics of C. gattii Patients in the United States, 2004–2010

Characteristic n N %
Age (mean, median, range), years 52, 55 (15–95)
. 18 3 64 5
19–29 3 64 5
30–49 23 64 36
50–69 27 64 42
70+ 8 64 13
Symptoms
Headache 32 55 58
Nausea 26 50 52
Cough 29 57 51
Dyspnea 24 51 47
Fever 25 57 44
Vomiting 22 50 44
Fatigue 17 40 43
Weight loss 21 50 42
Loss of appetite 15 46 33
Chills 16 52 31
Muscle pain 15 52 29
Chest pain 14 52 27
Neck stiffness 11 49 22
Night sweats 6 44 14
Photophobia 5 49 10
Seizure 4 45 9
Blurry vision 2 45 4
Any respiratory 39 55 71
Any central nervous system 19 44 43
Clinical findings
Pneumonia 27 49 55
Meningitis 26 51 51
Crypto lung 18 50 36
Crypto head 6 22 27
Outcomes of infection
Hospitalized 52 57 91
Died 16 52 31

differences by genotype and patient type. Although a relatively small number of non-outbreak-strain infections have been reported, significant differences between these and outbreak-strain infections have been noted in the proportion of infected patients who were immunocompromised and the propensity for respiratory and CNS symptoms (Table A9-2). It seems likely that different genotypes of C. gattii might infect different patient types; in addition, clinical manifestation of C. gattii infection might differ either by infecting strain genotype, patient immune status, or both.

Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
×

Table A9-2 Comparison Between Outbreak-Strain (VGIIa/b/c) and Other Genotypes of Infection with C. gattii, United States, 2004–2010

Symptoms, Patient Characteristics, Outcomes VGIIa/b/c Other genotypes RR p
Respiratory symptoms
   Cough 58% 11% 5.3 0.01
   Dyspnea 53% 12% 4.3 0.05
   Any respiratory symptom 76% 44% 1.7 0.10
Central nervous system symptoms
   Seizure 3% 50% 0.05 0.01
   Blurry vision 8% 43% 0.19 0.04
   Any central nervous system symptom 34% 100% 0.04 0.00
Preexisting medical condition 78% 33% 7.1 0.01
Hospitalized 90% 100% 0.9 1.00
Died from or with C. gattii 32% 16% 2 0.65

NOTES: p: Fisher’s exact p-value; RR: Relative risk

Historical Isolates of C. gattii in the United States

In spite of extensive press coverage in the United States during 2010 about the “new deadly fungus” in the United States and British Columbia (Discover Magazine, 2010; Hutchison, 2010; Park, 2010), C. gattii infections are not entirely novel in the United States: in particular, the infection repeatedly has been found in California in the past. In a 1984 study, C. gattii was found in 46 of 315 clinical isolates of Cryptococcus from the United States; 30 (65 percent) of the 46 C. gattii isolates were from patients residing in Southern California. Among all 71 Cryptococcus isolates from Southern California, C. gattii represented 42 percent, compared with 6 percent in the rest of the continental United States (Kwon-Chung and Bennett, 1984b). The same group reported in a different paper that both of the two clinical Cryptococcus isolates from Hawaii were C. gattii (Kwon-Chung and Bennett, 1984a). Another study, which examined cryptococcal isolates from HIV-infected patients from Los Angeles in the early 1990s, found C. gattii in 12 percent of these isolates (Chaturvedi et al., 2005). Genotyping of the isolates showed that 28 of 30 isolates were of subtype VGIII; 1 was VGI, and 1 was of subtype VGII (Byrnes et al., 2010b).31C. gattii was also found in three of 358 genotyped isolates collected from around the United States during surveillance from 1992 to 1994; two isolates were from San Francisco (VGI and VGIII) and one was from Atlanta (VGI) (Brandt et al., 1996). Finally, two isolates of genotype VGIIa, indistinguishable by multilocus sequence typing (MLST) from the major strain in Vancouver Island, were collected from the sputum of a Seattle man in the 1970s (Diaz et al., 2000) and from a eucalyptus tree in San

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31 Also see contributed manuscript by Heitman in Appendix A (pages 226–248).

Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
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Francisco in 1992 (Fraser et al., 2005). Taken together, these data suggest that Southern California, but probably not the rest of the country, has long been an endemic area for C. gattii. In addition, sporadic infections appear to be occurring from other states, although the travel history of these patients and their potential exposure site is unknown. Recently, environmental isolates of C. gattii have also been found in Puerto Rico from a variety of cacti and tree material (Loperena-Alvarez et al., 2010).

C. gattii in the PNW vs. C. gattii in Other Areas of the World

Globally, the most common infecting strains of C. gattii and the immune status of patients infected appear to vary. In endemic Australia and Papua New Guinea, C. gattii (usually type VGI) appears to occur primarily in apparently immunocompetent patients and cause CNS disease (Seaton et al., 1996a,b; Speed and Dunt, 1995). However, in several African countries, VGIV-type infections are most frequent, and are reported exclusively from HIV-infected patients (Litvintseva et al., 2005; Steele et al., 2010). By contrast, in Venezuela (Villanueva et al., 1989), Brazil (Santos et al., 2008), Paraguay, Argentina (Castanon-Olivares et al., 2000; Kwon-Chung and Bennett, 1984a,b), Peru (Bustamante Rufino and Swinne, 1998; Kwon-Chung and Bennett, 1984a,b), and Colombia (Escandon et al., 2006), infections (most commonly genotype VGII, although different from the PNW outbreak strains) occur in both HIV-uninfected and HIV-infected patients. In Mexico, all four C. gattii genotypes have been reported (Olivares et al., 2009), primarily from HIV-uninfected persons (Castanon-Olivares et al., 2000; Lopez-Martinez et al., 1996). In Asia, C. gattii (primarily VGI and VGII) has been reported from Vietnam, Cambodia, Thailand, Korea, Japan, Malaysia, India, China, and Nepal (Chen et al., 2008; Choi et al., 2010; Kwon-Chung and Bennett, 1984a,b; Lui et al., 2006; Okamoto et al., 2010), in both immunocompetent and immunocompromised persons (Chau et al., 2010; Ngamskulrungroj et al., 2010).

The outbreak of C. gattii in B.C. and the PNW is qualitatively different in genotype, presentation, disease course, and outcome than has been seen with C. gattii in other locations. In contrast to findings from areas outside of North America, C. gattii patients in both B.C. and the PNW most commonly present with respiratory rather than CNS symptoms (Galanis and MacDougall, 2010; Harris et al., 2010). In addition, a high proportion of infections in both the PNW and B.C. occur in immunocompromised (but usually HIV-uninfected) patients. In B.C., 38 percent of patients are immunocompromised (Galanis and MacDougall, 2010); using the same case definition for immunocompromised state, in the United States, approximately 59 percent of patients are immunocompromised (CDC, unpublished data).

Interestingly, differences also might exist between C. gattii patients in the United States and in B.C. U.S. patients were younger, more likely to be hospital-

Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
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ized, and more likely to die from or with their infection than were B.C. patients (Harris et al., 2010). The reasons for these differences are unclear, but might include differences in case ascertainment in the PNW compared with B.C., which has led to capture of a lower proportion of non-hospitalized C. gattii patients in the PNW. Alternately, they could relate to the different genotypes seen in the PNW compared with B.C. A comprehensive review of patient medical charts is under way. It might help elucidate the differences and provide some insight into whether these differences are real, and if so, why they exist.

The differences in U.S. C. gattii infections in the PNW, U.S. infections outside the PNW, and infections occurring in other areas of the world might be a function of C. gattii subtype, tropism, environmental distribution, or surveillance bias. That is, C. gattii infections might not be reported completely from other areas because they have not been looked for systematically elsewhere. Regardless of the reasons for the outbreak in North America, reports of the outbreaks do not appear to be exclusively due to temporal changes in surveillance. Retrospective speciation studies of isolates from B.C. before 1999 (Fyfe et al., 2008) and from the Seattle area before 2004 (Upton et al., 2007) suggested that the increase in reported cases represents a true increase in infections in the region. It remains to be seen whether the U.S. C. gattii infections outside of the PNW are sporadic, travel associated, or linked to part of a larger emerging health issue in other areas of the United States. Below is a discussion about efforts to conduct surveillance for C. gattii infections outside of the PNW.

Surveillance for Human C. gattii Infections Outside the PNW

To address the question of how frequently C. gattii infections are occurring outside of the PNW, in October 2010 the CDC issued an alert through ClinMicroNet (Dwyer, 2003), a listserv sent to directors of U.S. clinical microbiology laboratories. The alert described the ongoing outbreak in the Pacific Northwest, and requested that any unspeciated Cryptococcus isolates (or those already speciated as C. gattii) be sent to the Mycotic Diseases Branch laboratory at the CDC, with isolation date, source city, and source state. Isolates were speciated at CDC and C. gattii isolates were genotyped. As of January 2011, 32 isolates, isolated between 2006 and 2010, from patients in seven states had been submitted to the CDC. Of these, 10 were C. gattii; half were from California (Table A9-3). Collection of isolates is ongoing and cases continue to be reported to the CDC, albeit rarely, from states outside of Washington and Oregon. The recent commercial availability of specialized culture medium (CGB medium) that enables rapid discrimination between C. neoformans and C. gattii will hopefully facilitate this process (Butler-Wu and Limaye, 2011).

Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
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Table A9-3 Sources and Species of Isolates of Cryptococcus Submitted Following a Request Through ClinMicroNet, United States, October 2010–February 2011

State C. neoformansisolates C. neoformansisolates Total isolates
AL 3 0 3
CA 1 5 6
GA 1 2 3
HI 0 1 1
IL 14 0 14
OR 0 1 1
TX 3 0 3
UT 0 1 1
Total 22 10 32

Clinical Aspects of C. gattii Infection and Differences from C. neoformans

Several attempts have been made to carry out head-to-head comparisons of clinical disease caused by C. gattii and C. neoformans. In 1995, Speed and Dunt (1995) published a paper comparing clinical symptoms among hospitalized C. gattii and C. neoformans patients. In addition to noting that fewer than 10 percent of C. neoformans infections in Australia occurred in otherwise healthy patients while 100 percent of C. gattii infections occurred in healthy patients, the authors also reported that C. gattii more frequently involved cerebral and meningeal sites, had neurologic sequelae, required CNS or thoracic surgery to resect cryptococcomas, and required longer periods of treatment (Speed and Dunt, 1995). In addition, C. gattii infections more frequently resulted in relapse than C. neoformans infections. The authors commented that the nearly three-fold longer therapy times required for patients with C. gattii compared with patients with C. neoformans infections stemmed from the difficulty in reducing the size of the cryptococcomas and the inability to rapidly control infection in patients with C. gattii. However, they also noted that both bloodstream infections and mortality were exclusively limited to patients with C. neoformans (Speed and Dunt, 1995).

The same year, Mitchell et al. (1995) published a report directly comparing the two cryptococcal species in immunocompetent patients with cerebral disease (Mitchell et al., 1995). Similar to the findings by Speed and Dunt (1995) and later by Chen et al. (2000), the authors reported that both lung and brain cryptococcomas were more common among patients with C. gattii than C. neoformans infections. In addition, they reported that immunocompetent patients with C. gattii infection were significantly more likely to have a “poor outcome”—defined as moderate to major sequelae or death—than immunocompetent patients with

Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
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C. neoformans infections. When they compared outcomes among patients with meningitis but normal brain imaging at initial presentation, they found no differences by infecting species; however, they did find that C. gattii patients presenting with mass lesions on initial intracranial scan were more likely to have poor outcomes than those who did not (Mitchell et al., 1995). Taken together, these data suggested that the epidemiology of C. neoformans and C. gattii in Australia was different even when controlling for patient immune status.

However, a similar paper, published in 2010, compared infection with C. gattii and C. neoformans in immunocompetent patients in Vietnam (Chau et al., 2010) and failed to demonstrate differences in clinical phenotype by infecting species. The authors of this report, in contrast to Mitchell et al. (1995), suggested that host immune status was more influential on clinical course and outcome than was infecting species. This was also a conclusion drawn by Chen et al. (2000), who compared cryptococcal infections among both immunocompetent and immunosuppressed patients in Australia and New Zealand and demonstrated that immunocompetent hosts were more likely to present with lung infections, species type notwithstanding, than were immunocompromised hosts. Chen et al. (2000) also noted that C. gattii infections were more likely to occur in the brain than C. neoformans infections, but that cryptococcomas were associated both with infection with C. gattii and with immunocompetent status.

Lui et al. (2006) also compared cryptococcal infection in immunocompetent and immunosuppressed patients in China, noting elevated proportions of immunocompetent patients presenting with meningitis compared with immunocompromised patients, more intense inflammatory responses, and a lower risk of death. Although the paper also indicated that immune status might influence clinical course more than infecting species, the small numbers of C. gattii infections made evaluation of the effect of infecting species difficult.

Only two studies have directly compared C. gattii infections to C. neoformans infections exclusively in AIDS patients. In Botswana, Steele et al. (2010) compared C. gattii and C. neoformans infections in AIDS patients with cryptococcal meningitis, and found few differences in terms of clinical presentation or in-hospital mortality (Steele et al., 2010). Morgan et al. (2006) found similar results among South African patients with cryptococcal meningitis. It is possible that, among severely immunosuppressed patients who have a low capacity to respond immunologically to infection, such as late-stage AIDS patients, the infecting cryptococcal species is irrelevant to outcomes, while infecting species have a stronger influence when patients are mildly to moderately immunosuppressed or not immunosuppressed. These studies did not carry out extensive brain or thoracic imaging to evaluate the presence of cryptococcomas.

The poor prognosis of immunocompetent patients infected with C. neoformans has been studied previously, and has been suggested to be due to the delay in diagnosis, inappropriate treatment, and potentially the presence of an intact immune system that might provoke an immune reconstitution inflammatory

Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
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syndrome (IRIS) (Ecevit et al., 2006; Lui et al., 2006). IRIS is a paradoxical clinical deterioration that is well documented during treatment of cryptococcosis following initiation of antiretroviral therapy in AIDS patients (Woods et al., 1998) and is thought to be due to an overzealous “rebound” immune response in the presence of significant amounts of infecting pathogen. An IRIS-like syndrome has also been documented in patients infected with C. gattii, where the syndrome was suggested to be due to concomitant immune rebound and decreases in IL-10 (Einsiedel et al., 2004). These same factors may contribute to the severity of C. gattii infection in immunocompetent hosts. At least one report exists of a patient whose condition improved with steroid treatment, suggesting that an overly functional immune system could confound treatment efforts in some patients (Lane et al., 2004).

Discussion

The described outbreaks of C. gattii infection in the temperate climates of B.C. and the PNW demonstrate a much less restrictive geographic range for C. gattii than previously thought, and a broader range of persons who are susceptible to infection. In particular, a compromised immune status now appears to be a significant risk factor for at least some subtypes of C. gattii infection (CDC, 2010; Galanis and MacDougall, 2010). In addition, data from patients associated with these outbreaks suggest that different C. gattii genotypes might infect different types of patients, and/or demonstrate different clinical courses resulting from infection. The mere existence of an outbreak associated with C. gattii, never previously reported, suggests that genetic components might be important for pathogen spread in ways that are still poorly understood. More than ever, collecting data is important that disease recognition and optimal treatment of C. gatiii infections can be investigated. Several existing challenges now face the field.

Existing Challenges

One existing challenge is diagnosis of infection. Although several methods exist to identify cryptococcal infections, including culture, India Ink stains of cerebrospinal fluid or sputum (Cohen, 1984), and commercially available cryptococcal antigen (CrAg) test kits (Saha et al., 2008), these methods cannot distinguish between C. neoformans and C. gattii. A simple way to confirm whether or not a cryptococcal isolate is species gattii is to plate the isolate on canavanineglycine bromothymol blue (CGB) agar (Klein et al., 2009; Kwon-Chung et al., 1982), where C. neoformans will leave the medium unaffected in color (yellow to green) due to a failure to grow, and C. gattii will turn the medium blue due to use of glycine as a carbon source. This medium is currently available from at least one commercial supplier, but is not widely used in U.S. clinical microbiology labs. Thus, many C. gattii infections likely are being misdiagnosed as

Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
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C. neoformans. Ensuring that clinicians are aware of C. gattii infection and the possible need for clinical differentiation from C. neoformans, and that their reference laboratories are able to speciate Cryptococcus isolates (and have an interest in doing so), is critical to evaluate fully the geographic spread of disease and the clinical spectrum of infections.

Investigating whether or not the most recent findings warrant modified treatment guidelines is an additional challenge. The Infectious Diseases Society of America published guidelines in 2010 (Perfect et al., 2010) that refer to differences in the treatment of C. gattii infections, compared with C. neoformans infections: specifically, C. gattii infections might require lengthier, more aggressive treatment when compared with C. neoformans infections. The increased propensity for C. gattii to form cryptococcomas is also noted (Perfect et al., 2010). However, the guidelines were largely based on data from C. gattii infections occurring in Australia and Papua New Guinea. Increasingly, our data suggest that even among C. gattii infections, not all cryptococcal infections are alike. However, it is unclear which factors—infecting species, infecting subtype, host immune status, or perhaps even host genetics—are most influential on patient presentation and infection. Data from rigorous clinical studies are of utmost importance in ensuring that clinician guidelines provide sufficient guidance to optimize patient care. To this end, a large-scale, longitudinal chart review of C. gattii infections is ongoing as a collaborative effort among Australia, B.C., and the United States, designed to address some of these questions. Results are expected sometime in 2012.

Finally, the development of prevention messages is a challenge. Unlike C. neoformans, which grows in pigeon feces, C. gattii appears to live in association with trees and soil surrounding them (Springer and Chaturvedi, 2010). The tree type appears to be less important than the presence of a wood substrate for growth, and C. gattii has to date been associated with more than 50 tree species (Randhawa et al., 2001; Springer and Chaturvedi, 2010). It has also been found in air and water samples. These findings notwithstanding, C. gattii has not been found ubiquitously around the globe in a distribution similar to C. neoformans, and thus we can postulate that at least some environmental restrictions remain in place for this organism. Environmental organisms present a specific challenge for public health prevention because infections are usually relatively rare, and difficult to avoid without draconian measures (e.g., staying indoors and purifying air). This represents a quandary for public health officials. It remains to be seen whether “hot spots” of infection exist in the PNW for which generalized recommendations can be made that would benefit patient health, perhaps for subgroups of higher risk patients. The benefits of outdoor activity would need to be weighed against any risk calculated for these patients, and such recommendations are bound to be controversial.

Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
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Where Did C. gattii Come From, and Where Is It Going?

How did C. gattii arrive in the temperate areas of North America? Kidd et al. (2007) demonstrated, during an environmental sampling study for C. gattii in B.C., that anthropogenic activities could spread the pathogen, showing the presence of C. gattii on the shoes of human samplers and wheel wells of sampling vehicles as they traveled from one sampling site to the next. In addition, they showed that the pathogen was present in highly trafficked areas of Vancouver Island; that it could be found in the air, in freshwater, and in seawater around the area; and that the spores could survive for more than a year in many of these media. Thus, it is not difficult to hypothesize several methods by which the pathogen could have found itself in temperate B.C., and easier still to imagine mechanisms by which it could be transported into the United States. However, the question of whether new, cold-tolerant strains of C. gattii were brought to B.C. or whether they were formed there, through recombination of two or more preexisting or “seeded” strains, is still unresolved. It is also possible that the pathogen was always tolerant of temperate-weather climates, but existed in caches too small in these regions to sustain human infection or to establish permanent habitats; repeated seeding of these regions through global materials trafficking (e.g., wood or trees) could have created a sustainable niche for the pathogen. Alternately, changes in the global climate could be facilitating the optimal habitat development and spread of C. gattii, providing minimum conditions under which the pathogen can successfully propagate. Whole-genome sequencing is currently being carried out with C. gattii isolates obtained from patients associated with this outbreak, as well as more historical isolates (Lockhart et al., 2010), which could shed some light on the origin of the current outbreak and provide ideas for where it might move next.

Conclusion

The increase in the number of reports during the past decade related to the occurrence of C. gattii infections outside of traditional endemic tropical and subtropical regions has provided excellent opportunities to learn more about this important pathogen. The differences between individual cryptococcal infections appear to be linked not only to patient immune status and infecting species, but also to genetic subtypes within a species. It is unclear if the species and subtypes have preferences for infection among certain patient types, possibly due to a need for host immune support (or lack thereof) for replication, or if differences in environmental colonization patterns might influence the type of patient infected. For example, a pathogen with a ubiquitous distribution and a preference for immunocompromised patients will have a much higher infection rate and a much higher immunocompromised to immunocompetent patient ratio than would a pathogen with “hot spot distribution,” which would infect fewer patients overall, but be limited to patients living in its area of environmental distribution (most of

Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
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whom are immunocompetent). Thus, the immunocompromised to immunocompetent ratio might be nothing more than a function of the degree of distribution of a Cryptococcus species in the environment and the types of patients living in its area of distribution. Determining what governs the range of distribution of C. gattii—and understanding when it reaches a stable environmental equilibrium in new areas of emergence—is critical for understanding this relationship.

In spite of the recent flood of reports about C. gattii, much remains unknown. The epidemiologic curve has not yet stabilized in the United States, and the trajectory of future infections is unknown. The lack of comprehensive surveillance, both within North America and without, and the genetic variety inherent in C. gattii, has limited our current understanding of pathogen spread and pathogenesis. The conditions that favor pathogen colonization and propagation are not known. Evidence shows that infections in the United States and particularly the PNW are qualitatively different from those occurring elsewhere, but it is unclear whether or not these differences warrant modifications to existing treatment guidelines. Continued collection of robust surveillance data will assist in answering some of these questions. The coming years should see increasing amounts of information on C. gattii infections globally, which should shed light on genotype- and subtype-specific differences among C. gattii infections.

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Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
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Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
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Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
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Laurenson, I. F., D. G. Lalloo, S. Naraqi, R. A. Seaton, A. J. Trevett, A. Matuka, and I. H. Kevau. 1997. Cryptococcus neoformans in Papua New Guinea: A common pathogen but an elusive source. Journal of Medical and Veterinary Mycology 35(6):437–440.

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Lopez-Martinez, R., J. L. Soto-Hernandez, L. Ostrosky-Zeichner, L. R. Castanon-Olivares, V. Angeles-Morales, and J. Sotelo. 1996. Cryptococcus neoformans var. gattii among patients with cryptococcal meningitis in Mexico. First observations. Mycopathologia 134(2):61–64.

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MacDougall, L., S. E. Kidd, E. Galanis, S. Mak, M. J. Leslie, P. R. Cieslak, J. W. Kronstad, M. G. Morshed, and K. H. Bartlett. 2007. Spread of Cryptococcus gattii in British Columbia, Canada, and detection in the Pacific Northwest, USA. Emerging Infectious Diseases 13(1):42–50.

Mitchell, D. H., T. C. Sorrell, A. M. Allworth, C. H. Heath, A. R. McGregor, K. Papanaoum, M. J. Richards, and T. Gottlieb. 1995. Cryptococcal disease of the CNS in immunocompetent hosts: Influence of cryptococcal variety on clinical manifestations and outcome. Clinical Infectious Diseases 20(3):611–616.

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Morgan, J., K. M. McCarthy, S. Gould, K. Fan, B. Arthington-Skaggs, N. Iqbal, K. Stamey, R. A. Hajjeh, and M. E. Brandt. 2006. Cryptococcus gattii infection: Characteristics and epidemiology of cases identified in a South African province with high HIV seroprevalence, 2002–2004. Clinical Infectious Diseases 43(8):1077–1080.

Ngamskulrungroj, P., C. Serena, F. Gilgado, R. Malik, and W. Meyer. 2010. Global VGIIa isolates are of comparable virulence to the major fatal Cryptococcus gattii Vancouver Island outbreak genotype. Clinical Microbiology and Infection 17(2):251–258.

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Olivares, L. R., K. M. Martinez, R. M. Cruz, M. A. Rivera, W. Meyer, R. A. Espinosa, R. L. Martinez, and G. M. Santos. 2009. Genotyping of Mexican Cryptococcus neoformans and C. gattii isolates by PCR-fingerprinting. Medical Mycology 20:1–9.

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Park, B. J., K. A. Wannemuehler, B. J. Marston, N. Govender, P. G. Pappas, and T. M. Chiller. 2009. Estimation of the current global burden of cryptococcal meningitis among persons living with HIV/AIDS. AIDS 23(4):525–530.

Perfect, J. R., and A. Casadevall. 2011. The history of cryptococcus and cryptococcosis. In Cryptococcus: From human pathogen to model yeast, pp 17–26, edited by J. Heitman, T. R. Kozel, K. J. Kwon-Chung, J. R. Perfect, and A. Casadevall. Washington, DC: ASM Press.

Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
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Perfect, J. R., W. E. Dismukes, F. Dromer, D. L. Goldman, J. R. Graybill, R. J. Hamill, T. S. Harrison, R. A. Larsen, O. Lortholary, M. H. Nguyen, P. G. Pappas, W. G. Powderly, N. Singh, J. D. Sobel, and T. C. Sorrell. 2010. Clinical practice guidelines for the management of cryptococcal disease: 2010 update by the Infectious Diseases Society of America. Clinical Infectious Diseases 50(3):291–322.

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Upton, A., J. A. Fraser, S. E. Kidd, C. Bretz, K. H. Bartlett, J. Heitman, and K. A. Marr. 2007. First contemporary case of human infection with Cryptococcus gattii in Puget Sound: Evidence for spread of the Vancouver Island outbreak. Journal of Clinical Microbiology 45(9):3086–3088.

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Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
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A10

SEXUAL REPRODUCTION, EVOLUTION, AND ADAPTATION OF CRYPTOCOCCUS GATTII IN THE PACIFIC NORTHWEST OUTBREAK

Joseph Heitman,32,33Edmond J. Byrnes III32,34and John R. Perfect32,33

Abstract

How microbial pathogens emerge to cause outbreaks and become established as agents of disease in humans involves genetic exchange, zoonotic transmission, and perturbations of ecosystems and habitats. The threat of emerging infectious diseases is particularly poignant for eukaryotic pathogens, the fungi, and parasites, given that these microbes are more difficult to treat and have complex genomes and lifecycles. A sobering recent development has been the emergence and reemergence of several fungal pathogens in both humans and other animals, including Geomyces destructans in bats, Batrachochytrium dendrobatidis in amphibians, Nosema ceranae in bees (colony collapse disorder), and Cryptococcus gattii in humans and other animals in the Pacific Northwest. Here we review issues surrounding the C. gattii outbreak that began on Vancouver Island in 1999 and has expanded into the United States in Washington, Oregon, and California and has the potential to expand further. The focus will be on the emergence of C. gattii in the United States, including the appearance of a novel, highly virulent genotype and the potential role of sexual reproduction in the emergence of novel pathogens and their dispersal via airborne spores.

Introduction

The early history of cryptococcosis was documented in single or small series of cases. From an initial case of tibial osteomyelitis with the encapsulated Cryptococcus neoformans yeast in 1895 until a seminal monograph on this disease by Littman and Zimmer in 1956, the entire repertoire of reports in the medical literature numbered less than 300 cases (Littman and Zimmer, 1956). This was a humble beginning for this cosmopolitan, encapsulated basidiomycete that has now emerged into an outbreak mode in the new millennium. In the first half-century of its known existence, many of the clinical features of cryptococcosis were well described, including its propensity to invade the central nervous

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32 Department of Molecular Genetics and Microbiology, Duke University Medical Center.

33 Division of Infectious Diseases, Department of Medicine, Duke University Medical Center.

34 Current address: Division of Infectious Diseases, Johns Hopkins University School of Medicine, Baltimore, MD, USA 21287-0005.

Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
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system. The occurrence of outbreaks of mastitis (persistent inflammation of the udders) in dairy herds in which hundreds of animals were infected brought the realization that cryptococcal infection outbreaks can occur in mammals (Pounden et al., 1952; Simon et al., 1953). These outbreaks in animals and the link to the environment again were emphasized with reports of goats developing Cryptococcus gattii infection in Spain temporally linked to the importation of Eucalyptus trees (Baro et al., 1998). These two outbreaks in animals vividly demonstrated that cryptococcal infections could change from sporadic to outbreak as the endemic status changes. However, the first prescient report that recognized the future emergence of human cryptococcosis was from Kaufman and Blumer at the Centers for Disease Control and Prevention (CDC) when they called cryptococcosis “an awakening giant” mycoses (Kaufman and Blumer, 1978). As clinical mycologists in a major reference laboratory, they observed increasing numbers of cases as the immunosuppressed population dramatically increased due to the use of immunosuppressive therapies in modern medicine. In 1983 to 1984, reports of opportunistic cryptococcal infections coinciding with the early natural history of HIV infection provided insights into the emerging association of HIV infection and cryptococcosis (Lerner and Tapper, 1984; Vieira et al., 1983). In a landmark epidemiologic work in 2009, Park and colleagues from the CDC estimated that, in association with the AIDS pandemic, there were approximately 1 million cases of cryptococcosis per year worldwide, with at least 600,000 deaths per year in the past 5 years due to cryptococcosis (Park et al., 2009). While this massive outbreak continues, on another front, an outbreak of C. gattii infections on Vancouver Island has been observed over the past decade and has now migrated down into the Northwest United States, infecting humans and other mammals (Byrnes and Heitman, 2009; Byrnes et al., 2009, 2010; DeBess et al., 2010; Fraser et al., 2005; Kidd et al., 2004; MacDougall et al., 2007; Upton et al., 2007).35 Cryptococcosis started as a medical curiosity in a few patients, but because of medical interventions with immunosuppression, an immunosuppressive viral pandemic (HIV/AIDS), and a change in local climates, this yeast has become more prevalent in clinical medicine. Its present impact supports its title as a major emerging fungal disease or, to be more direct, “the giant is fully awake.”

Vancouver Island C. gattii Outbreak and Expansion into the United States

Our focus in this chapter will be the C. gattii outbreak that began on Vancouver Island in 1999 and has now expanded into the Canadian mainland in British Columbia and into the United States. A considerable body of knowledge is available with respect to the life and virulence cycles for this pathogenic yeast (Heitman, 2011). C. neoformans and C. gattii are closely aligned species. C. neoformans is prevalent in the environment globally, and C. gattii has been

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35 See also the contributed manuscript by Harris in Appendix A (pages 207–225).

Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
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thought to be geographically restricted to tropical and subtropical regions until its recent emergence in the relatively temperate climate on Vancouver Island. We are exposed to both organisms from the environment; C. neoformans is typically associated with pigeon guano and less commonly with trees, whereas C. gattii is commonly isolated from trees and soil, and also present in the air on Vancouver Island. We are exposed by inhaling spores and desiccated yeast cells, both of which can cause an initial pulmonary infection. This can be cleared, recede into a dormant latent form, cause fulminant pneumonia, or even make its way to the central nervous system via the bloodstream to infect both the covering of the brain (meninges) and the brain itself (meningoencephalitis). For reviews of the virulence and lifecycles, see Idnurm et al. (2005) and Kronstad et al. (2011).

Of particular importance are recent studies from the CDC’s Park and colleagues (2009) that reveal that Cryptococcus has reached pandemic proportions. More than a million cases occur globally each year. This is largely in the context of the AIDS pandemic and results in more than 600,000 attributable mortalities, approximately a third of all AIDS-associated deaths. This is thought to be largely attributable to C. neoformans, but there is also likely to be a more substantial burden of C. gattii infection occurring globally than is currently appreciated given that few clinical microbiology laboratories routinely assign Cryptococcus species status. It is in this global context that we consider the expanding and ongoing outbreak of C. gattii in the Pacific Northwest (Figure A10-1).

Cryptococcus is a species complex, and it is important to know which pathogen you are dealing with in the context of infection. For example, this is particularly important with E. coli, in which various strains are causing infections such as EPEC, EHEC, UPEC, and VTEC (enteropathogenic E. coli, enterohaemorrhagic E. coli, uropathogenic E. coli, and verotoxin-producing E. coli). This is also true with fungal pathogens. Two species are currently recognized: C. neoformans and C. gattii (Figure A10-2). The two can be readily distinguished in clinical microbiology labs on L-canavanine glycine bromothymol blue (CGB) agar, exploiting the ability of C. gattii to cause a pH change on this media, resulting in a blue chromogenic reaction (Figures A10-2A and B). We now appreciate that the group of isolates currently recognized as C. gattii spans four cryptic species groups. Currently, these are recognized as the VGI, VGII, VGIII, and VGIV molecular types (Figure A10-2C) based on molecular phylogenetic analyses that show each as a distinct, well-defined group (Bovers et al., 2008; Fraser et al., 2005; Ngamskulrungroj et al., 2009a). We also know from the whole-genome analysis for the representative VGI (WM276) and VGII (R265) isolates, which has just been completed at the Broad Institute and the University of British Columbia in Vancouver by Jim Kronstad and colleagues, that at a whole-genome level the different molecular types are not exchanging genetic information (D’Souza et al., 2011). Similarly, the molecular types are four genetically isolated cryptic species based on robust analysis of “molecular barcodes” throughout their genomes us-

Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
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img-229

Figure A10-1 The C. gattii outbreak expanded into, and emerged within, the United States (n = 56). BC = British Columbia.

ing multilocus sequence typing (MLST) (Bovers et al., 2008; Fraser et al., 2005). This distinction of cryptic species is important because the VGII cryptic species is causing the outbreak in the Pacific Northwest, whereas the VGI lineage is more prevalent and more commonly causing disease in Australia (see Heitman, 2011).

The outbreak of C. gattii in the Pacific Northwest began in 1999 and prior to this, no C. gattii infections were reported as causing disease in this region of the world. The first cases came to the attention of astute clinicians and veterinarians on the southeastern shores in Naniamo and Parksville on Vancouver Island (Duncan et al., 2005; Hoang et al., 2004; Stephen et al., 2002). Over the past decade, there have been approximately 260 cases with an approximately 10 percent attributable mortality rate, and many infections in animals. Interested read-

Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
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img-230

Figure A10-2 Cryptococcus pathogenic species complex. Panel A shows growth on niger seed media that detects production of the pigment melanin (+) or its absence (–), production of urease (+) as a pink color on Christensen’s agar, and growth and a blue color change (+) on CGB agar. Panel B shows a summary of the phenotypic data shown in panel A. Panel C is a phylogenetic tree of the relationships between the molecular types in the species complex. C. neoformans: opportunistic, AIDS, global, pigeon guano. C. gattii: primary, non-AIDS, tropical, arboreal. CGB = L-canavanine-glycine-bromothymol blue.
SOURCE: Panel A modified from Byrnes et al., PLoS Pathogens, 2010 (figure S2). Panel B courtesy of Heitman. Panel C modified from Chapter 22, Figure 2 of Carter et al. (2011).

Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
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are referred to a reprint36 included in this volume from Karen Bartlett, who played a critical role in identifying the environmental source of the organism on Vancouver Island (Bartlett et al., 2008), and to the manuscript from Julie Harris from the CDC Cryptococcus working group.37 Our charge in this article was to consider two aspects of the outbreak: first, its expansion into the United States, and second, the possible roles of sexual reproduction in the origin of the outbreak isolates and the ongoing production of airborne infectious spores.

The first cases associated with the Vancouver Island outbreak in patients from mainland British Columbia with no travel history to the island appeared in 2003–2004 (Bartlett et al., 2008). Environmental sampling studies provided evidence that C. gattii expanded across the water to a broader niche, including the Canadian mainland (MacDougall et al., 2007). The southern tip of Vancouver Island is very close to the U.S. border, and therefore a key question was if and when the outbreak might spread into the United States. The San Juan Archipelago is a part of Washington state, located as near as 5 km from the gulf islands off the coast of Vancouver Island. The first C. gattii index case in the United States was an elderly patient with leukemia on Orcas Island, Washington, who presented with a pulmonary C. gattii nodule in January 2006 (Upton et al., 2007). Since then, the outbreak has expanded into the United States from Canada, and we summarize here the evidence and key findings.

From 2006 to 2010, approximately 70 cases in patients have been reported, and the most complete records are attributable to the efforts of the CDC Cryptococcus working group. The cases in humans and animals are shown in Figure A10-1, and the icons represent cases for which we have isolates in the laboratory that have been molecularly analyzed. Two types of isolates are circulating on Vancouver Island: the VGIIa/major genotype, which causes approximately 95 percent of the infections, and the VGIIb/minor genotype, which represents the remaining 5 percent of isolates (Fraser et al., 2005). Both of these genotypes have been found in the environment on Vancouver Island and cause infection in both patients and animals, and both have now emerged within the United States. Of particular interest is a completely novel genotype that has emerged in Oregon: VGIIc, or the novel genotype (Byrnes et al., 2009, 2010). VGIIc has not yet been found in Washington state, Vancouver Island, or anywhere else in the world; thus, it is a completely new emergence from 2005 to 2010 in Oregon.

We can identify genotypes by applying MLST of genetic barcodes throughout the genome. In this technique, coding and non-coding regions of genes are PCR amplified, sequenced, and assigned a unique allele number. The alleles are then color coded and organized in a tabular format in which each line represents a different isolate associated with the outbreak or a global isolate in the strain collection. This allows one to appreciate that there has been what appears to be a large clonal expansion of the VGIIa/major genotype in the region that dominates

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36 See contributed manuscript by Bartlett in Appendix A (pages 101–116).

37 See contributed manuscript by Harris in Appendix A (pages 207–225).

Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
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the outbreak on Vancouver Island and its expansion into Puget Sound and beyond in Washington state. There are fewer isolates of the VGIIb/minor genotype, but these have been found on Vancouver Island, in Oregon, and most recently in Washington state. The VGIIc/novel genotype has thus far been found only in Oregon and has several, unique alleles not found to date in any other isolates examined from global sources. The CDC has identified one isolate from a patient in Idaho that appears to be closely related to the VGIIc/novel genotype, but it may be the result of travel exposure (DeBess et al., 2010).

Where did these VGII outbreak genotypes originate? The VGIIa/major genotype is indistinguishable across 30 MLST loci and several variable number of tandem repeat loci from the NIH444 strain, which was isolated in the early 1970s from a sputum sample from a patient in Seattle (Fraser et al., 2005). This isolate is the type strain for C. gattii and is molecularly indistinguishable from the VGIIa/major outbreak strain at all loci examined thus far. Therefore, the major outbreak strain appears to have been in this geographic region for at least 40 years and possibly even longer. The VGIIb/minor genotype isolates are indistinguishable from isolates from a fully recombining sexual population in Australia (Campbell et al., 2005a,b; Fraser et al., 2005). The VGIIc/novel genotype has thus far only been identified in isolates collected in Oregon, and we have not observed it in a large collection of more than 200 C. gattii isolates collected globally. It appears as if the VGIIc/novel genotype either emerged locally in Oregon or, alternatively, it may be present in an undersampled environmental niche that remains to be discovered. Further evidence that this new genotype in Oregon is novel involves haplotype network mapping. In this approach, the MLST alleles are compared and we apply a computer algorithm to predict the ancestral allele. The alleles are then organized into a diagram rooted with the ancestral allele, and alleles derived from it by mutations and genetic drift are indicated with lines and circles. In this analysis alleles that are arisen from the ancestral allele long ago lie closest to the ancestral allele, whereas alleles that arose recently lie distal. The MLST alleles that are private to the VGIIc genotype are distal in these haplotype networks, suggesting that they arose recently. The alleles that the VGIIc alleles are derived from come from isolates that originated from either Australia or South America (Byrnes et al., 2010). This analysis then suggests that those alleles might represent possible sites of origin of at least some of the genetic material in the Oregon VGIIc/novel outbreak isolate.

We have conducted mammalian virulence studies in a mouse inhalation model, both at the Wadsworth Center in Albany with our collaborators Ping Ren, Sudha Chaturvedi, and Vishnu Chaturvedi and also at Duke University (Byrnes et al., 2010; Fraser et al., 2005). The VGIIa/major genotype and the VGIIc/novel genotype isolates are both highly virulent in the mouse model used, whereas the minor VGIIb genotype is considerably attenuated by direct comparison (Byrnes et al., 2010). Isolates collected globally that share many but not all markers with the VGIIa/major genotype are considerably less virulent than VGIIa (Byrnes

Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
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et al., 2010). This includes, for example, isolates from both California and South America. Of note, the NIH444 type strain (a VGIIa/major genotype isolate) is also somewhat less virulent than contemporary outbreak VGIIa/major genotype strains, which may reflect either attenuation as a result of long-term storage and passage; variation in virulence even among the VGIIa/major isolates; or unknown genetic differences between NIH444 and the VGIIa/major outbreak genotypes in regions of the genome not yet analyzed. Therefore, it appears as if virulence has increased with the emergence of the VGIIa/major outbreak genotype and the VGIIc/novel genotype from their original source strains.

Several investigators have been addressing why outbreak isolates are more virulent than other genotypes. There have been two prescient studies. Jim Kronstad and colleagues at the University of British Columbia in Vancouver have shown that there is less protective inflammation in the lungs of mice infected with the outbreak isolates (Cheng et al., 2009). This may then lead to increased virulence if the host fails to mount a sufficient, protective immune response. In addition, the work from Robin May’s lab at the University of Birmingham has described an increased intracellular proliferation rate of the VGIIa and VGIIc outbreak isolates in macrophages and linked mitochondrial function to the robustness of their interaction with host immune cells (Byrnes et al., 2010; Ma et al., 2009). Given that Cryptococcus is a pathogen able to grow either outside or inside of host immune cells, an enhanced proliferation rate in the context of the macrophage intracellular milieu may lead to enhanced virulence. These studies are excellent starting points to use to begin to dissect the virulence mechanisms at the interface with host immune cells in the lung.

To summarize, this outbreak was originally restricted geographically to Vancouver Island and then spread to the mainland of British Columbia. Starting in 2006 (and maybe earlier in one case in Oregon in 2005), it emerged within the United States. Thus, there clearly appears to have been a geographic expansion from Vancouver Island across Puget Sound to reach Washington, mostly involving the VGIIa/major genotype. However, there is more diversity in strains from Oregon, involving both the VGIIa/major and VGIIb/minor genotypes observed on Vancouver Island, but also including the novel VGIIc genotype that has not been identified on Vancouver Island. In essence, this looks like an outbreak within an outbreak, which may be of independent origins. It is as though two pebbles have been dropped into a pond at different times, one earlier than the other, and they have generated concentric waves that are now expanding outward and intersecting. There is considerable concern that this outbreak will continue to expand geographically given that there are cases and isolates now from Idaho and California (Personal communication, Shawn Lockhart, CDC Mycotic Diseases Branch, May 2011). There is also a well-documented risk to travelers to the endemic region, who then return to locations around the world with an infection of the VGIIa/major isolate and present with a very unusual fungal infection that is not commonly described in those regions and that might confuse clinicians

Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
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(Chambers et al., 2008; Georgi et al., 2009; Hagen et al., 2010; Lindberg et al., 2007).

The Role of Sexual Reproduction in Pathogen Emergence and Outbreaks

The second charge of this article was to consider the role of sexual reproduction in the emergence of pathogens and how it might contribute to adapting to a novel niche. We might start with just a very general question of, why sex? For those investigators who focus on bacteria or other prokaryotes, their mechanisms for genetic transfer typically involve horizontal gene transfer. But for the eukaryotic pathogens, including fungi, parasites, and the oomycete plant pathogens such as Phytophthora infestans (the cause of potato blight), their genetic exchange mechanisms involve sexual reproduction. The general theme that emerges for all of these groups of eukaryotic pathogens is that sexual reproduction plays a critical role in their diversity and, in many cases, also in their infection cycles (Heitman, 2006, 2010).

What benefits are conferred by sexual reproduction? This process enables the exchange of genetic information and generates diversity, and it can also purge the genome of deleterious mutations. It also allows these organisms to stay one step ahead of their transposable elements, which might otherwise accumulate to litter the genome.

We know a great deal about the sexual reproduction of Cryptococcus because of the pioneering work of June Kwon-Chung at the National Institutes of Health some 30 years ago (Kwon-Chung, 1975, 1976a,b). There are two mating types, called a and α (Hull and Heitman, 2002; McClelland et al., 2004). A panoply of signals regulates the interactions of cells of opposite mating type, allowing fusion and completion of their sexual cycle, thereby generating infectious spores. We know from both historic and recent experimental studies that these spores are indeed infectious propagules (Botts and Hull, 2010; Botts et al., 2009; Giles et al., 2009; Sukroongreung et al., 1998; Velagapudi et al., 2009; Zimmer et al., 1984).

Two of the many signals that regulate the C. gattii sexual cycle are interactions with plants and extreme desiccation (Xue et al., 2007). As noted by Karen Bartlett, there is more Cryptococcus found in the air in Vancouver Island in July when it is very hot and dry (Bartlett, 2010). Indeed, these are also the ideal conditions for stimulating the sexual cycle, at least under laboratory conditions. Thus, Cryptococcus may mate more in the hot and dry conditions of July than in other times of the year.

Sex in Cryptococcus produces spores, which are infectious. There is also a link of the mating type to virulence that has focused interest on its sexual cycle (Kwon-Chung et al., 1992; Nielsen et al., 2003, 2005a,b; Okagaki et al., 2010). One of the most curious features of this species is that most of the Cryptococcus population is just one mating type (α), even though the sexual cycle that was originally defined in the laboratory requires both opposite mating types (Hull

Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
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and Heitman, 2002). This fact raised a central conundrum for the entire field of cryptococcal pathogenesis: If there is a well-maintained a-α opposite-sex sexual cycle, but the a mating type is extremely rare, if not completely absent in many populations, how then can a sexual cycle be an important step of the infectious cycle for this fungus? It might be that it is not important, such that these unisexual populations were just clonally reproducing mitotically as yeasts. However, we discovered that C. neoformans can undergo an unusual sexual cycle involving only one of the two mating types (Lin et al., 2005).

The opposite-sex cycle that we have known about for 30 years is depicted in the lower panel of Figure A10-3. Then in 2005, an alternative sexual cycle was reported called unisexual reproduction or same-sex mating, and this is depicted in the upper panel of Figure A10-3 (Lin et al., 2005; Wang and Lin, 2011). This alternative sexual cycle only involves α cells. They can fuse with another α cell from the population, or they can, in extreme cases, fuse with themselves, undergo meiosis, and produce spores.

You might wonder, what would be the point of undergoing sexual reproduction with yourself? There is no genetic diversity to admix in this circumstance. We always think about sexual reproduction as involving two parents with very

img-235

Figure A10-3 Cryptococcus neoformans can reproduce unisexually and bisexually.

Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
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different genetic compositions. It turns out that mating with yourself can also lead to the generation of genetic diversity. Sex itself can serve as something of a mutagen to generate genotypic and phenotypic diversity. This strategy turns completely on its head what we traditionally think of as the primary role of sexual reproduction. An analogy might be the appearance of mismatch repair mutator mutations in bacterial pathogens, which arise to result in the generation of genome-wide mutations and are then swept from the population as a consequence of their concomitant deleterious effects.

As an example, in preliminary studies we isolated 100 progeny produced by this same-sex mating cycle and looked for phenotypic diversity. We have found within this set of just 100 isolates clear examples of azole resistance, temperature-sensitive growth, hyperfilamentous growth, and increased melanin production. By comparison, analysis of 100 progeny produced by mitotic clonal growth yielded no such examples. Many of the phenotypically distinct isolates turn out to be aneuploid in one way or another. This is based on comparative genome hybridization analysis showing that one of these morphologically distinct isolates now has an extra copy of chromosome 10. The presence of an extra copy of a chromosome is termed aneuploidy. We often associate aneuploidy with very deleterious consequences. We do not have to go further than Down syndrome as an example to think about what might be bad about aneuploidy. On the other hand, in the microbial kingdom, aneuploidy can be a rich source of phenotypic diversity. There are well-documented studies in Candida albicans that an a special type of chromosome formed by duplicated arms of a chromosome (termed an isochromosome) can form and drive fluconazole resistance (Selmecki et al., 2006, 2008, 2009). Similarly, we also know that azole heteroresistance in Cryptococcus is driven by aneuploidy for chromosome 1 (Hu et al., 2008; Sionov et al., 2009, 2010). A variety of other experimental evolution studies have brought to the forefront the idea that aneuploidy might be a driving force for genotypic and phenotypic plasticity (Pavelka et al., 2010; Rancati et al., 2008; Torres et al., 2007, 2010). So this may be one mechanism by which a same-sex mating cycle could engender phenotypic and genotypic diversity in a population without needing a partner that is genetically divergent.

Another way to view same-sex mating is that it is a mechanism for selfing. Selfing/inbreeding is common in fungi and the paradigm is mating type switching in S. cerevisiae, which allows mother–daughter cell mating. Same-sex mating is another route to self, but not via mating type switching. It is a question at some level of outcrossing vs. inbreeding. Same-sex mating superimposes much more limited genetic diversity on a well-adapted fungal genome that has run the gauntlet of natural selection. As such, unisexual reproduction may confer one of the benefits of sex (generation of diversity), but at the same time avoid one of its costs (breaking apart well-adapted genomic/genetic configurations). For organisms that are well adapted to a niche, this more limited genetic diversity may better answer subtle changes in selection pressures.

Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
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We have alluded to the fact that there are restricted geographic areas where fungal sex is extant. Why is that? Why would you have these little areas and pockets in Africa and that is where they have sex, and in the rest of the world they don’t have sex except for the unusual unisexual cycle? At least for Cryptococcus, what it looks like is, where they have opposite-sex mating seems to be where there are restricted populations where both mating types still exist. We know the most about this from C. neoformans, where there is an extant population that is on a very specific indigenous tree in South Africa, in Botswana (Litvintseva et al., 2011). But everywhere else in the world, you find just the alpha mating type (Hull and Heitman, 2002). So there is a niche in nature where opposite-sex mating is occurring. Until 5 years ago everyone presumed everywhere else it was asexual and mitotic. But what we are coming to appreciate is that there is just a different version of the sexual cycle occurring in these other populations where there is just one mating type.

This is interesting as another classic example is Phytophthora infestans. In Mexico and in Peru and other areas of South America, that is where the sexual cycle occurs, new versions arise, and then they are often exported on potato crops. That was the source of the Irish potato famine. But why there is a sexual cycle in such a restricted place is not really clear. Toxoplasma gondii might be another interesting pathogen to consider. It only has its sexual cycle in cats and other felids, not in other animals of any sort (Heitman, 2006). So what is it about the gastrointestinal tract of a cat that is so different from a mouse or a human that allows this parasite to undergo its sexual cycle there? The parasite sexual cycles are even more bizarre than some of the fungal ones, and restricted to extreme niches (Heitman, 2006, 2010).

What about the specific example of C. gattii sexual reproduction? June Kwon-Chung elucidated the C. gattii sexual cycle more than 30 years ago (Kwon-Chung, 1976a,b). Spores were produced in her classic studies via an a-α opposite-sex mating cycle that she defined. We were approached by several of the Australian groups working on the VGI genotypes in the late 1990s, including Wieland Meyer and Dee Carter, and we spent more than 5 years recapitulating this sexual cycle under laboratory conditions. This required a great deal of heroic effort on the part of several individuals in various laboratories, in large part because fecundity can be reduced with long-term passage and storage and because the VGI molecular type that is predominant in Australia is more recalcitrant in mating assays compared to the VGII and VGIII C. gattii molecular types. We were finally able to recapitulate a sexual cycle for C. gattii involving the formation of dikaryotic hyphae with special cells linking the hyphal cells (fused clamp cells) culminating in the production of basidia and basidiospores, all of the morphological features of the sexual cycle, including meiotic recombination (Campbell et al., 2005a,b; Fraser et al., 2003). Based on this advance, we were able to show that the vast majority of the outbreak isolates from Vancouver Island are fertile in laboratory crosses (Byrnes et al., 2010; Campbell et al., 2005a;

Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
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Fraser et al., 2003). These are a-α matings that were conducted, but we stress the point that every single isolate that is associated with the outbreak is of the α mating type. No one has found any a isolates occurring anywhere on Vancouver Island or in Washington or Oregon.

How might mating occur on Vancouver Island and in the Pacific Northwest in this unisexual population? In previous studies we observed that an association of Cryptococcus with plants can stimulate the fungal sexual cycle (Xue et al., 2007). Part of the idea in testing for this phenomenon in the first place was that plants, and more specifically trees, are the environmental niche for C. gattii (Heitman et al., 2011). For the plant pathogenic fungus Ustilago maydis, which infects corn, the infectious form is the filamentous dikaryotic hyphae produced by the sexual cycle, which is stimulated by interaction with the plant. In fact, U. maydis has to be in its sexual form to infect the plant host. We found something very similar with Cryptococcus. We were unable to infect a plant efficiently with the haploid yeast, but the filamentous dikaryotic state will infect plants and stimulate a plant defense response (Xue et al., 2007). In turn, if we put seedlings distant from a fungal mating mixture on a plate, the plants secrete small molecules, such as inositol, that stimulate completion of the sexual cycle and production of spores (Xue et al., 2007, 2010). We observed this with seedlings of Arabidopsis or Eucalyptus and are now exploring this with Douglas fir because of the link to this indigenous tree species in the Pacific Northwest outbreak. This line of investigation suggests that this may be one niche in nature where the sexual cycle is occurring to produce spores as small airborne infectious propagules.

Another critical line of investigation involves population genetic studies conducted by Dee Carter from the University of Sydney, Australia. She has focused on the sexual cycle that may be occurring in Eucalyptus tree hollows. She discovered that in populations with both mating types, they can engage in opposite-sex mating, but in other tree hollows where only α isolates are found, they are also sexually recombining (Saul et al., 2008). So it seems as if the organism has two extant sexual cycles, one opposite sex and one same sex, and whom you mate with depends on who your neighbors are in the population. All of her studies have focused on the VGI cryptic species, which has very limited genetic diversity and an extremely high level of apparent inbreeding (Campbell and Carter, 2006; Campbell et al., 2005a,b; Carter et al., 2007, 2011). All of the isolates on Vancouver Island associated with the clonal outbreak are of a different cryptic species, VGII. However, based on Dee Carter’s studies of VGI sexual reproduction, the presumption is that there may be both forms of sexual reproduction occurring also for the VGII isolates in nature, associated with the environmental niche in trees or plants for the VGII lineage and involving both opposite and same-sex mating depending on where a isolates are found in nature.

We have advanced the population genetic analysis in two ways with our global isolates to look for measures of sexual reproduction and recombination occurring in the population. One of these is a simple test called an allele com-

Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
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patibility test. The basic premise is that if you cross two strains that differ at two markers, you obtain four types of progeny produced by sexual processes by reassorting two unlinked loci (i.e., AB by ab yields AB, ab, Ab, and aB) (Carter et al., 2011), which we all know from studying Punnett squares. So the multilocus sequence loci can be analyzed as two unlinked alleles in the population. If you find all four possible combinations, this is indicative of sexual reproduction and recombination occurring in the population. By looking at the multilocus sequence markers, we find that these measures of recombination are rampant throughout the VGII global population (Byrnes et al., 2010). In addition, if we just examine the sequences of the multilocus sequence alleles themselves, which represent approximately 1 kilobase of sequence each, we can find examples of hybrid recombinant alleles that implicate isolates as potential parents or potential offspring (Byrnes et al., 2010). A lot of mitotic recombination must be occurring to see evidence of recombination within just a random 1-kilobase sequence in a 20-megabase genome. The potential parents that are identified by this type of analysis originate from South America, Africa, and Australia (Byrnes et al., 2010). These findings suggest that sexual reproduction is occurring globally in multiple environments and locations.

We went on to examine these global isolates for fertility in the lab and documented, by scanning with electron microscopy, the production of meiotic spores, which are the products of sex that may be infectious propagules found in the air on Vancouver Island. The majority of the α strains we analyzed are fertile. We have found a very limited number of a strains in the global population. For example, we found 6 out of 200 (~3 percent) are this minor mating type. Five out of six of these a strains are fertile in the laboratory, and thus they may be undergoing opposite-sex mating in nature. Because five of these six a isolates are from South America, it may be a site in which opposite-sex mating is still extant in the population (Escandon et al., 2007; Fraser et al., 2005; Ngamskulrungroj et al., 2008). We know that C. neoformans has also retained extant opposite-sex mating, but that it is geographically restricted to Botswana and South Africa and occurring in the VNB C. neoformans lineage (Litvintseva et al., 2003, 2007).

When we return to consider the VGIIb minor genotype and its relationship to global isolates, this provides critical insight. For instance, we looked at 30 MLST alleles, the VGIIb outbreak isolates are completely indistinguishable from isolates from a fertile, unisexual, sexually recombining population in Australia that was identified by Dee Carter and colleagues (Campbell et al., 2005a,b). It suggests that this population may be the ancestral source for the VGIIb/minor outbreak genotype isolates now circulating in the Pacific Northwest. While one might posit just the opposite (transfer from Vancouver Island to Australia), the diversity of the population in Australia supports this as the ancestral rather than the derived population. Thus, the most parsimonious model is that the isolates from the outbreak originated from Australia (Figure A10-4).

Based on a broad comparison of the isolates on Vancouver Island, the VGIIa/

Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
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img-240

FIGURE A10-4 Sexual reproduction and the origin of an outbreak.

major and the VGIIb/minor genotypes share half of their genetic markers and differ at the other half. So in a very simple model, they might be progeny from a genetic cross (i.e., siblings), or one might be a parent and the other an offspring. In this simple conceptual framework, one could imagine an isolate undergoing mating with an unknown isolate in nature to produce the outbreak isolates. However, the VGIIa/major and the VGIIb/minor genotypes on Vancouver Island are all of the same mating type, α. When we look in detail at the mating type locus, we can see that they are molecularly distinct from each other (Fraser et al., 2005) and are not identical by descent. So, in essence, they have two different ancestries for this region of their genome that dictates their mating type. A more parsimonious model then is that genetic crosses that led to the production of these isolates would have involved two parents with different α mating alleles in a same-sex mating cycle. It is also possible that both types of crosses have occurred (opposite-sex and same-sex mating) and that there has been more than one cross involved here in the genesis of these isolates that are responsible for the outbreak and also for the global isolates to which they are related.

Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
×

Conclusions and Perspective

To summarize, three molecular genotypes are found circulating in the Pacific Northwest. The most prominent is the VGIIa/major genotype (Figure A10-1 in yellow), and we can date its origin to at least the early 1970s to an isolate from a patient in Seattle. The VGIIb/minor genotype is shown in red and it is molecularly indistinguishable from isolates from a fertile recombining population in Australia. In green is a completely novel VGIIc genotype that has emerged in Oregon. Two of these genotypes (VGIIa, VGIIc) are highly virulent in both macrophage and mouse models (Byrnes et al., 2010).

From where do these VGII isolates originate? We find VGII isolates globally: in Africa, Australia, and South America. But based on available evidence to date, the most parsimonious model is that at least one of these genotypes (VGIIb/minor) came from a population in Australia, given that isolates from the two are indistinguishable (Figure A10-4). In terms of the crosses hypothesized to have been involved in the origin of the outbreak, all of the populations that have been identified are all α. There are no a mating types of this lineage that have been found on Vancouver Island, in the Pacific Northwest, or in Australia. The only places where VGII mating type a isolates have been found so far are South America and Greece. So it may be that in Australia and the Pacific Northwest that same-sex mating drives diversity in the population, but opposite-sex mating is a possibility in South America. In the context of the outbreak, we also envision that same-sex mating is contributing to the production of infectious spores, which are the aerosolized small particles detected with Anderson air samplers. It is entirely possible that those propagules are desiccated yeast cells, spores, or a mixture of both. Experiments to test if these are actually spores in nature are being planned, and if spores are present in the air on Vancouver Island, it seems likely that they are being produced by same-sex mating occurring in the environment, given that the population is α unisexual.

Disease caused by C. gattii vs. C. neoformans has been the subject of several reviews, and despite the impression that C. gattii frequently causes infection in apparently immunocompetent hosts and commonly presents with focal cryptococcomas in the lung or brain (Chen et al., 2000; Mitchell et al., 1995; Speed and Dunt, 1995), it has been difficult to clinically distinguish between C. neoformans and C. gattii disease. In fact, the presentation of C. gattii infections in Vancouver and the Pacific Northwest outbreak appear similar to C. neoformans infections, and disease is not limited to the immunocompetent host. C. gattii infections such as those caused by the VGIII molecular type/cryptic species can be even found in HIV-infected individuals in endemic areas (Byrnes et al., 2011; Chaturvedi et al., 2005). Another aspect that has been reported to distinguish these two species is their response to therapy. It has been suggested that C. gattii infections require longer therapy (West et al., 2008). However, in vitro susceptibility testing has produced inconsistent results; some investigators have found similar minimum inhibitory concentrations (MICs) whereas others have found higher MICs to azoles for

Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
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C. gattii compared to C. neoformans (Thompson et al., 2009; Torres-Rodriguez et al., 2008). It is always difficult to be precise in determining the response to therapy when in a normal host; a component of immune reconstitution inflammatory syndrome may influence clinical judgment of treatment failure. A recent study primarily in HIV-infected patients demonstrated that there was no difference in the outcome of C. gattii vs. C. neoformans infections in a single medical center (Steele et al., 2010). This is interesting and may be due to small numbers, but early results comparing C. gattii infections in the Vancouver Island vs. the Pacific Northwest outbreaks suggested a higher mortality in the United States (Kluger et al., 2006). After review of present data on outcomes in the literature, in vitro drug susceptibility studies, and clinical experience, the 2010 Infectious Diseases Society of America Cryptococcal Guidelines support the treatment of C. gattii infections in a manner similar to those of C. neoformans (Perfect et al., 2010). However, C. gattii infections may produce more cryptococcomas in the brain or lung, and chronic conditions such as hydrocephalus and a slow response to antifungal therapy may be prominent features of disease with this species.

Despite the lack of precise differences in C. gattii vs. C. neoformans disease clinically, which would require immediate identification of the species, there are differences in epidemiology regarding the range of infections. It is also suggested on a pathobiological basis that C. gattii may present more with acute disease rather than reactivation disease. This hypothesis is based on (1) antibody studies in children where C. neoformans, but not C. gattii, antibodies are frequently detected, and (2) the higher percentage of disease that is observed in immunocompetent patients with C. gattii (Goldman et al., 2001). It has also been shown that C. gattii may induce a different host immune response compared to C. neoformans (Cheng et al., 2009). Furthermore, the Vancouver Island outbreak represents an ideal arena to check for genetic susceptibility to C. gattii infection because a vast number of individuals were exposed to high numbers of infectious propagules, but only a minority of those exposed was identified with clinical disease. However, it is clear through both animal studies and transplant cases that C. gattii infections can reactivate (Dromer et al., 1992; Kluger et al., 2006). Finally, although major virulence factors (melanin, capsule, high-temperature growth) are similar, some of the networks such as the trehalose pathway connecting these virulence factors are different in these two species (Ngamskulrungroj et al., 2009b; Petzold et al., 2006). The possibility that differences in both host and microbe genetics may influence the course and management of infections will drive studies on this unique example of a fungal outbreak in humans as we continue its analysis.

Acknowledgments

We thank Wenjun Li and Yonathan Lewit for their contributions to the analysis of the outbreak; Dee Carter, Vishnu Chaturvedi, Jim Kronstad, Kieren Marr,

Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
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and Robin May for collaboration; Rory Duncan, Chris Lambros, and Dennis Dixon from the National Institute of Allergy and Infectious Diseases (NIAID) and Victoria McGovern from the Burroughs Wellcome Fund for their support; and Stephen Johnston for his prescient questions. We also explicitly thank all of the Forum members and the Institute of Medicine for shining a very bright light on the fungal kingdom, and for the invitation to participate. Our research is supported by NIH/NIAID R37 grant AI39115.

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Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
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Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
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Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
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Litvintseva, A. P., I. Carbone, J. Rossouw, R. Thakur, N. P. Govender, T. G. Mitchell. 2011. Evidence that the human pathogenic fungus Cryptococcus neoformans var. grubii may have evolved in Africa. PLoS One 6(5):e19688.

Ma, H., F. Hagen, D. J. Stekel, S. A. Johnston, E. Sionov, R. Falk, I. Polacheck, T. Boekhout, and R. C. May. 2009. The fatal fungal outbreak on Vancouver Island is characterized by enhanced intracellular parasitism driven by mitochondrial regulation. Proceedings of the National Academy of Sciences, USA 106(31):12980–12985.

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Nielsen, K., G. M. Cox, A. P. Litvintseva, E. Mylonakis, S. D. Malliaris, D. K. Benjamin, Jr., S. S. Giles, T. G. Mitchell, A. Casadevall, J. R. Perfect, and J. Heitman. 2005a. Cryptococcus neoformans α strains preferentially disseminate to the central nervous system during coinfection. Infection and Immunity 73(8):4922–4933.

Nielsen, K., R. E. Marra, F. Hagen, T. Boekhout, T. G. Mitchell, G. M. Cox, and J. Heitman. 2005b. Interaction between genetic background and the mating-type locus in Cryptococcus neoformans virulence potential. Genetics 171(3):975–983.

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Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
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Petzold, E. W., U. Himmelreich, E. Mylonakis, T. Rude, D. Toffaletti, G. M. Cox, J. L. Miller, and J. R. Perfect. 2006. Characterization and regulation of the trehalose synthesis pathway and its importance in the pathogenicity of Cryptococcus neoformans. Infection and Immunity 74(10):5877–5887.

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Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
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A11

YEAST INFECTIONS—HUMAN GENETICS ON THE RISE38

Steven M. Holland39and Donald C. Vinh39

We lead inextricably mycotic lives: yeasts leaven our bread, ferment our wine and beer, and inhabit our skins, mouths, and gastrointestinal tracts; however, not all is harmony. Hippocrates reported aphthous ulcers consistent with thrush in patients with severe debilitation, but it was not until the 1840s that in the newly emerging field of clinical experimental medicine that thrush—as well as other mycotic conditions, including ringworm—was recognized as being caused by transmissible fungi. In 1923 Christine Marie Berkhout named what we now call Candida albicans, for the white robe, toga candida, worn by Roman senators and senatorial candidates (Emmons et al., 1977). Fungal infections in general, and candida infections in particular, are important markers of innate or acquired immune dysfunction. However, despite the estimated 1.5 million species of fungi in the world, precious few cause human disease, and those that do (in the

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38 Originally published as Holland, Steven M; Vinh, Donald C.2009. Yeast Genetics on the Rise. New England Journal of Medicine. Article DOI: 10.1056/NEJMe0907186. Copyright © 2009 Massachusetts Medical Society. Available at: http://www.nejm.org/doi/full/10.1056/NEJMe0907186.

39 Steven M. Holland, M.D., and Donald C. Vinh, M.D. From the Laboratory of Clinical Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD.

Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
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absence of iatrogenic factors) usually cause inapparent or mucocutaneous infection. Therefore, genetic factors in both the pathogen and the host must be key to an understanding of who gets disease and why.

Despite the ubiquity and importance of fungi, little is known about specific human genetic predispositions to them. There are three distinct categories of fungi: filamentous molds (e.g., aspergillus, mucor, and trichophyton), dimorphic fungi (e.g., the endemic mycoses, histoplasma, blastomyces, coccidioides, and sporothrix, a non-endemic pathogen), and yeasts (e.g., candida, cryptococcus, and trichosporon). In the absence of iatrogenic factors, visceral infections of filamentous mold occur almost exclusively in patients with chronic granulomatous disease, whose capacity to generate phagocyte superoxide is defective. Severe invasive aspergillosis develops in these patients (who have normal lungs), as do, on occasion, infections with invasive candida; interestingly, mucocutaneous candidiasis is not encountered in chronic granulomatous disease. In contrast, development of most other cases of pulmonary aspergillosis that are unrelated to immunosuppression requires previous airway damage, such as bronchiectasis or bullous disease. Dimorphic fungi cause inapparent or relatively mild disease in the vast number of people who are otherwise normal. Severe pneumonia and disseminated disease occur infrequently, as in cases of advanced human immunodeficiency virus (HIV) and in patients with defects of the interferon-γ–interleukin-12 axis, indicating that intracellular killing plays a crucial role in the body’s defense against these infections. Mucocutaneous candidiasis, vaginal candidiasis, thrush, and onychomycosis occur in a variety of disparate conditions, ranging from diabetes mellitus to the autoimmune polyendocrinopathy–candidiasis–ectodermal dystrophy (APECED) syndrome, to HIV, to Job’s (hyper-IgE) syndrome. Our recent identification of mutations in signal transducer and activator of the transcription 3 gene (STAT3) as the cause of Job’s syndrome unexpectedly provides a link to the important articles by Ferwerda et al. (2009) and Glocker et al. (2009) in this issue of the Journal.

Whereas mucocutaneous candidiasis has long been recognized as a consequence of profound lymphocyte dysfunction, or lymphopenia, it occurs in Job’s syndrome in patients with a relatively normal number of functioning lymphocytes. Mutations in the gene encoding transcription factor STAT3 impair numerous pathways, including the generation of interleukin-17–committed T lymphocytes (Th17 cells). Th17 cells fill a gap in previous knowledge about lymphocyte-mediated killing: If Th1 (interferon-γ–producing) lymphocytes are controlling intracellular infection and Th2 (interleukin-4–producing) lymphocytes are directing antibody-mediated protection against extracellular infection, which cells protect the exposed mucosa and epithelium? Among the products of Th17 cells is interleukin-22, which synergizes with interleukin-17 in the epithelial synthesis of cationic antimicrobial peptides, such as defensins. STAT3 is also central to the induction and signaling of itself, interleukin-17, and interleukin-22 (Fig. A11-1) (Minegishi et al., 2009).

We encounter most microbes first at epithelial surfaces through cell-

Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
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Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
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Figure A11-1 (facing page). Mechanisms of Fungal Sensing and Control. The β-glucan on the budding yeast forms of candida bind to dectin-1 on epithelial cells and phagocytes, leading to activation of the CARD9 signaling complex (along with Bcl-10–MALT1) (Panel A). This in turn produces cytokines that help drive CD4+ T lymphocytes toward the Th17 phenotype, a STAT3-dependent process. Th17 lymphocytes elaborate interleukin-17 (augmenting neutrophil production and recruitment) and interleukin-22, which are synergic in the STAT3-dependent production of antimicrobial peptides by epithelial cells. The generation of superoxide by the phagocytic NADPH oxidase system is crucial for the killing of filamentous molds such as aspergillus (Panel B). Interactions between macrophages and lymphocytes are required for the control of intracellular infections such as the dimorphic fungi. In response to fungal infection, macrophages release interleukin-12, which acts on its cognate receptor on T lymphocytes and natural killer (NK) lymphocytes. Interleukin-12–stimulated lymphocytes release interferon-γ, which acts on macrophages through STAT1 to kill intracellular fungi. Interferon-γ also augments tumor necrosis factor α (TNF-α), which acts in part through the nuclear factor κB (NF-κB) to kill fungi and drive inflammatory responses. Molecules with identified mutations relevant to fungal susceptibility are shown in red.

receptors. These receptors couple the binding of specific microbial components to signal transduction pathways, which results in local and systemic immune responses, including activation of the central activator of inflammation, nuclear factor κB. Specific immunodeficiencies involving receptor molecules and their signaling have been characterized for herpes simplex virus encephalitis, Neisseria meningitidis, and Streptococcus pneumoniae (Beutler, 2009). As expected, the closer the defect is to the initial contact with the pathogen, the more likely it is that the susceptibility will be microbiologically narrow. In contrast, defects that occur at the convergence of several pathways tend to be broader and more severe. Dectin-1 is the cell-surface receptor for β-glucan, a major component of the budding yeast-cell wall, and its signaling travels through a series of molecules, including caspase-recruitment–domain (CARD) protein 9, leading to activation of nuclear factor κB.

Ferwerda et al. identified a Dutch family with impaired in vitro responses to β-glucan. The spectrum of disease was limited to nails and mucosa. Interestingly, the ages at clinical disease presentation were 10 to 12 years for the homozygous daughters but 40 and 55 years for the heterozygous mother and father, respectively, suggesting both hormonal and gene-dose effects. Generation of interleukin-6 and interleukin-17 was impaired only in response to the dectin-1 ligand, β-glucan. The specific stop codon the authors identified in dectin-1 is remarkably common in some parts of Africa and Europe (allele frequency, 3 to 7%), suggesting that unrecognized forces maintain it in populations. Glocker et al. identified an extended Iranian family with predominantly mucocutaneous but also fatal candidiasis of the central nervous system caused by mutations in the critical dectin-1 signal transduction molecule, CARD9, impairing both dectin-1 signaling and Th17 production. CARD9 also funnels a variety of other cell-

Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
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surface and intra-cellular signals, including the p38 mitogen-activated protein kinase (MAPK) and Jun N-terminal kinase (JNK) pathways, possibly accounting for its greater clinical severity as compared with isolated dectin-1 deficiency. The CARD9 mutation appears to be rare, and its rarity is commensurate with its severity.

Important caveats concerning both reports are that the key manifestations are mucocutaneous, whereas the functional studies were performed on leukocytes. It remains to be proven whether the key mechanisms in these cases of severe candidiasis consist of impaired dectin-1 signaling at the epithelial level or impaired leukocyte activation of epithelium, mediated through cytokines such as interleukin-17.

The long and largely happy coexistence of humans and fungi has necessitated the existence of ways to detect and control fungi, keeping them in their place. Apparently, these pathways are different for candida, aspergillus, and mucor. Although we do not yet understand the entire conversation between leukocytes and epithelium, with these two reports we have now overheard some of the key words that will enable us to listen more thoughtfully.

No potential conflict of interest relevant to this article was reported.

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A12

THE INCREASED RISK OF GLOBAL WHEAT RUST PANDEMICS: PUTTING YELLOW RUST INTO PERSPECTIVE

Mogens Støvring Hovmøller40

Infectious diseases of humans are receiving great attention due to their direct and immediate impact on human mortality (King et al., 2006; WHO, 2008),

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40 Aarhus University, Faculty of Agricultural Sciences, Department of Integrated Pest Management, Flakkebjerg, 4200 Slagelse, Denmark. Mogens.hovmoller@agrsci.dk.

Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
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whereas the diseases of crop plants are subject to much less publicity although they are threatening crop productivity, food security, and thereby the livelihood of billions of people around the world. Currently, wheat rust fungi are among the top 10 constraints for food production in many parts of the world, such as sub-Saharan Africa, East Asia, and South Asia (FAO, 2010; Waddington et al., 2010), mainly due to their epidemic potential and transboundary spread by wind (Brown and Hovmøller, 2002).

According to U.S. Department of Agriculture statistics, global wheat production was generally lower in 2010 compared to the previous year (USDA, 2011). The reduced productivity was caused by several factors, such as unfavorable weather conditions, resulting in large-scale flooding, droughts, and bushfires in different parts of the world, as well as severe yellow rust epidemics (USDA, 2010).

Rust diseases have been recognized as being harmful to wheat since ancient Greece. Theophrastus of Eressus (371–286 BC), the founder of botany and a pupil of Aristoteles, noted in his book ΠΕΡΙ ΦΥΤΩΝ ΑΙΤΙΩΝ (The Causes of Plants) that some plants of barley were more susceptible to the rusts than others, the most susceptible denoted “Achilles Barley,” and he recognized the importance of sowing in due time to reduce rust diseases on different crop plants such as wheat, barley, peas, and beans (Theophrastus, 1990). Wheat rust may be caused by three plant pathogenic fungi, Puccinia graminis (Leonard and Szabo, 2005), Puccinia triticina (Bolton et al., 2008), and Puccinia striiformis (Hovmøller et al., 2011). The corresponding diseases are termed black (stem) rust, brown (leaf) rust, and yellow (stripe) rust, respectively (synonymous terms in brackets) (Figure A12-1).

The three wheat rust fungi are biotrophic and heteroaecious, meaning they generally require a primary wheat host for asexual reproduction and an alternate host to complete sexual reproduction. Berberis spp. have been known to serve as an alternate host for stem rust for more than two centuries. For instance, Schøler (1818), who was a school teacher in Denmark, demonstrated a link between stem rust on barberry and some cereals via repeated infection experiments. His results started a so-called “barberry quarrel,” where the usefulness of barberry eradication programs was discussed. Later, de Bary (1865, 1866) demonstrated the complete life cycle of the stem rust fungus. However, the problems related to barberry adjacent to cereal fields were observed by farmers in Europe much earlier, with the first local laws against barberry being implemented in France in the 17th century and in the Americas in the 18th century (Roelfs, 1982). Surprisingly, the discovery of barberry as an alternate host for yellow rust infecting wheat and Poa grass, respectively, was not made until very recently (Jin et al., 2010). The alternate host of P. triticina depend on the primary host: Isolates from cultivated wheat and wild emmer have Thalictrum speciosissimum (in the Ranunculaceae) as alternate host, whereas several species in the Boraginaceae, such as Anchusa aggregata, Anchusa italica, Echium glomeratum, and Lycopsis arvensis, may

Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
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img-254

Figure A12-1 Typical macroscopic symptoms of rust infections on adult wheat plants. Puccinia striiformis Westend (yellow/stripe rust) [left photo], Puccinia triticina (brown/leaf rust) [center photo], and Puccinia graminis tritici (black/stem rust) [right photo].

serve as alternate host for P. triticina from wild wheat and rye (Anikster et al., 1997). Although this paper deals with all three wheat rusts, the primary focus is yellow rust, which has spread at unprecedented scales in recent years and caused severe epidemics even in areas where the disease was previously non-significant or absent (Hovmøller et al., 2010).

Yellow Rust Epidemiology

Wheat rusts are highly epidemic on susceptible cultivars in a rust-favorable environment. In the past, yellow rust was considered most harmful in cool and wet climates, whereas leaf rust and stem rust epidemics were favored by warmer temperatures. They are all characterized by passive spreading of airborne urediniospores carried by the wind, potentially across hundreds of kilometers (Hovmøller et al., 2002; Kolmer, 2005; Leonard and Szabo, 2005; Zadoks, 1961). Since 2000, epidemics of yellow rust in particular have accelerated in many areas (Hovmøller et al., 2010). In the United States, annual losses due to wheat yellow

Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
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rust exceeded 1 million metric tons over several years, and in each of the years 2003 and 2010 they mounted to 2.4 million tons (Long, 2000–2010), despite increased and widespread use of agrochemicals already in 2003 (Chen, 2005). In China, yellow rust is considered the most damaging disease on wheat, which is grown on more than 20 million hectares (Wan et al., 2004). In three yellow rust epidemic years, annual losses varied between 1.8 and 6 million tons; in 2002, losses of 1.3 million tons were recorded and 1.9 million tons were saved by treating at least 6 million hectares with fungicides (Wan et al., 2004). Yellow rust epidemics reached record levels in northern Africa in 2009 (Ezzahiri et al., 2009) and also in central and western Asia (Mboup et al., 2009), where more than 90 percent of important wheat varieties were susceptible to the disease (Sharma et al., 2009). Areas particularly affected by yellow rust epidemics in 2009 and 2010 are illustrated in Figure A12-2.

New genetic variability of the pathogen, resulting from natural population dynamic forces such as mutation, recombination, and migration, may be followed by host-induced selection, which is a major driver of changes in gene and genotypic frequencies of biotrophic plant pathogens (Hovmøller et al., 1997).

img-255

Figure A12-2 Map indicating the distribution of global wheat production and regions of recent yellow rust epidemics. Each red dot is equivalent to 20,000 tons of wheat grain production. The blue and orange circles indicate some of the areas in which severe yellow rust epidemics on wheat were reported in 2009 and 2010. Yellow rust severity varied greatly from field to field within the highlighted areas, depending on susceptibility of actual host cultivar, local environmental conditions, pathogen race distribution, and disease management practices. The epidemics in Northwest Europe in 2009 (dotted circle) were restricted to relatively few cultivars of wheat and triticale.
SOURCE: Adapted from Trethowan et al. (2002). Dr. Dave Hodson, Food and Agriculture Organization of the United Nations, Rome, is acknowledged for kind permission to reprint the map.

Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
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In agricultural systems, where crop cultivars with similar or identical resistance specificities are cultivated across large areas, the role of selection is greatly enhanced (Bayles et al., 2000; Hovmøller et al., 1997; McDonald and Linde, 2002). Therefore, crop varieties that are resistant when first deployed in agriculture may become susceptible after some years in cultivation. This phenomenon, which is often referred to as the boom-and-bust cycle, has been reported in numerous cases for the wheat rusts (e.g., Bayles et al., 2000; Chen, 2005; Enjalbert et al., 2005; Hovmøller, 2001; Wellings, 2007). The boom-and-bust cycle is closely related to resistance genes in the host and two categories of pathogen traits, virulence and aggressiveness.

Virulence and Aggressiveness

The yellow rust fungus is specialized on the cereal host at both species and cultivar levels (Hovmøller et al., 2011). The former refers to variation in the infection and reproduction capacity of the pathogen on different host genera (e.g., wheat, barley, rye, tricicale), whereas the second level of specialization occurs within the host genus, that is, at the cultivar level. The latter is typically based on the interaction between host resistance gene products and pathogen avirulence gene products in a gene-for-gene relationship (Flor, 1956). Mutation in the avirulence gene may result in virulence, which can be defined as the qualitative ability of the pathogen to cause disease by compromising a matching resistance gene in the host. In case the host cultivar possesses more than a single resistance gene, the virulence phenotype, representing a specific combination of different virulences, becomes critical in determining the fate of host susceptibility (e.g., Hovmøller and Henriksen, 2008; Johnson, 1992; Line and Qayoum, 1992). The virulence phenotype can be resolved from the outcome of a race analysis, which is carried out by inoculating a rust isolate onto a set of host differential lines carrying known resistance genes, for details (see, e.g., Hovmøller and Justesen, 2007a).

The study of race dynamics in P. striiformis populations has often been an important part of early warning and yellow rust strategies in Europe (Hovmøller and Henriksen, 2008; Johnson, 1992), North America (Chen and Penman, 2005; Line and Qayoum, 1992), China (Wan et al., 2007), and Australia (Wellings, 2007). Since 1967, where a national race survey was established in the United Kingdom (Johnson, 1992), similar programs were established in Germany (Flath and Bartels, 2002), France (De Vallavieille-Pope et al., 1990), and Denmark (Hovmøller, 2001). In recent years, European data on P. striiformis virulence dynamics have been made publicly available via the Eurowheat database at www.eurowheat.org, which also describes options for disease control, such as cultural practices and fungicide efficacies (Jørgensen et al., 2010). Systematic race surveys were initiated in the United States in the late 1960s after a series of severe yellow rust epidemics on wheat (Line and Qayoum, 1992). Systematic race surveys have also been implemented in China (Chen et al., 2009; Wan et al., 2004),

Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
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India (Prashar et al., 2007), Australia (Wellings, 2007; Wellings and McIntosh, 1990), and South Africa (Boshoff et al., 2002).

In addition to the virulence phenotype, pathogen aggressiveness, which is a quantitative measure of the ability of a virulent isolate to cause disease on a susceptible host plant, is a key determinant for the rate of pathogen spread and evolution. Some of the recent epidemics since 2000 have been ascribed to the emergence of yellow rust strains with increased aggressiveness and tolerance to warm temperatures (Hovmøller et al., 2008). In this context, strain was defined as a group of P. striiformis isolates sharing both molecular marker phenotype and virulence phenotype. For instance, Milus et al. (2009) demonstrated a significantly increased aggressiveness at both high and low temperatures, compared with isolates of pre-2000 races from North America and Europe (Milus et al., 2009). At the low-temperature regime, gradually changing from 10°C (night) to 18°C (day), the aggressive strains produced more than 70 percent more spores per infected leaf area compared to reference isolates from before 2000. At the high-temperature regime (12°C at night to 28°C during the day), isolates of the aggressive strains produced approximately 150 percent more spores per infected leaf area. Milus and colleagues (2009) concluded that the aggressive strain had most likely enhanced the yellow rust epidemics in North America and contributed to the spread of yellow rust into areas that were previously considered too warm for yellow rust epidemics. The aggressive strain detected in North America was indistinguishable from a strain detected as exotic incursion in Australia in 2002 (Hovmøller et al., 2008; Wellings et al., 2003). In Europe, a nearly identical strain was first detected in 2000, whereas first appearance of this strain in the Red Sea area and in western and central Asia is unknown (Hovmøller et al., 2008). According to time and area, these observations may be comparable to the famous panglobal spread of the Irish potato famine fungus Phytophthora infestans (Goodwin et al., 1994).

The distinction between virulence and aggressiveness may be subject to controversy because virulence toward a resistance gene with minor effects may be manifest by increased disease progress and spore production. These are the same variables used to measure aggressiveness. However, in case of virulence, there should be evidence for a significant host–pathogen interaction, whereas in the case of aggressiveness, the host–pathogen interaction is ideally non-significant. This implies that for a range of host cultivars with varying levels of rust susceptibility, they should rank the same for aggressive and non-aggressive isolates.

The Need for Coordinated Global Action

The number of foreign incursions of wheat rust races has increased significantly in recent years. Park et al. (2010) reported nine foreign incursions of wheat stem rust and wheat leaf rust into Australia between 1925 and 2005. Of these seven had occurred since 1969. Hovmøller et al. (2011) documented at least six

Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
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exotic incursions of yellow rust at continental scales since 1979, often resulting in the spread of yellow rust epidemics to new regions or continents. Scherm and Coakley (2003) noted that the rate of exotic pathogen invasion in the United States had increased from about five instances per decade from 1940 to 1970 to more than three times that number during the 1990s. This increasing rate of incursions of the yellow rust fungus may partly be ascribed to the emergence of new, highly aggressive rust strains combined with increasing human travel and commerce. However, regardless of the reasons for the increasing spread, the situation escalates, emphasizing the relevance of pathogen surveys covering larger areas and a need for global coordination. The use of crop cultivars with similar or identical resistance specificities across large wheat-growing areas supports this conclusion.

The accelerating wheat yellow rust epidemics requires coordinated multinational action to complement the ongoing initiatives of the Borlaug Global Rust Initiative (www.globalrust.org) to fight the multivirulent strain of wheat stem rust, known as Ug99 (Northoff, 2007, 2008). The speed by which threatening new strains can be diagnosed and reported widely is essential, and the precision by which traits such as virulence and aggressiveness are diagnosed is another bottleneck (Hovmøller et al., 2011). Precise diagnosis of both traits require pure and correct identification of the seed stocks of standard differential wheat varieties used for the race assays, genetically pure pathogen isolates, and well-defined experimental conditions for temperature, humidity, light, and plant nutrition, as well as trained staff who can assess and interpret the virulence phenotype and aggressiveness. The acknowledgment of these challenges led to the establishment of a Global Rust Reference Center (GRRC) in 2008 targeting yellow rust, supported by the International Center for Agricultural Research in the Dry Areas (ICARDA), the International Maize and Wheat Improvement Center (CIMMYT), and Aarhus University (Denmark) (Hovmøller et al., 2010). GRRC is accessible for receiving yellow rust samples on a year-round basis, which is a major advance complementing the capacities of existing regional and national rust diagnostic laboratories. At the Borlaug Global Rust Initiative (BGRI) Coordination Meeting in Syria in September 2009 (Dold, 2009), a proposal was made to extend the activities of GRRC to all wheat rust fungi, that is, yellow rust, brown rust, and stem rust.

The long-term aims of a Global Rust Reference Center would be the tracking of new wheat rust incursions and assessment of ongoing pathogen evolution at global scales, supplying data to online early-warning systems for farmers, breeders, and policy makers, like the current “Rust Spore” monitoring system run by the Food and Agriculture Organization of the United Nations (FAO, 2010). Another main activity would be to assist in training of students and junior scientists in rust pathology, and maintenance and extension of a unique world wheat rust collection to facilitate resistance breeding efforts. The activities should complement ongoing regional and national survey activities and research efforts

Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
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by BGRI partners, national counterparts, and advanced research institutions, resulting in stronger global efforts to mitigate the consequences of the inherent pathogen dynamics. At present, the activities at GRRC are limited to race analysis of relatively few pathogen samples per year in addition to training activities. To become sustainable in the long term, GRRC must contain a considerable portfolio of activities to reduce vulnerability due to potential changes in staff, management, and political awareness.

The identification of sources of resistance from cultivated and wild crop relatives to wheat is probably the most urgent task to reduce the threats posed by the wheat rusts, including yellow rust. Plants have an immunity system with several layers. Race-specific resistance provides protection only during one or few steps of the infection process and is therefore considered vulnerable to pathogen evolution whereas partial resistance, based on several or many sources of resistance, is considered more durable. Nevertheless, breeding for increased partial and non-host resistance protecting wheat against a broad spectrum of pathogen species and races can be done by large-scale field screening of thousands of breeding lines by applying targeted pathogen species and strains.

Prospects for New Research

The serious impacts and characteristics of wheat yellow rust epidemics should remind society about the vulnerability of global food production and the need to understand the interactions between agricultural crops, their pathogens, and the environment. Until recently, investigations of the basic biological and molecular mechanisms of yellow rust evolution have been hampered by the biotrophic lifestyle of yellow rust, precluding the use of molecular tools used for fungi that can be cultured on artificial media, and by the lack of an experimental system for genetic studies. These limitations may soon be overcome by the significant advances of molecular technologies, new insights into pathogen biology, and the cloning of host resistance genes, which were achieved in recent years. Molecular markers are probably the best established tools for resolving recent as well as past dispersal and evolutionary events in P. striiformis (Hovmøller and Justesen, 2007b; Hovmøller et al., 2008; Mboup et al., 2009). Next generation sequencing is currently the most promising tool for the identification of genetic changes related to virulence in fungal pathogens (Stergiopoulos and de Wit, 2009). A Danish–British genome sequencing initiative was started in 2009 (Walter et al., 2009), followed by a more comprehensive U.S.-led initiative (Broad Institute, 2009) aiming to resolve the whole genome sequence of P. striiformis. Such knowledge will most certainly speed up the discovery of genes involved in pathogenicity. The recent discovery of barberry as sexual host for P. striiformis (Jin et al., 2010), which has solved a century-old mystery about the yellow rust lifecycle, represents another major breakthrough that can serve as the basis for the development of an experimental system allowing classical genetic studies.

Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
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Functional characterization of isolated rust genes is still a great challenge because of the biotrophic lifestyle, which is hampering genetic transformation of P. striiformis. The identification of sources of resistance in wheat, especially for resource-poor areas in Africa and Asia, is probably the most urgent task to reduce the threats posed by wheat rust pathogens. However, this will require a long-term commitment and combined efforts of breeders, pathologists, and biologists to keep pace with the wheat rusts, which can evolve rapidly to compromise genetic resistance of the wheat host (Hovmøller and Justesen, 2007b; Wellings and McIntosh, 1990).

Concluding Remarks

The fight against infectious crop diseases has become a collective responsibility and requires a collective investment with a global, long-term political and scientific commitment. Such a global network will need to establish the physical and human resources to support the progress of wheat rust management technologies, that is, pathogen monitoring and resistance breeding, training programs for farmers and pathologists, and scientific progress. Hence a major priority of a global network should be to link researchers and plant breeders (1) with each other and with appropriate partners in resource-poor countries that are affected most by sudden wheat rust epidemics, and (2) with political authorities to allow for rapid actions. The establishment of the BGRI in 2006, inspired by Dr. Norman Borlaug, the pioneer of the Green Revolution and the front leader of cereal rust resistance breeding, represents a major milestone in this respect. The real challenge is to ensure sustained activities beyond ongoing, short-term projects. In the long term, surveillance and prevention measures of wheat rust epidemics will only be successful if performed on a coordinated, global scale. Borlaug used to say: “Rust never sleeps”—and events of recent years have shown how right he was.

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Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
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Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
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Wan, A., Z. Zhao, X. M. Chen, Z. He, S. Jin, Q. Jia, G. Yao, J. Yang, B. Wang, and G. Li. 2004. Wheat stripe rust epidemic and virulence of Puccinia striiformis f. sp. tritici in China in 2002. Plant Diseases 88:896–904.

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Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
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A13

FUNGAL PATHOGENESIS IN PLANTS AND ANIMALS: SIMILARITIES AND DIFFERENCES

Barbara Howlett41

Introduction

Although the vast majority of fungal species do not cause disease, ones that affect plants are responsible for significant economic losses (Skamnioti and Gurr, 2009). In the past couple of decades, fungal diseases of humans have become an increasing threat, especially for people who are immunologically compromised (Sexton and Howlett, 2006). Consequently there has been a rapid explosion in knowledge about fungal pathogenesis of animals and this has been taken up by people studying plant pathogenic fungi. Pathogenesis involves the interaction of two partners with input from the environment, a concept described as the “disease triangle” in plant pathology. The “damage-response” concept developed for animal pathogens emphasizes that the outcome of an interaction is determined by the amount of damage incurred on the host (Casadevall and Pirofski, 2003). These concepts are useful reminders of the complexity of the interaction and the interdependence of host and pathogen.

Tools to Study Fungal Pathogenesis

The large number of fungi with sequenced genomes and recent advances in genetic manipulation of fungi are leading to an improved understanding of mechanisms associated with disease. Comparative genomics is being used to identify candidate genes involved in disease. Amplification of particular gene families within a genome is consistent with lifestyles: For instance, many plant pathogens have large gene families that encode enzymes to degrade the cuticle and cell wall, which coats plant cells, while animal pathogens (e.g., Coccidioides immitis, the cause of valley fever) have gene families encoding enzymes that degrade proteins of the skin, which is usually the initial barrier to invasion (Sharpton et al., 2009). Transcriptomics can show which genes are turned on; microarrays have been used extensively and many datasets are publicly available (Cairns et al., 2010). Proteomic analysis of culture filtrates provides information about secreted proteins (secrotome) that are accessible to the host and thus may play a role in the interaction; this approach has been applied to identify such proteins of Leptosphaeria maculans, the blackleg pathogen of canola (Vincent et al., 2009).

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41 The University of Melbourne.

Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
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The generation of fungal mutants via tagged random insertional mutagenesis allows identification of novel genes involved in disease, while targeted gene knockout or gene silencing allows functional analysis of candidate pathogenicity genes. Analyses of human pathogenic fungi generally rely on cell lines and animal models with different immunosuppression regimes. By contrast, plant pathogens can be studied directly on their hosts. Many more fungal species infect plants compared to animals and thus more plant fungal systems than animal fungal systems are studied, but with a shallower focus. Plants obviously can be manipulated with fewer ethical issues than those associated with animal experimentation. Findings from model systems can often be applied to other host–pathogen interactions. The best studied plant fungal system for ascomycetes is rice and Magnaporthe oryzae (cause of rice blast) (Ebbole, 2007) and for basidiomycetes is maize and Ustilago maydis (cause of corn smut) (Brefort et al., 2009). Model animal fungal systems include immune-suppressed mice and Aspergillus fumigatus, Candida albicans, or Cryptococcus spp; recently amoeboid and non-vertebrate animal model systems, particularly the insect Galleria mellonella, have been used (Mylonakis et al., 2007). General similarities and differences between fungal pathogens of plants and animals are described in Table A13-1 and are discussed in more detail later in this chapter.

The Invasion and Disease Process

The disease process can be considered as a series of consecutive steps beginning with recognition between host and fungus, penetration of the host, colonization and avoidance of host defenses, development of disease symptoms, and finally fungal reproduction and dispersal. These steps are developmentally regulated, and fungal mutants can be generated that are arrested at particular stages (for review see Sexton and Howlett, 2006). The mutated genes are often referred to as pathogenicity genes and are broadly categorized as to where they appear to act in the disease pathway. This is an arbitrary classification for convenience. Clearly there are pathogenicity genes with pleiotropic effects at many steps in the pathway, as some processes (e.g., recognition, pH regulation, oxidative burst, signaling) occur several times. Pathogenicity genes of fungi that attack plants have been reviewed recently (Van de Wouw and Howlett, 2011). Interestingly the steps related to symbiotic relationships between plant and fungi are similar to those involved in pathogenesis; thus the classification of plant–fungus interactions as pathogenic or symbiotic is indistinct.

Fungal inoculum of plants is usually spores (sexual or asexual) that then germinate on the surface of the plant. Animal pathogenic fungi can exist in forms such as yeasts, conidia, or hyphal fragments. Some such fungi undergo dimorphic switching. In some cases, the yeast form is more pathogenic than the hyphal phase, while in others the converse is true. A key class of hydrophobic proteins, hydrophobins coat the fungal cell wall, which is composed generally of

Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
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Table A13-1 General Similarities and Differences Between Fungal Pathogens of Plants and Animals

Feature Plants Animals
Number of fungal pathogens Many Few
Importance Cause 25–30% crop losses Usually affect immunocompromised animals
Lifestyle and environmental niche Often biotroph; necrotroph; also saprophytes Often soil saprophytes; not obligates
Experimental systems Many; often shallow focus Few; different host cell and immunosuppression regimes of non-humans
Host specificity Often host species or cultivar specificity Sometimes species specific, not genotype specific
Mode of entry into hosts Via stomata, or breaching surface barriers via enzymes or pressure created by infection structures Via inhalation, ingestion, or wounds in skin
Dispersal Between hosts Not usually between hosts
Reproduction Sexual and asexual Usually asexual (ascomycetes)
Overall pathogen requirements Often complex set of physical and molecular barriers to overcome Inocula small enough to enter host, survive at 37°C and avoid immune responses

chitin, a polymer of β 1,4 N-acetyl glucosamine, β 1,3 glucans, as well as other polysaccharides and proteins.

Plants have more complex physical barriers to invasion than animals do. Epidermal cells on the plant surface have a cuticle comprising epoxy fatty acids, and a cell wall that is composed of a matrix of proteins and interacting carbohydrates, including β 1,3 and β 1,4 glucans such as cellulose (Carpita and Gibeaut, 1993). By contrast the animal surface, the stratum corneum, is much thinner and the cells are highly keratinized (Proksch et al., 2008). Fungi enter animals by inhalation, ingestion, or wounds. Basically, for a fungus to be an animal pathogen, its major features include being small enough to enter tissue, able to survive at 37°C, and able to evade host immune responses (Sexton and Howlett, 2006). Indeed, the temperature optimum for many fungi is significantly lower than 37°C, which has been proposed as an explanation for why relatively few fungi are important pathogens of mammals (Robert and Casadevall, 2009). The mammalian characteristics of maintenance and close regulation of body temperature are proposed to have a selective advantage in conferring resistance to many fungal pathogens (Bergman and Casadevall, 2010).

Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
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Fungi enter plants via the stomatal apertures, where air exchange occurs; by digesting the cuticle and cell wall with hydrolytic enzymes; or by developing infection structures, appressoria, which accumulate high concentrations of glycerol and puncture the surface due to high turgor pressure (Van de Wouw and Howlett, 2011). As mentioned above, fungi enter animals through the skin or via inhalation or wounds; an exception is Histoplasma capsulatus, which enters the host via receptor-mediated endocytosis (Woods, 2003).

The first basal or innate layer of defense is very similar in plants and animals. It involves binding of pathogen associated molecular patterns (PAMPs) to pattern recognition receptors on the host membrane surface. This triggers signaling pathways that induce a range of defense responses, including production of reactive oxygen species and sometimes programmed cell death. This response is termed “pathogen triggered immunity.” PAMPs common to plant and animal pathogenic fungi include wall carbohydrate fragments, such as chitin oligosaccharides or β 1,3 glucans. As well as conservation among PAMPs, there is also conservation in structural domains of Pattern Recognition Receptors (for review see Zipfel, 2009).

In plants often the responses triggered by innate basal immunity are not strong enough to stop pathogen invasion. Consequently a second round of recognition occurs involving effector-triggered immunity (Jones and Dangl, 2006). Often there is a “gene for gene” interaction between avirulence (effector) genes in the pathogen and resistance genes in the plant, such that the pathogen is unable to attack host genotypes with the corresponding resistance genes. Thus there is usually a high degree of host specificity with plant diseases, with only particular varieties (genotypes) of a single species being susceptible. In contrast to plant diseases, most animal diseases do not display host genotype specificity, although some fungi only cause disease in certain animal species. Apart from innate basal immunity, the immune system of mammals is very different from that of plants. The responses that come into play if the pathogen is not stopped by the innate basal immunity response include the innate complement system, circulating cells such as phagocytes that can internalize and destroy pathogen cells, and adaptive antibody-mediated defenses (Speth et al., 2004).

Fungal plant pathogens have a range of lifestyles and nutritional requirements. Many have a saprophytic phase, and some are obligate, surviving only on their hosts. Some (biotrophs) require hosts to be living, while others (necrotrophs) kill plant tissues. Some fungi are both biotrophs and necrotrophs at different stages during growth in planta. Some fungi such as mycorrhizae and endophytes colonize plants in a symbiotic relationship deriving carbon from photosynthesis by the plant and often conferring drought tolerance on the plant. Fungi that infect animals often have a saprophytic lifestage in the soil, and few if any are obligate.

After invasion, fungi then need to colonize the host, derive nutrition, and avoid or subvert host defense responses. In many plant–pathogen interactions, defense responses include the hypersensitive response, whereby an oxidative burst

Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
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by the plant generates reactive oxygen species associated with the programmed cell death of host cells. This can arrest pathogen growth, particularly that of biotrophs. Necrotrophic fungi can subvert this defense process to derive nutrition from the dead host tissue. Toxins produced by necrotrophic fungi also kill plant tissue. However, they are generally not often important disease determinants. This is also often the case for secondary metabolite toxins produced by fungi that attack animals. Their role may be to protect fungi against predators such as insects, nematodes, and amoebae during their saprophytic growth phase in the soil (Kempken and Rohlfs, 2010).

To complete its lifecycle, a fungus must reproduce and exit the host to find another host. Many animal pathogenic ascomycetes do not have a sexual phase. However, the sexual cycle of basidiomycetes such as Cryptococcus spp. is extremely important in virulence, as discussed by Heitman.42 Both animal- and plant-pathogenic fungi reproduce mitotically within the host. Although plant-to-plant transmission of fungal disease is very common, direct transmission of fungal pathogens between mammalian hosts is unusual. In plant pathogens, conidia (vegetative spore) production usually occurs after infection has been established and lesions have developed. Conidia are spread from plant to plant by wind or in water droplets. However, many pathogens of mammals are transmitted by inhalation as hyphae or as arthroconidia, in dust and wind (e.g., C. immitis) or soil. Increasingly, biofilms, whereby a community of microorganisms attaches to a solid surface, mediate dispersal of fungi in hospital environments (Ramage et al., 2009).

A Fungal Pathogen That Infects Plants and Animals

Given some of the similarities in strategies that fungi use to cause disease in plants and animals, it is perhaps surprising that few organisms have been reported to infect both plants and animals. A single isolate of Fusarium oxysporum f. sp. lycopersici can infect both plants and animals. The animal experimental system involves injection of the tail vein of immunodepressed mice and the plant system involves incubation on intact tomato roots (Ortoneda et al., 2004). Mutants in several genes of F. oxysporum f. sp. lycopersici have been tested for their virulence in the animal and plant models. A mitogen activated protein kinase gene (fmk1) and an Rho1 GTPase, which are both involved in signaling, are not required for virulence in mice, but are essential for virulence in tomatoes. In contrast, the transcription factor PacC, which mediates the environmental pH signal, and the photosensor WC-1, which interacts with a light-sensitive transcription factor, WC-2, are necessary for full virulence in mice, but are not essential for virulence in tomatoes (Martínez-Rocha et al., 2008; Ortoneda et al., 2004; Ruiz-Roldan et al., 2008). These findings highlight redundancy in some signaling pathways

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42 See contributed manuscript by Heitman in Appendix A (pages 226–248).

Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
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Table A13-2 Fungicides Used to Control Plant and Animal Diseases

Fungicide Group Target Plants Animals
Azoles Ergosterol biosynthesis in fungal membrane Widely used Widely used
Echinocandins β 1,3 glucan synthesis Not used New drug
Polyenes: Amphotericin B Ergosterol biosynthesis Not used Yes
Nikkomycin Z Chitin synthesis CHS I Not used Trialed against C. immitis
Strobulurins Mitochondrial cytochrome bc complex Used but resistance can develop: G143A mutations in cytochrome bc Not used; toxic
Bion Salicylic acid analog: Mimics systemic acquired resistance in plants Used in horticulture; expensive Not effective as dependent on plant defense responses

and the complexity of these interactions. It will be interesting to compare global transcriptional analyses of tomato and mouse tissue infected with this fungus to further identify networks and signaling components regulated in response to interaction with two extremely different host types.

Control of Fungal Diseases

Animal diseases are controlled mainly by a well-functioning immune system of a potential host. There are no approved vaccines against fungi, and fungicides often are not efficacious (Ostrosky-Zeichner et al., 2010). Plant diseases are controlled by a combination of approaches. These include management of inocula; for instance, removing infected stubble (crop trash) at the end of the growing season; using fungicides; and introgressing (breeding) resistance genes, often from related species, into varieties. For diseases of both types of hosts, a range of fungicides is employed; choices depend on toxicity and cost/benefit ratio (Table A13-2). Azoles target ergosterol biosynthesis in the fungal membrane and are the most common group of fungicides used for diseases on both types of hosts. Echinocandins, which target synthesis of β 1,3 glucans in the fungal cell wall, are now being used to combat fungal diseases of humans, but would not be effective against fungal plant pathogens given that plant cell walls also have β 1,3 glucans. Nikkomycin Z, which targets chitin synthesis, is being used to control C. immitis infections (Ostrosky-Zeichner et al., 2010),43 while strobilurins,

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43 See article by Galgiani in Appendix A (pages 196–207).

Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
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which target mitochondrial cytochrome bc complex, are deployed against plant diseases, but resistance to them can develop and these molecules have a degree of toxicity toward animals (Bartlett et al., 2002). Bion, which is a salicyclic acid analogue, can induce systemic acquired resistance in some plants (Beckers and Conrath, 2007). This molecule is used more in horticulture than broad-acre grain crops due to its cost.

Deploying resistance genes is an important strategy in controlling plant disease. When corresponding avirulence genes are mutated or deleted, the fungus is not recognized by the plant, and infection ensues. Thus resistance is overcome, often resulting in severe yield losses. Some fungi can more readily overcome resistance than others; such fungi are deemed to have “high evolutionary potential” and they usually outcross prolifically, producing large numbers of wind-borne sexual spores as inocula (McDonald and Linde, 2002). These properties enable such fungi to adapt to selection pressures imposed by extensive sowing of crop varieties with resistance conferred by single genes. Thus the frequency of virulent isolates will increase and resistance in the plant can break down. An example of such a breakdown of resistance occurred in 2003 when resistance in canola to blackleg disease caused by L. maculans broke down (Sprague et al., 2006). This resulted in up to 90 percent yield losses in regions of Australia, costing farmers up to $20 million (B. J. Howlett, unpublished). The resistance had been conferred by a canola gene named Rlm1. The corresponding avirulence effector gene, AvrLm1, has been cloned and is located in a very “plastic” part of the L. maculans genome (Gout et al., 2006).

Recently the genome sequence of this fungus has been acquired (Rouxel et al., 2011). The fungus has a unique structure. More than one third comprises repetitive elements composed of degenerated transposons. Gene-rich regions with high GC content alternate with gene-poor regions with high AT content with sharp boundaries between them. Disease-related genes such as effectors are interspersed in the gene-poor regions among repetitive DNA. This organization contributes to the readily changeable nature of the fungal genome and enables effector genes to be readily gained, lost, or mutated (Rouxel et al., 2011; Van de Wouw et al., 2010). Markers are now available for several other avirulence effector genes (Fudal et al., 2007; Parlange et al., 2007), and thus the frequency of virulent isolates within fungal populations in the field that are virulent toward a particular resistance gene can be monitored. This information allows farmers to rotate the disease resistance genes in the canola varieties that they sow from year to year. This strategy, which can be applied to other plant diseases, can help prevent outbreaks and maximizes the duration of effectiveness of resistance genes.

Closing Remarks

The development of fungal diseases of plants and animals has many parallels. A fungus must overcome many hurdles to successfully invade a plant and cause

Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
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disease. There is a continuum from disease to damage to symbiosis with plant–fungal interactions. The most important properties of a human pathogen are to be able to survive at 37°C and to avoid the immune responses. The use of fungicides to control disease is made on the basis of specificity and after cost/benefit analyses. Such decision making about a broad acreage crop is very different from that about a human patient. An important strategy to control plant disease is to incorporate resistance genes. However, virulence toward this resistance can be selected for, due to the high evolutionary potential of some fungi. Thus resistance genes have to be managed (rotated) by farmers to maximize longevity of resistance.

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Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
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Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
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A14

CLIMATE, GLOBALIZATION, AND TRADE: IMPACTS ON DISPERSAL AND INVASION OF FUNGAL PLANT PATHOGENS

Michael Jeger,44Marco Pautasso,44and James Stack45

Climate change is likely to become a major issue in plant pathology over the coming years. In this overview, we provide recent evidence for this statement. Moreover, we point out the importance for future plant disease management of climate change interactions with other global change drivers, such as increased long-distance trade. Recent advances in aerobiology, together with new molecular tools used in landscape and geographical genetics, can help in addressing the challenges posed by an increasing number of emerging plant diseases worldwide. There is increasing evidence that climate change will be a key issue in how plant pathogens will affect food security and ecosystem health.

Less knowledge is available on the potential impacts of climate change on biological control of exotic fungal plant pathogens. Network theory is a promising tool to improve biosecurity in the face of the increased volumes of traded plants coupled with climate warming. Although there are now many reviews of the literature on the topic of climate change and plant diseases, there is a need to keep up with the rapid development of the subject.

Introduction

The emergence of fungal plant pathogens can occur as the result of at least three processes: (1) the evolution of new genotypes within a pathogen population that is endemic to a habitat or location (e.g., through genetic recombination); (2) the introduction of an exotic pathogen genotype into a new, receptive habitat or location; and/or (3) the natural selection of new pathogen genotypes from an endemic population as a result of changes in host genotype, host species, or external pressures (e.g., environmental conditions, fungicide applications, changes in plant cultural practices). Emergence is a function of introduction (via dispersal or evolution), establishment (i.e., adaptation to the habitat or location), and spread

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44 Division of Biology, Imperial College London, Silwood Park, Ascot, SL5 7PY, U.K.

45 Department of Plant Pathology, Kansas State University, 4024 Throckmorton Plant Sciences Center, Manhattan, KS 66506-5502.

Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
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(i.e., dispersal via natural or human-mediated mechanisms). Weather variables, including wind patterns and storms, and the global trade in plants and plant products facilitate the dispersal of plant pathogens and thus provide opportunities for the emergence of fungal plant pathogens. Further, weather is a primary regulator of invasion into new habitats and locations by plant pathogens. Consequently, climate change may provide unprecedented opportunities for the emergence of fungal plant pathogens through the dispersal of fungal pathogens to new locations and/or through habitat modifications for both pathogens and plants.

Climate, globalization, and trade are major drivers of the dispersal and invasion of fungal plant pathogens, affecting agricultural and horticultural crops, plantation and forest trees, and plants in natural/semi-natural environments. Patterns of weather, notably large-scale air movements, have been implicated in long-range dispersal of several economically important pathogens between and within continents. In some cases, continental dispersal of plant pathogens is tracking and catching up with previous movements of crop plants, in turn accompanying human population migrations that occurred over centuries, sometimes millennia. The rate of movement of humans, crop plants, and plant pathogens has intensified markedly in recent decades with the increased globalization of the world economy. Expansion of trade pathways and technological innovations make possible the production, harvesting, storage, marketing, shipping, and air freight of food crops, ornamental plants, and plant products to an extent that previously was not possible (Figure A14-1).

From a broad perspective, climate and global change are drivers of large-scale ecological perturbations that facilitate novel “biomixing” and “ecological fitting” (Agosta et al., 2010). These phenomena can lead to rapid host switching, the emergence of hybrid pathogens, and invasion of new infectious diseases and pests (Brasier, 2008; Palm, 1999). Changes in plant phenology occur in a variety of ways, depending on species and geography. Such changes impact on plant interactions with fungi in general (Gange et al., 2011; Kauserud et al., 2010) and with fungal pathogens in particular (Grulke, 2011; Marçais et al., 2009). The consequence is that multiple species at many sites need to be studied in order to understand and predict regional change and impact (Ibanez et al., 2010). An alternative strategy has been to focus on multiple drivers of global change on a single species (Baeten et al., 2010; Matesanz et al., 2009; Paajanen et al., 2011). Global environmental change arises from CO2 enrichment, increased nitrogen deposition, climate shifts, biotic interactions, and land use change (Tylianakis et al., 2008). These factors have pervasive effects on antagonistic and mutualistic interactions between plants and fungi and in some cases increase the severity of pathogen infection while weakening mutualisms. Interactions must be expected between tree genetic diversity, variation in phenology, resistance to defoliators and fungal pathogens, increased CO2, and ozone concentration affecting tree growth and mortality; some of these interactions have been reviewed by Pautasso (2009).

Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
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img-275

Figure A14-1 The increase in goods (109 tons × km) moved in the United Kingdom from the 1930s to the 1990s.
SOURCE: Schulz (2004).

In this overview we consider the impact of climate, globalization, and trade on dispersal and invasion of fungal plant pathogens in the broader ecological context described above. We make a distinction between dispersal that is mediated by natural means, mostly atmospheric, and that which is mediated by human intervention of one form or another. This is partly a matter of convenience: we recognize that at some scales of dispersal both means can be important. With respect to pathogen emergence, dispersal is ineffective without establishment, spread, and persistence—the elements of invasion.

Natural Dispersal of Fungal Plant Pathogens

Fungal spores that escape the boundary layer of crop canopies can be transported over long distances subject to their biophysical characteristics and meteorological conditions. The study of such transport belongs firmly in the domain of aerobiology, a discipline that owes much of its development to the effect of aero-allergens on human health (Dallafior and Sesartic, 2010). An account of an early pioneer of aerobiology, Philip Gregory, and the link with fungi of agricultural and human health concern, can be found in Lacey et al. (1997). Weather has

Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
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significant effects on the incidence of aero-allergens, including the abundance and biodiversity of spores of the fungal plant pathogen Alternaria alternata (Magyar et al., 2009). Under conditions predicted by climate change, changes in planting practices and modified crop management may be required to keep allergen concentrations under control (Beggs, 2010).

Even if climate change is becoming important in allergen aerobiology (D’Amato and Cecchi, 2008), vegetation normally will be the main source of fungal spores in the atmosphere. Thirty-two genera of fungi arising from vegetation sources were found across both cultivated and urban areas in three regions in Egypt, with a clear association with weather conditions and many implications for the spread of human and plant diseases (Awad, 2005). Fungal concentration in the atmosphere may not be the best indicator of health risk, which may be more associated with the predominant aero-allergen present (Awad, 2005). The effects of meteorological factors on atmospheric dispersal, in biophysical terms relevant for plant pathogens (including viruses and bacteria), was provided in a recent review (Jones and Harrison, 2004).

In some cases, the dispersal of fungal plant pathogens has been modeled explicitly using biophysical principles. A recent example has been the dispersal of Phakopsora pachyrhizi, causing soybean rust (Andrade et al., 2009). The American continent was free of P. pachyrhizi until 2001. After its introduction in Paraguay, the pathogen rapidly became established throughout Bolivia, Brazil, and Argentina, possibly due to a combination of large-scale cultivation of the plant host, international movement of infected material, and long-distance natural dispersal. Urediniospores of this fungus have been shown to remain viable long enough to be able to travel hundreds of kilometers (Savage et al., 2010). In 2004, Asian soybean rust was reported in the continental United States (Goellner et al., 2010). The fungus now overwinters in warm southern U.S. locations. Spore escape was modeled and combined with a standard large-scale transport model to forecast spore deposition over U.S. soybean production areas. Canopy turbulence and canopy porosity were found to be key determinants of spore escape.

Some effort has been made to test the validity of transport models (Skelsey et al., 2009). Spijkerboer et al. (2002) evaluated the Gaussian plume model (GPM) for predicting and describing spore dispersal over a potato crop. The main purpose was prediction of Phytophthora infestans, but for experimental purposes they used a commonly used fern spore. They concluded that the GPM was not applicable in risk assessments, unless combined with site-specific information at the source, such as spore escape in relation to wind speed.

Other more empirical approaches can also be used for specific purposes. High-speed imaging showed that, by synchronizing the ejection of thousands of spores, ascomycete fungi such as the pathogen Sclerotinia sclerotiorum form an air flow that carries spores around intervening obstacles to atmospheric currents and new infection sites (Roper et al., 2010). Mundt et al. (2009) used simple empirical relationships based on the inverse power law to describe the spread of

Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
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plant diseases such as wheat stripe rust, wheat stem rust, potato late blight, and Southern corn leaf blight. Much of the earlier literature on this approach is cited by the authors. They found that the estimated power law parameter varied little over five orders of magnitude on a distance scale. Evidence was found to support the hypothesis that disease advances through accelerating, rather than constant, waves. Integration of (unmanned) aerial measurements of spore concentration at various distances from the source with simulation of spore flight trajectories is important to develop reliable decision support systems to predict risk of disease spread, as shown for Phytophthora infestans in potato fields in Virginia (Aylor et al., 2011; Techy et al., 2010).

A feature of recent work on dispersal has been the integration with population genetics aspects of pathogen diversity (Hovmøller et al., 2008; Montarry et al., 2010). This can operate at the level of host specialization and biotrophy and the ways in which wind dispersal acts as a survival mechanism—what has been termed “Oases in the desert” (Brown et al., 2002). Equally, the strongly stochastic nature of long-distance dispersal can lead to founder effects in the pathogen population (Brown and Hovmøller, 2002). In such cases, pathogen genotypes that successfully establish in new regions and/or on new cultivars may be different from those at the source—the so-called founder effect. Such effects were found in the migration of the fungus causing black Sigatoka of bananas, Mycosphaerella fijiensis, from Southeast Asia to Africa and Latin America (Brown and Hovmøller, 2002; Rivas et al., 2004). In both of the new regions founder effects were present in the new invasions. Unresolved for this pathogen is the relative importance of air-dispersed ascospores (probably limited) and the movement of infected plant material (largely unrecorded). The fungus Corynospora cassiicola has a wide geographical range in the tropics and subtropics and many plant hosts. Common fungal lineages were widely distributed geographically, indicating long-distance dispersal of clonal lineages, but also previously unrecognized genetic diversity involving some degree of host specialization on some hosts (Dixon et al., 2009). The advent of modern molecular tools in epidemiology provides a step change in both the tracking of dispersal of novel fungal genotypes and in risk assessment of emerging fungal diseases in plants and in animals (Gladieux et al., 2011; Moslonka-Lefebvre et al., 2011).

Climate and Plant Diseases

Weather and climate generally have major impacts on diseases caused by fungal plant pathogens. This topic has had extensive historical coverage that it is not possible to cover in this overview. What is more significant and of immediate concern is how climate change will impact the distribution and severity of diseases caused by known pathogens and the emergence of new invasive pathogens. This topic has also had its fair share of literature reviews: recently, e.g., in the context of structural change in the international horticultural industry (Dehnen-

Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
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Schmutz et al., 2010); the evolution of the phytosanitary regulatory framework (MacLeod et al., 2010); forest health and adaptive management (Parks and Bernier, 2010); the impacts on plant health and carbon sequestration in Australia (Singh et al., 2010); cool season grain legume crops and their diseases (Thomas, 2010); urban trees and their pathogens (Tubby and Webber, 2010); diseases in tropical plantation crops (Ghini et al., 2011); the disease triangle and changes in plant phenology (Grulke, 2011); rice diseases and pests (Haq et al., 2011); diseases of food crops (Luck et al., 2011); the geographical distribution of plant pathogens (Shaw and Osborne, 2011); and plant pathogens in Sweden (Roos et al., 2011).

Here, we refer to climate change to include trends in air composition as well as trends in global warming. Air composition in terms of SO2 concentration has been shown to be associated with the relative prevalence of two important fungal pathogens of wheat (Fitt et al., 2011), a finding made possible by the application of molecular analytical tools to archived plant material from the long-term Broad-balk experiments at Rothamsted Research in the United Kingdom. Elevated atmospheric CO2 and ozone concentrations decreased the incidence of downy mildew disease in soybean, but increased the severity of disease caused by Fusarium virguliforme (Eastburn et al., 2009). Changes in precipitation and temperature resulted in increased disease severity for both diseases, and there were indirect effects due to treatment effects on canopy structure and leaf age. Similar kinds of results were obtained in studies of four pepper diseases (Shin and Yun, 2010). Elevated CO2 and temperature treatments increased the rate of progress for two bacterial diseases, but not for a stramenopile disease (Phytophthora capsici) or a fungal disease (Colletotrichum acutatum).

The relationship between climate change, plant diseases, and food security (Chakraborty and Newton, 2011) considers international cooperation and integrated solutions, including disease management issues, to be essential to meet the food demands of the growing world population. Plant breeding for climate-related traits such as drought avoidance or tolerance (Khan et al., 2010) must also take into account disease resistance. The arable sector will be critical in this respect, with mitigation and adaptation strategies with respect to plant disease control likely to become a key area (Fitt et al., 2010). Because of the variation in crop growth and pathogen environmental requirements, geographical divides in crop yield and productivity may become more pronounced (Butterworth et al., 2010). Climate change will also impact on the incidence of mycotoxins in food (Russell et al., 2009), a much-neglected topic in relation to food security and human and animal health.

Forest health is one area where the impact of climate change and biological invasions is providing a clear signal (Kliejunas et al., 2008; Sturrock et al., 2011; Woods et al., 2010). In turn, emerging forest diseases under climate change can lead to a positive feedback by reducing the carbon stocks of affected forests (Peltzer et al., 2010), as seen in British Columbia with the developing Dothis-

Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
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troma needle blight epidemic following the devastating mountain pine beetle outbreaks. The resilience of northern boreal forests to rapid climate change can be questioned (Chapin et al., 2010), as can forest establishment under conditions of permafrost thaw where fungal pathogens may affect seedling survival (Camill et al., 2010). In forest tree nurseries in Finland, rust, powdery mildew, and other fungal leaf diseases are already causing more problems because of climate warming (Lilja et al., 2010). A more optimistic outlook due to improved adaptive management practices is presented with respect to white pine blister rust (Hunt et al., 2010), where white pines have broad ecological ranges and are less likely to be maladapted thus succumbing to the disease, and hence may be more resilient in the long term. Similar issues will need to be faced with regard to urban trees (Tubby and Webber, 2010) and Mediterranean forests (Attorre et al., 2011; La Porta et al., 2008), where the impact of non-native insect pests and fungal pathogens introduced through trade pathways (see later sections) is already being observed.

The above discussion concerns general issues regarding climate change and plant diseases. There have been many accounts of global/climate change impacts on diseases caused by specific fungal plant pathogens in recent years. A selection of these is cited with summary comments in Table A14-1.

Climate and Beneficial/Biocontrol Fungi

Compared with studies on plant pathogenic fungi, there have been relatively few studies on the effects of climate change on beneficial plant-associated fungi. There has been some consideration of mycorrhizal associations and endophytic fungi, but little on tritrophic mycoparasitic interactions (Singh et al., 2009). In a review of the results of 135 studies, Compant et al. (2010) found that elevated CO2 had a positive influence on the incidence of arbuscular and ectomycorrhizal fungi, whereas the effects on endophytic fungi were more variable. Effects of temperature were idiosyncratic, with positive, neutral, and negative effects equally common. Plant growth-promoting fungi (as with bacteria) positively affected plants subject to a degree of drought stress. Considerable research has been done with tritrophic interactions among arthropod pests and their natural enemies (Thomson et al., 2009), including fungal entomopathogens; similar research is lacking for biocontrol of plant pathogens (Ghini et al., 2008). The expectation must be that prediction will be difficult because of the indirect and direct effects on biocontrol fungi, unless there is a good understanding of tritrophic interactions (Thomson et al., 2009). Biological control of weeds using fungi is simply a case of a plant pathogenic fungus being used for a beneficial purpose. Cirsium arvense is a troublesome weed with world-wide distribution. Climate change will exacerbate invasion and persistence of the weed and make biological control using a variety of pathogenic fungi more difficult (Tiley, 2010).

Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
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Table A14-1 Selected Papers Illustrating the Effects of Climate and Global Change Factors on Specific Pathogen–Host Systems

Pathogen Host Reference Comments
Valsa Alder Worrall Previous oscillation period in damage of ~21 years
melandiscus   et al. (2010) likely to dampen with warmer summers and with no period of recovery
Erisiphe necatrix Grapevine Pugliese et al. (2010) Increase in CO2 concentration did not affect incidence possibly due to increased photosynthesis with higher CO2 and temperature
Phytophthora citricola Beech Fleishmann et al. (2010) Elevated CO2 and low N supply enhanced susceptibility, but host compensation followed
Cercospora spp. Red bud and Sweet bud McElrone et al. (2010) Incidence/severity under elevated CO2 greater with above average rainfall and temperature (one species), but mitigated by higher photosynthetic efficiency
Leptosphaeria maculans and L. biglobosa Oilseed rape Stonard et al. (2010) Geographic variation in species may be exacerbated by climate change with more damaging species expanding in range
Didymella rabiei Chickpea Frenkel et al. (2010) Isolates infecting crop and wild Cicer show different adaptation to high temperature, with the potential for hybrids infecting both hosts over a broader temperature range
Phakopsora pachyrhizi Soybean Del Ponte and Esker (2008) Integrating epidemiological and meteorological factors suggest no restricted overwintering areas in Brazil
Seiridium cardinale Mediterranean cypress Zocca et al. (2008) Pathogen is associated with insect vectors, which are able to reach the range margin and thus the continuous threat of arrival in the expanding range
Cronartium ribicola White pine Frank et al. (2008) Pathogen arrived in New Mexico in ~1970 with upper flow synoptic classification indicating early June 1969 most favourable
Fusarium spp. Wheat Xu et al. (2008) Environmental conditions differentially affect infection and colonization process and the comparative abundance of six toxigenic species in the head blight disease
Fusarium spp. Maize Horvath (2003) Damage by beetles provides conducive growing conditions for the toxigenic fungi, with feeding behavior changing under drought conditions
Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
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Human-Mediated Dispersal of Fungal Plant Pathogens

Thus far we have considered natural dispersal and invasion of fungal plant pathogens, where the main drivers are weather variables, or more generally climate. Although pathogens have always accompanied humans and their crops over the centuries, most notably with the historical migrations of human populations (Guillemaud et al., 2011; Money, 2007), there have been instances where crops moved to new areas have escaped, at least temporarily, the pathogens endemic in the original distributional range of the crops (Mitchell and Power, 2003). Equally, there are cases where crops grown in a new environment have been exposed to novel pathogens. These movements have taken place over centuries with periods of time for adaptation (assisted through plant breeding) or for the pathogen to reencounter the host in a new environment. With the globally connected world that now exists, these time scales are much shorter—decades or, in some cases, much less. The consequence has been the emergence of new fungal plant pathogen species or levels of subspecific variants that were previously unrecorded. For example, since the publication of Erwin and Ribeiro’s 1996 Handbook on the genus, 39 new species of Phytophthora and two species of hybrids have been formally described (Ersek and Ribeiro, 2010). It is unlikely that this increase is due solely to the advent of modern molecular taxonomic techniques. More likely it is due to the ability of this genus to adapt to new hosts in new environments, through encounters made possible by new pathways.

Less spectacular has been the historical accumulation of non-indigenous forest pests in the United States, where some 450 insect and pathogen species have colonized since European settlement (Aukema et al., 2010). Some 16 pathogenic species have caused substantial damage to trees. This finding is more in keeping with analyses over 10 taxonomic groups of alien species (including fungi), which suggested a historical legacy going back at least a century (Essl et al., 2011). This result would imply that Britain is more at risk for invasion of new exotic species than other European countries (as has happened, for instance, for Phytophthora ramorum), given its historical links to its Commonwealth (Figure A14-2). However, even where that is the case, the corollary is that the impact of current global activity will be even more manifest in the decades to come (Crooks, 2005).

Dispersal Through Trade Pathways

Even in a country with an efficient and visible plant quarantine service, such as Australia, there are problems in defining what is present and what is absent (Hyde et al., 2010). These authors make a case for a reinventory of Australia’s plant pathogens and consider five fungal groups in which what were thought to be species were in fact species complexes. Without this level of discrimination concerning what is in the country, it will not be possible to operate an effective quarantine and plant protection service. The often tortuous relationship between plant quarantine and trade barriers can be a problematic political issue (MacLeod

Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
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img-282

Figure A14-2 The world in 1897, with British possessions marked in red.
SOURCE: Cambridge University Library available at Wikimedia Commons.

et al., 2010; Perrings et al., 2010). Emerging plant diseases can be seen as negative externalities deriving from the international trade in plants (Lansink, 2011). The removal of trade barriers, however, can be beneficial and in some cases desirable. For example, seed trade legislation was designed primarily to protect trade and return royalties to contemporary plant breeders. Increasingly, the importance of exploiting the genetic diversity present in cereal land races has been recognized, but to exploit their use fully, changes in legislation will be required (Newton et al., 2010). Also the genetic diversity of target plant pathogens should be used in building comprehensive collections to allow efficient, reliable, and specific diagnostic and detection tools in the national and international trade (Barba et al., 2010).

The Sanitary and Phytosanitary Agreement of the World Trade Organisation specifies that countries cannot regulate against unknown pests, yet many invasive alien forest insects and pathogens were new to science when first recorded in a new environment (Britton et al., 2010). To counter this, effective surveillance systems are required to facilitate early detection; these are lacking in many nations. Britton et al. (2010) propose a global network of sentinel plantings based in historical gardens and arboreta to enable early detection and rapid response

Suggested Citation:"Appendix A: Contributed Manuscripts." Institute of Medicine. 2011. Fungal Diseases: An Emerging Threat to Human, Animal, and Plant Health: Workshop Summary. Washington, DC: The National Academies Press. doi: 10.17226/13147.
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to such incursions. To support early detection, diagnostic capability and capacity are needed to provide rapid and accurate identification of emerging or introduced pathogens. Progress has been made in establ