Fungal Pathogens of Plants

In addition to contributing heavily to annual losses in global crop production,¹⁷ fungal plant pathogens are associated with many notable episodes of human suffering and economic and ecological loss, including:

• Irish Potato Famine: The mid-19th-century epidemic of potato late blight in Ireland led to the Irish Potato Famine, which caused or contributed to the starvation and death of well over 1 million people and the emigration of another 1 million (Money, 2007; Vurro et al., 2010). At the time of the Potato Famine, one-third of Ireland’s population of eight million was dependent upon the potato as a primary food source (Money, 2007) (Figure WO-3). See also Large (1965) and Woodham-Smith (1962).
• Southern corn leaf blight: The 1970 southern corn leaf blight epidemic led to the loss of 710 million bushels of corn—valued at more than $1 billion at the time, or about $5.6 billion in 2009 dollars (Tatum, 1971).
• Dutch elm disease: The impact of Dutch elm disease extends well beyond the death of 100 million mature elm trees in the midle of the 20th century. It not only transformed the landscape of cities and forests, but it has continued to alter associated ecosystem dynamics to this day through reduced food sources and nesting sites for wildlife, altered tree composition, and density (Loo, 2009; Money, 2007).

FIGURE WO-3

Depiction of starving Irish children in 1847 potato famine. by Cork artist James Mahony (1810–1879) SOURCE: Wikimedia Commons.

Fungal plant diseases have far-reaching health impacts that extend beyond the infected plant species—including, but not limited to, negative impacts on associated flora and fauna (Giraud et al., 2010; Loo, 2009). As the Irish Potato Famine illustrated, crop losses can have devastating impacts on populations that are heavily, or solely, dependent on a single food source for their caloric needs. Speaker Jim Stack of Kansas State University observed that with 59 percent of calories consumed by humans being derived from just four plant species (rice, wheat, maize, and potatoes), fungal diseases in these staple crops may catastrophically threaten local and global food security (Strange and Scott, 2005; Vurro et al., 2010). (Dr. Stack’s contribution to the workshop summary report can be found in Appendix A, pages 273–296.)

Fungal Pathogens of Humans and Animals

Given the ubiquity and diversity of fungi, it is perhaps surprising that, of the nearly 1,400 recognized human pathogens, a little more than 20 percent (~ 325) are fungal, and fewer than a dozen are associated with “life-threatening” disease (Casadevall, 2007; Woolhouse and Gaunt, 2007). Historically, fungal diseases of humans have had a lower disease burden than bacterial, viral, or parasitic infections, although this disease burden may be changing. It has been noted that fungal diseases are increasing in incidence in the growing populations of immunocompromised human hosts (Romani, 2004). Once established, fungal diseases are often difficult to treat (Casadevall, 2007; Romani, 2004).

Disease in humans most often results from opportunistic¹⁸ infections (Shoham and Levitz, 2005). Only a few fungal diseases (e.g., coccidioidomycosis, histoplasmosis) are caused by “primary” fungal pathogens¹⁹ that induce symptomatic disease in otherwise healthy people (Casadevall, 2007; Cutler et al., 2007). The “apparent” resistance of humans to fungal disease may be a reflection of the host immune response, coupled with the high basal temperature of mammals, which often exceeds the thermotolerance²⁰ range for many fungi (Casadevall, 2005; Garcia-Solache and Casadevall, 2010; Robert and Casadevall, 2009).

Primary fungal pathogens of humans can also infect other mammals, such as domesticated livestock and companion animals. These diseases are generally not considered contagious and are acquired via inhalation of aerosolized infectious propagules²¹ from environmental reservoirs, such as soil or trees (Casadevall and Pirofski, 2007). According to speaker Luis Padilla of the Smithsonian Conservation Biology Institute, wildlife are also affected by opportunistic and primary fungal pathogens, but the epidemiology of these diseases in wildlife is not well understood. (Dr. Padilla’s contribution to the workshop summary report can be found in Appendix A, pages 296–312.) Two fungal diseases of wildlife, amphibian chytridiomycosis and bat white-nose syndrome, emerged rapidly and unexpectedly over the past several decades. These diseases are associated with unprecedented local and global population declines of amphibian and bat species, and pose serious threats to biodiversity and ecosystem stability (Frick et al., 2010; Wake and Vredenburg, 2008).

Fungal Pathogens as “Invasive Species”

“Fungi are the only group of organisms that have been convincingly shown to cause extinction,” Casadevall remarked, referring to the extinction of the land snail Partula turgida by a parasitic microsporidian fungus (Cunningham and Daszak, 1998). As Casadevall observed, this capacity for destruction may be due, in part, to the fact that “when [fungal pathogens] get into an ecosystem—a vertebrate host, for example—they simply don’t care. They have no need for that host in order to go forward. They will take down every last member of the species.” In contrast, most newly introduced viral and bacterial pathogens in a naïve host eventually attenuate their virulence such that infection does not kill the host. Such adaptations are beneficial to both the host and pathogen in that the host survives and the pathogen avoids an “evolutionary dead end” (IOM, 2009). As noted by Casadevall and Pirofski (2007), the host independence of “environmental” microbes,²² including many fungi, may confer advantages that promote survival and virulence in other niches, including new ecosystems and novel host species.

The term “invasive species” is used to describe “non-native”²³ plants and animals that, when introduced to new environments, reproduce or spread so aggressively that they harm their adopted ecosystems (Carlton, 2004; Dybas, 2004). They compete with native organisms for food and habitat, act as predators or parasites of native species, and cause or carry diseases, often with devastating ecological and economic consequences (Pimentel et al., 2005). As observed by Morse (2004), infectious diseases represent another form of biological invasion—often arising “out of nowhere” with devastating effects.

Discussions during the workshop illuminated the capacity of many fungal pathogens to persist in environmental reservoirs and to readily adapt to new environmental niches and host species. Like invasive species, these fungal pathogens have been able to thrive in new environments and are changing the ecosystem in ways that are difficult to anticipate and even more daunting to prevent (Desprez-Loustau et al., 2007; Giraud et al., 2010; Rizzo, 2005). Given both the links and similarities between invasive species and many pathogenic fungi, it may be useful to view the origins of disease emergence, and the strategies deployed to prevent or mitigate the threats associated with fungal pathogens, through the larger lens of biological invasiveness.

Trade, Travel, and Tourism

The increase in international transportation, travel, and trade associated with globalization in the 20th century has amplified the frequency of interactions between people, plants, animals, and microbes—providing novel opportunities for the rapid introduction, emergence, and spread of infectious diseases (IOM, 2010). The explosive growth of globalization—with dramatic increases in both the quantity and diversity of goods—has been enabled by a simultaneous decrease in travel time (IOM, 2010). Goods can be transported between most places in the world in less time than the incubation period for most infectious diseases (Cliff and Haggett, 2004; IOM, 2010). A study of factors associated with the emergence of diseases in crop plants demonstrated that the majority were spread via trade and travel (Anderson et al., 2004). Stack said this should not be surprising: In 2007 alone, the United States imported more than 48 million tons of agricultural products, only 1–2 percent of which were inspected for possible pathogens and other pests (Becker, 2009; Stack, 2010).²⁸

Local and global transportation of ornamental plants, trees, and timber also contribute to the introduction and spread of fungal diseases. The pathogens responsible for Dutch elm disease and chestnut blight were transported to America in shipments of beetle-infested timber imported from Asia and live chestnut trees imported from Japan, respectively (Money, 2007). Molecular epidemiological analyses of many P. ramorum isolates support the hypothesis that nursery plants infected with Phytophthora ramorum were the initial “source” for the epidemic of sudden oak death that began in California in 1995 (Mascheretti et al., 2008). P. ramorum has since emerged in the United Kingdom and Europe and now infects more than 100 plant species (Grünwald et al., 2008). Stack noted that potential pathogens can be transported in plants, plant associated material (e.g., soil), seeds, and objects manufactured using plant products, such as wooden instruments and packing materials.

Human spatial mobility has increased at least 1,000-fold in the past 200 years, with more people traveling faster, farther, and less expensively than ever (Figure WO-5) (Cliff and Haggett, 2004; Hufnagel et al., 2004).

FIGURE WO-5

Global aviation network. A geographical representation of the civil aviation traffic among the 500 largest international airports in 100 countries is shown. Each line represents a direct connection between airports. The color reflects the number of passengers (more...)

Travelers are now able to easily explore once-remote areas that serve as both sources and sinks for emerging infectious diseases (Choffnes, 2008; IOM, 2010). Adventure travelers intrude on once-remote environments and often make contact with exotic wildlife, encountering microbes that have never before been recognized as human pathogens in the “developed” world (IOM, 2010). These ecotourists become unwitting vectors of disease when they bring these exotic infectious diseases back with them—on their person/clothing/luggage, etc.—when they return to their home countries. If the conditions are favorable, an introduced pathogen may persist and spread (Wilson, 2003). White-nose syndrome, which is currently decimating New World bat populations in the United States, may have been accidentally introduced by recreational cavers from Europe²⁹ (Wibbelt et al., 2010).

Infectious disease pandemics have also been associated with the legal and illegal trade in and transportation of animals (IOM, 2010; Karesh et al., 2005; Smith et al., 2009). Between 2000 and 2006, the United States traded approximately 1.5 billion animals, according to speaker and Forum member Peter Daszak of EcoHealth Alliance. (Dr. Daszak’s contribution to the workshop summary report can be found in Appendix A, pages 188–196.) These animals come from a wide range of species, and most animal imports into the United States come from emerging infectious disease “hot spots” (see Table WO-1) (Jones et al., 2008; Smith et al., 2009).

TABLE WO-1

Number of Individual Animals Traded by the United States (2000–2006).

The international amphibian trade is thought to have contributed to the emergence and global spread of amphibian chytridiomycosis (Catenazzi et al., 2010; Daszak et al., 2003; Fisher and Garner, 2007; Schloegel et al., 2010; Weldon et al., 2004). Since the 1990s, this fungal disease has been implicated in the widespread population declines—including some local extinction events—of more than 200 species of frogs, toads, and salamanders (Fisher et al., 2009; Kilpatrick et al., 2009; Lips et al., 2006; Schloegel et al., 2006; Skerratt et al., 2007).

Speaker Ché Weldon of North-West University of South Africa noted the many points in the global amphibian trade pathway where traded species from different origins come into contact including collector-supplier facilities, breeding facilities, end-user facilities, etc. (Dr. Weldon’s contribution to the workshop summary report can be found in Appendix A, pages 355–367.) Weldon discussed two widely traded amphibian species—the African clawed frog, Xenopus laevis, and the American bullfrog, Rana catesbeiana—that are “asymptomatic” carriers of the causative agent of amphibian chytridiomycosis Bd. X. laevis has been traded internationally since the 1930s (Weldon et al., 2007). Between 1998 and 2004 alone, more than 10,000 specimens of X. laevis were exported from South Africa to over 100 institutions in more than 30 countries worldwide (Weldon et al., 2007). Rana catesbeiana is one of more than 200 amphibian species in the international food trade, which altogether moves tens of millions of individual amphibians around the globe every year (Schloegel et al., 2010). He added that both of these species have now established feral populations in import countries, placing “native” species at risk of exposure to Bd (Weldon et al., 2007).

Aerial Dispersal—Winds and Extreme Weather Events

Limited dispersal of fungal spores carried by the wind is common and is considered a key factor in the local spread of some fungal diseases. Sporadic outbreaks of valley fever have occurred when spores of Coccidioides spp. are swept up from the soil and carried by winds to be inhaled by susceptible hosts. Long recognized as a threat to the health of military personnel stationed in arid regions of California, Valley Fever outbreaks have also been associated with land use changes and occupational or recreational exposures to dust (Chiller et al., 2003; Crum-Canflone, 2007; Warnock, 2006). In 1994, the magnitude 6.7 Northridge earthquake led to an outbreak of valley fever in southern California (Figure WO-7A) (Schneider et al., 1997). More recently, the massive dust storm that swept through Arizona on July 5, 2011 is predicted to cause a similar increase in cases of valley fever (Chan, 2011) (Figure WO-7B). Speaker John Galgiani,³¹ of the University of Arizona, explained that even small winds or soil disturbances can easily loft spore-laden dusts into the air. (Dr. Galgiani’s contribution to the workshop summary report can be found in Appendix A, pages 196–207.) Galgiani further observed that inhalation of a single spore “at the right time” can cause disease. Approximately 40 percent of infected persons develop symptoms, which initially manifest as pneumonia (i.e., cough, chest pain, fever, and weight loss); fatigue; bone and joint pains (“desert rheumatism”); or skin rashes (Hector and Laniado-Laborin, 2005; Tsang et al., 2010). While dust storms and environmental disturbances are clearly an important driver of the spread of Coccidioides spp., Galgiani said that simply living in an endemic region,³² without any direct contact with the soil, puts one at risk of exposure. Yet the fungus is only sparsely distributed. Galgiani noted that, “you can do a lot of desert digging and disrupting and not even be close to the fungus.”³³

FIGURE WO-7

Environmental disturbances and dust storms contribute to the dispersal of fungal spores. (A) Dust from landslides caused by a 5.6 magnitude aftershock of the 1994 Northridge earthquake blows out of the Santa Susana Mountains into the Simi Valley. An outbreak (more...)

Airborne spore dispersal may also synergize with intercontinental trade and travel to rapidly spread diseases between and within continents (Figure WO-6 [blue]). Yellow rust (Puccinia striiformis f. sp. tritici) is believed to have been introduced into Western Australia from southern Europe, in 1979, as an adherent spore on an air traveler’s clothing (Wellings, 2007). Once introduced into Australia, the pathogen spread across Australia’s wheat belt and into New Zealand via wind dispersal (Brown and Hovmøller, 2002). Indeed, winds allow many agriculturally important fungal plant diseases to gradually expand their geographic range (Brown and Hovmøller, 2002).

Pandemics caused by intercontinental aerial dispersal of spores can and do occur—often facilitated by hurricanes and other extreme weather events. Examples include:

• Sugarcane rust (Puccinia melanocphala) is believed to have been introduced from West Africa into America by cyclonic winds (Figure WO-6 [red]) (Brown and Hovmøller, 2002).
• Coffee leaf rust (Hemileia vastatrix) may have been transported via transatlantic winds between Angola to Bahia in Brazil in 1970 (Figure WO-6 [red]) (Brown and Hovmøller, 2002).
• Asian soybean rust was brought into the United States from South America by Hurricane Ivan in 2004 (Schneider et al., 2005).

Some scientists are concerned that the frequency and duration of such extreme weather events could increase with global climate change, which in turn could influence the incidence and intensity of fungal disease outbreaks (Garrett et al., 2006; Greer et al., 2008).

Temperature, Humidity, and Climate Change

Like most microorganisms, fungi are highly sensitive to changes in weather and climate³⁴—particularly temperature, humidity, and wind—that can directly influence their growth, spread, and survival (Harvell et al., 2002). One of the most tragic outcomes of a weather-induced fungal disease outbreak was the Irish Potato Famine, in which a sustained pattern of cool, rainy weather enabled the emergence and spread of the “fungus-like” oomycete,³⁵ Phytophthora infestans, the causative agent of potato late blight (Fry and Goodwin, 1997; Large, 1965; Woodham-Smith, 1962). In 1845 and 1846, late blight led to yield reductions of 40 and 90 percent, respectively, in the potato—at that time Ireland’s staple food crop (Money, 2007). As previously noted, the resulting “Great Famine” led to the death of more than 1 million and the emigration of over 1 million more Irish people, primarily to the United States (Strange and Scott, 2005; Vurro et al., 2010).

When combined with reduced genetic diversity in the host plant, weather can contribute to a “perfect storm” for a devastating agricultural disease epidemic (Rosenzweig et al., 2001; Vurro et al., 2010). Unusually warm, moist weather, coupled with a wholly susceptible host, provided the ideal conditions for the emergence and spread of Helminthosporium maydis (also known as Cochliobolus heterostrophu and Bipolaris maydis), the causative agent of Southern corn leaf blight (SCLB) (Rosenzweig et al., 2001). Over the course of the 1970–1971 growing season, the SCLB epidemic spread from the tip of Florida up to Alberta, Canada, destroying a significant proportion of the corn crop in its path (Ullstrup, 1972). Yield reductions were most severe in the southern states, with many farms experiencing total crop loss. Average yield loss in the Corn Belt states³⁶ was 20–30 percent, with some parts of Illinois and Indiana reporting yield losses of 50–100 percent (Ullstrup, 1972). In the 1970 season alone, the SCLB epidemic led to the loss of 710 million bushels of corn—valued at more than $1 billion at the time (or about $5.6 billion in 2009 dollars) (Tatum, 1971).

Compton Tucker, of the National Aeronautics and Space Administration (NASA) Goddard Space Flight Center, presented data from a variety of satellite and ground sources³⁷ documenting increases in global temperatures worldwide, as well as changes in the atmospheric concentration of carbon dioxide. (Dr. Tucker’s contribution to the workshop summary report can be found in Appendix A, pages 324–342.) He also explained how general circulation models, which simulate the atmosphere, accounting for wind, humidity, clouds, temperature, composition of the atmosphere (e.g., presence of trace gases), and other weather-related variables, can be used to predict where on the surface of the earth (both land and water) temperature and precipitation levels are likely to change.³⁸ According to Tucker, these models predict that over the next century, average surface temperatures will increase by 2–5°C, and regions of the world will get wetter or drier (Figure WO-8).

FIGURE WO-8

Change in precipitation between the 1971–2000 average and the 2091–2100 average in inches of liquid water/year. SOURCE: Geophysical Fluid Dynamics Laboratory, National Oceanic and Atmospheric Administration.

Fungal diseases are influenced by weather fluctuations and display “seasonality”—suggesting the possible influence of long-term climate changes (IOM, 2003, 2008a; Rosenzweig et al., 2001). Stack noted that the onset of potato late blight has been occurring earlier and earlier over the past 20 years in some regions of the world and has resulted in more severe losses and greater mitigation challenges (Hannukkala et al., 2007). In part, this is due to changing temperatures and increased frequency of precipitation (Hannukkala et al., 2007).

Stack observed that modeling studies predict many negative impacts on plant health in response to climate change, including shifts in the range, timing, and severity of fungal diseases of plants³⁹ (Jeger and Pautasso, 2008; Pautasso et al., 2010). A 3°C increase in temperature, for example, is anticipated to alter the phenology⁴⁰ and conditions of the host species enough to result in expansion of the geographic range of Phytophthora cinnamomi, which has already decimated forests across southeastern Australia (Lonsdale and Gibbs, 1996). An enormous effect is predicted for the severity of phoma stem canker (Leptosphaeria maculans) on oilseed rape, with many regions of the United Kingdom expected to experience a 40–50 percent yield loss by 2050 (Butterworth et al., 2010).

Modeling studies predict that it is not just the plant pathogens themselves that are likely to be impacted by continued climate change, Stack observed, but host species as well (Loustau, 2006; Pautasso et al., 2010). Stack remarked that while modeling studies forecast climate change effects on the distribution or severity of many fungal plant pathogens, for most crop plants the future is uncertain—both with regard to plant disease occurrence and the associated impacts on food security.

Host Defenses in Plants and Animals

As discussed previously, fungal diseases of plant or animal hosts involve several common steps (see “Fungi as Pathogens”). When it comes to host defenses, remarked Howlett, animals and plants have several important similarities and differences. Basal innate immunity⁴¹ is an important defense system shared by organisms that infect plants, animals, and insects, with immunity activated by recognition of pathogen-associated molecular patterns (PAMPs) (Nürnberger et al., 2004). Other plant defense systems include a complex physical barrier (a thick and impervious cuticle and cell wall), a repertoire of pathogen-specific resistance genes, and systemic acquired resistance (i.e., if one leaf is infected and the plant does not die, the plant mounts a strong immune defense in the event that another leaf is infected later). By contrast, animals have a less complex physical barrier (i.e., the skin and respiratory surface) and distinct innate immune system components (with the complement system and phagocytes and other circulating cells) as well as a battery of adaptive,⁴² antibody-mediated, immune system defenses (i.e., T and B cells) (Sexton and Howlett, 2006). “Most serious human fungal diseases occur in immunocompromised hosts,” noted Howlett, suggesting that “the mammalian immune defense system is very effective.”

Fungal Disease and the Mammalian Immune System

Fungal disease in humans usually reflects some underlying immune dysfunction (Holland and Vinh, 2009). Speaker Steven Holland of the National Institute of Allergy and Infectious Diseases noted several examples of fungal diseases in otherwise healthy individuals that were ultimately associated with previously unknown primary immune disorders.⁴³ (Dr. Holland’s contribution to the workshop summary report can be found in Appendix A, pages 248–252.) Holland described a healthy and young individual with no previously recognized immunodeficiency who presented to an emergency department with acute shortness of breath that rapidly progressed to severe respiratory distress. The woman was eventually diagnosed with chronic granulomatous disease (CGD).⁴⁴ Infection by the ubiquitous fungus, Aspergillus fumigatus, probably occurred when she was handling soil and plant debris and led to the onset of symptoms (Siddiqui et al., 2007). Holland also reviewed the discovery of rare genetic immune deficiencies that underlie two serious diseases associated with fungal infection: Job’s syndrome⁴⁵ and severe coccidioidiomycosis (Buckley et al., 1972; Davis et al., 1996; Holland et al., 2007; Vinh et al., 2009).

The incidence of opportunistic fungal infections⁴⁶ has increased recently and is associated with the growing populations of vulnerable, immunocompromised individuals (e.g., people living with HIV/AIDS, recent organ transplant recipients) (Romani, 2004). In Casadevall’s opinion, the period since the 1950s should be viewed as a transition decade in which “fungi become more important to human health” (Figure WO-9).

FIGURE WO-9

Incidence of systemic fungal disease has increased since the 1950s. Over the same period, the use of medical technology and the HIV/AIDS pandemic have led to an increased number of immunocompromised individuals. The emergence of systemic fungal disease (more...)

In the 1950s, only about 100 reported cases of disease were caused by Cryptococcus neoformans, Casadevall observed; today, there are about 1 million cases worldwide, mostly among persons with HIV/AIDS (Park et al., 2009). The yeast infection caused by Candida spp. was also uncommon until the 1950s. Many have associated the increase in Candida infections to the increased number of immunocompromised individuals (Dixon et al., 1996). Casadevall speculated that this may also be linked to the introduction of antibiotics, which altered the microbial flora in the human host.

Forum member Fred Sparling of the University of North Carolina, Chapel Hill, remarked “that there was significant cryptococcal disease⁴⁷ in the pre-HIV era,” and that he continued to observe the disease in apparently healthy individuals. Holland agreed and noted that he expected that “we may find new mechanisms for susceptibility that might not be ‘Mendelian,’⁴⁸ because it is not familial, but something that comes on, typically, in adulthood.”

Casadevall considers host immune status in humans so important in the development of fungal disease that, in his opinion, fungal virulence can only be properly defined as a function of it. Casadevall went on to explain that pathogenicity is not an invariant, absolute quality in an infectious disease agent, but that the pathogenicity of a microorganism varies depending on the host and over time (Casadevall, 2007). He reviewed the “damage response framework”—illustrated in Figure WO-10—that was developed by Pirofski and Casadevall as a way to illustrate these concepts (Casadevall and Pirofski, 2003; Pirofski and Casadevall, 2008).

FIGURE WO-10

Damage response framework. When the framework was proposed in the late 1990s, conventional thinking was that the stronger the immune system response, the less damage to the host, as depicted in the two lefthand graphs (Cryptococcus spp. on the top, Candida (more...)

Host damage can derive from either the pathogen (e.g., among immunocompromised individuals with weak immune systems) or the host (e.g., among healthy host individuals whose microbial flora has been disturbed by antibiotic use, triggering a disproportionately strong immune response) (Casadevall and Pirofski, 2003). This model predicts that not just immunocompromised individuals are at risk of disease from fungal infection(s), but also healthy hosts who mount a disproportionately strong immune response (Casadevall and Pirofski, 2003).

Casadevall also suggested that there may be additional “subtle” effects of fungal infection that we are just beginning to observe on a population level. He noted that “we are dealing with things now that we never saw 30–40 years ago. The elimination of many viral and bacterial exposures, especially early in life, without the concomitant elimination of fungal diseases could be a factor in asthma and other atopic diseases.” Relman added, “We are not good at measuring subtle damage. If there are fundamentally important but less obvious forms of damage going on in the environment due to the emergence of fungi, we are not going to be very swift at detecting them, or insightful about understanding their implications.” Infectious propagules⁴⁹ of C. neoformans spp. are everywhere, Casadevall stated, and “we are all exposed to them.” Despite this high level of exposure, Casadevall observed, C. neoformans infection rarely causes illness in non-immunocompromised individuals. Casadevall went on to suggest that asthma in children may be linked to an immune system that has been thrown out of immunological balance by chronic exposure to C. neoformans. To support this hypothesis, Casedevall pointed to two studies: Goldman et al. (2001) reported that C. neoformans infects the majority of immunocompetent children ages 2 and older,⁵⁰ and animal studies that demonstrated that even when asymptomatic, chronic cryptococcal infection predisposes an individual to asthma (Goldman et al., 2006).

Microbial Flora

Host immune defenses against fungal disease extend to their microbial flora. As speaker Vance Vredenburg of San Francisco State University explained, amphibians “wear their defenses on their skin” (e.g., glands produce defensive toxins). (Dr. Vredenburg’s contribution to the workshop summary report can be found in Appendix A, pages 342–355.) Indeed, Brucker et al. (2008) isolated a strain of bacteria (Janthinobacterium lividum) from the skin of the red-backed salamander (Plethodon cinereus) and demonstrated that the bacteria produced antifungal metabolites at concentrations lethal to the causative agent of amphibian chytridiomycosis (Bd) (Figure WO-11).

FIGURE WO-11

Microbial flora as a host defense. Components of a host’s microbial flora can be beneficial to the host by competing for resources or by secreting compounds that affect the survival of other, potentially harmful microbial components of the flora. (more...)

Host Behavior and Thermal Tolerance

Other host characteristics including individual or group behavior can contribute in unexpected ways to disease establishment and spread. Several participants noted that behaviors such as clustering for warmth (amphibians) or during hibernation (bats) may increase opportunities for pathogen transmission between animals. According to Casadevall, having a body temperature that exceeds the thermal tolerance of fungi⁵¹ may also be a significant host defense (Robert and Casadevall, 2009). Casadevall explained that most fungi thrive in the temperature range of 12°C to 30°C. The mammalian body temperature of 37°C, he speculated, may represent a balance between warding off fungal infection (not too cold, or too close to ambient) and keeping metabolic costs down (not too hot) (Bergman and Casadevall, 2010).

Pathogen Adaptation

Many fungi do not need a living host to survive. Fungi are well adapted to exploit winds and water as a means for their dispersal. Moreover, a variety of “environmental” cues trigger fungal growth, sexual and asexual reproduction, sporulation, and continued existence during adverse environmental conditions (Bahn et al., 2007; Judelson and Blanco, 2005; Kauserud et al., 2008; Kumamoto, 2008). In response to environmental stimuli, such as heat or drought, fungal organisms can become “dormant”—an inactive state during which growth and development cease but from which the organisms can be revived—or transform into forms that are resilient to heat, drought, and winds. As discussed at the meeting, the environment and environmental stimuli may also serve as a reservoir and trigger for fungal pathogen adaptation and evolution (Casadevall, 2007; Lin and Heitman, 2006; Stukenbrock and McDonald, 2008).

Sexual reproduction in fungi typically requires the presence of two different mating types (Heitman, 2006). Two signals that regulate the sexual cycle of C. gattii are interactions with plants and extreme desiccation (Lin and Heitman, 2006; Xue et al., 2007). According to Heitman, evidence suggests that when only one mating type is present in an environment, C. gattii will adopt a “same-sex” mating strategy for reproduction (Fraser et al., 2005; Lin et al., 2005; Saul et al., 2008). This adaptability may be a widespread phenomenon, one that enables recombination, the generation of genetic diversity, and the geographic expansion of fungi (Heitman, 2006, 2009).⁵²

Same-sex mating may have contributed to the expansion of C. gattii’s geographical range to Vancouver Island and the U.S. Pacific Northwest (Fraser et al., 2005). Heitman also discussed how recombination between C. gattii lineages of the same “sex” may have resulted in a “hypervirulent” recombinant genotype associated with the outbreak. Two of the three pathogen genotypes associated with the C. gattii outbreak (VGIIa and VGIIc) are considered highly virulent (Byrnes et al., 2010; Fraser et al., 2005). Moreover, VGIIa is considerably more virulent than VGIIa isolates from other parts of the world (Fraser et al., 2005). Although the reason why the VGIIa and VGIIc genotypes are so virulent is unclear, there may be a link between the capacity for mating and production of spores and virulence (Byrnes et al., 2010; Fraser et al., 2005; Lin and Heitman, 2006).

Howlett explained that plant breeders consider fungal pathogens to have a “high evolutionary potential” if organisms undergo prolific sexual reproduction and produce large numbers of genetically diverse spores that then act as inoculum. This capacity leads to frequent breakdowns in a host plant’s resistance to infection by particular strains of a fungal pathogen. Howlett also explained how the agricultural environment plays a role in the breakdown of resistance.

Agricultural crops are large swaths of genetically identical plants that “exert high levels of selection pressure” on populations of fungal strains produced during sexual reproduction. Of the billions of offspring produced, the few fungal strains that can infect these “resistant” plant strains will be amplified with each subsequent disease cycle. As the frequency of virulent pathogens increases, host resistance to disease eventually breaks down. Howlett noted that this is exactly what happened with Leptosphaeria maculans, the causative agent of blackleg in canola. In 2000, a new cultivar⁵³ of L. maculans with a major resistance gene was released on the Eyre Peninsula, Australia. Within 3 years, the fungal pathogen had developed the capacity to overcome the host species’ genetic resistance resulting in yield losses of more than 90 percent (Sprague et al., 2006).

The environment can also serve as a reservoir for pathogen adaptation and evolution. Fungal pathogens that are free living in the environment may acquire what Casadevall called “accidental virulence.” He noted that the soil can be an extreme environment and that soil-dwelling microbes must adapt to rapidly changing, often harsh, conditions (Casadevall and Pirofski, 2007). Traits acquired in this environment, which allow fungal species to survive predation from amoeba and other protozoan organisms, may also contribute to virulence capabilities in hosts never before encountered by fungal pathogens. Casadevall suggested that the concept of “accidental virulence” might best describe how environmentally acquired fungi can be so virulent in “new” mammalian and other host organisms (Casadevall, 2007; Casadevall and Pirofski, 2007).

Phenomenology

C. gattii is a basidiomycetous yeast that colonizes tree bark, decaying wood, and nearby soil and is a cause of cryptococcosis, a potentially fatal infection in humans and animals (Galanis and MacDougall, 2010; Levitz, 1991; Lin and Heitman, 2006; MacDougall et al., 2007). Before the current outbreak in British Columbia, Canada and the Pacific Northwest, the environmental source with which C. gattii had been most often associated was the wood, bark, and detritus of eucalyptus trees (Levitz, 1991). More recent and widespread global surveillance has established that the fungus also colonizes other tree species (Lin and Heitman, 2006).⁵⁴

Individuals become exposed to C. gattii by inhaling the organism or its spores from soils or trees that have been colonized by the fungus (Lin and Heitman, 2006; Sorrell, 2001). Once inhaled, C. gattii can cause severe infection of the lungs and brain, including pneumonia, meningoencephalitis, and cryptococcomas. Unlike C. neoformans, which has become a major cause of death in HIV-infected individuals around the world, C. gattii also infects apparently healthy, immunocompetent individuals (Galanis and MacDougall, 2010). The disease affects a wide variety of humans and animals, but no case of transmission between animals and/or humans has ever been documented (CDC, 2010; Datta et al., 2009a).

Timely diagnosis of a C. gattii infection can be difficult. Patients infected with this fungal pathogen often remain asymptomatic for 6 months or more. When symptoms do present, fungal agents are not commonly considered by physicians when evaluating pulmonary disease in an otherwise healthy patient (Knox, 2010). Treating infected individuals can also be challenging because the disease tends to require prolonged antifungal therapy, sometimes with multiple drug courses (Iqbal et al., 2010; Sorrell, 2001). Some have suggested that when compared with C. neoformans, C. gattii infections tend to require more prolonged and invasive treatment (Sorrell, 2001). Harris remarked that “existing data suggest that not all cryptococcal infections are alike” and “it is not clear which factors are the most influential on the patient’s presentation—the species, subtype, host immune status, or host genetics, or some combination of factors.”⁵⁵

Discovery and Spread

Veterinarians and clinicians first observed cases of C. gattii infections in animals and humans on Vancouver Island in 1999 (Datta et al., 2009a). Until 2004, all known human cases of C. gattii infection in the region occurred in individuals who either resided on or visited the island, specifically its eastern coast (Bartlett et al., 2007). In 2004, cases of C. gattii infections emerged on the British Columbia mainland in humans and animals that had not visited Vancouver Island, suggesting an expansion of the endemic zone of the fungus (Datta et al., 2009a; MacDougall et al., 2007). Also in 2004, cases of humans infected with C. gattii who had not traveled to British Columbia emerged in Washington and Oregon, marking the southern expansion of the fungus into the United States (CDC, 2010; Datta et al., 2009a; MacDougall et al., 2007). The emergence and recognition of a new, more virulent strain of the fungus accompanied C. gattii’s expansion into Oregon (Byrnes et al., 2009, 2010). Recently, investigators found evidence that the outbreak may have also expanded into other states, as researchers have collected isolates from humans and animals in California and Idaho (Iqbal et al., 2010).

Animal sentinels were instrumental to the study of C. gattii emergence and spread in British Columbia (Bartlett et al., 2007). Bartlett noted that “human cases, in all cases, were preceded by veterinary cases.” There are at least three to four times as many pet cases as human cases, she continued, and “it was a veterinarian that tipped us off that we had an outbreak.” Harris remarked that C. gattii “is not a picky pathogen,” infecting a wide variety of animals, including, but not limited to dogs, cats, dolphins, porpoises, elks, llamas, Bactrian camels, alpacas, horses, and sheep.

The exact means by which C. gattii spread from Vancouver Island to the British Columbia mainland, and south to the United States, remains unknown. Researchers suspect that a number of factors may be responsible, including human-mediated dispersal. A 2007 sampling study in British Columbia found C. gattii in areas of its endemic zone that were subject to high foot and vehicle traffic. These observations, combined with the finding of positive fungal samples on peoples’ shoes and in the wheel wells of vehicles that had been driven in fungal endemic regions, support the contention that dispersal may be partially anthropogenic (Kidd et al., 2007). Forestry activities could also facilitate C. gattii dispersal by aerosolizing fungus particles during tree cutting and/or mechanically “seeding” the fungus during the transfer of cut tree products, such as mulch, to new areas (Kidd et al., 2007). In addition to these human-mediated dispersal methods, Kidd and colleagues (2007) suggested that birds and animals might also play a role by passively transporting the fungus during migration.

As reviewed by Bartlett, environmental sampling of endemic areas has helped to describe how C. gattii is distributed in the environment and how easily it might spread. Sampling revealed high levels of C. gattii in the soil as well as its presence in the air, freshwater, saltwater, trees, and even dead wood, such as fence posts, Bartlett remarked. Sampling results have also illustrated the effects of forestry activities on the abundance of C. gattii in the air (measured in colony-forming units,⁵⁶ or CFUs). According to Bartlett, air samples taken in endemic areas, where trees were being removed, revealed a baseline concentration on the order of 100 CFU/m³, compared to 10,000 CFU/m³ during tree chainsawing and wood chipping in the same area (Kidd et al., 2007). Once it is in the air, Bartlett observed “the organism can travel 10 kilometers, easily, probably further than that.” The organism is also resilient: Bartlett noted that she can still isolate viable propagules from sawdust samples taken in 2001.

Origins of the Outbreak in the Pacific Northwest

The origins of the Pacific Northwest outbreak of C. gattii remain a mystery (Datta et al., 2009a). Some investigators have suggested that the fungus was introduced through the importation of contaminated trees, shoes, wooden pallets, or shipping crates (Kidd et al., 2007). Supporting this hypothesis is the finding that the VGIIb minor subtype found in British Columbia and the Pacific Northwest is similar and may be related to VGIIb strains found in Australia (Byrnes et al., 2010). Another origin hypothesis suggests that the VGIIa subtype has existed in the Pacific Northwest for some time. The latter hypothesis is supported by a case reported in 1971 of a patient in Seattle, Washington, who was infected with a VGIIa strain of C. gattii similar to the strain in the current outbreak (Byrnes et al., 2010; Datta et al., 2009a). However C. gattii was introduced, it is clear that the fungus is now established in the region and appears to be evolving into new, more virulent strains, as evidenced by the newly discovered and highly virulent VGIIc strain (Byrnes et al., 2010; Knox, 2010).

Perhaps more interesting than the question of how C. gattii was introduced to British Columbia and the Pacific Northwest is why it has now colonized the region. Bartlett emphasized that the fungus previously was endemic only in areas with tropical or subtropical climates—never in a temperate rainforest. Researchers have speculated that global warming may play a role, with the temperature in the region having increased enough for the fungus to become established (Bartlett et al., 2007; Kidd et al., 2004). Indeed, between 1998 and 2004, British Columbia experienced six consecutive seasons of above-average temperatures, with increases of more than 3°C in some seasons (Kidd et al., 2004).

Another possible explanation for C. gattii’s establishment in British Columbia and the U.S. Pacific Northwest is that the organism itself has adapted such that it can now successfully colonize a “novel” environment. Investigators have found environmental isolates of C. gattii in trees that had never previously been found to harbor the fungus, such as the Douglas fir and Western hemlock (Datta et al., 2009a). Bartlett remarked that the environmental sampling data may help to define C. gattii’s ecological niche in British Columbia. The distribution of C. gattii in the environment, thus far, appears heterogeneous, with colonization levels differing significantly in regions such as the west and east coasts of Vancouver Island, which, observed Bartlett, are “dramatically different in terms of rainfall, soil type and vegetation” (Figure WO-13).

FIGURE WO-13

Environmental sampling for Cryptococcus gattii in British Columbia (2001–2009). Colonization levels of C. gattii reflect a heterogeneous distribution of C. gattii in the environment. Biogeoclimatic zones are also indicated. SOURCE: Bartlett (2010). (more...)

Further research into the reasons why C. gattii emerged in the temperate rain-forest of the Pacific Northwest is needed because it could help researchers predict the fungus’s future spread and further the scientific community’s understanding of how environmental pathogens establish themselves in new environmental niches.

Molecular Epidemiology, Virulence, and Drug Resistance

Heitman explained that C. gattii spans four genetically isolated species groups: VGI, VGII, VGIII, and VGIV. Examination of the molecular genotypes of fungal isolates from infected patients reveals that nearly all of the observed infections in British Columbia and the U.S. Pacific Northwest have been caused by one molecular subtype of C. gattii—the VGII type (Byrnes et al., 2009). In other regions of the world where C. gattii is endemic, two other molecular subtypes predominate—VGI and VGIII (Byrnes et al., 2009; Kidd et al., 2004). The VGII genotype is further subdivided into three subtypes: the majority genotype VGIIa, which is unique to the Pacific Northwest region and not found in other endemic regions; the less common VGIIb genotype; and the VGIIc subtype, which has appeared in Oregon within the past several years (Byrnes et al., 2010; Kidd et al., 2004).

Some researchers have suggested that the predominant VGIIa and the newly discovered VGIIc C. gattii subtypes are more virulent than strains found in other endemic countries such as Australia (Byrnes et al., 2010). This is supported by the high rate of C. gattii infections in the current outbreak, which, at 25.1 cases/million people on Vancouver Island, is among the highest in the world (Galanis and MacDougall, 2010). Bartlett cautioned, however, that the high rate of C. gattii infection could be a result of increased surveillance or exposure, not increased virulence.

Recently, researchers have compared the drug susceptibility of the three VGII subtypes found in British Columbia and the Pacific Northwest to the more common VGI and VGIII genotypes. The VGIIc strain was found to be significantly more drug resistant to nearly all of the tested antifungal compounds (voriconazole, fluconazole, flucytosine, and amphotericin B) than the VGI or VGIII genotypes. The VGIIa and VGIIb strains were also observed to be more resistant to some antifungal drugs (fluconazole, flucytosine, and amphotericin B for VGIIa; fluconazole for VGIIb) than the VGI and VGIII strains, though their levels of resistance were lower than those of the VGIIc strain (Iqbal et al., 2010).

Phenomenology

Geomyces destructans hyphae⁵⁸ and conidia⁵⁹ invade the hair follicles and sebaceous and sweat glands of bats hibernating in caves and mines with seasonal temperature ranges between 2°C and 14°C (Blehert et al., 2009). The skin of affected bats does not typically show signs of inflammation or an immune response at the site of fungal invasion (Meteyer et al., 2009). Hibernating bats infected with WNS often have severely depleted fat reserves, which are critical for successful hibernation (Blehert et al., 2009).⁶⁰

Researchers have not yet confirmed whether Geomyces destructans is the primary pathogen that causes WNS and the eventual death of affected bats, or if it is an opportunistic infection that invades animals already immunocompromised by some other, yet to be defined pathogen (Puechmaille et al., 2010). How WNS kills bats is also unclear. Infection by the pathogen may irritate the animals, rousing them from hibernation, and causing them to deplete their fat reserves to such an extent that they are unable to survive through the winter (Blehert et al., 2009; Cryan et al., 2010; FWS, 2011). Speaker David Blehert of the National Wildlife Health Center at the U.S. Geological Survey presented recent research suggesting that the fungus causes damage to the wing epidermis and skin structures that help protect against water loss, causing bats to lose too much water to survive their winter hibernation (Cryan et al., 2010). (Dr. Blehert’s contribution to the workshop summary report can be found in Appendix A, pages 167–176.)

Discovery and Spread

WNS was first documented in February 2006 in a single cave near Albany, New York (FWS, 2011). Since then, WNS has spread rapidly across the United States and Canada, killing more than a million bats of six different species, making it the “worst wildlife health crisis in memory” (FWS, 2011). WNS is thought to spread primarily through bat-to-bat contact. However, human cavers and tourists may also be contributing to the spread of this pathogen by inadvertently transporting spores of Geomyces destructans from cave to cave on their clothing and equipment (Frick et al., 2010; FWS, 2011).

During routine hibernacula surveys in 2006–2007, biologists with the New York Department of Environmental Conservation discovered bats exhibiting signs of WNS in five caves, all within a 15-km radius of what is now recognized as the likely “index” site near Albany, New York, recounted Blehert. The following winter (2007–2008), researchers reported the discovery of WNS at 33 sites across Connecticut, Massachusetts, New York, and Vermont—all within a 210 km radius of the index site (Blehert et al., 2009). By the end of the 2010–2011 hibernation season, the disease had been confirmed in bats in 18 U.S. states (FWS, 2011). The disease has also spread north into Ontario, Quebec, New Brunswick, and Nova Scotia in Canada (FWS, 2011) (Figure WO-15).

FIGURE WO-15

Spread of bat white-nose syndrome (WNS) in North America as of June 6, 2011. SOURCE: Figure courtesy of Bat Conservation International, www.batcon.org.

Blehert remarked that the magnitude of the mortality events being observed is not only unprecedented among U.S. bat species but also among the 1,100-plus bat species worldwide. Indeed, a recent modeling study predicted that for one of the most common bat species in North America—the little brown bat (Myotis lucifugus)—there is a 99 percent chance of regional extinction within the next 16 years as a result of mortality from white-nose syndrome (Frick et al., 2010).

Affected New World bat species include the big brown bat (Eptesicus fuscus), Eastern small-footed bat (Myotis leibii), little brown bat (M. lucifugus), Northern long-eared bat (M. septentrionalis), endangered Indiana bat (M. sodalis), and tricolored bat (Perimyotis subflavus) (Figure WO-16). Although disease pathology has not been confirmed, DNA from G. destructans has been detected on skin samples from the endangered grey bat (M. grisescens), the southeastern bat (M. austroriparius) and the cave bat (M. veilfer) (FWS, 2011). Investigators are concerned that the endangered Virginia big-eared bat (Corynorhinus townsendii virginianus) may also be “at risk.” Although the fungus has been detected on other bat species that share their hibernacula, there has yet to be confirmed case of an infected animal (FWS, 2011).

FIGURE WO-16

Species affected by bat white-nose syndrome (WNS). SOURCE: Merlin D. Tuttle, Bat Conservation International, www.batcon.org.

Investigators are concerned that WNS could eventually spread to infect all 25 of the hibernating bat species native to the United States, threatening more than 50 percent of the native U.S. bat populations (Bat Conservation International, 2010). In late May 2010, FWS officials reported that a live bat from Oklahoma was PCR-positive for G. destructans DNA. This finding alarmed many, because the infected species, the cave bat M. velifer, frequently shares hibernacula with other bat species with migratory ranges that extend across the western United States into Mexico, increasing the potential for further spread of the disease to the west and south (Bat Conservation International, 2010; Oklahoma Department of Wildlife Conservation and FWS, 2011). This finding, however, was not confirmed by fungal culture or histopathology; and, to date, WNS has not been confirmed in states west of the Mississippi River.

Bat researchers and human cavers may also be contributing to the rapid spread of this pathogen within and across states. Blehert noted that the index site for WNS, Howes Cave, is connected to Howe Caverns, a commercial tourist cave that entertains up to a quarter-million visitors per year. Although there are no data supporting (or refuting) the hypothesis that humans are serving as transmission “vectors,” Blehert noted that the U.S. Fish and Wildlife Service recommends that all people who enter caves (e.g., researchers and speleologists) employ decontamination protocols for potentially contaminated clothes and equipment.

Origins

The origins of WNS and its relationship to G. destructans remain unknown (Qaummen, 2010). However, recent evidence from Europe and characterization of the newly described pathogen may provide clues. In early 2010, researchers published several reports confirming that a number of bats (all Myotis spp.) in France, Hungary, Germany, and Switzerland, while infected with G. destructans, remained healthy (Puechmaille et al., 2010; Wibbelt et al., 2010). Speaker Gudrun Wibbelt, of the Leibniz Institute for Zoo and Wildlife Research, reported that not all of the European hibernacula surveyed have infected bats, and of those that do, often only a small number of bats within the colony are infected. (Dr. Wibbelt’s contribution to the workshop summary report can be found in Appendix A, pages 368–403.) Researchers have now confirmed the presence of G. destructans on 8 species of Old World bats in 12 countries in Europe (Puechmaille et al., 2011).⁶¹ In addition, photographic evidence suggests that the fungus was present on bats in Europe at least as early as the 1980s (Wibbelt et al., 2010). These findings, which have important implications for future WNS research, could help explain the origins of the disease and may also provide clues for understanding the mechanisms of the infection.

One possible interpretation of these data is that G. destructans may have originated in Europe and that Old World bats and this pathogen may have co-evolved (Puechmaille et al., 2011; Wibbelt et al., 2010). This hypothesis is supported anecdotally by reports from routine winter bat surveys in Europe from the past 30 years. The surveys occasionally noted the presence of a white fungus similar in appearance to G. destructans on otherwise healthy bats, Wibbelt noted. If European bats have coevolved with the fungus, they might be able to muster a sufficient immune response to control and survive infection by G. destructans (Puechmaille et al., 2010). Some have proposed the possibility that the microbial flora of bat skin or other abiotic surfaces in European hibernacula “may have also coevolved to incorporate G. destructans as a non pathogenic component of the microbial community” (Wibbelt et al., 2010). Wibbelt also reported the possibility that Old World bats may only be colonized in a superficial fashion on the outer epidermis without any invasion into deeper tissues.

A second possible interpretation of the discovery of healthy European bats infected with G. destructans is that disease transmission in Old World bat populations may be affected for some biological or behavioral reason (Puechmaille et al., 2010, 2011; Wibbelt et al., 2010). European bats tend to hibernate in relatively small groups (rarely more than 100 individuals per cluster), Wibbelt explained. This might make it more difficult for the disease to spread. In the United States, bats hibernate in groups that can reach into the hundreds of thousands (Wibbelt et al., 2010).

Additional explanations for why European bats infected with G. destructans do not succumb to WNS include the possibility that the fungal strain in the United States is more virulent than the strain in Europe or that G. destructans is not the primary cause of death in WNS. However, Blehert did note that his team’s and others’ diagnostic investigations of infected New World bats had ruled out toxins, parasites, and known viral and bacterial pathogens as associated with WNS (Blehert et al., 2009; Chaturvedi et al., 2010; Gargas et al., 2009). Wibbelt observed that G. destructans isolates from North America and Europe appear identical in morphology and in the sequence of two genes (ITS and SSU) commonly used as a marker to distinguish between different species (Puechmaille et al., 2011). Further research will be necessary to determine the true cause of differences between North American and European bats infected with G. destructans.

Phenomenology

B. dendrobatidis is an aquatic chytrid fungus—an early diverging class of fungi—that infects keratinized epidermal cells of amphibians, causing rapidly progressing and deadly chytridiomycosis in susceptible species (Fisher et al., 2009). First identified in 1998 and characterized in 1999, Bd is unique among other chytrids (Berger et al., 1998; James et al., 2006; Longcore et al., 1999). It is one of only two known chytrid fungi to parasitize vertebrates and the only known species to infect the keratinized skin of living amphibians (Berger et al., 1998; Fisher et al., 2009). Bd’s asexual spore is a “free living” and motile zoospore, possessing a single flagellum that allows the spore to travel small distances (usually less than 2 cm) and thrive in aquatic habitats such as streams and ponds (Fisher et al., 2009; Kilpatrick et al., 2009; Kriger and Hero, 2007; Rosenblum et al., 2010).

According to speaker Vance Vredenburg, from San Francisco State University, investigators believe that amphibians become infected with Bd through both casual contacts with zoospores in the water as well as through direct animal-to-animal transmission. Once infected, the susceptible animals carry Bd for 24 to 220 days before they succumb to chytridiomycosis (Lips et al., 2006). Vredenburg remarked that the susceptibility to Bd colonization and subsequent development of chytridiomycosis varies widely across species, populations, and individuals. Indeed, laboratory experiments have found mortality rates from 0 to 100 percent, depending on temperature, species, and age of the infected animals (Berger et al., 2005; Daszak et al., 2004; Kilpatrick et al., 2009; Lamirande and Nichols, 2002; Woodhams et al., 2003).

Many of the most notable and rapid declines in amphibia have occurred among those populations living at high altitudes in mountainous regions, leading some to associate outbreaks of fatal chytridiomycosis with cooler climates and high altitudes (see Fisher et al., 2009). Several amphibian species that live near sea level also have experienced notable population declines from this fungal disease, however, suggesting that this association may be an “oversimplification of a complex host–pathogen relationship” (Fisher et al., 2009). The preponderance of the evidence supports the observation that Bd does prefer cooler temperatures, growing and reproducing between 4°C and 25°C (Berger et al., 2004; Drew et al., 2006; Kilpatrick et al., 2009; Kriger and Hero, 2007). Indeed, the available evidence suggests that the virulence of Bd is inversely related to temperature, perhaps in a species-dependent manner (Fisher et al., 2009; Walker et al., 2010).

The specific mechanisms by which some species suffer rapid declines when Bd is introduced while others are able to tolerate varying levels of infection without the development of disease—or even resist infection entirely—remain unknown. Nevertheless, chytridiomycosis apparently results from the complex interplay of pathogen, host, and environmental factors (Briggs et al., 2010; Rosenblum et al., 2010; Vredenburg et al., 2010; Walker et al., 2010). Recounting his investigations of disease dynamics in multiple populations of Bd-infected amphibians (Rana muscosa and Rana sierrae) in Sequoia and Kings Canyon National Parks in the Californian Sierra Nevada, Vredenburg noted that even populations of the same species can have “very different outcomes of infection, depending on where you are in the Sierra Nevada.”⁶²

Although numerous studies have established that Bd is the proximate cause of the observed declines in amphibian populations around the world, the specific ways by which chytridiomycosis causes death remains a mystery (Figure WO-18).

FIGURE WO-18

A chytridiomycosis outbreak in southern mountain yellow-legged frogs (Rana muscosa), Sixty Lake Basin, Kings Canyon National Park, CA, USA. SOURCE: Photo kindly provided by V. Vredenburg (August 15, 2006).

In susceptible animals, chytridiomycosis causes a thickening of the skin (hyperkeratosis), abnormal proliferation of epidermal cells (hyperplasia), and sometimes increased skin shedding (Berger et al., 1998, 2005). Only rarely do infected animals have visible skin lesions or other pathologies generally associated with lethal infections. Bd may disrupt the normal regulatory functions of amphibian skin, causing osmotic imbalances, electrolyte depletion, and ultimately death (Rosenblum et al., 2010). Although some investigators have suggested that Bd might release lethal toxins, no specific toxin has been identified (Fisher et al., 2009; Rosenblum et al., 2010). As discussed by Vredenburg, investigators are also exploring the possibility that other microbial components of the amphibian skin microbiome can contribute to disease mitigation (Harris et al., 2009).

Is Bd a Newly Emergent or Previously Endemic Pathogen?

One of the many unresolved debates among scientists who study this disease is whether Bd is a newly emerging pathogen that recently spread across the globe (known as the novel pathogen hypothesis) or if it has existed for some time as a commensal or symbiont of amphibians, and only recently became more virulent as a result of environmental changes that altered its relationship with its hosts (the endemic pathogen hypothesis) (Fisher and Garner, 2007; Fisher et al., 2009; Rachowicz et al., 2005; Rosenblum et al., 2009, 2010).

What is known is that Bd has existed at a low prevalence in some populations of amphibians since at least the 1930s, but massive species declines were not reported until recently (Weldon et al., 2004). A number of studies established significant associations between Bd, declines in amphibian populations, and global warming (Fisher et al., 2009; Pounds et al., 2006). Fisher remarked that more research is needed to illuminate the specific mechanisms through which climate change or other environmental factors might influence the host–pathogen dynamic between Bd and certain species of amphibians (Fisher et al., 2009). The preponderance of the evidence generated to date supports the novel pathogen hypothesis—Bd appears to be a newly emergent disease (Fisher et al., 2009; James et al., 2009; Rosenblum et al., 2009, 2010).

The most compelling evidence in support of the novel pathogen hypothesis comes from recent studies mapping the genome of Bd isolates from around the world (Rosenblum et al., 2009). All global diversity of Bd can be explained by a single ancestral diploid strain that subsequently spread across the world, diversifying through mitotic and meiotic recombination (James et al., 2009; Morgan et al., 2007). According to Fisher, investigators still do not know the origin of this single diploid strain; however, all known Bd strains most likely came from a small, genetically “bottlenecked” ancestral population (James et al., 2009). These data provide strong, inferential evidence in support of the theory that Bd is a recently emergent fungal pathogen that rapidly expanded its geographic range across the globe in the first half of the 20th century (Fisher et al., 2009; Rosenblum et al., 2009).

Origins and Spread

Despite its widespread “footprint,” little is known about Bd’s origins and how it has spread across the planet (Fisher et al., 2009; Kilpatrick et al., 2009; Rosenblum et al., 2009, 2010). Bd clearly is widely distributed, but its distribution is not homogeneous (Fisher et al., 2009). Indeed, a number of areas around the world—most notably Madagascar—have a rich diversity of amphibian species, but Bd has not spread there yet (Fisher et al., 2009; Weldon et al., 2008).

Skin samples from the African clawed frog (Xenopus laevis) collected in 1938 on the Western Cape of South Africa provide the earliest evidence of Bd-associated amphibian skin infections (Weldon et al., 2004). As noted by Weldon, X. laevis is able to asymptomatically carry the fungus without developing chytridiomycosis and has not experienced any population declines as a result of Bd infections. In the early 20th century (between the 1930s and 1960s), the frogs were globally marketed and used in human pregnancy assays (Weldon et al., 2004). X. laevis is also widely used as a model organism in developmental biology because its metamorphosis from zygote to tadpole can be easily observed in the laboratory (Weldon et al., 2004). The global trade in African clawed frogs has led investigators to suggest that Bd originated in Africa and that X. laevis served as the natural reservoir host for this fungal pathogen (Weldon et al., 2007). The fungus then spread to new hosts and environmental niches as a result of the human-mediated movement of amphibians, globally and locally (Weldon et al., 2004).

Some studies have cast doubt on the hypothesis that the fungus originated in Africa (Fisher et al., 2009; Rosenblum et al., 2009, 2010). As noted by Fisher, studies of the genetic diversity of Bd in different species found that fungal isolates from North American bullfrogs (Rana catesbeiana), another species that is widely traded and is able to carry Bd without any associated morbidity, are significantly more genetically diverse than isolates from African clawed frogs (James et al., 2009). This is contrary to what researchers would expect if African clawed frogs were the original reservoir for Bd and suggests that the fungus origins may be outside of Africa (Fisher et al., 2009; Rosenblum et al., 2010). Fisher observed that genomic sequencing of global Bd isolates reveals that “what we have been calling Bd actually consists of at least three highly divergent lineages.”

A number of theories have been put forward to explain Bd’s spread around the globe (Fisher and Garner, 2007). As Weldon observed, many believe the spread was at least partially human-mediated, the result of international trading and transportation of amphibians that were infected with the fungus, but did not show signs of obvious illness. Anthropogenic spread through the amphibian trade, however, cannot explain how the fungus was introduced into and spread across environments where there has been minimal human activity.

Phenomenology

P. ramorum is a fungus-like oomycete⁶³ or “water mold” that thrives in the cool, wet climate of California coastal forests (Kliejunas, 2010). In contrast to most species of Phytophthora, P. ramorum exhibits a remarkably broad host range, infecting a variety of tree and non-tree species—ranging from hardwood and conifer trees, to shrubs and leafy plants⁶⁴ (Brasier and Webber, 2010; Grünwald et al., 2008). P. ramorum colonizes the leaves of many plants and the inner bark and sapwood of trees. The fungus can also survive in a dormant state in decaying matter, such as leaf litter on the forest floor, and in the soil (Parke and Lucas, 2008). Rizzo remarked that within forest ecosystems in California, “once we started looking, we started finding it on just about every plant we looked at, ranging from ferns to redwood trees.”

Infection occurs when spores and zoospores—dispersed by winds or water—come into contact with susceptible plants. Moisture is not only essential for the production of infectious propagules, but free water on plants—from fog, dew, or rainfall—enhances infection and dispersal (Kliejunas, 2010; Rizzo and Garbelotto, 2003). Indeed, monitoring streams that are “baited” with Rhododendron leaves, Rizzo said, has been an effective method for early detection of this pathogen in forest ecosystems. Disease manifests differently depending on the plant species and the part of the host plant that is infected (Grünwald et al., 2008) (Figure WO-19 and Table WO-2). Bleeding lesions and stem cankers develop in forest trees, followed by rapid declines and “sudden” death. Ramorum blight causes shoot-tip dieback and leaf spots in woody shrubs and ornamental trees. Unlike sudden oak death, ramorum blight rarely kills its host (Grünwald et al., 2008).

FIGURE WO-19

Sudden oak death and ramorum blight. (A) Aerial view of a forest in Humbolt County with patches of trees dying of sudden oak death; (B) canker on tanoak; (C) signs of ramorum blight on a variety of host plant species. SOURCE: Rizzo (2010).

TABLE WO-2

Disease Types and Associated Symptoms Caused by P. ramorum.

Discovery and Spread

Observers first spotted symptoms of sudden oak death and ramorum blight in the mid-1990s in Marin County, California, and in European nurseries (Mascheretti et al., 2009). In 2000, researchers identified the common causative fungal pathogen for these diseases, P. ramorum (Garbelotto and Rizzo, 2005; Kliejunas, 2010). Trade and transportation of ornamental plants enabled the introduction of this novel pathogen into the United States and Europe. P. ramorum has since spread north to southern Oregon and south to Big Sur in California, where tree mortality rates are among the highest (Kliejunas, 2010). In 2009, symptoms of the disease were first detected in Japanese larch trees in the English counties of Devon, Cornwall, and Somerset (Forestry Commission, 2010). By August 2010, the disease had spread to Japanese larch trees in the counties of Waterford and Tipperary in Ireland (Brasier and Webber, 2010; Forestry Commission, 2010). Many now consider this disease to be a serious threat to Japanese larch and possibly other tree species in Europe (Brasier and Weber, 2010).

The ornamental trade and other anthropogenic factors continue to serve as a source for global and local spread of P. ramorum. In 2004, “pre-symptomatic” Rhododendron plants infected with the fungal pathogen were discovered in a Southern California plant nursery, but not before the nursery had shipped potentially infected plants to more than 40 states (Figure WO-20) (Goss et al., 2009).

FIGURE WO-20

P. ramorum “migration” pathways. The ornamental plant trade continues to serve as a major pathway for the spread of P. ramorum. Arrows indicate confirmed P. ramorum-positive nursery trace forwards. Blue arrows are 2004 trace forwards and (more...)

As Rizzo noted, many of the fungicides used in the nursery trade do not kill the pathogen, but rather suppress symptoms; this is probably how P. ramorum spreads over very long distances via the plant trade. Leaves, flowers, and stems of infected plants carry the pathogen, which is also spread by the transportation of plant or associated plant materials, including soil (Kliejunas, 2010). Waterways are an effective means of spreading P. ramorum. Investigators have detected the pathogen in streams contaminated with run-off from infected nurseries (Kliejunas, 2010). P. ramorum may also provide an interesting case study of humans acting as vectors for fungal disease, as sudden oak death “has potentially been spread to new areas by hikers, mountain bikers, and equestrians” (IOM, 2010). Rizzo also pointed to the movement of “green waste,” that is, compost, firewood, mulch, and other plant matter, as an another possible means of spreading the fungus within and across ecosystems.

As of 2009, the U.S. Department of Agriculture (USDA) Animal and Plant Health Inspection Service reported detection of P. ramorum in 11 states (Alabama, California, Georgia, Maryland, Mississippi, New Jersey, North Carolina, Oregon, Pennsylvania, South Carolina, and Washington) at 30 sites (24 nurseries and 6 in the landscape) (Kliejunas, 2010). The pathogen’s eastward spread has placed Eastern native forests at risk and has led many experts to worry that P. ramorum could have ecological consequences comparable to those of chestnut blight and Dutch elm disease (Goss et al., 2009).

The distribution of sudden oak death in California forests is heterogeneous, Rizzo noted. Weather—including winds, temperature, humidity—contribute to the pathogen’s establishment and spread within a new environment (Kliejunas, 2010). Infected tanoaks do not contribute significantly to disease spread, but, as Rizzo observed, many other plants in forested areas (e.g., understory and canopy trees) are also host species for P. ramorum. The pathogen’s very broad host range, therefore, is also an important transmission factor for disease. In California forests, the California bay laurel (Umbellularia californica) drives transmission of the pathogen (Kliejunas, 2010; Rizzo and Garbelotto, 2003). Leaf tips of these trees serve as a prolific source of inocula. Dissemination of spores can occur via wind and rainsplash (Rizzo and Garbelotto, 2003). In the United Kingdom, Japanese larch trees not only developed canker and died but the leaves also served as foliar hosts and the source of massive amounts of inocula (Brasier and Weber, 2010).

Origins

The lack of reports of P. ramorum in the United States before the mid-1990s, combined with its aggressiveness and limited geographic range relative to its hosts’ distribution, suggests that P. ramorum was only recently introduced into the United States (Grünwald et al., 2008). Genetic evidence supports the hypothesis that the U.S. and European strains are distinct and that both strains likely originated from a third, as yet unknown, source (Grünwald et al., 2008). Molecular studies have identified three main lineages: (1) EU1—found in both North American and European nurseries and some European woodlands; (2) NA2—found in California and Washington nurseries; and (3) NA1—found in North American nurseries and in California and Oregon forests (Grünwald et al., 2008).

An alternative explanation is that P. ramorum may have existed in California for many years, but only recently emerged because of changes in the environment (e.g., increased temperatures, fire suppression, modifications in land use patterns) that have led to an increased prevalence and aggressiveness of the pathogen (Rizzo and Garbelotto, 2003). Native P. ramorum or other Phytophthora sp. may also have evolved into a more virulent form, may have undergone a change in host specificity or preference, or may represent an entirely new hybrid species (Rizzo and Garbelotto, 2003). It has been reported that a novel Phytophthora hybrid—a cross between P. cambivora, an oak pathogen, and P. fragariae-like isolates, a strawberry pathogen—emerged in Europe in the 1990s and has killed thousands of alder trees (Alnus spp.) (Brasier et al., 1999). Given the large number of Phytophthora spp. in California agricultural and horticultural environments, a hybrid origin for P. ramorum is certainly a possibility; although other explanations are also possible (Rizzo and Garbelotto, 2003; Tyler et al., 2006).

Phenomenology

Puccinia striiformis f. sp. tritici (hereafter, P. striiformis) is an obligate, basidomycetous, pathogen of wheat. It infects the green tissues of host plants, causing damage to leaf blades, and reducing the yield and quality of produced grains and seeds (Chen, 2005). Severe infections of yellow rust also stunt the growth of wheat plants. Average reported yield losses due to yellow rust range from 10 to 70 percent; yield losses in highly susceptible cultivars can reach 100 percent (Chen, 2005). Weather—including humidity, rainfall, temperature, and wind—is critical for developing favorable conditions for fungal infection and growth (Chen, 2005).

Named for the yellow pustules of powdery spores (urediniospores) that appear as “stripes” on the leaf blades of infected plants, yellow “stripe” rust is an important “cooler climate” wheat disease (Chen, 2005) (Figure WO-22). Disease can occur early in the growing season, when temperatures are low. Areas affected by this disease tend to be in temperate regions and high-elevation areas in the tropics (Chen, 2005). P. striiformis depends on a living host for survival and produces huge numbers of airborne spores that are carried by wind from one susceptible host to another (Brown and Hovmøller, 2002).

FIGURE WO-22

Yellow “stripe” rust on wheat. SOURCE: Hovmøller (2010).

Discovery and Spread

The most widespread yellow rust epidemic in the United States occurred in 2000 (Milus et al., 2006). Before that time, yellow rust in the United States was restricted to the temperate regions of California and the Pacific Northwest, where cool and moist weather patterns prevail (Chen, 2005). Suddenly, “overnight, more or less,” remarked Mogens Støvring Hovmøller of Aarhus University, the disease appeared in the warmer and drier wheat belt. (Dr. Hovmøller’s contribution to the workshop summary report can be found in Appendix A, pages 252–263.) For the first time, severe epidemics were reported east of the Rocky Mountains—in South Dakota, Nebraska, Kansas, Oklahoma, and Texas (Figure WO-23) (Milus et al., 2009).

FIGURE WO-23

Presence of “trace” and “severe” levels of yellow rust in North America since 2000. SOURCE: Adapted from Chen (2005).

Similarly, between 2002 and 2003, yellow rust swept across Western Australia, where yellow rust was absent until 2002—an area once considered too warm for severe epidemics of yellow rust (Milus et al., 2009). In central and northern Europe, epidemics on wheat at that time were less pronounced, the majority remaining resistant to the disease. According to Hovmøller, two P. striiformis strains of almost identical DNA-fingerprints were responsible for these epidemics (Hovmøller et al., 2008). Human activity was likely the main driver for pathogen introduction in North America and Australia, Hovmøller remarked, given the rapid spread of the pathogen to distant continents in less than 3 years and the indistinguishable DNA fingerprints of prevalent pathogen isolates. Once the pathogen arrived, the disease spread rapidly via airborne spores, he said. According to Hovmøller, the presence of large areas of susceptible crop varieties likely amplified and accelerated the spread of these yellow rust strains.

The emergence of yellow rust in new warmer regions concerns researchers because it suggests a change in its epidemiology. Indeed, the strains of P. striiformis associated with recent outbreaks of disease are more aggressive,⁶⁷ more heat tolerant, and able to produce two to three times more spores in less time than other strains (Milus et al., 2009). These adaptations appear to improve the fitness of these strains and may explain their rapid spread on a global scale (Hovmøller et al., 2008). In Australia and the United States, these newer, more aggressive strains appear to have replaced older strains and have continued to thrive in subsequent seasons (Milus et al., 2009). According to Hovmøller, while the epidemic has apparently slowed down in North America from 2006 to 2009, major yellow rust epidemics occurred in northern and eastern Africa, Western and Central Asia, China, and the Middle East (Hovmøller et al., 2010). In 2010, according to Hovmøller, observers reported disease outbreaks in northern and eastern Africa, Asia, and the Middle East, and the epidemics reappeared in the United States.

Origins

The exact geographic origin of these more aggressive strains of P. striiformis is unknown, although phylogenetic analyses reveal that West and Central Asia may be the evolutionary origin. Hovmøller remarked that the dramatic change in phenotype (e.g., spore production, heat tolerance), coupled with the sudden appearance of these strains suggests a recombination event somewhere, rather than evolution through a series of mutations.

Unlike many of the fungal diseases discussed at the workshop, yellow rust is well known. Epidemics of this disease have plagued the world’s farmers for centuries, and P. striiformis is familiar to plant breeders. In the 1940s, Norman Borlaug⁶⁸ developed new “rust-resistant” wheat strains that also dramatically increased global crop yields (Rust in the bread basket, 2010). These advances inspired the “Green Revolution” that brought these techniques and disease-resistant wheat into widespread use. According to Brown and Hovmøller (2002), these techniques have helped to keep many crop diseases under control, but now, relatively few crop varieties (with specific resistance genes) are sometimes used across large areas at “continental scales.” A reduced crop diversity in modern agriculture, according to Hovmøller, increases the potential impact posed by the global movement of new, more virulent forms of plant pathogens (Brown and Hovmøller, 2002; Stukenbrock and McDonald, 2008).⁶⁹

International Regulations and Coordination

International regulations that support infectious disease surveillance and detection activities include: the International Health Regulations (IHRs) and the Sanitary and Phytosanitary Measures (Baker and Fidler, 2006; Cash and Narasimhan, 2000; MacLeod et al., 2010; WHO, 2008). In 2008, the OIE added amphibian chytridiomycosis to its list of “notifiable”⁷² aquatic animal diseases, (Schloegel et al., 2010). As several participants observed, however, this reporting requirement only applies to OIE member states, and only to animals traded internationally. Indeed, the effectiveness of these formal and informal reporting regimes has yet to be demonstrated, and many have suggested that fear of adverse economic consequences (e.g., trade and tourism restrictions) will limit their usefulness as an early warning disease reporting network (Cash and Narasimhan, 2000; Fidler, in IOM, 2010; Hueston, in IOM, 2007; Perrings et al., 2010).

Guidelines on hygiene and quarantine procedures for captive and wild animals have also been developed by the conservation community to reduce the spread of zoonotic diseases, but these guidelines are considered underused or difficult to enforce (Daszak et al., 2000). Two conventions developed to address the international wildlife trade and the conservation of biodiversity, the Convention for International Trade of Endangered Species and the Convention on Biological Diversity are based entirely on voluntary agreement, noted speaker Weldon. While these agreements reflect noble aspirational goals, according to Weldon, there is limited opportunity to actually implement the measures.

Several participants emphasized the limitations of current capacity to detect emerging pathogenic fungi. As speaker Fisher observed, national strategies are limited by their focus on known threats to humans and agriculturally important species, and international strategies are nearly nonexistent or very slow moving. This is particularly true of wildlife surveillance, which Fisher said is “completely under the radar.” Forum member Roger Breeze of Lawrence Livermore National Laboratory agreed, noting that “we are not very good at looking for things we know about, even those diseases that are economically important, such as foot and mouth disease.” Breeze continued that “what we are talking about over the last few days is broadening the number of organisms involved and the number of areas of economic life that are involved.” He went on to note that many organisms discussed during the workshop, such as ornamental plants, do not currently fall under any one organization’s regulatory responsibility. “We have a huge international failure in biosecurity,” according to Breeze, and the problem “needs to be approached in a different manner.”

No single agency or multilateral organization is solely focused on infectious diseases in humans, plants, and animals. Several workshop participants observed that the creation of a single entity that was responsible for collecting and analyzing data from across the “threat” spectrum and ensuring that disease interventions are based on the input of professionals working with humans, domestic animals, and wildlife could significantly enhance current disease surveillance and response capabilities (Choffnes, 2008; GAO, 2010; Hueston, in IOM, 2007; Perrings et al., 2010).

Improved coordination of disease surveillance and response activities, Daszak noted, would “benefit all sectors—whether it is food production, travel and trade, or human and environmental health.” Moreover, sectors may benefit in unanticipated ways. Blehert remarked that “wildlife health is important to world health. Not just with regard to disease surveillance, but also with regard to basic research. There is much that we can learn from emerging diseases of wildlife such as WNS or amphibian chytridiomycosis that likely have significant implications with regard to ecosystem integrity and function. Only by incorporating domestic animal health, wildlife health, and human health into the same model can we fully understand the ecology of infectious disease.”

Several participants suggested that within the United States, an interagency task force could link together the plant, animal, and human health communities. Forum member Russell added that the Department of Defense is now one of several interagency partners involved in a forum on emerging pandemic threats as a sub-Interagency Policy Committee (IPC) of the U.S. government’s Global Health Initiative (GHI). Among its other activities, this sub-IPC assembled an interagency working group that developed a document detailing the U.S. response to the revised IHRs. He suggested that this interagency forum could serve as an effective model for coordinating the U.S. government activities in areas of common concern.

Forum member Edward McSweegan, from the National Institutes of Allergy and Infectious Diseases, added that a previous interagency program that was focused on international infectious diseases was orchestrated by the U.S. Department of State and the Office of Science and Technology Policy (OSTP). Forum Vice-Chair, James Hughes of Emory University, noted that this effort was established under the aegis of the Committee on International Science, Engineering, and Technology Policy of President Clinton’s National Science and Technology Council and involved many agencies: the National Institutes of Health (NIH), CDC, Food and Drug Administration, U.S. Agency for International Development, and DoD, among others. He said that many of the recommendations from their 1995 report are “still relevant to today’s world.”⁷³ McSweegan also suggested that the funding of cross-disciplinary research on emerging fungal diseases might be modeled after the success of the NIH–National Science Foundation (NSF) Ecology of Infectious Diseases Initiative,⁷⁴ perhaps as a collaboration of the NIH, USGS, and USDA.

Informal Disease Reporting Networks

Informal disease reporting networks are an increasingly important component of global disease surveillance. Examples include ProMED-mail, which is administered by the International Society for Infectious Diseases, and Bd-Maps, which was developed at Imperial College London. Both are platforms that allow contributors anywhere in the world to report and access disease observations—even via cell phone applications (Brownstein et al., 2009; Fisher et al., 2009).

Since its founding in 1994, ProMED-mail⁷⁵ (Program for Monitoring Emerging Diseases, PMM) has served as an important platform for rapid communication about emerging infectious diseases of humans, animals, and plants (IOM, 2007). Speaker Larry Madoff, of the Massachusetts Department of Public Health and University of Massachusetts Medical School, explained that in contrast to the traditional, hierarchical approach to public health reporting, ProMED collates information from a wide variety of unofficial or informal sources⁷⁶ and distributes reports to members in near real time (Brownstein et al., 2009; Madoff, 2004; Morse et al., 1996). A recent quantitative assessment of the effect of informal source reporting on the global capacity for infectious disease detection concluded that ProMED-mail and other informal disease reporting resources improve the timeliness of detection and reporting, although the effect varies geographically (Chan et al., 2010).

ProMED-mail is a free service, with all reports screened by a panel of expert moderators before being posted to over 54,000 subscribers from more than 180 countries. ProMED-mail emphasizes a One Health approach to disease surveillance, Madoff remarked, by reporting on plant (mostly threats to food crops), animal (both agricultural and zoonotic threats), and human pathogens to all subscribers. Pointing to C. gattii as an example of a recently emerged fungal human pathogen with many non-human hosts, Madoff reminded the audience that the risk of disease emergence in humans is greater among pathogens with many non-human hosts (Woolhouse and Gowtage-Sequeria, 2005).

Informal efforts to aggregate information on the presence or absence of disease in the field also contribute to improving the speed and broadening the scope of current disease surveillance. Fisher described the Bd Global Mapping Project⁷⁷ (Bd-Maps) and its associated activity, RACE (Risk Assessment of Chytridiomycosis to European amphibian biodiversity). Bd-Maps collects information from groups of researchers in the field and national surveillance data from several European countries, including the Netherlands, Spain, Switzerland, and the United Kingdom, to create a shared database that provides information on where Bd has been detected, globally and locally. Fisher hopes this information will not only aid in the prediction of where Bd will likely emerge in the future, but also encourage at-risk areas to implement appropriate biosecurity controls.

The Bd-Maps project has a public website with a map detailing the incidence of positive Bd reports and, for each report, links to data. There is also an embargoed (private) website to encourage participation of scientists who prefer that their data remain private. As of December 2010, the publicly available database reported about 6,500 Bd-positive animals out of 30,000 sampled among 3,500 sites worldwide, with 49 of 74 countries and 440 species of amphibians with known Bd infections. The data come from multiple sources, including contributions directly from the field using a smart phone application called EpiCollect.⁷⁸ Fisher observed that these data may be used to assess either global or country-level trends and to detect broad-level associations (e.g., the data show that Bd is present in many areas where species richness has declined without any [other] explanation). Fisher noted that the project has provided a means for communicating important information rapidly among interested parties.

Astute Observers in the Field

Human capacity is needed for more effective surveillance and detection of fungal and other infectious diseases of humans, animals, and plants, remarked many participants. Weldon noted the importance of field biologists in discovering the accelerated loss of amphibian biodiversity and in initiating investigations on the possible causes—from climate change and habitat destruction to chemical pollutants. Blackwell remarked that the causative agent of amphibian chytridiomycosis (Bd) was recognized as a fungus rather late in the epidemic by one of the few mycologists who study the fungal phylum Chytridiomycota. Howlett urged more training in classical mycology: “While molecular systematics and phylogenomics has helped to advance understanding of mycology, these methods need to be complemented by field studies and identification of the causative agent of a disease by symptoms or pathogen morphology.” Forum member Fletcher agreed, adding that more classically trained plant pathologists are needed: “These are the scientists who can go out into the field and identify pathogens of any type, fungal or otherwise.” Many senior plant pathologists are near retirement, but not many younger pathologists have the skills and knowledge to take their place, she said. Rizzo agreed, noting that in the 1980s, seven researchers in California specialized in forest diseases; by the time that sudden oak death emerged as a major problem in California, there were none. He further observed that agricultural departments in colleges across the United States continue to be downsized.

Speakers Galgiani and Holland emphasized the importance of improved education of physicians and other front-line healthcare workers in the diagnosis and treatment of fungal diseases. Holland noted that fungal diseases due to previously undiagnosed primary immune deficiencies are not frequent, but that a few cases happen “every year, in every country.” He further cautioned that, “if you don’t think about them and you don’t recognize these diseases as fungal, the patients don’t survive.” Even in areas with endemic fungal disease, the correct differential diagnosis is often missed, commented Galgiani. In the case of symptomatic Valley Fever, which often initially presents as a community-acquired pneumonia, specific antibody testing is required to discriminate Coccidioides infections from other causes of pneumonias. Even then, Galgiani noted, with early infections conventional serological testing produces false positives in an estimated one third to two thirds of all infected patients.

Rapid and Accurate Tools for Detection and Diagnosis

The lack of sensitive and specific tools for the diagnosis of emerging diseases limits the effectiveness of disease surveillance and treatment efforts (IOM, 2007). In the ornamental plant trade, surveillance focuses on preventing the introduction of plant pathogens. This occurs at the international, national, and regional levels, Rizzo explained. In Europe and North America, strict controls have been placed on nurseries to quickly contain and eradicate outbreaks of P. ramorum (Kliejunas, 2010). In the United States, plants in nurseries are subject to routine inspections, and plants that are sold across state or county boundaries are closely monitored. In states such as Oregon, Washington, and California, they are quarantined to ensure they are free from infection (Kliejunas, 2010). Despite these measures, P. ramorum continues to spread via ornamental plant trade pathways (see sudden oak death case example). In part, this is due to the lack of effective detection tools. Asymptomatic infections and the use of fungicides can limit the effectiveness of quarantine protocols based only on visual inspection.

To track the spread of the C. gattii outbreak, public health officials in British Columbia and the U.S. Pacific Northwest have listed C. gattii–associated disease as a reportable disease. However, speaker Julie Harris of the CDC remarked that C. gattii surveillance has been limited by overreporting of the most severe cases and underreporting of all cases. In cases where samples are sent to the laboratory for identification, not all labs are using the canavanine-glycine-bromothymol blue (CGB) agar test⁸¹ or genetic sequencing⁸² that are needed to differentiate between C. gattii and C. neoformans infections. Harris noted that underreporting is due to a number of factors including the fact that many of the smaller laboratories across Washington and Oregon:

• may not be aware of the need for culture to make an accurate diagnosis;
• may not be aware that they should be sending isolates to their respective state health departments for confirmatory testing; or
• may lack the capacity for fungal culture and testing.

Harris observed that labs with mycology capacity are not as common as labs with viral or bacterial capacity, and requests for additional training or capacity need to come from the local level. She noted that the C. gattii Public Health Working Group, formed in 2008 by the CDC and state and local public health departments and laboratories and the British Columbian Centre for Disease Control, is working to standardize surveillance by increasing clinician awareness of C. gattii infection and working with global laboratories to characterize genetic and phenotypic variety in C. gattii.

Padilla, from the Smithsonian Conservation Biology Institute (SCBI), identified fungal diagnostic capacity as a particularly challenging area of emerging fungal disease threat management in wild animals and one in need of further research. Too often, new fungal pathogens in wildlife are either misdiagnosed or undiagnosed. The limited diagnostic capacity leads to the “clumping” of information under known fungal disease syndromes, Padilla remarked, and this often precludes the recognition of true emerging fungal diseases and prevents further investigation of true host–pathogen dynamics. Fungal infections are often identified only to the genus, not the species level, making it difficult to understand host–species relationships. The clumping of information could also result in dangerous management decisions—when assumptions about one host are based on what is known about another host. For example, he reported that preliminary findings by researchers working at the SCBI suggested that the bacterium Janthinobacterium lividum does not provide the same anti-chytridiomycosis protection in Bd-infected captive-bred Panamanian golden frogs (Atelopus zeteki), as it does in Bd-infected yellow mountain frogs (Rana muscosa). In fact, J. lividum seems limited in its ability to colonize the skin of A. zeteki and thus is also limited in its ability to play the same anti-fungal role that it does in R. muscosa. However, the understanding of a bacterium–host system conferring anti-fungal protective properties suggests that other species-specific host-adapted bacteria could confer the same protection to A. zeteki. Padilla expressed hope that these findings will lead to more appropriate treatments for this particular frog species.

The Fungal “Background”

Limited understanding of fungal biodiversity and biogeography can impede surveillance, detection, and discovery efforts, noted Blehert. It has been reported that an abundance of closely related Geomyces species have been found in the same soil that harbors G. destructans (Lindner et al., 2010). These related species are currently confounding the ability to conduct routine soil analysis as a mode of surveillance for WNS, Blehert said. Rizzo added that “when we find something new, it’s very difficult to know whether it is exotic and something to be concerned about or an interesting new native organism that we weren’t aware of.”

Russell asked participants for their views on effective techniques for the “discovery” of fungal pathogens (i.e., detection of an unknown disease agent). Heitman suggested that the fungal nuclear ribosomal internal transcribed spacer (ITS⁸³) sequences might be useful for determining if genetic material isolated by researchers is fungal in origin. Blackwell mentioned that mycologists now use non-culture–based molecular tools coupled with field explorations to identify new fungal species (Jones et al., 2011; Jumpponen and Jones, 2009; Lara et al., 2010; Porter et al., 2008; Schadt et al., 2003) and to characterize the distribution of fungal species (Daughtrey et al., 1996). These techniques are increasingly being used because many fungal species are not easily cultured. Culturing techniques and pathology investigations, however, are still needed to characterize an organism: these are “hard and laborious things to do,” observed Daszak, but the “value of the product is so much better, it is orders of magnitude better, because you can do something with it.” You can “send it to others for consultation or determine an organism’s biology or how it causes disease in host species,” Daszak said.

Blackwell noted that the NSF has had several programs in systematics⁸⁴ and biodiversity for some years. In 2004, the NSF created the “Assembling the Tree of Life” program with the goal of constructing an evolutionary history for all major lineages of life (See glossary for more information). These and other related programs have increased support for research on the evolution and diversity of Kingdom Fungi, which has been helpful for improving detection, diagnosis, and discovery methods (see Blackwell et al., 2006; Hibbett et al., 2007). A database of 40,000 fungal species (with an emphasis on fungal plant pathogens) developed by the USDA Agricultural Research Service includes information such as host range, geographic distribution, relevant scientific literature, and for some species, descriptions and illustrations (Rossman and Palm-Hernandez, 2008).

Responding to Fungal Diseases of Plants—from Agriculture to Landscapes

In plants, disease eradication strategies include clear-cutting or controlled burning of infected plants (Rizzo et al., 2005). Fungicides are often used to protect high-value plants, but their widespread and frequent use is often not economically feasible (Rizzo et al., 2005; Scheffer et al., 2008). The development of resistant cultivars or strains, which may take years to decades, is currently the most successful disease control strategy in plants, particularly agricultural crops (Vurro et al., 2010).

Developing strains of wheat that are resistant to the newly emerging and more aggressive forms of yellow rust is the primary strategy for limiting the devastating effects of P. striiformis on wheat. In the meantime, early detection is essential to reduce crop yield losses due to yellow rust, Hovmøller said. In the short term, options for control are limited to fungicide sprays which may be unavailable or not affordable to farmers in the developing world. The replacement of susceptible wheat with locally adapted, resistant, or less susceptible varieties can also slow disease spread, he remarked.

As Hovmøller noted, when it comes to wheat rust, “what’s going on in one continent may be your problem the following day.” To prevent long-term damage, intensified international collaboration is needed to build wheat rust surveillance, detection, and response capacity. Several promising developments on the international scale were reported by Hovmøller. In 2008, a Global Rust Reference Center (GRRC) for yellow rust was established to improve yellow rust management in countries where facilities and expertise are scarce. GRRC is supported by Aarhus University in Denmark, the International Center for Agricultural Research in the Dry Area, and the International Maize and Wheat Improvement Center (CIMMYT). In 2011 the activities will be extended to wheat stem rust (Puccinia graminis) via projects facilitated by the Borlaug Global Rust Initiative. GRRC is complementing existing national diagnostic laboratories, which cannot receive rust samples year round from all countries. The primary goals of GRRC are to conduct virulence and race⁸⁵ analyses, secure isolates for future resistance breeding and research, facilitate research and training, and provide information for a global wheat rust early warning system. The Borlaug Global Rust Initiative, which was established in response to the stem rust Ug99 outbreak in East Africa, now deals with all three wheat rusts.⁸⁶

Disease management is considerably more difficult when dealing with plants of limited economic value (i.e., non-timber and non-crop plants), despite their significant ecological value, Rizzo noted. Management of sudden oak death and ramorum blight, according to Rizzo, is “scale dependent.” One can manage individual trees, landscapes, or entire regions (Rizzo et al., 2005). At the individual level, fungicides are available that can be injected into a tree or sprayed on the bark to prevent infection (Garbelotto et al., 2002). In forests, containment, including cutting and controlled burnings in areas with infected trees, is the primary means of infection control. When asked whether there is a possibility for developing treatments for sudden oak death that could be applied at the landscape level, Rizzo said the options are limited. In Oregon, there have been attempts to conduct aerial spraying with phosphonate (a chemical fungicide), but it is unlikely that any type of aerial spraying would be acceptable in California. Rizzo also cautioned that it took many decades to breed genetic resistance for Dutch elm disease. The complexity of oak genetics also makes it very challenging to use breeding to develop oaks resistant to P. ramorum infection. Rizzo went on to note that research on potential biocontrol agents, such as viruses, is at a very early stage.

Landscape-level management also uses predictive modeling. For sudden oak death, models based on host species distribution, climate, and other factors identify areas at risk for invasion by the pathogen. These areas can then be surveyed using “aerial imaging, plot-based monitoring, and stream sampling to determine the presence of P. ramorum or signs of infected trees” (IOM, 2008b). Eradication methods are only effective if the disease is detected early enough. For areas where the pathogen is established, management approaches seek to avoid negative ecological consequences, such as the growth of invasive plant species. Ultimately, Rizzo said, we are trying to develop methods to “live with the pathogen.”

Treatment Options for Fungal Diseases of Humans and Animals

Available antifungal therapies are generally of limited value due to toxicity problems (Figure WO-26) (Ostrosky-Zeichner et al., 2010). The lack of accurate diagnostics further limits the effectiveness of existing fungal treatments. Approaches using antibody therapy and vaccines (for certain endemic pathogens) remain challenging due to the ongoing evolution of pathogens (Cox and Magee, 2004; Galgiani, 2007, 2008; Ostrosky-Zeichner et al., 2010). Overall, there are few new therapeutic agents in the development pipeline with the potential for broad antifungal effects (Ostrosky-Zeichner et al., 2010).

FIGURE WO-26

Mechanisms of action of selected antifungals. An illustration of the mechanism of action of currently available antifungals as well as selected antifungals under development. (A) Echinocandins and nikkomycin Z inhibit the formation of the fungal cell (more...)

The development of new treatments for fungal diseases has also been slowed by an underappreciation for the effects that fungal diseases can have on human health, Galgiani asserted. In endemic areas of Arizona and California, about a third of all coccidioidomycosis cases lead to illnesses requiring medical attention⁸⁷ (Tsang et al., 2010). While oral therapy with azole antifungal drugs is safe and convenient, many patients do not respond to treatment (20–40 percent failure rate). Moreover, many patients who initially respond to treatment experience relapses after treatment ends (Galgiani, 2007; Hector and Laniado-Laborin, 2005). Galgiani reviewed current efforts at the University of Arizona to develop a new antifungal known as nikkomycin Z, a competitive inhibitor of chitin synthase that interferes with cell wall construction (Galgiani, 2007). Discovered in an antifungal discovery program by Bayer in the 1970s, the compound demonstrated antifungal activity in mice in the 1980s (see Hector et al., 1990). Only after the University of Arizona acquired the compound in 2005, have clinical trials resumed (Galgiani, 2007).⁸⁸

In the absence of a vaccine or other preventive measures for C. gattii infection of humans and animals, officials concede the public can do little to protect themselves from infection (Knox, 2010). Additional research is needed to clarify the epidemiology and drug susceptibilities of the various strains of C. gattii present in the region to help inform treatment guidelines. Moreover, researchers need to learn more about the natural history and pathogenicity of the fungus to further prevention, treatment, and intervention efforts (Datta et al., 2009a,b). Heitman noted that research is ongoing to determine the nature of hypervirulence (D’Souza et al., 2011); the differences between immune responses to C. gattii and C. neoformans infections (Cheng et al., 2009); and why C. gattii can so readily invade the cells of immunocompetent individuals (Kronstad et al., 2011; Ma et al., 2009; Voelz and May, 2010).

The challenge of developing effective treatments for fungal diseases is compounded by the scale and complexity of treating diseases of wildlife. Fungicidal treatment protocols are being explored for amphibian chytridiomycosis and include methods to alter the skin microbiome⁸⁹ (Fisher et al., 2009; Harris et al., 2009) (Figure WO-27). For WNS, some researchers are investigating whether treating bats with antifungal agents might improve their survival (Platt, 2010), while others are exploring the possibility of developing a vaccine against this fungal pathogen (Buchen, 2010). Adapting these protocols to large and dispersed wild animal populations, while minimizing unanticipated ecosystem impacts, is challenging and may continue to limit conservation efforts (Fisher et al., 2009). In addition to captive breeding programs, conservation efforts target habitat preservation, limiting the spread of infected species, and protecting endangered species. Further research is also needed to fill in some gaps in knowledge that still exist. Researchers need to gain a better understanding of the biology of both Bd and G. destructans and their respective hosts to answer questions related to the determinants of virulence, the hallmarks of effective immune response, and the specific mechanisms by which these pathogens kill their hosts. Added to this is the unique challenge of managing disease in hibernating animals in delicate underground ecosystems. Biology of infectious diseases, however, is not part of the traditional wildlife ecology education curriculum, Blehert remarked. Nor are speleologists, tourists, recreational cavers, or hikers required to have such knowledge.

FIGURE WO-27

Frogs in the Sierra Nevada region, being treated in baths containing a fungicidal bacterium in hopes of eliminating infection by the fungal pathogen (Bd) associated with the deadly disease: amphibian chytridiomycosis. SOURCE: Photo by Anand Varma.

Buying Time Through the Conservation of Threatened Wildlife Populations

Captive breeding programs at the SCBI⁹⁰ have helped to rescue species that were on the verge of extinction, endangered by habitat loss or by the introduction of disease into areas with naïve and susceptible host populations. According to Padilla, captive propagation programs can also serve two additional functions in the management and mitigation of emerging fungal diseases⁹¹:

• Fungal diseases identified in a captive animal population can be an early indication of an emerging threat in the wild. Based on Padilla’s experience, there is a wide range of opportunistic and primary fungal diseases that have been observed in captive animals.
• Captive populations are also established for the purpose of studying a fungal disease that would otherwise be difficult or impossible to study. An example of this important work is the captive population of Japanese giant salamanders (Andrias japonicus) established by the Smithsonian National Zoological Park, in which the presence of Bd and the efficacy of itraconazole treatment can be studies and monitored over time in ways that could not be possible in their wild counterparts.

SCBI has used captive breeding to save several species from extinction. The endangered black-footed ferret population was revived from just 18 individuals in 1988 to a current population of 800 to 1,000 in the wild (Weidensaul, 2000). Other animal species, including the golden lion tamarin, California condors, Przewalski’s horses, and the scimitar-horned oryx, have also benefited from the SCBI’s captive breeding program’s success.

Despite success with multiple species, establishing and maintaining captive breeding programs is technically challenging. In the fall of 2010, SCBI developed a captive colony of the endangered Virginia big-eared bats (Corynorhinus townsendii virginianus) in response to the threat posed by G. destructans. Specialist insect-eating bats, such as the Virginia big-eared bat, are notoriously difficult to keep in captivity. But, Padilla noted, in light of the possible extinction of this endangered subspecies, SCBI decided to take on the “high risk” project of developing a captive colony of these bats. However, Padilla said, although the bats did not die of WNS, the colony of 40 bats experienced extremely high mortality (90 percent) in the first 200 days of captivity.

The Amphibian Ark⁹² is a global network of captive breeding programs working in the short term to protect amphibian species at immediate risk of extinction (IUCN, 2005). The Smithsonian’s National Zoo currently houses a fifth of the world’s Panamanian golden frog populations (Figure WO-28). It is hoped that the Smithsonian’s expertise in captive breeding will contribute to the preservation of amphibians and New World bats that are currently at risk of local or global extinction.

FIGURE WO-28

Panamanian golden frog (Atelopus zeteki). The Smithsonian National Zoo has established a captive breeding program to help rescue this critically endangered species. SOURCE: Photo by Brian Gratwicke, Wikimedia commons.

Weldon highlighted a number of successful conservation programs that target amphibian populations outside of captive breeding programs.⁹³ These included:

• The population management of Alytes obstetricans in Peńalara Natural Park, Spain, in response to annual Bd outbreaks;
• Australia’s Bd Threat Abatement Plan, which was initiated in 2006, with the goal of preventing amphibian populations and regions that are currently free of chytridiomycosis from becoming infected; and
• Madagascar’s Early Detection Plan, which monitors high-risk areas (e.g., ports of importation, areas visited by tourists, areas of high biodiversity) and builds facilities for captive breeding and research in the event that Bd does reach the island (Weldon et al., 2008).

Importantly, Weldon noted, these programs include disease prevention as a prioritized conservation measure.

Weldon also recounted one instance in which conservation efforts unintentionally contributed to the spread of diseases. Population decline of amphibians on the island of Mallorca were linked to Bd infection in 2008 (Walker et al., 2008). The “source” of infection was traced to a project designed to boost populations of the island’s midwife toad (Alytes muletensis). Cross-contamination is thought to have occurred between two species that were cohoused at the breeding facility: the midwife toad and imported frogs from South Africa (Xenopus gilli) that were infected with Bd. Captive midwife toads reintroduced into the wild served as vectors that brought the pathogen to other amphibian populations on the island (Rosenblum et al., 2009; Walker et al., 2008). Weldon said, “This illustrates that if we are to proceed with the reintroduction programs, great caution should be taken, because you could potentially be introducing the pathogens with the species that you are trying to conserve.”