Emerging Infectious Diseases
[Volume 5 No.6 / November - December 1999]
   Perspectives
   Emerging Infectious Diseases and Amphibian Population
   Declines
   Peter Daszak,* Lee Berger, Andrew A. Cunningham,
   Alex D. Hyatt, D. Earl Green, and Rick Speare
   *Institute of Ecology, University of Georgia, Athens,
   Georgia, USA; Australian Animal Health Laboratory,
   Commonwealth Scientific Industrial Research
   Organization, Geelong, Victoria, Australia; School of
   Public Health and Tropical Medicine, James Cook
   University, Townsville, Queensland, Australia;
   Institute of Zoology, Zoological Society of London,
   London, United Kingdom; and National Wildlife Health
   Center, U.S. Geological Survey, Madison, Wisconsin, USA
    We review recent research on the pathology,
    ecology, and biogeography of two emerging
    infectious wildlife diseases,
    chytridiomycosis and ranaviral disease, in
    the context of host-parasite population
    biology. We examine the role of these
    diseases in the global decline of amphibian
    populations and propose hypotheses for the
    origins and impact of these panzootics.
    Finally, we discuss emerging infectious
    diseases as a global threat to wildlife
    populations.
   Emerging infectious diseases have been reported
   increasingly as causes of death in free-living wild
   animals (1). These diseases are a particular threat to
   wildlife species whose population, habitat, or range
   has been diminished or artificially manipulated to
   promote species survival (e.g., captive breeding,
   translocation, and release programs) (2-4). An early
   example of an emerging disease panzootic was the
   introduction of rinderpest in African domestic cattle
   in 1889 (5). More recently, epizootics and panzootics
   of wildlife have been increasingly reported in
   terrestrial (1) and marine (6) habitats and are
   probably underreported (1,4,7-9). Recent advances in
   theoretical and experimental host-parasite ecology have
   demonstrated a major role for infectious agents in the
   population biology of wild animals (10,11). We discuss
   recent data on two newly emerging infectious diseases
   of amphibians and, by reference to host-parasite
   ecology, propose hypotheses to explain their origin and
   impact.
   Amphibian Population Declines
   Global declines in amphibian population are perhaps one
   of the most pressing and enigmatic environmental
   problems of the late 20th century (12-19). While some
   declines are clearly due to habitat destruction, others
   are not associated with obvious environmental factors.
   Causal hypotheses include the introduction of predators
   or competitors, increased ultraviolet (UV-B)
   irradiation, acid precipitation, adverse weather
   patterns, environmental pollution, infectious disease,
   or a combination of these. Transdermal water uptake and
   gaseous exchange and a biphasic life cycle are
   important aspects of amphibian biology. These factors
   led to the hypothesis that amphibians act as sentinels
   for global environmental degradation (12,18). However,
   this role has yet to be demonstrated, and many causal
   factors may be present (12,19,20).
   Of particular concern are population declines in
   ecologically pristine areas, such as the montane
   tropical rain forests of Australia and Central America,
   where human impact from agriculture, deforestation, or
   pollution is thought to be negligible. Here, long-term
   data demonstrate recent and catastrophic amphibian
   population declines, often resulting in the complete
   loss of amphibian species (local extinction of multiple
   species) from large swaths of habitat (20-25). These
   declines include the disappearance and presumed
   extinction of the recently discovered golden toad (Bufo
   periglenes) of Costa Rica (23) and as many as seven
   Australian amphibian species, including two species of
   gastric-brooding frog (Rheobatrachus spp.) (20). These
   data, along with recent findings of amphibian mass
   deaths in these areas, suggest that such local
   extinctions are not normal population fluctuations or
   metapopulation dynamics.
   Investigations in Australia, the United Kingdom, and
   North and Central America (26-31) have repeatedly found
   two diseases as the causes of amphibian mass deaths
   globally (Table 1): chytridiomycosis in the rain
   forests of Australia and Central and South America and
   some parts of North America (28-32) and iridoviral
   infections in the United Kingdom, the United States,
   and Canada (26,27,33-36). Both diseases have been
   classified as emerging (1). The parasitic infection
   recently implicated as the cause of amphibian
   deformities in North America has not been associated
   with mass deaths or population declines (31).
Table 1. Mass deaths caused by chytridiomycosis and ranaviral disease in wild populations of amphibians
Chytridiomycosisan Emerging Panzootic Fungal Disease of Amphibians
Chytridiomycosis is a fungal disease first
described in 1998 from moribund and dead adult
amphibians collected at sites of mass deaths in
Australia and Panama from 1993 to 1998 (28).
Here, long-term ecologic study sites reported
catastrophic amphibian population declines in
Big Tableland, Queensland (39,40), and Fortuna
and Cerro Pando, Panama (24,25,28). No
significant pathogens were found on routine
parasitologic, bacteriologic, mycologic,
virologic examinations of tissue samples (28).
Fresh skin smears and histologic sections of
the epidermis, however, consistently contained
large numbers of developing and mature
sporangia of a new genus of chytrid fungus
(phylum Chytridiomycota) (Figures 1, 2).
Sporangia were also present within the keratinized mouthparts, but not the epidermis, of sympatric tadpoles (tadpoles lack epidermal keratin) (28,29). No significant morphologic differences between chytrids infecting
Australian and Central American amphibians were
found by transmission electron microscopy, and
the pathogen was identified as a member of the
order Chytridiales by analysis of zoospore
ultrastructure and 18s rDNA sequence data (28). Chytrids are
ubiquitous fungi that develop without hyphae and are found in aquatic habitats and moist soil, where they degrade cellulose, chitin, and keratin (41). Parasitic chytrids mainly infect plants, algae, protists, and invertebrates (41); the amphibian pathogen is the only example of a chytrid parasitizing vertebrates (28).
Figure 1. Figure 1a: Ventral abdominal skin of Bufo haematiticus from western Panama. The superficial keratinized
layer of epidermis (stratum corneum) contains numerous intracellular spherical-to-ovoid sporangia
(spore-containing bodies) of Batrachochytrium sp. The mature sporangia (sp, arrows) are 12-20 m (n =
25) in diameter and have refractile walls 0.5-2.0 m thick. Most sporangia are empty, having discharged
all zoospores, but a few sporangia contain two to nine zoospores. This stratum corneum is markedly
thickened adjacent to groups of parasitized cells and in some cases, the superficial layer has become
detached. No chytrids are present in the stratum spinosum, stratum basale, dermis, dermal glands, and
blood vessels. Note the absence of hyphae and lack of an inflammatory cell response in the deeper layers
of epidermis and the dermis. Hematoxylin and eosin stain. Bar = 35 m. 1b. Ventral skin of upper hind
limb of Atelopus varius from western Panama. Two sporangia containing numerous zoospores are visible
within cells of the stratum corneum. Each flask-shaped sporangium has a single characteristic discharge
tube (arrow) at the skin surface. Exiting zoospores are visible in the discharge tubes of both sporangia.
Hyperkeratosis is minimal in this acute infection. Tissues were fixed in neutral-buffered 10% formalin,
paraffin-embedded, sectioned at 6 m thick and stained with hematoxylin and eosin. Bar = 35 m.
Clinical signs of amphibian chytridiomycosis include abnormal posture, lethargy, and loss of righting reflex. Gross lesions, which are usually not apparent, consist of abnormal epidermal sloughing and (more rarely) epidermal ulceration; hemorrhages in the skin, muscle, or eye; hyperemia of digital and ventrum skin, and congestion of viscera (29). Diagnosis is by identification of characteristic intracellular flask-shaped sporangia and septate thalli within the epidermis (Figures 1, 2).
Its occurrence solely in keratinized tissue suggests that the chytrid uses amphibian keratin as a nutrient. A hyperkeratotic and hyperplastic response of the epidermis to infection (restricted to the stratum corneum and stratum granulosum) usually coincides with the immediate location of chytrid developmental stages. Inflammatory cell response is negligible. An isolate cultured from captive dendrobatid frogs has recently been used to fulfil Koch's postulates
as a fatal pathogen of frogs and has been described as a new genus and species, Batrachochytrium dendrobatidis (42). Three mechanisms by which chytridiomycosis causes death have been proposed (28): 1) epidermal hyperplasia impairs essential cutaneous respiration or osmoregulation; 2) a fungal toxin is absorbed systemically (although
a lack of clinical signs in infected larvae suggests otherwise); and 3) these factors are combined (28,37).
Figure 2.Scanning electron micrograph of digital skin of a wild frog (Litoria lesueuri, from Queensland,
Australia) that died of cutaneous chytridiomycosis. Many cells within this area of the superficial layer of
the epidermis contain mature sporangia, and unopened discharge tubes are visible protruding through
infected cells. The skin was fixed in 2.5% glutaraldehyde, postfixed in 1% osmium tetroxide, dehydrated,
critical-point-dried, sputter coated with gold, and examined with a JEOL JSM 840 scanning electron
microscope at 5 kV. All specimens are from animals that were naturally infected and died due to
chytridiomycosis in montane rain forest regions of Panama and Australia. Bar = 5 m. Photo courtesy of L.
Berger, reprinted with permission from A. Campbell (29).
Comparison of histologic, ultrastructural, and
18s rDNA sequence data indicates that the
chytrids found in wild Australian (28) and
captive Central American (T.Y. James, D.
Porter, J.E. Longcore, pers. comm.) amphibians  enlarged
belong to the genus Batrachochytrium, are  image
probably conspecific, and form a distinct
monophyletic clade within the Chytridiales.
Emergence of Chytridiomycosis
Retrospective histologic surveys of museum
specimens of montane, riparian anurans from
protected sites in Central America and
Australia, conducted 1 to 10 years before the
population declines, showed no evidence
chytrid infection, which suggests that
chytridiomycosis has recently emerged on two continents (28). The relatively synchronous discovery of chytridiomyosis in Australia and Central America in association with amphibian population declines is striking. The data suggest that Batrachochytrium 1) may
be endemic to these regions and the amphibian deaths and declines attributed to it have only recently been discovered; 2) may be endemic and has recently become pathogenic (e.g., through an increase in the organism's prevalence or virulence, or a decrease in the host's defenses), or 3) may have been introduced recently into
these geographic regions and is now parasitizing novel host species.
The pattern of amphibian deaths and population declines associated with chytridiomycosis is characteristic of an introduced virulent pathogen dispersing through a nave population (7,10,39). In Australia, a distinct geographic and temporal progression in population declines has occurred (20), moving northward at a mean rate of 100 km per year (39). In Central America, a progression from northern Costa Rica to western Panama occurred from 1996 to 1998
(24,25). The uneven progression of declines in Australia (40) may reflect gaps in surveillance. Small-scale irregularities, however, characterize the epidemiology of many pandemics (43,44), within which individual epidemics progress at different rates in different areas. In amphibian populations, unevenness may be due to differences in ecologic factors (e.g., population density, habitat,
age structure); differences among pathogen strains; stochastic factors, such as the time of introduction; or a combination of these factors. In Australia and Central America, population declines have been catastrophic, occurring over a few months, with dramatic population loss and high rates of adult deaths (20,24,25,28,39,40,45). Such high depopulation rates are characteristic of introduced virulent pathogens (10). Conversely, in coevolved host-pathogen relationships, a degree of herd immunity to
the pathogen and lower virulence (infectivity and death rates) are normally observed. The low host specificity of amphibian chytridiomycosis (more than 30 species of wild amphibians from seven families in Central America and Australia [28,29]) also suggests that the disease was not enzootic in those montane rain-forest populations. Ability to infect a range of host species is a characteristic of many invading pathogens (10) and is less common in
endemic microparasites that have coevolved with their hosts.
The most parsimonious hypothesis for the origin of chytridiomycosis panzootics in Australia and Central America is the introduction of disease into populations of previously unexposed amphibians. Introduction of pathogens, termed pathogen pollution (1), is increasingly recognized as a significant threat to global biodiversity (1,6,46) and forms an integral part of human history (10,47). There are precedents for the introduction of fungal pathogens (including chytrid parasites) that cause high death rates
(41,48). Mechanisms by which wildlife pathogens can be introduced are common; for example, a consequence of the increasing mobility of humans is the global translocation of wildlife, plants, soil, and ballast water (1,4,49,50). Freshwater fish and amphibians are also transported globally. In Australia, chytridiomycosis-infected cane
toads (Bufo marinus), a recently introduced species, have been found (29), and in North America, bullfrogs (Rana catesbeiana) and other species of amphibians have been translocated or introduced widely. Some authors have suggested that tourists or fieldworkers surveying
amphibian populations may have facilitated the dissemination of Batrachochytrium (19), although this has not been demonstrated. Batrachochytrium may have coevolved with some amphibians (e.g., lowland) species, populations of which remain unaffected. Recent disturbances of rain forest habitats may have introduced this parasite into nave populations in Central America and Australia, leading to mass deaths. This range of disease outcomes parallels
many diseases of humans, e.g., measles and smallpox, which produce a range of effects on persons in disease-endemic regions and cause massive deaths when introduced into nave populations (48).
The occurrence of chytridiomycosis in free-living North American  amphibians (Table 1) suggests a less obvious pattern of dissemination than in Central America and Australia. This irregularity may be due to a paucity of data, the pathogen's being enzootic to the United States, or the pathogen's introduction a number of years before. Historical reports of declines in the United
States include postmetamorphic death syndrome, which progressed in waves through populations of amphibians, causing 90% to 100% death rates in recently metamorphosed animals and low death rates in larvae (50). Recent reports of chytridiomycosis-linked die-offs in Bufo boreas markedly resemble these previous die-offs. This observation and the finding that chytridiomycosis caused similar die-offs (of B. canorus and R. pipiens) in the 1970s (Table 1) (32),
support the last of the above hypotheses. In the United States an amphibian pathogen (histologically very similar to Batrachochytrium but identified as Basidiobolus ranarum) has been described in wild Wyoming toads (Bufo hemiophrys baxteri) (51) and captive dwarf African clawed frogs (Hymenochirus curtipes) (52). As the latter species was widely introduced in ornamental garden ponds throughout
the United States in the late 1980s, it may be involved in the dissemination of Batrachochytrium.
Chytridiomycosis as the Cause of Population Declines
The ability of a pathogen to cause local population declines
resulting in local host extinction requires a mechanism of
persistence at low host densities. In epidemiologic models, highly virulent parasites rapidly suppress the host population density below a threshold value required to maintain transmission, resulting in the pathogen's extinction and recovery of the host population
(7,10). Microparasites such as Batrachochytrium, with their
relatively short duration of infection and high death rates, have an increased threshold population density and are usually less able to persist. Many parasites have evolved life history strategies for persistence (10) and the presence of reservoir hosts may augment the impact of other introduced wildlife diseases on host populations (46). The aclinical presence of Batrachochytrium in the keratinized
mouthparts of amphibian larvae implicates this life-cycle stage as a reservoir host for the pathogen. This form of infection may enable Batrachochytrium to persist in reduced amphibian populations (Figure 3). In both Australia and Central America, chytrid infection was observed in larval mouthparts months after initial adult deaths: the
larvae of many tropical amphibian species survive 12 to 18 months and some temperate species as long as 3 years before metamorphosing. Examples of larval infection enhancing pathogen-mediated population declines and leading to host population extinctions have been reported for invertebrates (53).
Figure 3. Diagrammatic representation of the range of disease outcomes in populations of amphibians
affected by a Batrachochytrium-like pathogen. Factors that hypothetically predispose some amphibian
populations to declines are illustrated. In this model, host ecologic traits (left side of pyramid) and
parasite biologic traits (right side of pyramid) combine to produce declines in a specific group of
amphibian species that have low fecundity, are stream-breeding habitat specialists, and occur in montane
regions. These characteristics predispose them to population declines after introduction of a waterborne
pathogen with a low preferred developmental temperature and ability to persist at low host population
densities.1
1Note that the relative number of mass death events decreases with increasing impact on population.
Persistence may be further enhanced by
saprophytic development (Figure 3).
Batrachochytrium can be cultured in vitro on
tryptone agar without the addition of keratin
or its derivatives (37,42), and it will grow for at least one generation on cleaned epidermal keratin or on amphibians that have died of the infection (42). Batrachochytrium
may survive and reproduce as a saprophytic
organism in the environment, at least for
short periods. Keratin (from decaying
carcasses, shed skin, and other sources) is
widely distributed in the environment, and
chytrids that use this substrate are well
known (42). Furthermore, the ability to
develop and reproduce saprophytically is
common to many other fungal (54) (including
chytrid [41]) and bacterial (55) pathogens. An epidemiologic model of a host-parasite system for pathogens that can reproduce saprophytically clearly shows a lowering of the host threshold population, allowing the pathogen to drive the host to extinction (55). Development of Batrachochytrium for even short periods outside its amphibian host may greatly increase its impact and accelerate population declines. Long-term presence as a saprophyte may explain
the lack of recolonization of streams from which amphibians have been extirpated in both Australia (29) and Central America (24,25).
The impact of chytridiomycosis may be enhanced by the ecologic characteristics of certain host species. In Australia, chytridiomycosis-linked deaths have occurred in both declining and nondeclining species (28,29). Species with declining populations belong to a similar ecologic guild: regionally endemic rain forest specialists with low fecundity that reproduce in streams and live at high altitudes (22). These characteristics, which are largely shared by declining Central American amphibians (24,25), are predictors of increased impact from chytridiomycosis. Species that reproduce in streams are probably more susceptible to a waterborne pathogen than terrestrial breeders. Low fecundity (56) and habitat specialization
indicate a reduced ability to recover from population declines caused by stochastic events, including disease introduction. Laurance, McDonald, and Speare (39) suggested that the relation between high-altitude populations and declines may be due to a pathogen with a lower preferred developmental temperature. Preliminary data on cultured Batrachochytrium are consistent with this hypothesis: it develops most rapidly at 23oC in culture, with slower growth at 28oC and reversible cessation of growth at 29oC
(42). The growth rate of Batrachochytrium in the skin (and therefore virulence) and the survival of zoospores outside the host (and therefore transmission rate) are likely to be lower in amphibians from the warmer lowland regions. The ability of the pathogen to survive saprophytically in the environment and for the disease to persist may also be enhanced in the cooler montane regions. These
laboratory data may explain why chytridiomycosis has been associated with population declines in North American amphibians in montane localities (31,32) and after periods of cool weather at many U.S. and Australian sites (29,31).
Potential Environmental Cofactors in the Emergence of
Chytridiomycosis
Multiple factors (host, pathogen, environmental) may be involved in chytridiomycosis emergence. Some authors have hypothesized that infectious disease is only the proximate cause of declines and that environmental factors such as increased UV-B, chemical pollution, climate change, or stress may have predisposed amphibian populations
to opportunistic pathogens (13,32,57). Recent work at Monteverde, Costa Rica, suggests that atmospheric warming, with a resultant elevation of the average altitude of the base of the orographic cloud bank and an increase in dry periods, is causally linked to amphibian declines at this site (57). Although no pathologic studies of amphibians were undertaken, overcrowding during periods of drought may have allowed chytridiomycosis to cause substantial deaths (57). Further work is required to test this hypothesis, since
a drier climate would also predict a lower overall impact from chytridiomycosisa disease transmitted by flagellated, waterborne zoospores. The evidence suggests that cofactors are not required for chytridiomycosis to cause amphibian mass deaths. Chytridiomycosis is highly pathogenic to captive-bred amphibians exposed in captivity where control animals remained healthy (28,42). Further experimental
infections using extremely small inocula (100 zoospores) also proved fatal (29).
Figure 4. Transmission electron micrographs of iridovirus cultured from the liver of a naturally diseased
common frog (Rana temporaria) by using a fathead minnow epithelial cell line. 4a. Virus-infected cell.
Large isocahedral viruses are conspicuous within the cytoplasm (arrows). Bar = 2 m. 4b. Paracrystalline
array of iridovirus. Bar = 200 m.
Some deaths among wild amphibians have been attributed to immunosuppression, predisposing them to infectious disease (13,32). In the chytridiomycosis-related deaths,
chytridiomycosis was consistently found as the cause of death, and the range of opportunistic infections expected to occur in immunocompromised animals was not found (28,29). An increase in UV-B irradiation may influence amphibian declines (58), but in the subtropical regions of Australia and Central America, data demonstrate no significant    increase in UV irradiation (29,59). Even so, the potentional effect of increased irradiation on montane riparian rain forest amphibians is uncertain, since these animals lay eggs under rocks or in sand banks and adults are exposed to direct sunlight (24,29). Furthermore, in these regions, the species most likely to be affected by UV increases (arboreal
amphibians, which bask or lay exposed eggs) are not in decline (20). Despite extensive research, chemical pollution (20,25), habitat destruction (22), or climate change (57) have not, so far, been causally linked either to the Australian declines or to those at Central American sites other than Monteverde. No other possible cofactors, such as sympatric pathogens, have been found.
Emerging Viral Diseases of Amphibians
Iridoviruses have been implicated as the cause of amphibian mass deaths worldwide, with novel iridoviruses of amphibians recently identified from a number of regions (Tables 1, 2). The Iridoviridae encompass five recognized genera: Iridovirus, Chloriridovirus, Ranavirus, Lymphocystivirus, and goldfish virus 1-like viruses (71). Of these, the genus Ranavirus contains pathogens of fish, amphibians, and reptiles (Table 2; Figure 4).
Table 2. Iridovirusesa,b of herpetofauna (34)
Characteristics of Ranaviral Disease
Ranaviruses are often highly virulent and cause systemic infections in amphibians. Experiments with Bohle iridovirus and Gutapo virus suggest that tadpoles are the most susceptible developmental stage for ranavirus infection, and death rates of 100% occur (72). Infected metamorphs die without overt signs of infection, and infected adults show either no overt signs or, occasionally, a general weakness. Histologically, acute necrosis of hematopoietic and lymphoid tissues and of leukocytes occurs in most organs of infected animals (72). Epizootiologic data on tadpole edema virus
infections of North American amphibians are scanty, although the virus was isolated mostly from diseased animals (60-62).
Experimental infections show that bullfrog (R. catesbeiana) tadpoles infected with tadpole edema virus develop an acute lethal disease after a short incubation. The virus invades the liver, kidneys, and digestive tract and causes hemorrhage in skeletal tissue, pathologic findings similar to those described in B. marinus infected with the
ranaviruses frog virus 3, Bohle iridovirus, or Gutapo virus (72). Tadpole edema virus was uniformly lethal to three species of experimentally infected North American toad (61,62). Experimental inoculation with tadpole edema virus caused high rates of deaths in toads (100%, 4 to 17 days postinfection in young and adult Bufo sp. and newly metamorphosed Scaphiopus sp.), 40% death rates in
metamorphosing bullfrogs (R. catesbeiana), and no deaths in newly hatched salamanders (Ambystoma sp.). For the last two species, the low virulence suggests either some prior exposure and acquired immunity or a degree of innate resistance.
Pathologic data from ranaviral infections in wild amphibians may be complicated by secondary bacterial infection. Cunningham et al. (26) described two syndromes in Rana temporaria collected at sites of mass deaths in the United Kingdom: a hemorrhagic syndrome affecting the skeletal musculature and the alimentary and reproductive tracts
and an ulcerative skin syndrome with dermal ulceration and often necrosis of the distal limbs, but without hemorrhage in the muscles or viscera. In some areas, frogs were found with lesions common to both syndromes (26). These lesions are characteristic of red-leg, a syndrome thought to be caused by bacterial infection; however, Cunningham et al. (26) concluded that the lesions they described were caused by primary iridoviral infection, with or without secondary bacterial infection. Secondary bacterial infection was
also reported in wild-collected, diseased A. t. stebbinsi infected with Ambystoma tigrinum virus (27). This infection resulted in rapid onset (5 to 7 days) of apparent epidermal hyperplasia, then dermal hemorrhage, followed by visceral hemorrhage and death rates of up to 45% (27).
Factors Associated with the Emergence of Ranaviruses
The epizootiology of ranaviral disease in amphibians is poorly understood. Data from closely related ranaviral infections of fish, however, suggest a number of factors which may explain their recent emergence. Epizootic hematopoietic necrosis virus is a ranaviral pathogen of fish and the causative agent of epizootic hematopoietic
necrosis, a notifiable disease under the Office Internationale des Epizooties (73,74). Sequencing data suggest that this disease is unique among the ranaviruses, is probably indigenous to Australia, and has a wide geographic range. Dissemination may be partly due to
the virus's ability to remain infectious under adverse conditions and for prolonged periods (Table 3).
 Table 3. Longevity of infectious epizootic
 hematopoietic necrosis virus (72,74,75)
The resistant nature of epizootic hematopoietic necrosis virus suggests that amphibian iridoviruses may survive long periods at the bottom of ponds, particularly during winter. Jancovich et al. (27) demonstrated that water containing Ambystoma tigrinum virus-infected Ambystoma became uninfective after 2 weeks at 25oC, and and epizootics of Ambystoma tigrinum virus and regina ranavirus often
followed periods of cold weather (27,36). Epizootic hematopoietic necrosis virus may be spread by fomites such as fishing nets, boats, and fishing rods and through artificial stocking of ponds for recreational fishing (76). Birds have the potential to mechanically transfer virus on their feathers, feet, or bills, or by regurgitation of ingested infected material (76). Similar modes of
spread are likely to occur with the amphibian ranaviruses. The occurrence of epizootic hematopoietic necrosis may be correlated with poor husbandry, including overcrowding, inadequate water flow, and fouling of local environments with feed from fish farms (77).
Recent movements of amphibians may have disseminated ranaviral diseases. The North American bullfrog (Rana catesbeiana), the host of tadpole edema virus, has been widely introduced in the western United States and South America. In the United Kingdom, ranaviruses may be disseminated by the common practice of translocation of
amphibian egg masses and larvae by humans. Zupanovic et al. (65,78) reported the existence of ranaviruses and antibodies to ranaviruses in the cane toad (Bufo marinus) in Venezuela. In Australia, where B. marinus was introduced to Queensland in 1935, ranaviral antibodies can be identified in this species throughout its range (78). The range of B. marinus has expanded rapidly in Australia, and the toad threatens environmentally sensitive wetlands in the Kakadu National
Park. The effects of toxicity, predation, and competition of this relentlessly expanding species are well documented, and its potential to disseminate novel viral pathogens to native amphibians should also be considered in evaluating its ecologic impact. So far, a ranavirus (Bohle iridovirus) has been isolated from only one native Australian amphibian, the ornate burrowing frog (Limnodynastes ornatus) (79).
Experimental infection with Bohle iridovirus causes illness and death in a range of frogs and toads, fish, and reptiles (L. Owens, pers. comm.) (80,81). Gutapo virus, isolated from Venezuelan B. marinus (65), is also able to infect other amphibian species (72). Despite Bohle iridovirus' low host specificity, current levels of surveillance have not implicated it in diseases of free-ranging Australian animals other than those from which it was originally isolated. The ability of Bohle iridovirus to infect fish implicates the widespread introduction of fish for recreational purposes as a potentially significant factor in the dissemination of amphibian ranaviruses. Similarly, Redwood Creek virus, a ranavirus found to cause death of the endangered red-legged frog (67), can infect both amphibians and fish.
Impact of Ranaviral Disease on Amphibian Populations
The link between amphibian population declines and ranaviral disease is less clear than that with chytridiomycosis. However, these highly virulent ranaviruses are a potential threat to amphibian populations, especially populations isolated from previous disease outbreaks (and thus lacking specific immunity) and species with low fecundity. In particular, the high death rates of tadpoles infected
by ranaviruses predict a negative impact on populations. Using the model of Lampo and De Leo (82), a tadpole death rate of 80% to 90% would remove 80% to 90% of the animals that would otherwise survive metamorphosis, resulting in an approximately 80% reduction in adult populations (Lampo, pers. comm.). Although such an epizootic might not endanger the long-term survival of a highly adaptable, highly
fecund species such as B. marinus, species that inhabit specialized ecologic niches or have low fecundity might be adversely affected.
Recent outbreaks of iridoviral disease have been particularly notable in Rana temporaria (United Kingdom) and Ambystoma tigrinum (Arizona, Saskatchewan). These outbreaks involved of extensive deaths and the annual recurrence of the diseases. Common ecologic themes occur for these amphibian populations. The natural habitat of A. t. stebbinsi has been entirely destroyed by drainage after human colonization of the southwestern United States, and relict populations are now confined to manmade watering holes for cattle. Other outbreaks of ranaviral disease in A. tigrinum have been associated with altered habitats and artificial ponds. Similar habitat destruction has occurred in the United Kingdom, where most outbreaks of iridovirus infection are reported from artificial ponds (16). High population densities of frogs (16) and salamanders (27)
occur in these bodies of water, facilitating the transmission of viruses and predisposing the amphibian populations to mass deaths.
Implications of Emerging Infectious Diseases for Amphibian
Population Declines and Wildlife Conservation
The geographic spread of chytridiomycosis threatens populations of endemic and endangered amphibians in rain forests of Australia and Central and South America. Chytridiomycosis has now been reported from 38 amphibian species in 12 families, including ranid and hylid
frogs, bufonid toads, and plethodontid salamanders
(28,32,37,38,42,83). Although many of these records are from captive animals, the data demonstrate that chytridiomycosis is fatal to species originating from Europe, Africa, Madagascar, the Americas, and Oceania and attest to its potential impact should the disease be introduced into these areas. The emergence of amphibian ranaviruses raises similar concerns. Their ability to infect a wide range of amphibian and fish hosts (different vertebrate classes), global
distribution, and high virulence clearly establish them as a global threat to amphibian populations. Despite the recent geographic spread and impact of chytrids and ranaviruses, these diseases probably do not account for all the reports of declining amphibian populations, and evidence exists for other causal factors, such as habitat loss (12). Although chytridiomycosis causes high death rates in a range of species and habitats, it has caused population declines of amphibians only in certain species confined to montane
rain forests, while the emergence of ranaviral disease seems to be associated with disturbed or degraded habitats.
Hypotheses regarding the origins and impact of emerging infectious diseases on amphibians are being tested. A number of Batrachochytrium and iridovirus isolates have been cultured, and analysis of their phylogenetic and pathologic features in natural and experimental infections, as well as their biologic characteristics (e.g., ability to survive in the environment, evolution of virulent strains) has already begun. Basic host and parasite ecologic data, such as prevalence and duration of infection and presence of reservoir hosts, are being collected. Data from these studies may enable the formulation of management plans to
limit the diseases' impact. Development of chemotherapeutic regimens and diagnostic tests (enzyme-linked immunosorbent assay, in situ hybridization) may enable rapid progress toward these goals (29). National and international structures for the rapid dissemination of information between scientists, politicians, and the public may be
crucial in combating the threat of these globally emergent
pathogens. However, large geographic areas (e.g., Africa and much of Asia) have not yet been surveyed for declining amphibian populations or for the occurrence of these pathogens. Raising awareness of this threat should be one of the highest priorities for the immediate future.
Many introduced pathogens have affected human populations (10,47). However, far fewer studies of introduced diseases among wildlife have been published, and usually only those producing obvious population losses are studied vigorously (1-6). The loss of amphibian populations to the point of local extinction is a striking effect of chytridiomycosis in Australia and Central America. However, the effects of introduced wildlife diseases may be more far-reaching and subtle, with knock-on (ripple) effects permeating throughout the ecosystem (1,2,7,82,84). In many tropical and some temperate areas, amphibians make up a significant proportionsometimes mostof these vertebrate biomass (85), and their loss is likely to have unpredictable effects on populations of other species. For example, loss of herbivorous amphibian larvae may cause an overgrowth of algae in montane tropical streams, with further knock-on effects. In Australia and Central America, certain species
of snakes prey exclusively on amphibians and are likely to suffer significant population declines, in the same way that the red fox population was drastically reduced in the United Kingdom after the introduction of myxomatosis (10). Such insidious effects following disease introduction and an underestimation of their historical incidence suggest that this pathogen pollution may be as serious a conservation threat as habitat destruction and chemical pollution
(1).
Acknowledgments
The authors thank Trent K. Bollinger, Cynthia Carey, V. Greg
Chinchar, Doug Docherty, Timothy Y. James, M. Lampo, Karen Lips, Joyce E. Longcore, Don K. Nichols, David Porter, L. Owens, and Allan P. Pessier for access to unpublished data and papers in press. and Mark A. Farmer, and David Porter, University of Georgia and members of the Infectious Diseases Pathology Activity, Centers for Disease Control and Prevention, for their hospitality.
Dr. Daszak is a parasitologist at the Institute of Ecology,
University of Georgia, Georgia, USA, investigating amphibian
chytridiomycosis and other emerging pathogens of wildlife. He is interested in the pathology, cell biology, and host-parasite ecology of emerging diseases of wildlife.
Address for correspondence: Peter Daszak, Institute of Ecology, University of Georgia, Ecology Building, Athens, GA 30602, USA; fax: 706-542-4819; e-mail:  daszak@arches.uga.edu.
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Emerging Infectious Diseases
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