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History and recent progress on chytridiomycosis in amphibians

Article in Fungal Ecology · February 2016

DOI: 10.1016/j.funeco.2015.09.007

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History and recent progress on chytridiomycosis

in amphibians

Lee BERGERa,*, Alexandra A. ROBERTSa, Jamie VOYLESb,

Joyce E. LONGCOREc, Kris A. MURRAYd,e, Lee F. SKERRATTa
a
One Health Research Group, College of Public Health, Medical and Veterinary Sciences, James Cook University,
Townsville, Queensland, 4810, Australia
b
Department of Biology, University of Nevada, Reno, NV, 89557-0325, USA
c
School of Biology and Ecology, University of Maine, Orono, ME, 04469-5722, USA
d
Grantham Institute e Climate Change and the Environment, Imperial College London, Exhibition Road,
London SW7 2AZ, UK
e
School of Public Health, Imperial College London, Norfolk Place, London, W2 1PG, UK

article info abstract

Article history: Batrachochytrium dendrobatidis (Bd) emerged in the 1970s in Australia and the Americas,
Received 25 March 2015 causing rapid and catastrophic declines and extinctions of na€ıve amphibian populations as
Revision received 4 September 2015 it spread through remote rainforest and alpine regions. The description of chy-
Accepted 9 September 2015 tridiomycosis in 1998 stimulated a large and diverse global research effort, including
Available online 6 October 2015 studies on phylogeny, distribution, ecology, and virulence e but mitigating its effect
Corresponding editor: remains a major challenge. In 2010 a second Batrachochytrium species, B. salamandrivorans
€ rlocher
Felix Ba (Bsal), emerged after spreading to Europe from Asia and has decimated fire salamanders in
the Netherlands and Belgium. Bsal appears to be restricted to salamanders and newts
Keywords: whereas Bd can infect all amphibian orders. These cases show that despite the current
Batrachochytrium dendrobatidis advanced state of globalisation, severe pathogens are still spreading and some may cur-
B. salamandrivorans rently be excluded by geographic barriers, hence biosecurity still has potential to mitigate
Wildlife spread of undiscovered and unpredictable pathogens of wildlife.
Extinction ª 2015 Elsevier Ltd and The British Mycological Society. All rights reserved.
Emerging infectious disease
Population decline
Conservation
Chytridiomycosis

Introduction diversity when introduced into na€ıve host populations (Scott,

The emergence and spread of Batrachochytrium species in
amphibians has overturned a traditional view that pathogens
do not significantly affect wildlife populations (Skerratt et al.,
2007; Martel et al., 2014). On the contrary, pathogens, even
when acting alone, can cause catastrophic losses of wildlife
1988; Tompkins et al., 2015). Chytridiomycosis caused by
Batrachochytrium dendrobatidis (Bd) has generated a huge
research effort since it was first reported in 1998. Here we
focus on Bd with a brief section on the recently discovered
B. salamandrivorans (Bsal), which also causes amphibian chy-
tridiomycosis. Past reviews of Bd have covered immunology

* Corresponding author.
E-mail address: lee.berger@jcu.edu.au (L. Berger).
http://dx.doi.org/10.1016/j.funeco.2015.09.007
1754-5048/ª 2015 Elsevier Ltd and The British Mycological Society. All rights reserved.
90
(Rollins-Smith et al., 2011; Voyles et al., 2011), pathology,
diagnosis (Berger et al., 2009), treatment (Berger et al., 2010;
Baitchman and Pessier, 2013), probiotics (Bletz et al., 2013),
genetics (Rosenblum et al., 2009) emergence and impacts
(Skerratt et al., 2007; Fisher et al., 2009b; Kilpatrick et al., 2010),
and management (Woodhams et al., 2011; Scheele et al.,
2014b; Skerratt et al., accepted for publication). In this review,
we describe the discovery of Bd and summarise recent
research on phylogeny, distribution, ecology, and virulence in
the global effort to mitigate this pandemic. Lastly, we discuss
Batrachochytrium species in the context of emerging fungal
diseases in wildlife. This review is not intended to be
exhaustive, and we tend to cite Australian studies as
examples.
Discovery and impacts of Bd
Some of the most dramatic amphibian population declines
began in the late 1970s, particularly in remote and protected
mountain regions of Australia and Central America. It took
about a decade, however, for this global catastrophe to gain
attention because amphibian populations can fluctuate dra-
matically and because mass mortality events were rarely
observed. Determining the cause of amphibian “dis-
appearances” became an urgent conservation task with
researchers testing hypotheses on pollutants, increased UVB
radiation, feral predators and climate change (Alford and
Richards, 1999; Stuart et al., 2004). Habitat loss is a major cause
of amphibian declines but was not linked to these “enigmatic”
population crashes that were occurring in pristine and pro-
tected areas (Stuart et al., 2004).
In Australia, Keith McDonald (chief ranger of the Queens-
land Government) recruited Rick Speare (epidemiologist
employed in public health at James Cook University) to
examine the pattern of declines in his spare time. They pro-
posed that an introduced fatal infectious disease of frogs had
spread north across Queensland between 1979 and 1993, with
a mean rate of spread of w100 km/yr 1, wiping out pop-
ulations of adults (but not tadpoles) within months of arrival
(Laurance et al., 1996). McDonald and Speare conducted
intense monitoring of abundant rainforest frog populations in
the north where they predicted this disease would spread to
next, including the last population of the, now extinct, sharp
snouted dayfrog (Taudactylus acutirostris). The effort to mon-
itor this population led to the detection of a die-off in late 1993
as it decimated the population (Laurance et al., 1996). A team
of investigators with expertise in detecting emerging infec-
tious diseases collected dead frogs and used pathology,
microbiology, electron microscopy and DNA sequencing to
diagnose a novel epidermal infection that they named chy-
tridiomycosis (Berger et al., 1998).
While these diagnostic investigations were underway, an
initial experiment was conducted to verify the pathogenicity
of this superficial skin infection. This experiment involved
exposing frogs to infected skin pieces and resulted in 100 %
mortality within 3 weeks (Berger et al., 1998). Histopathology
explained why tadpoles were not included in the mass mor-
talities; although tadpoles can carry infection in their kerati-
nised mouthparts, the infections are not usually fatal until
L. Berger et al.
after metamorphosis when the rest of the skin becomes ker-
atinised and infection becomes widespread (Berger et al., 1998;
Marantelli et al., 2004). In 1997, researchers had also found
rainforest frogs dying during an outbreak of chytridiomycosis
in Panama, confirming that this disease was the global culprit
for widespread declines (Berger et al., 1998). Contempora-
neously, Bd was discovered in metamorphs of South Ameri-
can dendrobatid frogs that died at the National Zoological
Park, Washington, USA. Bd was isolated into pure culture from
these frogs, studied and described as a new genus and species
(Longcore et al., 1999; Pessier et al., 1999).
The spread of declines in areas of North and Central
America enabled researchers to monitor Bd at remaining sites
with abundant amphibians. The disease arrived in well stud-
ied sites in both Panama and California in 2004 and
researchers documented a steep rise in Bd prevalence as
amphibian populations crashed (Lips et al., 2006; Vredenburg
et al., 2010). Transmission experiments with pure cultures
involving even low numbers of zoospores resulted in fatal
infections (Berger et al., 1999, 2004; Woodhams et al., 2003;
Carey et al., 2006). It has since become accepted that the
spread of chytridiomycosis has caused hundreds of species
worldwide to decline with numerous extinctions, and chy-
tridiomycosis is now regarded as the most devastating disease
on record to impact vertebrate biodiversity (Skerratt et al.,
2007). In Australia, extinctions include iconic species such as
the southern and northern gastric brooding frogs (Rheoba-
trachus silus, R. vitellinus), last seen in 1981 and 1985,
respectively.
Chytridiomycosis has become endemic through many
regions where it can usually be found at more than 5 %
prevalence and where disease outbreaks may occur season-
ally (Berger et al., 2004; McDonald et al., 2005; Skerratt et al.,
2010). Persisting amphibian species exhibit different respon-
ses in post-epidemic systems; some have lower abundance
and smaller distributions and some are continuing to decline
and suffer significant annual mortality from chy-
tridiomycosis, even decades after introduction (McDonald and
Alford, 1999; Murray et al., 2009; Pilliod et al., 2010; Phillott
et al., 2013; Brannelly et al., 2015a). For example, annual
mortality of the alpine tree frog (Litoria verreauxi alpina) results
in almost complete population turnover after breeding
(Scheele et al., 2015). Some critically endangered species now
have very small populations and will need intervention to
become secure. In Australia, six species have been placed in
this category, including the corroboree frog (Pseudophryne
corroboree), which relies on continual reintroductions to per-
sist (Skerratt et al., accepted for publication). Populations of
some susceptible species, however, are recovering in abun-
dance and distribution albeit at levels lower than before chy-
tridiomycosis was introduced (McDonald et al., 2005; Newell
et al., 2013; Scheele et al., 2014a).
Taxonomy and phylogeny of Bd
Bd is a member of the Chytridiomycota, an early diverging
lineage of true fungi whose members reproduce by forming
uniflagellate zoospores. Although early studies (Berger et al.,
1998; Longcore et al., 1999) placed the genus Batrachochytrium
History and recent progress on chytridiomycosis
in the Chytridiales, molecular studies have since identified
additional lineages in what was earlier considered one order.
Batrachochytrium is now placed in the order Rhizophydiales
(Letcher et al., 2006). Members of this order have a chitin-
walled zoosporangium plus rhizoids that anchor to the sub-
stratum and also increase the surface area for enzyme
excretion and nutrient absorption (Alexopoulos et al., 1996).
Bd zoosporangia grow inside host epidermal cells (Berger
et al., 2005a; Greenspan et al., 2012). Zoospores, the asexual
reproductive spores, form within the zoosporangium follow-
ing mitosis and are released through a discharge tube to the
epidermal surface. Zoospores are surrounded by a membrane
and are motile but spread in water largely via passive move-
ment. They encyst on the skin surface and infect deeper cells
via a germ tube (Greenspan et al., 2012). The development of
sporangia matches the differentiation and turnover of epi-
dermal cells so that by the time sporangia are mature they
have been carried to the skin surface (Berger et al., 2005a).
A global, virulent, panzootic lineage of B. dendrobatidis (Bd-
GPL) occurs throughout amphibian inhabited continents and
is thought to have formed from recombination between pre-
viously geographically isolated strains (Farrer et al., 2011).
Based on analyses of 36 genetic loci, Bd-GPL can be subdivided
into a North American clade (Bd-GPL-1) and a more globally
dispersed clade (Bd-GPL-2) (Schloegel et al., 2012). Regional
lineages also exist and population genetics based on rese-
quencing data has indicated the existence of lineages that are
likely specific to South Africa (Cape lineage, Bd-CAPE) and
Switzerland (Bd-CH) (Farrer et al., 2011) e regions without
known population declines caused by Bd (Weldon et al., 2004;
Spitzen-Van Der Sluijs et al., 2014). Based primarily on anal-
yses of the rDNA internal transcribed spacer region (ITS)
haplotypes, an Asian Bd lineage is also present in Japan (Goka
et al., 2009), China (Bai et al., 2012), India (Dahanukar et al.,
2013), and Korea (Bd-Korea; Bataille et al., 2013) while Brazil
harbours an endemic lineage (Bd-Brazil), as well as a hybrid
between Bd-GPL and Bd-Brazil (Schloegel et al., 2012). Con-
tinental endemic lineages are usually sparsely detected,
whereas Bd-GPL lineages are common worldwide. One
hypothesis explaining this observation is that Bd-GPL has out-
competed local endemic strains.
Not only does Bd have a complex population genetic
structure, but the species is interesting in that strains are
subject to aneuploidy (Farrer et al., 2013; Rosenblum et al.,
2013). Copy number of chromosomal regions (contigs) within a
single isolate can range from one to four. Although chromo-
somal copy number is not a predictor of relatedness (Rose-
nblum et al., 2013), copy number of some genes may affect
pathogenicity.
An isolate with the genetic signature of both Bd-GPL and
Bd-Brazil from one site in Brazil suggests that sexual repro-
duction is possible in Bd (Schloegel et al., 2012), but such
recombination events may be rare. In many Chy-
tridiomycetes, sexual reproduction is accompanied by the
formation of thick-walled, resistant sporangia (resting spores)
that can withstand drying. Resting spores, however, most
commonly form asexually and although they have not been
seen in Bd, they do occur in pure cultures of other species in
the Order Rhizophydiales (e.g., Longcore, 2004; Letcher and
Powell, 2005). Hence it is possible both sexual and asexual
91
formation of resting spores by Bd may occur though they have
not been detected under conditions in skin and culture
examined so far, and experiments with culture have shown
Bd is susceptible to heat and desiccation (Johnson et al., 2003).
Recently another species, B. salamandrivorans (Bsal), has
been added to the genus Batrachochytrium (Martel et al., 2013)
(additional information below). These two species are unusual
as they are the only known chytrid pathogens of vertebrates.
Their closest known relative is Homolaphlyctis polyrhiza, a
saprobe isolated from a cellulosic substrate (Longcore et al.,
2011).
Global distribution
A global survey of wild and archived amphibian populations
has revealed that Bd is now widespread and has an excep-
tionally broad host range, detected in over 520 amphibian
species (including frogs, toads, caecilians, salamanders and
newts) from 56 countries (see collation of databases at www.
bd-maps.net; Murray et al., 2010; Gower et al., 2013; Olson
et al., 2013). Bd has been found in a huge variety of habitats
and under a wide range of climatic condition, but thrives in
relatively cool, wet, diverse habitats in alpine, temperate and
tropical areas (e.g., Kriger and Hero, 2007b; Murray et al., 2010;
Skerratt et al., 2010; Ocock et al., 2013). In global and Aus-
tralian analyses, a large temperature range is correlated with
lower risk of Bd infection (Murray et al., 2011a; Murray and
Skerratt, 2012; Liu et al., 2013) and in Australia and Costa Rica
low rainfall and high temperatures reduce Bd occurrence
(Puschendorf et al., 2009, 2011; Murray et al., 2011a). Excep-
tions to this occur such as seen with die-offs due to chy-
tridiomycosis in the stream-dwelling Arizona desert lowland
leopard frog (Lithobates yavapaiensis) (Savage et al., 2011).
Severe population impacts leading to decline and extinc-
tion are more likely in cooler, wetter, less thermally variable
regions (Bielby et al., 2008; Murray et al., 2011b). In addition,
exposure, infection risk and potential for severe population
effects are strongly mediated by host species’ ecological and
life history traits (Murray et al., 2010, 2011b; Murray and
Skerratt, 2012) as well as the presence of other extrinsic fac-
tors. For example, within the distribution of Bd, local envi-
ronmental factors other than climate and weather that reduce
the effects of chytridiomycosis include salinity (Heard et al.,
2014; Stockwell et al., 2015b) and suitability for aquatic
microfauna (Schmeller et al., 2014).
Retrospective diagnoses show that Bd strains have occur-
red historically (>80 yrs) in parts of Asia, Africa and Brazil, and
in these regions frogs have not undergone declines associated
with mass mortality (Weldon et al., 2004; Goka et al., 2009;
Rodriguez et al., 2013; Tarrant et al., 2013). A survey of 15 Asian
countries found only 2.35 % Bd prevalence (Swei et al., 2011).
The general lack of population effects combined with genomic
studies revealing endemic Bd lineages, are consistent with
Asia, Africa and Brazil having a long history of infections and
these regions are possible origins of the species (Bataille et al.,
2013; Rodriguez et al., 2013; Tarrant et al., 2013). The unex-
pected detection of Bd by PCR on museum samples collected
since 1888 in Illinois requires confirmation by alternative tests,
but may represent a US endemic strain (Talley et al., 2015).
92
Distribution models show that Bd has likely already spread
to most climatically suitable regions (Fisher et al., 2009b;
Ro€ dder et al., 2010; Murray et al., 2011a), with a notable
exception being West Africa (Penner et al., 2013). It is possible
that inhospitable non-forested terrain to the west of Nigeria
has been a barrier to spread in Africa (Penner et al., 2013). Of
immediate concern is Madagascar, where Bd has recently
been detected by PCR at low intensities in sites scattered
among otherwise Bd-negative populations (Bletz et al., 2015;
Kolby et al., 2015). However, such an important diagnosis
should be confirmed by visualising sporangia by histology
and/or immunohistochemistry and/or fungal culture (Skerratt
et al., 2008, 2011).
Within infected countries smaller areas may be free of
chytridiomycosis. For example, in Australia, na€ıve pop-
ulations in apparently suitable environment exist at the
extremes of Bd distribution in Australia, including in south-
west Tasmania and Cape York Peninsula as well as on an
island 3 km off the east coast (Berger et al., 2004; Murray et al.,
2010, 2011a; Skerratt et al., 2010; Stockwell et al., 2015a).
Ecology
Temperature effects
Temperature plays an important role in the disease ecology of
chytridiomycosis and can largely explain seasonality and
distribution (based on both altitude and latitude) of infections
in many regions (Berger et al., 2004; McDonald et al., 2005;
Skerratt et al., 2007; Murray et al., 2009, 2011a,b, 2013; Phillott
et al., 2013). Temperature affects both the ectothermic
amphibian hosts (e.g., by mediating immune responses) and
growth of Bd which is restricted to between w4  Ce26  C
(Berger et al., 2004; Piotrowski et al., 2004).
In laboratory studies, Bd reproduced more slowly at lower
temperatures but zoospores were active for longer, and iso-
lates adapted to lower temperatures (4  C) over time and
exhibited shifts in reproductive traits (Voyles et al., 2012).
Evidence for adaptation to temperatures was also seen in
differences in growth among Bd isolates collected across a
latitudinal gradient (Stevenson et al., 2013).
Prevalence and intensity of infection are associated with
seasonal changes in environmental temperatures (McDonald
et al., 2005; Murray et al., 2009, 2013; Phillott et al., 2013). The
disease is mitigated by high temperatures and hence is gen-
erally more severe in moderate climates, such as winter in
tropical north-eastern Australia, spring in centraleeastern
Australia and summer in temperate southern Australia
(Berger et al., 2004; Kriger and Hero, 2007a; Skerratt et al., 2010;
Aziz et al., 2011).
Differential host susceptibility and resistance
Amphibians vary in their innate susceptibility to chy-
tridiomycosis, both within and among populations and spe-
cies (Berger et al., 2009; Savage and Zamudio, 2011; Gahl et al.,
2011; Brannelly et al., 2012). Resistant species include some
major invasive amphibians, including North American bull-
frogs (L. catesbeianus) and African clawed frogs (Xenopus laevis)
L. Berger et al.
(Kilpatrick et al., 2010). This differential tolerance among hosts
(e.g., with some species acting as Bd reservoirs) is problematic
for more vulnerable sympatric species (D. Hunter, unpub).
In laboratory inoculation experiments, mortality rates
have differed dramatically among species, ranging from 0 to
100 %, with many species exhibiting an intermediate level of
susceptibility. Additionally, within a species susceptibility to
Bd may vary between populations and clutches, and immu-
nological studies are aimed at understanding the mechanisms
for these innate differences (Savage and Zamudio, 2011; Elli-
son et al., 2014a,b; Bataille et al., 2015). The increasing lon-
gevity of Fleay’s barred frog (Mixophyes fleayi) post decline
(Newell et al., 2013) provides hope that innate immunity may
be evolving in some species.
Amphibian behaviour and life history also affect disease
outcome; for example, species that bask or inhabit warmer
and drier microclimates have higher survival rates than oth-
ers within the same habitats, while population impacts have
been most commonly observed in species with greater asso-
ciation with water (Bielby et al., 2008; Murray et al., 2011b;
Rowley and Alford, 2013). Additionally, in some species, pop-
ulation persistence is related to high recruitment rates of
juveniles despite high mortality, and there is some evidence of
compensatory recruitment (Muths et al., 2011; Phillott et al.,
2013; Scheele et al., 2015).
Although adaptive immune responses have been detected,
highly effective acquired immunity towards Bd in vivo has not
been demonstrated in the species tested, and frogs became re-
infected even after multiple exposures (Ramsey et al., 2010;
Cashins et al., 2013; McMahon et al., 2014). Hence developing a
vaccine may be challenging, given the difficulties in over-
coming the immunosuppressive effects of B. dendrobatidis (see
Virulence Section below).
Reservoirs and non-amphibian interactions
The possibility that Bd can grow or survive away from
amphibians (either as a saprobe, by forming a resting spore, by
infecting an alternative host or in being transmitted by a non-
amphibian vector) has important implications for under-
standing the distribution and persistence of Bd. Although
amphibians appear to be the key habitat for Bd, some evidence
suggests that amphibian-independent persistence of Bd is
possible. For example, Bd DNA has been detected in non-
amphibian samples such as crayfish, lizards and water fowl
(Kilburn et al., 2011; Garmyn et al., 2012; Brannelly et al.,
2015b). Most of these preliminary studies rely on positive PCR
results, which only indicate presence of Bd DNA, not infectious
zoospores or replication within host tissue. The strongest
evidence for a non-amphibian reservoir of Bd is provided in a
study of crayfish with Bd detected in gut contents (McMahon
et al., 2013). Experimentally exposed crayfish had increased
mortality and could also transmit Bd to amphibian tadpoles.
Bd DNA has also been detected in water and sediments, but
the inability to isolate Bd from the environment has limited
investigation on saprobic life stages (Kirshtein et al., 2007;
Hyman and Collins, 2012). In experiments, Bd persisted at low
levels on moist soil or sediment for weeks, with greater sur-
vival under sterile conditions (Johnson and Speare, 2003;
Walker et al., 2007), and the possibility of extended survival
History and recent progress on chytridiomycosis
away from the host has not been ruled out. Bd appears to be
easily outcompeted by other microbes, however, and is
inhibited by secretions from many bacteria (Bletz et al., 2013).
Aquatic zooplankton, including crustacea (e.g., Daphnia), cil-
iates and rotifers may also consume Bd zoospores (Buck et al.,
2011; Schmeller et al., 2014). Predation by zooplankton
reduced Bd zoospores and infection rates in Pyrenean
mountain lakes and also experimentally reduced infection in
tadpoles (Schmeller et al., 2014). Hence, altering aquatic
microfauna provides hope for biological control, particularly
in higher nutrient lakes rather than clear lower nutrient
streams. In addition, altering microbiota on amphibian skin
by exposing animals to inhibitory bacterial species has been
shown to reduce Bd infection rates in the laboratory and is
being trialed in the field (Bletz et al., 2013).
Bd appears to spread naturally through suitable environ-
ments, but can jump across barriers such as deserts or oceans,
probably via pet or produce trade (Kolby et al., 2014).
Virulence
Much research has focused on identifying and understanding
mechanisms of Bd virulence, with the aim to develop targeted
approaches to attack Bd or enhance host resistance. Inves-
tigations of virulence have been directed by: (1) knowledge of
effects on hosts and (2) analysis of Bd strain variation.
Infection with Bd appears to cause immunosuppression in
some species (Fites et al., 2013; Young et al., 2014), which
presumably enables infection to progress. Zoosporangia, but
not zoospores, produce one or more immunosuppressive
metabolites (Fites et al., 2013). These metabolites inhibit
lymphocyte proliferation in vitro and cause splenocyte apop-
tosis by inducing caspase signalling pathways, but do not
affect macrophage or neutrophil activity. In vivo, Bd metabo-
lites inhibit localised adaptive skin immune responses, but
not innate leucocyte proliferation (Fites et al., 2014). This
pathogen-mediated immunosuppression may help explain
the contrasting results of host immunity proteomic studies, as
splenic T cell activation and signalling genes are more sup-
pressed in susceptible frog species (Ellison et al., 2014a,b).
Interestingly, even frogs with severe chytridiomycosis do not
have obvious signs of immunosuppression such as secondary
infections (Berger et al., 2004). Further work on Bd’s immu-
nosuppressant and virulence will help uncover the precise
mechanisms of resistance and susceptibility to
chytridiomycosis.
Additionally, high Bd burdens disrupt skin function and
electrolyte homeostasis in frogs. During Bd infection, epi-
thelial ion transport is inhibited causing a 25e70 % depletion
of plasma electrolytes (Voyles et al., 2009; Marcum et al., 2010).
This depletion of ions most likely affects cardiac electrical
function, ultimately leading to cardiac arrest and death
(Voyles et al., 2009). It is not yet known whether specific ion
channel inhibitors are responsible for this phenomenon, or
whether it is the result of general skin degradation. Activity-
based studies have pointed to the role of proteases in the
disruption of host skin integrity. Incubation of zoospore
supernatants with X. laevis skin explants caused the dis-
ruption of intracellular junctions and detachment-induced
93
cell apoptosis (Brutyn et al., 2012). Bd supernatants contain
proteolytic enzymes with various functions, including elas-
tolytic and tryptic activity (Symonds et al., 2008; Moss et al.,
2010), although the exact role of specific enzymes in patho-
genesis has not been determined. Gene expression studies
also support the role of proteases in colonisation and patho-
genicity, as several fungalysin metallopeptidases, aspartyl
peptidases and serine proteases were differentially expressed
between zoospore and sporangial life stages, or when Bd was
grown on amphibian substrata (Rosenblum et al., 2008, 2012).
Differences in morbidity and mortality in experimentally
infected amphibians indicate that Bd virulence can vary
between strains of the same and different lineages (Berger
et al., 2005b; Farrer et al., 2011). Increased Bd growth rate,
zoospore production and sporangial size in pure culture, have
been linked with increased host mortality and immunosup-
pressive activity (Fisher et al., 2009a; Farrer et al., 2011; Piovia-
Scott et al., 2014; Voyles et al., 2014a). Differential gene
expression between (a) the two life stages, (b) different growth
substrates, or (c) different Bd strains have identified many
putative Bd virulence factors that may help Bd invade host
cells or evade immune responses, including proteases, adhe-
sins, lipases, and crinkler- (CRN)-like effector genes and fun-
gal proteins similar to those involved in vertebrate signal
transduction, immune regulation and intracellular trafficking
systems (Rosenblum et al., 2008, 2012; Fisher et al., 2009a;
Garland et al., 2011). Further studies, however, are required to
validate and assign mechanistic functions to these candidate
genes.
Genome analysis suggests that the most rapidly evolving
gene regions in Bd encode putative virulence factors such as
proteases and CRN-like effectors (Farrer et al., 2013). The
ability of Bd to rapidly adapt has been seen in vitro with growth
rates and virulence changing over successive passages (Brem
et al., 2013; Langhammer et al., 2013; Voyles et al., 2012, 2014a).
The potential for evolution of virulence raises concerns
regarding the likelihood and severity of chytridiomycosis
epizootics into the future.
Comparison to B. salamandrivorans
A second species of Batrachochytrium, B. salamandrivorans
(Bsal), was recently discovered, causing severe mortality and
declines of fire salamanders (Salamandra salamandra) in
Europe. In 2010, the Netherlands’ fire salamander population
experienced a sudden 96 % decline. Half the group of 39 ani-
mals that were brought into captivity began dying and Bsal
was cultured from these hosts and described (Martel et al.,
2013). Bsal spread to Belgium causing outbreaks in 2013 and
2014. Global surveys and infection experiments combined
with genetic work showed that it most likely originated in East
Asia (Thailand, Vietnam, and Japan), where it does not cause
obvious disease (Martel et al., 2014). The lack of detection of
Bsal outside Europe or Asia means there is a high threat of Bsal
introduction into populations of na€ıve salamanders, such as
those in North America which is a hotspot of salamander
diversity, and has relatively poorly regulated but high volume
amphibian trade (Yap et al., 2015). Mitigating this threat is one
of the highest current priorities for animal conservation.
94
Batrachochytrium salamandrivorans has the same zoospore
ultrastructural features as Bd but has slightly different
developmental morphology (such as the occurrence of germ
tubes in culture and a greater proportion of colonial thalli in
skin) and infects different amphibian hosts. Genetically, Bsal
is sufficiently divergent that it is not detected by PCR primers
that amplify a relatively conserved section of the ITS region of
all Bd lineages (Blooi et al., 2013). Analysis of sequences from
18S and 28S rDNA indicates a large genetic distance between
the two species, but groups them together with strong support
(Martel et al., 2013). In experimental results thus far, Bsal
occurs only in urodeles (salamanders and newts) and not
anurans (frogs and toads) (Martel et al., 2014). Like Bd, Bsal also
infects epidermal cells but invades into deeper layers, result-
ing in a more erosive and ulcerative disease than caused by Bd
(Berger et al., 2005a; Martel et al., 2013). Another difference
between the two species is a lower temperature preference for
Bsal, which grows best between 10  C and 15  C and dies at
25  C (Martel et al., 2013).
Emerging fungi of wildlife
Another recently emerging fungal disease that is devastating
to biodiversity is white nose syndrome, caused by Pseudo-
gymnoascus destructans (Minnis and Lindner, 2013), and which
has spread across North America killing millions of hiber-
nating cave bats (Eskew and Todd, 2013). Similarly to Batra-
chochytrium spp., it infects skin, has broad host specificity and
has emerged due to spread by human transport e it likely
originated in Europe where it causes aclinical infections in Old
World bats. Snake fungal disease (caused by Ophidiomyces
ophiodiicola) has also recently emerged in the eastern US. It
causes a fatal skin disease and population impacts are sus-
pected but are still unclear (Allender et al., 2015). These recent
cases add to other fungal wildlife diseases such as in coral,
bees and platypus as well as oomycete infections in aquatic
animals. Some diseases are multifactorial and in many cases
spread to naive hosts is the key driver of emergence (Connolly,
2009; Fisher et al., 2012; Tompkins et al., 2015). These exam-
ples show that the effect of fungi on animal populations, as on
plants, can be severe and should not be overlooked. Fisher
et al. (2012) conclude that attributes of fungi that make them
dangerous differ among species and may include: (1) broad
host range, (2) reservoirs in carrier species, saprobic stages or
long lived environmental spores, (3) rapid growth and viru-
lence similar to bacteria or viruses, and (4) well developed
toxin production. Most mammals, however, have higher body
temperatures than ectotherms, platypus (32  C) and hiber-
nating bats (7  C). A higher body temperature is suspected to
be an advantage against fungal infections and may explain
why fungal disease in mammals is usually opportunistic and
secondary to other co-factors (Robert and Casadevall, 2009).
Conclusions
Since being recognised as a cause of global amphibian
declines, Bd has become one of the most well studied wildlife
pathogens, with much research aiming towards the greater
L. Berger et al.
challenge of mitigation (Scheele et al., 2014b). However,
wildlife diseases are complex to investigate and to manage. As
the ecology of Bd is affected by many host and environment
variables, factors that influence population survival are con-
text specific. Hence, conservation solutions will be diverse and
necessitate species-specific research in many instances.
Interventions based on current knowledge are being trialled
(Scheele et al., 2014b), but it is hoped that further discoveries
(e.g., of host resistance or pathogen virulence) will be able to
achieve more sustainable disease control. Captive and wild
assurance colonies and attempts to improve survival rates of
critically endangered species are the highest priorities, while
helping less threatened species re-establish their former dis-
tributions and abundances is next. When selecting optimal
frog habitat to preserve, including environmental refugia from
chytridiomycosis has become a high priority (Puschendorf
et al., 2011).
For Bsal, there is still a chance to stop further international
spread, and overwhelming evidence supports a ban on sala-
mander trade (Martel et al., 2014; Yap et al., 2015).
The catastrophic emergences of Batrachochytrium species
(in w1979 and 2010) show that despite the current advanced
state of globalisation, severe pathogens may still be retained
by historical bio-geographic boundaries; hence, continental
and even country level biosecurity is crucial because it can
prevent the spread of undescribed pathogens. This is partic-
ularly the case given the potential for strain recombinations to
result in increased virulence via the trade in wildlife. Although
environmental changes can precipitate disease outbreaks,
pandemics are most commonly caused by the spread of a
pathogen to na€ıve regions (Skerratt et al., 2007; Tompkins
et al., 2015). The emergences of Bd and Bsal highlight the
difficulty of detecting and predicting which pathogens are
dangerous. Neither seems to have caused obvious disease in
its hosts of origin, in which they would have appeared as
harmless skin microbes if surveys had been conducted. There
are other examples of diseases impacting biodiversity that
may not be predictable based on past events (e.g, trans-
missible Tasmanian devil facial tumour disease). This dem-
onstrates the need for incorporating uncertainty into
biosecurity and risk assessments, especially when dealing
with relatively undescribed systems like wildlife health. Many
countries have rigorous biosecurity for livestock which should
be adapted for wildlife, however, current processes focus on
specific diseases and hence do not incorporate the unpre-
dictability of emerging diseases. In addition, a reserve
capacity of diverse and flexible teams is needed to address
wildlife disease outbreaks should biosecurity fail; similar to
crises in general. Hence collecting baseline knowledge of
wildlife disease is important, especially in terms of early
detection of incursions (Grogan et al., 2014).
The discovery of Bd has led to a paradigm shift in attitude
to wildlife disease as a conservation issue. The initial unwill-
ingness to accept that an introduced pathogen could be the
sole cause of population decline and extinction has now been
overcome. As a result, there is now more rapid recognition of
the impact of pathogens in wildlife disease outbreaks, such as
Bsal and white nose syndrome (Voyles et al., 2014b). The next
step is to ensure this evidence is used in policy decisions
(Langwig et al., 2015).
History and recent progress on chytridiomycosis
Acknowledgements
This work was supported by Australian Research Council
grants (FT100100375, LP110200240 and DP120100811), a
Queensland Accelerate Fellowship (14-218) and the Taronga
Conservation Science Initiative. Thanks to Rick Speare for
discussions.
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