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Successful elimination of a lethal wildlife infectious disease in nature

Authors:
  • Biodiversity Research Institute (IMIB)

Abstract

Methods to mitigate the impacts of emerging infectious diseases affecting wildlife are urgently needed to combat loss of biodiversity. However, the successful mitigation of wildlife pathogens in situ has rarely occurred. Indeed, most strategies for combating wildlife diseases remain theoretical, despite the wealth of information available for combating infections in livestock and crops. Here, we report the outcome of a 5-year effort to eliminate infection with Batrachochytrium dendrobatidis affecting an island system with a single amphibian host. Our initial efforts to eliminate infection in the larval reservoir using a direct application of an antifungal were successful ex situ but infection returned to previous levels when tadpoles with cleared infections were returned to their natal sites. We subsequently combined antifungal treatment of tadpoles with environmental chemical disinfection. Infection at four of the five pools where infection had previously been recorded was eradicated, and remained so for 2 years post-application.
rsbl.royalsocietypublishing.org
Research
Cite this article: Bosch J, Sanchez-Tome
´
E,
Ferna
´
ndez-Loras A, Oliver JA, Fisher MC, Garner
TWJ. 2015 Successful elimination of a lethal
wildlife infectious disease in nature. Biol. Lett.
11: 20150874.
http://dx.doi.org/10.1098/rsbl.2015.0874
Received: 18 October 2015
Accepted: 28 October 2015
Subject Areas:
health and disease and epidemiology
Keywords:
chytridiomycosis, Batrachochytrium
dendrobatidis, mitigation, Alytes muletensis,
Mallorca
Author for correspondence:
Jaime Bosch
e-mail: bosch@mncn.csic.es
These authors contributed equally to this
study.
Electronic supplementary material is available
at http://dx.doi.org/10.1098/rsbl.2015.0874 or
via http://rsbl.royalsocietypublishing.org.
Conservation biology
Successful elimination of a lethal wildlife
infectious disease in nature
Jaime Bosch
1,†
, Eva Sanchez-Tome
´
1
, Andre
´
s Ferna
´
ndez-Loras
1
, Joan A. Oliver
2
,
Matthew C. Fisher
3
and Trenton W. J. Garner
4,
1
Museo Nacional de Ciencias Naturales, CSIC, Jose
´
Gutie
´
rrez Abascal 2, Madrid 28006, Spain
2
Conselleria de Medi Ambient i Mobilitat, Govern de les Illes Balears, Gremi Corredors 10, Polı
´
gon Son Rossinyol,
Palma 07009, Spain
3
Department of Infectious Disease Epidemiology, Imperial College London, St Mary’s Hospital, Norfolk Place,
London W2 1PG, UK
4
Institute of Zoology, Regent’s Park, London NW1 4RY, UK
Methods to mitigate the impacts of emerging infectious diseases affecting
wildlife are urgently needed to combat loss of biodiversity. However, the
successful mitigation of wildlife pathogens in situ has rarely occurred.
Indeed, most strategies for combating wildlife diseases remain theoretical,
despite the wealth of information available for combating infections in live-
stock and crops. Here, we report the outcome of a 5-year effort to eliminate
infection with Batrachochytrium dendrobatidis affecting an island system with
a single amphibian host. Our initial efforts to eliminate infection in the larval
reservoir using a direct application of an antifungal were successful ex situ
but infection returned to previous levels when tadpoles with cleared
infections were returned to their natal sites. We subsequently combined anti-
fungal treatment of tadpoles with environmental chemical disinfection.
Infection at four of the five pools where infection had previously been
recorded was eradicated, and remained so for 2 years post-application.
1. Introduction
Emerging infections are on the increase, incurring extraordinary economic and
health costs and globally degrading our natural capital. In response, several
efforts to eradicate animal pathogens are underway, however with few suc-
cesses reported [1,2]. Research on livestock pathogens predominates and
provides insight as to how pure wildlife pathogens may be combated for host
conservation purposes [1,2]. Delivery of an efficient and practical intervention
is a cornerstone of any scheme to eliminate infectious diseases, and the direct
application of antimicrobials to infected hosts or immunization can be used
effectively to control pathogen replication within a host and to reduce the like-
lihood of transmission to susceptible individuals [3]. However, for these types
of interventions to be effective, control of environmental reservoirs of (re)infec-
tion must also be achieved. Local control of pathogens through the use of
environmental chemical treatments has been effectively used to disinfect
areas where environmental transmission of parasites can occur, but the
impact of chemical treatment on transmission and maintenance of infection
in concert with antimicrobial treatments has rarely been examined [4].
Amphibian chytridiomycosis, a disease predominantly caused by the aquatic
chytrid fungus Batrachochytrium dendrobatidis (Bd) has driven population declines,
local extirpations and species extinctions across five continents [5]. The pathogen
is an extreme generalist, infecting over 700 amphibian species (http://www.bd-
maps.net). Strategies developed to ameliorate the impacts of chytridiomycosis are
predominantly geared towards disease-free maintenance of captive assurance
colonies, and multiple methods have been developed to treat captive amphibians
&
2015 The Author(s) Published by the Royal Society. All rights reserved.
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against infection with Bd [68]; however, most attempts at
immunization have failed [9]. The remaining approaches that
hold promise for in situ control include bioaugmentation
with bacteria, direct application of antifungal drugs and
environmental application of anti-Bd chemicals. Although
not without promise, research on the application of bioaug-
mentation so far describes complex interactions between
host, beneficial bacteria, the broader microbiota and pathogen
that are strongly dependent upon environmental context and
amphibian community structure [10,11]. For this reason,
bioaugmentation strategies are unlikely to converge on an
intervention that can be generalized across amphibian commu-
nities and ecosystems. The immediacy of the epizootic of
chytriomycosis calls for an intervention that can be applied
across systems, so we chose to explore direct application of
antifungal drugs to infected hosts and environmental appli-
cation of chemicals as strategies to eliminate Bd from a
simple, single host system [12].
2. Material and methods
Biannual surveys at five permanent ponds (3 Torrent des Fer-
rerets, 2 Coco
´
de sa Bova; Mallorca, Spain) were undertaken
from 2008 and are ongoing. We sampled Mallorcan midwife
toad (Alytes muletensis) tadpoles, as terrestrial stages are rarely
captured as they take refuge in inaccessible locations. Tadpoles
of this and other Alytes sp. are recognized as reservoirs of infec-
tion [13,14]. To sample, we swabbed tadpole mouthparts
following established protocols [12,13]. All ponds affected by
chytridiomycosis on the island were included in the study and
none was left as untreated controls owing to conservation
requirements. However, chemical disinfection efforts at Torrent
de Ferrerets preceded those at Coco
´
de sa Bova, affording us
the opportunity to compare across sites.
Swabs were processed according to standard extraction and
quantitative PCR (qPCR) methods [15] in duplicate and run
against negative controls and positive controls (0.1, 1, 10 and
100 zoospore genomic equivalents, GE).
For antifungal treatments, tadpoles were collected and trans-
ported in plastic bottles containing pond water. We used air
pumps and tubes with aeration stones to ensure tadpole survival
during the outward hikes. Tadpoles were then transported to the
laboratory and kept in several cooled, glass aquaria. All tadpoles
were bathed daily for 7 days in aged tapwater containing
1.0 mg l
21
itraconazole (Sporanox, Janssen-Cilag Inc.) and
returned to aquaria after each treatment. Aquaria water was
replaced every day during the 7 days treatment. After treatment,
tadpoles were returned to the collection sites by helicopter, either
immediately if ponds were not drained or after ponds were
refilled by autumn rain. In these cases, subsets of 40 tadpoles
from each aquarium were swab-sampled 15 days post-treatment.
Environmental disinfection was done using Virkon S
(DuPont Inc.) at 1% final concentration and a single application
applied ad libitum to the environment. The disinfectant was lib-
erally applied to all rock, gravel, crevice and vegetated areas that
surrounded the immediate environs of each breeding site.
3. Results
W e initially attempted mitigation by trea ting in 2009 A. muletensis
tadpoles inhabiting two permanent pond sites in one of the
two infected drainages, Coco
´
de sa Bova (electronic sup-
plementary material, figure S1), with the antifungal
itraconazole. We used a treatment protocol previously
shown to eliminate infection in tadpoles [7]. Treatments
were applied ex situ, and prior to post-treatment release the
two ponds were completely drained of water and naturally
dried by the arid environment that typifies Mallorca. We
had previously determined that Bd is absent from the other
two ephemeral water bodies in this drainage, and environ-
mental Bd is not thought to persist during periods of
drying [16]. The two ponds naturally refilled during the
autumn rainy season. At no point during this prolonged
period of captivity did we detect any evidence of infection
in the treated tadpoles. The following spring, qPCR analysis
showed that all treated animals had contracted infections not
significantly different from what had been recorded at the
location before treatment [17] (figure 1). Repeating the proto-
col in the spring of 2012, this time without draining the
breeding sites, and with tadpole release only 7 days after
treatment, was again not associated with reduction in the
prevalence of infection or reduced burdens of infection in
the following spring (figure 1).
In contrast, at three breeding sites used by the species in the
second drainage, Torrent des Ferrerets (electronic supplemen-
tary mate rial, figure S2), we could not detect infection in any
animals sampled in 2013 after trea tment of tadpoles and wha t-
ever terrestrial A. muletensis life stages we could capture with
itra conazole, draining the sites and then treating the environ-
ment with Virkon S (electronic supplementary material, figures
S3 and S4; figure 1). Replication of this pr otocol a t Coco
´
de sa
Bova in 2013 and applica tion of Virkon S solution to the rock cre-
vices located around the ponds where metamorphosed
A. muletensis reside again cleared infection in the larger popu-
lation of tadpoles resident in the larger pond at this location.
Residual infection was detected in tadpoles occupying the smal-
ler permanent pond site. Data fr om samples take n at Torrent des
Ferrerets 2 y ears a ft er chemical disinfection showed tha t the
effect of environmental application of Virkon S twinned with
itra conazole treatment of tadpoles carried over across years, as
again no evidence of infection was detected in 2014 (figure 1).
4. Discussion and conclusion
W e cannot sa y with certainty why dir ect trea tm ent of tadpoles
with antifungals without environmental disinfection failed
to resolve infection at Coco
´
de sa Bova, but the most likely
explana tion is that infection reinvaded tadpoles from post-
metamorphic animals that we could not a ccess in their
terres trial refuges. We do occasionally discover corpses of juven-
iles exhibiting a str ong molecular signal of infection. Lik e other
amphibian species, Alytes spp. tadpoles scavenge from corpses,
and this process is presumed to be a factor in transmission of Bd
from corpses to tadpoles in another species [18,19]. Irrespectiv e of
this, our application of Virko n S at Torr ent des F err er ets pro vided
proof-of-principle that environmental application of fungicides
and other chemical treatments may be a better approa ch when
combined with antimicrobial trea tment of infected hosts. This
initial conclusion was reinfor ced when we recapitulated our
result by clearing infection in C oco
´
de sa Bova the following
year. In our case, combining chemical disinfection twinned
with antifungal treatment of tadpoles pro v ed the better stra tegy,
eliminating infection and prev enting spill-back over the short
term at four of the five pools where we attempted mitigation.
The development of disinfection strategies alone cannot
eliminate the threat of chytridiomycosis, as evidence
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continues to accumulate that lethal amphibian-associated
chytrid fungi are frequently being introduced into Europe
and beyond [12,20]. Clearing site-level infection is no guaran-
tee against pathogen reintroduction or the introduction of
novel pathogens. However, to cope with the existing, recur-
ring and future threats of chytridiomycosis, rapid response
strategies require cheap, simple and transferrable methods
for mitigating infection that can be employed as soon as the
threat has been identified. We acknowledge that Virkon S is
a controversial chemical to use environmentally and our
use of it was driven by the urgency of midwife decline on
Mallorca [21]. Virkon S is only one of several chemical treat-
ments known to have antifungal properties against chytrid
fungi [22,23] and antifungal treatments do not require exten-
sive investment in time and effort. We argue that research
informing efforts to combat chytridiomycosis should include
in-depth investigations of the impact of antifungals and anti-
Bd chemicals on amphibian health without discarding
attempts to develop immunization and other methods of dis-
ease control. Research on the application of these chemicals
for control of wildlife diseases must also include investigation
of the potential impacts of chemical application to other bio-
diversity, the environment and associated ecosystem services.
Ethics.
The work was carried out under the Govern de les Illes
Balears’s permit no. CEP 43/2015.
Data accessibility. Data are available in the electronic supplementary
material.
Authors’ contributions. J.B., T.W.J.G. and M.C.F. designed and wrote the
paper, with contributions from E.S.-T. Data were collected and/
or analysed by E.S.-T., A.F.-L. and J.A.O; all authors provided
intellectual input and edited/approved the manuscript.
Competing interests. We declare we have no competing interests.
Funding. This work was funded by Fundacio
´
n General CSIC, Banco
Santander and BiodivERsA project RACE.
Acknowledgements. We thank S. Pinya, X. Manzano, C. Serrano,
A. Dı
´
az-Guerra, E. M. Albert, S. F. Walker, D. Daversa,
S. Ferna
´
ndez-Beaskoetxea, J. Vo
¨
ro
¨
s, B. R. Schmidt, B. Tapley and
J. Bielby for field assistance, the people working at Paratge Natural
de la Serra de Tramuntana and Conselleria de Medi Ambient
(especially J. Mayol and E. Moragues), and the owner of the Mossa
property for field site access.
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... Although small in number, PIs conducted to date have involved a range of species, pathogens, regions and purposes and provide an informative evidence base to draw from ( Figure 1). Data from published PIs demonstrate their worth for disease management across diverse scenarios; for example, oral praziquantel baiting of red foxes Vulpes vulpes reduced Echinococcus multilocularis egg contamination among urban areas in Switzerland (Hegglin et al., 2003); topical Pseudomonas fluorescens application protected Myotis lucifugus bats from white nose syndrome (WNS) associated mortality in North America (Hoyt et al., 2019); and, itraconazole treatment of Alytes muletensis tadpoles and environmental disinfection eradicated Batrachochytrium dendrobatidis (Bd) from ponds in Spain (Bosch et al., 2015). Lessons learned can also inform future improvements (Langwig et al., 2015). ...
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... Strategies for mitigating the impact of chytridiomycosis on wild amphibian populations would also be valuable. Diverse avenues have been explored, such as antifungal treatment of tadpoles coupled with environmental disinfection (Bosch et al. 2015), and probiotic therapy through bioaugmentation of microbes that confer defense against chytrids (Bletz et al. 2013;Kearns et al. 2017;Woodhams et al. 2020); but the broad applicability and scalability of these methods have yet to be determined. Additional strategies discussed by the group included biological control using mycoviruses, which has been applied against the fungal agent responsible for chestnut blight (Rigling and Prospero 2018), and the introduction of nonpathogenic chytrid lineages to outcompete virulent lineages. ...
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... It is also possible that Virkon ® Aquatic could be used to treat the environment. In an isolated aquatic system in Spain, Bd was eradicated by treating tadpoles with itraconazole and the environment with 1% Virkon ® S (Syndel, Ferndale, WA, USA) [80]. However, similar to plant-derived fungicides, the toxicity of Virkon ® Aquatic on amphibians and other aquatic organisms needs to be evaluated [81,82]. ...
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Emerging infectious diseases are a serious threat to wildlife communities, and the ability of pathogens to survive in the environment can exacerbate disease impacts on hosts and increase the likelihood of species extinction. Targeted removal or control of these environmental reservoirs could provide an effective mitigation strategy for reducing disease impacts but is rarely used in wildlife disease control. We examined the effectiveness of managing environmental transmission to reduce impacts of an emerging infectious disease of bats, white-nose syndrome. We used a chemical disinfectant, chlorine dioxide (ClO 2 ), to experimentally reduce Pseudogymnoascus destructans , the fungal pathogen causing WNS, in the environment. We conducted laboratory experiments followed by three years of field trials at four abandoned mines to determine whether ClO 2 could effectively reduce P. destructans in the environment, reduce host infection, and limit population impacts. ClO 2 was effective at killing P. destructans in vitro across a range of concentrations. In field settings, higher concentrations of ClO 2 treatment sufficiently reduced viable P. destructans conidia in the environment. The reduction in the environmental reservoir at treatment sites resulted in lower fungal loads on bats compared to untreated control populations. Survival following treatment was higher in little brown bats ( Myotis lucifugus ), and trended higher for tricolored bats ( Perimyotis subflavus ) compared to untreated sites. These findings support the management of environmental reservoirs as an effective control strategy for wildlife disease and provide a valuable tool for ongoing conservation efforts. More broadly, these results highlight how the intensity of environmental reservoirs can have cascading impacts on host infection and population declines.
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Emerging infectious diseases are reducing biodiversity on a global scale. Recently, the emergence of the chytrid fungus Batrachochytrium salamandrivorans resulted in rapid declines in populations of European fire salamanders. Here, we screened more than 5000 amphibians from across four continents and combined experimental assessment of pathogenicity with phylogenetic methods to estimate the threat that this infection poses to amphibian diversity. Results show that B. salamandrivorans is restricted to, but highly pathogenic for, salamanders and newts (Urodela). The pathogen likely originated and remained in coexistence with a clade of salamander hosts for millions of years in Asia. As a result of globalization and lack of biosecurity, it has recently been introduced into naïve European amphibian populations, where it is currently causing biodiversity loss.and remained in coexistence with a clade of salamander hosts for millions of years in Asia. As a result of globalization and lack of biosecurity, it has recently been introduced into naïve European amphibian populations, where it is currently causing biodiversity loss.
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Emerging infectious diseases are reducing biodiversity on a global scale. Recently, the emergence of the chytrid fungus Batrachochytrium salamandrivorans resulted in rapid declines in populations of European fire salamanders. Here, we screened more than 5000 amphibians from across four continents and combined experimental assessment of pathogenicity with phylogenetic methods to estimate the threat that this infection poses to amphibian diversity. Results show that B. salamandrivorans is restricted to, but highly pathogenic for, salamanders and newts (Urodela). The pathogen likely originated and remained in coexistence with a clade of salamander hosts for millions of years in Asia. As a result of globalization and lack of biosecurity, it has recently been introduced into naïve European amphibian populations, where it is currently causing biodiversity loss.and remained in coexistence with a clade of salamander hosts for millions of years in Asia. As a result of globalization and lack of biosecurity, it has recently been introduced into naïve European amphibian populations, where it is currently causing biodiversity loss.
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Wildlife diseases pose an increasing threat to biodiversity and are a major management challenge. A striking example of this threat is the emergence of chytridiomycosis. Despite diagnosis of chytridiomycosis as an important driver of global amphibian declines 15 years ago, researchers have yet to devise effective large-scale management responses other than biosecurity measures to mitigate disease spread and the establishment of disease-free captive assurance colonies prior to or during disease outbreaks. We examined the development of management actions that can be implemented after an epidemic in surviving populations. We developed a conceptual framework with clear interventions to guide experimental management and applied research so that further extinctions of amphibian species threatened by chytridiomycosis might be prevented. Within our framework, there are 2 management approaches: reducing Batrachochytrium dendrobatidis (the fungus that causes chytridiomycosis) in the environment or on amphibians and increasing the capacity of populations to persist despite increased mortality from disease. The latter approach emphasizes that mitigation does not necessarily need to focus on reducing disease-associated mortality. We propose promising management actions that can be implemented and tested based on current knowledge and that include habitat manipulation, antifungal treatments, animal translocation, bioaugmentation, head starting, and selection for resistance. Case studies where these strategies are being implemented will demonstrate their potential to save critically endangered species.
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Amphibian chytridiomycosis, caused by the chytrid fungus Batrachochytrium dendrobatidis (Bd), is an emerging infectious disease that widely threatens amphibian biodiversity. However, population-level outcomes following the introduction of the pathogen are highly context dependent and are mediated by a broad suite of biotic and abiotic variables. Here, we examine the effect of the introduction of Bd on native island populations of the IUCN red-listed amphibian species Alytes muletensis, the Mallorcan midwife toad. We show that the outcome of pathogen introduction is not only dependent on biotic factors, but is also dependent on environmental factors that vary across local scales. Our experimental infections confirm that the genotype of Bd occurring on Mallorca is hypovirulent in A. muletensis when compared against the lineage found occurring on mainland Iberia. Long-term population data show that A. muletensis populations on the island are increasing overall, but trends in highly infected populations are conflicting. We use mathematical models and field data to demonstrate that this divergence in population response to infection can be explained by local environmental differences between infected sites, whereas pathogen genetics, host genetics, and intrinsic epidemiological dynamics driven by fungal load are less likely to be the cause of these differing population trajectories. Our results illustrate the need to take into account the appropriate environmental scale and context when assessing the risk that an emerging pathogen presents to a naive population or species.