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87
Batrachochytrium dendrobadis
Ambystoma andersoni, A. dumerilii
A. mexicanum
Christopher J. Michaels1, Mahew Rendle1, Cathy Gibault2, Javier Lopez3, Gerardo Garcia3,
Mahew W. Perkins4, Suzea Cameron5 & Benjamin Tapley1
1 Zoological Society of London (ZSL) London Zoo, Regent's Park, London, NW1 4RY, UK
2 Parc de Thoiry, Rue du Pavillon de Montreuil, 78770, Thoiry, France
3 Chester Zoo, Moston Rd, Upton-by-Chester, Upton, Chester, CH2 1EU, UK
4 ZSL Instute of Zoology, Moston Rd, Upton-by-Chester, Upton, Chester, CH2 1EU, UK
5Birch Heath Veterinary Clinic, Birch Heath Road, Tarporley CW6 9UU, UK
Herpetological Journal SHORT NOTE
Correspondence: Christopher Michaels (
christopher.michaels@zsl.org
)
Volume 28 (April 2018), 87-92
Published by the Brish
Herpetological Society
Batrachochytrium dendrobatidis (Bd; Longcore et
al., 1999) is a major fungal pathogen known to
infect all orders of amphibian (Gower et al., 2013)
and is a driver of population declines (Bosch et al.,
2001; Lips et al., 2006; Briggs et al., 2010; Scheele
et al., 2017). The recently described congener
B. salamandrivorans (Bsal; Martel et al., 2013) also
causes disease and its spread is a cause for major concern
(Martel et al., 2014; Richgels et al., 2016; Spitzen-van
der Sluijs 2016; Laking et al., 2017). Bsal is thought to
be less cosmopolitan than the globally distributed Bd
(Fisher et al., 2009; Sabino-Pinto et al., 2016); however,
surveys of captive and free-living populations are far
from comprehensive.
In capvity, a variety of chemical treatments as well
as heat therapy (27-37 ˚C) have been used to treat the
chytrid infecons caused by both agents (e.g. Berger et
al., 2004; Bowerman et al., 2010; Chaield and Richards-
Zawacki 2011; Martel et al., 2011; Woodhams et al.,
2003, 2012; Brannelly et al., 2012; Blooi et al., 2015a;
b). However, urodelan amphibians tend to exhibit low
tolerances to elevated temperatures (e.g. Liu et al.,
2006; Bury, 2008), which oen precludes heat treatment
as a viable method to clear Bd infecon. It is therefore
imperave that the ecacy of other treatment opons
is tested.
Successful therapy using itraconazole has been
documented in numerous amphibian species from all
three orders (Forzan et al., 2008; Tamukai et al., 2011;
Brannelly et al., 2012; Rendle et al., 2015), but dierent
species respond differently to the drug. Itraconazole
toxicosis has been reported in some amphibian species
treated at ‘routine’ doses of the drug (Garner et al.,
2009; Woodhams et al., 2012). It has therefore been
recommended to record the effects of Bd on host
animals and to develop and test protocols for treang
this widespread pathogen in a variety of species of
amphibian (Woodhams et al., 2012).
We present data following Bd diagnosis and
subsequent treatments of 40 Ambystoma dumerilii,
26 A. mexicanum and 10 A. andersoni in capvity (see
Supplementary Materials for details). Animals were
maintained according to the protocols outlined in Table 1
at the Zoological Society of London (ZSL), Chester Zoo (CZ),
Parc de Thoiry (PT) and a private breeder in the UK (PB).
Salamanders were sampled inially and post treatment
(see Table 1) for Bd/Bsal screening by swabbing dorsum,
In order to beer understand the impacts and treatment
of infecon with Batrachochytrium dendrobadis (Bd) and
Batrachochytrium salamandrivorans (Bsal) it is important to
document host species, the eect of infecon and response
to treatment protocols. Here we report asymptomatic
Bd infecon detected through duplex qPCR screening of
three Mexican ambystomad salamanders; Ambystoma
andersoni, Ambystoma dumerilii and Ambystoma
mexicanum at three zoo collecons, and A. andersoni and
A. mexicanum in a private collecon. Bsal was tested for
but not detected. We also report the eecveness and side
eects of ve treatment protocols in these species. Using
the anfungal agent itraconazole, A. dumerilii were cleared
of infection without side-effects using the granulated
preparaon (Sporanox). Morbidity and mortality occurred
when A. dumerilii and A. andersoni were treated using
a liquid oral preparaon of the itraconazole (Itrafungol);
infecon was successfully cleared in surviving specimens of
the laer species. Ambystoma mexicanum was successfully
cleared without any side-eects using Itrafungol. Mortality
and morbidity were likely caused by toxic eects of some
component on the liquid preparaon of itraconazole, but
aspects of water quality and husbandry cannot be ruled
out.
Key words: Bd, axolotl, Ambystoma, chytridiomycosis,
itraconazole
88
ventrum, cloaca, lips, tail base and plantar aspect of the
feet using a sterile dry swab (see Table 1 for numbers
of swabs collected and Supplementary Materials for
swabbing methods). Duplex qPCR was used to test for
the presence of Bd and Bsal DNA following protocols
developed by Hya et al. (2007) and Blooi et al. (2013)
(see Supplementary Materials). Itraconazole baths were
used for treatment in all instuons using the protocols
outlined in Table 2 and detailed in Supplementary
Materials. Results of initial and post-treatment qPCR
tesng, including Genomic Equivalent (GE) values, are
presented in Table 1; all species presented with at least
some animals infected with Bd, but Bsal was not detected.
All animals that survived treatment tested negave for
both pathogens aer treatment (Table 2). Idencaon
of the lineage or strain of Bd infecng animals (Retallick
& Miera, 2007) was beyond the scope of this work.
All surviving salamanders in all collecons repeatedly
tested negave for Bd post treatment (see Table 2). At
ZSL and PT, there were no observed adverse side eects
to treatment with Sporanox in A. dumerilii. Water quality
was monitored at ZSL and PT, and remained good (see
Table 1). At CZ, there was 100% mortality of animals
shortly aer exposure to the treatment regimen using
Itrafungol. Water quality was not monitored at CZ. At CZ
on day 7 of treatment with Itrafungol, six A. dumerilii were
found dead. The remaining animals exhibited excessive
mucus production, cloudy eyes, erratic movements
and inappetence. At post mortem examination and
subsequent histopathology, the salamanders were thin
and presented acute dermas (somemes ulcerave
or necrotic) and branchitis. Some specimens showed
hepatocyte vacuolation. Treatment was stopped but
aer two days, the condion of the remaining animals
had connued to worsen and they were euthanased on
welfare grounds. No CZ animals completed the treatment
protocol.
At PB, A. mexicanum showed no clinical adverse
eects of treatment with Itrafungol. In A. andersoni, 50%
mortality was encountered when treated with Itrafungol.
Ambystoma dumerilii A. mexicanum A. andersoni
P 3 indi-
vidual swabs
11 indi-
vidual swabs
2 pooled
swabs, 8 individuals each
2
pooled swabs, eight indi-
viduals each
5
pooled swabs, 3 individuals
each; 1 individual swab
13
individual swab
N/A
1
pooled swab for 4 A.
mexicanum
1 pooled swab
for 4 A. mexicanum
1
pooled swab for 2 A.
andersoni
1 pooled swab
for 2 A. andersoni
3/3 +ve,
6.48,
12.6, 2964.12 GE
3/3 -ve
1/2 two pooled swabs
+ve.
2/2 pooled swabs
-ve.
6/13 +ve,
31, 41.64, 84.72,
97.44, 114.72,
704.76 GE
13/13 -ve
+ve
-ve
+ve
-ve
3-4 animals held in 100
x 30 x 30 cm aquaria
Aquaria ltered using
air-stream sponge
lters.
5 animals in a 100 x 50
x 60 cm aquarium; 11
animals individually in 40
x 30 x 30 cm plasc boxes.
Large aquarium lter with
internal lter. Small boxes
unltered; 100% water
change performed daily.
4-5 animals held in
400L aquaria.
Large plasc boxes (varying capacity).
No ltraon.
Daily 100% water changes and disinfecon
of enclosures.
c. 8
+ 0 -
0.03mg/L (with two
brief instances of c.
0.5mg/L)
2
0-0.04mg/L (with one
instance of c. 0.5mg/L
<10 mg/l
175-
200mg/L
15-17 ˚C
6.8 - 7.2
2 0mg/L
50 - 75 mg/l
370 micro
Siemens.
18 ˚C
Water parameters
not recorded.
7.9.
16-20 ˚C.
Husbandry and swabbing protocols for Ambystoma salamanders reported in this study
C. Michaels et al.
89
Bd infection and treatment in axolotls
At PB, A. mexicanum showed no clinical adverse eects
of the Itrafungol treatment. In A. andersoni, however,
animals lost vigour during treatment and within one week
of compleon of treatment, ve animals had died (50%
mortality). Animals exhibited shrinking gill branches, loss
of gill laments both of which were noceable in living
and dead animals. Animals also showed reduced feeding
behaviour. Ten days post treatment, surviving animals
were removed from the established aquarium into
which they had been placed and maintained in a 160L
plasc box with 100% daily water changes. Following this
intervenon, mortality stopped and animals recovered
to normal appearance and behaviour. No histological
data are available from these animals.
Bd infecon has not previously been reported from A.
dumerilii or A. andersoni, but has been recorded in other
neotenic Mexican Ambystoma (namely A. altamirani, A.
granulosum, A. mexicanum, A. rivulare, A. ordinarium
and A. velasci; Forzan et al., 2008; Frias-Alvarez et al.,
2008; Galindo-Bustos et al., 2014; Spitzen-van der Sluijs
et al., 2011). Animals of all three species in all four
collecons were apparently able to carry Bd infecon
without clinical disease. This has been reported in other
ambystomad salamanders (Spitzen-van der Sluijs et al.,
2011; Davidson et al., 2003; Padge-Flohr, 2008) and
this may be linked to the producon of skin pepdes
that inhibit the growth of Bd (Sheafor et al., 2008). Our
data suggest that all three species invesgated here may
be able to act as reservoirs of Bd, at least within capve
populaons and, potenally, in nature.
In A. dumerilii, infecon loads, in terms of GE per
sample, were broadly similar overall between Chester
Zoo and ZSL (Table 1), but variaon within species was
substanal. All but one swabbed animal (ZSL; 2964.12
GE) had very low loads. Ambystoma mexicanum in a Bd
posive laboratory colony were reported to have loads
of 0 - 1726.29 GE (Frias Alvarez et al., 2008). Although
the highest infecon load in A. dumerilii is approximately
40% higher than the maximum infecon load reported
in A. mexicanum, no measure of variaon was given by
Frias-Alverez et al. (2008) and so direct comparison with
our data is not possible. The use of pooled swabbing at
Parc de Thoiry for A. dumerilii, and for A. andersoni and
A. mexicanum in the private collecon precluded any
esmaon of infecon intensity and so comparisons with
the literature are not possible.
For unknown reasons, Bd was not detected on some
A. dumerilii individuals within Bd posive groups; this can
probably be regarded as represenng the boom end of
the detected variaon in infecon loads between infected
salamanders. This observaon mirrors circumstances
reported in colonies of A. mexicanum in both laboratory
(Frias Alvarez et al., 2008) and zoo (Galindo-Bustos et
al., 2014) sengs. Labial swabs, alongside samples from
other sites, were collected as some larval ambystomad
salamanders possess keranized jaw sheaths that may
act as infecon foci for Bd (Venesky et al., 2010) as well
as keran elsewhere on the body (Bosch and Marnez-
Solano, 2006), and so it is likely that swabbing was as
ecient as possible for the collecon of chytrid DNA.
Although these results are likely to reect real negaves,
extremely low infecon burdens below the detecon
threshold are also possible.
All animals in this study tested negative for Bsal
infecon, although the animals were maintained within
the opmal temperature range for this fungus (Martel
et al., 2013; Blooi et al., 2015a). Negave results are
important in delineating the overall presence of Bsal
Ambystoma dumerilii A. mexicanum A. andersoni
Itraconazole (Sporanox;
Janssen Pharamceuca N.V.,
Beerse B-2340, Belgium).
Itraconazole (Itrafungol; Elanco,
Division Eli Lilly Canada Inc.,
150 Research Lane, Suite 120,
Guelph, ON, N1G 4T2, Canada)
Itraconazole (Itrafungol)
Therapeuc
itraconazole
concentraon,
duraon and tem-
perature
0.01%. 15 minute baths
daily for eleven days at c.
16 ˚C.
0.01%. 7 minute
baths daily for seven days.
0.005%. 15
minute baths daily for seven days
Both versions at c. 18˚C.
0.01%. 5 minute baths daily
for ten days, followed by 10
rest days and then a further
ten days of 5 minute baths.
Treatment course not com-
pleted due to mortality.
Water temperature not
recorded.
0.01% in buered with one
tsp NaHCl/5L tap water to
maintain pH 7.
5 minutes per day, daily
over six days.
16-20 ˚C.
Treatment protocol Animals were moved to
individual c. 1L containers
of itraconazole soluon.
Filtered aquaria were not
sterilised between treat-
ments in order to preserve
biological ltraon.
Animals were to be bathed in 1
litre of soluon in a clear plasc
bag. Aquaria and lters sterilized
with 1:500 F10 disinfectant aer
5 and 10 days of treatment.
Treatment was not completed.
Animals bathed in individual 1L containers. Enclosures
sterilised between treatments.
Mortality 0% 100% (animals either died from
presumed toxicosis or were
euthanased)
A. mexicanum: 0% A. andersoni: 50%
Bd negave post
treatment?
Y Animals did not survive treat-
ment
Y Y
Protocols for and outcomes of itraconazole treatment in Ambystoma salamanders reported in this study.
90
in capve populaons. These results contribute to the
current belief that Bsal infecon is sll relavely rare in
capve urodelans (Sabino-Pinto et al., 2016).
Our results show that Bd infecon can be eliminated
using the established an-fungal chemical itraconazole
in neotenic A. dumerilii, A. mexicanum and A. andersoni.
Treatment with itraconazole of conrmed Bd infecon in
neotenic Ambystoma has not been previously reported.
Metamorphosed A. grinum were successfully cleared
of Bd infecon using a similar protocol to that described
here (10 minute 0.01% itraconazole (Itrizole oral soluon)
baths every other day for seven treatments; Tamukai et
al., 2011). The variaons employed by Thoiry and the
private keeper demonstrate that Bd infection can be
treated, at least in this case, by using a lower itraconazole
soluon (0.005%) and shorter bath duraon (5 minutes)
than the 0.01% and 10-minute immersion me typically
used for treang Bd infecon (Jones et al., 2012). This is
congruent with trials in the anurans Litoria caerulea and
Anaxyrus baxteri (0.005% itraconazole baths in Sporanox
form; Jones et al., 2012) and indicates that low dosage
and short immersion me may be useful in a wide range
of amphibian taxa, at least with low infecon loads.
Dierent preparaons of itraconazole appear to have
different effects and efficacy in different Ambystoma
species. The granule (Sporanox) preparation of
itraconazole can apparently be used without deleterious
side eects in A. dumerilii (this study) and A. mexicanum
(Forzan et al., 2008), while liquid preparations are
apparently safe for use in A. mexicanum (this study;
Itrafungol) and in A. grinum (Itrizole Oral Soluon 1%,
Janssen Pharmaceucal K.K., Tokyo, Japan; Tamukai et
al., 2011). However, our data suggest that use of the
liquid preparaon (Itrafungol) of itraconazole may be
linked to rapid morbidity and mortality in A. dumerilii
and A. andersoni. As water quality was not measured
in collecons using Itrafungol, and as other aspects of
husbandry including disinfecon of lter media co-varied
with treatment regimen, it is possible that detrimental
eects observed were caused by factors other than the
drug. However, deleterious side-eects have also been
recorded in anuran tadpoles treated with Itrafungol
(Garner et al., 2009). We found no evidence in the
literature suggesng either safe use or toxic eects of
any non-itraconazole ingredient (see Supplementary
Materials) of Itrafungol on amphibians. Although
the acve ingredient is the same in both compounds
(itraconazole), there may also have been interactive
eects between itraconazole and other compounds in
the drug, for example through eects on bioavailability
of the active compound. The Itrafungol solution was
buered at PB, but not at CZ. This may have an eect on its
ecacy against Bd (e.g. pH aects Bd growth; Piotrowski
et al., 2004) and any side-eects on the salamanders
themselves, but this preparaon led to morbidity and
mortality in both collecons. We recommend that the
use of this preparaon should probably be treated with
cauon in ambystomad salamanders.
The deleterious side-eects reported here represent
only impacts on health that can be detected in the short
term. It is possible that other eects on health may be
more subtle or require more longitudinal studies to
detect. Furthermore, the treatment designs used here
were based on previous reports of successful treatments
and do not represent targeted or evidence-based
approaches. The use of data from in vitro exposures of
fungus to candidate treatment regimens could inform
the selecon of the lowest dose and shortest exposures
possible to successfully eliminate the pathogen (e.g.
Martel et al., 2011). Such an approach may avoid negave
side-effects and reduce the chance of unforeseen
negative outcomes of treatment attempts, although
suscepbility of the fungus in vitro does not necessarily
equate to successful therapy in vivo (Berger et al., 2010).
We also demonstrate that Bd infection can be
successfully treated without sterilisaon of biological
filters. This is congruent with Rendle et al. (2015)
and reinforces that a balance can be struck between
eecve therapy and the maintenance of appropriate
environmental parameters. By disinfecting biological
filters between itraconazole treatments, and unless
other methods of dealing with nitrogenous waste (e.g.
chemical ltraon) are employed, the environment in
which animals are kept may rapidly become toxic due to
the accumulaon of waste products. As Bd can, on the
basis of these and other data, be eliminated without the
disinfecon of the environment, biological lters may be
le intact during the treatment of aquac amphibians for
Bd. Bd can survive outside amphibian hosts (Johnson &
Speare, 2003). We were unable to determine if infecve
colonies of Bd survived on the lter media post treatment,
but repeated and long term negave Bd results suggest
that such colonies were either absent or at least unable
to re-infect salamanders.
The authors would like to extend our thanks to keeping
and veterinary sta at ZSL (Iri Gill, Luke Harding, Daniel
Kane, Joe-Smiley Capon-Doyle, Martin Franklin, Nic
Masters, Heather Macintosh, Sophie Sparrow, Mary-
Anne Jones and Jo Korn), at Chester Zoo and at Parc de
Thoiry (Roseline Chambaud, Fanny Grados and Guillaume
Vialaret). We would also like to thank Mark Pickstock,
Debbie Moore and Pinmoore Animal Laboratory Services
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Accepted: 12 February 2018
C. Michaels et al.
