Detection of Batrachochytrium dendrobatidis in Mexican Bolitoglossine Salamanders Using an Optimal Sampling Protocol

Abstract

The role of the chytrid fungus Batrachochytrium dendrobatidis (Bd), which is the causal agent of chytridiomycosis, in the declines of Central American bolitoglossine salamanders is unknown. Here we establish a swabbing protocol to maximize the detection probability of Bd in salamanders. We then used this protocol to examine captive and wild Mexican bolitoglossine salamanders of 14 different species for the presence of Bd. Of the seven body parts sampled, the pelvic region, hindlimbs, forelimbs, and the ventral side of the tail had the most Bd per surface area and thus might provide the best sampling regions of salamanders to detect Bd infections. Sixteen out of 33 (48%) of the dead captive salamanders had Bd infections and epidermal hyperkeratosis, whereas none of the 28 clinically healthy captive animals were infected. Nine out of 17 (53%) of the wild salamanders carried low zoospore loads of Bd but had no clinical signs of disease. The high prevalence of Bd in dead captive salamanders, its absence in clinically healthy living ones and its presence in wild salamanders is consistent with Bd being involved in recent bolitoglossine population declines, but further studies would be required to draw a causal link.

Full text

Detection of Batrachochytrium dendrobatidis in Mexican
Bolitoglossine Salamanders Using an Optimal Sampling
Protocol
Pascale Van Rooij,
1
An Martel,
1
Joachim Nerz,
2
Sebastian Voitel,
3
Filip Van Immerseel,
1
Freddy Haesebrouck,
1
and Frank Pasmans
1
1
Laboratory of Bacteriology and Mycology, Department of Pathology, Bacteriology and Avian Diseases, Faculty of Veterinary Medicine,
Ghent University, Salisburylaan 133, 9820 Merelbeke, Belgium
2
Ja
¨gerstraße 50, 71032 Bo
¨blingen, Germany
3
Spangenbergstraße 81, 06295 Eisleben, Germany
Abstract: The role of the chytrid fungus Batrachochytrium dendrobatidis (Bd), which is the causal agent of
chytridiomycosis, in the declines of Central American bolitoglossine salamanders is unknown. Here we
establish a swabbing protocol to maximize the detection probability of Bd in salamanders. We then used this
protocol to examine captive and wild Mexican bolitoglossine salamanders of 14 different species for the
presence of Bd. Of the seven body parts sampled, the pelvic region, hindlimbs, forelimbs, and the ventral side of
the tail had the most Bd per surface area and thus might provide the best sampling regions of salamanders to
detect Bd infections. Sixteen out of 33 (48%) of the dead captive salamanders had Bd infections and epidermal
hyperkeratosis, whereas none of the 28 clinically healthy captive animals were infected. Nine out of 17 (53%) of
the wild salamanders carried low zoospore loads of Bd but had no clinical signs of disease. The high prevalence
of Bd in dead captive salamanders, its absence in clinically healthy living ones and its presence in wild
salamanders is consistent with Bd being involved in recent bolitoglossine population declines, but further
studies would be required to draw a causal link.
Keywords: Batrachochytrium dendrobatidis, bolitoglossine, salamander, swabbing protocol, captive, wild
Since the 1960s, significant declines and extinctions of
amphibians have been observed worldwide (Stuart et al.
2004) and potential causes include overexploitation, habitat
loss, climate change, and infectious diseases (Stuart et al.
2004; Rohr et al. 2008;Lo
¨tters et al. 2009; Rohr and Raffel
2010). Several population declines have been linked to the
presence of a pathogenic chytrid fungus, Batrachochytrium
dendrobatidis (Bd; La Marca et al. 2005; Lips et al. 2006),
which invades keratinized epithelial cells and can cause
increased tissue growth (hyperplasia) and thickening of the
cornified skin layers (hyperkeratosis) (Berger et al. 1998).
Salamanders of the family Plethodontidae, commonly
called ‘‘lungless salamanders’’, contribute significantly to the
biological diversity of Central America (Larson et al. 2006;
Wake and Vredenburg 2008), but are experiencing signifi-
cant declines that might be caused by chytridiomycosis
(Parra-Olea et al. 1999; Lips et al. 2006; Rovito et al. 2009).
For instance, Bd was recently detected in plethodontid
Correspondence to: Pascale Van Rooij, e-mail: pascale.vanrooij@ugent.be
EcoHealth
DOI: 10.1007/s10393-011-0704-z
Short Communication
Ó2011 International Association for Ecology and Health

salamander populations that are declining in Mexico and
Guatemala (Cheng et al. 2011). Here we establish a swabbing
protocol to maximize the detection probability of Bd in
salamanders and then used this protocol to examine captive
and wild Mexican bolitoglossine salamanders of 14 different
species for the presence of Bd.
Thirty-three dead captive adult bolitoglossine sala-
manders (Bolitoglossinae, Plethodontidae, Caudata), each
preserved in 70% ethanol in a separate container, were used
to establish an efficient swabbing protocol for Bd. The
specimens had not been fixed in formalin prior to storage
in ethanol. To determine whether these animals were
infected with Bd, a pelvic tissue sample was taken from
each specimen. To prevent possible cross-contamination,
disposable gloves and dissection material were changed
between containers. For each sample, DNA was extracted
from the tissue using proteinase K digestion, following the
protocol of Bandi et al. (1994), with 1:10 dilutions stored at
-20°C. Quantitative PCR (qPCR) assays were performed
on a CFX96 Real Time System (BioRad Laboratories,
Hercules, CA, USA), with amplification conditions and
primer and probe concentrations according to Boyle et al.
(2004). For each sample, qPCR assays were performed in
duplicate. Amplification standards of 1000, 100, 10, 1, and
0.1 zoospore genomic equivalents (GEs) were included
within each assay, as well as 3 negative and 1 positive
control sample. A result was considered positive when
values higher than 0.1 GEs were obtained twice.
Animals with high Bd loads were selected for the
sampling of seven selected body sites: chin, plantar side of
forelimb and hindlimb, dorsum, dorsal and ventral sides of
the tail, and the abdomen (Fig. 1; Table 1). Each of these
sites was rubbed 5 times with separate sterile synthetic
swabs (160 C, Copan Italia S.p.A., Brescia, Italy). DNA was
extracted according to Hyatt et al. (2007) and Bd was
quantified as described above. To standardize the surface
area sampled, surface area morphometry of each body part
was performed using Optimas 6.5 image analysis software
(Media Cybernetics Inc., Bethesda, MD, USA) so that GEs
of Bd could be quantified per cm
2
of skin. To validate the
qPCR results, histological slides were examined for the
presence of Bd. To do so, for each salamander, two sam-
pling sites, one with the minimum and the other with the
maximum GEs, were excised, fixed in 10% neutral buffered
formalin, embedded in paraffin and stained with hema-
toxylin and eosin (HE) and with an immunoperoxidase
(IPX) stain for Bd,as described by Berger et al. (2002).
Thirty-three dead adult and 28 healthy adult captive
Mexican plethodontid salamanders from two private collec-
tions were screened for the presence ofBd. Then, the swabbing
protocol was used on 17 healthy (no clinical signs of chy-
tridiomycosis) adult wild bolitoglossine salamanders during
an opportunistic field survey conducted in the Ve
´racruz
and Me
´xico regions of Mexico in August 2010 (Fig. 2). These
regions were selected because they represent the natural
habitat of the examined captive species. All of the animals
were sampled with a sterile synthetic swab over the seven
aforementioned body regions as described above. An overview
of all the animals sampled, together with their species desig-
nation and sampling localities is given in Table 2.
Data were log transformed to correct for non-nor-
mality. A one-way ANOVA was used to determine whether
the abundance of Bd (GE/cm
2
) differed according to the
body part sampled. A post hoc Tukey HSD test was used to
Figure 1. Assessing the effect of the body part sampled on the detection of Bd in Caudata. Schematic presentation of the body sites that were
swab sampled: 1chin, 2plantar side forelimb, 3ventral side abdomen, 4plantar side hindlimb, 5ventral side tail, 6dorsum, 7dorsal side tail.
P. Van Rooij et al.

identify which body parts were significantly different from
one another. All tests were performed in SPSS (Version 17;
SPSS Inc., Chicago, IL, USA).
Based on the analysis of pelvic tissue, evidence of Bd
infection was found in sixteen out of 33 (48%) dead
captive bolitoglossine salamanders, comprising 6 species:
Bolitoglossa platydactyla,Bolitoglossa rufescens,Pseudoeu-
rycea belli,Pseudoeurycea cephalica,Pseudoeurycea leprosa,
and Pseudoeurycea longicauda. Tail loss or tail autotomy
was observed in 8 out of the 16 infected salamanders
belonging to the species Bolitoglossa platydactyla,
B. rufescens,P. cephalica, and P. leprosa, suggesting that
infections might induce tail autotomy. Animals with GEs
of Bd in pelvic skin samples equal to or higher than 20
were used to determine the effect of the body part sampled
on the detection of Bd (Table 1). Eleven animals belong-
ing to the species B. platydactyla (n= 2), B. rufescens
(n= 6), P. cephalica (n= 1), and P. leprosa (n= 2) were
included in this study. All seven sampling sites
tested positive in the infected salamanders, except for 2
Table 1. Density of Batrachochytrium dendrobatidis in different locations on the bodies of infected bolitoglossine salamanders
Sampling site Sample
type
Mean GE ±SE/cm
2
(log 10)
Min. GE/cm
2
(log 10)
Max. GE/cm
2
(log 10)
No. of
positives
No. of
samples
Prevalence
(%)
Significance
level (P)
Chin Swab 2.31 ±3.70 Neg. 4.23 9 11 82 0.046*
Plantar side forelimb Swab 3.45 ±3.58 1.76 4.04 11 11 100 0.920
Dorsum Swab 2.55 ±2.77 Neg. 3.28 10 11 91 0.002*
Dorsal side tail Swab 3.06 ±3.30 Neg. 3.76 11 11 100 0.034*
Ventral side abdomen Swab 2.92 ±3.36 Neg. 3.89 9 11 82 0.002*
Plantar side hindlimb Swab 3.89 ±4.14 Neg. 4.68 10 11 91 0.818
Ventral side tail Swab 3.06 ±3.38 Neg. 3.92 11 11 100 0.038*
Pelvic region Tissue 4.43 ±4.49 2.45 5.01 11 11 100 N/A
Neg. samples where Bd was not detected, N/A not applicable.
* Significant difference between a swabbed site compared to the pelvic tissue samples.
Figure 2. Localities in Mexico
surveyed for the presence of
Batrachochytrium dendrobatidis.
The stars indicate localities where
Bd was detected; the circles indi-
cate localities where Bd was not
detected. The numbers correspond
with the species mentioned in
Table 2;thenumbers indicated
with an asterisk represent the
species found to be positive for
Bd.
Detection of Bd in Bolitoglossine Salamanders

Table 2. Overview of all bolitoglossine species examined for the presence of Batrachochytrium dendrobatidis using the swab protocol for
Caudata
Species Site Latitude
(°N)
Longitude
(°W)
IUCN Red
List Category
Wild/
captive
No. infected/
No. examined
[prevalence %
(95% CI)]
GE
Bolitoglossa
rufescens
1
Cordoba, Veracruz 18 52.200 97 00.200 LC Wild 1/1 8
Bolitoglossa
rufescens
2
Orizaba, Veracruz 18 50.600 97 01.800 LC Wild 1/1 5
Chiropterotriton
chiropterus
3
Zempoala, Veracruz 19 03.892 99 18.814 CR Wild 0/1
Chiropterotriton
lavae
4
La Joya, Me
´xico 19 37.426 97 01.862 CR Wild 0/1
Chiropterotriton
orculus
5
Rio Frio, Me
´xico 19 04.400 98 41.300 VU Wild 0/1
Chiropterotriton
orculus
6
Popocatepetl, Me
´xico 19 02.800 99 52.500 VU Wild 0/1
Pseudoeurycea
cephalica
7,8
Zempoala, Veracruz 19 03.900 99 18.800 NT Wild 2/24,19
Pseudoeurycea
cephalica
9
Rio Frio, Me
´xico 19 22.400 98 39.400 NT Wild 1/16
Pseudoeurycea
cephalica
10
Hidalgo, Me
´xico 20 11.300 98 44.800 NT Wild 1/1 6
Pseudoeurycea
firscheini
11
Orizaba, Veracruz 18 42.900 97 20.500 EN Wild 1/118
Pseudoeurycea
leprosa
12
Rio Frio, Me
´xico 19 21.873 98 40.651 VU Wild 0/1
Pseudoeurycea
leprosa
13
Zempoala, Veracruz 19 04.219 99 20.430 VU Wild 0/1
Pseudoeurycea
leprosa
14
Rio Frio, Me
´xico 19 22.400 98 39.400 VU Wild 1/11
Pseudoeurycea
leprosa
15
Popocatepetl, Me
´xico 19 04.400 98 41.300 VU Wild 1/112
Pseudoeurycea
longicauda
16
N/A 19 26.254 100 10.683 EN Wild 0/1
Pseudoeurycea
robertsi
17
Nevado de Toluca,
Me
´xico
19 02.800 99 52.500 CR Wild 0/1
Wild total 9/17 [52.9%
(28.5–77.4)]
Bolitoglossa
platydactyla
NT Captive 0/2
Bolitoglossa
platydactyla

NT Captive 2/2 379, 3191
Bolitoglossa
rufescens

LC Captive 6/7 1, 5, 68, 152,
386, 589
Chiropterotriton
chiropterus
CR Captive 0/2
P. Van Rooij et al.

B. rufescens specimens (3/7 and 6/7 sites positive) and 1
Pseudoeurycea leprosa specimen (6/7 sites positive) (data
not shown).
Based on the qPCR results, the plantar side of the
forelimbs and hindlimbs and the ventral side of the tail
consistently had the highest Bd densities, and the dorsum
and chin had the lowest Bd densities. In 9 out of the 11
samples with high GE values of Bd, the histological slides
showed few to abundant sporangia embedded in the upper
keratinized layers of the epidermis (Fig. 3), coinciding with
hyperkeratosis but not epidermal hyperplasia. In 10 out of
the 11 samples with low GEs, no histological lesions could
be observed.
We detected a significant main effect of body part on
Bd density (P= 0.025). However, Tukey’s post hoc mul-
tiple comparison test did not detect significant differences
Table 2. continued
Species Site Latitude
(°N)
Longitude
(°W)
IUCN Red
List Category
Wild/
captive
No. infected/
No. examined
[prevalence %
(95% CI)]
GE
Chiropterotriton
chiropterus
CR Captive 0/6
Chiropterotriton
chiropterus

CR Captive 0/5
Chiropterotriton
multidentatus

EN Captive 0/1
Pseudoeurycea
belli

VU Captive 2/4 1, 9
Pseudoeurycea
belli
VU Captive 0/2
Pseudoeurycea
cephalica
NT Captive 0/1
Pseudoeurycea
cephalica

NT Captive 2/3 18, 117
Pseudoeurycea
leprosa
VU Captive 0/14
Pseudoerycea
leprosa

VU Captive 3/3 41, 151, 1331
Pseudoeurycea
longicauda

EN Captive 1/4 2
Pseudoeurycea
nigromaculata

CR Captive 0/1
Pseudoeurycea
robertsi

CR Captive 0/2
Pseudoeurycea
sp. nov.*
N/A Captive 0/1
Thorius
troglodytes

EN Captive 0/1
Captive total 16/61 [26.2%
(15.1–37.4)]
Species and numbers indicated in bold are qPCR positive for Bd and are presented with the corresponding genomic equivalents (GEs); the indexed numbers
refer to the localities were the wild species were sampled (Fig. 2).
LC least concern, NT near threatened, VU vulnerable, EN endangered, CR critically endangered, N/A no data available.

Specimens from the species are dead.
* Species are under description by Parra-Olea & Wake.
Detection of Bd in Bolitoglossine Salamanders

among the body parts. To determine whether swabbing
might be a viable alternative to destructive tissue sampling,
we compared Bd densities in skin samples from the pelvic
region to Bd densities from swab samples of the seven body
parts. For every body part, the mean Bd density was lower
on the swab sample than it was in the tissue sample, sug-
gesting that swabbing underestimates Bd densities. How-
ever, some caution should be used interpreting these results
given that there is variation in Bd densities among body
parts and all swab samples were compared to a single tissue
sample from the pelvic region. Although all the swab
samples had less Bd than the tissue sample, a multiple
comparison test revealed that Bd density in the tissue
sample was not significantly different from Bd densities in
the swab samples of the forelimbs (P= 0.920) or hindlimbs
(P= 0.818). Hence, in salamanders, swab sampling of the
limbs might provide a reliable, nondestructive alternative
to tissue sampling for Bd quantification.
Bd was not detected in any of the 28 healthy, living
Mexican bolitoglossine salamanders, whereas 48% (16/33)
of the dead captive animals carried high zoospore loads of
up to 3191 GEs (Table 2). Fifty-three percent of the wild
animals (9/17) sampled were positive for Bd,and Bd was
detected in seven localities (Fig. 2). None of the wild ani-
mals showed obvious clinical signs of chytridiomycosis, and
the positive wild animals carried rather low zoospore loads.
A high incidence of Bd infections was found in the
sampled bolitoglossine salamanders with the pelvic region,
the forelimbs and hindlimbs and the ventral side of the tail
having the highest Bd densities. To maximize the chances
of detecting Bd in Caudata, we recommend sampling these
body parts. In contrast, in Anura (frogs and toads) the
ventral skin and the toes seem more predisposed to high Bd
densities (Berger et al. 2005; Puschendorf and Bolan
˜os
2006).
There is a paucity of data demonstrating susceptibility
of plethodontid salamanders to Bd infection. We revealed
that clinically healthy captive salamanders were free of Bd
infection, but that in half of the dead captive salamanders
Bd was present, combined with hyperkeratosis, a hallmark
of clinical Bd infections. This result is consistent with
recent infection trials revealing high virulence of Bd to
B. rufescens and P. leprosa (Cheng et al. 2011) and the
report of Pasmans et al. (2004) demonstrating lethal Bd
infection in captive Bolitoglossa dofleini. Bd was detected in
half of the wild Mexican bolitoglossine salamanders sam-
pled but was not associated with any obvious clinical signs
of disease. Although the high prevalence of Bd in dead
captive salamanders, its absence in clinically healthy living
ones, and its presence in wild salamanders is consistent
with Bd being involved in recent bolitoglossine population
declines, further studies will be necessary before Bd is
conclusively demonstrated to be a causal factor in the
decline of Mexican bolitoglossine salamanders.
ACKNOWLEDGMENTS
This study was funded by a research grant from Ghent
University to Pascale Van Rooij (BOF08/24J/004). Poly-
clonal antibodies against B. dendrobatidis were kindly
provided by Dr. Alex D. Hyatt (Australian Animal Health
Laboratory, CSIRO, Victoria, Australia). We are grateful to
Arnaud Jamin and Eike Amthauer for kindly providing
Plethodontid specimens, David Van Rooij (RCMG, Ghent
University, Belgium) for providing help in mapping of the
sampling localities and two anonymous referees for
providing helpful suggestions that greatly improved the
manuscript.
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Detection of Bd in Bolitoglossine Salamanders