Herpetological Review 48(2), 2017
360 AMPHIBIAN AND REPTILE DISEASES
Herpetological Review, 2017, 48(2), 360–363.
© 2017 by Society for the Study of Amphibians and Reptiles
Prevalence of Batrachochytrium dendrobatidis and
B. salamandrivorans in the Gulf Coast Waterdog,
Necturus beyeri, from Southeast Louisiana, USA
The globally widespread amphibian fungal pathogen
Batrachochytrium dendrobatidis (Bd) has been linked to
amphibian declines worldwide (Lips et al. 2006; Skerratt et
al. 2007). In Louisiana, USA, Bd has been found in several
amphibian species (Chatfield et al. 2012; Rothermel et al. 2008),
but to our knowledge no population-level die-offs have been
observed. Published literature on Bd prevalence in Louisiana is
scant for some amphibian species and completely absent for
many others. This trend is likely driven by the perception that Bd
is not a major problem in this area due to a lack of observed die-
offs attributable to chytridiomycosis.
Batrachochytrium salamandrivorans (Bsal) is an emerging
amphibian fungal pathogen and was first described after
populations of European Fire Salamanders (Salamandra
salamandra) were decimated in the Netherlands (Martel et al.
2013). Since the observations in the Netherlands, Bsal has been
found in wild salamander populations in neighboring Germany
and Belgium (Spitzen-van der Sluijs et al. 2016), and has been
linked to animals in captivity (Cunningham et al. 2015) and trade,
with a proposed origin of Asia (Martel et al. 2014). Occurrence of
Bsal has not been documented in any wild salamanders in North
America, but due to the prolific trade in Asian salamanders, there
is reason to think that it could arrive or may already be in North
America. Prudent surveillance is vital, as a Bsal introduction in
the United States could be devastating to this global hotspot of
salamander diversity (Gray et al. 2015).
The Gulf Coast Waterdog, Necturus beyeri, is a permanently
aquatic neotenic salamander that inhabits sandy spring-fed
streams along the Gulf Coastal Plain, and its global conservation
status is listed by NatureServe as ‘apparently secure’ (G4). In
Louisiana, the Gulf Coast Waterdog is ranked S3 by the Louisiana
Department of Wildlife and Fisheries, which indicates the species
is rare and local throughout the state (21 to 100 known extant
populations). Very little is known about prevalence rates of Bd
in this species. Of two individuals examined by Chatfield et al.
(2012), one tested positive for Bd. To our knowledge, this species
has never been tested for Bsal, and no species of Necturus was
used in challenge experiments by Martel et al. (2014). Therefore,
it is unknown if members of this genus are susceptible to Bsal
infection. Our aim was to conduct additional sampling for Bd
and Bsal in N. beveri in Louisiana.
Gulf Coast Waterdogs were captured in southeast Louisiana
with unbaited minnow traps at sites along ca. 12 km of Bayou
Lacombe as part of an occupancy modelling study (Fig. 1). Al-
though sampling took place over three weeks, all swabs used
in this study were collected in one round of checking all traps
and thus it is certain that each represents a unique individual.
Here we report Bd and Bsal presence and pathogen load, as de-
termined using quantitative polymerase chain reaction (PCR)
results of swabs taken from 76 waterdogs captured 31 March
through 2 April 2015 (Table 1). All captures were of live individu-
als and no other amphibians were captured in the traps.
To test for Bd and Bsal presence, waterdogs were placed
in clear zip-top plastic bags and swabbed by gently rubbing
a rayon-tipped swab (MWE113, Advantage Bundling SP, LLC,
Durham, NC, USA) five times each on the following areas: the
dorsum, each side, the venter, and the bottom of each foot. New
nitrile gloves and new zip-top bags were used for each animal.
Genomic DNA was extracted from the swabs using the
Qiagen DNeasy Blood & Tissue Kit (Qiagen, Inc., Valencia, CA,
USA) and the manufacturer’s recommended protocol for animal
tissue with the following modifications: swabs were incubated
for one hour and were vortexed and spun in a centrifuge after 30
minutes and again at the end of the incubation period; samples
were eluted twice using 100 uL of elution buffer each time instead
of 200 uL once. A multiplexed quantitative (real-time) PCR assay
was used to detect the presence of Bd and Bsal DNA, following
Boyle et al. (2004) and Blooi et al. (2013). Bovine serum albumin
(0.7 mL) was added to each reaction well, as this has been shown
to aid in overcoming problems with inhibition (Garland et al.
2010). All samples were run in triplicate and scored as positive
if at least one replicate tested positive for Bd or Bsal. To confirm
that reactions were not inhibited, an internal positive control
(Applied Biosystems, Inc.) was added to one replicate of each
sample (Hyatt et al. 2007). A seven-fold dilution series of plasmid
standards from Pisces Molecular (Boulder, CO, USA) was run on
every qPCR plate to enable quantification of pathogen load for
both Bd and Bsal. As each assay used only 5 uL of the 200 uL of
genomic DNA extracted, per assay load values were multiplied
by 40 to estimate whole swab pathogen loads.
BRAD M. GLORIOSO
J. HARDIN WADDLE*
U.S. Geological Survey, Wetland and Aquatic Research Center,
Lafayette, Louisiana 70506, USA
CORINNE L. RICHARDS-ZAWACKI
Department of Ecology and Evolutionary Biology, Tulane University,
New Orleans, Louisiana 70118, USA; Present address:
Department of Biological Sciences, University of Pittsburgh,
Pittsburgh, Pennsylvania 15213, USA
*Corresponding author; e-mail: firstname.lastname@example.org
taBle 1. Prevalence of Batrachochytrium dendrobatidis (Bd) infection
by size (SVL = snout–vent length) and sex in Necturus beyeri in south-
east Louisiana, USA.
Sex Size range No. No. Bd Prevalence
(SVL, mm) sampled positive
Juvenile Unknown 63–83 7 2 0.29
Adult Male 91–128 52 23 0.44
Female 95–122 17 8 0.47
Total 76 33 0.43
Herpetological Review 48(2), 2017
AMPHIBIAN AND REPTILE DISEASES 361
Fig. 1. Map of the study area showing the sites where Necturus beyeri were captured for assessment of infection with Batrachochytrium den-
drobatidis (Bd) or B. salmandrivorans along Bayou Lacombe, St. Tammany Parish, Louisiana, USA. Solid circles represent sites where at least
one individual tested Bd-positive (Bd+). Open circles represent sites where no individuals tested Bd-positive. The embedded table gives
greater site-level detail: N = no. individuals sampled; Prev = Bd prevalence among individuals sampled at the site. The inset map indicates the
location of the study area within Louisiana.
Herpetological Review 48(2), 2017
362 AMPHIBIAN AND REPTILE DISEASES
Overall, 33 of 76 (43%) waterdogs tested positive for Bd (Glo-
rioso and Waddle 2017). Sixteen of 33 positive results were posi-
tive in all three runs, whereas the remaining 17 were positive in
two of three runs. No difference in prevalence was observed by
adult sex (Table 1). Eleven of 12 traps with three or more wa-
terdogs captured and swabbed contained a mix of positive and
negative results (Fig. 1). Pathogen loads were typically low in Bd-
positive individuals (mean = 364, SD = 298; range 84–1212 plas-
mid equivalents). No waterdogs tested positive for Bsal, and no
clinical signs of chytridiomycosis were observed in any captured
Our prevalence estimate for Bd in Gulf Coast Waterdogs was
nearly twice the rate of all Necturus (N = 17) samples from Chat-
field et al. (2012). The timing of our study coincided with the re-
ported peak infection period found by others for semi-aquatic
species in the southeast (e.g., Kinney et al. 2011; Rothermel et al.
2008). However, the timing of this study also coincides with the
peak time to capture these animals, and captures of this species
in mid-summer are rare. The lower Bd prevalence observed in
Chatfield et al. (2012) may be attributable to warmer water tem-
peratures experienced in summer by some of the permanently
aquatic salamanders they studied (e.g., Amphiuma and Siren).
Water temperatures where these animals live routinely exceed
the optimal growth range of Bd (17°–25°C) in the hottest months
in the Gulf Coastal Plain (Piotrowski et al. 2004).
The relatively cool stable environment that waterdogs inhab-
it suggests that they may serve as year-round hosts of Bd (Chat-
field et al. 2012). Additionally, Bd has been shown to complete
its life cycle in crawfish and transmit Bd infection to amphibians
(McMahon et al. 2013). Crawfish were commonly captured in our
traps and are a known primary prey item of Gulf Coast Waterdogs
in at least some populations (Shoop and Gunning 1967). More
work is needed examining the thermal relationship of Bd, craw-
fish, and Gulf Coast Waterdogs to clarify the possible year-round
Currently it is not known if Necturus is susceptible to Bsal.
Given that the optimal growth range of Bsal (10–15°C) is lower
than that of Bd (Martel et al. 2013) and that the spring-fed lotic
waters that Necturus beyeri inhabit provide suitable tempera-
tures for Bsal for at least part of the year, there would be cause
for concern if Necturus beyeri were shown to be susceptible to
Bsal. More research into the effect of Bd on Necturus as well as
increased monitoring for Bsal across the range of all taxa in the
genus is warranted.
Acknowledgments.—We thank L. Muse and S. Godfrey for as-
sistance with field work. Waterdogs were captured under Louisiana
Department of Wildlife and Fisheries Scientific Collecting Permit
LNHP-15-010002E. All handling of animals was conducted in ac-
cordance with approved IACUC protocols (USGS WARC FY2008-1).
Any use of trade, firm, or product names is for descriptive purposes
only and does not imply endorsement by the U.S. Government. This
is contribution number 578 of the U.S. Geological Survey Amphibian
Research and Monitoring Initiative (ARMI).
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© 2017 by Society for the Study of Amphibians and Reptiles
Batrachochytrium dendrobatidis and Batrachochytrium
salamandrivorans Surveillance in Salamanders of
Southeastern Virginia, USA
The United States (US) has the highest diversity of
salamanders in the world, and many of these are endemic to
only the southeastern states making the US the global hotspot
for salamander biodiversity (Gray et al. 2015). For this reason,
managing disease threats to US salamanders is a high priority
(Grant et al. 2015). Chytridiomycosis is a disease caused by the
pathogenic chytrid fungi Batrachochytrium dendrobatidis (Bd)
and B. salamandrivorans (Bsal). Bd is an emerging infectious
disease that has had a devastating impact on amphibian
populations across Central America, Europe, North America,
and Australia (Skerratt et al. 2007; Cheng et al. 2011; Martel et
al. 2013). All amphibian orders (Anura, Urodela, Gymnophiona)
have experienced disease symptoms and population declines
from (Bd) (Skerratt et al. 2007; Martel et al. 2013; Martel et
al. 2014). Bsal is newly discovered and initial experimental
challenges have reported affects on some urodeles (Martel
et al. 2014; Van Rooij et al. 2015). While Bd has been present
in the US for many years, Bsal has only been found to cause
clinical disease in wild salamanders in Europe to date (Martel
et al. 2013, Richgels et al. 2016), however Bsal surveillance of
wild amphibian populations has been limited elsewhere. Bsal
is significantly affecting both captive and free ranging fire
salamander (Salamandra salamandra) populations in northern
Europe (Martel et al. 2014; Van Rooij et al. 2015). Bsal appears
to have originated in Asia where it likely has remained in
coexistence with salamander hosts for millions of years; spread
to Europe was likely human-mediated through international
trade (Martel et al. 2014). Herein we report the results of
combined Bd and Bsal surveillance of native, wild salamanders
in southeastern Virginia, US.
Salamanders at three sites (Fig. 1) were sampled on
three days in mid- to late-October 2016 for Bd and Bsal: 1)
Sandy Bottom Nature Park, Hampton, Virginia (37.0624°N,
76.4317°W), is a 185-ha public park built around former
borrow pits with created wetlands and a mixed hardwood
and pine forest, and surrounded by suburban development;
2) Grafton Ponds Natural Area Reserve, Yorktown, Virginia
(37.1816°N, 76.5177°W), a 152-ha mixed hardwood and pine
forested wetland complex contiguous with a larger public
park and watershed protection area; and 3) Northwest River
Park, Chesapeake, Virginia (36.5795°N, 76.1529°W), a 309-ha
riverside public park with a mixed hardwood and pine forest.
We opportunistically captured salamanders by searching
under natural cover objects, such as logs and fallen bark, and
restrained specimens in separate moistened plastic bags.
We examined each salamander and collected morphometric
data (e.g., body weight and SVL) (Table 1). Coordinates were
determined using mobile applications, Apple and Google Maps,
with GPS and cellular network assisted locations. We swabbed
salamanders along their ventrum (feet, thighs, abdomen) with
a fine-tipped rayon swab with a plastic handle and placed swabs
Virginia Zoo, 3500 Granby Street, Norfolk, Virginia 23504, USA
Virginia Herpetological Society, 11837 Rock Landing Drive,
Suite 300, Newport News, Virginia 23606, USA
*Current address: ZSL London Zoo, Regent’s Park London,
England NW1 4RY; e-mail: email@example.com
Fig. 1. Surveillance sites in southeastern Virginia, USA for Batracho-
chytrium dendrobatidis and B. salamandrivorans in native, wild sala-
manders. GPNAR: Grafton Ponds Natural Area Reserve, SBNP: Sandy
Bottom Nature Park, and NRP: Northwest River Park.