See discussions, stats, and author profiles for this publication at: https://www.researchgate.

net/publication/321685598

A Survey of Batrachochytrium dendrobatidis Occurrence in Amphibians of

Walker and Floyd Counties, Georgia, USA

Article  in  Herpetological Review · December 2017

CITATIONS READS

0 183

2 authors:

Michael B Morgan Christopher B. Mowry

Berry College Berry College
16 PUBLICATIONS   559 CITATIONS    21 PUBLICATIONS   511 CITATIONS   

SEE PROFILE SEE PROFILE

Some of the authors of this publication are also working on these related projects:

Atlanta Coyote Project (https://atlantacoyoteproject.org) View project

Effects of estradiol on Aiptasia View project

All content following this page was uploaded by Michael B Morgan on 08 December 2017.

The user has requested enhancement of the downloaded file.

 AMPHIBIAN AND REPTILE DISEASES 777

infection loads are suppressed in hosts exposed to salt. Oecologia compromises PCR detection of amphibian chytrid fungus
177:901–910. Batrachochytrium dendrobatidis DNA. Dis. Aquat. Org. 81:93–
Van Sluys, M., K. M. Kriger, A. D. Philott, R. Campbell, L. F. Skerratt, 97.
and J.-M., Hero. 2008. High-temperature storage of samples

Herpetological Review, 2017, 48(4), XX–XX.

© 2017 by Society for the Study of Amphibians and Reptiles

A Survey of Batrachochytrium dendrobatidis

Occurrence in Amphibians of Walker and Floyd
Counties, Georgia, USA
Previous studies have detected the fungal pathogen specimens were opportunistically caught using nets, although
Batrachochytrium dendrobatidis (Bd) in a variety of anuran and we increased our sample size of tree frogs (Hyla cinerea and H.
caudate species in the southeastern USA (Rothermel et al. 2008; chrysoscelis) at Berry College by using open-ended PVC tube traps
Timpe et al. 2008; Chatfield et al. 2009; Gonynor et al. 2011; Huang mounted on trees and poles near water (Fig. 2). Every attempt
and Wilson 2013). Within this region of the country, Georgia is was made to minimize the potential for cross-contamination of
fortunate to have a particularly rich amphibian community, Bd between individuals and sites. Amphibians were individually
with at least 57 salamander and 31 frog species found within the placed in new Ziploc bags while they were being processed.
state’s borders (CNAH 2012). Although Bd has caused declines or Fresh nitrile gloves were used to handle each animal while it was
extinctions in over 200 amphibian species worldwide (Pounds et measured (snout–vent length) and sampled with a polyester swab
al. 2006), clinical signs of the disease chytridiomycosis caused following procedures in Pessier and Mendelson (2010). Separate
by it have been low in the southeastern USA (Bd-maps.net). nets were used at different sampling sites and all equipment
However, individuals can harbor the fungus without showing was disinfected with a 3% bleach solution between sites. Swabs
signs of the disease. It is important to maintain surveillance were allowed to air dry and the tips were broken off and placed
of Bd-infected amphibian populations over time, especially in sterile microfuge tubes, which were subsequently stored at
as the Bd-host relationships may change with environmental -20ºC until DNA extraction was performed. All amphibians were
conditions, such as climatic conditions, particularly within the released at their collection site within 30 minutes of capture.
temperate zones of the Northern Hemisphere (Olson et al. 2013; Genomic DNA was extracted from swabs using Qiagen DNA Easy
Xie et al. 2016). Our aim was to investigate the current prevalence kit (Valencia, California, USA). Chytrid DNA was also isolated
of the Bd fungus in amphibians in two previously surveyed sites from a laboratory culture (provided by J. E. Longcore). Samples
in northwestern Georgia.
Timpe et al. (2008) looked for the occurrence of Bd across
Georgia’s five physiographic provinces from 2006 to 2008.
Therefore, we chose to revisit two of their northwestern sites
for our study beginning in 2012: 1) Berry College (Ridge and
Valley province) and 2) Crockford-Pigeon Mountain Wildlife
Management Area (Cumberland Plateau province) (Fig. 1).
Amphibians were collected from multiple locations within
each of our two sites (Table 1). We sampled post-metamorphic
amphibians at Berry College from July-December 2012 and again
in June 2013. Sampling at Crockford-Pigeon Mountain occurred
from May-August 2013 and in August 2014. The majority of

CHRISTOPHER B. MOWRY*
CHRISTOPHER M. KEENE
STEPHANIE E. PRISLAND
BRETT D. TYLER
ANDREW A. MONTGOMERY
ALEXANDER P. MOWRY
REBECCA A. MARTIN
SARAH STEVENS
JOSEPH ELLWANGER
MICHAEL B. MORGAN
Department of Biology, Berry College, Box 430,
Mt. Berry, Georgia 30149, USA Fig. 1. Locations and physiographic provinces where amphibians
were sampled for Batrachochytrium dendrobatidis in Georgia, USA.
*Corresponding author; e-mail: cmowry@berry.edu
Red = Berry College site; white = Crockford-Pigeon Mountain site.

Herpetological Review 48(4), 2017

 778 AMPHIBIAN AND REPTILE DISEASES
PHOTO BY A. MONTGOMERY
Fig. 2. Although most frogs were opportunistically captured using
nets for Batrachochytrium dendrobatidis sampling, open-ended PVC
tube traps mounted on trees and poles near water at Berry College,
Georgia, USA were also used to capture tree frogs (shown here: Green
Treefrogs, Hyla cinerea).
Table 1. Coordinates for sites surveyed for Batrachochytrium dendro-
batidis in northwestern Georgia, USA. WMA = Wildlife Management
Area.
Site Latitude (°N) Longitude (°W)
Berry College P. glutinosus 0 5
Victory Lake 34.298737 85.202305
Possum Trot Rd. 34.310810 85.246442
Morgan-Deerfield 34.291052 85.194043
Crockford-Pigeon Mountain WMA positive. The only other Bd-positive E. lucifuga that we are aware
Petty John’s Cave 34.664835 85.362826
The Pocket Falls 34.709060 85.375524
Lost Wall waterfall 34.665369 85.372466
Dougherty Gap 34.613770 85.456395
Rape Gap Pond 34.641383 85.412737
Sag Pond 34.680325 85.395622
were screened in three independent PCR reactions with chytrid-
specific PCR primers and conditions described by Boyle et al.
(2004) and Annis et al. (2004).
We sampled a total of 96 amphibians representing 17 species
(five families) for the presence of Bd DNA (Table 2). Using a
2%TBE agarose gel, twenty-seven samples had evidence of
amplification using Boyle et al. (2004) primers, but 26 of those
27 exhibited the false-positive amplicon described by Wunder
et al. (2012). Confirmation of Bd DNA detection was ultimately
identified in samples that exhibited correct amplicon sizes for
both Boyle (146bp) and Annis (300bp) primers. Only one sample,
a Cave Salamander (Eurycea lucifuga) collected at Crockford-
Pigeon Mountain, exhibited the predicted amplicon for both
Boyle et al. (2004) and Annis et al. (2004) primers. We did not
observe clinical signs of chytridiomycosis in any amphibians
during our study.
Our results are consistent with Timpe et al.’s (2008) previous
study that found a low occurrence (< 2%) of Bd-positive post-
metamorphic amphibians in northwestern Georgia. Timpe et al.
(2008) also detected Bd at Crockford-Pigeon Mountain, although
Herpetological Review 48(4), 2017
Table 2. Number of individuals per amphibian species surveyed for
Batrachochytrium dendrobatidis in two physiographic provinces
in Georgia, USA. BC = Berry College (Ridge and Valley) and C-PM =
Crockford-Pigeon Mountain Wildlife Management Area (Cumber-
land Plateau). ( ) represents subset of individuals on which Bd was
detected.
Species BC C-PM
Anura
Acris crepitans 0 1
Bufo americanus 2 0
Gastrophryne carolinensis 2 0
Hyla chrysoscelis 3 1
H. cinerea 33 1
H. gratiosa 0 2
Lithobates catesbeianus 8 1
L. clamitans 0 4
L. palustris 0 5
L. sphenocephalus 9 0
Caudata
Aneides aeneus 0 1
Desmognathus conanti 0 6
Eurycea cirrigera 0 1
E. longicauda 0 1
E. lucifuga 0 6 (1)
Plethodon petraeus 0 4
none of the three Eurycea lucifuga tested in that study was Bd-
of came from a site in the southern Appalachian Mountains in
Wise County, Virginia (Davidson and Chambers 2011; Bd-maps.
net). Neither our survey nor Timpe et al. (2008) detected Bd
at Berry College. These joint results lead us to believe that Bd
prevalence is not increasing in this area.
None of the other five salamanders that we sampled
concurrently from Petty John’s Cave tested positive for Bd.
These included two additional Eurycea lucifuga, two Plethodon
glutinosus, and one P. petraeus. Moreover, the Bd-positive
Eurycea lucifuga individual did not exhibit signs of clinical
chytridiomycosis. However, our detection of a Bd-positive
Eurycea lucifuga salamander in Petty John’s Cave at Crockford-
Pigeon Mountain is of some concern because this ecologically
unique site has heavy human traffic, which potentially increases
the likelihood of spreading the pathogen to other sites in the
area. As with white-nose syndrome in bats, accidental movement
of fungal pathogens into and out of caves by human visitors can
lead to devastating consequences (Foley et al. 2011). Petty John’s
Cave, along with other cave-associated amphibian communities,
warrants further investigation of spatio-temporal patterns of Bd
prevalence in the southeastern region of the USA. Additionally,
the endemic Pigeon Mountain salamander (P. petraeus) is listed
as Rare in Georgia (http://georgiawildlife.com/species) and
therefore is a species of conservation concern.
It is also noteworthy that the air temperature inside Petty
John’s Cave at the time of our sample collection was significantly
colder than it was outside the cave (13.3ºC vs. 23ºC). Bd occurrence
and host susceptibility in seasonally variable temperate zones
has previously been linked to cooler temperatures. For example,

Kriger and Hero (2007) found a significant negative relationship
between chytridiomycosis prevalence and mean air temperature
in an Australian study of Litoria wilcoxii. Savage et al. (2011)
found similar results in populations of Lithobates yavapaiensis
in Arizona, USA, although Huang and Wilson (2013) did not find
such a relationship when comparing amphibians in Georgia’s
Blue Ridge and Piedmont provinces.
Acknowledgments.—Funding was provided by a Berry College
Development of Undergraduate Research grant (to CBM and MBM)
and a Richards Undergraduate Research Support grant (to RAM and
JE). Amphibian sampling was conducted under a Berry College IA-
CUC permit. Thanks to D. Gregory for facilitating access to Crock-
ford-Pigeon Mountain WMA. For field assistance, we thank T. Moore
and B. Pierson. J. Mendelson and L. Smith provided valuable guid-
ance in initiating the study. We also thank C. Lillard, D. Olson, and
an anonymous reviewer for helpful comments, and K. Clements for
assistance in figure preparation.
Literature Cited
Annis, S. L., F. P. Dastoor, H. Ziel, P. Daszak, and J. E. Longcore. 2004.
A DNA-based assay identifies Batrachochytrium dendrobatidis in
amphibians. J. Wildl. Dis. 40:420–428.
Bd-maps.net. 2017. <http://www.bd-maps.net/maps/> Accessed 13
June 2017.
Boyle, D. G., D. B. Boyle, V. Olsen, J. A. T. Morgan, and A. D. Hyatt. 2004.
Rapid quantitative detection of chytridiomycosis (Batrachochytri-
um dendrobatidis) in amphibian samples using real-time Taqman
PCR assay. Dis. Aquat. Org. 60:141–148.
Center for North American Herpetology (CNAH). 2012. The amphib-
ians, reptiles, turtles, and crocodilians of Georgia. <http://www.
cnah.org> Accessed 13 June 2017.
Chatfield, M. W. H., B. B. Rothermel, C. S. Brooks, and J. B. Kay. 2009.
Detection of Batrachochytrium dendrobatidis in amphibians from
the Great Smoky Mountains of North Carolina and Tennessee,
USA. Herpetol. Rev. 40:176–179.
Davidson, S. R. A., and D. L. Chambers. 2011. Occurrence of Batracho-
chytrium dendrobatidis in amphibians of Wise County, Virginia,
USA. Herpetol. Rev. 42:214–215.
Foley, J., D. Clifford, K. Castle, P. Cryan, and R. S. Ostfeld. 2011. In-
vestigating and managing the rapid emergence of white-nose
Herpetological Review, 2017, 48(4), XX–XX.
© 2017 by Society for the Study of Amphibians and Reptiles
Two Ranavirus-Associated Mass-Mortality Events
among Larval Amphibians in Illinois, USA
LUCAS J. KIRSCHMAN*
Department of Zoology, Southern Illinois University,
Carbondale, Illinois, USA
JOHN G. PALIS
Palis Environmental Consulting, P.O. Box 387,
Jonesboro, Illinois 62952, USA
KELLEY A. FRITZ
KYLE ALTHOFF
ROBIN W. WARNE
Department of Zoology, Southern Illinois University,
Carbondale, Illinois, USA
*Corresponding author; e-mail: l.j.kirschman@alaska.edu
Herpetological Review 48(4), 2017
View publication stats
AMPHIBIAN AND REPTILE DISEASES 779
syndrome, a novel, fatal, infectious disease of hibernating bats.
Conserv. Biol. 25:223–231.
Gonynor, J. L., M. J. Yabsley, and J. B. Jensen. 2011. A preliminary sur-
vey 
of Batrachochytrium dendrobatidis exposure in hellbenders
from a stream in Georgia, USA. Herpetol. Rev. 42:58–59. 

Huang, R. and L. Wilson. 2013. Batrachochytrium dendrobatidis in
amphibians of the Piedmont and Blue Ridge Provinces in North-
ern Georgia, USA. Herpetol. Rev. 44:95–98.
Kriger, K. M., and J. M. Hero. 2007. Large-scale seasonal variation in
the prevalence and severity of chytridiomycosis. J. Zool. 271:352–
259.
Olson, D. H., D. M. Aanensen, K. L. Ronnenberg, C. I. Powell, S. F. Walk-
er, J. Bielby, T. W. J. Garner, G. Weaver, The Bd Mapping Group, and
M. C. Fisher. 2013. Mapping the global emergence of Batracho-
chytrium dendrobatidis, the amphibian chytrid fungus. PLoS ONE
8(2):e56802.
Pessier, A. P., and J. R. Mendelson (eds.). 2010. A Manual for Control
of Infectious Diseases in Amphibian Survival Assurance Colonies
and Reintroduction Programs. IUCN/SSC Conservation Breeding
Specialist Group, Apple Valley, Minnesota. http://www.cbsg.org.
Pounds, J. A., M. R. Bustamante, L. A. Coloma, J. A. Consuegra, M. P.
Fogden, P. N. Foster, E. La Marca, K. L. Masters, A. Merino-Viteri,
and R. Puschendorf. 2006. Widespread amphibian extinctions from
epidemic disease driven by global warming. Nature 439:161–167. 

Rothermel, B. B., S. C. Walls, J. C. Mitchell, C. K. Dodd, L. K. Irwin, D. E.
Green, V. M. Vazquez, J. W. Petranka, and D. J. Stevenson. 2008. Wide-
spread occurrence of the amphibian fungus Batrachochytrium
dendrobatidis in the southeastern USA. Dis. Aquat. Org. 82:3–18.
Savage, A. E., M. J. Sredl, and K. R. Zamudio. 2011. Disease dynamics
vary spatially and temporally in a North American amphibian.
Biol. Conserv. 144:1910–1915. 

Timpe, E. K., S. P. Graham, R. W. Gagliardo, R. L. Hill, and M. G. Levy.
2008. Occurrence of the fungal pathogen Batrachochytrium den-
drobatidis in Georgia’s amphibian populations. Herpetol. Rev.
39:447–449.
Wunder, J., N. Lampazzi, K. Acre, N. Bent, S. Canter, A. Chapman, M. Da-
vies, D. Kashan, J. Keiley, and R. MacIntyre. 2012. Promoting amphib-
ian conservation through the college classroom: detection of Ba-
trachochytrium dendrobatidis among local amphibians. Herpetol.
Conserv. Biol. 7:462–469.
Xie, G.Y., D.H. Olson, a and A.R. Blaustein. 2016. Projecting the global
distribution of the emerging amphibian fungal pathogen, Batra-
chochytrium dendrobatidis, based on IPCC climate futures. PLoS
ONE 11(8):e0160746.
Growing evidence suggests that ranaviruses are globally
distributed pathogens that infect a diversity of ectothermic
vertebrates, and can cause sudden mass-mortality events,
especially in larval amphibians (Brunner et al. 2004, 2015; Gray
et al. 2009; Warne et al. 2016). Ranavirus epizootics in larval
amphibians can also amplify viral titers in the environment
(Hall et al. 2016) and potentially export sublethally infected
metamorphs to the surrounding ecosystem, creating a possible
transmission route to sympatric hosts (Brunner et al. 2007).
Despite a growing awareness of disease threats to amphibians,
ranavirus epizootics are likely undetected and underreported
because they are often rapid and cryptic (Brunner et al. 2015).
Given the broad range of hosts threatened by ranaviruses (Duffus