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Batrachochytrium dendrobatidis in Adult Notophthalmus viridescens in North-Central Alabama, USA



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Herpetological Review 41(1), 2010
Service State Wildlife Grant, contract number E2-08-WDS13. Special
thanks to Katie Smith and Ron Ronk, Indiana Department of Natural
Resources, for enabling this project.
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This section offers a timely outlet for streamlined presentation of research
exploring the geographic distribution, host range, and impact of emerging am-
phibian pathogens, especially the amphibian chytrid fungus Batrachochytrium
dendrobatidis (Bd) and ranaviruses. Bd is an emerging pathogen linked to mass
mortality and declines of amphibians worldwide, yet Bd has also been detected
in amphibians without disease. Ranaviruses also cause mass mortality, but have
not yet been linked to large-scale declines. We know relatively little about their
global distribution, host range, or impacts on host populations. To improve our
understanding of the scope of this issue, we encourage submission of studies that
illuminate the geographic distribution, host ranges, and impact of these pathogens
on amphibian populations, including research on individual species or groups
of species, wild or captive animals, native or non-native species, live animals
or museum specimens, environmental samples, and, provided there is sufficient
, reports of non-detections.
We ask authors to: 1) restrict the Introduction of their paper to a maximum of
two paragraphs to highlight the context of their study; 2) briefly include both field
and laboratory Methods; 3) present Results in a Table, although a map might also
be useful, and limited text; and 4) have a short discussion of a maximum of three
paragraphs to touch upon key findings. Please include the following information
in submissions as appropriate: coordinates and description of sampling areas (or
please note if locations are extremely sensitive to reveal, and provide general area
instead); species name(s) and life history stages examined, as well as other spe-
cies present; whether samples were collected randomly or just from dead or mori-
bund animals; date of specimen collection; evidence of unusual mortality; num-
bers of positive and negative samples; disposition of voucher specimens; name
of collaborative laboratory or researcher conducting histological sections or PCR
analyses; and names of cooperative land owners or land management agencies.
We encourage researchers to conduct post-mortem examinations when possible
to identify the cause of death when reporting mortalities. We aim to expedite the
review and publication process! Please e-mail submissions directly to Associate
Editor, Dr. Dede Olson:
If a sample of 30 individuals of a particular life history stage of a particular
species yields no positive results, and the diagnostic test is highly sensitive, one
can conclude that the prevalence of infection is less than 10% with 95% con-
fidence. With a sample of 10 an infection in one of four individuals could go
undetected. We encourage researchers to collect sufficient samples that negative
results are meaningful.
Herpetological Review, 2010, 41(1), 45–47.
© 2010 by Society for the Study of Amphibians and Reptiles
Batrachochytrium dendrobatidis in Adult
Notophthalmus viridescens in North-Central
Alabama, USA
Department of Biological and Environmental Science, Samford University
800 Lakeshore Drive, Birmingham, Alabama 35229, USA
Wildlife Disease Laboratories
San Diego Zoo’s Institute for Conservation Research
P.O. Box 120551 San Diego, California 92112-0551, USA
*Corresponding author; e-mail:
Batrachochytrium dendrobatidis (Bd) has devastated many
amphibian populations, especially in tropical areas (Berger et al.
1998; Daszak et al. 1999). However, the geographic distribution of
Herpetological Review 41(1), 201046
this emerging pathogen is not well known (see http://www.spatia- There is currently
only one published report of Bd in Alabama, USA, at Horseshoe
Bend National Park located in Tallapoosa Co. (Byrne et al. 2008);
the affected species was the semi-terrestrial Eurycea cirrigera
(Southern Two-lined Salamander). Rothermel et al. (2008) found
Bd across the southeastern United States, but Alabama was not
sampled. However, they found Bd-infected Notophthalmus viri-
descens (Eastern Newt) in Georgia, North Carolina, and Virginia.
Additional reports have detected Bd in anurans and caudates in
eastern Georgia (Timpe et al. 2008) and southwestern Tennessee
(Venesky and Brem 2008). We report on a haphazard collection
of dead adult Notophthalmus viridescens discovered during a field
zoology class trip conducted by KAB in Birmingham, Alabama.
Methods.—On 25 February 2009, seine and dipnet sampling
were conducted at Red Lakes, Ruffner Mountain Nature Preserve,
Birmingham, Alabama (33.562N 86.6900ºW). Many (30–40) live
adult Eastern Newts in breeding condition (smooth skin; males with
nuptial pads) were sampled. We also noticed a number of recently
deceased newts. The dead animals were slightly bloated, presum-
ably due to dying in the water, were floating just above the bottom
of the shallow pond (ankle to thigh deep, 15–90 cm), but otherwise
appeared normal (no loose or peeling skin, no discoloration, no
signs of trauma). Twelve dead newts were collected, and we saw
at least another 15 dead newts. Nine newts were fixed in formalin,
then stored in 70% ethanol. Three newts were preserved by freezing
at -10ºC. Ambystoma maculatum (Spotted Salamander) egg masses
were present in the pond where the dead newts were collected and
two large adult Spotted Salamanders (appeared healthy) were found
in the woods approximately 40 m away from the pond and released
after examination. Presumably, this pond also is used by anurans
as a breeding site because it is fishless and periodically dries due
to evaporation when there is a hot, dry summer.
Two formalin-fixed newts were examined by APP. The fixed
carcasses were demineralized in hydrochloric acid (RDO Rapid
Decalcifier, Apex Engineering Corporation, Aurora, IL USA),
followed by preparation of serial transverse histological sections
through the entirety of head and body and longitudinal sections
through the hindlimbs and feet. Body, limb and foot slices were
then automatically processed for histology, embedded in paraffin,
sectioned at 5-6 μm and stained with hematoxylin and eosin. Liver
from 2 frozen newts was submitted to the Amphibian Disease
Laboratory at the San Diego Zoo for real-time PCR for ranaviruses
using previously described techniques (Pallister et al. 2007).
Results.—Lesions in the two formalin-fixed newts examined
histologically were limited to the skin. Changes were diffuse and
involved over 90% of the skin surfaces examined (which included
multiple sections of dorsal and ventral skin from the head, torso,
legs, feet and tail) and consisted of mild to moderate orthokeratotic
hyperkeratosis and epidermal hyperplasia with moderate numbers
of fungal organisms in cells of the stratum corneum (superficial
keratinized skin layers) typical of Batrachochytrium dendroba-
tidis. Features of these organisms considered to be diagnostic of
Bd included colonial thalli and flask-shaped zoosporangia with
prominent discharge papillae (Longcore et al. 1999). Skin lesions
were significantly more severe than the relatively focal and minimal
lesions observed in Bd infected, but healthy American bullfrogs
(Hanselmann et al. 2004) and were similar to those associated with
mortality in naturally and experimentally infected anurans (Nichols
et al. 2001). Lesions were also subjectively more severe than those
previously observed by one of the authors (APP) in other sala-
mander species (Davidson et al. 2003; Vasquez et al. 2009). Other
tissues examined histologically, including but not limited to, brain,
liver (including subcapsular hematopoietic tissue), kidney, spleen,
gastrointestinal tract, bone and skeletal muscle were considered to
be within normal limits. Specifically, no lesions typical of a lethal
Ranavirus infection (Green et al. 2002) or of the Ichthyophonus-
like infections previously reported in Notopthalmus viridescens
(Raffel et al. 2006) were observed. The examined newts were
determined to be in good nutritional condition based on abundant
coelomic cavitary fat stores. Real-time PCR for ranaviruses was
negative. Based on these findings, chytridiomycosis was deter-
mined to be the likely cause of death. However, the small number
of newts sampled for diagnostic investigation means that other
potential causes for this mortality event, including but not limited
to, other unidentified infectious agents and events such as chemi-
cal exposure or altered water quality. Chemical and environmental
changes especially may not result in distinctive lesions that can be
identified by histologic examination.
Discussion.—This report is significant in documenting the pres-
ence of Bd in another salamander species 110 km from the nearest
known locality, Tallapoosa Co. Alabama. This is the first report of
a mortality event associated with Bd in Alabama. Ruffner Moun-
tain is currently used as a nature education center and is also the
second largest urban nature preserve in the United States (S. Mc-
Cracken, pers. comm.). However, iron ore mining was conducted
here from 1886-1953 and it served as a limestone quarry through
the 1920’s (White 1981). Red Lakes (there were once six, now
two remain) where the infected newts were found, was originally
a settling pond for the water used in a beneficiation process that
separated high quality from low quality ore (S. McCracken, pers.
comm.). The water is a rust-red color and stains field equipment.
Although we did not test for industrial contaminants such as
heavy metals, pH was 8.4 (water temp = 10.1ºC, air temp 13.4ºC,
RH = 52.5%). The pH optimum for Bd is 6-7 with less growth at
a pH of 8, the highest pH tested (Piotrowski et al. 2004). Future
work should examine potential linkages between environmental
contaminants and the incidence and/or severity of Bd infections
in aquatic amphibians.
Acknowledgments.—The Amphibian Disease Laboratory at the Zoologi-
cal Society of San Diego is supported in part by grant LG-25-08-0066
from the Institute of Museum and Library Services. Any views, findings,
conclusions or recommendations expressed in this publication do not nec-
essarily represent those of the Institute of Museum and Library Services.
Thanks to Ruffner Mountain Nature Preserve for use of their pond, S.
McCracken for providing history of the area, and J. R. Mendelson III for
suggesting the collaboration.
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Herpetological Review, 2010, 41(1), 47–49.
© 2010 by Society for the Study of Amphibians and Reptiles
Batrachochytrium dendrobatidis Detected in
Amphibians from National Forests in
Eastern Texas, USA
Southern Research Station, Forest Service, U.S. Department of Agriculture
506 Hayter Street, Nacogdoches, Texas 75965-3556, USA
Stephen F. Austin State University, Department of Biology
1936 North Street, Nacogdoches, Texas 75962-3050, USA
*Corresponding author; e-mail:
The amphibian disease chytridiomycosis, caused by the patho-
genic fungus Batrachochytrium dendrobatidis (Bd, Longcore et
al. 1999), is well known as a major threat to amphibians resulting
in mass die-offs and population declines throughout the world
(Berger et al. 1998; Blaustein and Keisecker 2002; Daszak et al.
2003; McCallum 2005; Rachowicz et al. 2006). Batrachochytrium
dendrobatidis has been detected on amphibians from sites across
North America (Ouellet et al. 2005; Woodhams et al. 2008) and
appears to be most prevalent in the western and the northeastern
United States (Longcore et al. 2007; Schlaepfer et al. 2007).
Whereas infected anurans also have been found throughout the
southeastern US (Green and Dodd 2007), there have been no
reports of Bd from amphibians in eastern Texas, a broad area
encompassing 10,000,000 ha. We sampled amphibians for the
presence of Bd in four National Forests in eastern Texas (approxi-
mately 31°N latitude).
Amphibians were sampled for Bd from 9 January to 27 May 2009
in the Angelina, Davy Crockett, and Sabine National Forests, and
the Stephen F. Austin Experimental Forest (Fig. 1). The Stephen
FIG. 1. Locations of the Angelina, Davy Crockett, Sabine National
Forests, and the Stephen F. Austin Experimental Forest where six of
18 amphibian species tested positive for the amphibian chytrid fungus,
Batrachochytrium dendrobatidis.
... Previous studies have documented sub-clinical infection of adult eastern newts in the wild with Bd (Rothermel et al. 2008, Groner & Relyea 2010, Raffel et al. 2010 and Rv (Duffus et al. 2008, Glenney et al. 2010, Todd-Thompson 2010, Richter et al. 2013. Chytrid-associated mortality events involving small numbers of N. viridescens have been reported from sites in southwestern Virginia (Rothermel et al. 2008) and north-central Alabama (Bakkegard & Pessier 2010). However, few newt species have been screened for more than one pathogen (but see Bovero et al. 2008, Glenney et al. 2010, Richter et al. 2013 and pathogen prevalence among different life stages has not been examined. ...
Long-term monitoring of amphibians is needed to clarify population-level effects of ranaviruses (Rv) and the fungal pathogen Batrachochytrium dendrobatidis (Bd). We investigated disease dynamics of co-occurring amphibian species and potential demographic consequences of Rv and Bd infections at a montane site in the Southern Appalachians, Georgia, USA. Our 3-yr study was unique in combining disease surveillance with intensive population monitoring at a site where both pathogens are present. We detected sub-clinical Bd infections in larval and adult red-spotted newts (Notophthalmus viridescens viridescens), but found no effect of Bd on body condition of adult newts. Bd infections also occurred in larvae of 5 anuran species that bred in our fishless study pond, and we detected co-infections with Bd and Rv in adult newts and larval green frogs (Lithobates clamitans). However, all mortality and clinical signs in adult newts and larval anurans were most consistent with ranaviral disease, including a die-off of larval wood frogs (Lithobates sylvaticus) in small fish ponds located near our main study pond. During 2 yr of drift fence monitoring, we documented high juvenile production in newts, green frogs and American bullfrogs (L. catesbeianus), but saw no evidence of juvenile recruitment in wood frogs. Larvae of this susceptible species may have suffered high mortality in the presence of both Rv and predators. Our findings were generally consistent with results of Rv-exposure experiments and support the purported role of red-spotted newts, green frogs, and American bullfrogs as common reservoirs for Bd and/or Rv in permanent and semi-permanent wetlands.
... Few studies have investigated Bd infection in N. viridescens, but they suggest that newts can be infected. One study in Alabama found two dead newts that were confirmed positive for Bd, and it was suggested that these individuals might have died as a result of chytridiomycosis (Bakkegard and Pessier 2010). In western Pennsylvania, a survey detected Bd infection in newts at six locations with varying prevalence (Groner and Relyea 2010), and in central Pennsylvania, infected individuals were found at 12 of 16 ponds (Raffel et al. 2010). ...
... Catastrophic dieoffs of amphibians attributable to Bd have occurred in montane regions of Central America (Lips et al. 2003) and western North America (Daszak et al. 1999; Rachowicz et al. 2006; Vredenburg et al. 2010). In contrast, there have been very few cases of Bd-associated mortality in the eastern United States (e.g., Todd-Thompson et al. 2009; Bakkegard and Pessier 2010), despite high prevalence of Bd in ranids, hylids, and salamandrids (Longcore et al. 2007; Rothermel et al. 2008). Ranaviral disease has caused many more mass mortality events of amphibians in the eastern United States, mostly involving ranid frogs and ambystomatid salamanders (Green et al. 2002; Dodd 2004; Electronic supplementary material: The online version of this article (doi: 10.1007/s10393-013-0843-5) contains supplementary material, which is available to authorized users. ...
Full-text available
The interactive effects of environmental stressors and emerging infectious disease pose potential threats to stream salamander communities and their headwater stream ecosystems. To begin assessing these threats, we conducted occupancy surveys and pathogen screening of stream salamanders (Family Plethodontidae) in a protected southern Appalachians watershed in Georgia and North Carolina, USA. Of the 101 salamanders screened for both chytrid fungus (Batrachochytrium dendrobatidis) and Ranavirus, only two exhibited low-level chytrid infections. Prevalence of Ranavirus was much higher (30.4% among five species of Desmognathus). Despite the ubiquity of ranaviral infections, we found high probabilities of site occupancy (≥0.60) for all stream salamander species.
Mucosal defenses are crucial in animals for protection against pathogens and predators. Host defense peptides (antimicrobial peptides, AMPs) as well as skin-associated microbes are key components of mucosal immunity, particularly in amphibians. We integrate microbiology, molecular biology, network-thinking, and proteomics to understand how host and microbially derived products on amphibian skin (referred to as the mucosome) serve as pathogen defenses. We studied defense mechanisms against chytrid pathogens, Batrachochytrium dendrobatidis (Bd) and B. salamandrivorans (Bsal), in four salamander species with different Batrachochytrium susceptibilities. Bd infection was quantified using qPCR, mucosome function (i.e., ability to kill Bd or Bsal zoospores in vitro), skin bacterial communities using 16S rRNA gene amplicon sequencing, and the role of Bd-inhibitory bacteria in microbial networks across all species. We explored the presence of candidate-AMPs in eastern newts and red-backed salamanders. Eastern newts had the highest Bd prevalence and mucosome function, while red-back salamanders had the lowest Bd prevalence and mucosome function, and two-lined salamanders and seal salamanders were intermediates. Salamanders with highest Bd infection intensity showed greater mucosome function. Bd infection prevalence significantly decreased as putative Bd-inhibitory bacterial richness and relative abundance increased on hosts. In co-occurrence networks, some putative Bd-inhibitory bacteria were found as hub-taxa, with red-backs having the highest proportion of protective hubs and positive associations related to putative Bd-inhibitory hub bacteria. We found more AMP candidates on salamanders with lower Bd susceptibility. These findings suggest that salamanders possess distinct innate mechanisms that affect chytrid fungi. IMPORTANCE How host mucosal defenses interact, and influence disease outcome is critical in understanding host defenses against pathogens. A more detailed understanding is needed of the interactions between the host and the functioning of its mucosal defenses in pathogen defense. This study investigates the variability of chytrid susceptibility in salamanders and the innate defenses each species possesses to mediate pathogens, thus advancing the knowledge toward a deeper understanding of the microbial ecology of skin-associated bacteria and contributing to the development of bioaugmentation strategies to mediate pathogen infection and disease. This study improves the understanding of complex immune defense mechanisms in salamanders and highlights the potential role of the mucosome to reduce the probability of Bd disease development and that putative protective bacteria may reduce likelihood of Bd infecting skin.
It has been reported that sialic acid containing oligosaccharides play an important rolein the adhesion between cancer cells and endothelial cells and metastatic potential of tumorcells is proportional to cell surface sialylation. In the present study, we investigatedwhether there is a change in serum total and lipid bound sialic acid levels of patientswith benign and non-melanom malignant skin tumors and, evaluated whether the measurementof sialic acid levels may be useful clinically in distinguishing patients with benignskin tumors from those with non-melanom malignant skin tumors. In this study, 27patients with malignant skin tumors (16 men, age 50.78 ± 12.46 years), 39 patients withbenign skin tumors (17 men, age 48.59 ± 16.23 years) were included. Serum total andlipid bound sialic acid determination was performed by the thiobarbituric acid methoddescribed by Warren and, the resorsinol method described by Katopodis, respectively.Student’s T test and Z test were used to analyze the results. The mean serum total andlipid bound sialic acid levels were found to be 63.01 ± 11.89 mg/dl and 15.77 ± 2.44mg/dl, respectively in patients with benign skin tumors; and 65.95 ± 7.30 mg/dl and16.70 ± 3.80 mg/dl, respectively in patients with non-melanom malignant skin tumors.Serum total sialic acid and lipid bound sialic acid of patients with benign skin tumorswere not different from those of patients with non-melanom malignant skin tumors. Thepercentage of lipid bound sialic acid was found to be 25.3 in patients with non-melanommalignant skin tumors, 25 in patients with benign skin tumors. There was no significantdifference between the value of lipid bound sialic acid percentage in these groups. Ourfinding that there is no significant difference between serum total and lipid-bound sialicacid levels of patients with non melanom malignant skin tumors which are not able tometastase and patients with benign skin tumors supports the studies reporting that sialicacids play an important role in metastases.
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Captive and wild frogs from North and Central America and Australia recently have died with epidermal infections by chytridiomycete fungi. We isolated a chytridiomycete into pure culture from a captive, blue poison dart frog that died at the National Zoological Park in Washington, D.C. Using this isolate, we photographed developmental stages on nutrient agar, examined zoospores with transmission electron microscopy, and inoculated test frogs. This inoperculate chytrid develops either monocentrically or colonially and has thread-like rhizoids that arise from single or multiple areas on the developing zoosporangium. The taxonomically important features of the kinetosomal region of the zoospore indicate that this chytrid is a member of the Chytridiales but differs from other chytrids studied with transmission electron microscopy. Its microtubule root, which begins at kinetosome triplets 9-1 and extends parallel to the kinetosome into the aggregation of ribosomes, is distinctive. Histologic examination of test frogs revealed that the pure culture infected the skin of test frogs, whereas the skin of control frogs remained free of infection. The fungus is described as Batrachochytrium dendrobatidis gen. et sp. nov.
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A chytrid fungus, Batrachochytrium dendrobatidis, was found in salamanders, Am- bystoma tigrinum stebbinsi, collected in southern Arizona, USA. The chytrid was iso- lated and cultured, and Koch's postulates were satisfied by infection of metamor- phosed salamanders with pure culture and subsequent reisolation from these sala- manders. We used the salamander strain and a strain isolated from lowland leopard frogs in Arizona, Rana yavapaiensis, to infect metamorphosed A. tigrinum, R. yava- paiensis, and R. boylii. All three species became infected, but none of the infected salamanders died within 60 days, and mortality of infected frogs did not differ significantly from controls, although sample size was small. Chytrid infection could not be detected by light histology in most of the infected frogs and one of the infected salamanders 60 days after infection. To date, there are three records of chytridiomycosis in salamanders on websites; ours is the first complete report of occurrence and pathology of chytridiomycosis in field-collected North American sal- amanders. Our results also demonstrate that chytridiomycosis does not always lead to mortality. Individuals within a species vary in susceptibility to infection, animals appear to recover from the infection, and syntopic salamanders and frogs may act as reciprocal pathogen reservoirs for chytrid infections.
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Chytridiomycosis is an emerging fungal disease of amphibians responsible for mass mortalities and population declines globally. One hypothesis for its recent emergence is anthropogenic introduction of the causative agent Batrachochytrium dendrobatidis through trade in amphibians for pets, food and biocontrol. In this study, we examined histological samples from apparently healthy American bullfrogs Rana catesbeiana that have been introduced into the Venezuelan Andes. B. dendrobatidis was present in 96% (46/48) of the individuals examined. In contrast to cases of chytridiomycosis outbreaks, the majority (44/46) of frogs had few, small lesions consistent with little or no clinical disease and no unusual mortality was observed. These findings have implications for amphibian declines in Venezuela and elsewhere. First, the high prevalence of Batrachochytrium but lack of clinical signs or chytridiomycosis-related mortality suggests that R. catesbeiana may be a good reservoir for this parasite in Venezuela. Second, the presence of this emerging pathogen in an introduced amphibian species suggests that trade and introduction of amphibians should be monitored and controlled to halt the spread of this pathogen nationally and internationally.
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Batrachochytrium dendrobatidis is a pathogen of amphibians that has been implicated in severe population declines on several continents. We investigated the zoospore activity, physiology and protease production of B. dendrobatidis to help understand the epidemiology of this pathogen. More than 95% of zoospores stopped moving within 24 h and swam less than 2 cm before encysting. Isolates of B. dendrobatidis grew and reproduced at temperatures of 4-25 C and at pH 4-8. Growth was maximal at 17-25 C and at pH 6-7. Exposure of cultures to 30 C for 8 d killed 50% of the replicates. B. dendrobatidis cultures grew on autoclaved snakeskin and 1% keratin agar, but they grew best in tryptone or peptonized milk and did not require additional sugars when grown in tryptone. B. dendrobatidis produced extracellular proteases that degraded casein and gelatin but had no measurable activity against keratin azure. The proteases were active against azocasein at temperatures of 6-37 C and in a pH range of 6-8, with the highest activity at temperatures of 23-30 C and at pH 8. The implications of these observations on disease transmission and development are discussed.
A telemetric system suitable for radio tracking anuran species is described. Its characteristics are given in detail, which will enable electrical engineers to construct the various elements. The system proved to be successful in field tests with Bufo bufo bufo L.
Several pathogens affect amphibians, but a chytridiomycete fungus, Batrachochytrium dendrobatidis, is of particular interest because this pathogen is linked to localized amphibian population declines and extinction of species. Species-specific infections and pathogen distribution are poorly understood, particularly in members of Caudata. We found B. dendrobatidis in adult Eurycea cirrigera (Southern Two-lined Salamander), a species not previously reported as susceptible to this fungal pathogen, and this report is the first record of B. dendrobatidis occurrence in Alabama.
Serious systemic disease in fish and amphibians is associated with the ranaviruses, epizootic haematopoietic necrosis virus (EHNV) and Bohle iridovirus (BIV) in Australia, and European sheatfish virus (ESV) and European catfish virus (ECV) in Europe. EHNV, ESV and ECV are recognized causative agents of the OIE (Office International des Epizooties) notifiable systemic necrotizing iridovirus syndrome and are currently identified by protein-based assays, none of which are able to rapidly identify the specific agents. The aim of this study was to develop TaqMan real-time PCR assays that differentiated these viruses using nucleotide sequence variation in two ranavirus genes. A conserved probe representing 100% sequence homology was used as a reference for virus-specific probes. The virus-specific probes produced a similar signal level to the conserved probe while those probes binding to non-target viral DNA produced an altered fluorescent curve. The pattern of probe binding was characteristic for each virus. Sensitivity, specificity and dynamic range of the assay were assessed. The test is currently useful as a research and initial screening tool, with the potential to become a sensitive and specific method for detection and differentiation of ranaviruses with further development.