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2012 SOUTHEASTERN NATURALIST 11(1):59–64
Prevalence of Salmonella sp. Isolated from Cryptobranchus
alleganiensis alleganiensis in Eastern Tennessee
Bridgid Lammers1, David Bemis2, Phil Colclough3, and Marcy J. Souza2,*
Abstract - Cryptobranchus alleganiensis alleganiensis (Eastern North American Hellbender)
were collected from the Little River of the Great Smoky National Park (n = 22)
and the Hiwassee River of the Cherokee National Forest (n = 30), TN. The cloaca of
each animal was swabbed for Salmonella culture and identification. No Salmonella was
identified from any of the samples. The prevalence of Salmonella in Hellbenders may be
higher than reported here since feces were not collected and animals were only sampled
once. Park visitors should still take precautions to protect themselves from water-borne
The enteric bacterium Salmonella presents a challenge to public health (Herikstad
et al. 2002). Many Salmonella serotypes have been isolated from a variety
of reptile and amphibian hosts and have been linked to the occurrence of disease
in humans (Burnham et al. 1998, CDC 2009, Clarkson et al. 2010, Lockhart et
al. 2008, Mermin et al. 2004, Rosenstein et al. 1965, Srikantiah et al. 2004, Van
Meervenne et al. 2009, Woodward et al. 1997). Annually in the United States,
approximately 74,000 people are infected with Salmonella via contact with an
amphibian or reptile.
Historically, amphibians have had a lower prevalence of Salmonella than
reptiles (Chambers and Hulse 2006, Pfleger et al. 2003). However, amphibians
worldwide have been shown to carry Salmonella at least occasionally
(Bartlett et al. 1977, Chambers and Hulse 2006, Everard et al. 1979, Kourany
et al. 1970, Pfleger et al. 2003, Sharma et al. 1974, Thomas et al. 2001,
Trust et al. 1981). Studies have linked human salmonellosis with amphibian
reservoirs (CDC 2009, Clarkson et al. 2010, Mermin et al. 2004, Pfleger et al.
2003, Srikantiah et al. 2004).
Although the occurrence of Salmonella infections in humans and domestic
animal reservoirs has been monitored, few studies have examined the prevalence
of this pathogen in wildlife, and in particular amphibians (Chomel et al.
2007). Even less is known about Salmonella in salamander species. Chambers
and Hulse (2006) found that among wild-caught native amphibians, salamanders
had a lower prevalence (11%) of Salmonella than did all amphibian
species combined (39%). There are no data available on the prevalence of
1University of Tennessee College of Veterinary Medicine, Knoxville, TN, 37996. 2Department
of Biomedical and Diagnostic Sciences, University of Tennessee College of
Veterinary Medicine, Knoxville, TN 37996. 3Knoxville Zoological Gardens, PO Box
6040, Knoxville, TN 37914. *Corresponding author - email@example.com.
60 Southeastern Naturalist Vol. 11, No. 1
Salmonella in Cryptobranchus alleganiensis alleganiensis (Daudin) (Eastern
North American Hellbender).
The Eastern North American Hellbender may sit on the top of the food chain
in the aquatic ecosystems that it inhabits (Nickerson and Mays 1973). A great
diversity of Salmonella has been found in natural ecosystems, especially in rivers,
and the cool, well-oxygenated, alkaline streams that Hellbenders are found
in may provide an ideal media for the growth of Salmonella (Barzily and Kott
1991, Baudart et al. 2000, Morse and Duncan 1974, Nickerson et al. 2003). It has
been demonstrated that direct contact with a shedding animal is not necessary to
transfer Salmonella from animal to human, and the pathogen can remain viable
in water even after the animal has been removed (Bartlett et al. 1977, Rosenstein
1965, Srikantiah et al. 2004, Van Meervenne et al. 2009).
There has been no published research examining the potential contamination
of waterways with Salmonella from native amphibian species; this contamination
could potentially lead to human salmonellosis in park visitors. This study
examined the prevalence of cloacal Salmonella in two distinct populations of
Hellbenders in East TN.
Materials and Methods
Hellbender populations are present in the Little River (LR) of the Great
Smoky Mountain National Park (GSMNP), TN and Hiwassee River (HR) of the
Cherokee National Forest (CNF), TN. Both rivers are used for many recreational
purposes such as boating, fishing, swimming, and sightseeing/camping along
the bank. The GSMNP and the CNF have millions of visitors annually, with an
unknown portion of these visitors engaging in water-related activities.
River sites were chosen based on accessibility, appropriate habitat of shallow,
cool, fast moving waters, and the presence of known Hellbender populations
from historical surveys. Sites were surveyed from April to September 2010.
Animal collection and sampling
Hellbenders were collected by hand in both rivers. Disposable nitrile
gloves were worn by investigators and were changed after handling and sampling
each animal. Animals were placed in cloth or plastic bags with water
for holding. Cloth bags were not re-used until laundered with detergent in hot
water; plastic bags were disposed of after one use. A swab (BBL™ Culture-
Swab Plus™, Becton Dickinson and Co., Sparks, MD) was inserted through
the vent into the cloaca and rotated 3–4 times. The swab was then placed
into the sterile transport media and kept cool. Morphometrics (total length
[cm], snout-to-vent length [cm], mass [g], and age class) were recorded. Age
class (adult, sub-adult, juvenile, larva) determination was based on length,
mass, and observer experience. PIT tags were placed in each animal over 40 g
2012 B. Lammers, D. Bemis, P. Colclough, and M.J. Souza 61
for identification, and GPS coordinates were recorded for each animal’s collection
Cloacal swabs underwent a routine bacteriology screen for Salmonella. This
procedure included MacConkey’s agar (MAC), Hektoen (HE) agar, and Selenite
broth incubated at 35° Celsius. At 24 hours, any colonies seen were recorded; if
no growth or only very small colonies were observed, the plates were incubated
for an additional 24 hours. If lactose negative colonies were identified, they were
tested for oxidase. All oxidase negative colonies were screened with Triple sugar
iron (TSI) agar, urea agar, and Motility Indole Ornithine (MIO) agar. If the selenite
broth was cloudy at 24 hours, the contents were subcultured to HE agar. If
selenite broth appeared clear at 24 hours, it was incubated for 48 hours before
subculture. All negative plates were held for 48 hours before discarding.
Prevalence and 95% confidence intervals (CI) were calculated for each of the
two Hellbender populations. Odds-ratio with confidence intervals was calculated
to determine if significant differences in prevalence between the two populations
were present (P < 0.05).
A total of 52 Hellbenders were sampled from both rivers (LR, n = 22; HR, n =
30). Adults (LR, n = 8; HR, n = 11), sub-adults (LR, n = 0; HR, n = 1), juveniles
(LR, n = 5; HR, n = 17) and larvae (LR, n = 9; HR, n = 1) were collected from
both rivers. Morphometrics are shown in Table 1.
Table 1. Morphometric data of Eastern North American Hellbenders (Cryptobranchus alleganiensis
alleganiensis) caught and sampled in two eastern Tennessee rivers, the Little and Hiwassee. Age
class, snout-to-vent length (SVL; cm), total length (TL; cm) and mass (g) are reported according
to which river animals were collected.
SVL mean ± SD TL mean ± SD Mass mean ± SD
River Age class (n) (range) (range) (range)
Hiwassee Adult (11) 20.2 ± 3.4 30.3 ± 4.4 183.8 ± 78.3
(15.6–26) (23.5–36) (86–330)
Sub-adult (1) 19.1 33.0 197
Juvenile (17) 13.8 ± 4.8 21.2 ± 5.9 80.2 ± 62.2
(3.2–22.2) (6.5–31.8) (8–235)
Larvae (1) 2.5 4.0 1.0
Little Adult (8) 21.0 ± 5.1 32.2 ± 8.5 206.0 ± 106.5
(12.7–26.7) (19.7–41.3) (36–337)
Juvenile (5) 8.8 ± 3.4 13.7 ± 5.0 19.8 ± 14.6
(3.8–12.4) (6.4–19.7) (2–35)
Larvae (9) 4.6 ± 1.3 6.7 ± 2.2 3.8 ± 2.5
(3–7.6) (4–11.4) (2–8)
62 Southeastern Naturalist Vol. 11, No. 1
The prevalence of cloacal Salmonella isolated from Hellbenders was 0% [95%
CI = 0–6.85] from the Little River and Hiwassee River. Because no Salmonella
was isolated from either population, no further statistics were performed.
The lack of cloacal Salmonella isolated from Hellbenders in eastern TN suggests
a small risk of transmission to humans in the Little and Hiwassee Rivers.
Although the prevalence was zero, various factors could have led to a lower
measured level than the true prevalence.
Salmonella can be shed intermittently by a carrier animal, and fecal samples
are typically collected over the course of 3–5 days for culture and identification.
Fecal samples are generally preferred over swabs to isolate Salmonella. Sampling
a wild animal over numerous consecutive days is not feasible, and fecal
samples are not reliably produced by animals. Animals were only held captive for
10–15 minutes during this study. Therefore, single, cloacal swabs were collected
from each animal. The true prevalence of Salmonella in Hellbender populations
may be higher than that found in this study.
The total number of Hellbenders in either of the rivers is not known, and
sampling a larger percentage of the population would certainly increase the
power of the study. These animals were being collected as part of another study
and were sampled opportunistically for Salmonella. It is difficult to know if the
sampled population is representative of the entire population of Hellbenders in
There are few studies examining the prevalence of Salmonella in wild amphibians,
and none in Hellbenders. Future studies may keep animals captive for
longer periods of time in order to collect a fecal sample; the stress associated with
extended capture would need to be evaluated. Collecting more animals would increase
the likelihood of accurately sampling the entire population. Outbreaks of
human salmonellosis associated with amphibians have been reported, and monitoring
of Salmonella in various amphibian species is warranted. Additionally,
humans coming in contact with amphibians or water potentially contaminated by
amphibians should be warned to wash themselves after contact and to not drink
untreated water. These precautions will reduce the risk of contracting Salmonella,
as well as other zoonotic pathogens found in untreated water such as Giardia
This research was approved by the University of Tennessee IACUC. We would like
to thank Arnold Saxton, the volunteers that aided in collection of animals, and the staff
of the University of Tennessee College of Veterinary Medicine (UTCVM) Microbiology
Laboratory. This study was funded in part by the UTCVM Center of Excellence Summer
2012 B. Lammers, D. Bemis, P. Colclough, and M.J. Souza 63
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