PROCEEDINGS OF THE
NORTH AMERICAN VETERINARY CONFERENCE
VOLUME 20
JANUARY 7-11, 2006
ORLANDO, FLORIDA
SMALL ANIMAL EDITION
Reprinted in the IVIS website (http://www.ivis.org) with the permission of the NAVC.
For more information on future NAVC events, visit the NAVC website at www.tnavc.org
Exotics — Wildlife
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VETERINARY ADVANCES THAT IMPACT
AMPHIBIAN CONSERVATION
Kevin Wright, DVM
University Animal Hospital
Tempe, AZ
HYPOVITAMINOSIS A
Hypovitaminosis A was first recognized in captive
Wyoming toads, Bufo baxteri, an endangered species
that is still the subject of an intense captive propagation
program to produce specimens for reintroduction to the
wild. Many captive-raised toads appeared unable to
prehend their prey, a disorder called short tongue
syndrome. The condition appeared to be acquired rather
than inherited, with clinical signs usually occurring in
subadult animals and slowly worsening over time.
Affected toads eventually require force feeding as they
become completely unable to capture prey.
The underlying cause of short tongue syndrome was
determined to be squamous metaplasia of the mucus
glands of the tongue rather than an actual shortening of
the tongue. (Pessier et al, 2002). As a result, the lingual
mucus glands were no able to produce the mucus
needed to adhere a cricket to the surface of the toad’s
tongue. In a 4-year period, over 50% of the captive
Wyoming toads necropsied had squamous metaplasia of
their tongues. Free-ranging toads had significantly
higher liver retinol (44–164 µg/g) than captives with short
tongue syndrome (undetectable to 7.3 µg/g). This finding
strongly supported the presumptive diagnosis of
hypovitaminosis A since squamous metaplasia of the
mucus glands is consistent with this nutritional disorder
in other vertebrates. Since the documentation of this
lesion in Wyoming toads, several other amphibians have
been noted with lingual squamous metaplasia.
Squamous metaplasia has been noted in the urinary
bladder and kidney. About 44% of the necropsied
Wyoming toads had opportunistic bacterial and fungal
infections suggestive of an impaired immune system,
another sign consistent with hypovitaminosis A.
Captive leopard frogs developed white swellings in
the lower eyelid. Histologic exam of the eye did not
reveal metaplasia and histologic examination of the
tongue of one afflicted frog was inconclusive, suggesting
decreased mucus production but without obvious
squamous metaplasia. It turned out the commercial
multivitamin supplement they were being fed had no
vitamin A in it! The frogs responded to supplementation
with vitamin A and antibiotic treatment. Poison dart frogs
with similar lesions nonresponsive to antibiotics resolved
with topical vitamin A.
Hypovitaminosis A in captive amphibians is likely due
to the use of inappropriate vitamin supplements or
supplements that have been stored improperly or have
expired. Many pet owners believe apocryphal Internet
postings about vitamin A being poisonous and then
intentionally feed inappropriate diets. It seems likely that
there are species-specific dietary needs for vitamin A.
Vitamin A supplementation is recommended for any
clinically ill amphibian. Compounding pharmacies may
be able to make an injectable solution that is dilute
enough for use in small patients. With a suggested dose
of 2 IU/g bodyweight every 72 hr, an injectable solution
should be prepared with a concentration of about
200 IU/ml. Continue treatment until clinical signs resolve.
A human-grade vitamin A supplement can be diluted
with vegetable oil or propylene glycol to an appropriate
concentration and orally or topically administered at a
dose of 1 IU/g daily for 2 weeks or until clinical signs
resolve.
Review the feeding practices used for any amphibian
presented for physical examination and pay particular
attention to how vitamins are stored and time of feeding.
For example, if a nocturnal frog is offered vitamin-dusted
crickets in the day, the crickets may lose the vitamins
before they are eaten. There is no evidence that betacarotene will be converted into vitamin A by amphibians,
so it should not be used as the major source of “vitamin
A” in the diet.
It is important to collect samples from all organs,
particularly the whole tongue, bladder, kidneys,
reproductive organs, and eyelids and conjunctiva during
an amphibian necropsy. The affected lingual mucus
glands of the tongue typically show disease at the rostral
tip of the tongue first so a longitudinal section of the
tongue, rather than a cross-section, should be evaluated
histologically. A frozen section of the liver may be
submitted for vitamin A analysis. Squamous metaplasia
and liver retinol levels below 40 µg/g suggest clinically
relevant hypovitaminosis A.
CHYTRIDIOMYCOSIS
The fungus Batrachochytridium dendrobatidis has
been implicated in catastrophic amphibian population
declines on several continents. This fungus feeds on
keratin found in amphibian skin and tadpole beaks.
Known as chytridiomycosis, this epidermal infection
results in a dermatitis that often results in deaths of
untreated amphibians. Typically frogs will develop a
brownish cast to their skin and being to shed more than
normal. Some may have erythema of the drink patch,
the ventral skin and feet, and may have discolored digits.
Some amphibians die without obvious signs of illness.
Infected frogs may crouch with a curved spine; some
may spend more time in water than normal. Some
species of amphibians can be asymptomatic carriers.
Carriers are often a source of reinfection in a captive
collection and may have devastating impacts if inhabiting
wetlands that support susceptible species. Tadpoles lack
keratinized skin except for their beaks, which become
deformed with infection. Froglets often quickly succumb
to chytridiomycosis after completing metamorphosis.
Chytrid may be detected using light microscopy to
examine a wet mount of the shed skin or skin scraping of
a heavily infected amphibian. The tips of toes, the drink
patch (if present), and sections of the skin with
excessive shedding or other lesions often have
sufficiently high densities of chytrid organisms to be
detected histologically. Histology does not consistently
detect early or light infections or heavy infections that
are not sampled appropriately. PCR analysis of skin
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The North American Veterinary Conference — 2006
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scraping is the recommended way to detect chytrid as it
detects the fungus’ ribosomal DNA. It is sensitive
enough so that it can detect a sample with only a few
zoospores (the motile life stage of chytrid). PCR samples
are collected by scraping a sterile wood applicator
across the skin of the amphibian. Several areas of the
skin should be scraped, including the ventral abdomen,
obvious lesions, and the tips of the digits. The sample
should be preserved in 1.0 ml of 70% ethanol and
submitted for analysis. The drawback is that the sheer
volume of samples being submitted for testing means a
timelag of 4 or more weeks between submission and
report. This test is available through Pisces Molecular
[2200 Central Avenue, Suite F, Boulder, CO 80301.
Phone (303) 546-9300. FAX (303) 546-9400.].
I recommend that any ill amphibian should be
immediately treated for chytridiomycosis since it is a
major source of morbidity and mortality in wild and
captive amphibians. Rapid treatment is essential for
cure. Entire collections may need to be treated to
eliminate silent carriers of the infection.
A proven treatment regimen uses itraconazole.
A veterinarian that treats amphibians should have a 1%
stock solution available at all times. The 1% stock
solution of itraconazole should be diluted with 0.6%
saline to form a 0.01% therapeutic solution. (To make
0.6% saline, combine 100 ml of “full strength” 0.9%
saline with 50 ml of distilled water or simply add 6 g
noniodized table salt to 1 liter of distilled water. To make
0.01% itraconazole solution, add 1 ml of 1% itraconazole
solution to 99 ml of 0.6% saline. This “therapeutic”
solution should be discarded within 24 hrs of
constitution. The amphibian is bathed in the 0.01%
therapeutic solution for at least 5 minutes daily for
11 consecutive days. Successful treatments have used
different dosing frequencies, such as a 5-minute bath
2 days in a row followed by baths every other day for five
treatments.
Itraconazole has caused deaths in tadpoles and small
postmetamorphic frogs and toads and elevated
environmental temperature (see below) may be a better
option. Always treat one or two specimens of a new
species to assess the impact of treatment before
exposing the whole population.
Infected amphibians may be dehydrated and
supportive fluid therapy such as soaking in amphibian
Ringer’s solution may be helpful. (To make amphibian
Ringer’s solution, adding 6.6 g NaCl, 0.15 g KCl, 0.15 g
CaCl2, and 0.2 g NaHCO3 to 1 liter of distilled water.
It may be stored for up to 30 days, or it may be
autoclaved and stored in sterile containers. Up to 10 g of
glucose may be added to the solution, too, but this
reduces the shelf life of unsterilized solution to 24 hrs.)
Behavioral fever may be the key to the survival of
some free-ranging amphibians. Chytrid infections of
captive red-eyed Australian tree frogs, Litoria chloris,
were cured by maintaining the frogs at 37°C for
16 hours. Shorter periods of time may be efficacious,
too. Lower temperatures, such as 27°C, are not 100%
effective in curing an amphibian. While not appropriate
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for amphibians that are cool-adapted, elevating the
environmental temperature may be a relatively easy way
to treat large collections and enclosures. Make sure that
there is adequate humidity and open water available
during heat treatment so the amphibians do not
desiccate during treatment.
Wild populations of amphibians have developed
apparent resistance to chytrid infection. The reason for
this is unknown but suggests inherited resistance to the
pathogen.
Equipment exposed to chytrid can be sterilized by any
of the following methods: (1) rinsing with 1% sodium
hypochlorite (household bleach); (2) 20-second
exposure to 70% ethanol or 1 mg/ml benzalkonium
chloride; (3) desiccation and exposure to 50–60°C heat
for 30 minutes; (4) 0.008% quaternary ammonium
compound Wax-128 (Waxie Sanitary Supply, 9353
Waxie
Way,
San
Diego
CA,
800-995-4466,
www.waxie.com), which contains 6.8% didecyl dimethyl
ammonium chloride (DDAC) as the active ingredient.
Additional Reading
1.
2.
3.
4.
5.
6.
7.
8.
Berger L, Speare R, Daszak P, et al.
Chytridiomycosis causes amphibian mortality
associated with population declines in the rain
forests of Australia and Central America. Proc Natl
Acad Sci USA 1998;95:9031-9036
Berger L, Speare R, Hines HB, et al. Effect of
season and temperature on mortality in amphibians
due to chytridiomycosis. Austral Vet J 2004;
82(7):31-36.
Johnson ML, Berger L, Philips L, Speare R.
Fungicidal effects of chemical disinfectants, UV light,
desiccation and heat on the amphibian chytrid
Batrachochytrium dendrobatidis. Dis Aquatic
Organisms 2003;57:255-260.
Longcore
JE,
Pessier
AP,
Nichols
DK..
Batrachochytrium dendrobatidis gen. et. sp. nov., a
chytrid pathogenic to amphibians. Mycologia
1999;91:219-227.
Muths E, Corn PS, Pessier AP, Green DE.
Evidence for disease-related amphibian decline in
Colorado. Biol Conserv 2003;110:357-365
Nichols DK, Lamirande EW. Treatment of cutaneous
chytridiomycosis in blue-and-yellow poison dart
frogs
(Dendrobates
tinctorius)
(abstract),
Proceedings: Getting the Jump on Amphibian
Disease, Cairns, Australia, August 2000, p 51.
Nichols OK, Lamirande EW, Pessier AP, Longcore
JE. Experimental transmission and treatment of
cutaneous chytridiomycosis in poison dart frogs
(Dendrobates auratus and Dendrobates tinctorius)
(abstract), Proceedings: Joint Conf Am Assoc Zoo
Vet and Int Assoc Aquatic Anim Med, New Orleans,
September 2000, pp 42-44.
Nichols DK, Lamirande EW, Pessier AP, Longcore
JE. Experimental transmission of cutaneous
chytridiomycosis in two species of dendrobatid
frogs. J Wildlife Dis 2001;37(1):1-11.
Exotics — Wildlife
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9.
Pessier AP, Roberts DR, Linn M, et al. “Short
tongue
syndrome,”
lingual
squamous
metaplasia and suspected hypovitaminosis A in
captive Wyoming toads. Proc Assoc Reptilian
Amphibian Vet 2002, pp 151-153.
10. Retallick RWR, McCallum H, Speare R.
Endemic infection of the amphibian chytrid
fungus in a frog community post-decline. PLoS
Biology 2004;2(11):1965-1971
11. Taylor SK, Williams ES, Thorne ET, et al.
Causes of mortality in the Wyoming toad.
J Wildl Dis 1999;35:49-57
12. Woodhams DC, Alford RA, Marantelli G.
Emerging disease of amphibians cured by
elevated body temperature. Dis Aquatic
Organisms 2003;55:65-67
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