Experimental Transmission and Treatment of Cutaneous Chytridiomycosis in Poison Dart Frogs (Dendrobates auratus and Dendrobates tinctorius)
Species of fungi in the Phylum Chytridiomycota (chytrids) commonly occur in water and moist soil. A
fatal skin disease associated with cutaneous infection by chytrids has been recently described in many species of captive
and wild anuran amphibians in the United States, Central America, South America, and Australia.1,2,4,6
Cutaneous chytridiomycosis appears to be an emergent disease that may be contributing to the global decline of wild
A previously undescribed genus and species of chytrid, Batrachochytrium dendrobatidis (B.
dendrobatidis), was originally isolated from a captive blue poison dart frog (Dendrobates azureus) that died
at the National Zoological Park.3 This chytrid has also been isolated from other species of frogs and toads
(J. Longcore, unpubl. data). The purposes of this series of experiments were to prove that B. dendrobatidis can
cause fatal skin disease in dendrobatid frogs and to determine one or more methods that can be used to successfully treat
cases of cutaneous chytridiomycosis.
Two species of captive-bred, juvenile frogs were used in this study: green-and-black poison dart
frogs (GBF; Dendrobates auratus) and blue-and-yellow poison dart frogs (BYF; Dendrobates tinctorius). Frogs
were housed in plastic storage containers that contained plastic, grass-like carpet for substrate and plastic leaves to
provide hiding places. The containers were slightly inclined to provide a pool of filtered water at one end. Temperatures
were timer-controlled so that the containers warmed to 25°C for 8 hr during the day and gradually cooled to ambient
room temperature (20-22°C) at night. Containers were cleaned every 2-3 days by thoroughly rinsing the container,
carpet substrate, and plastic leaves with copious amounts of filtered water. Frogs were provided fruit flies
(Drosophila melanogaster and/or Drosophila hydei) ad libitum.
Cultures of B. dendrobatidis were grown on 2% tryptone agar (Difco Laboratories, Detroit, MI)
and in 1% tryptone broth (Difco Laboratories). Blocks of agar with chytrid colonies containing active zoospores were
rinsed with sterile 1% tryptone broth. The agar rinse was then mixed with an equal volume of tryptone broth culture
containing good chytrid colony growth. This mixture of agar rinse and broth culture was used as the B.
dendrobatidis inoculum for the experiments. Rinses of sterile tryptone agar plates were combined with equal volumes
of sterile tryptone broth and then used to sham-inoculate negative control frogs.
In Experiment 1, 200 µl of the inoculum was dripped onto the backs and hindlegs of two BYF each
day, and three BYF were sham-inoculated daily with 200 µl of the sterile inoculum. In Experiment 2, the experimental
group consisted of six GBF and the control group contained three frogs from this species. The frogs in this experiment
were inoculated once per day for 5 days, for up to 4 wk. For Experiment 3, three GBF were individually housed and the
pool of water in each cage was inoculated with 1 ml of the B. dendrobatidis inoculum for either 1, 3, or 5
Twelve BYF were divided into four groups of three frogs in Experiment 4. Each frog was inoculated
with 200 μl of the B. dendrobatidis inoculum daily for four consecutive days. Once clinical signs of
chytridiomycosis were present (14 days PI), frogs in groups 1-3 were topically treated with different antimicrobial
drugs; these drugs were diluted with 0.6% saline to the final concentrations. Group 1 frogs were immersed in a 0.01%
solution of miconazole (Conofite lotion, 1% miconazole nitrate, Schering-Plough Animal Health Corporation, Union, NJ) for
5 min each day for 8 consecutive days. Group 2 frogs were immersed in a 0.01% suspension of itraconazole (Sporanox,
Janssen Pharmaceutica, Inc., Titusville, NJ) for 5 min daily for 11 consecutive days. Frogs in group 3 were immersed for
5 min daily in a 0.1% solution of trimethoprim-sulfadiazine (Tribrissen 48% injection for i.v. use in horses,
Schering-Plough Animal Health Corporation) for 11 consecutive days. The frogs in group 4 served as positive controls and
were not treated.
In each experiment, all frogs exposed to B. dendrobatidis developed cutaneous
chytridiomycosis. Clinical signs consisted of anorexia, lethargy, and excessive shedding of skin. All infected frogs in
Experiments 1-3 and the untreated frogs in Experiment 4 died within 35 days of initial chytrid exposure. Typical
histologic lesions were epidermal hyperkeratosis and acanthosis associated with the presence of chytrid organisms in the
keratinized cell layers. Cultures of skin from one frog in Experiment 1 and two frogs in Experiment 2 resulted in
re-isolation of B. dendrobatidis from all three animals.
Clinical signs resolved completely in those frogs treated with miconazole and those treated with
itraconazole. These frogs were euthanatized and necropsied 63 days after initial chytrid inoculation. There was no gross
or histologic evidence of chytrid infection in these animals at the time of necropsy.
Frogs treated with the trimethoprim-sulfadiazine solution continued to shed excessive amounts of skin
and cytologic examination of shed skin pieces revealed chytrids within epidermal cells. One of these frogs was found dead
18 days after drug therapy ended, and another frog died 2 days later. Necropsy confirmed that these frogs died from
chytridiomycosis. The third frog in this group was euthanatized 63 days after initial chytrid inoculation; at necropsy,
this frog had relatively mild skin lesions associated with low numbers of chytrid organisms.
The results from Experiments 1 and 2 fulfilled Koch's postulates regarding the association between an
infectious agent and a disease, thereby proving that Batrachochytrium dendrobatidis can cause fatal cutaneous
infections in dendrobatid frogs. Experiment 3 demonstrated that frogs may become infected through exposure to
environments that have been contaminated with B. dendrobatidis.
Experiment 4 showed that effective treatment of cutaneous chytridiomycosis is possible. Both groups
of frogs that were topically treated with imidazole anti-fungal compounds were cleared of chytrid infections. The primary
anti-fungal activity for all imidazoles is inhibition of ergosterol synthesis leading to instability of fungal cell
walls.7 The frogs tolerated itraconazole therapy much better than treatment with miconazole; however, this was
likely a result of the formulation of the miconazole solution used in this experiment rather than a direct effect of the
Trimethoprim-sulfadiazine appeared to have a fungistatic effect on B. dendrobatidis. Frogs
that were treated with this compound continued to shed excessive amounts of skin that was infected with chytrids. These
frogs survived longer than those in the untreated positive control group; however, after the daily
trimethoprim-sulfadiazine treatments ended, two of the frogs died of chytridiomycosis. Similar results were seen in a
captive colony of arroyo toads (Bufo microscaphus californicus) that was affected with
One frog that had been treated with trimethoprim-sulfadiazine survived until the end of the study and
was found to have a low-level chytrid infection at necropsy. This suggests that a carrier state can exist and that other
factors may influence the pathogenesis of cutaneous chytridiomycosis. Further studies on possible co-factors that can
affect the interaction between chytrids and amphibians should be conducted.
A Senior Post-doctoral Fellowship (98-3545-A) from the Friends of the National Zoo (FONZ) supported
Dr. Pessier. Vince Rico, Ed Smith, and the rest of the staff at the National Zoo's Department of Amazonia provided the
fruitflies used to feed the frogs and were valuable sources of advice about frog husbandry procedures. Paul Miles
generously donated some of the frogs used in these experiments.
1. Berger L, R Speare, P Daszak, DE Green, AA Cunningham, CL Goggin, R Slocombe, MA Ragan,
AD Hyatt, KR McDonald, HB Hines, KR Lips, G Marantelli, H Parkes. 1998. Chytridiomycosis causes amphibian mortality
associated with population declines in the rain forests of Australia and Central America. Proc. Natl. Acad. Sci.
2. Daszak P, L Berger, AA Cunningham, AD Hyatt, DE Green, R Speare. 1999. Emerging
infectious diseases and amphibian population declines. Emerg. Infect. Dis. 5:735-748.
3. Longcore JE, AP Pessier, DK Nichols. 1999. Batrachochytrium dendrobatidis gen. et
sp. nov., a chytrid pathogenic to amphibians. Mycologia 91(2): 219-227.
4. Nichols DK, AP Pessier, JE Longcore. 1998. Cutaneous chytridiomycosis: an emerging
disease? Proc. Am. Assoc. Zoo Vet. Omaha, Nebraska. Pp. 269-271.
5. Nichols DK, AJ Smith, CH. Gardiner. 1996. Dermatitis of anurans caused by fungal-like
protists. Proc. Am. Assoc. Zoo. Vet. Puerto Vallerta, Mexico Pp. 220-221.
6. Pessier AP, DK Nichols, JE Longcore, MS Fuller. 1999. Cutaneous chytridiomycosis in
poison dart frogs (Dendrobates spp.) and White's tree frogs (Litoria caerulea). J. Vet. Diag. Invest.
7. Prescott JF. 1999. Antimicrobial chemotherapy, In: Hirsh, D.C. and Y.C. Zee (eds.).
Veterinary Microbiology. Blackwell Science, Malden, Massachusetts. Pp. 28-45.