Abstract

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 amphibian populations.^1,2,4

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the National Zoological Park.³ This chytrid has also been isolated from other species of frogs and toads (J. Longcore, unpublished data).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 hours 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 weeks. 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 consecutive days.

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 IV 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 drug.

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 chytridiomycosis.⁵

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.

Acknowledgments

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 fruit flies 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.

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