Experimental Transmission of Cutaneous Chytridiomycosis in Dendrobatid Frogs
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Journal of Wildlife Diseases, 37(1), 2001, pp. 1–11 EXPERIMENTAL TRANSMISSION OF CUTANEOUS CHYTRIDIOMYCOSIS IN DENDROBATID FROGS Donald K. Nichols,1,4 Elaine W. Lamirande,1 Allan P. Pessier,1,3 and Joyce E. Longcore2 1 Department of Pathology, National Zoological Park, Smithsonian Institution, 3000 Connecticut Avenue NW, Washington, DC 20008, USA 2 Department of Biological Sciences, University of Maine, Orono, Maine 04469, USA 3 Current address: Department of Pathology, Zoological Society of San Diego, P.O. Box 120551, San Diego, California 92112, USA 4 Corresponding author (e-mail: [email protected]) ABSTRACT: In a series of three experiments during March–October, 1998, two species of captive- bred poison dart frogs (Dendrobates tinctorius and D. auratus) were exposed to Batrachochytrium dendrobatidis, a recently-described chytridiomycete fungus (chytrid) that was originally isolated from a blue poison dart frog (D. azureus). All frogs exposed to the chytrids developed a fatal skin disease, whereas none of the control frogs developed skin lesions. The most consistent clinical sign in chytrid-exposed frogs was excessive shedding of skin. Gross lesions were subtle, usually affected the legs and ventrum, and consisted of mild skin thickening and discoloration. Micro- scopic examination of shed skin pieces and/or skin imprints demonstrated the presence of chytrids and was used for ante mortem and post mortem confirmation of chytrid infection. Histologically, there was epidermal hyperkeratosis, hyperplasia, and hypertrophy associated with low to moderate numbers of chytrids in the keratinized layers. These experiments demonstrated that Batracho- chytrium dendrobatidis can be a fatal pathogen in poison dart frogs. The experimentally-induced disease in these frogs resembled cases of cutaneous chytridiomycosis that have recently been described in several other species of captive and wild amphibians. Key words: Batrachochytrium dendrobatidis, chytrid, cutaneous chytridiomycosis, Dendro- bates auratus, Dendrobates tinctorius, experimental infection, fungus, poison dart frog. INTRODUCTION frogs using Batrachochytrium dendrobati- Most species in the fungal class Chytri- dis, a recently described chytrid species diomycetes (chytrids) live on decaying or- (Longcore et al., 1999). The study consist- ganic material (Barr, 1990). Although ed of a series of three experiments per- some species of chytrids were known to formed during March–October 1998. The parasitize fungi, plants, and invertebrate purposes of Experiment 1 were to develop animals, none had been reported to be a techniques for infecting frogs with chytrids pathogen of vertebrates until recently. and to monitor the progression of any dis- Berger et al. (1998) and Pessier et al. ease that resulted. The purpose for Ex- (1999) described a novel skin disease in periment 2 was to determine if the results several species of anuran amphibians, as- of Experiment 1 were repeatable, using sociated with the presence of epidermal frogs from a different species. Experiment infection by chytrids. Deaths of captive 3 was conducted to determine if frogs frogs (Pessier et al., 1999) and the decline could be infected by a different method of of many wild populations of frogs and exposure to chytrids and, if so, what effects toads in Australia and Panama (Berger et this had on the progression of the disease. al., 1998) have been attributed to this dis- MATERIALS AND METHODS ease. Although chytrids were clearly pre- sent in the skin lesions of the dead ani- Experiment 1 mals, it had not been conclusively dem- In March 1998, eight captive-bred juvenile onstrated that infection by these organisms (2–3 mo post metamorphosis; body weight was the cause of the lesions. range 0.67–0.94 g) blue-and-yellow poison dart frogs (Dendrobates tinctorius) were obtained This paper describes the experimental from a privately owned collection of poison transmission of fatal cutaneous chytridi- dart frogs (The Boa Barn, Long Green, Mary- omycosis in two species of dendrobatid land, USA) that had no history of chytridiomy- 1 2 JOURNAL OF WILDLIFE DISEASES, VOL. 37, NO. 1, JANUARY 2001 cosis or any other skin disease. These frogs to completely new cages set up as previously were housed two or three per cage in 5.7 l plas- described. The frogs were divided into two tic storage boxes (Sterilite Corporation, Town- groups; the control group consisted of three send, Massachusetts, USA). To allow for ade- frogs (B1, B2, and B3) and the experimental quate ventilation, a 26 ϫ 13 cm rectangular group had two frogs (B4 and B5). section was removed from the middle of the lid The chytrid species used for this experiment, of each container and silkscreen fabric was Batrachochytrium dendrobatidis, was isolated taped over these areas to prevent the frogs from a blue poison dart frog (Dendrobates from escaping. A 15 ϫ 25 cm piece of polypro- azureus) that died at the National Zoological pylene grass-like carpet (Bretlin Inc., Calhoun, Park in September 1997 (Longcore et al., Georgia, USA) was placed in the bottom of 1999). Cultures of this organism were grown on each container as substrate and two or three 2% tryptone agar (Difco Laboratories, Detroit, leaves from a plastic aquarium plant were add- Michigan, USA) and in 1% tryptone broth (Dif- ed to provide hiding places. The containers co Laboratories). For this experiment, these were inclined by setting one end on 2.5 cm cultures were kept at the same fluctuating tem- plastic blocks. Approximately 100 ml of tap wa- peratures (20–25 C) present in the frogs’ in- ter that had been passed through a reverse os- cubators. For each experimental exposure, a mosis water filter (RO water) was added to 1 ϫ 2 cm block of agar with chytrid colonies each container, resulting in the formation of a containing active zoospores (viewed with an in- pool of water at the lower end. The containers verted microscope) was removed with a sterile were placed inside incubators (Thermocare scalpel and rinsed 10 times with 0.5 ml of ster- Model W1 Intensive Care Warmers, Thermo- ile 1% tryptone broth. The agar rinse was trans- care Inc., Incline Village, Nevada, USA) that ferred to a sterile vial and mixed with an equal were timer-controlled so that the containers volume of tryptone broth culture containing warmed to 25 C for 8 hr during the day and good chytrid colony growth (based on turbidity gradually cooled to ambient room temperature of broth for cultures between 9- and 19-days- (20–22 C) at night. Containers were cleaned old). Using a pipette, a 200 l aliquot of this every 2–3 days by thoroughly rinsing the con- mixture was dripped onto the back and hind- tainer, carpet substrate, and plastic leaves with legs of each frog in the exposure group. Each copious amounts of RO water. Frogs were fed frog in the control group was similarly exposed ad libitum fruitflies (Drosophila melanogaster to 200 l of a solution prepared in the same vestigial wing) (Carolina Biological Supply manner from tryptone agar plates and tryptone Company, Burlington, North Carolina, USA) broth that did not contain chytrids. Frogs were that were raised on a commercial fruitfly food exposed once a day for 30 days or until they (Formula 4–24 Instant Drosophila Medium, died. Carolina Biological Supply Company). The flies Frogs were observed several times a day were coated with a fine dust of a vitamin-min- throughout the experiment for clinical signs of eral mixture (Miner-All O, Sticky Tongue disease. Pieces of shed skin (keratinized epi- Farms, Menifee, California, USA) just before dermis) that were found in the cages were re- being added to the frog cages. moved, placed on glass slides to air dry, and The frogs were acclimated to the above con- then stained (DipQuick, Jorgensen Laborato- ditions for 3 wk before beginning the experi- ries Inc., Loveland, Colorado, USA) for micro- ment. Three frogs died between days 17 and scopic examination. Frogs that were found 21 of the acclimation period. A complete nec- dead were removed from the cages for post ropsy, including histologic examination of skin mortem examination. One frog was found mor- and all major organs, was performed on each ibund and was euthanized by immersion in a dead frog. Necropsy revealed that the frogs had solution of RO water containing a high concen- decreased body fat stores and numerous oxy- tration of tricaine methane sulfonate (TMS, urid and strongyle nematodes within the intes- Cresent Research Chemicals, Phoenix, Arizona, tinal tract; the cause of death in each case was USA). At the end of the experiment (5 days attributed to nematode parasitism and poor after the last frog in the experimental group body condition. Each of the remaining frogs died), the surviving control frogs were eutha- was weighed and treated once orally with 0.1 nized in the same manner. mg fenbendazole (Panacur, Hoechst-Roussel, A necropsy was performed on each frog and Somerville, New Jersey, USA); this dose was the coelomic cavity organs were removed for approximately 100 mg/kg (actual dose:106–149 fixation in 10% buffered formalin. One hindleg mg/kg). Four days later, each frog was orally from the first frog that died in the experimental treated with 0.25 g of ivermectin (Ivomec, group was removed placed on ice packs, and Merck and Company, Rathway, New Jersey, shipped to one of the authors (JEL) for chytrid USA; actual dose: 266–373 g/kg) and moved isolation and identification as previously de- NICHOLS ET AL.—EXPERIMENTAL CHYTRIDIOMYCOSIS IN DENDROBATID FROGS 3 scribed (Longcore et al., 1999). The remainder of disease. Pieces of shed skin that were found of each carcass was fixed intact in 10% formalin in the cages were removed, placed on glass and then decalcified in 5% trichloroacetic acid slides to air dry, and stained as before for mi- (Fisher Scientific, Fair Lawn, New Jersey, croscopic examination.