Amphibian chytridiomycosis, caused by the fungus Batrachochytrium dendrobatidis (Bd), has been implicated in the decline and extinction of amphibian species worldwide, in addition to catastrophic losses of animals in captivity. Conservation of threatened amphibians, including captive breeding and maintenance of animals in zoos, research facilities, and private collections, requires effective control of pathogens. Several chemical compounds, including Formalite III®, itraconazole, and chloramphenicol, have been used to treat amphibians infected with Bd, with varying levels of success. Here, we report successful clearance of Bd in five species of post-metamorphic anurans and one caudate species using terbinafine hydrochloride (HCl) in alcohol, which is available over the counter as Lamisil AT™ (Novartis Pharmaceuticals Inc., New York, NY). Treatments consisting of 5 min soak in fresh 0.01% or 0.005% terbinafine HCl in alcohol for either five consecutive days or for six treatments spread across 10 days successfully cleared Bd from 100% of 81 test subjects in eight trials. Our results indicate that terbinafine HCl in alcohol has a high therapeutic index as a treatment for Bd infection in living post-metamorphic amphibians.
Amphibians appear to be in decline worldwide (Wake, 1991; McCallum, 2007) and are considered the most imperiled group of vertebrates on the planet (Johnson, 2006). At least 122 species have disappeared since 1980, and a third of all living species are currently threatened with extinction (Stuart et al., 2004; Wake and Vredenburg, 2008). One of the most immediate global threats to amphibians appears to be chytridiomycosis (Skerratt et al., 2007), caused by the zoosporic fungal pathogen Batrachochytrium dendrobatidis (Bd) (Longcore et al., 1999). Bd has now been detected on all continents that are inhabited by amphibians, and numerous global declines and extinctions have been linked to amphibian chytridiomycosis epizootics (Berger et al., 1998; Lips, 1999; Bradley et al., 2002; Weldon et al., 2004; Une et al., 2008). Chytrid fungus appears to have become endemic in some geographic areas of the United States too (Padgett-Flohr and Hopkins, 2009).
Clinical signs associated with chytridiomycosis include lethargy, anorexia, and excessive skin sloughing, although these signs may not appear for a month or more after infection and may precede death by as little as a few hours (Kriger et al., 2007). Thus, signs of disease may come too late to be of clinical use. Bd infection, as opposed to amphibian chytridiomycosis as a disease, can be detected either through histological examination (Berger et al., 1999; Hyatt et al., 2007) or DNA analysis via PCR (Annis et al., 2004; Kriger et al., 2006; Hyatt et al., 2007). Further, some species, such as bullfrogs (Lithobates [Rana] catesbeiana), may carry Bd but remain asymptomatic (Hanselmann et al., 2004) and, thus, can provide a reservoir for infection of sensitive species within a region or within herpetological collections.
In addition to threatening wild amphibian populations worldwide, Bd is a major concern in captive breeding efforts and the maintenance of zoo and private collections (Daszak and Cunningham, 1999; Daszak et al., 2000). A number of compounds, including Formalite III® (a mixture of formalin and malachite green), fluconazole, F10SC Veterinary Disinfectant®, itraconazole, ketoconazole, chloramphenicol, and benzalkonium chloride, have been evaluated for the treatment of Bd in live amphibians, with varying results (Nichols and Lamirande, 2000; Taylor, 2001; Parker et al., 2002; Young et al., 2007; Berger et al., 2008; Bishop et al., 2009; Garner et al., 2009).
Terbinafine hydrochloride (TBF, C21H26ClN, molecular weight 327.9 g/mol, CAS 78628-80-5) is an antifungal compound currently used to treat fungal diseases in some vertebrates, including humans (Kotnik, 2002; Ghannoum et al., 2009). TBF acts by blocking ergosterol biosynthesis in the fungal cell wall; specifically, it inhibits squaline epoxidase and, subsequently, causes a toxic accumulation of squalene in the fungal cell (Petranyi et al., 1987). We tested the efficacy of TBF dissolved in alcohol on post-metamorphic amphibians infected with Bd using topical applications of TBF and evaluated amphibian tolerance to TBF in alcohol.
Amphibian collection and husbandry—This study was conducted under permits from the Oregon Department of Fish and Wildlife (Scientific Taking Permits 090-08 and 037-09), the California Department of Fish and Game (Scientific Collecting PermitSC-000852),andtheU.S.PRTTE0066112-4. We collected 36 juvenile bullfrogs (Lithobates catesbeiana) from Thousand Trails Pond, Deschutes County, Oregon (43°49′15 N, 121°27′07 W), a site previously shown to have a high prevalence of Bd infection (Pearl et al., 2009). Prior to treatment, we housed bullfrogs communally in two 10-gal (38-L) glass tanks in 10 L of well water for two weeks, fed animals to satiation on house crickets (Acheta domesticus) twice weekly, and changed tank water weekly. We obtained five additional Bd-infected (as confirmed by histological examination of skin scrapes) species from three sources: laboratory-raised California tiger salamanders (Ambystoma californiense) and foothill yellow-legged frogs (Rana boylii) from Southern Illinois University (Padgett-Flohr, 2008), field-collected black-eyed litter frogs (Leptobrachium nigrops) and Malaysian horned frogs (Megophrys nasuta) from an animal importer, and captive-bred Cranwell's horned frogs (Ceratophrys cranwelli) from a commercial breeder.
Prior to treatment, we communally housed 20 individuals of each of these taxa in glass tanks appropriately sized to species: 5.5 gal (20.8 L), 10 gal (38.0 L), or 20 gal (77.0 L). We divided the tanks into terrestrial and aquatic portions and fed all animals house crickets and mealworms (Tenebrio molitor) ad libitum. During treatment periods, we housed six or seven individuals of each species in 1.5-gal (5.7-L) plastic containers lined with moist paper towels. Following treatment, we maintained all animals in 1.5-gal (5.7-L) tubs for three weeks.
Bd assay pretreatment—We tested each animal for Bd immediately prior to treatment using individual PCR sample kits provided by Pisces Molecular, LLC (Boulder, CO); the protocol we used to collect the samples was provided by Pisces. Each individual animal was handled using fresh vinyl gloves. We wiped a sterile cotton-tipped wood swab across the skin of the lower abdomen 10 times and then across the ventral surface of each thigh five times; and finally, we wiped once across the webbing between each toe on each rear foot. We placed each swab into a leakproof vial of 70% ethanol, coded the vials for blind assay, and submitted the samples to Pisces for PCR testing for the presence of Bd. All individuals used in the efficacy experiments tested positive for Bd prior to treatment.
Bd assay post-treatment—At the completion of each trial, all animals were individually swabbed as described above. The swabs from all members of each replicate were pooled for DNA processing. Thus, if any animal within a trial replicate was positive, the replicate would be considered to have failed to clear Bd. All treatment replicates were tested again at three or four weeks post-treatment to check for latent infection that recurred after treatment.
Preparation of treatment solutions—We added 1% TBF in alcohol to distilled water to achieve treatment baths with final TBF concentrations of 0.01%, 0.005%, or 0.0005% (final alcohol concentrations of 1%, 0.5%, and 0.05%, respectively). In the trials with bullfrogs, we used Lamisil AT spray, which comes as a 1% TBF solution (10 mg/g) of TBF in ethanol. All other trials started with a stock solution of TBF obtained by dissolving 1 g of generic TBF in 99 g of solvent-grade ethanol (CH3CH2OH; EMD Chemicals) diluted with distilled water to achieve final concentrations immediately prior to treatment. The pH of treatment solutions was approximately 7.0 at all concentrations tested.
Experimental trials: Treatment 1—We randomly assigned 56 Bd-positive juvenile bullfrogs to one of five treatments consisting of six or seven frogs each (Table 1). Treatment 1A (14 individuals in two replicates) consisted of 5 min soaks in 0.01% TBF/alcohol bath for five consecutive days. Treatment 1B (18 individuals in three replicates) received 5 min baths in 0.005% TBF/alcohol on days one, two, five, six, eight, and 10. Treatment 1C was identical to 1B, but frogs were maintained in water with 0.0005% TBF between treatments. Treatment 1D and 1E were controls, identical to treatments 1A and 1B but without addition of TBF/alcohol to treatment baths. Between treatments, we maintained each group of frogs together in 10 L of unchlorinated well water in plastic buckets. Prior to the experiment, and again on each day of treatment, we disinfected each holding container with 10% bleach (NaClO) solution, after which containers were triple rinsed and refilled with distilled water or fresh well water. We housed each replicate group together following the final treatment, placing them in freshly disinfected glass tanks for four weeks, with tank disinfection and water changes after two weeks. Four weeks after treatment, again we swabbed each animal and pooled swabs from each replicate to test for the presence of Bd, as previously described.
Experimental trials: Treatment 2—We randomly assigned six or seven Bd-positive individuals of each of five amphibian species (California tiger salamanders, Cranwell's horned frogs, black-eyed litter frogs, Malaysian horned frogs, and foothill yellow-legged frogs) to one of three treatment groups (Treatments 2A, 2B, and 2C; Table 2), yielding two replicates with the tiger salamander and one replicate for each of the other species. Each treatment group was housed in 5.7-L plastic tubs during trials as described above. Treatment group 2A received 0.005% TBF/alcohol; 2B received 0.0005% TBF/alcohol; and 2C, the control, received distilled water, applied as a 5 min bath daily for five days. For treatment, we removed the amphibians from their housing containers and placed them in a concentration-specific treatment container, which we disinfected between treatments as previously described. We prepared fresh treatment solutions immediately prior to use. We used enough solution to partially cover animals and then gently rocked the treatment container to ensure complete coverage of the animals. Housing containers were washed and disinfected before animals were returned following treatments. Following the five days of treatment, we allowed the amphibians to remain undisturbed in their housing containers for a day and then swabbed all individuals for the Bd DNA assay and pooled swabs from each treatment group under the assumption that detection of Bd within a group indicated a treatment failure. We repeated Bd sampling again three weeks post-treatment.
Amphibian tolerance to TBF/alcohol—To further investigate amphibian tolerance to TBF/alcohol, we subjected 14 species of anurans and one caudate (for list of species, see Appendix 1) to TBF/alcohol as described in Experiment 2A–C above but did not test these amphibians for the presence of Bd. Husbandry of animals tested for TBF/alcohol tolerance and protocols for exposure to TBF/alcohol were as described for treatments 2A–C above. In each case, we exposed five individuals of a species to either 0.005% or 0.01% TBF solution for 5 or 15 min, every 24 hours, for five or 10 days. We monitored all animals exposed to TBF during the testing period and for 60 days following exposure.
We found that daily 5 min baths in 0.005% or 0.01% TBF dissolved in ethanol for five consecutive days, or a total of six 5 min treatments administered over 10 days, successfully cleared detectable levels of Bd from infected amphibians, as determined by PCR testing (see Tables 1 and 2). We observed noticeable brightening of skin color and increased activity levels in treated individuals following treatment. Amphibians cleared of detectable Bd by this technique tested negative for the presence of Bd after 3–4 weeks, whereas all control animals remained Bd-positive. TBF in ethanol was well tolerated by all species we tested. We observed no mortalities or adverse impacts from exposure to TBF concentrations up to 0.01% solution (final alcohol concentration up to 1%) for treatments lasting 15 min per day for 10 days.
TBF dissolved in alcohol and applied in a series of daily 5 min baths can successfully clear detectable levels of Bd in post-metamorphic amphibians. The variety of species that we tested suggests that this compound should be similarly effective for other species as well. We acknowledge that we did not run a control using ethanol at equivalent concentrations (0.5% or 1%) without TBF. Although ethanol is an effective disinfectant against Bd at 70% concentration (Johnson et al., 2003), fungicidal effectiveness of ethanol declines with decreasing concentration and exposure time. Indeed, Kruse et al. (1963, 1964) showed that fungal disinfection of laboratory surfaces required >30% EtOH and >10 min immersion. Thus, it is unlikely that repeated 5 min exposures to 0.5–1% ethanol would eliminate or kill chytrid zoospores.
TBF has several advantages over other currently used treatments for Bd. TBF is a readily available over-the-counter treatment for human fungal infection (Lamisil AT). Although itraconazole has been shown to be effective against Bd infection (Nichols and Lamirande, 2000; Garner et al., 2009), it is a prescription drug within the United States, is more expensive than terbinafine, and is more complicated to prepare if using the protocol of Nichols and Lamirande (2000). Chloramphenicol, an antibiotic recently shown to clear Bd from infected frog larvae (Bishop, 2009), has been implicated as a cause of fatal human aplastic anemia (Wallerstein et al., 1969), and thus, is to be used with caution. Therefore, with respect to cost, efficacy, and ease of use, TBF appears to be a highly satisfactory treatment for Bd infection of living amphibians and is readily available to professionals and home herpetoculturists alike.
Funding was provided by grants from the Oregon Community Foundation and Sunriver Nature Center and Observatory. We especially want to thank Scott Hendricks for assistance in procuring essential supplies and Steven Busch and Janet Emery for assistance in obtaining animals and testing treatment solutions as well as for their continued advice and insight.
APPENDIX 1: SPECIES TESTED FOR TOLERANCE TO TERBINAFINE HCL IN ALCOHOL
Black-eyed litter frog (Leptobrachium nigrops); black-webbed tree frog (Rhacophorus reinwardtii); bullfrog (Lithobates [Rana] catesbeiana); California tiger salamander (Ambystoma californiense); Cranwell's horned frog (Ceratophrys cranwelli); eastern banjo frog (Limnodynastes dumerili); false tomato frog (Dyscophus guineti); foothill yellow-legged frog (Rana boylii); green tree frog (Litoria caerulea); Indonesian spotted frog (Rana signata); Malayan horned frog (Megophrys nasuta); Malayan flying frog (Rhacophorus prominanus); spotted litter frog (Leptobrachium hendricksonii); Vietnamese blue tree frog (Polypedates dennysii); western toad (Bufo [Anaxyrus] boreas).