Woodhams DC et al. (2012), Treatment of amphibians infected with chytrid f...

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Dis Aquat Organ 2012 Feb 17;981:11-25. doi: 10.3354/dao02429.

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Treatment of amphibians infected with chytrid fungus: learning from failed trials with itraconazole, antimicrobial peptides, bacteria, and heat therapy.

Woodhams DC , Geiger CC , Reinert LK , Rollins-Smith LA , Lam B , Harris RN , Briggs CJ , Vredenburg VT , Voyles J .

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Amphibian conservation goals depend on effective disease-treatment protocols. Desirable protocols are species, life stage, and context specific, but currently few treatment options exist for amphibians infected with the chytrid fungus Batrachochytrium dendrobatidis (Bd). Treatment options, at present, include antifungal drugs and heat therapy, but risks of toxicity and side-effects make these options untenable in some cases. Here, we report on the comparison of several novel treatments with a more generally accepted antifungal treatment in experimental scientific trials to treat Bd-infected frogs including Alytes obstetricans tadpoles and metamorphs, Bufo bufo and Limnodynastes peronii metamorphs, and Lithobates pipiens and Rana muscosa adults. The experimental treatments included commercial antifungal products (itraconazole, mandipropamid, steriplantN, and PIP Pond Plus), antimicrobial skin peptides from the Bd-resistant Pelophylax esculentus, microbial treatments (Pedobacter cryoconitis), and heat therapy (35°C for 24 h). None of the new experimental treatments were considered successful in terms of improving survival; however, these results may advance future research by indicating the limits and potential of the various protocols. Caution in the use of itraconazole is warranted because of observed toxicity in metamorphic and adult frogs, even at low concentrations. Results suggest that rather than focusing on a single cure-all, diverse lines of research may provide multiple options for treating Bd infection in amphibians. Learning from 'failed treatments' is essential for the timely achievement of conservation goals and one of the primary aims for a publicly accessible treatment database under development.

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Genes referenced: SART3

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	Fig. 1. Bufo bufo. Newly metamorphosed B. bufo treated with antimicrobial peptides (AMPs) either before exposure to Batrachochytrium dendrobatidis (Bd) or after experimental infection. Peptides were harvested non-destructively from the skin of the chytridiomycosis-resistant species Pelo phylax esculentus (see ‘Materials and methods’). (A) Kaplan-Meier survival curve of toads in each treatment throughout the experiment (log-rank test on censored survival data: χ2 = 15.179, df = 2, p = 0.004). (B) Mean (±SE) change in weight throughout the experiment in all frogs weighed at least twice (ANOVA: F = 3.632, df = 4, p = 0.012). Identical letters above bars indicate homogeneous subsets (Tukey test)
	Fig. 2. Rana muscosa. Results of treating adult R. muscosa naturally infected with Batrachochytrium dendrobatidis (Bd) with the antifungal itraconazole or the probiotic bacterium Pedobacter cryoconitis. (A) Kaplan-Meier survival curve of frogs after treatments (log-rank test on censored survival data: χ2 = 1.494, df = 2, p = 0.474). (B) Intensity of infection with Bd throughout the experiment. (C) Mean (±SE) change in weight throughout the experiment in all frogs weighed at least twice (ANOVA: F = 3.455, df = 2, p = 0.041). Identical letters below bars indicate homogeneous subsets (Tukey test)
	Fig. 3. Rana muscosa. Skin pH of R. muscosa infected with Batrachochytrium dendrobatidis (Bd). (A) Mean (±SE) pH of ventral and dorsal skin surfaces. (B) Infection intensity shows a slight but significant correlation with ventral skin pH (light gray line) but not dorsal skin pH (dark gray line)
	Fig. 4. Limnodynastes peronii. Striped march frogs that had naturally acquired infections with Batrachochytrium dendrobatidis (Bd) as tadpoles remained infected upon metamorphosis. (A) A histological section of the skin of an infected L. peronii stained with hematoxalin and eosin, showing clear hyperkeratosis of the epidermis and typical Bd zoosporangia. (B) A newly metamorphosed L. peronii with clinical signs of chytridiomycosis
	Fig. 5. Alytes obstericans. The effects of 3 commercial antifungal treatments applied to larval A. obstericans on Batrachochytrium dendrobatidis (Bd) infection intensities (Bd load = log zoospore equivalents). (A) No significant effect of the PIP Pond Plus treatment on Bd load (ANOVA: F = 0.7148, df = 24, p = 0.5528). (B) No significant effect of the Steriplant N treatment on Bd load (ANOVA: F = 0.3056, df = 24, p = 0.8210). Box plots show the medium value (line), 25 and 75% quantiles (box), 5 and 95% quantiles (whiskers), and outliers (s). (C) No significant effect of the mandipropamid treatment on the Bd load (linear regression: p = 0.0638). Five animals cleared infection, including 2 in the control group. The tadpole with the highest possible water-soluble dosage (4 mg l−1) was still infected with Bd. (D) Thresholds of Bd zoospore intensity affect the interpretation of treatment results. All individuals with zoospore counts smaller than the threshold are considered uninfected. When no threshold is employed, almost all frogs regardless of treatment are considered infected. At higher thresholds, treatment seems to have a larger effect. Here, Treatments P100, P50, and P25 represent different dosages of the agent PIP Pond Plus (see ‘Treating Alytes obstericans with commercial antifungals’)