Providing evidence to improve practice

Action: Use antifungal treatment to reduce chytridiomycosis infection Amphibian Conservation

Key messages

  • Twelve of 16 studies (including four randomized, replicated, controlled studies) in Europe, Australia, Tasmania, Japan and the USA found that antifungal treatment cured or increased survival of amphibians with chytridiomycosis. Four studies found that treatments did not cure chytridiomycosis, but did reduce infection levels or had mixed results.
  • Six of the eight studies (including two randomized, replicated, controlled studies) in Japan, Tasmania, the UK and USA testing treatment with itraconazole found that it was effective at curing amphibians of chytridiomycosis. One study found that it reduced infection levels and one found mixed effects.
  • Six studies found that specific fungicides caused death or other negative side effects in amphibians.

 

Supporting evidence from individual studies

1 

A replicated, controlled study of captive amphibians in the USA (Groff et al. 1991) found that benzalkonium chloride was more effective at reducing chytrid infection (misdiagnosed as Basidiobolus ranarum (Berger, Speare, Pessier, Voyles & Skerratt 2010)) than copper sulphate or formalin-malachite green in dwarf African clawed frogs Hymenochirus curtipes. Mortality at day 24 was lower for 2 mg/l benzalkonium chloride (10%), compared to 4 mg/l benzalkonium chloride (16%), 1 mg/l copper sulphate (30%) and formalin (10 mg/l)-malachite green (0.8 mg/l; 25%). In the control group 74% died. Frogs treated with 2 mg/l benzalkonium chloride that survived had only mild infections compared to moderate to severe infections following the other two treatments. A group of 135 frogs from an infected population was bathed in each treatment. Frogs were bathed for 30 minutes on alternate days over six days, this was repeated after eight days. There was an untreated control group of 130 frogs. Five frogs from each group were examined for infection before treatment and on days 1, 3, 5, 10 and 15 after treatment had started. The study ended after 24 days.

 

2 

A replicated, controlled study in a laboratory (Nichols & Lamirande 2001) found that experimentally infected blue-and-yellow poison dart frogs Dendrobates tinctorius treated with miconazole or itraconazole were cured of chytridiomycosis. However, frogs were intolerant to miconazole (possibly due to ethyl alcohol in the solution). Juveniles were experimentally infected with the chytrid fungus. Once excessive skin shedding had started, frogs were treated with miconazole (0.01% solution) or itraconazole (0.1% suspension). Frogs were bathed in the treatments daily for five minutes for eight or 11 days respectively. Controls were untreated. Frogs were then killed humanely and examined.

 

3 

A replicated study of captive amphibians at the University of California, Berkeley, USA (Parker et al. 2002) found that western clawed frog Xenopus tropicalis treated with formalin-malachite green solution were cured of chytridiomycosis. Five frogs died within the first 48 hours of treatment. However, following the last treatment, all 10 surviving frogs gradually improved in health. The four examined at three weeks, one and two months showed no signs of infection and the remaining six frogs had regained normal body weight within four months. Fifteen naturally infected frogs were treated four times with formalin-malachite green solution (25 parts per million formalin and 0.10 mg/L malachite green) at a dilution of 0.007 ml/L of tank water for 24 hours every second day. Following treatment, four were selected at random and killed humanely at either three weeks, one month or two months for examination for infection.

 

4 

A replicated, controlled study in 2004 at the University of Alexandria, Egypt (Essawya et al. 2005) found that when fluconazole was swallowed by square-marked toads Bufo regularis there were significant changes in blood cells, similar to the effects of a carcinogen. White blood cell structure changed in 60% of the toads force-fed with fluconazole and 80% fed with a carcinogen. Controls showed no change. Most white blood cells showed changes such as nuclear abnormalities, vacuolated cytoplasm and reduced organelles. Red blood cells were anaemic with fragmented or degenerated nuclei, long cytoplasmic projections and vacuolated cytoplasm. Fifty adults were force-fed one of the following treatments for 20 weeks: fluconazole daily at a therapeutic dose level (0.26 mg in 0.5 ml saline), a carcinogenic chemical 7,12-dimethylbenz(a) anthracene (0.5 mg in 0.2 ml olive oil) twice/week, a control of 0.2 ml of olive oil or of 0.5 ml saline. Blood samples were obtained from the heart and examined after 20 weeks.

 

5 

A before-and-after study of an established collection of amphibians in Cheshire, UK (Forzán, Gunn & Scott 2008) found that frogs, axolotls Ambystoma mexicanum and Kaup’s caecilians Potymotyphlus kaupii treated with itraconazole were cured of chytridiomycosis. Approximately 20 individuals had died before treatment (following introduction of new individuals), but once treated there were no further cases of chytridiomycosis for 60 days. The collection was therefore considered disease free. Amphibians were kept in clear plastic boxes at 19–23°C in quarantine (with strict sterilization protocols). Frogs (mainly poison frogs Dendrobates, Epipedobates and Phyllobates spp.) were bathed or soaked daily in itraconazole (10 mg/ml) for five minutes over 11 days. Axolotls and caecilians were treated with itraconazole directly in their tank water (concentration 0.01%) for 30 minutes every five days for four treatments. Following treatment, itraconazole was removed from tanks by filtering.

 

6 

A replicated, controlled study of captive amphibians in Melbourne, Australia (Berger et al. 2009) found that although treatment with benzalkonium chloride or fluconazole resulted in increased survival times for juvenile green tree frogs Litoria caerulea, mortality rate was still 100%. All treated and untreated frogs died and all uninfected frogs survived. Treatments significantly increased survival time (benzalkonium chloride: 43–44 days, range 21–67; fluconazole: 44 days, range 29–76) compared to untreated frogs (38 days, range 30–67). Time until death did not differ significantly between treatments. Eighteen experimentally infected frogs were sprayed twice a day and kept in a solution with benzalkonium chloride at 1 mg/L and 18 with fluconazole at 25 mg/L. Half were treated for three days and half for seven days. Fourteen were untreated.

 

7 

A randomized, replicated, controlled study in England, UK (Garner et al. 2009) found that treatment with itraconazole cured all captive Mallorcan midwife toad Alytes muletensis tadpoles of chytridiomycosis, but caused depigmentation. All treated tadpoles tested negative for chytrid infection. However, tadpoles showed significant depigmentation in all treatments and some controls. Fifteen of 17 infected control tadpoles tested positive for infection over 21 days. Tadpoles were infected over two weeks then randomly assigned to treatments. Nine treatment groups of six tadpoles were treated with itraconazole baths of 0.5, 1.0 or 1.5 mg/L over 7, 14 or 21 days. Tadpoles were killed humanely one week later. Three control groups of 4–5 infected tadpoles were euthanized at 14, 21 or 28 days post-treatment to test for infection.

 

8 

A review in 2010 describing a replicated controlled study (Berger et al. 2010) found that treatment with benzalkonium chloride, fluconazole or methylene blue did not cure great barred frog Mixophyes fasciolatus tadpoles of chytridiomycosis. Although they did not cure infections, benzalkonium chloride and fluconazole reduced infection levels. However, at concentrations above 1 mg/L (2–10 mg/L) benzalkonium chloride caused death of tadpoles (over 29%). Methylene blue at concentrations of 12–24 mg/L also caused high mortality. Fifty-six tadpoles were bathed daily in benzalkonium chloride (1 mg/L; 3 hrs) for three days, repeated five days later, or in fluconazole (7 mg/L; 6 hrs) for seven days, or methylene blue (3 or 6 mg/L) for three days. There were 57 controls. Frogs were tested 18 days after treatment. Other studies included in this review have been summarized individually.

 

9 

A replicated, controlled study of six amphibian species naturally infected with chytridiomycosis in the USA (Bowerman et al. 2010) found that treatment with terbinafine hydrochloride in ethanol was effective at curing infection in all animals. All bullfrogs Rana catesbeiana, California tiger salamanders Ambystoma californiense, foothills yellow-legged frogs Rana boylii, black-eyed litter frogs Leptobrachium nigrops, Malaysian horned frogs Megophrys nasuta and Cranwell’s horned frogs Ceratophrys cranwelli treated with 0.01% or 0.005% solutions tested negative for chytrid after 3–4 weeks. However, those treated with 0.0005% solution and all control animals remained infected. There were no adverse effects from daily exposure to solution up to 0.01% for up to 15 minutes over 10 days. Amphibians were tested for chytrid before and after treatment. Wild-caught bullfrogs were randomly assigned to four treatments comprising a five minute bath in terbinafine HCl in ethanol: at 0.01% for five consecutive days (n = 14), at 0.005% for six treatments over 10 days (n = 18), as the previous treatment but kept in a 0.0005% solution between treatments, and a control group. Six or seven individuals of the five other (captive or wild caught) species received five minute baths on five consecutive days of: 0.005%, 0.0005% or distilled water.

 

10 

A before-and-after study in 2009–2010 of a pond in Mallorca (Lubick 2010) found that treating resident Mallorcan midwife toads Alytes muletensis with itraconazole and drying out the pond reduced the prevalence but did not eradicate chytridiomycosis. All samples from tadpoles came back positive for the chytrid fungus the spring after treatment and pond drying. However, the number of spores detected on each swab was lower than the previous year, suggesting a lower level of infection. Healthy-looking toads were seen breeding in the pond following treatment. Over 2,000 toad tadpoles were removed from the pond in March–August 2009. They were taken to a laboratory and completed a week-long treatment of daily five minute baths in itraconazole. Tadpoles were held in captivity for up to seven months. The pond was emptied and left to dry over the summer. Once the pond refilled in autumn, tadpoles were released. The following spring tadpoles were swabbed to test for chytridiomycosis.

 

11 

A replicated, controlled study of captive amphibians in Europe (Martel et al. 2011) found that Iberian midwife toads Alytes cisternasii and poison dart frogs (Dendrobatidae) sprayed with voriconazole were cured of chytridiomicosis. All five infected poison dart frogs treated were cured. Infection was eliminated from all but one midwife toadlet sprayed with voriconazole at 1.3 mg/L, but only four of seven sprayed at 0.13 mg/L. The one toad treated with 1.3 mg/L that was not cured was sprayed five (rather than one) months after infection. All toadlets housed on tissue soaked in voriconazole remained infected. No toxic side effects were seen. One week after experimental infection with the chytrid fungus, 14 toadlets were sprayed daily with voriconazole (1.3 or 0.13 mg/L water) and five were kept on paper towels soaked in voriconazole (1.3 mg/L) for seven days. Six animals were controls. Five months after experimental infection a further 20 toadlets were sprayed with voriconazole (1.3 mg/L) for 7 days. Animals were tested weekly for infection. A colony of 52 poison dart frogs, five positive for chytridiomycosis, was sprayed daily with voriconazole (1.3 mg/L) for seven days. Frogs containers were sterilized by heating to 45°C for three days.

 

12 

A randomized, replicated, controlled study in 2011 of captive amphibians in the USA (Brannelly, Richards-Zawacki & Pessier 2012) found that Australian green tree frogs Litoria caerulea and coastal-plain toads Incilius nebulifer treated with itraconazole were cured of chytridiomycosis. Itraconazole at 0.01, 0.005 and 0.003 but not 0.001% cured infection. Survival was highest with 0.003% itraconazole. However, itraconazole caused death, loss of appetite, lethargy and skin discolouration, particularly at 0.01 and 0.005%. Survival did not differ between infected animals treated for six or 11 days with 0.003% or six days with 0.005% itraconazole and untreated animals. However, treatment with all other concentrations for 11 days resulted in reduced survival (0.01%: 66–100% mortality) compared to infected untreated animals. Nine separately housed green froglets and 9–17 communally housed toadlets were randomly assigned to each treatment: infection with chytrid, infection and itraconazole baths for 5 minutes for six or 11 days and an uninfected control. Skin swabs were taken for four weeks after treatment.

 

13 

A randomized, replicated, controlled study in 2010 of captive amphibians in Tennessee, USA (Hanlon, Kerby & Parris 2012) found that southern leopard frog tadpoles Lithobates sphenocephalus treated with thiophanate-methyl (TM) were cured of chytridiomycosis. All treated tadpoles tested negative for the infection at day 60, as did controls. All infected untreated tadpoles tested positive. By day 60, treated tadpoles were significantly heavier (TM + chytrid: 2.0; TM: 1.1; controls: 0.8–0.9 g) and longer (TM + chytrid: 22; TM: 18; controls: 17 mm). The same was true for metamorphosis mass (TM + chytrid: 1.1; TM: 0.9; controls: 0.5–0.7 g) and length (TM + chytrid: 23; TM: 22; controls: 18–19 mm). Ten tadpoles were randomly assigned to each treatment: thiophanate-methyl treatment of chytrid infected tadpoles, thiophanate-methyl treatment alone, chytrid infection alone and an uninfected control group. Tadpoles were bathed in thiophanate-methyl (0.6 mg/L) and water was changed every three days. Animals were measured and tested for infection at day 60 and measured on tail resorption.

 

14 

A replicated study in 2009 of captive amphibians in the USA (Jones et al. 2012) found that reduced-dose itraconazole was an effective treatment for natural infections of chytridiomycosis in Wyoming toads Anaxyrus baxteri, White’s tree frogs Litoria caerulea and African bullfrogs Pyxicephalus adspersus. Although 15 infected toads and one tree frog died during treatment, all animals surviving at the end of treatment tested negative for chytrid for five or 13 months. Before treatment, 70% of Wyoming toads, 45% of tree frogs and both bullfrogs tested positive for chytridiomycosis. Eighty Wyoming toads were bathed for 5 minutes with itraconazole at 100 mg/L for three days, 5 mg/L for six days and then 50 mg/lLon the last day. Eleven tree frogs and two African bullfrogs were treated daily with itraconazole at 50 mg/L for 5 minutes over 10 days. Toads were tested for chytrid monthly for five months after treatment and frogs every two weeks for two months and once at 13 months. Animals were not rinsed following baths.

 

 

15 

A replicated, controlled study in a laboratory in Australia (Stockwell, Clulow & Mahony 2012) found that exposing Peron’s tree frogs Litoria peronii to low concentrations of sea salt significantly lowered chytrid infection loads and increased survival rates. Infection loads were significantly lower with concentrations of 1–4 parts per trillion (ppt) of sodium chloride compared to 5 ppt or no salt. Frogs exposed to 3 ppt had significantly higher survival rates (100%) than at lower (1 ppt: 37; 2 ppt: 63%) or higher concentrations (4 ppt: 72%; 5 ppt: 54%) or with no salt (37%). Survival and weight gains were not reduced with salt. Concentrations of 0–5 ppt sodium chloride did not reduce chytrid fungus survival, but 4–5 ppt significantly reduced growth (10–12 vs 18–22 developing zoospores) and motility (3–7 vs 27%) compared to controls. Frogs were housed with water containing: 0, 1, 2, 3, 4 or 5 ppt sea salt. Chytrid in solution (1 mL) was added to half of each salt treatment (11 replicates/treatment). After 30 days body mass was measured and at 120 days swabs were tested for chytrid infection. Chytrid culture (100 ml) was added to 10 replicates of 0, 1, 2, 3, 4 or 5 ppt sea salt and incubated at 22°C for 11 days. Fungus survival, growth and motility were assessed.

 

16 

A small replicated study in Japan (Une et al. 2012) found that Japanese giant salamanders Andrias japonicus treated with itraconazole were cured of chytridiomycosis. By day five of treatment all four previously infected salamanders tested negative for the disease. Tests remained negative for two weeks. Four naturally infected salamanders were bathed daily in 0.01% itraconazole for 5 minutes over 10 days. Animals were tested for chytrid before treatment, on treatment days five and 10 and seven and 14 days after treatment.

 

17 

Randomized, replicated, controlled studies in 2007–2009 of amphibians with chytridiomycosis in the USA and Tasmania (Woodhams et al. 2012) found that treatment with itraconazole cured northern leopard frogs Lithobates pipiens, did not increase survival of mountain yellow-legged frogs Rana muscosa and was highly toxic to striped marsh frog Limnodynastes peronii metamorphs. All four treated leopard frogs were cured, although one control frog died with signs of toxicity. Eight treated marsh frogs died by the third day of treatment. Although treatment did not increase survival of yellow-legged frogs (treated: 30%; controls: 39%), it reduced weight loss (0.2 vs 0.4 g/week) and cleared infection in surviving frogs. Frogs were randomly assigned to treatments. Ten wild-caught naturally infected yellow-legged frogs, four infected leopard frogs and eight wild-caught naturally infected marsh frogs were bathed with itraconazole (100 mg/L) for 5 minutes daily and then rinsed for 11, five or three days respectively. There were 13 control yellow-legged frogs, seven marsh frogs (bathed in water) and eight leopard frogs. Yellow-legged frogs were tested for infection at seven and 13 days after treatment and leopard frogs before and 17 days after treatment.

 

18 

A randomized, replicated, controlled study in 2010 in Switzerland (Woodhams et al. 2012) found that common midwife toad Alytes obstetricans tadpoles treated with three commercial antifungal treatments were not cured of chytridiomicosis. All but one tadpole treated with PIP Pond Plus and all those treated with Steriplant N remained infected. Only three of 18 treated with Mandipropamid (at 0.1, 1.4 and 1.6 mg/L) were cured. Wild-caught tadpoles were randomly assigned to treatments. Twenty-eight were treated daily with PIP Pond Plus (probiotic bacteria, enzymes and isopropanol) in doses of 0, 25, 50 or 100 μg/ml added to their water for seven days. Twenty-eight were treated with Steriplant N (water and 0.04% oxidants) on day 0 (control), one (5 parts per million), two (10 parts per million) or three (15 parts per million). Twenty-one tadpoles were treated with Mandipropamid (phenylglycinamides and mandelamides) at 18 different doses from 0.01 to 4 mg/L (in acetone), with three controls. Tadpoles were swabbed and tested a week after treatment.

 

Referenced papers

Please cite as:

Smith, R.K., Meredith, H. & Sutherland, W.J. (2017) Amphibian Conservation. Pages 9-65 in: W.J. Sutherland, L.V. Dicks, N. Ockendon & R.K. Smith (eds) What Works in Conservation 2017. Open Book Publishers, Cambridge, UK.