Chytridiomycosis in captive amphibians: Bd, Bsal, and the treatment pathway

Bottom line

Chytridiomycosis in a captive amphibian is treatable, and the first decision is which pathogen you have. For Batrachochytrium dendrobatidis (Bd), Merck's professional guidance gives itraconazole as a "0.01% bath for 5 min/day for 10-11 days" [1] — but that concentration for 11 days caused 100% mortality in recently metamorphosed Incilius nebulifer, which is why reduced-concentration protocols exist [2]. A caudate changes the plan: Batrachochytrium salamandrivorans (Bsal) is a separate species [3] against which Bd protocols fail therapeutically [4]. Diagnose by qPCR on a skin swab and treat a single negative as inconclusive.

The two pathogens are not interchangeable

The pathogen was identified in 1998 in sick and dead anurans from montane rain forests in Queensland and Panama during mass mortality events, described then only as "a chytridiomycete fungus (Chytridiomycota; Chytridiales)" [5]; it was named Batrachochytrium dendrobatidis the following year by Longcore, Pessier and Nichols. Bsal was described in 2013 from collapsing fire salamander (Salamandra salamandra) populations in the Netherlands; it sits in a distinct clade alongside Bd, causes erosive skin disease and rapid mortality in experimentally infected fire salamanders, and tolerates lower temperatures [3].

A 2014 screen of more than 5,000 amphibians concluded that Bsal is restricted to, but highly pathogenic for, salamanders and newts (Urodela) [6]. Later work widened that: across 35 North American species from 10 families, "Bsal caused infection in 74% and mortality in 35% of species tested," and "Both salamanders and frogs became infected and developed Bsal chytridiomycosis" [7]. Caudates die of Bsal, but anurans sharing a collection are not epidemiologically irrelevant. Both organisms infect keratinocytes in adult skin and tadpole mouthparts [1].

Pathophysiology: an osmoregulatory death

The infection is confined to keratinised epidermis, and the animal dies of what that does to skin function. In clinically diseased green tree frogs (Litoria caerulea), "electrolyte transport across the epidermis was inhibited by >50%, plasma sodium and potassium concentrations were respectively reduced by approximately 20% and approximately 50%, and asystolic cardiac arrest resulted in death" [8]. The organism "infects keratinized epithelial cells of amphibian skin and causes disease and mortality by interfering with important skin functions, especially electrolyte balance" [9].

An antifungal bath alone is therefore not a plan for a collapsing patient: "Supportive treatment of the clinically ill patient is necessary for success, with particular attention to electrolyte therapy" [9].

Clinical presentation

Signs are nonspecific and arrive late. Merck's professional text lists "brown to red discoloration of the skin, excessive shedding and mucus production, muscle incoordination, and acute death," and in tadpoles "depigmentation of toothrows or acute death" [1]. Expect also lethargy, anorexia, abnormal postures such as limbs held away from the body or the ventrum lifted off the substrate, and a slowed or absent righting reflex near death. None of that separates chytridiomycosis from ranavirus, bacterial dermatosepticaemia or a water-quality problem, so test rather than assume.

Diagnosis: qPCR on a skin swab

Real-time PCR on swabs of the integument, or on pieces of skin, is diagnostic [1]. The assay "can accurately detect and quantify one zoospore in a diagnostic sample," and replaced toe-clip histology, "invasive and insensitive particularly at early stages of infection when treatment may be possible" [10].

Technique determines the result. Sample where sporangia concentrate: pelvic (drink) patch, ventral abdomen, ventral thighs, hind-foot webbing and the ventral surfaces of the digits, with firm repeated strokes on each site. Restrain ventrum-up in a clean single-use glove, change gloves between animals, and do not rinse first. In larvae the keratinised mouthparts are the infected tissue [1].

A negative swab is weak evidence in a low-burden animal. Comparing swabs with filtered water from the same Bombina orientalis, "Many subjects, despite being diagnosed as Bd-negative by conventional methods, released Bd zoospores into collection containers and thus must be considered infected," and loads from filtered water were "at least 1000 times higher than those estimated from swabs" [11]. A single negative rules out a heavy burden only; serial testing clears an animal. Skin histopathology is useful post-mortem but is the wrong tool for the early, treatable, live case.

Treatment: itraconazole baths for Bd

Itraconazole bathing is the mainstay, and the concentration is the most dangerous number here: the same figure is well tolerated in one species and lethal in another. Note first that 0.01% w/v is 100 mg/L — the percentage in Merck's guidance and the mg/L figure used in most of the literature and in compounding requests are one concentration, not two options. The ladder converts as 0.01% = 100 mg/L, 0.005% = 50 mg/L, 0.0025% = 25 mg/L, 0.002% = 20 mg/L, 0.0005% = 5 mg/L. Each protocol below is quoted with its population.

Protocol as published	Same concentration, both units	Population	Source
Itraconazole "0.01% bath for 5 min/day for 10-11 days"	0.01% = 100 mg/L — see the caveat on the row below, which is the same concentration	Captive amphibians, general professional guidance	[1]
Itraconazole baths at "100 mg l-1 is commonly used in captive amphibians, but side effects are observed in some amphibian species and life stages"	100 mg/L = 0.01% — the identical concentration to the row above, and to the 100%-mortality arm below	Captive amphibians	[12]
"0.0025% itraconazole for 11 days, and both 0.005% and 0.0025% itraconazole for six days were all found to be effective," using 5 min baths	0.0025% = 25 mg/L; 0.005% = 50 mg/L	Incilius nebulifer, recent metamorphs	[2]
"0.01% itraconazole treatment for 11 days caused 100% mortality in I. nebulifer, and half that concentration (0.005% itraconazole for 11 days) caused 60% mortality"	0.01% = 100 mg/L; 0.005% = 50 mg/L	Incilius nebulifer, recent metamorphs	[2]
"Tadpole treatment has been successful using a much lower concentration (0.0005%) of itraconazole, but was associated with depigmentation"	0.0005% = 5 mg/L	Tadpoles	[2]
Itraconazole (Itrafungol) "0.002% to 0.0025% solution ... for 5 min each day for 10 d"	0.002-0.0025% = 20-25 mg/L	Ambystoma mexicanum and Taricha granulosa adults	[13]
Terbinafine "0.01% bath, buffered using bicarbonate to a pH of 7.2-7.4, for 5 min/day for 5 days"	0.01% = 100 mg/L, but of terbinafine, not itraconazole	Captive amphibians, general professional guidance	[1]
"raising environmental temperatures for captive populations to >23°C"	not a concentration	Captive populations, Bd	[1]

The low-concentration protocols exist because the original concentration harmed animals: that trial cut "the treatment concentration from 0.01-0.0025% and ... the treatment duration from 11-6 days of 5 min baths," reporting cure "with fewer side effects and less treatment-associated mortality" [2]. So the 100 mg/L a clinician is most likely to order compounded is 0.01%, the concentration at which "side effects are observed in some amphibian species and life stages" [12] and the one that killed every recent metamorph in that trial [2]. Tadpoles and recent metamorphs have the least margin. All of this is extra-label use.

In the axolotl and rough-skinned newt colony treated on that schedule (Itrafungol, an oral solution used as a bath), 2 of 46 axolotls died during the 10 days and all 48 newts survived, though some newts "presented with lethargy and closed eyes beginning on the 3rd to 5th day of treatment"; PCR at 7, 28 and 180 days was negative in both species [13].

Treatment when the organism is Bsal

Bd protocols fail against Bsal, partly because antimycotic minimum inhibitory concentrations differ between the two organisms [4]. Both published approaches are temperature-dependent. Heat alone can clear it: "Colonization of salamanders by B. salamandrivorans occurred at 15°C and 20°C but not at 25°C," and "Exposing B. salamandrivorans infected salamanders to 25°C for 10 days resulted in complete clearance of infection" in fire salamanders [14]. That suits a heat-tolerant caudate, not a cool-adapted species.

The drug route is combination therapy, and ambient temperature is part of the prescription. Voriconazole or itraconazole alone, or with polymyxin E, at 15 °C for 10 days "decreased fungal loads but did not clear Bsal infections. However, topical treatment of Bsal infected animals with a combination of polymyxin E (2000 IU/ml) and voriconazole (12.5 μg/ml) at an ambient temperature of 20 °C resulted in clearance of Bsal infections." Treatment ran twice a day for 10 days and cleared all 12 fire salamanders in a field outbreak [4].

Supportive care and prognosis

Electrolyte therapy is what the clinical review identifies as necessary in the ill patient [9]: shallow amphibian-appropriate electrolyte baths rather than plain dechlorinated water, corrected temperature and water quality, secondary-infection cover, and minimal handling. Caught before collapse the prognosis is good; an animal already incoordinate or unable to right itself is guarded to poor.

Biosecurity, quarantine and reporting

Reporting obligations are jurisdiction-dependent. These fungi are "reportable to the World Organisation for Animal Health (OIE)" [1], and WOAH lists chytridiomycosis (B. dendrobatidis) [15], but whether a positive obliges you to notify a competent authority varies by country and may differ between Bd and Bsal.

Trade rules also vary. The United States listed 201 salamander species as injurious wildlife under the Lacey Act, effective January 28, 2016, restricting importation and interstate transport of live and dead animals including parts, while "Owners of any of the animals listed as injurious will be allowed to keep them under this rule" [16]. That is one country's rule, not a universal one.

The private trade is a real reservoir: contact-tracing from Bsal in UK captive amphibians identified 16 linked collections across Western Europe; animals from 11 were tested, Bsal was detected in seven, and deaths occurred in five [17]. Quarantine incoming amphibians separately with dedicated equipment, test on arrival and again before release, and treat a shipment as one unit.

For equipment, complete kill of Bd zoosporangia in culture followed 20-second exposure to 70% ethanol, 1 mg Virkon ml-1, or 1 mg benzalkonium chloride ml-1; bleach "was effective at concentrations of 1% sodium hypochlorite and above"; heat gave 100% mortality at "4 h at 37 degrees C, 30 min at 47 degrees C and 5 min at 60 degrees C"; and UV light at 1000 mW m-2 at 254 nm "was ineffective at killing B. dendrobatidis in culture" [18].

Never release or rehome an exposed or treated animal on clinical recovery alone; clearance is a serial-PCR finding.

Frequently Asked Questions

Can I use the 0.01% (100 mg/L) itraconazole bath protocol in any amphibian? No, and note first that 0.01% w/v and 100 mg/L are the same concentration, not two protocols. Merck's professional guidance gives itraconazole as a 0.01% bath for 5 min/day for 10-11 days [1]; Jones and colleagues describe baths at 100 mg/L as commonly used but with side effects in some amphibian species and life stages [12]; and in Brannelly's treatment trial that same 0.01% for 11 days caused 100% mortality in recently metamorphosed Incilius nebulifer, while 0.005% (50 mg/L) for 11 days caused 60% mortality in the same animals [2]. Match the concentration to a protocol published in your patient's species and life stage.

My patient is a newt or salamander — does that change anything? Yes. Bsal is a distinct species described by Martel and colleagues in 2013 from fire salamanders [3], and Blooi and colleagues showed that Bd protocols fail therapeutically against it [4]. Request a Bsal assay, not only a Bd assay, whenever the patient is a caudate.

Does a negative qPCR swab mean the animal is free of Bd? No. In Shin and colleagues' comparison, many subjects diagnosed as Bd-negative by swabbing released Bd zoospores into their containers and had to be considered infected, and loads from filtered water were at least 1000 times higher than swab estimates [11]. Use serial testing before clearing an animal or declaring cure.

Why does an animal with a superficial skin infection die? Because amphibian skin is an osmoregulatory organ. In Voyles and colleagues' clinically diseased green tree frogs, electrolyte transport across the epidermis was inhibited by more than 50%, plasma sodium and potassium fell by approximately 20% and approximately 50%, and asystolic cardiac arrest caused death [8] — which is why Baitchman and Pessier call electrolyte therapy necessary in the ill patient [9].

Is raising the temperature enough on its own? For Bsal in a heat-tolerant caudate it can be: in Blooi and colleagues' work, exposing Bsal-infected salamanders to 25°C for 10 days produced complete clearance, and colonisation did not occur at 25°C at all [14]. For Bd, Merck's professional guidance describes raising captive environmental temperatures to above 23°C [1]. Check thermal tolerance first — several cool-adapted caudates are stressed at 25°C.

Do I have to report a positive result? That depends on your jurisdiction, so confirm locally. Merck's professional text states these fungi are reportable to the World Organisation for Animal Health [1], and WOAH lists chytridiomycosis caused by B. dendrobatidis [15]. Trade rules differ too: the US Fish and Wildlife Service listed 201 salamander species as injurious wildlife under the Lacey Act effective January 28, 2016 [16].