SEPARC Information Sheet #18R

BATRACHOCHYTRIUM SALAMANDRIVORANS

By: Kenzie E. Pereira and Dr. J. Patrick W. Cusaac

Amphibian Die-offs:

The skin disease chytridiomycosis has been identified as a major driver of amphibian declines and extinctions worldwide (Berger et al., 2016) and is caused by chytrid fungal pathogens, Batrachochytrium dendrobatidis (Bd; see Information Sheet #2R) (Longcore et al., 1999) and Batrachochytrium salamandrivorans (Bsal; Martel et al., 2013). Unlike Bd, which emerged in the 1970’s and has been linked to catastrophic population declines and extinctions globally having had the greatest impact among anuran , Bsal was only recently described in 2013 following the mass die-off of fire (Salamandra salamandra) in Bunderbos, Netherlands (Martel et al., 2013). Bsal has since spread to other regions of Europe including Belgium, Germany, and the United Kingdom where it has been identified in both wild and captive populations (Cunningham et al., 2015; Martel et al., 2013; Sabino-Pinto et al., 2015; Spitzen-van der Sluijs et al., 2016). To date, Bsal has not been detected among North American amphibians (Bales et al., 2015; Glorioso et al., 2017; Klocke et al., 2017; Parrott et al., 2017). For the most recent data regarding the global spread of Bd and Bsal, please visit https://amphibiandisease.org/.

Though occurrence of Bsal appears to be limited to regions of East Asia, where it has not caused apparent disease (Laking et al., 2017; Martel et al., 2014), and areas of Europe, Bsal poses a significant threat to North American amphibian biodiversity, especially salamander species inhabiting the southeastern United States (U.S.; Yap et al., 2015, 2017). Further, an epidemiological model based on Bsal outbreaks among S. salamandra suggests that once introduced, Bsal can rapidly spread (estimated 11 kilometers per year) causing population crashes within weeks or months (Schmidt et al., 2017).

Figure 1. Mapping the threat of Bsal to North American salamanders. Salamander Bsal vulnerability model illustrating the high susceptibility of salamanders inhabiting the southeastern United States to Bsal. Black squares indicate major ports for salamander imports. Port 6 represents Mexico City (Yap et al. 2015).

Pathogen Characteristics:

Chytridiomycosis is caused by two chytrid taxa: Bd (Longcore et al., 1999) and Bsal (Martel et al., 2013). The life cycle of Bd is characterized by two stages: a motile zoospore stage (i.e., infective stage) and a sedentary zoosporangium stage (i.e., growth stage; Berger et al., 2005a). Bsal also has an infective stage and a growth stage, but unlike Bd, Bsal has two spore forms in the infective stage: a motile zoospore and an encysted spore containing a cell wall (Figure 2; (Stegen et al., 2017)). Spores of Bsal are released from discharge tubes of colonial (thalli containing multiple zoosporangia) or monocentric (single thallus forming a single zoosporangium) thalli (Figure 3). In vivo, mature zoosporangia of Bsal form germ tubes, and colonial thalli tend to be more abundant than observed in Bd cultures (Figure 3). Bsal also has lower thermal growth characteristics than Bd and experiences optimal growth between 10 ˚C and 15 ˚C. Death of Bsal in vitro occurs at temperatures equal to or above 25 ˚C (Martel et al., 2013). However, salamanders have been captured in Vietnam with detectable Bsal DNA at temperatures exceeding this hypothetical threshold, suggesting the thermal tolerance range of Bsal in vivo is wider than predicted. (Laking et al., 2017).

Figure 2. Transmission electron micrograph of spore forms of Bsal. (A) encysted spore contained within zoosporangium and free within the superficial epidermis. (B) motile zoospore. * = lipid globule; black arrow = cell wall; F = flagellum; M = mitochondrion; N = nucleus; R = ribosomal mass (Stegen et al., 2017).

Figure 3. Culture of Bsal in vitro grown on TGhL broth at 15 ˚C. (A) Colonial thalli indicated by black arrow and discharge tubes indicated by white arrow. (Scale bar, 100 µm). (B) Scanning electron micrograph of mature zoosporangium showing rhizoids (R), discharge tubes (D), and germ tube formation (Scale bar, 10 µm) (Martel et al., 2013).

Persistence of Bsal in environmental media varies by spore type. For example, encysted spores incubated in sterile pond water can be infective for up to 31 days post-release, and are more resistant to predation by micropredators (e.g., rotifer species) than zoospores (90 vs. 50 % survival after four hours, respectively; Stegen et al., 2017). This is likely due to differing ecologies of the spore types; zoospores move throughout the water column actively pursuing hosts with the aid of a single posterior flagellum, whereas encysted spores float at the air-water interface. Persistence in soil appears to be much lower, but still sufficient to result in infection of susceptible hosts up to 48 hours after spores were released onto soil (Stegen et al., 2017).

Transmission:

Bsal can be transmitted across salamander hosts via direct contact (Martel et al., 2014), as well as environmental media (e.g., water and soil containing Bsal spores; Stegen et al., 2017). New world salamanders including members of (e.g., Notophthalmus and Taricha) and Plethodontidae (e.g., Hydromantes) appeared to be highly susceptible to Bsal and experienced rapid mortality when exposed to pathogen concentrations of 5,000 zoospores/mL (Martel et al., 2014). Other species, such as the Japanese fire-bellied newt (Cynops pyrrhogaster), Blue-tailed fire-bellied newt (Cynops cyanurus), and Tam Dao salamander ( deloustali) may serve as reservoir hosts because these species were able to limit or clear infection by Bsal (Martel et al., 2014). Recent data suggest that the susceptible host range is broader than predicted by these initial experiments, and includes anuran hosts, which can maintain subclinical infections sufficient for transmission to more susceptible hosts (e.g., S. salamandra; Stegen et al., 2017). Additionally, encysted spores are highly adhesive and can adhere to scales and feet of avian vectors (e.g., geese; Stegen et al., 2017), which may translocate the pathogen across long distances to naïve systems.

Signs of Disease and Diagnostic Testing:

Salamanders suffering from Bsal-induced chytridiomycosis exhibit anorexic, apathetic, and ataxic behaviors (Martel et al., 2013). Unlike Bd-induced chytridiomycosis which is characterized by epidermal hyperkeratosis (i.e., increased thickness of the stratum corneum) and hyperplasia (Berger et al., 2005b), Bsal-induced chytridiomycosis can generally be discerned by the presence of multifocal skin erosions and deep ulcerations across the body (Martel et al., 2013) (Figure 4.A). Necrotic keratinocytes (i.e., epidermal cells) containing colonial or monocentric thalli are often found along the periphery of skin lesions (Figure 4.B and 4.C; Martel et al., 2013). In some highly susceptible species, epidermal necrosis is systemic, and in some cases thalli can penetrate through the epidermal layer into the underlying tissue (D. L. Miller, unpublished data).

Based on the infectivity experiments conducted by Martel et al. (2014), mortality of infected salamanders was frequent and occurred within 10 to 54 days post-inoculation (Martel et al. 2014, Supplementary Table S1), but infection is generally at detectable levels within days of exposure.

Figure 4. (A) Dorsal view of fire salamander (S. salamandra) exhibiting skin lesions caused by Bsal infection (Blooi et al. 2015); (B) Micrograph of the skin of a fire salamander that died from infection with Bsal (A) Immunohistochemical staining of 5 µm skin section. Intracellular colonial thalli abundant throughout the epidermis surrounding erosive skin lesions. (Scale bar, 20 µm). (B) Transmission electron micrograph of an intracellular colonial thallus of Bsal within a keratinocyte (Scale bar, 4 µm; Martel et al., 2013).

Diagnosis of Bsal can be achieved using immunohistochemical staining and/or molecular methodology. Blooi et al. (2013) provide a protocol for the rapid simultaneous detection of Bd and Bsal using Real-Time PCR (or quantitative PCR, qPCR), and Dillon et al. (2017) developed a chytrid-specific antibody which can be used to quantify Bd and Bsal using a lateral-flow assay. Because the antibody is not specific to either chytrid fungus, this method should be used in conjunction with qPCR. Diagnosis of clinical disease (i.e., chytridiomycosis) was previously defined as an infection load of 10,000 genetic (zoospore) equivalents per swab (Blooi et al., 2013; Kinney et al., 2011). However, chytridiomycosis can more definitively be diagnosed through a combination of positive qPCR and histological evidence of Bsal-induced epidermal necrosis (Thomas et al., 2018).

Factors Contributing to Emergence:

The first mass-mortality event associated with Bsal infection occurred in 2010 among a wild salamander population (S. salamandra) in the Netherlands (Martel et al., 2013). Bsal has since been linked to the mortality of captive salamanders in the United Kingdom (Cunningham et al., 2015) and Germany (Sabino-Pinto et al., 2015). Further, Bsal has spread to other regions of Europe and has been detected among wild populations of Fire salamander (S. salamandra) Alpine newt (Ichthyosaura alpestris), and Smooth newt (Lissotriton vulgaris) (Spitzen-van der Sluijs et al., 2016). Though population declines have been observed for European species in which Bsal has been detected, additional research is needed to establish a causal relationship (Spitzen-van der Sluijs et al., 2016).

It is hypothesized that Bsal originated in East Asia and has spread to naïve host populations through the international trade of Asian amphibians (Martel et al., 2014). In a robust study conducted by Martel et al. (2014), over 5,000 wild amphibian specimens from Asia, Africa, North America and Europe were screened for Bsal using qPCR. Positive test results for Bsal were found only in from East Asia (Thailand, Vietnam, and Japan), where apparent disease has not been observed, and European samples. Additional surveys revealed that Bsal is the predominant chytrid fungus among Vietnamese salamanders (genera: Tylototriton, Paramisotriton; (Laking et al., 2017) and is prevalent among those in southern China (genera: Cynops, Pachytriton, Paramisotriton, Tylototriton, and Andrias; (Yuan et al., 2018)). Combined, these data suggest that Bsal is widely spread throughout East Asia. In addition, molecular data suggest that Bsal has long been endemic to the area. The historical presence of Bsal within East Asian amphibian communities may explain why some species (e.g., Cynops pyrrhogaster, C. cyanurus, Paramesotriton deloustali) are able to tolerate or clear infections following experimental exposure to Bsal serving as potential reservoir hosts (Martel et al., 2014).

Commercial trade of Asian amphibians is the greatest factor contributing to the spread of Bsal and has been implicated in pathogen transmission to naïve salamander hosts (Martel et al. 2014). Over 750,000 Asian salamanders were imported into the United States during the years 2010 to 2014 (Yap et al. 2015). The majority of imported salamanders were of the genera Cynops and Paramesotriton (Yap et al. 2015). Previously, it was thought that Bsal was vectored primarily through the trade of Asian salamanders (Martel et al., 2014) resulting in the banning of salamander importation in the U.S. and Canada. However, recent data suggest that heavily traded anurans (e.g., Bombina spp.) may also contribute to the increased spread of Bsal (Nguyen et al., 2017).

Preventative Measures:

To date, Bsal has not been detected in the U.S. To minimize the risk of pathogen spillover from imported amphibians into native ecosystems, the U.S. Fish and Wildlife Service amended the Lacey Act prohibiting the importation of 201 salamander species (Yuan et al., 2018; for complete rule see: https://www.fws.gov/policy/library/2016/2016-00452.pdf). While this list includes Asian salamanders known to carry Bsal, recent evidence suggests that anurans including the Hubei fire-bellied toad (genus: Bombina), a genus prevalent in the U.S. pet trade (Herrel and van der Meijden, 2014), may also be an important vector for the spread of Bsal (Nguyen et al., 2017).

To reduce the risk of human-mediated pathogen transmission of Bsal to native amphibian populations, it is imperative that proper hygiene protocols are followed. Phillott et al. (2010) and Grant et al. (2017) provide a thorough review of hygiene protocols and biosafety procedures for field-based studies. Additional information on disinfection protocols for field equipment can be found in Information sheet #10R. Efficacy of chemical disinfectants in killing Bsal and sister species, Bd can vary (Van Rooij et al., 2017). Van Rooij et al. (2017) provide the concentrations and minimal exposure times required to kill Bsal zoospores and zoosporangia for a number of chemical disinfectants (Table 1). It is noteworthy that hydrogen peroxide (concentrations: 0.5 to 6%) was ineffective against Bsal and should not be used for the disinfection of field equipment (Van Rooij et al., 2017).

Table 1. Efficacy of chemical disinfectants in killing Bsal zoospores and zoosporangia. ADAC: C12-C16-alkyl dimethyl ammonium chloride, BAC: benzalkonium chloride, DDAC: didecyl dimethyl ammonium chloride, DcDAC: dicoco dimethyl ammonium chloride, EDTA: ethylenediaminetetraacetic acid, IPA: isopropyl alcohol, MEK: methyl ethyl ketone, PAA: peracetic acid, PHMB: polyhexamethylene biguanide, QAC: quaternary ammonium compounds (Van Rooij et al., 2017)

Disinfectant Active Ingredient (AI) Concentration AI Minimal Exposure Time for 100% Killing Ethanol (EtOH) EtOH 70% 30 seconds Disolol® 95% EtOH, denatured with 2% IPA and 2% Undiluted 30 seconds MEK Hibiscrub® 4% chlorohexidine digluconate 0.25, 0.5, 0.75% 30 seconds Chloramine-T® N-Chloro-4-methylbenzenesulphonamide 0.5% 5 minutes sodium salt 1% 2 minutes Concentrated Bleach 8% sodium hypochlorite (NaOCl) 1:5 dilution 5 minutes 4% 30 seconds Kickstart® 5% PAA, 20% H2O2, 10% acetic acid 0.05% 5 minutes 0.1% 2 minutes Potassium KMnO4 1% 10 minutes permanganate 2% 5 minutes (KMnO4) Virkon S® < 50% potassium monopersulphate 0.5% 5 minutes 1% 2 minutes Dettol medical® 4.8% chloroxylenol, 1-2% IPA, 5-15% pine 1:20 dilution 5 minutes oil Biocidal® < 0.1% DDAC, ADAC, DcDAC, and < 2% Undiluted 30 seconds EDTA Safe4® 3-5% BAC Undiluted 30 seconds F10® 1-10% BAC, 0.01-0.1% PHMB 1:100 dilution 30 seconds hydrochloride, 1-10% C10 alcohol 1:250 dilution 30 seconds ethoxylate 1:500 dilution 30 seconds 1:1000 dilution 30 seconds Sodium Chloride NaCl 10% 10 minutes (NaCl)

In the event of a disease outbreak in wild or captive amphibians, the Association of Fish and Wildlife Agencies (AFWA): Amphibian and Reptile Conservation Committee and the National Bsal Task Force released a Bsal Rapid Response Plan template (available at: http://www.salamanderfungus.org/june-2018/). Because early detection and containment are key to preventing the establishment of Bsal in North America, the Partners of Amphibian and Reptile Conservation (PARC) Disease Task Team has also created a system for reporting amphibians with suspected Bsal infections (details at: http://www.salamanderfungus.org/help/).

In the lab, captive salamanders can be effectively cleared of Bsal infections by exposing animals to 25˚C for 10 days (Blooi et al., 2015). However, host thermal tolerance must be considered prior to thermal treatment (Bury, 2008; Hutchison, 1961). Blooi et al. (2015) provide a detailed protocol for an alternative treatment appropriate for temperature sensitive species using voriconazole, polymyxin E combined with low temperature exposure (protocol link: http://www.nature.com/article-assets/npg/srep/2015/150630/srep11788/extref/srep11788- s1.pdf).

Author’s Affiliation and Contact Information: Kenzie Pereira Duquesne University Bayer School of Natural and Environmental Sciences [email protected]

Dr. J. Patrick W. Cusaac Iatric Manufacturing Solutions, LLC [email protected]

Recommended Citation:

Pereira, K.E, Cusaac, J.P.W. 2019. Batrachochytrium salamandrivorans: An emerging amphibian pathogen. Southeastern Partners in Amphibian and Reptile Conservation, Disease, Pathogens and Parasites Task Team, Information Sheet #18R.

Reviewed and edited by Dr. Deb Miller, Dr. Jennifer Ballard, and Dr. Amanda Duffus (co-chairs of the Diseases, Pathogens, and Parasites Task Team of Southeastern PARC).

Previous Version:

Pereira, K.E. 2015. Batrachochytrium salamandrivorans: An emerging amphibian pathogen. Southeastern Partners in Amphibian and Reptile Conservation, Disease, Pathogens and Parasites Task Team, Information Sheet #18.

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