Batrachochytrium salamandrivorans Can Devour More than Salamanders

The chytrid fungi Batrachochytrium salamandrivorans (Bsal) and Batrachochytrium dendrobatidis (Bd) cause chytridiomycosis, an often fatal skin disease that has been associated with the decline of more than 500 amphibian species worldwide (Scheele et al. 2019). Though Bsal has been isolated from wild anurans in Vietnam, where the fungus is thought to have originated (Nguyen et al. 2017), clinical chytridiomycosis caused by Bsal has previously been observed exclusively in urodele amphibians (i.e., salamanders; Martel et al. 2014; Stegen et al. 2017). For example, experimental exposures to Bsal showed that a European frog species (Alytes obstetricans) could be an asymptomatic carrier but did not develop chytridiomycosis (Stegen et al. 2017). To evaluate whether North American frogs could become infected with Bsal, we challenged adult wild-caught Osteopilus septentrionalis (Cuban treefrogs) with zoospore doses known to infect salamanders (Carter et al. 2020) and used a multimodal diagnostic approach to determine their susceptibility to infection. This frog is an invasive species in at least four states in the US and 14 countries (Cottrell and Ventosa 2018) and is commonly found in the pet trade (Global Invasive Species Database 2008; Hedges et al. 2019); hence, it could play a role in global dissemination of Bsal. All procedures followed approved University of Tennessee Institutional Animal Care and Use Committee protocol no. 2395.

Frogs were captured in the wild in Florida, US, and transported to the University of Tennessee, Knoxville, US. They were housed individually at 30 C for 10 d to clear any preexisting Bd infections (Chatfield and Richards-Zawacki 2011) then acclimated over 2 wk to 15 C, the approximate optimal growth temperature for Bsal (Martel et al. 2013). We assume that stress associated with heat treatment was negligible, because ambient temperatures of 30 C or greater occur in the native subtropical distribution of Cuban treefrogs and in Florida.

Zoospores used for exposure were harvested from tryptone gelatin hydrolysate agar plates after 6 d of growth. Plates were flooded with 7 mL of autoclaved dechlorinated water and filtered with a 20-µm sieve to isolate zoospores. Zoospores were enumerated with a hemocytometer and verified by flow cytometry. We exposed four animals per treatment group in 100-mL plastic cylindrical containers with 9 mL of autoclaved dechlorinated water and 1 mL of the randomly assigned Bsal zoospore dose (5×10³, 5×10⁴, 5×10⁵, or 5×10⁶). Two control animals were treated identically but exposed to 10 mL of autoclaved dechlorinated water. After 24 h, we removed frogs from the inoculation tubes and placed them in individual 710-cm³ vivariums containing a moist paper towel and polyvinyl chloride cover object. We fed animals crickets (2% of their body mass daily) and replaced each animal's container, cover object, and paper towel every 3 d. Animals were housed in environmental chambers on a 12-h light, 12-h dark cycle, at 15 C and >90% humidity.

We monitored twice daily for signs of Bsal chytridiomycosis, including lethargy, focal lesions, ulcerations, increased skin sloughing, hemorrhage, anorexia, and loss of righting response (Martel et al. 2013; Carter et al. 2020). Animals were humanely euthanatized when they lost righting ability and recorded as a mortality event for data analyses. We swabbed each animal every 6 d, starting 4 d postexposure to Bsal, using standardized swabbing protocols for Bd and Bsal (Blooi et al. 2013). We extracted genomic DNA from each swab with Qiagen DNeasy Blood and Tissue kits (Qiagen, Hilden, Germany). To detect the presence and estimate the quantity of Bsal DNA on each swab, we performed quantitative PCR with an Applied Biosystems QuantStudio 6 Flex (Thermo Fisher Scientific, Waltham, Massachusetts, USA) quantitative PCR instrument (Blooi et al. 2013). We ran all swab samples in duplicate and considered a sample positive if both replicates reached cycle threshold before 50 cycles. We used a standard curve of synthetic Bsal DNA (gBlock) to estimate the number of Bsal zoospore copies per microliter in each sample. Swabs collected at the first timepoint after Bsal exposure and at postmortem examination were also tested for Bd DNA.

We confirmed Bsal colonization in histologic cross sections of epidermal tissues stained with H&E. We used RNAscope® (Advanced Cell Diagnostics, Newark, California, USA) in situ hybridization staining to evaluate and highlight the distribution of Bsal zoosporangia in infected tissues and verify that frogs were Bd-negative (Ossiboff et al. 2019). Although it is possible that the frogs had exposure to Bd in the wild, there is no evidence that prior Bd exposure affects susceptibility of hosts to Bsal infection (Longo et al. 2019; Greener et al. 2020). To demonstrate that Bsal was the causative agent of clinical signs in exposed frogs, we reisolated and cultured Bsal from infected toe tissue collected from a diseased frog (Martel et al. 2013). After obtaining a pure Bsal culture verified by quantitative PCR and microscopically, we grew, harvested, and enumerated Bsal zoospores and exposed adult eastern newts (Notophthalmus viridescens) to 5×10⁶ zoospores from the reisolated culture with the same methods as for the frog experiment. Because adult newts are aquatic, we housed them individually in 2-L plastic containers holding 300 mL of water and a polyvinyl chloride cover object (Malagon et al. 2020). Monitoring, cleaning, and feeding was on the same schedule as described for the frogs.

All statistical analyses were performed in RStudio version 3.5.3 (R Core Team 2020). We analyzed the survival data shown by performing Kaplan-Meier survival analysis and Cox proportional hazard models with the survival package (Goel et al. 2010). To compare food consumption among zoospore doses, we fit a binomial generalized linear mixed effects model with a logit link function. We included fixed effects for number of days postexposure, treatment (five levels: control, 5×10³, 5×10⁴, 5×10⁵, and 5×10⁶), and the interaction between days postexposure and treatment. We included a random effect of individual, and we allowed both the intercept and the effect of days postexposure (i.e., the slope) to vary by individual. We fit the model with the brms packages in R with uninformative priors (Bürkner 2018) and used the posterior predictions from the model to test whether percent food consumption differed between the control group and the four dose treatments after 40 d postexposure. We performed an analysis of variance comparing log-transformed Bsal zoospore genomic equivalents per microliter postmortem among exposure doses. When the analysis of variance was significant (α=0.05), post hoc Bonferroni corrected t-tests were performed to evaluate pairwise differences in Bsal zoospore load.

The skin of all frogs tested positive for Bsal DNA after exposure at the three highest doses and contained high genomic copies (Figs. 1, 2). All animals were negative for Bd DNA. The frogs experienced dose-dependent survival; individuals exposed to 5×10⁵ and 5×10⁶Bsal zoospores showed 75% and 100% mortality, respectively (Fig. 3). No frogs in the lowest two doses or controls died by the end of the 75-d experiment. Median survival duration for 5×10⁵-exposed frogs was 47 d postexposure (range 46–75 d); median survival duration for 5×10⁶-exposed frogs was only 25 d (range 15–45 d). Median survival duration of frogs exposed to 5×10⁶Bsal zoospores was similar to frogs exposed to 10⁶Bd zoospores at the same temperature (Raffel et al. 2013).

Infected Cuban treefrogs developed erythema and hemorrhage on their feet and ventrums and excessive shedding on their feet (Fig. 4A). On the dorsum, spots of darkened pigmentation developed that progressed to hemorrhages (Fig. 4B). Histologic examination in conjunction with in situ hybridization (Ossiboff et al. 2019) revealed multiple variably sized, necrotizing, crater-like epidermal lesions randomly distributed throughout the body and containing numerous intralesional Bsal thalli (Fig. 4C, D). These gross and histologic findings are consistent with Bsal chytridiomycosis (Thomas et al. 2018). All animals were confirmed negative for Bd chytridiomycosis with in situ hybridization. Infected frogs consumed less invertebrate prey as disease progressed (Fig. 5); median food consumption for animals exposed to 5×10³, 5×10⁴, 5×10⁵, and 5×10⁶, 40 d after exposure, decreased by 54% (95% confidence interval [CI], 14–81), 51% (95% CI, 8–80), 50% (95% CI, 7–81), and 92% (95% CI, 60–99), respectively, compared with control animals. Anorexia has been associated with amphibian chytridiomycosis caused by both Bd and Bsal (Martel et al. 2013; Van Rooij et al. 2015).

Zoosporangia of Bsal were successfully reisolated from toe tissue collected from a morbid 5×10⁵-exposed frog. In cell culture, we observed formation of zoosporangia and motile spores (Supplementary Material Videos S1, S2) diagnostic of chytrid fungi (Van Rooij et al. 2015). The isolate also tested positive for Bsal DNA by quantitative PCR, and exposure to 5×10⁶ zoospores caused 100% infection and mortality in <10 d in eastern newts, a species susceptible to Bsal (Longo et al. 2019). Histopathology and in situ hybridization (Fig. 4E, F) confirmed Bsal chytridiomycosis in the newts, fulfilling Koch's postulates.

Our results demonstrate that Bsal chytridiomycosis is not limited to urodele host species. Therefore, current amphibian import bans focusing largely on stopping the trade of urodele species may be insufficient to prevent introduction of Bsal into the US and elsewhere. Because anurans constitute 99% of global amphibian trade (Can et al. 2019), outright trade bans to prevent possible entry of infected amphibians into Bsal-free nations could be met with significant resistance by industry. We suggest as an alternative that animal health certifications or other programs that support sustainable clean trade be considered as intervention strategies. Furthermore, our results show that species in the most diverse anuran family, Hylidae (IUCN 2019), that have experienced multiple species extinctions because of Bd (Scheele et al. 2019), may be susceptible to Bsal, as well. Additional research into the susceptibility of more anuran species is warranted to formulate a complete picture of the threat Bsal poses to amphibian species worldwide.

The authors thank Peter Iacono, Eric Suarez, and Brooke Talley of the Florida Fish and Wildlife Conservation Commission for collecting the Cuban treefrogs used in this study. The University of Tennessee (UT) and University of Florida histology laboratories assisted with slide preparations, and Melissa Brown helped with in situ hybridization. We thank Bobby Simpson and Alex Anderson of the UT Institute of Agriculture East Tennessee Research and Education Center for laboratory and logistic support. We also thank Bailee Augustino for assistance with animal care and data collection. This work was partially supported by the National Science Foundation Division of Environmental Biology (EEID grant 1814520) and US Department of Agriculture National Institute of Food and Agriculture (Hatch Project 1012932) awarded to M.J.G. and D.L.M. The UT College of Veterinary Medicine Center of Excellence program provided salary support to A.E.T. Authors declare no competing interests. All data are available in the main text. Code for statistical analyses is available in the public repository, TRACE (https://trace.tennessee.edu).

Supplementary material for this article is online at http://dx.doi.org/10.7589/JWD-D-20-00214.