Batrachochytrium salamandrivorans: The Next Threat to North American Biodiversity (hosted by TWS)

The next great threat to North America’s biodiversity is on the doorstep – an emerging fungal pathogen, called Bsal (Batrachochytrium salamandrivorans). This pathogen is believed to be from Asia and responsible for decimating salamander populations in Europe. The current lack of regulations that require clean trade of amphibians or translocation of zoospores by humans on fomites may result in the emergence of Bsal in North America, possibly within the next decade. Wildlife biologists in North America have the unique opportunity to prepare for the emergence of a pathogen before it can cause serious impacts on biodiversity and ecological processes. This symposium will review current plans of the North American Bsal  Task Force to formulate effective options for Bsaldetection, response, and management. A series of research talks by North American scientists will familiarize the audience with our current understanding of species susceptibility and risk, host immune defenses, possible ecological factors that could contribute to emergence, and different management strategies. This information will be key to effectively respond to and manage a possible Bsal outbreak event by providing the best available data, which can inform objective, science-based decision-making. The symposium will include 13 presentations by amphibian pathogen experts and two panel discussions for opportunities to interact with the audience.

8:00AM North American Bsal Strategic Plan: Plans to Prevent an Invasion
  Jacob Kerby
Chytridiomycosis is thought to be one of the greatest threats to amphibian biodiversity today. With the recent discovery of a second species of pathogenic chytrid fungus, Batrachochytrium salamandrivorans, there are critical concerns over the introduction of this pathogen into North America. The Bsal Task Force was formed in 2015 to address this primary concern. The task force is made up of experts from the US, Canada, and Mexico from a variety of backgrounds in many fields including: academia, wildlife and disease agencies, and industry. In this talk, I will highlight the recent release of a Bsal Strategic Plan that outlines efforts to prevent Bsal invasion and highlights timely and appropriate responses to a detection of Bsal in North America. In particular, I will outline plans from the task force’s seven working groups: Surveillance/Monitoring, Diagnostics, Data Management, Response, Outreach/Communication, Research, and Decision Support. Subsequent talks in the symposium will specifically detail some of these group’s accomplishments and plans, but this introductory talk will provide the overall framework for these efforts. It is our hope that the increased awareness of this strategic plan can improve our ability to quickly deal with any potential invasion of this deadly pathogen into North America.
8:20AM Susceptibility of North American Amphibian Species to Batrachochytrium Salamandrivorans
  Jonah Piovia-Scott, Davis Carter, John Romansic, Joseph Cusaac, Louise Rollins-Smith, Laura Reinert, Debra Miller, Allan Pessier, Reid Harris, Lori Williams, Andrea Upchurch, Priya Nanjappa, Matthew Gray
The recently described fungal pathogen Batrochochytrium salamandrivorans (Bsal) poses a serious threat to native amphibians throughout North America. Bsal is thought to have originated in East and Southeast Asia and spread to Europe through the pet trade, where it is responsible for dramatic declines in wild Fire Salamander (Salamandra salamandra) populations. Bsal is not known to have colonized North America, a global center for salamander biodiversity. Risk models based on environmental suitability suggest that Bsal poses a high risk to North American amphibian biodiversity, especially in the southeastern and northwestern USA. Our study seeks to expand and refine these projections of Bsal risk by evaluating the susceptibility of more than 30 native North American amphibians using controlled laboratory exposure experiments. Experimental animals were exposed to one of four Bsal zoospore doses (103-106), and we checked animals for signs of chytridiomycosis and swabbed animals to estimate Bsal infection intensity for at least six weeks after exposure. Of the species tested, approximately 70% became infected and 30% developed Bsal chytridiomycosis. Infection prevalence and mortality were generally dose-dependent. Susceptible species that developed clinical Bsal chytridiomycosis and mortality included: Chiropterotriton sp., Aquiloeurycea cephalica, Ensatina eschscholtzii, Aneides aeneus, Eurycea wilderae, Pseudotriton ruber, Notophthalmus viridescens, N. meridionalis, N. perstriatus, and Taricha granulosa. Tolerant species that maintained low intensity Bsal infections over several successive swabs included at least seven caudates and five anurans. Resistant species also included both caudates and anurans. Our results suggest that most North American amphibian communities will be composed of a combination of amplification, tolerant, and resistant host species, which could facilitate the emergence, spread and maintenance of Bsal in the western hemisphere.
8:40AM Studies of the Skin Secretions of Eastern Newts in Defense Against Batrachochytrium Salamandrivorans
  Louise Rollins-Smith, Laura Reinert, Mitchell Le Sage, E. Davis Carter, Debra Miller, Matthew Gray
Amphibians have been declining around the world for more than four decades. Contributing to these declines are the chytrid fungi, Batrachochytrium dendrobatidis (Bd) and Batrachochytrium salamandrivorans (Bsal), which cause the disease chytridiomycosis. Amphibians have complex immune defenses against fungi, and we continue to investigate more of these defenses. Here, we present preliminary evidence that innate skin secretions of Eastern newts, Notophthalmus viridescens, contain a mixture of proteins that reduce the viability of infectious Bsal zoospores. The proteins appear to be more abundant when induced from animals held in cold conditions (6 °C) in comparison with warmer newts (at 14 °C or 22 °C). In Bsal exposure trials, pathogen loads were also reduced at 6 °C in comparison with animals exposed at 14 °C or 22 °C. Although cold temperature did not impair production of the defensive proteins, they were only partially protective for Bsal-exposed newts. Newts at 6 °C developed chytridiomycosis and died more slowly than at 14 °C; however, mortality was similar between temperatures. Thus, it appears that temperature may plan a critical role in innate skin defenses against Bsal chytridiomycosis, and those defenses alone are insufficient to prevent development of chytridiomycosis in this susceptible host species. Funding NSF IOS-1557634, IOS-155759, DEB-1814520.
9:00AM Testing the Adaptive Microbiome Hypothesis in Eastern Red-Spotted Newts Exposed to Batrachochytrium Salamandrivorans
  Douglas Woodhams, Molly Bletz
Pathogens encounter complex microbiomes at host mucosal surfaces, but little is known about the ecology of microbiome colonization and maintenance of symbiosis. While colonization of the host sometimes leads to disease, we propose that symbiotic microbiomes are adaptive: enrichment of the microbial community with anti-pathogen members is a result of competitive microbial interactions coupled with host immune responses. According to this adaptive microbiome hypothesis, upon recovery from infection, enriched microbial communities are primed for a second exposure, and like immune memory cells, or a seed bank, are able to quickly bloom in response to pathogen exposure. Rather than dysbiosis, perturbations of the microbiome may represent adaptive responses, or a healthy defensive state. This adaptive microbiome defense blurs the distinction between innate and adaptive immunity and helps explain the curiously high diversity of symbionts hosted by eukaryotes which may depend on high diversity to enable the functional capacity of the microbiome for host defense. Here, we examine this hypothesis in amphibian populations and hosts interacting with emerging pathogens. Amphibians are an excellent model system because their moist skin surfaces are easily sampled in time series, and because they retain vertebrate immune capacities representative of the human gastrointestinal tract and lung. Here we use an updated amphibian Antifungal Isolates Database which includes greater than 6000 cultures from hundreds of host species tested against the pathogen Batrachochytrium dendrobatidis (Bd) to predict functional response across datasets showing microbiome shifts upon Bd exposure. Defensive isolates commonly upregulated upon exposure were identified. At a time when industrialization has been linked to disappearing microbiota important for host health, applications of microbial therapies such as probiotics may contribute to conservation efforts for one third of the world’s amphibian species currently threatened by the emergence of chytridiomycosis.
9:20AM Antifungal Properties of Salamander Skin Secretions Are Not Always Related to in-Vivo Susceptibility or Equally Effective Against Both Chytrid Fungal Pathogens.
  Kenzie Pereira, Sarah Woodley
Infectious diseases afflict wildlife worldwide. Understanding how environmental and host-factors influence host susceptibility is key to developing effective conservation and management strategies to prevent further biodiversity loss. Chytridiomycosis, a lethal skin disease caused by chytrid fungi, Batrachochytrium dendrobatidis (Bd) and B. salamandrivorans (Bsal), has devastated amphibian populations worldwide. While Bd has been linked to anuran declines and extinctions, Bsal now poses a similar threat to salamanders. To date, Bsal has decimated European salamander populations and is predicted to soon emerge within North America. To better understand host-factors influencing salamander susceptibility to Bsal and to inform the development of proactive mitigation strategies, we investigated whether species-specific differences in the antifungal properties of skin secretions were related to in-vivo susceptibility to Bsal. Because the skin is the first line of defense against chytrid fungi and anuran antifungal skin secretions have been correlated with reduced susceptibility to Bd, we predicted that the skin secretions of salamanders with no or low susceptibility to Bsal would inhibit fungal growth in-vitro and would be equally effective against Bd and Bsal. Potential antifungal peptides were concentrated from the skin secretions of four salamander species using solid phase extraction, combined with Bd or Bsal within a 96-well plate, and assessed for antifungal activity by measuring changes in optical density. We found that the antifungal properties of salamander skin secretions varied among species, was not always related to in-vivo susceptibility, and did not always have similar effects on Bd and Bsal. These findings suggest that while skin secretions may be important for protecting some species from chytridiomycosis, other species may be protected by alternative mechanisms. Further, the antifungal properties of skin secretions may be pathogen-specific. The results of this study highlight the complex nature of host-pathogen systems and warrant additional research to elucidate factors driving disease susceptibility in wildlife.
9:40AM The Pathology of Bsal Chytridiomycosis: Documenting Anatomical Changes and Exploring What They Might Mean Clinically
  Debra Miller, Agata Grzelak, Anastasia Towe, Robert Ossiboff, Carolyn Cray, Rajeev Kumar, Kurt Ash, E. Davis Carter, Brittany Bajo, Markese Bohanon, Anna Peterson, Matthew Gray
Batrachochytrium salamandrivorans (Bsal) is a recently discovered pathogen that is of global concern because of its potential to cause high mortality in amphibians, especially salamanders. Experimental challenges are underway to estimate species susceptibilities in order to understand invasion risk and develop disease mitigation strategies to minimize the possibility of Bsal emerging in North America. To date, 29 amphibian species (22 salamander, 7 anuran) have been tested. For each challenge, individuals were exposed to Bsal in a water bath at one of five concentrations (0, 5 x 103, 104, 105, 106) for 24 hours, and their condition monitored for six weeks. Gross changes varied from increased skin sloughing to discrete ulcerations. Histologically, Bsal organisms were seen from superficial to full thickness epidermis, and in discrete crater formations or diffuse laminar patterns. These changes were seen in salamanders and anurans. For a similar fungus, B. dendrobatidis (Bd), changes in blood electrolytes that lead to host paralysis and cardiac arrest have been reported. Therefore, we hypothesized that species experiencing Bsal chytridiomycosis would have similar changes in electrolytes, because like Bd, Bsal damages the epidermis hence may affect skin osmoregulation – a key physiological process that influences blood chemistry. To test this hypothesis, we collected heart blood from a larger susceptible species, Taricha granulosa, at necropsy using heparinized capillary tubes. A blood smear was prepared, and a portion of whole blood was added to Natt Herrick’s solution for total blood cell counts. The remaining blood was centrifuged and plasma collected for blood chemistry profiles and protein electrophoresis. Initial biochemical results revealed decreases in sodium, chloride, and potassium in terminal stages of the disease (5×106 dose) similar to Bd. Identifying biochemical changes associated with Bsal chytridiomycosis will help elucidate pathogenesis and be key to developing disease mitigation strategies and exploring treatment options in susceptible species.
09:40AM Break
1:10PM Winter Is Coming: Temperature Dependent Virulence of Batrachochytrium Salamandrivorans
  Davis Carter, Debra Miller, Brittany Bajo, Anna Peterson, Markese Bohanon, Kurt Ash, Pattarawan Watcharaanantapong, Matthew Gray
Batrachochytrium salamandrivorans (Bsal) is a recently emergent fungal pathogen that has caused dramatic population declines of European fire salamanders (Salamandra salamandra). Initial in vitro growth experiments suggest Bsal has an optimum growth temperature between 10 – 15 C and an upper thermal limit of 25 C. Preliminary risk analyses suggest temperature will be an important factor driving Bsal invasion in the United States if the pathogen is introduced. In order to understand how Bsal virulence and host susceptibility are affected by temperature, we performed Bsal-host challenge experiments at three environmentally relevant temperatures. We exposed adult eastern newts (Notophthalmus viridescens) to a sham inoculation of autoclaved water (control) or one of four Bsal zoospore doses (5 x 10e3-6) at one of three ambient temperatures, 6 C, 14 C or 22 C. Following exposure, we monitored animals twice daily for signs of disease. We also swabbed each animal weekly to monitor infection intensity via qPCR. At 14 C, all eastern newts became infected and died within one-month post-exposure. Median survival duration at 14 C was 26, 20, 14 and 7 days for 5x10e3, 5x10e4, 5x10e5 and 5x10e6, respectively. No individuals became infected or died when exposed to Bsal at 22 C. At 6 C, results were intermediate, with survival durations approximately one to two weeks longer than 14 C. Newts that died from chytridiomycosis at 6 C had lower zoospore loads than at 14 C, suggesting lower infection tolerance at lower temperature. Our results suggest that Bsal invasion in the United States may be geographically narrower than indicated in previous risk models, with the greatest likelihood of emergence occurring at mid- to northern latitudes and at higher elevations, often where salamander diversity is the greatest. Seasonal fluctuations in disease emergence are expected, with autumn and winter months experiencing greatest mortality.
1:30PM Host Density and Habitat Structure Influence Contact Rates and Transmission of Batrachochytrium Salamandrivorans
  Matthew Gray, Daniel Malagon, Luis Melara, Olivia Prosper, Suzanne Lenhart, Davis Carter, Anna Peterson, Debra Miller
Batrachochytrium salamandrivorans (Bsal) is an invasive fungal pathogen that is emerging in Europe and is highly pathogenic to some salamander species, especially those in the family Salamandridae. Given its widespread distribution and high abundance, the eastern newt (Notophthalmus viridescens) has the potential to significantly contribute to Bsal’s epidemiology if the pathogen emerges in North America. For pathogens that transmit via direct contact between hosts, management strategies that reduce contact rates can limit invasion potential and outbreak size. Thus, we designed two studies to estimate: 1) contact rates given different host densities and levels of habitat structure, and 2) the probability of transmission from infected to susceptible individuals as Bsal chytridiomycosis progressed. Using parameter estimates from these experiments, we modeled infection and disease outcomes for a population of eastern newts using a system of ordinary differential equations. Bsal transmission was very efficient between newts even at early stages of infection. For example, one contact of one-second duration between an infected and susceptible newt resulted in 100% transmission only 12 days after the infected newt was initially exposed to Bsal zoospores. Contact rates between newts were density dependent – reducing host density 4-fold reduced contact rates by 5 – 15X. At higher newt densities, adding plants to mesocosms reduced contacts by 3X. Our simulations show rapid transmission of Bsal among newts, resulting in >95% infection prevalence in one month and >80% mortality in three months, illustrating the potential for severe population impacts. Our results demonstrate that if Bsal is introduced into North America, the eastern newt has the potential to play a major role in its epidemiology. Additionally, reducing newt density or increasing habitat structure at Bsal positive sites might reduce transmission and outbreak size.
1:50PM Multiple Transmission Pathways in Mathematical Models of Bsal
  Angela Peace, Md Rafiul Islam, Matthew Gray
Epidemic dynamics of infectious diseases with multiple routes of transmission are complex. Mathematical models can be used to determine invasion potential and identify which transmission pathway is dominant and can ultimately help identify appropriate intervention strategies. We developed compartmental host-pathogen models to examine the transmission dynamics of an emerging fungal pathogen on an amphibian population. Multiple stages of infection are incorporated into the model, allowing disease-induced mortality and zoospore shedding rates to vary as the disease progresses. Parameter sensitivity analysis shows that the recovery rate and environmental zoospore degradation rates are influential parameters. Calculation of the basic reproductive number (R0) highlights the virulence of this pathogen and is used to determine that direct transmission is the dominant transmission pathway for small population densities, while environmental transmission dominates in large populations.
2:10PM Chytrid Fungal Co-Infections in Amphibians: What Do We Know and What Should We Expect?”
  Ana Longo
Invasive fungal pathogens Batrachochytrium dendrobatidis (Bd) and B. salamandrivorans (Bsal) are causing mortality events and population declines in amphibians around the world. B. salamandrivorans has not been found in the Americas but is predicted to emerge there given the presence of competent hosts, high volume import pathways, and lack of effective biosecurity measures. Eastern newts (Notophthalmus viridescens) are the most abundant and widespread salamanders in United States with known susceptibility to both fungi. However, little is known about how their current Bd infections will interact with novel Bsal infections. Here, we ran a series of experiments in which we exposed newts to each fungal pathogen, combinations of both fungi, and three Bd isolates to determine if natural exposure to Bd provides protection against Bsal and quantify the effect of co-infections on newt survival. We found that hosts exposed under simultaneous co-infections experienced 78% mortality over a period of 18 weeks, which was driven by the persistence of Bsalbecause newts cleared Bdinfection within a month. We also investigated the responses of several species of Plethodontid salamanders to co-infections. We found high variation in disease responses from complete resistance to 100% mortality. Our findings identify species that could serve as potential superspreaders of Bsal in North America.
2:50PM Refreshment Break
3:00PM Chytridiomycosis in Mexico: Strains, Infection and Potential Risk
  M. Basanta, Allison Q. Byrne, Erica Bree Rosenblum, Eria A. Rebollar, Gabriela Parra-Olea
Mexico hosts the fifth largest amphibian fauna in the world and almost 58% of its species are under a category of threat. One of the principal threats is the chytridiomycosis caused by Batrachochytrium dendrobatidis (Bd) and Batrachochytrium salamandrivorans (Bsal). Previous studies in Mexico identified 74 amphibian species with Bd presence and also identified several potential high-risk zones if Bsal arrives in the country. The aim of this work was to evaluate the potential risk of Bd and Bsal in Mexico considering Bd genotypes and Bsal presence. We used positive Bd amphibians’ skin swabs and chytrid isolates from the north, central and south Mexico to determine Bd’s genotypes and Bsal presence using the Fluidigm Access Array platform. We obtained 90 isolates and 89 skin swabs from 35 amphibian species, of which 60 samples are from potential high-risk zones if Bsal arrives in the country. Our work increased the known Bd distribution in Mexico with eleven new amphibian species and seven new Mexican states with Bd presence, and it is the first study that analyzed Bsal presence and genetic variability of Bd for Mexico. This study will help to understand the dynamics of the disease between chytrids and native amphibians in the country to prevent, mitigate and conserve the amphibian species from the disease.
3:20PM Using Edna Technology to Detect Bsal – Implications for Surveillance
  Christian Yarber, Caren Goldberg, Allan Pessier, Jonah Piovia-Scott, John Romansic, Jesse Brunner
Batrachochytrium salamandrivorans (Bsal), an emerging pathogen that threatens global salamander diversity, spread into Europe via the international trade of live amphibians. While it has not been detected outside of Europe or Asia, its continued expansion is likely without strong biosecurity and surveillance practices. However, the volume of animals shipped around the world is immense (e.g., millions of live amphibians into US yearly) making surveillance with individual-based methods of detection unfeasible. Environmental DNA (eDNA)-based detection offers the promise of collecting and screening a handful of samples from a shipment to have reasonable certainty of detecting Bsal or other pathogens. We conducted a series of laboratory experiments to evaluate eDNA’s potential for Bsal surveillance in trade. We aimed to determine the 1) best materials to sample from, 2) best sample processing methods, 3) limits of detection, and 4) ability to detect infected animals. Our initial findings suggest filtering results in >10-fold higher yields of Bsal DNA than centrifugation and the 95% limit of detection for Bsal when filtering is around 200 zoospores. We were able to detect Bsal DNA in animals’ substrates and holding water with high concordance with direct swabs of individual animals. Ongoing experiments will determine best practices and diagnostic sensitivity in real-world trade scenarios. We conclude with discussions of how eDNA-based detection might be applied to the trade of live animals.
3:40PM Proactive Conservation: Developing Strategies to Combat the Salamander-Eating Fungus
  Molly Bletz, Priya Nanjappa, Douglas Woodhams
Arrival of the recently discovered chytrid fungus, Batrachochytrium salamandrivorans (Bsal), looms over the United States – a hotspot of salamander diversity rivaled by no other around the world. This ‘salamander-eating’ fungus is decimating fire salamander populations in multiple European countries and poses immense threat to global salamander diversity. Therefore, understanding the risk of Bsal invasion in the US and preparing for both how to respond if it emerges, as well as how to support population resilience through an invasion, is essential. Three key strategies include prevention, management, and mitigation. Preventing or minimizing the risk of entry of the pathogen through amphibian trade is critical; the 2016 USFWS listing of 201 salamander species as injurious under the Lacey Act was a first step in prevention. However, management of this deadly pathogen will require additional policy and legislative updates, coupled with implementable science-based mitigation strategies to slow disease emergence and reduce spread. Disease response strategies in the wild can include physical, chemical and biological measures, including actions such as altering water chemistry or temperature, modifying micropredator communities, applying fungicides, vaccination, and probiotic bioaugmentation. We have characterized susceptibility across populations of two of the most at-risk species in the US: eastern newts and two-lined salamanders, finding that populations differ in their mucosal protection against Bsal. This crucial step aids to identify geographic regions that are at greatest threat if Bsal invades, which in turn helps direct surveillance and management response actions. Promising preliminary evidence a from controlled-laboratory experiment suggests that bacteria that produce volatile antifungal compounds (VOCs) may be able to limit infection buildup in hosts simply by being present in the environment. Continued development of proactive and integrative, multi-pronged approaches will be essential to combat significant biodiversity losses if Bsal emerges in the United States.
4:00PM Disease Decisions Can be Rational Choices
  Evan Grant, Riley Bernard
Despite calls for improved responses to emerging infectious diseases in wildlife, management is seldom considered until a disease has been detected in a population. Emerging diseases have the potential to affect social, economic and ecological interests of North American resource managers, who are entrusted by society to manage protected areas and wildlife populations. Although preventing the arrival of a pathogen is most effective for controlling emerging infectious diseases, prevention is not failsafe. Resource managers must consider multiple social, economic and ecological objectives, which result in difficult trade-offs for any given disease management strategy (i.e., an optimal action for managing a wildlife disease may result in declines in recreational or economic values). Complexity arises in balancing numerous and competing demands on land managers, and this effectively limits our ability to identify and implement proactive management – representing a major challenge for developing management strategies for Bsal and other emerging infectious diseases. The goal of the Decision Support Working Group (DSWG) of the North American Bsal Task Force is to support management decisions regarding Bsal through the facilitation of decision-making processes, identification and collation of information needed to make decisions, development of models to predict the outcomes of different management options, and the evaluation of tradeoffs and risk to overcome impediments to optimal decision-making. We present work of the group in support of the Bsal Task Force.
4:20PM Panel Discussion

Organizers: Jamie Voyles, Matthew Gray
Supported by: North American Bsal Task Force – Jake Kerby; Wildlife Disease Association – Dave Jessup; Wildlife Disease Working Group, The Wildlife Society – Krysten Schuler

Location: Reno-Sparks CC Date: September 30, 2019 Time: 8:00 am - 5:00 pm