The application of disease management strategies is context dependent and typically targets the host, environment, pathogen, or combinations thereof. A suite of disease management strategies focuses on reducing pathogen spread within a population (Woodhams et al., 2018). These strategies often attempt to reduce environmental persistence of pathogens or contact rates between hosts (Garner et al., 2016; Woodhams et al., 2018; Thomas et al., 2019). Identifying key transmission pathways is a fundamental step to understanding which management strategies are likely to be most effective at reducing infection and disease in populations (Islam et al., 2021).

Amphibian diseases are one of the leading causes for observed amphibian population declines around the globe (Canessa et al., 2018; Scheele et al., 2019). Given the pronounced effect diseases have on amphibians, successful management of amphibian populations hinges on the effective management of disease. Currently, there are three notifiable amphibian pathogens recognized by the World Organization for Animal Health (OIE), which have been designated as notifiable due to the known threat each poses to amphibians. These notifiable pathogens include viruses in the genus Ranavirus and two chytrid fungus (Batrachochytrium) species. Ranaviruses and Batrachochytrium dendrobatidis (Bd) have caused declines in amphibian populations across the globe for several decades. Recently, a new chytrid fungus (B. salamandrivorans, Bsal) was identified associated with enigmatic declines of salamanders in Europe (Brunner et al., 2015; Olson et al., 2021).

In 2013, Bsal was first described after declines of European Fire Salamanders (Salamandra salamandra) were observed in the Bunderbos region of Netherlands (Annemarieke Spitzen-van der et al., 2013; Martel et al., 2013). Bsal kills a host by creating necrotic ulcerations through the epidermis, thereby allowing bacterial infiltration and interrupting important physiological processes, such as osmoregulation (Bletz et al., 2018; Sheley et al., 2023). Mortality due to Bsal chytridiomycosis can occur less than two weeks post-exposure to the pathogen in highly susceptible hosts (Carter et al., 2021), suggesting that Bsal transmission and epidemics can occur rapidly (Islam et al., 2021).

A key to understanding Bsal epidemiology and devising effective disease management is identifying transmission pathways (Peace et al., 2019; Islam et al., 2021). Bsal can be transmitted directly between infected and susceptible individuals via as little as one second of contact (Malagon et al., 2020), and exposure to motile Bsal zoospores in water can result in transmission and mortality (Carter et al., 2021). For highly susceptible hosts in aquatic ecosystems, such as eastern newts (Notophthalmus viridescens), the functional form of Bsal transmission among all possible pathways is density independent (Tompros et al., 2022). One contributor to density independent transmission is an environmental reservoir for the pathogen. Environmental reservoirs identified for chytrid fungi include alternative hosts or growth substrates, such as invertebrates or decaying organic matter (McMahon et al., 2013; Stegen et al., 2017; Canessa et al., 2019). Recent evidence suggests that Bsal can be saprophytic (Kelly et al., 2021). Thus, it is possible that infected individuals that experience disease-induced mortality could continue to be a source of transmission as a carcass decays or until it is removed from the environment by scavengers (Le Sage et al., 2019). If carcasses are a source of Bsal transmission, it also suggests they could be removed as part of a disease management plan to reduce the effects of an outbreak.

Carcasses have been evaluated for their importance in other disease systems, yet they have not been researched as a source of transmission for either Bd or Bsal. Throughout human history, infected corpses have been suspected sources of disease transmission dating back to bubonic plague outbreaks, although the route of transmission was often incorrectly attributed to miasma released by their decay (Smith, 2013). A more contemporary account of disease spread from corpses has been noted with Ebola outbreaks (Prescott et al., 2015). In non-amphibian wildlife disease systems, carcasses have been recognized as a source of transmission for African swine-fever and chronic wasting disease (Jennelle et al., 2009; Pepin et al., 2020). Transmission of ranaviruses by carcasses can be an important pathway contributing to infection and disease (Brunner et al., 2007; Le Sage et al., 2019; Peace et al., 2019). Given post-mortem transmission of pathogens is known for other disease systems, we designed an experiment to evaluate whether and for how long Bsal could be spread from infected carcasses to uninfected, susceptible individuals through either direct contact or via indirect transmission from the shedding of infectious zoospores. We also estimated zoospore shedding rates from infected carcasses, which do not exist for Bd or Bsal (DiRenzo et al., 2014; Maguire et al., 2016; Islam et al., 2021). We hypothesized that infected carcasses would remain infectious for a short duration (<48 hrs) and lose their ability to shed zoospores, hence contributing minimally to post-mortem Bsal epidemiology.

Our carcass transmission experiment demonstrates the potential importance of infected carcasses to Bsal epidemiology. All susceptible newts paired with carcasses became infected during the cohousing timepoints, even when only indirect, waterborne transmission was allowed. We found newt carcasses can be infectious for at least three days post-mortem, hence extending the infectious duration of a host, which likely would influence the overall reproductive number of Bsal. Furthermore, carcasses likely contribute to frequency-dependent transmission, as they extend the infectious period of infected animals (Tompros et al., 2022). The median duration of survival for susceptible newts paired with carcasses was 26.5 days (95% CI = 24, 31). Based on the survival time of susceptible newts from this experiment, carcasses could extend the duration of infectiousness and zoospore shedding by at least 10% (26.5 days + 3 additional days as an infectious carcass). While we did perform a heat treatment method to clear Bd infections in all individuals used in our experiments, we did not clear them of all possible pathogens or parasites which could influenced their susceptibility. That said, we did not observe any gross signs of other infections, and other than Bd, there have been no co-infection experiments performed with Bsal, so the possible directional effects are unknown. Bsal loads on the infected carcasses and the number of shed zoospores released by carcasses did not decline over the 72-hr experiment duration. Further, greater Bsal loads were detected on infected carcasses 72 hrs post-mortem compared to those sampled 24 hrs after death. The greater pathogen loads measured 72 hrs post-mortem may be the result of the carcass epidermis deteriorating making swabbing efforts more successful. Alternatively, any adaptive or innate immune defenses against Bsal would have ceased function following death, facilitating unregulated pathogen growth. We also observed greater rates of mortality in susceptible newts paired with carcasses at 24 – 48 and 48 – 72 hrs post-mortem than for hosts paired with carcasses immediately after death. These results collectively suggest that the contribution of carcasses to Bsal epidemiology may extend beyond 72 hrs and warrants additional investigation. The rate of carcass decay is likely an important factor which would impact the overall contribution of carcasses to secondary infections. If carcasses persist in the environment for longer durations, they would likely continue to increase the overall R 0 of Bsal (assuming Bsal continues to grow on the carcasses). Collectively, this study indicates that carcasses likely play an important role in Bsal transmission dynamics for aquatic salamander hosts, and Bsal should be considered “necronotic” (nekro = corpse, nosos = disease) given the fact carcasses may contribute substantially to the overall basic reproductive number within a Bsal disease system.

Our results demonstrate that Bsal epidemiology models should include a carcass compartment in addition to the traditional susceptible, exposed, and infected classes (Fischer et al., 2013). In ranavirus systems, carcasses can transmit virions by contact and through the water (similar to Bsal), but also via necrophagy (Pearman et al., 2004; Harp and Petranka, 2006; Le Sage et al., 2019; Peace et al., 2019). Unlike scavenging anuran tadpoles, necrophagy may not be an important transmission pathway for Bsal in caudates, because larval and adult salamanders are insectivores (Wells, 2007). However, necrophagy may play a role in transmission of Bsal to anurans, considering some frog species are susceptible to Bsal, and larval amphibians can become infected by the pathogen (Nguyen et al., 2017; Nguyen et al., 2017; Gray et al., 2023). Bd infections in anuran tadpoles are often observed in the keratinized mouthparts prior to metamorphosis (McMahon and Rohr, 2015). Given the rasping nature of tadpole feeding, it is possible their mouthparts could become infected while feeding on Bsal-infected carcasses. Following introduction to a tank, carcasses shed approximately 1850 zoospores in the first 24 hrs, 999 zoospores in the next 24 hrs, and 1224 spores in the third 24-hr period. These shedding rates resulted in highly efficient indirect transmission (100%) and 83% mortality of susceptible newts that became infected in the partitioned cohousing chambers. These estimates represent the first quantification of zoospore shedding rates for Bsal-infected newts. Ranavirus virion and Bd zoospore shedding rates have been estimated previously in live animals (DiRenzo et al., 2014; Peace et al., 2019). DiRenzo et al. (DiRenzo et al., 2014) estimated Bd zoospore shedding rates by Atelopus zeteki immediately before euthanasia were equivalent to 23.55 zoospores genomic equivalents per minute which is nearly 500X the shedding rate measured in our study for recently dead carcasses. The greater shedding rate measured by DiRenzo et al. (DiRenzo et al., 2014) may be related to the high pathogen loads on their frogs, which were >4x10⁶ Bd zoospore equivalents. It is unclear from our study how long carcasses would continue contributing to the zoospore pool during a Bsal outbreak. However, we observed no decline in Bsal shedding over the 72-hr period indicating that carcasses may shed for longer durations. With carcasses lacking any immunological control of pathogen growth, competitive growth of other microbes or deterioration of growth substrate (i.e., skin for Bsal) are likely processes that would eventually decrease pathogen shedding from carcasses.

Our results indicate that Bsal response plans should consider strategies that reduce carcass density, such as physical removal of infected individuals or carcasses. Introduction of or implementation of habitat management strategies that enhance invertebrate scavenger populations might facilitate carcass removal (Le Sage et al., 2019) and reduce pathogen spread. However, it also is possible that scavenging could release zoospores as carcasses are consumed and shredding of infected tissues releases fragmented epidermal cells containing zoosporangia. Some scavenging species also could become infected with Bsal or mechanically transmit it to susceptible hosts. For example, crayfish are known to become infected with Bd (McMahon et al., 2013) and as scavengers may readily ingest amphibian carcasses. Habitat manipulations, such as dewatering a wetland, could be used to accelerate carcass decomposition and reduce indirect transmission rates from carcasses. However, this strategy would likely have negative effects on other resident aquatic species. We recommend all Bsal response strategies be conducted in consultation with experts and appropriate regulatory authorities (Grant et al., 2017; Hopkins et al., 2018).

If Bsal becomes established in naïve regions (such as the United States), eradication will be challenging due to the pathogen’s ability to infect multiple host species (Martel et al., 2014; Carter et al., 2019; Friday et al., 2020), environmentally stable life-stages (Stegen et al., 2017), and recent evidence suggesting that at least some Bsal isolates may have saprophytic abilities (Kelly et al., 2021). Unless the pathogen is introduced into a heavily monitored amphibian population, initial detections could occur years following introduction, making containment difficult. If Bsal eradication is difficult to achieve, as has been the case in Europe (Stegen et al., 2017; Canessa et al., 2018; Martel et al., 2020), we must be willing to use disease management strategies to prevent potentially catastrophic losses to amphibian diversity and preserve the valuable ecosystem services these species provide. Evaluating the contribution of all Bsal transmission routes is a critical first step to identifying effective strategies. Successful management of Bsal will likely require alteration of transmission routes and implementation of strategies that increase host resistance or tolerance to the pathogen (Wilber et al., 2017; Canessa et al., 2018; Islam et al., 2021).

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

The animal study was approved by The University of Tennessee Institutional Animal Care and Use Committee under protocol #2623. The study was conducted in accordance with the local legislation and institutional requirements.

EC: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Resources, Visualization, Writing – original draft, Writing – review & editing. JD: Conceptualization, Data curation, Formal analysis, Methodology, Resources, Writing – original draft, Writing – review & editing. MW: Conceptualization, Formal analysis, Investigation, Supervision, Writing – original draft, Writing – review & editing. DM: Conceptualization, Funding acquisition, Investigation, Supervision, Writing – original draft, Writing – review & editing. MG: Conceptualization, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Supervision, Writing – original draft, Writing – review & editing.