Europe: Western Europe (France, Germany, UK, The Netherlands, Belgium, Spain)

The Americas: not known to be introduced yet (high at risk)

Africa: not known to be introduced yet

Australia: not known to be introduced yet

Introduction and Justification for Study Region

Batrachochytrium salamandrivorans (Bsal) is a fungal pathogen threatening global salamander populations, with significant concern for the U.S. west coast due to its high salamander diversity (Yap et al., 2017). The region harbors numerous endemic species, including those of the Salamandridae family, known to be highly susceptible to Bsal. The west coast’s extensive trade in amphibians increases the risk of Bsal introduction, making this region a priority for mitigation strategies (Gray et al., 2015; Richgels et al., 2016).

Justification for Itraconazole Use

Itraconazole is a broad-spectrum antifungal that has shown effectiveness in treating chytrid fungi, including Bsal. Studies indicate that itraconazole can successfully clear Bsal infections in salamanders. Plewnia et al. (2023) demonstrated the clearance of Bsal in Salamandra salamandra using daily 10-minute baths in a 0.01% itraconazole solution over 11 days, with no observed adverse effects. This evidence supports itraconazole as a potential emergency response tool for Bsal outbreaks. However, their study focused exclusively on S. salamandra, a European species, and did not evaluate the effects of itraconazole on North American salamanders, which may have different physiological responses. Our study will assess the efficacy and potential adverse effects of itraconazole in native west coast species to ensure its applicability for conservation interventions in this region.

Justification for Study Species Selection

The study will focus on the California newt (Taricha torosa) and the Ensatina salamander (Ensatina eschscholtzii)due to their ecological importance and potential susceptibility to Bsal.

1.     Ecological Relevance and Conservation Concerns

o   Taricha torosa is a key species in freshwater ecosystems, displaying high site fidelity and reliance on aquatic breeding, which increases its exposure to Bsal. Additionally, it faces threats from habitat degradation, climate change, and pollution (Kats & Ferrer, 2003). Laboratory trials have shown that Taricha torosa experiences lethal infections when exposed to Bsal, supporting its classification as a high-risk species (Richgels et al., 2016).

o   Ensatina eschscholtzii is a widespread terrestrial salamander, dependent on moist microhabitats. Although not fully aquatic, its potential to act as a Bsal carrier requires investigation. Studying both aquatic (T. torosa) and terrestrial (E. eschscholtzii) species allows for a broader understanding of how Bsal spreads and persists across different habitats.

2.     Management and Conservation Implications

o   If T. torosa proves highly susceptible, controlled treatment in captivity or in-situ itraconazole baths could be developed as a mitigation strategy.

o   If E. eschscholtzii exhibits lower susceptibility, differential treatment strategies may be necessary depending on the species and habitat type.

Research Questions – aims of the study

1.     What is the efficacy of itraconazole in clearing Bsal infections in Taricha torosa and Ensatina eschscholtzii?

2.     Does itraconazole treatment have adverse effects on the health and behavior of treated salamanders?

3.     What is the optimal treatment regimen (concentration and duration) for effective Bsal clearance?

Study Design

Location: The study will be conducted at a research facility in California, ensuring controlled conditions for infection and treatment trials.

Sample Size: A total of 60 individuals per species (120 total) will be divided into three groups of 20 (numbers choice: good statistical power):

·       Treatment group: receiving itraconazole baths

·       Control group: receiving water baths

·       Sham treatment group: receiving placebo baths

Methods:

·       Infection Protocol: Salamanders will be experimentally infected with a standardized dose of Bsal zoospores (Martel et al., 2013).

·       Treatment Regimen: Following infection confirmation via qPCR, the treatment group will receive daily 10-minute baths in a 0.01% itraconazole solution for 11 days (Plewnia et al., 2023).

·       Monitoring: Individuals will be monitored for clinical signs of chytridiomycosis, behavioral changes, and adverse reactions. Body weight and skin integrity will be recorded.

Data Collection and Analysis

·       Infection Status: Skin swabs will be collected at baseline, post-infection, mid-treatment, end of treatment, and weekly for four weeks post-treatment. Quantitative PCR will determine Bsal load (Boyle et al., 2004).

·       Health Assessments: Data on body weight, skin condition, and behavior will be statistically analyzed.

·       Statistical Analysis: Infection clearance rates and health parameters will be compared using ANOVA and chi-square tests (p<0.05).

Integration into management actions

Findings from this study will inform wildlife agencies on itraconazole’s practicality as an emergency response tool. If effective, treatment protocols could be applied in field settings through temporary captivity or in-situ treatment stations. Additionally, this study will contribute to developing targeted conservation strategies for high-risk species.

• Bsal is shown to most likely be transmitted through direct contact with infected individuals (Schmidt et al., 2017), with water-borne and soil transmission pathways also being a possibility. In this way, host population density has been found to play a significant role in the transmission dynamics of the disease where increasing population density can increase the probability of transmission and thus the potential for an outbreak to ensue (Chowdhury et al., 2025). Experimental studies on eastern newts to date have shown that at densities of 2 newts/sq meter, extinction still occurs (Chowdhury et al., 2025) . However, lower densities than this have not been investigated, where if transmission was greatly reduced could guide management of vulnerable populations in the face of a potential outbreak.

2. Species selected for the study:

• Large-blotched Ensatina (Ensatina eschscoltzii klauberi): This species can be found mainly in the peninsular ranges of southern California and part of the eastern San Bernadino Mountains. This ensatina prefers moist, shaded forest and oak woodlands, acting as a key mesopredator and prey species in these ecosystems. The species is already of special concern in California and experimental studies have shown that they are highly vulnerable to virulent Bsal infection (Gray et al., 2023).

3. Overall research objective:

   • To investigate whether a host density of less than two individuals per square meter can significantly reduce transmission rates of Bsal in the target species.

4. Study design:

   • Sample size (n=300)

   • Methodology:

     - Ensatinas will be housed in temperature-controlled tanks at different densities ranging from 0.5 to 8 individuals per m²

     - Individuals will be selected at random to initiate each epidemic after being exposed to a high dose of Bsal zoospores based on known ID-50 for the species (Gray et al., 2023).

     - Every 3 days, individuals are collected and swabbed to check Bsal infection status and infection load on the skin using qPCR.

   • Statistical analysis - Kaplan-Meier Analysis (Dudley et al., 2016)

5. Implications for future management: If found that at a certain density threshold that transmission is greatly reduced then this could be a viable option for in-situ management of a population during an outbreak. For example, translocating individuals such that the population reduces below the minimum threshold required for disease propagation. This would be monitored by continual population counts in these regions.

   
   

   This could also help improve understanding of transmission dynamics at more natural densities.

It has been recorded that BSal evolves rapidly during an outbreak, gaining new genetic functions, for example a saprotrophic ability.

Sequencing during an epidemic would allow analysis of these changes and therefore predictions to be considered regarding potential environmental contamination, environmental niches, changes between infections in captive and wild species and isolate specific life cycles. As Bd shows a variety of of environmental impacts associated with different strains, so it can be hypothesized that BSal will show the same.

2) Methods:

Sample wild-caught and captive (zoo/domestic owned) animals showing signs of infection. Enrichment for and sequencing of genomes would give results that could then be matched with previously reported data. Sequence capture technology has been shown to be useful in these studies (K Mulder et al 2024).

Samples would be taken at varying temporal intervals, ie 0, 1, 7, 30 and 100 days during an outbreak. This is to assess how rapidly the genome may or may not change. Ideally individuals are sampled are either captured or at least marked, so that repeated samples from the same individuals, and therefore direct comparison of the Bsal genome, can be made.

Enough individuals should be sampled so that trends in any genome changes can be monitored, ideally at least 100.

The individual characteristics including severity of signs, body condition, environmental conditions etc should also be recorded against each Bsal genome recording, as this will allow for trends to be studied. I.e. are individuals within a certain humidity/temperature microclimate more susceptible to certain genome sequences, are certain genome changes more virulent, etc.

3) Findings' effect on management options:

Early detection and determination of variant could allow for more effective mitigation measures to be put in place.

(M Kelly et al, 2021, K Mulder et al, 2024)

For example, this may also help dictate certain susceptibilities to anti fungal treatments such as itraconazole (see other research question), predict potential reservoir hosts, and allow us to know optimum temperature ranges for the fungus. This knowledge would therefore aid management in controlling environmental temperatures, trying to separate species populations, etc., in order to prevent spread as much as possible.

Refs.

Diversity, multifaceted evolution, and facultative saprotrophism in the European Batrachochytrium salamandrivorans epidemic, Moira Kelly, Frank Pasmans, Jose F. Muñoz, Terrance P. Shea, Salvador Carranza, Christina A. Cuomo & An Martel 

Nature Communications volume 12, Article number: 6688 (2021)

Sequence capture identifies fastidious chytrid fungi directly from host tissue

Author links open overlay panel. Kevin P. Mulder ^{a b}, Anna E. Savage ^c, Brian Gratwicke ^d, Joyce E. Longcore ^e, Ed Bronikowski ^d, Matthew Evans ^d, Ana V. Longo ^f, Naoko P. Kurata ^{b g h}, Tim Walsh ^d, Frank Pasmans ^a, Nancy McInerney ^b, Suzan Murray ^d, An Martel ^a, Robert C. Fleischer ^b, Fungal Genetics and Biology

Volume 170, February 2024, 103858