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Systematic approach to isolating Batrachochytrium dendrobatidis.

Anthony W Waddle1, Marlai Sai, Joshua E Levy

  • 1School of Life Sciences, University of Nevada, Las Vegas, 4505 S. Maryland Parkway, Las Vegas, NV 89154, USA.

Diseases of Aquatic Organisms
|March 9, 2018
PubMed
Summary

We developed a new protocol to successfully isolate the amphibian chytrid fungus, Batrachochytrium dendrobatidis (Bd), from frogs. Focusing on foot tissue significantly improved isolation success, aiding Bd infection research.

Keywords:
AmphibianChytridiomycosisFungusIsolationProtocol

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Area of Science:

  • Herpetology
  • Mycology
  • Infectious Diseases

Background:

  • The amphibian chytrid fungus, Batrachochytrium dendrobatidis (Bd), causes chytridiomycosis, a devastating disease affecting amphibian populations worldwide.
  • Accurate detection and isolation of Bd are crucial for understanding its transmission dynamics and developing effective conservation strategies.
  • Previous studies suggest Bd concentrates in specific skin regions, but standardized isolation protocols are lacking.

Purpose of the Study:

  • To develop and validate a reliable protocol for isolating Batrachochytrium dendrobatidis (Bd) from anuran skin samples.
  • To compare the efficacy of Bd isolation from different anatomical regions (thigh, abdomen, foot) in treefrogs.
  • To assess the impact of infection intensity on the success rate of Bd isolation.

Main Methods:

  • Developed a novel protocol involving skin tissue sampling from ventral regions of Pseudacris regilla and P. triseriata.
  • Utilized a combination of four antibiotics to minimize bacterial contamination during fungal isolation.
  • Quantified Bd infection levels using zoospore equivalents (ZE) via swabbing and quantitative real-time PCR.
  • Isolated Bd through culturing on specific media.

Main Results:

  • Successfully isolated Bd from 68.9% of sampled frogs across both species.
  • Achieved low contamination rates (9.7%), primarily fungal.
  • Demonstrated a positive correlation between Bd infection intensity (≥40 ZE) and successful isolation.
  • Observed significantly higher Bd isolation success rates from foot tissue compared to thigh and abdomen samples.
  • Isolation success reached 74% for P. triseriata and 100% for P. regilla at higher infection intensities.

Conclusions:

  • The developed protocol is effective for isolating Bd from anurans, particularly from foot tissue.
  • Sampling frog feet, especially webbing, is recommended for maximizing Bd isolation success.
  • This method aids in disease surveillance and research on Bd epidemiology and impacts on amphibian populations.