to BioTechniques free email alert service to receive content updates.
Tracking a Bat Killer in Caves

10/31/2012
Sarah C.P. Williams

Last year, researchers identified the fungus responsible for white-nose syndrome in bats. Since then, they have continued studying it with the hope of saving dwindling bat populations. To do so, they’ve turned to a set of familiar tools: real-time PCR, culturing, and sequencing.  


Last year, researchers made a clear causal link between the fungus Geomyces destructans and white-nose syndrome, a disease that’s been sweeping through bat colonies in the northeastern part of North America since 2006, leaving bat casualties in its wake. But much about the fungus and how it causes disease in bats is still unknown. Now, a team of scientists has improved protocols using real-time PCR to detect the fungus more reliably—from both bat skin and the soil in bat caves. The techniques will help analyze how the fungus spreads and where it thrives.

Bats in a cave in New York have white noses indicative of infection by Geomyeces destructans. Source: New York Department of Environmental Conservation

Researchers studying Geomyces destructans over the past few years have relied on PCR primers complementary to the internal transcribed spacer (ITS) region of Geomyces destructans rRNA. However, because of fungi with similar ITS regions, they had to couple the PCR with sequencing of PCR products to confirm a diagnosis.

“Using that method, we had a few instances of cross-reaction with other fungi on bat skin which required follow-up DNA sequencing to resolve,” said Jeffrey Lorch of the University of Wisconsin-Madison. “And then we started screening environmental samples for the fungus, and with those tests we saw a lot more cross-reaction due to the presence of other species of fungi closely related to Geomyces destructans.”

With the overall goal of a more specific test that would work in soil samples as well as on samples from bats, Lorch and collaborators at the U.S. Geological Survey and U.S. Forest Service searched for a new portion of the genome to target with real-time PCR. After comparing Geomyces destructans rRNA sequences to that of other Geomyces species, the team chose the intergenic spacer (IGS) region as the new target. The new test correctly identified Geomyces destructans in 91 samples from bats with a known infection by the fungus, and discriminated the fungus from 54 closely related species (1).

Lorch and his colleagues went on to improve on previous soil tests for Geomyces destructans by culturing each of 24 samples—which originated from caves that bats hibernate in, both within the known range of white-nose syndrome as well as outside the range. PCR with follow-up DNA sequencing was used to identify each fungus that grew in culture. A total of 332 fungal isolates were recovered from the samples, including Geomyces destructans and as many as 35 other Geomyces species. This proof-of-concept experiment paves the way for future studies that require isolating the fungi from soil (2).

“Being able to use the environment as a way to survey whether the fungus is present in a cave is one aspect of it,” said Lorch. But the technique isn’t optimized for that use yet—researchers don’t yet know whether the presence of Geomyces destructans in cave soils will inevitably result in bats developing disease or whether caves themselves serve as the source of the infectious fungus.

The test will also be used to study the basic biology of Geomyces destructans. “We want to know whether the environment serves as a reservoir for this fungus,” he said. “We can now attempt to trace what happens to the fungus in the caves when the bats leave for the summer, for example.”

The team is planning additional studies to characterize how the interplay of bats, the environmental conditions in the caves they inhabit, and the pathogen Geomyces destructans lead to the development of white-nose syndrome, and why Geomyces destructans causes disease while other closely related fungal species appear to be harmless to bats.

References

  1. Muller LK, Lorch JM, Lindner DL, O’Connor M, Gargas A, Blehert DS. 2012. Bat white-nose syndrome: a real-time TaqMan polymerase chain reaction test targeting the intergenic spacer region of Geomyces destructans. Mycologia. Epub ahead of print, September 2012.
  2. Lorch JM, Lindner DL, Gargas A, Miller LK, Minnis AM, Blehert DS. 2012 A culture-based survey of fungi in soil from bat hibernacula in the eastern United States and its implications for detection of Geomyces destructans, the causal agent of bat white-nose syndrome. Mycologia. Epub ahead of print, October 2012.

Keywords:  PCR sequencing