Experimental evaluation of genomic DNA degradation rates for the pathogen Pseudogymnoascus destructans (Pd) in bat guano

Abstract

Pseudogymnoascus destructans (Pd), the causative agent of white-nose syndrome in bats (WNS), has led to dramatic declines of bat populations in eastern North America. In the spring of 2016, WNS was first detected at several locations in Washington State, USA, which has prompted the need for large scale surveillance efforts to monitor the spread of Pd. Pd is typically detected in bats using invasive methods requiring capturing and swabbing individual bats. However, Pd can also be detected in guano, which may provide an efficient, affordable, and noninvasive means to monitor Pd in bats across North America. The widespread implementation of Pd surveillance in guano is hindered by substantial uncertainty about the probability of detecting Pd when present, and how this probability is influenced by the time since defecation, local environmental conditions, the amount of guano sampled, and the original concentration of DNA shed in the guano. In addition, the expected degradation rate of Pd DNA depends on whether the Pd DNA found in guano represents extracellular DNA fragments, intracellular DNA from dead Pd fungal cells, or from intracellular and viable Pd cells. While this is currently unknown, it has been posited that most environmental DNA, such as Pd found in guano long after defecation, is fragmented extracellular DNA. Using non-viable isolated DNA at precise quantities, we experimentally characterized the degradation rates of Pd DNA in guano samples. We spiked 450 guano samples with Pd gDNA in a 10-fold dilution series from 1 million to 1,000 fg and placed them in variable environmental conditions at five sites at Mount Rainier National Park in Washington State, which is a priority location for Pd surveillance. We evaluated DNA degradation over 70 days by quantifying the amount of DNA in samples collected every 14 days using real-time quantitative PCR (qPCR). Our sampling period was from July 10th to September 17th 2018 which overlaps with bat movement between summer roosts as well as movement from maternity colonies fall swarms. We detected Pd DNA in guano 56 and 70 days after inoculation with 1 million and 100,000 fg respectively, while the lowest quantity (1,000 fg) was detected until 42 days. Detection probability was variable among sites and lower where samples were left exposed without overhead cover. If Pd is shed as extracellular DNA in guano at quantities above 1,000 fg, then guano collection is likely to provide an effective tool for environmental screening of Pd that can be employed in an early detection and rapid response framework throughout Washington and other regions where this disease is rapidly emerging.

Pseudogymnoascus destructans growth in wood, soil and guano substrates

“…Detection of Pseudogymnoascus destructans through monitoring and surveillance is performed by detecting DNA extracted from swabs or environmental samples using qPCR. However, samples can be qPCR positive either because of extracellular DNA 39 , DNA from dead cells, or DNA from live fungus, but only live fungus is infectious. Therefore, assessing the growth and viability of pathogens such as Pd on different substrates can provide insight about the role of environmental substrates as reservoirs to inform management of potential fomites implicated in the spread of the disease.…”

“…We interpreted an increase in DNA concentration as evidence of Pd growth and stable levels as evidence of Pd persistence. Detection of Pd DNA does not necessarily imply that Pd is alive and pathogenic 39 . For each day corresponding to a sampling event (i.e.…”

“…Pd is persistent in hibernacula in the United States while in Eurasian hibernacula the prevalence and pathogen loads decrease due to a seasonal decay of Pd during summer that leads to delayed infection, shorter period of Pd growing on bats and low loads 22 . Although transmission of Pd has been linked to colony size and clustering 32 , the role played by particular substrates as environmental reservoirs on disease impacts is only partially understood 39 .…”

Population genetics as a tool to elucidate pathogen reservoirs: Lessons fromPseudogymnoascus destructans, the causative agent of white-nose disease in bats

“…These conditions have been shown to allow the persistence of viable P. destructans spores on abiotic substrate for at least 2 years (Fischer et al, 2020). In addition to walls being likely abiotic reservoirs, sediments as well as bat guano have been suggested to be possible sources of P. destructans environmental infection (Lorch et al, 2013; Urbina et al, 2020). However, biotic interactions between fungi and other microorganisms are most likely stronger in sediments/guano compared to hibernacula walls, making sediments/guano a challenging environment for P. destructans to survive in and grow (Zhang et al, 2014, Wilson et al, 2017, Urbina et al, 2021).…”

Population genetics as a tool to elucidate pathogen reservoirs: Lessons fromPseudogymnoascus destructans, the causative agent of White‐Nose disease in bats

“…In addition to walls, sediments as well as bat guano have been suggested to be possible sources of P. destructans environmental infection (Lorch et al, 2013;Urbina et al, 2020). However, biotic interactions between fungi and other microorganisms are most probably stronger in sediments/guano compared to hibernacula walls, making sediments/guano a challenging environment for P. destructans to survive in (Urbina et al, 2021;Wilson et al, 2017;Zhang et al, 2014).…”