Department of Fisheries and Wildlife, Oregon State University, 2820 SW Campus Way, Nash Hall, Corvallis, OR, 97331, USA.
National Park Service, Mount Rainier National Park, Ashford, WA, USA.
Sci Rep. 2021 Jan 12;11(1):763. doi: 10.1038/s41598-020-80707-1.
Understanding how a pathogen can grow on different substrates and how this growth impacts its dispersal are critical to understanding the risks and control of emerging infectious diseases. Pseudogymnoascus destructans (Pd) causes white-nose syndrome (WNS) in many bat species and can persist in, and transmit from, the environment. We experimentally evaluated Pd growth on common substrates to better understand mechanisms of pathogen persistence, transmission and viability. We inoculated autoclaved guano, fresh guano, soil, and wood with live Pd fungus and evaluated (1) whether Pd grows or persists on each (2) if spores of the fungus remain viable 4 months after inoculation on each substrate, and (3) whether detection and quantitation of Pd on swabs is sensitive to the choice to two commonly used DNA extraction kits. After inoculating each substrate with 460,000 Pd spores, we collected ~ 0.20 g of guano and soil, and swabs from wood every 16 days for 64 days to quantify pathogen load through time using real-time qPCR. We detected Pd on all substrates over the course of the experiment. We observed a tenfold increase in pathogen loads on autoclaved guano and persistence but not growth in fresh guano. Pathogen loads increased marginally on wood but declined ~ 60-fold in soil. After four months, apparently viable spores were harvested from all substrates but germination did not occur from fresh guano. We additionally found that detection and quantitation of Pd from swabs of wood surfaces is sensitive to the DNA extraction method. The commonly used PrepMan Ultra Reagent protocol yielded substantially less DNA than did the QIAGEN DNeasy Blood and Tissue Kit. Notably the PrepMan Ultra Reagent failed to detect Pd in many wood swabs that were detected by QIAGEN and were subsequently found to contain substantial live conidia. Our results indicate that Pd can persist or even grow on common environmental substrates with results dependent on whether microbial competitors have been eliminated. Although we observed clear rapid declines in Pd on soil, viable spores were harvested four months after inoculation. These results suggest that environmental substrates and guano can in general serve as infectious environmental reservoirs due to long-term persistence, and even growth, of live Pd. This should inform management interventions to sanitize or modify structures to reduce transmission risk as well early detection rapid response (EDRR) planning.
了解病原体如何在不同基质上生长，以及这种生长如何影响其传播，对于理解新发传染病的风险和控制至关重要。假丝酵母菌（Pd）会导致许多蝙蝠物种患上白鼻综合征（WNS），并能在环境中存活并传播。我们通过实验评估了 Pd 在常见基质上的生长情况，以更好地了解病原体的持久性、传播和生存机制。我们将活的 Pd 真菌接种到消毒的粪便、新鲜粪便、土壤和木材中，并评估了以下内容：（1）Pd 是否在每种基质上生长或存活；（2）如果将真菌孢子接种到每种基质上 4 个月后，孢子是否仍然具有活力；（3）在两种常用 DNA 提取试剂盒中，选择哪种试剂盒对拭子上的 Pd 检测和定量更敏感。在将每种基质接种 46 万个 Pd 孢子后，我们每隔 16 天从每个基质中收集约 0.20 克粪便和土壤，并从木材上采集拭子，通过实时 qPCR 随时间量化病原体负荷。我们在整个实验过程中都检测到了所有基质上的 Pd。我们观察到，在经过消毒的粪便上，病原体负荷增加了十倍，且保持了持久性，但在新鲜粪便上则没有出现生长。在木材上，病原体负荷略有增加，但在土壤中下降了约 60 倍。四个月后，我们从所有基质中收获了显然具有活力的孢子，但从新鲜粪便中收获的孢子未能发芽。我们还发现，从木材表面拭子中检测和定量 Pd 对 DNA 提取方法很敏感。与 QIAGEN DNeasy Blood and Tissue Kit 相比，常用的 PrepMan Ultra Reagent 方案产生的 DNA 要少得多。值得注意的是， PrepMan Ultra Reagent 未能检测到许多被 QIAGEN 检测到的木材拭子中的 Pd，而这些拭子随后被发现含有大量活的分生孢子。我们的结果表明，Pd 可以在常见的环境基质上存活或甚至生长，其结果取决于是否已经消除了微生物竞争者。尽管我们观察到 Pd 在土壤中的快速下降，但在接种四个月后仍收获了有活力的孢子。这些结果表明，由于 Pd 的长期存活和甚至生长，环境基质和粪便通常可以作为传染性环境储库。这应告知管理干预措施，以进行消毒或修改结构，以降低传播风险以及早期检测快速反应（EDRR）规划。
