DETERMINING THE ENVIRONMENTAL SPORE LOAD OF PSEUDOGYMNOASCUS DESTRUCTANS A Thesis Presented to The Graduate Faculty of The University of Akron In Partial Fulfillment of the Requirements for the Degree Master of Science Charbel Elie Cherfan July, 2018 DETERMINING THE ENVIRONMENTAL SPORE LOAD OF PSEUDOGYMNOASCUS DESTRUCTANS Charbel Elie Cherfan Thesis Approved: Accepted: ______________________________ ______________________________ Advisor Department Chair Dr. Hazel Barton Dr. Stephen Weeks ______________________________ ______________________________ Committee Member Dean of Arts & Sciences Dr. Richard Londraville Dr. Linda Subich ______________________________ ______________________________ Committee Member Dean of the Graduate School Dr. Joel Duff Dr. Chand Midha ______________________________ Date ii ABSTRACT White Nose Syndrome (WNS) is a fungal disease that is causes high mortality in cave and mine hibernating bats. The disease is caused by a fungal pathogen Pseudogymnoascus destructans (Pd), which has spread throughout North America since its discovery in 2006. While Pd has been detected in the absence of bats, there are little data examining the role of humans’ act as a vector for the disease. To assess their role, I collected cave sediment, shoe and cloth samples and performed DNA analysis to establish the amount of detectable Pd in the samples examined by microscopy. While microscopy only detected Pd in two samples, qPCR detected Pd in all WNS positive sites. In all cases, the samples contained Pd loads below the current WNS decontamination guidelines. My data suggests that qPCR is semi-quantitative for identifying Pd in the environment. It is unable to distinguish between non-infectious vegetative cells and infectious spores and therefore an as effective an approach as microscopy to determine the potential for WNS infection. Levels of Pd found on clothing and shoe samples, and the inability of Pd to survive outside of the cave environment suggest that humans are unlikely to be effective vectors for Pd transport. iii TABLE OF CONTENTS Page LIST OF TABLES………………………………………………………………………...v LIST OF FIGURES………………………………………………………………………vi CHAPTER I. REVIEW OF LITERATURE………………………………….……………….....1 II. MATERIALS AND METHODS……………………………………..……...........7 Quantitative Polymerase Chain Reaction/Real-Time PCR (qPCR)…………..…..10 III. RESULTS………………..………………………………………………………12 IV. DISCUSSION…………………………………………………………..……..…22 LITERATURE CITED…………………………………………………………………..26 iv LIST OF TABLES Table Page 1. qPCR detection Efficiency IGS gene.……….…………………………………….......14 2. qPCR detection Efficiency Spores….. …………………………………….……….…16 v LIST OF FIGURES Figure Page 1. WNS Map………………………………………………………………………………5 2. Pd IGS region…………………………………………………………………………11 3. IGS gene standard……………………………………..………………………………13 4. Spore standard……………….….……………………………………………………..15 5. Amplification Plot……………………………………………………………………..17 6. MACA qPCR vs microscopy......……………………………………………………...19 7. CUGA qPCR vs microscopy…………………………………………………….....…20 8. Shoe spore load………………………………………………….……………...….….21 vi CHAPTER I REVIEW OF LITERATURE In the winter of 2006, during a routine bat survey in Howe Caverns, NY, scientists identified a large number of dead and dying bats (Veilleux 2008). In addition to dead bats, many bats displayed unnatural behavior, such as entrance roosting and flying outside of the cave in the middle of winter. All of the dead and diseased bats had a white fuzzy growth on their muzzle. The presence of this white material on infected bats led to the disease to be called White Nose Syndrome (WNS) (Blehert et al., 2009). This fuzz was later identified as a novel fungal pathogen, initially called Geomyces destructans, but later reclassified as a member of genus Pseudogymnascus (Blehert et al., 2009; Minnis and Lindner 2013). A defining characteristic of WNS is that it affects multiple species of bats; Pseudogymnoascus destructans (Pd) is a psychrophilic fungus, with preferred growth conditions between 2- 14 °C with a minimum relative humidity of 82%. As a result, the fungus is able to take advantage of any animal that hibernates under these conditions frequently found in mine and cave environments (Reynolds and Barton 2014). There are seven major North American bat species that have been demonstrated to develop WNS, Myotis lucifugus (Little Brown Bat), Myotis septentrionalis (Northern long-eared Bat), Myotis sodalis (Indiana Bat), Myotis grisescens (Gray Bat), Eptesicus fuscus (Big Brown Bat) and Perimyotis subflavus (Eastern pipistrelle) (Muller et al., 2013). All of these species that are susceptible to WNS hibernate in caves and mines 1 between 2-10°C, within the range for Pd growth (Blehert et al., 2009). Although other species appear to be infected, they do not develop the characteristic WNS pathology, these include Corynorhinus townsendii virginianus (Virginia Big Eared), Lasiurus borealis (Eastern red bat), Lassionycteris noctivagans (Silver-haired bat), Corynorhinus rafinesquii (Rafinesque’s big eared-bat), Myotis velifer (Cave bat) and Corynrhinus townsendii (Townsen’s big-eared bat). Possible reasons for low mortality rates include; the environment where the bats hibernate (for example, the Eastern red bat hibernates in trees and a natural immunity to infection (such as the Virginia big eared bat). Studies by Lorch et al., (2011) demonstrated that Pd is the causing agent of WNS using Koch’s Postulates. Koch’s Postulates are a series of tests that must be satisfied in order to demonstrate that a microorganism is responsible for a specific disease. The four tests are as follows: one, the microorganism must be present in all cases of the disease. Two, the microorganism can be isolated from the diseased host and grown in pure culture. Three, the pathogen from the pure culture must cause the disease when inoculated into a healthy host. Four, the pathogen must then be re-isolated from the new host and shown to be the same as the originally inoculated pathogen. Lorch et al. (2011) kept their environmental conditions as close to natural hibernacula conditions as possible, at 6.5°C and 82% humidity (Lorch et al., 2011). They inoculated wings of healthy bats with Pd, and used a sham-inoculated group of bats as a control that were placed in a separate cage (30 cm) from infected bats (Lorch et al., 2011). After 83 days, Pd infected bats began dying from the symptoms of WNS, whereupon the researchers were able to isolate pure culture of Pd from the diseased animals, satisfying 2 Koch’s postulates (Lorch et al., 2011). Despite their close proximity to the Pd infected bats, control bats had no symptoms of the disease (Lorch et al., 2011). The researchers then moved these controls bats into the same cage as the WNS-positive bats, and after an additional 83-100 days, these bats also started to die from WNS (Lorch et al., 2011). The Lorch et al. (2011) experiments demonstrated that Pd was the causative agent of WNS, but also suggested that Pd does not spread as easily as previously thought, and could only spread from an infected to un-infected animal that when they were in close proximity, through direct contact (Lorch et al., 2011). When researchers first identified WNS in bats it was unclear where it had come from, although French bat researchers had described a white, fuzzy material on the muzzles of bats as long ago as 1918 (Campana et al., 2017). This caused researchers in North America to compare Pd with the material found on the muzzles of European bats (Wernecke et al., 2012). The comparison revealed that the material on the European bats was also Pd, although no mass mortality rates are seen in European bats compared to North America bats (Puechmaille et al., 2011). However, the European strain of Pd still has a high mortality rate in North American bat species (Puechmaille et al., 2011). European bats are still infected with Pd, but without any of the pathology seen with WNS, besides epidermal white fuzz (Wernecke et al., 2012). This suggests that the European bats have an intrinsic resistance to WNS, possibly through a long evolutionary relationship between the pathogen and the host species (Wernecke et al., 2012). The close genetic relationship between European Pd and the ability of European isolates of Pd to kill North American bats supports the hypothesis that Pd was accidentally introduced to North America from Europe (Wernecke et al., 2012). 3 The experiments by Lorch et al. (2011) are the only evidence that direct contact is needed to spread WNS, and many researchers have questioned whether Pd could infect bats from the environment, or from contaminated clothing used by cave visitors (Reynolds and Barton 2014; Lorch et al., 2011). Studying the epidemiology of the spread of WNS suggests that it primarily follows the migration patterns of the bats, which follow the Appalachian Mountains south, suggesting predominantly bat-to-bat transfer (Figure 1); however, there have been some historic jumps of the pathogen, including outbreaks in West Virginia and Washington, which might be explained by vectored transport (Figure 1). 4 Figure 1: White Nose occurrence map (www.whitenosesyndrome.org) August 7th, 2017. 5 It remains unclear how Pd was first introduced to North America, but there are two hypotheses: the first is that a WNS infected bat found its way inside of a European shipping crate to North America; the second hypothesis is human vectored transport, in which cave visitors carried the fungus to North America on contaminated equipment, and infected either the hibernacula