ROADWAY-ASSOCIATED CULVERTS MAY SERVE AS A TRANSMISSION CORRIDOR FOR PSEUDOGYMNOASCUS DESTRUCTANS AND WHITE-NOSE SYNDROME IN THE COASTAL PLAINS AND COASTAL REGION OF GEORGIA, USA

Historically, pathogenic fungi have been associated with reduced plant health and crop loss, but only recently have they been recognized as a pressing threat to animal health (Daszak et al. 2000; Fisher et al. 2012, 2016). Fungal pathogens have diverse and resilient reproductive mechanisms, facilitating survival in harsh environments until a suitable host becomes available. This ability has enabled the wide spread of several fungal pathogens (Fisher et al. 2012). One emerging fungal pathogen is Pseudogymnoascus destructans, the causative agent of white-nose syndrome (WNS) in North American bats (Blehert et al. 2009; Fisher et al. 2012).

Pseudogymnoascus destructans is a psychrophilic ascomycete that is able to grow between 0 C and 20 C, with an optimal growth range of 12.5–15.8 C, making it well suited for the same environments bats use for hibernation (Blehert et al. 2009; Turner et al. 2011; Verant et al. 2012). First observed in the US in 2006, WNS has been detected in 34 American states and seven Canadian provinces. This fungal infection has been associated with precipitous declines in cave-dwelling bat species such as little brown bat (Myotis lucifugus), northern long-eared bat (Myotis septentrionalis), and tricolored bat (Perimyotis subflavus; Blehert et al. 2009). Because of WNS, M. septentrionalis is currently listed as federally threatened throughout its range, and similarly affected species such as P. subflavus are proposed for federal listing. Clinical manifestation of WNS initiates an array of physiologic responses leading to hyperkalemia, chronic respiratory acidosis, and premature depletion of the bat's energy reserves, which often is fatal (Verant et al. 2014). Models predicted that affected populations could experience a 99% regional population collapse, resulting in the loss of >5.5 million bats (Frick et al. 2010). It has been found that P. destructans is transmitted through bat-to-bat or bat-to-substrate contact (Lorch et al. 2011; Hoyt et al. 2015; Ballman et al. 2017), in addition to suspected human-mediated transmission. Therefore, bat roosting preference, especially in anthropogenic structures, is important because it has the potential to influence pathogen transmission. Hibernation strategies vary across species, each fulfilling a specific physiologic need (Brack 2007), with some hibernating species, such as M. lucifugus, roosting in large, tightly packed clusters to provide a warm, thermally stable environment to minimize the amount of energy required to rewarm during arousal. However, although transmission rates are probably elevated among species that roost in clusters, because of constant bat-to-bat contact during the hibernation season, transmission within hibernacula is complex and expands across species and roosting behaviors (Hoyt et al. 2018).

It has been well documented that various bat species, including P. subflavus, select anthropogenic structures such as roadway-associated bridges as winter roosting sites (Keeley and Tuttle 1999; Geluso and Mink 2009; Allen et al. 2011; Bergeson et al. 2015). Researchers in various parts of the US have observed large numbers of P. subflavus and other bat species roosting in roadway-associated culverts (Walker et al. 1996; Meierhofer et al. 2019). The characteristics of roadway-associated structures typically selected by various bat species for roosting include concrete construction material, location in relatively warm areas, and the presence of crevices (Keeley and Tuttle 1999). Roosting orientation of various bat species within anthropogenic structures has also been well documented: P. subflavus has been observed roosting primarily in free-hanging orientations in open spaces within anthropogenic structures, unlike the southeastern myotis (Myotis austroriparius) which has been observed roosting primarily in clusters within deep crevices (Keeley and Tuttle 1999; Ferrara and Leberg 2005). Roosting orientation of bat species within anthropogenic structures could be a key factor influencing disease manifestation and severity in P. destructans infected populations, because crevices within these structures have been observed to maintain higher, more stable temperatures (Keeley and Tuttle 1999; Meierhofer et al. 2019). These stable microclimates provide ideal conditions for P. destructans growth, which could result in severe infections in bats selecting to roost in crevices of anthropogenic structures (Meierhofer et al. 2019).

We investigated bat use of roadway-associated culverts and the suitability of culverts as roosting sites during the hibernation season, with a focus on P. subflavus, a species known to hibernate for more consecutive months than other species, and which has been heavily affected by WNS (Davis 1964; Fujita and Kunz 1984; Blehert et al. 2011). The species is also currently proposed for federal listing under the Endangered Species Act because of its rapid decline since the detection of WNS (USFWS 2017). Understanding how P. subflavus uses roadway-associated culverts during the hibernation months will help characterize critical hibernation habitat.

We selected the coastal plains and coastal region of Georgia, US, for investigation because of their regional location and topographic variation. These regions are located directly south of P. destructans–positive counties in northern Georgia and provide a northern border to counties in northern Florida where naive P. subflavus populations hibernate, often in caves that are conducive to WNS (Sirajuddin 2018). These regions also have been largely neglected in surveys for bats and are characterized by starkly different topographic features when compared with the northern region of the state. Traditional bat hibernacula are located in the Blue Ridge ecoregion of northern Georgia, and are characterized by numerous karst cave structures and forested habitat. Since WNS was first detected in Georgia in the winter of 2013–14, all P. destructans–positive detections have been in counties located in this region (Fig. 1). In contrast, the coastal plains and coastal region of Georgia are characterized by sandy soil, open pine forests, and minimal karst structures (Griffith et al. 2001). Given the varying topography between the northern and southern regions of the state, the risk of pathogen transmission was thought to be minimal. However, anthropogenic structures, such as roadway-associated culverts, may facilitate bat and P. destructans movement between the karst cavernous region of northern Georgia and the seemingly suboptimal bat habitat of coastal Georgia (Fig. 1).

Our objectives were to understand the manner in which populations of P. subflavus used roadway-associated culverts, determine whether environmental conditions of culverts are favorable for P. destructans growth, and explore the spatial spread of P. destructans in the coastal and coastal plains regions of Georgia. Understanding how WNS-susceptible species, such as P. subflavus, use roadway-associated structures is critical to understanding pathogen dispersal and identifying potentially vulnerable populations.

Culvert surveys were conducted from December 2017 to March 2018 in the coastal and coastal plains regions of Georgia along Interstate 16, Interstate 95, and Interstate 75. When conditions allowed, surveyors randomly selected nearby smaller roadways for additional surveys. All surveys were conducted with a standard Georgia Department of Natural Resources (GADNR) datasheet and associated guidelines. Every culvert encountered was surveyed unless the height was less than 0.75 m (2.5 ft) or the water level was too high to navigate safely. Data collected during each survey included bat presence, species, roosting orientation, and location within the structure. Decontamination was conducted after examination of each culvert in accordance with the US Fish and Wildlife Service (USFWS) decontamination protocol (White-nose Syndrome Disease Management Working Group 2017).

Throughout the 2017–18 hibernation season, we surveyed 96 culverts for the presence of bat species across the coastal plains and coastal region of Georgia. Three species of special consideration (state designation of rare, endangered, threatened, or of concern) were observed roosting in surveyed culverts in varying numbers: P. subflavus, M. austroriparius, and Rafinesque's big-eared bat (Corynorhinus rafinesquii). Bats were documented roosting hanging on the wall or ceiling of the culvert (referred to as a free-hanging [FH] orientation) or inside cylindrical holes in the culvert ceiling called weep holes (referred to as a weep hole [WH] orientation). The unique roosting orientation and species observed during the 2017–18 hibernation season served as criteria for selection of 12 representative culverts for environmental monitoring. Representative culverts were selected on the basis of P. subflavus observations, the number of individual bats recorded, the most frequently observed roosting orientation, and location within the coastal plains or coastal region. The selected culverts were grouped into four blocks by geographic region, with each block containing three representative culverts. One representative culvert in each block in which bats had been observed roosting inside weep holes, one in which bats had been observed roosting in an FH orientation on the wall or ceiling, and one in which no bats had previously been observed (Fig. 2). We used the term “control culvert” for previously surveyed culverts where no bats were present. Three blocks (nine representative culverts) were selected on the basis of P. subflavus observations, and one block (three representative culverts) was selected on the basis of M. austroriparius observations. Each of the 12 representative culverts were surveyed monthly for bat presence, from June 2018–May 2019, with some exceptions because of adverse weather conditions.

Three temperature data loggers (HOBO 64K Pendant, Onset Computer Corporation, Bourne, Massachusetts, USA) were placed within each culvert: one in the center and one at each end. The loggers were placed in orientations mimicking bat roosting positions previously observed. In FH culverts, three temperature loggers were attached to an approximately 15×7-cm, 2-cm-thick wooden block with an eye screw and a carabiner; the wooden blocks were adhered to the culvert walls with 100% silicone caulk (GE Silicone, General Electric, Boston, Massachusetts, USA; Fig. 3). In WH culverts, two temperature loggers were mounted on the culvert wall as described earlier, and one was placed in a weep hole with a bolt and toggle, with mesh securing the weep hole opening to prevent bats from entering. Loggers were programmed to record temperature at a 10-min interval by the HOBO-ware Pro software (3.17.16, Onset Computer Corporation). Recorded temperature data were downloaded during monthly surveys with a HOBO Waterproof Data Shuttle. Device battery capacity and free memory were monitored, and batteries were replaced as needed. Temperature loggers were deployed June and July 2018, and temperatures were recorded until May 2019.

All bat handling was conducted under the Federal Collection Permit held by Katrina Morris of the GADNR. Bats were handled during culvert surveys to swab for the presence of P. destructans. All bat handling was conducted following the Range-wide Indiana Bat Survey Guidelines for safe bat handling (USFWS 2020). Wings were examined for any signs indicative of WNS, such as lesions or chafing (Reichard 2017). Bats were banded by placing a numbered band on the forearm; all band numbers and associated information were submitted to the GADNR.

During each hibernation season (2017–18 and 2018–19), swabs (Medline MDS202000 6-inch sterile cotton tipped applicators, Medline Industries, Northfield, Illinois, USA) were collected of culvert structures (substrates) and of bats found within the culvert, in accordance with the US Geological Survey National Wildlife Health Center (2019) protocol. All swabs collected in 2017–18 were suspended in 250 µL cetyltrimethylammonium bromide DNA extraction buffer (OPS Diagnostics, Lebanon, New Jersey, USA) in sterile 2-mL Eppendorf tubes (BRAND® micro-centrifuge tube, Millipore Sigma, Burlington, Massachusetts, USA). All swabs collected in 2018–19 were stored dry in sterile 2-mL Eppendorf tubes. All swabs were stored in a cooler while in the field and transferred to a –80 C freezer on arrival at the laboratory. For each swab collected, the date, culvert site number, swab type (bat or substrate), species code, and band number (if present) were documented.

We used descriptive statistical analyses to determine the potential influence of site characteristics and roost orientations. A one-tailed t-test was used to assess any differences in the number of P. subflavus observed roosting in WH and FH orientations between surveyed culverts between June 2018 and May 2019. Data from each temperature logger was grouped into 24-h segments (i.e., each day), then averaged. Calculated daily averages were used for further analyses. A single-factor analysis of variance (ANOVA) was used to assess the variance observed between the three temperature loggers within each culvert. The average daily temperature recorded by WH loggers was compared with the average daily temperature recorded by FH loggers within each individual culvert by a two-tailed paired t-test. Variance analysis and a two-tailed paired t-test were used to compare the variances of average daily temperatures collected by all WH loggers and FH loggers. After establishing the novelty of the temperature collected inside weep holes, a single-factor ANOVA was used to examine any variance between the average daily temperatures collected by the four WH loggers. Tukey's test was used to determine which of the four WH data loggers contributed to a significant difference observed in the single-factor ANOVA. All statistical analyses were conducted by Excel (2019, 16.0, Microsoft Corporation, Redmond, Washington, USA) and SAS (version 5.2, SAS Institute, Cary, North Carolina, USA).

Surveys were conducted in the selected 12 representative culverts on a monthly basis between June 2018 and May 2019, except July and August 2018. Roosting P. subflavus were observed in roadway-associated culverts in all calendar months except June and September (1–38 individuals/mo), whereas M. austroriparius and C. rafinesquii were observed roosting during every calendar month in varying numbers. The highest number of P. subflavus was observed in January and February 2019 (Fig. 4).

Half of the selected 12 representative culverts were located in the coastal plains region and the other half in the coastal region of Georgia. A consistently higher number of P. subflavus were observed in roadway-associated culverts in the coastal region than in the coastal plains region. During the 2017–18 hibernation season, 76% of P. subflavus were observed roosting in a clustering orientation inside weep holes. In any given winter month, more P. subflavus were recorded clustering in weep holes (population mean µ=9.33) than hanging on the culvert wall (µ=1.78). In February 2019, significantly more P. subflavus was observed roosting inside weep holes than hanging on the culvert walls (P<0.05).

Temperature was recorded for 321 d between June 2018 and May 2019; equipment malfunction occurred in four data loggers, producing gaps in temperature data for four representative culverts. During December, January, and February, the average daily temperatures in all 12 representative culverts fell within the P. destructans growth range of 0–20 C (Blehert et al. 2009; Verant et al. 2012). Such temperatures were recorded for 163–200 d within each representative culvert, with a mean±SD of 172.7±11.7 d. Additionally, the average daily temperatures within all 12 culverts fell within the optimal range for P. destructans growth during October, November, and December 2018 and January, February, and March 2019. Temperatures were recorded in the optimal growth range for 50–80 d (57.4±9.3 d). Temperatures within the P. destructans optimal growth range accounted for approximately 28–43% of days (mean 33.2%) recording temperatures below 20 C. The mean number of days with temperatures 0–20 C were similar in the coastal plains and coastal regions: 176.7±3.2 d and 168.7±14.4 d, respectively. Almost an identical average number of days spent in the optimal growth range were observed in culverts in the coastal plains and coastal regions: 57.2 d and 57.7 d, respectively.

Temperatures recorded inside weep holes were within the P. destructans growth range for 126–194 d (164.6±28.8 d). Furthermore, temperatures recorded in weep holes were within the optimal growth range of P. destructans for 36–112 d (63.5±34.1 d). Within weep holes, approximately 26–57% of all days recording temperatures under 20 C were within the optimal growth temperature range. Temperature loggers deployed inside weep holes in the coastal plains region recorded temperatures within the P. destructans growth range for an average of 160 d, compared with an average of 169.5 d in the coastal region. Temperature loggers deployed inside weep holes in the coastal plains recorded temperatures within the optimal P. destructans growth range for an average of 74 d, compared with an average of 53 d in the coastal region. The average daily temperature measured within the weep hole of one coastal plains culvert was significantly higher than the average daily temperature measured within the other three WH culverts. The average daily temperature measured in the remaining three WH culverts were not significantly different from each other.

The temperatures inside all weep holes differed significantly (P<0.05) from their free-hanging counterparts (Fig. 5). The average daily temperature collected inside weep holes was consistently higher than free hanging. The variance measured between average daily temperatures collected inside a weep hole was significantly different from its hanging counterpart in only one culvert. However, a pattern of lower average daily temperature variance collected inside weep holes, compared with those collected on the culvert wall, held true for all representative culverts surveyed (Tables 1, 2).

We collected 249 swabs from bats and culverts surveyed in the coastal plains and coastal regions of Georgia between 2017 and 2019. None tested positive for P. destructans.

Historically, P. subflavus has been documented roosting solitarily (Fujita and Kunz 1984; Sandel et al. 2001; Brack 2007; Vincent and Whitaker 2007). We consistently observed P. subflavus roosting in clusters of 2–10 individuals inside weep holes. This observation was particularly unexpected because of the many descriptions specifically characterizing P. subflavus as a strictly solitary roosting species (e.g., Fujita and Kunz 1984; Sandel et al. 2001; Brack 2007; Vincent and Whitaker 2007). This roosting behavior by a species traditionally considered solitary raises serious concerns regarding the potential transmission of P. destructans and development of WNS within culverts. Pathogen transmission rates among species that roost in clusters are higher because of constant contact during the hibernation season, although solitary roosting species do exhibit pathogen transmission via cryptic connections within hibernacula (Hoyt et al. 2018). Species that commonly exhibit clustering behavior, such as M. lucifugus and M. septentrionalis, have experienced rapid pathogen transmission and population declines. In contrast, P. subflavus has experienced slower pathogen transmission, and associated population declines were not observed for several years after initial pathogen detection (Hoyt et al. 2018). Additionally, M. austroriparius were often recorded roosting in the same culvert as P. subflavus. Myotis austroriparius is commonly observed roosting in large clusters and may act as an asymptomatic carrier for P. destructans. This species remains active all year, rarely experiencing deep torpor and potentially spreading P. destructans conidia during the winter months.

Temperatures within roadway-associated culverts fell within the growth range of P. destructans for an equivalent combined duration of 5 mo (172 d), indicating the potential for fungal growth and host infection during almost half of a calendar year. Within this time, the equivalent of almost 2 mo (57 d) saw temperatures within the optimal range for P. destructans growth. As expected, the average daily temperatures of coastal plains culverts were typically lower than culverts in the coastal region (Grider et al. 2016). Further investigation with a larger sample size will be required to characterize temperature patterns within culverts related to specific geographic regions.

Our data suggest that bats roosting inside weep holes during the critical winter months are exposed to a unique microclimate that differs from the rest of the culvert. Weep holes also offer more thermal stability, an environmental characteristic often appealing to a torpid mammal relying on fat reserves for survival (Hayman et al. 2016). Although weep holes were warmer than the rest of the culvert, their average daily temperatures remained within the optimal growth range of P. destructans, with less fluctuation. Maintaining temperatures in the 12.5–15.8 C range for a multiweek duration provides an adequate environment for efficient P. destructans growth within the weep hole microhabitat (Verant et al. 2012). Thus, culvert weep holes may provide a suitable microhabitat for the proliferation of P. destructans and the development of WNS. However, punctuated temperatures outside the published growth range of P. destructans were documented within culverts. It is unclear how this environment might influence the growth of P. destructans and the development of WNS; therefore, it should be further investigated to fully appreciate the potential of these sites to support P. destructans growth and dispersal and the development of WNS. Studies on the potential for summer dispersal of viable P. destructans by euthermic bats, as well as human accessories, have recovered culturable P. destructans during July in Tennessee, Kentucky, Virginia, Indiana, and Ohio, suggesting that the thermal tolerances of P. destructans within natural systems may be broader than values reported for laboratory studies (Ballman et al. 2017).

The disease triangle is a concept that suggests an inherent relationship between the host, pathogen, and environment (McNew 1960). This model is used to explain how host susceptibility, environmental conditions and variation, and pathogen virulence influence disease occurrence and outcomes. The disease triangle model is crucial to developing a complete understanding of WNS by identifying environmental and host factors that exacerbate or preclude disease processes. This understanding will inform the development of meaningful mitigations and facilitate implementation in a targeted and effective manor. Clear identification of a potential roosting environment could aid in the identification of susceptible bat populations and the prediction of pathogen transmission corridors as potential P. destructans reservoirs, and preemptive implementation of mitigation efforts could increase survivorship of affected bat species, as well as help preserve P. destructans–free areas.

Our data confirm that these culvert structures provide a desirable roosting environment for a highly susceptible host species, P. subflavus, during the hibernation season and suggests that roadway-associated culverts in the southern region of Georgia can provide a suitable environment for the survival and proliferation of P. destructans and the development of WNS in susceptible species such as P. subflavus. Our data also suggest that the average daily temperature within culverts is highly conducive to P. destructans growth, although the influence of punctuated elevated temperatures on P. destructans is unknown and represents a principal limitation of our study. Disease severity could also be exacerbated because of the previously unreported clustering behavior observed in P. subflavus roosting under these conditions, resulting in increased risk of pathogen transmission. Although P. destructans was not detected during the course of this investigation, it is evident that roadway-associated culverts within the coastal plains and coastal region of Georgia provide ideal host and environmental conditions for pathogen establishment and disease development and may offer a unique and consequential pathogen transmission corridor between suitable habitat patches. This situation raises major concerns because these roadways directly connect WNS-positive counties in northern Georgia with counties in northern Florida containing P. subflavus populations currently unaffected by WNS.

Supplementary material for this article is online at http://dx.doi.org/10.7589/JWD-D-21-00069.