Experimental inoculation trial to determine the effects of temperature and humidity on White-nose Syndrome in hibernating bats

Abstract

Disease results from interactions among the host, pathogen, and environment. Inoculation trials can quantify interactions among these players and explain aspects of disease ecology to inform management in variable and dynamic natural environments. White-nose Syndrome, a disease caused by the fungal pathogen, Pseudogymnoascus destructans (Pd), has caused severe population declines of several bat species in North America. We conducted the first experimental infection trial on the tri-colored bat, Perimyotis subflavus, to test the effect of temperature and humidity on disease severity. We also tested the effects of temperature and humidity on fungal growth and persistence on substrates. Unexpectedly, only 37% (35/95) of bats experimentally inoculated with Pd at the start of the experiment showed any infection response or disease symptoms after 83 days of captive hibernation. There was no evidence that temperature or humidity influenced infection response. Temperature had a strong effect on fungal growth on media plates, but the influence of humidity was more variable and uncertain. Designing laboratory studies to maximize research outcomes would be beneficial given the high costs of such efforts and potential for unexpected outcomes. Understanding the influence of microclimates on host-pathogen interactions remains an important consideration for managing wildlife diseases, particularly in variable environments.

Figure 1

Schematic of the experimental design and sample sizes with 7 environmental chambers with fixed temperature and humidity conditions and two sets of connected chambers allowing bats to behaviorally select temperature (left) or humidity conditions (bottom) for the infection trial on tri-colored bats (Perimyotis subflavus). Water loss conditions were based on water vapor pressure deficit (VPD) levels set to 0.05 kPA to produce low potential evaporative water loss (pEWL) for high humidity, 0.10 kPa for medium pEWL and humidity, or 0.15 kPA for high pEWL and low humidity. Numbers are sample sizes of bats assigned to separate cages within each chamber. Bats in the low temperature and high humidity chamber were combined into a single cage after a camera failed at the start of the experiment (top right).

Figure 2

Signs of Pseudogymnoascus destructans (Pd) infection or white-nose syndrome (WNS) disease for tri-colored bats (Perimyotis subflavus) exposed to different temperature and humidity regimes. (A) Fraction of bats with Pd detected by qPCR; (B) Fraction of bats with signs of WNS disease by histology, and (C) Mean quantity of Pd on bats at the end of the experiment. There was no statistical support for differences between temperature or humidity treatments for any response metrics. Points are estimated means and vertical lines show binomial standard error for prevalence and standard errors for Pd load.

Figure 3

Examples demonstrate the process of measuring and estimating fungal growth of Pseudogymnoascus destructans (Pd) on media plates in temperature and humidity treatment conditions. (A) Examples of fungal growth on media plates measured at days 7, 14, 21, 28, and 34 from two of the treatment conditions (11 °C, 92% RH and 5 °C, 88% RH). (B) Examples of estimating maximum growth rate and latency variables from fungal growth measurements in panel A. We fit a sigmoidal curve to describe fungal growth (thick solid black line) to estimate the inflection point of the curve (vertical solid line). We calculated the slope (solid red line) at the inflection point of the curve to estimate maximum growth rate, and the days until total growth area reached 2.5 cm² (dashed red lines) as an estimate of latency.

Figure 4

Influence of temperature and humidity on maximum growth rate and latency of growth of Pseudogymnoascus destructans (Pd) estimated based on the total area of Pd fungal growth on media plates. Actual measurements (gray circles) are shown with mean and standard error (diamond with error bars) overlaid. (A) Maximum growth rate increased with temperature, and (B) latency of growth was significantly shorter at higher temperatures. (C) Maximum growth rate was highest at the highest humidity level (vapor pressure deficit at 0.05 kPa) but did not differ significantly from the lowest humidity level (Tukey’s pairwise comparison, P > 0.05), but (D) Latency was not significantly different among humidity treatments. Different letters denote that Tukey’s post hoc pair-wise comparisons were significantly different (α = 0.05).

Figure 5

Comparison of fungal loads of Pseudogymnoascus destructans (Pd) measured on little brown bats (Myotis lucifugus) and tri-colored bats (Perimyotis subflavus) in experimental infection trials using the same experimental inoculation protocols.