Specific Alterations in Complement Protein Activity of Little Brown Myotis (Myotis lucifugus) Hibernating in White-Nose Syndrome Affected Sites

White-nose syndrome (WNS) is the most devastating condition ever reported for hibernating bats, causing widespread mortality in the northeastern United States. The syndrome is characterized by cutaneous lesions caused by a recently identified psychrophilic and keratinophylic fungus (Geomyces destructans), depleted fat reserves, atypical behavior, and damage to wings; however, the proximate cause of mortality is still uncertain. To assess relative levels of immunocompetence in bats hibernating in WNS-affected sites compared with levels in unaffected bats, we describe blood plasma complement protein activity in hibernating little brown myotis (Myotis lucifugus) based on microbicidal competence assays using Escherichia coli, Staphylococcus aureus and Candida albicans. Blood plasma from bats collected during mid-hibernation at WNS-affected sites had higher bactericidal ability against E. coli and S. aureus, but lower fungicidal ability against C. albicans when compared with blood plasma from bats collected at unaffected sites. Within affected sites during mid-hibernation, we observed no difference in microbicidal ability between bats displaying obvious fungal infections compared to those without. Bactericidal ability against E. coli decreased significantly as hibernation progressed in bats collected from an affected site. Bactericidal ability against E. coli and fungicidal ability against C. albicans were positively correlated with body mass index (BMI) during late hibernation. We also compared complement activity against the three microbes within individuals and found that the ability of blood plasma from hibernating M. lucifugus to lyse microbial cells differed as follows: E. coli>S. aureus>C. albicans. Overall, bats affected by WNS experience both relatively elevated and reduced innate immune responses depending on the microbe tested, although the cause of observed immunological changes remains unknown. Additionally, considerable trade-offs may exist between energy conservation and immunological responses. Relationships between immune activity and torpor, including associated energy expenditure, are likely critical components in the development of WNS

Little brown myotis affected by WNS.

<p>Hibernating little brown myotis (<i>Myotis lucifugus</i>) with and without visible white fungal growth characteristic of white-nose syndrome.</p

Microbicidal ability against different microbe types.

<p>Microbicidal ability against the three different microbes <i>E. coli</i>, <i>S. aureus</i> and <i>C. albicans</i> of blood plasma collected from hibernating little brown myotis across all sites and stages of hibernation. Plasma from <i>M. lucifugus</i> was significantly better at killing <i>E. coli</i> compared to <i>S. aureus</i> (<i>n</i> = 168; <i>t</i> = 18.22; df = 167; <i>p</i><0.001), significantly better at killing <i>E. coli</i> compared to <i>C. albicans</i> (<i>n</i> = 75; Wilcoxon <i>Z</i> = −6.59; <i>p</i><0.001), and significantly better at killing <i>S. aureus</i> compared to <i>C. albicans</i> (<i>n</i> = 75; Wilcoxon <i>Z</i> = −2.94; <i>p</i> = 0.003). Lines indicate mean percent microbe killed and 95% confidence intervals.</p

Microbicidal ability across the hibernation period.

<p>Microbicidal ability, expressed as percent microbe killed, against <i>E. coli</i> (A) and <i>S. aureus</i> (B) in blood plasma collected from little brown myotis hibernating in the affected Aeolus Cave during the 2008–2009 hibernation period. Bactericidal ability of blood plasma against <i>E. coli</i> varied significantly throughout the hibernation period (early <i>n</i> = 18; mid <i>n</i> = 18; late <i>n</i> = 16; F<i>_{2, 49}</i> = 5.86; <i>p</i> = 0.005; R² = 0.19) and was greater during early hibernation than in late hibernation (<i>p</i> = 0.004). Bactericidal ability of blood plasma against <i>S. aureus</i> also varied significantly throughout the hibernation period (early <i>n</i> = 18; mid <i>n</i> = 18; late <i>n</i> = 17; F<i>_{2, 51}</i> = 31.7; <i>p</i><0.001; R² = 0.55), but was greater in early hibernation compared with mid-hibernation (<i>p</i><0.001) and greater in late hibernation than in early hibernation (<i>p</i> = 0.002) and mid-hibernation (<i>p</i><0.001). Lines indicate mean percent microbe killed ±1 SE.</p

Microbicidal ability of blood plasma from little brown myotis collected in WNS-affected and unaffected sites.

<p>Microbicidal ability, expressed as percent microbe killed, against <i>E. coli</i> (A), <i>S. aureus</i> (B), and <i>C. albicans</i> (C) in <i>M. lucifugus</i> collected from WNS-affected and unaffected sites during the winter of 2008–2009. Significantly more <i>E. coli</i> was killed by plasma collected from bats hibernating in WNS-affected sites compared with plasma collected from bats hibernating in unaffected sites (affected <i>n</i> = 54; unaffected <i>n</i> = 31; F<i>_{1, 80}</i> = 16.22; <i>p</i><0.001; R² = 0.17). Bactericidal ability against <i>S. aureus</i> was also significantly greater in bats collected from affected sites compared with bats from unaffected sites (affected <i>n</i> = 55; unaffected <i>n</i> = 31; F<i>_{1, 81}</i> = 9.05; <i>p</i> = 0.004; R² = 0.10), but fungicidal ability against <i>C. albicans</i> was significantly lower in bats collected at affected sites compared with bats collected at unaffected sites (affected <i>n</i> = 52; unaffected <i>n</i> = 25; Mann-Whitney U = 383; <i>p</i> = 0.004). Lines indicate mean percent microbe killed ±1 SE.</p

Sample sizes for winter 2008–2009 collections across multiple hibernacula and different stages within the hibernation period.

<p>Sample sizes for winter 2008–2009 collections across multiple hibernacula and different stages within the hibernation period.</p

Microbicidal ability and body condition.

<p>Microbicidal ability, expressed as percent microbe killed, against <i>E. coli</i> (A–C), <i>S. aureus</i> (D–F), and <i>C. albicans</i> (G, H) in little brown myotis plotted against BMI. After controlling for site, BMI was not significantly related to bactericidal ability against <i>E. coli</i> in early hibernation (F<i>_{1, 38}</i> = 0.12; <i>p</i> = 0.745) or mid-hibernation samples (F<i>_{1, 77}</i> = 2.87; <i>p</i> = 0.094) but was positively related to BMI in late hibernation samples (F<i>_{1, 48}</i> = 4.47; <i>p</i> = 0.04; R² = 0.085). After controlling for site, BMI did not correlate with bactericidal ability against <i>S. aureus</i> during any stage of hibernation. Fungicidal ability of blood against <i>C. albicans</i> was not associated with BMI during mid-hibernation, (<i>n</i> = 24; Spearman's ρ = 0.14, <i>p</i> = 0.527), but was significantly and positively associated with BMI during late hibernation (<i>n</i> = 51; Spearman's ρ = 0.29, <i>p</i> = 0.039). Open diamonds indicate samples collected from bats hibernating in WNS-affected sites and crosses indicate samples collected from bats hibernating in unaffected sites.</p