Lisa Warnecke

Inoculation of bats with European Geomyces destructans supports the novel pathogen hypothesis for the origin of white-nose syndrome.

White-nose syndrome (WNS) is an emerging disease of hibernating bats associated with cutaneous infection by the fungus Geomyces destructans (Gd), and responsible for devastating declines of bat populations in eastern North America. Affected bats appear emaciated and one hypothesis is that they spend too much time out of torpor during hibernation, depleting vital fat reserves required to survive the winter. The fungus has also been found at low levels on bats throughout Europe but without mass mortality. This finding suggests that Gd is either native to both continents but has been rendered more pathogenic in North America by mutation or environmental change, or that it recently arrived in North America as an invader from Europe. Thus, a causal link between Gd and mortality has not been established and the reason for its high pathogenicity in North America is unknown. Here we show that experimental inoculation with either North American or European isolates of Gd causes WNS and mortality in the North American bat, Myotis lucifugus. In contrast to control bats, individuals inoculated with either isolate of Gd developed cutaneous infections diagnostic of WNS, exhibited a progressive increase in the frequency of arousals from torpor during hibernation, and were emaciated after 3–4 mo. Our results demonstrate that altered torpor-arousal cycles underlie mortality from WNS and provide direct evidence that Gd is a novel pathogen to North America from Europe.

Pathophysiology of white-nose syndrome in bats: a mechanistic model linking wing damage to mortality.

White-nose syndrome is devastating North American bat populations but we lack basic information on disease mechanisms. Altered blood physiology owing to epidermal invasion by the fungal pathogen Geomyces destructans (Gd) has been hypothesized as a cause of disrupted torpor patterns of affected hibernating bats, leading to mortality. Here, we present data on blood electrolyte concentration, haematology and acid–base balance of hibernating little brown bats, Myotis lucifugus, following experimental inoculation with Gd. Compared with controls, infected bats showed electrolyte depletion (i.e. lower plasma sodium), changes in haematology (i.e. increased haematocrit and decreased glucose) and disrupted acid–base balance (i.e. lower CO2 partial pressure and bicarbonate). These findings indicate hypotonic dehydration, hypovolaemia and metabolic acidosis. We propose a mechanistic model linking tissue damage to altered homeostasis and morbidity/mortality.

Behaviour of hibernating little brown bats experimentally inoculated with the pathogen that causes white-nose syndrome

Pathogens can affect host behaviour in ways that influence disease transmission as well as survival and fitness for both host and pathogen. Hibernating bats with white-nose syndrome (WNS) show a number of unusual behaviours including increased frequency of arousal from torpor, altered roosting behaviour and premature emergence. However, mechanisms underlying these patterns are not understood, and the behaviour of bats with WNS has not been examined systematically. Three hypotheses could explain increased arousal frequency. Bats may arouse to (1) groom in response to skin infection, (2) drink to offset dehydration or (3) increase activity, possibly in an attempt to access resources, avoid a source of infection or limit the risk of infecting relatives. We tested these hypotheses with captive little brown bats, Myotis lucifugus, inoculated with Pseudogymnoascus destructans, the fungus that causes WNS. In contrast to predictions of all three hypotheses, bats inoculated with the fungus tended to be less active than controls during arousals from torpor and did not increase grooming or visits to the water source in their enclosures. However, bats showed a dramatic reduction in clustering behaviour as infection progressed. Reduced activity and clustering could represent adaptive, maladaptive or pathological responses. Reduced activity could be an energy-saving mechanism or a pathological consequence of infection while reduced clustering could have beneficial or detrimental consequences for transmission, energetics, water balance and survival. Our results highlight the need for studies of host behaviour to understand dynamics of wildlife infectious diseases.

Activation of Innate Immune-Response Genes in Little Brown Bats (Myotis lucifugus) Infected with the Fungus Pseudogymnoascus destructans

Recently bats have been associated with the emergence of diseases, both as reservoirs for several new viral diseases in humans and other animals and, in the northern Americas, as hosts for a devastating fungal disease that threatens to drive several bat species to regional extinction. However, despite these catastrophic events little Information is available on bat defences or how they interact with their pathogens. Even less is known about the response of bats to infection during torpor or long-term hibernation. Using tissue samples collected at the termination of an experiment to explore the pathogenesis of White Nose Syndrome in Little Brown Bats, we determined if hibernating bats infected with the fungus Pseudogymnoascus destructans could respond to infection by activating genes responsible for innate immune and stress responses. Lesions due to fungal infection and, in some cases, secondary bacterial infections, were restricted to the skin. However, we were unable to obtain sufficient amounts of RNA from these sites. We therefore examined lungs for response at an epithelial surface not linked to the primary site of infection. We found that bats responded to infection with a significant increase in lungs of transcripts for Cathelicidin (an anti-microbial peptide) as well as the immune modulators tumor necrosis factor alpha and interleukins 10 and 23. In conclusion, hibernating bats can respond to experimental P. destructans infection by activating expression of innate immune response genes.

Basking behaviour and torpor use in free-ranging Planigale gilesi

We investigated the importance of energy-conserving strategies for free-ranging Planigale gilesi in arid Australia. We monitored torpor use and basking behaviour using internal temperature-sensitive transmitters. Torpor was used every day; the maximum torpor bout duration was 18.2 h and the minimum body temperature was 10.5°C. Basking behaviour was observed during rewarming from torpor as well as during normothermia. The use of torpor and basking is likely to reduce the energy requirements of P. gilesi, thus helping it to survive in a harsh and unpredictable environment.

White-Nose Syndrome Disease Severity and a Comparison of Diagnostic Methods

White-nose syndrome is caused by the fungus Pseudogymnoascus destructans and has killed millions of hibernating bats in North America but the pathophysiology of the disease remains poorly understood. Our objectives were to (1) assess non-destructive diagnostic methods for P. destructans infection compared to histopathology, the current gold-standard, and (2) to evaluate potential metrics of disease severity. We used data from three captive inoculation experiments involving 181 little brown bats (Myotis lucifugus) to compare histopathology, quantitative PCR (qPCR), and ultraviolet fluorescence as diagnostic methods of P. destructans infection. To assess disease severity, we considered two histology metrics (wing area with fungal hyphae, area of dermal necrosis), P. destructans fungal load (qPCR), ultraviolet fluorescence, and blood chemistry (hematocrit, sodium, glucose, pCO2, and bicarbonate). Quantitative PCR was most effective for early detection of P. destructans, while all three methods were comparable in severe infections. Correlations among hyphae and necrosis scores, qPCR, ultraviolet fluorescence, blood chemistry, and hibernation duration indicate a multi-stage pattern of disease. Disruptions of homeostasis occurred rapidly in late hibernation. Our results provide valuable information about the use of non-destructive techniques for monitoring, and provide novel insight into the pathophysiology of white-nose syndrome, with implications for developing and implementing potential mitigation strategies.

Summer and winter torpor use by a free-ranging marsupial

Torpor is usually associated with low ambient temperatures (T(a)) in winter, but in some species it is also used in summer, often in response to limited food availability. Since the seasonal expression of torpor of both placental and marsupial hibernators in the wild is poorly documented by quantitative data, we investigated torpor and activity patterns of the eastern pygmy-possum Cercartetus nanus (17.4 g) over two seasons. We used radio telemetry to track animals during winter (n=4) and summer (n=5) in a warm-temperate habitat and found that torpor was used in both seasons. In winter all animals entered periods of short-term hibernation (from 5 to 20 days) containing individual torpor bouts of up to 5.9 days. In summer, torpor bouts were always <1 day in duration, only used by males and were not related to daily mean T(a). Pygmy-possums entered torpor at night as T(a) cooled, and rewarmed during the afternoon as T(a) increased. Individuals interspersed torpor bouts with nocturnal activity and the percentage of the night animals were active was the same in summer and winter. Our study provides the first information on torpor patterns in free-ranging C. nanus, and shows that the use of torpor throughout the year is important for energy management in this species.

Short-term movement patterns and diet of small dasyurid marsupials in semiarid Australia

Since little information is available on the spatial ecology of small arid-zone marsupials, we used radio-tracking to investigate the small-scale activity patterns of three dasyurid species in semiarid Australia. Sminthopsis crassicaudata, Planigale gilesi and S. macroura were equipped with miniature radio-transmitters to monitor choice of resting sites and daily movements. Resting sites were located within an area of 1.27 ± 0.36 ha, 0.12 ± 0.02 ha and 3.60 ± 0.95 ha, respectively and individuals returned to previously used resting sites regularly. We also analysed scat samples of S. crassicaudata and P. gilesi, and identified Araneae, Hymenoptera and Orthoptera as the major prey taxa for both species. Our study presents the first radio-tracking-based information on movements for these species in semiarid habitat, which indicates that, over a period of several weeks, resting sites are situated within small and defined areas.

Evidence of ‘sickness behaviour’ in bats with white-nose syndrome

Many animals change behaviour in response to pathogenic infections. White-nose syndrome (WNS) is a fungal skin disease causing rapid declines of North American bats. Infection with Pseudogymnoascus destructans causes hibernating bats to arouse from torpor too often, potentially causing starvation. Mechanisms underlying increased arousals are not understood but fungal invasion of the wings could trigger thirst to relieve fluid loss or grooming to relieve skin irritation. Alternatively, bats might exhibit ‘sickness behaviour’, a suite of responses to infection that save energy. We quantified behaviours of healthy and experimentally inoculated little brown bats ( Myotis lucifugus ) that could reflect active (i.e., drinking, grooming) or inactive (i.e., sickness behaviour) responses to infection. Infected bats groomed less and were less likely to visit their water dish compared to controls. These results are consistent with research suggesting that P. destructans causes sickness behaviour which could help bats compensate for energetic costs associated with infection.

A persistently infecting coronavirus in hibernating Myotis lucifugus, the North American little brown bat

Bats are important reservoir hosts for emerging viruses, including coronaviruses that cause diseases in people. Although there have been several studies on the pathogenesis of coronaviruses in humans and surrogate animals, there is little information on the interactions of these viruses with their natural bat hosts. We detected a coronavirus in the intestines of 53/174 hibernating little brown bats (Myotis lucifugus), as well as in the lungs of some of these individuals. Interestingly, the presence of the virus was not accompanied by overt inflammation. Viral RNA amplified from little brown bats in this study appeared to be from two distinct clades. The sequences in clade 1 were very similar to the archived sequence derived from little brown bats and the sequences from clade 2 were more closely related to the archived sequence from big brown bats. This suggests that two closely related coronaviruses may circulate in little brown bats. Sequence variation among coronavirus detected from individual bats suggested that infection occurred prior to hibernation, and that the virus persisted for up to 4 months of hibernation in the laboratory. Based on the sequence of its genome, the coronavirus was placed in the Alphacoronavirus genus, along with some human coronaviruses, bat viruses and the porcine epidemic diarrhoea virus. The detection and identification of an apparently persistent coronavirus in a local bat species creates opportunities to understand the dynamics of coronavirus circulation in bat populations.