Experimental infection of bats with Geomyces destructans causes white-nose syndrome.

White-nose syndrome (WNS) has caused recent catastrophic declines among multiple species of bats in eastern North America. The disease's name derives from a visually apparent white growth of the newly discovered fungus Geomyces destructans on the skin (including the muzzle) of hibernating bats. Colonization of skin by this fungus is associated with characteristic cutaneous lesions that are the only consistent pathological finding related to WNS. However, the role of G. destructans in WNS remains controversial because evidence to implicate the fungus as the primary cause of this disease is lacking. The debate is fuelled, in part, by the assumption that fungal infections in mammals are most commonly associated with immune system dysfunction. Additionally, the recent discovery that G. destructans commonly colonizes the skin of bats of Europe, where no unusual bat mortality events have been reported, has generated further speculation that the fungus is an opportunistic pathogen and that other unidentified factors are the primary cause of WNS. Here we demonstrate that exposure of healthy little brown bats (Myotis lucifugus) to pure cultures of G. destructans causes WNS. Live G. destructans was subsequently cultured from diseased bats, successfully fulfilling established criteria for the determination of G. destructans as a primary pathogen. We also confirmed that WNS can be transmitted from infected bats to healthy bats through direct contact. Our results provide the first direct evidence that G. destructans is the causal agent of WNS and that the recent emergence of WNS in North America may represent translocation of the fungus to a region with a naive population of animals. Demonstration of causality is an instrumental step in elucidating the pathogenesis and epidemiology of WNS and in guiding management actions to preserve bat populations against the novel threat posed by this devastating infectious disease.

White-nose syndrome initiates a cascade of physiologic disturbances in the hibernating bat host.

BACKGROUND: The physiological effects of white-nose syndrome (WNS) in hibernating bats and ultimate causes of mortality from infection with Pseudogymnoascus (formerly Geomyces) destructans are not fully understood. Increased frequency of arousal from torpor described among hibernating bats with late-stage WNS is thought to accelerate depletion of fat reserves, but the physiological mechanisms that lead to these alterations in hibernation behavior have not been elucidated. We used the doubly labeled water (DLW) method and clinical chemistry to evaluate energy use, body composition changes, and blood chemistry perturbations in hibernating little brown bats (Myotis lucifugus) experimentally infected with P. destructans to better understand the physiological processes that underlie mortality from WNS. RESULTS: These data indicated that fat energy utilization, as demonstrated by changes in body composition, was two-fold higher for bats with WNS compared to negative controls. These differences were apparent in early stages of infection when torpor-arousal patterns were equivalent between infected and non-infected animals, suggesting that P. destructans has complex physiological impacts on its host prior to onset of clinical signs indicative of late-stage infections. Additionally, bats with mild to moderate skin lesions associated with early-stage WNS demonstrated a chronic respiratory acidosis characterized by significantly elevated dissolved carbon dioxide, acidemia, and elevated bicarbonate. Potassium concentrations were also significantly higher among infected bats, but sodium, chloride, and other hydration parameters were equivalent to controls. CONCLUSIONS: Integrating these novel findings on the physiological changes that occur in early-stage WNS with those previously documented in late-stage infections, we propose a multi-stage disease progression model that mechanistically describes the pathologic and physiologic effects underlying mortality of WNS in hibernating bats. This model identifies testable hypotheses for better understanding this disease, knowledge that will be critical for defining effective disease mitigation strategies aimed at reducing morbidity and mortality that results from WNS.

Environmental transmission of Pseudogymnoascus destructans to hibernating little brown bats.

Pathogens with persistent environmental stages can have devastating effects on wildlife communities. White-nose syndrome (WNS), caused by the fungus Pseudogymnoascus destructans, has caused widespread declines in bat populations of North America. In 2009, during the early stages of the WNS investigation and before molecular techniques had been developed to readily detect P. destructans in environmental samples, we initiated this study to assess whether P. destructans can persist in the hibernaculum environment in the absence of its conclusive bat host and cause infections in naive bats. We transferred little brown bats (Myotis lucifugus) from an unaffected winter colony in northwest Wisconsin to two P. destructans contaminated hibernacula in Vermont where native bats had been excluded. Infection with P. destructans was apparent on some bats within 8 weeks following the introduction of unexposed bats to these environments, and mortality from WNS was confirmed by histopathology at both sites 14 weeks following introduction. These results indicate that environmental exposure to P. destructans is sufficient to cause the infection and mortality associated with WNS in naive bats, which increases the probability of winter colony extirpation and complicates conservation efforts.

Assessment of toxicity and potential risk of the anticoagulant rodenticide diphacinone using Eastern screech-owls (Megascops asio).

In the United States, new regulatory restrictions have been placed on the use of some second-generation anticoagulant rodenticides. This action may be offset by expanded use of first-generation compounds (e.g., diphacinone; DPN). Single-day acute oral exposure of adult Eastern screech-owls (Megascops asio) to DPN evoked overt signs of intoxication, coagulopathy, histopathological lesions (e.g., hemorrhage, hepatocellular vacuolation), and/or lethality at doses as low as 130 mg/kg body weight, although there was no dose-response relation. However, this single-day exposure protocol does not mimic the multiple-day field exposures required to cause mortality in rodent pest species and non-target birds and mammals. In 7-day feeding trials, similar toxic effects were observed in owls fed diets containing 2.15, 9.55 or 22.6 ppm DPN, but at a small fraction (<5%) of the acute oral dose. In the dietary trial, the average lowest-observed-adverse-effect-level for prolonged clotting time was 1.68 mg DPN/kg owl/week (0.24 mg/kg owl/day; 0.049 mg/owl/day) and the lowest lethal dose was 5.75 mg DPN/kg owl/week (0.82 mg/kg owl/day). In this feeding trial, DPN concentration in liver ranged from 0.473 to 2.21 µg/g wet weight, and was directly related to the daily and cumulative dose consumed by each owl. A probabilistic risk assessment indicated that daily exposure to as little as 3-5 g of liver from DPN-poisoned rodents for 7 days could result in prolonged clotting time in the endangered Hawaiian short-eared owl (Asio flammeus sandwichensis) and Hawaiian hawk (Buteo solitarius), and daily exposure to greater quantities (9-13 g of liver) could result in low-level mortality. These findings can assist natural resource managers in weighing the costs and benefits of anticoagulant rodenticide use in pest control and eradication programs.

Copper pellets simulating oral exposure to copper ammunition: absence of toxicity in American kestrels (Falco sparverius).

To evaluate the potential toxicity of copper (Cu) in raptors that may consume Cu bullets, shotgun pellets containing Cu, or Cu fragments as they feed on wildlife carcasses, we studied the effects of metallic Cu exposure in a surrogate, the American kestrel (Falco sparverius). Sixteen kestrels were orally administered 5 mg Cu/g body mass in the form of Cu pellets (1.18-2.00 mm in diameter) nine times during 38 days and 10 controls were sham gavaged on the same schedule. With one exception, all birds retained the pellets for at least 1 h, but most (69%) regurgitated pellets during a 12-h monitoring period. Hepatic Cu concentrations were greater in kestrels administered Cu than in controls, but there was no difference in Cu concentrations in the blood between treated and control birds. Concentration of the metal-binding protein metallothionein was greater in male birds that received Cu than in controls, whereas concentrations in female birds that received Cu were similar to control female birds. Hepatic Cu and metallothionein concentrations in kestrels were significantly correlated. Histopathologic alterations were noted in the pancreas of four treated kestrels and two controls, but these changes were not associated with hepatic or renal Cu concentrations, and no lesions were seen in other tissues. No clinical signs were observed, and there was no treatment effect on body mass; concentrations of Cu, hemoglobin, or methemoglobin in the blood; or Cu concentrations in kidney, plasma biochemistries, or hematocrit. Based on the parameters we measured, ingested Cu pellets pose little threat to American kestrels (and presumably phylogenetically related species), although the retention time of pellets in the stomach was of relatively short duration. Birds expected to regurgitate Cu fragments with a frequency similar to kestrels are not likely to be adversely affected by Cu ingestion, but the results of our study do not completely rule out the potential for toxicity in species that might retain Cu fragments for a longer time.

Diclofenac residues as the cause of vulture population decline in Pakistan.

The Oriental white-backed vulture (OWBV; Gyps bengalensis) was once one of the most common raptors in the Indian subcontinent. A population decline of >95%, starting in the 1990s, was first noted at Keoladeo National Park, India. Since then, catastrophic declines, also involving Gyps indicus and Gyps tenuirostris, have continued to be reported across the subcontinent. Consequently these vultures are now listed as critically endangered by BirdLife International. In 2000, the Peregrine Fund initiated its Asian Vulture Crisis Project with the Ornithological Society of Pakistan, establishing study sites at 16 OWBV colonies in the Kasur, Khanewal and Muzaffargarh-Layyah Districts of Pakistan to measure mortality at over 2,400 active nest sites. Between 2000 and 2003, high annual adult and subadult mortality (5-86%) and resulting population declines (34-95%) (ref. 5 and M.G., manuscript in preparation) were associated with renal failure and visceral gout. Here, we provide results that directly correlate residues of the anti-inflammatory drug diclofenac with renal failure. Diclofenac residues and renal disease were reproduced experimentally in OWBVs by direct oral exposure and through feeding vultures diclofenac-treated livestock. We propose that residues of veterinary diclofenac are responsible for the OWBV decline.

Apparent tolerance of turkey vultures (Cathartes aura) to the non-steroidal anti-inflammatory drug diclofenac.

The nonsteroidal anti-inflammatory drug diclofenac is extremely toxic to Old World Gyps vultures (median lethal dose -0.1-0.2 mg/kg), evoking visceral gout, renal necrosis, and mortality within a few days of exposure. Unintentional secondary poisoning of vultures that fed upon carcasses of diclofenac-treated livestock decimated populations in the Indian subcontinent. Because of the widespread use of diclofenac and other cyclooxygenase-2 inhibiting drugs, a toxicological study was undertaken in turkey vultures (Cathartes aura) as an initial step in examining sensitivity of New World scavenging birds. Two trials were conducted entailing oral gavage of diclofenac at doses ranging from 0.08 to 25 mg/kg body weight. Birds were observed for 7 d, blood samples were collected for plasma chemistry (predose and 12, 24, and 48 h and 7 d postdose), and select individuals were necropsied. Diclofenac failed to evoke overt signs of toxicity, visceral gout, renal necrosis, or elevate plasma uric acid at concentrations greater than 100 times the estimated median lethal dose reported for Gyps vultures. For turkey vultures receiving 8 or 25 mg/kg, the plasma half-life of diclofenac was estimated to be 6 h, and it was apparently cleared after several days as no residues were detectable in liver or kidney at necropsy. Differential sensitivity among avian species is a hallmark of cyclooxygenase-2 inhibitors, and despite the tolerance of turkey vultures to diclofenac, additional studies in related scavenging species seem warranted.

Experimental Infection of Tadarida brasiliensis with Pseudogymnoascus destructans, the Fungus That Causes White-Nose Syndrome.

White-nose syndrome (WNS) is causing significant declines in populations of North American hibernating bats, and recent western and southern expansions of the disease have placed additional species at risk. Understanding differences in species susceptibility and identifying management actions to reduce mortality of bats from WNS are top research priorities. However, the use of wild-caught susceptible bats, such as Myotis lucifugus, as model species for WNS research is problematic and places additional pressure on remnant populations. We investigated the feasibility of using Tadarida brasiliensis, a highly abundant species of bat that tolerates captivity, as the basis for an experimental animal model for WNS. Using methods previously established to confirm the etiology of WNS in M. lucifugus, we experimentally infected 11 T. brasiliensis bats with Pseudogymnoascus destructans in the laboratory under conditions that induced hibernation. We detected P. destructans on all 11 experimentally infected bats, 7 of which exhibited localized proliferation of hyphae within the epidermis, dermis, and subcutaneous tissue, similar to invasive cutaneous ascomycosis observed in M. lucifugus bats with WNS. However, the distribution of lesions across wing membranes of T. brasiliensis bats was limited, and only one discrete "cupping erosion," diagnostic for WNS, was identified. Thus, the rarity of lesions definitive for WNS suggests that T. brasiliensis does not likely represent an appropriate model for studying the pathophysiology of this disease. Nonetheless, the results of this study prompt questions concerning the potential for free-ranging, migratory T. brasiliensis bats to become infected with P. destructans and move the fungal pathogen between roost sites used by species susceptible to WNS.IMPORTANCE White-nose syndrome (WNS) is a fungal disease that is causing severe declines of bat populations in North America. Identifying ways to reduce the impacts of this disease is a priority but is inhibited by the lack of an experimental animal model that does not require the use of wild-caught bat species already impacted by WNS. We tested whether Tadarida brasiliensis, one of the most abundant species of bats in the Americas, could serve as a suitable animal model for WNS research. While T. brasiliensis bats were susceptible to experimental infection with the fungus under conditions that induced hibernation, the species exhibited limited pathology diagnostic for WNS. These results indicate that T. brasiliensis is not likely a suitable experimental model for WNS research. However, the recovery of viable WNS-causing fungus from experimentally infected bats indicates a potential for this species to contribute to the spread of the pathogen where it coexists with other species of bats affected by WNS.

Raptor mortality due to West Nile virus in the United States, 2002.

West Nile virus (WNV) has affected many thousands of birds since it was first detected in North America in 1999, but the overall impact on wild bird populations is unknown. In mid-August 2002, wildlife rehabilitators and local wildlife officials from multiple states began reporting increasing numbers of sick and dying raptors, mostly red-tailed hawks (Buteo jamaicensis) and great horned owls (Bubo virginianus). Commonly reported clinical signs were nonspecific and included emaciation, lethargy, weakness, inability to perch, fly or stand, and nonresponse to danger. Raptor carcasses from 12 states were received, and diagnostic evaluation of 56 raptors implicated WNV infection in 40 (71%) of these cases. Histologically, nonsuppurative encephalitis and myocarditis were the salient lesions (79% and 61%, respectively). Other causes of death included lead poisoning, trauma, aspergillosis, and Salmonella spp. and Clostridium spp. infections. The reason(s) for the reported increase in raptor mortality due to WNV in 2002 compared with the previous WNV seasons is unclear, and a better understanding of the epizootiology and pathogenesis of the virus in raptor populations is needed.

Attempts to identify the source of avian vacuolar myelinopathy for waterbirds.

Attempts were made to reproduce avian vacuolar myelinopathy (AVM) in a number of test animals in order to determine the source of the causative agent for birds and to find a suitable animal model for future studies. Submerged vegetation, plankton, invertebrates, forage fish, and sediments were collected from three lakes with ongoing outbreaks of AVM and fed to American coots (Fulica americana), mallard ducks and ducklings (Anas platyrhynchos), quail (Coturnix japonica), and laboratory mice either via gavage or ad libitum. Tissues from AVM-affected coots with brain lesions were fed to ducklings, kestrels (Falco sparverius), and American crows (Corvus brachyrhynchos). Two mallards that ingested one sample of Hydrilla verticillata along with any biotic or abiotic material associated with its external surface developed brain lesions consistent with AVM, although neither of the ducks had clinical signs of disease. Ingestion of numerous other samples of Hydrilla from the AVM affected lakes and a lake with no prior history of AVM, other materials (sediments, algae, fish, invertebrates, and water from affected lakes), or tissues from AVM-affected birds did not produce either clinical signs or brain lesions in any of the other test animals in our studies. These results suggest that waterbirds are most likely exposed to the causative agent of AVM while feeding on aquatic vegetation, but we do not believe the vegetation itself is the agent. We hypothesize that the causative agent of AVM might either be accumulated by aquatic vegetation, such as Hydrilla, or associated with biotic or abiotic material on its external surfaces. In support of that hypothesis, two coots that ingested Hydrilla sampled from a lake with an ongoing AVM outbreak in wild birds developed neurologic signs within 9 days (ataxia, limb weakness, and incoordination), and one of two coots that ingested Hydrilla collected from the same site 13 days later became sick and died within 38 days. None of these three sick coots had definitive brain lesions consistent with AVM by light microscopy, but they had no gross or histologic lesions in other tissues. It is unclear if these birds died of AVM. Perhaps they did not ingest a dose sufficient to produce brain lesions or the lesions were ultrastructural. Alternatively, it is possible that a separate neurotoxic agent is responsible for the morbidity and mortality observed in these coots.

Spatial and temporal patterns of avian paramyxovirus-1 outbreaks in double-crested cormorants (Phalacrocorax auritus) in the USA.

Morbidity and mortality events caused by avian paramyxovirus-1 (APMV-1) in Double-crested Cormorant (DCCO; Phalacrocorax auritus) nesting colonies in the US and Canada have been sporadically documented in the literature. We describe APMV-1 associated outbreaks in DCCO in the US from the first reported occurrence in 1992 through 2012. The frequency of APMV-1 outbreaks has increased in the US over the last decade, but the majority of events have continued to occur in DCCO colonies in the Midwestern states. Although morbidity and mortality in conesting species has been frequently reported during DCCO APMV-1 outbreaks, our results suggest that isolation of APMV-1 is uncommon in species other than DCCO during APMV-1 outbreaks and that the cause of mortality in other species is associated with other pathogens. Populations of DCCO do not appear to have been significantly affected by this disease; however, because at least 65% of the APMV-1 outbreaks in DCCO in the US have involved APMV-1 strains classified as virulent to poultry (virulent Newcastle disease virus), its persistence and increased occurrence in DCCO warrants continued research and surveillance.

The fungus Trichophyton redellii sp. Nov. Causes skin infections that resemble white-nose syndrome of hibernating bats.

Before the discovery of white-nose syndrome (WNS), a fungal disease caused by Pseudogymnoascus destructans, there were no reports of fungal skin infections in bats during hibernation. In 2011, bats with grossly visible fungal skin infections similar in appearance to WNS were reported from multiple sites in Wisconsin, US, a state outside the known range of P. destructans and WNS at that time. Tape impressions or swab samples were collected from affected areas of skin from bats with these fungal infections in 2012 and analyzed by microscopy, culture, or direct DNA amplification and sequencing of the fungal internal transcribed spacer region (ITS). A psychrophilic species of Trichophyton was isolated in culture, detected by direct DNA amplification and sequencing, and observed on tape impressions. Deoxyribonucleic acid indicative of the same fungus was also detected on three of five bat carcasses collected in 2011 and 2012 from Wisconsin, Indiana, and Texas, US. Superficial fungal skin infections caused by Trichophyton sp. were observed in histopathology for all three bats. Sequencing of the ITS of Trichophyton sp., along with its inability to grow at 25 C, indicated that it represented a previously unknown species, described herein as Trichophyton redellii sp. nov. Genetic diversity present within T. redellii suggests it is native to North America but that it had been overlooked before enhanced efforts to study fungi associated with bats in response to the emergence of WNS.

Peracute sodium toxicity in free-ranging black-bellied whistling duck ducklings.

From 23 to 25 July 2002, 98-103 newly hatched black-bellied whistling ducks (Dendrocygna autumnalis) were observed alive at an inland saline lake (La Sal Vieja) in Willacy County, Texas (USA). Seventy-one (71%) died after showing signs indicative of sodium toxicity within 5 hr of entering the water; some died within minutes. Six carcasses were sent to the United States Geological Survey, National Wildlife Health Center (Madison, Wisconsin, USA) for analysis, and brain sodium levels of all ducklings were above 2,000 parts per million wet weight. More black-bellied whistling duck ducklings are likely to have been affected, but they were not observed after hatching.

Mortality in Laysan ducks (Anas laysanensis) by emaciation complicated by Echinuria uncinata on Laysan Island, Hawaii, 1993.

In November 1993, unusual mortality occurred among endangered Laysan ducks on Laysan Island, one of the remote refugia of the Northwestern Hawaiian Islands National Wildlife Refuge (USA). Ten live ducks were emaciated, and blood samples documented anemia, heterophilia, and eosinophilia. Pathology in 13 duck carcasses revealed emaciation, marked thickening of the proventricular wall, abundant mucus, and nodules in the gastrointestinal tract. Histology revealed granulomata associated with nematodes in the proventriculus, small intestines, and body walls of nine of 10 ducks examined on histology. We suspect that low rainfall and low food abundance that year contributed to enhanced pathogenicity of parasite infection, either through increased exposure or decreased host resistance. Because the Laysan duck is found only on Laysan island and is critically endangered, translocation of this species to other islands is being considered. Given that we have not seen pathology associated with Echinuria spp. in native waterfowl on other Hawaiian Islands and given the parasite's potential to cause significant lesions in Laysan ducks, it will be important to prevent the translocation of Echinuria spp.

Diagnostic and molecular evaluation of three iridovirus-associated salamander mortality events.

In 1998 viruses were isolated from tiger salamander larvae (Ambystoma tigrinum diaboli and A. tigrinum melanostictum) involved in North Dakota and Utah (USA) mortality events and spotted salamander (A. maculatum) larvae in a third event in Maine (USA). Although sympatric caudates and anurans were present at all three sites only ambystomid larvae appeared to be affected. Mortality at the North Dakota site was in the thousands while at the Utah and Maine sites mortality was in the hundreds. Sick larvae were lethargic and slow moving. They swam in circles with obvious buoyancy problems and were unable to remain upright. On the ventral surface, near the gills and hind limbs, red spots or swollen areas were noted. Necropsy findings included: hemorrhages and ulceration of the skin, subcutaneous and intramuscular edema, swollen and pale livers with multifocal hemorrhage, and distended fluid-filled intestines with areas of hemorrhage. Light microscopy revealed intracytoplasmic inclusions, suggestive of a viral infection, in a variety of organs. Electron microscopy of ultra thin sections of the same tissues revealed iridovirus-like particles within the inclusions. These viruses were isolated from a variety of organs, indicating a systemic infection. Representative viral isolates from the three mortality events were characterized using molecular assays. Characterization confirmed that the viral isolates were iridoviruses and that the two tiger salamander isolates were similar and could be distinguished from the spotted salamander isolate. The spotted salamander isolate was similar to frog virus 3, the type species of the genus Ranavirus, while the tiger salamander isolates were not. These data indicate that different species of salamanders can become infected and die in association with different iridoviruses. Challenge assays are required to determine the fish and amphibian host range of these isolates and to assess the susceptibility of tiger and spotted salamanders to heterologous virus isolates.

Evaluation of monkeypox virus infection of black-tailed prairie dogs (Cynomys ludovicianus) using in vivo bioluminescent imaging.

Monkeypox (MPX) is a re-emerging zoonotic disease that is endemic in Central and West Africa, where it can cause a smallpox-like disease in humans. Despite many epidemiologic and field investigations of MPX, no definitive reservoir species has been identified. Using recombinant viruses expressing the firefly luciferase (luc) gene, we previously demonstrated the suitability of in vivo bioluminescent imaging (BLI) to study the pathogenesis of MPX in animal models. Here, we evaluated BLI as a novel approach for tracking MPX virus infection in black-tailed prairie dogs (Cynomys ludovicianus). Prairie dogs were affected during a multistate outbreak of MPX in the US in 2003 and have since been used as an animal model of this disease. Our BLI results were compared with PCR and virus isolation from tissues collected postmortem. Virus was easily detected and quantified in skin and superficial tissues by BLI before and during clinical phases, as well as in subclinical secondary cases, but was not reliably detected in deep tissues such as the lung. Although there are limitations to viral detection in larger wild rodent species, BLI can enhance the use of prairie dogs as an animal model of MPX and can be used for the study of infection, disease progression, and transmission in potential wild rodent reservoirs.

A review of episodes of zinc phosphide toxicosis in wild geese (Branta spp.) in Oregon (2004-2011).

Epizootic mortality in several geese species, including cackling geese (Branta hutchinsii) and Canada geese (Branta canadensis), has been recognized in the Willamette Valley of Oregon for over a decade. Birds are generally found dead on a body of water or are occasionally observed displaying neurologic clinical signs such as an inability to raise or control the head prior to death. Investigation of these epizootic mortality events has revealed the etiology to be accidental poisoning with the rodenticide zinc phosphide (Zn(3)P(2)). Gross and histologic changes are restricted to acute pulmonary congestion and edema, sometimes accompanied by distension of the upper alimentary tract by fresh grass. Geese are unusually susceptible to this pesticide; when combined with an epidemiologic confluence of depredation of specific agricultural crops by rodents and seasonal avian migration pathways, epizootic toxicosis may occur. Diagnosis requires a high index of suspicion, appropriate sample collection and handling, plus specific test calibration for this toxicant. Interagency cooperation, education of farmers regarding pesticide use, and enforcement of regulations has been successful in greatly decreasing these mortality events since 2009.

Pathology in euthermic bats with white nose syndrome suggests a natural manifestation of immune reconstitution inflammatory syndrome.

White nose syndrome, caused by Geomyces destructans, has killed more than 5 million cave hibernating bats in eastern North America. During hibernation, the lack of inflammatory cell recruitment at the site of fungal infection and erosion is consistent with a temperature-induced inhibition of immune cell trafficking. This immune suppression allows G. destructans to colonize and erode the skin of wings, ears and muzzle of bat hosts unchecked. Yet, paradoxically, within weeks of emergence from hibernation an intense neutrophilic inflammatory response to G. destructans is generated, causing severe pathology that can contribute to death. We hypothesize that the sudden reversal of immune suppression in bats upon the return to euthermia leads to a form of immune reconstitution inflammatory syndrome (IRIS). IRIS was first described in HIV-infected humans with low helper T lymphocyte counts and bacterial or fungal opportunistic infections. IRIS is a paradoxical and rapid worsening of symptoms in immune compromised humans upon restoration of immunity in the face of an ongoing infectious process. In humans with HIV, the restoration of adaptive immunity following suppression of HIV replication with anti-retroviral therapy (ART) can trigger severe immune-mediated tissue damage that can result in death. We propose that the sudden restoration of immune responses in bats infected with G. destructans results in an IRIS-like dysregulated immune response that causes the post-emergent pathology.

Histopathology confirms white-nose syndrome in bats in Europe.

White-nose syndrome, associated with the fungal skin infection geomycosis, caused regional population collapse in bats in North America. Our results, based on histopathology, show the presence of white-nose syndrome in Europe. Dermatohistopathology on two bats (Myotis myotis) found dead in March 2010 with geomycosis in the Czech Republic had characteristics resembling Geomyces destructans infection in bats confirmed with white-nose syndrome in US hibernacula. In addition, a live M. myotis, biopsied for histopathology during hibernation in April 2011, had typical fungal infection with cupping erosion and invasion of muzzle skin diagnostic for white-nose syndrome and conidiospores identical to G. destructans that were genetically confirmed as G. destructans.

Frequent arousal from hibernation linked to severity of infection and mortality in bats with white-nose syndrome.

White-nose syndrome (WNS), an emerging infectious disease that has killed over 5.5 million hibernating bats, is named for the causative agent, a white fungus (Geomyces destructans (Gd)) that invades the skin of torpid bats. During hibernation, arousals to warm (euthermic) body temperatures are normal but deplete fat stores. Temperature-sensitive dataloggers were attached to the backs of 504 free-ranging little brown bats (Myotis lucifugus) in hibernacula located throughout the northeastern USA. Dataloggers were retrieved at the end of the hibernation season and complete profiles of skin temperature data were available from 83 bats, which were categorized as: (1) unaffected, (2) WNS-affected but alive at time of datalogger removal, or (3) WNS-affected but found dead at time of datalogger removal. Histological confirmation of WNS severity (as indexed by degree of fungal infection) as well as confirmation of presence/absence of DNA from Gd by PCR was determined for 26 animals. We demonstrated that WNS-affected bats aroused to euthermic body temperatures more frequently than unaffected bats, likely contributing to subsequent mortality. Within the subset of WNS-affected bats that were found dead at the time of datalogger removal, the number of arousal bouts since datalogger attachment significantly predicted date of death. Additionally, the severity of cutaneous Gd infection correlated with the number of arousal episodes from torpor during hibernation. Thus, increased frequency of arousal from torpor likely contributes to WNS-associated mortality, but the question of how Gd infection induces increased arousals remains unanswered.