Robert J. Rudd

Bat White-Nose Syndrome: An Emerging Fungal Pathogen?

White-nose syndrome (WNS) is a condition associated with an unprecedented bat mortality event in the northeastern United States. Since the winter of 2006*2007, bat declines exceeding 75% have been observed at surveyed hibernacula. Affected bats often present with visually striking white fungal growth on their muzzles, ears, and/or wing membranes. Direct microscopy and culture analyses demonstrated that the skin of WNS-affected bats is colonized by a psychro-philic fungus that is phylogenetically related to Geomyces spp. but with a conidial morphology distinct from characterized members of this genus. This report characterizes the cutaneous fungal infection associated with WNS.

First Isolation of West Nile virus from a patient with encephalitis in the United States.

West Nile virus (WNV) was isolated from a patient who developed encephalitis while undergoing treatment with CHOP (cyclophosphamide, hydroxydoxorubicin, vincristine [Oncovin], predisone) and rituximab for a non-Hodgkin B-cell lymphoma. Both standard reverse transcription–polymerase chain reaction (RT-PCR) and Taqman RT-PCR established the diagnosis of WNV infection from cerebrospinal fluid (CSF). Several whole blood samples and one serum sample underwent further testing. CSF and serum samples were negative for WNV antibody; however, all samples were positive by both RT-PCR assays. Infectious virus was recovered from a blood sample, and its identity was confirmed by using a WNV-specific immunofluorescence assay. The complete WNV genomes determined from CSF and from the virus isolate adapted from cell culture were the same. The results represent the first complete WNV genome sequence obtained directly from human CSF and the first time that infectious WNV has been recovered from a patient with encephalitis in North America.

Morphological and molecular characterizations of psychrophilic fungus Geomyces destructans from New York bats with white nose syndrome (WNS).

Background Massive die-offs of little brown bats (Myotis lucifugus) have been occurring since 2006 in hibernation sites around Albany, New York, and this problem has spread to other States in the Northeastern United States. White cottony fungal growth is seen on the snouts of affected animals, a prominent sign of White Nose Syndrome (WNS). A previous report described the involvement of the fungus Geomyces destructans in WNS, but an identical fungus was recently isolated in France from a bat that was evidently healthy. The fungus has been recovered sparsely despite plentiful availability of afflicted animals. Methodology/Principal Findings We have investigated 100 bat and environmental samples from eight affected sites in 2008. Our findings provide strong evidence for an etiologic role of G. destructans in bat WNS. (i) Direct smears from bat snouts, Periodic Acid Schiff-stained tissue sections from infected tissues, and scanning electron micrographs of bat tissues all showed fungal structures similar to those of G. destructans. (ii) G. destructans DNA was directly amplified from infected bat tissues, (iii) Isolations of G. destructans in cultures from infected bat tissues showed 100% DNA match with the fungus present in positive tissue samples. (iv) RAPD patterns for all G. destructans cultures isolated from two sites were indistinguishable. (v) The fungal isolates showed psychrophilic growth. (vi) We identified in vitro proteolytic activities suggestive of known fungal pathogenic traits in G. destructans. Conclusions/Significance Further studies are needed to understand whether G. destructans WNS is a symptom or a trigger for bat mass mortality. The availability of well-characterized G. destructans strains should promote an understanding of bat–fungus relationships, and should aid in the screening of biological and chemical control agents.

Public health impact of reemergence of rabies, New York.

This report summarizes the spread of a raccoon rabies epizootic into New York in the 1990s, the species of animals affected, and human postexposure treatments (PET). A total of 57,008 specimens were submitted to the state laboratory from 1993 to 1998; 8,858 (16%) animals were confirmed rabid, with raccoons the most common species (75%). After exposure to 11,769 animals, 18,238 (45%) persons received PET, mostly because of contact with saliva or nervous tissue. We analyzed expenditure reports to estimate the cost of rabies prevention activities. An estimated

Comparing ONRAB® AND RABORAL V-RG® oral rabies vaccine field performance in raccoons and striped skunks, New Brunswick, Canada, and Maine, USA.

13.9 million was spent in New York State to prevent rabies from 1993 to 1998. Traditional prevention methods such as vaccinating pets, avoiding wildlife, and verifying an animal’s rabies status must be continued to reduce costly PET. To reduce rabid animals, exposures, and costs, oral vaccination of wildlife should also be considered.

Clonal genotype of Geomyces destructans among bats with White Nose Syndrome, New York, USA.

Control of rabies in mesocarnivore reservoirs through oral rabies vaccination (ORV) requires an effective vaccine bait. Oral rabies vaccine performance in the field may be affected by a variety of factors, including vaccine bait density and distribution pattern, habitat, target species population density, and the availability of competing foods. A field study in which these covariates were restricted as much as possible was conducted along the international border of the state of Maine (ME), USA, and the province of New Brunswick (NB), Canada, to compare the performance of two oral rabies vaccines in raccoons (Procyon lotor) and striped skunks (Mephitis mephitis). RABORAL V-RG® (vaccinia-rabies glycoprotein recombinant oral vaccine in fishmealcoated sachet) or ONRAB® (adenovirus-rabies glycoprotein recombinant oral vaccine in Ultralite bait matrix) were distributed in ME and NB, respectively, by fixed-wing aircraft at a density of 75 baits/km2 along parallel flight lines spaced 1.0 km apart. Sera were collected from live-trapped raccoons and skunks 5–7 wk post-ORV and assayed to determine antibody prevalence in each area. Duplicate serum samples were provided blind to two different laboratories for analyses by rabies virus serum neutralization assays (at both laboratories) and a competitive enzyme-linked immunosorbent assay (at one laboratory). There was no significant difference in the proportion of antibody-positive animals determined by the three serologic methods, nor was there a significant difference between ONRAB and RABORAL V-RG in the proportion of antibody-positive striped skunks observed post-ORV. In contrast, the proportion of antibody-positive raccoons was significantly higher in the ONRAB- versus the RABORAL V-RG–baited areas (74% vs. 30%; χ2=89.977, df=5, P<0.0001). These data support that ONRAB may serve as an effective tool for raccoon rabies control.

The resistance of a North American bat species (Eptesicus fuscus) to White-nose Syndrome (WNS).

The dispersal mechanism of Geomyces destructans, which causes geomycosis (white nose syndrome) in hibernating bats, remains unknown. Multiple gene genealogic analyses were conducted on 16 fungal isolates from diverse sites in New York State during 2008–2010. The results are consistent with the clonal dispersal of a single G. destructans genotype.

Effects of Aerosolized Rabies Virus Exposure on Bats and Mice

White-nose Syndrome (WNS) is the primary cause of over-winter mortality for little brown (Myotis lucifugus), northern (Myotis septentrionalis), and tricolored (Perimyotis subflavus) bats, and is due to cutaneous infection with the fungus Pseudogymnoascus (Geomyces) destructans (Pd). Cutaneous infection with P. destructans disrupts torpor patterns, which is thought to lead to a premature depletion of body fat reserve. Field studies were conducted at 3 WNS-affected hibernation sites to determine if big brown bats (Eptesicus fuscus) are resistant to Pd. Radio telemetry studies were conducted during 2 winters to determine the torpor patterns of 23 free-ranging E. fuscus hibernating at a site where Pd occurs. The body fat contents of free-ranging E. fuscus and M. lucifugus during hibernation at 2 different WNS-affected sites were also determined. The numbers of bats hibernating at the same site was determined during both: a) 4–7 years prior to the arrival of Pd, and, b) 2–3 years after it first appeared at this site. The torpor bouts of big brown bats hibernating at a WNS-affected site were not significantly different in length from those previously reported for this species. The mean body fat content of E. fuscus in February was nearly twice that of M. lucifugus hibernating at the same WNS-affected sites during this month. The number of M. lucifugus hibernating at one site decreased by 99.6% after P. destructans first appeared, whereas the number of E. fuscus hibernating there actually increased by 43% during the same period. None of the E. fuscus collected during this study had any visible fungal growth or lesions on their skin, whereas virtually all the M. lucifugus collected had visible fungal growth on their wings, muzzle, and ears. These findings indicate that big brown bats are resistant to WNS.

Oral Rabies Vaccination in Raccoons: Comparison of ONRAB® and RABORAL V-RG® Vaccine-Bait Field Performance in Québec, Canada and Vermont, USA

Between 1956 and 1977, 4 human cases of rabies virus infection were attributed to aerosolized rabies virus; however, little work has been done to address this topic since the late 1960s. Employing modern nebulization equipment coupled with serologic, cell culture, and molecular technology, we have continued the investigation into aerosolized rabies virus as a potential route of transmission. Laboratory mice and 2 species of bats were exposed, through aerosol, to 3 variants of rabies virus. All bats survived exposure to aerosolized rabies virus and produced rabies neutralizing antibody. Several mice died of rabies as a result of aerosol exposure. Antibody response was followed for 6 months before animals were given an intramuscular challenge of rabies virus. Poor protection from challenge was afforded in bats, despite the presence of neutralizing antibodies.

Naturally Acquired Rabies Virus Infections in Wild-Caught Bats

The control of rabies in raccoons (Procyon lotor) and striped skunks (Mephitis mephitis) in North America has been conducted mainly through aerial distribution of oral vaccine-baits. The effectiveness of the vaccine-bait used is therefore of prime importance for disease eradication. In a previous field comparison between the ONRAB® bait in the province of New Brunswick, Canada, and RABORAL V-RG® bait in the state of Maine, USA, the ONRAB bait produced a higher percentage of antibody-positive raccoons under nearly identical bait distribution for the two vaccines. The main objective of the present study was to conduct a similar cross-border comparison of these two vaccine-baits using raccoon sera collected during post–oral rabies vaccination monitoring in Québec, Canada, and Vermont, USA, where ONRAB and V-RG, respectively, were distributed aerially at a targeted density of 150 baits/km2. A comparison of the equivalency of two serologic tests used in Canada and the USA was also conducted using sera from raccoons and striped skunks. Rabies virus neutralization assay (USA) yielded similar results to the competitive enzyme-linked immunosorbent assay (Canada), with agreement between the two tests of 92% for raccoon sera and 96% for skunk sera. With both assays, the percentage of antibody-positive raccoons was greater with ONRAB (51%, n=265) than with V-RG (38%, n=66). These new results support the conclusion from the previous study, that ONRAB vaccine-baits may be more effective for the control of rabies in raccoons.