Susceptibility and pathogenesis of little brown bats (Myotis lucifugus) to heterologous and homologous rabies viruses.

Rabies virus (RABV) maintenance in bats is not well understood. Big brown bats (Eptesicus fuscus), little brown bats (Myotis lucifugus), and Mexican free-tailed bats (Tadarida brasiliensis) are the most common bats species in the United States. These colonial bat species also have the most frequent contact with humans and domestic animals. However, the silver-haired bat (Lasionycteris noctivagans) RABV is associated with the majority of human rabies virus infections in the United States and Canada. This is of interest because silver-haired bats are more solitary bats with infrequent human interaction. Our goal was to determine the likelihood of a colonial bat species becoming infected with and transmitting a heterologous RABV. To ascertain the potential of heterologous RABV infection in colonial bat species, little brown bats were inoculated with a homologous RABV or one of two heterologous RABVs. Additionally, to determine if the route of exposure influenced the disease process, bats were inoculated either intramuscularly (i.m.) or subcutaneously (s.c.) with a homologous or heterologous RABV. Our results demonstrate that intramuscular inoculation results in a more rapid progression of disease onset, whereas the incubation time in bats inoculated s.c. is significantly longer. Additionally, cross protection was not consistently achieved in bats previously inoculated with a heterologous RABV following a challenge with a homologous RABV 6 months later. Finally, bats that developed rabies following s.c. inoculation were significantly more likely to shed virus in their saliva and demonstrated increased viral dissemination. In summary, bats inoculated via the s.c. route are more likely to shed virus, thus increasing the likelihood of transmission.

Presence of cross-reactions with other viral encephalitides in the indirect fluorescent-antibody test for diagnosis of rabies.

The antemortem diagnosis of rabies in humans employs techniques that require accuracy, speed, and sensitivity. A combination of histochemical analysis, in vitro virus isolation, immunological methods, and molecular amplification procedures are utilized in efforts to diagnose the disease. Modern medicine now offers potentially life-saving treatment for a disease that was considered invariably fatal once clinical signs develop. However, medical intervention efforts require a rapid and accurate diagnosis as early in the course of clinical disease as possible. Indirect fluorescent-antibody (IFA) testing on cerebrospinal fluid and serum specimens provides rapid results, but the specificity of the assay has not been well studied. Because false-positive IFA results could significantly affect patient treatment and outcomes, it is critical to understand the specificity of this assay. In this study, IFA testing was performed on 135 cerebrospinal fluid and serum specimens taken from patients with viral encephalitis or a presumed viral infection involving an agent other than rabies virus. Results indicate that false-positive results can occur in interpreting the rabies IFA test. Staining patterns morphologically similar to antirabies staining were observed in 7 of the 135 cerebrospinal fluid specimens examined. In addition, a majority of the cerebrospinal fluid specimens tested from patients with encephalitis presented immunoglobulin that bound to antigens present in the cell culture substrate. Of marked concern was the frequent presence of cross-reactive antibodies in encephalitis cases associated with West Nile and Powassan flaviviruses. Because IFA testing for rabies on human specimens may result in false-positive results, it should not be used as the sole basis for initiating antirabies treatment.

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

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.

Rapid real-time PCR assay for culture and tissue identification of Geomyces destructans: the etiologic agent of bat geomycosis (white nose syndrome).

Geomyces destructans is the etiologic agent of bat geomycosis, commonly referred to as white nose syndrome (WNS). This infection has caused severe morbidity and mortality in little brown bats (Myotis lucifugus) and has also spread to other bat species with significant decline in the populations. Currently, G. destructans infection is identified by culture, ITS-PCR, and histopathology. We hypothesized that a real-time PCR assay would considerably improve detection of G. destructans in bats. The 100 bp sequence of the Alpha-L-Rhamnosidase gene was validated as a target for real-time PCR. The assay sensitivity was determined from serial dilution of DNA extracted from G. destructans conidia (5 × 10(-1)-5 × 10(7)), and the specificity was tested using DNA from 30 closely and distantly related fungi and 5 common bacterial pathogens. The real-time PCR assay was highly sensitive with detection limit of two G. destructans conidia per reaction at 40 PCR cycles. The assay was also highly specific as none of the other fungal or bacterial DNA cross-reacted in the real-time PCR assay. One hundred and forty-seven bat tissue samples, suspected of infection with G. destructans, were used to compare the real-time PCR assay to other methods employed for the detection of G. destructans. Real-time PCR was highly sensitive with 80 of 147 (55%) samples testing positive for G. destructans DNA. In comparison, histopathology examination revealed 64/147 (44%) positive samples. The internal transcribed spacer (ITS)-PCR yielded positive amplicon for G. destructans from 37 tissue samples (25%). The least sensitive assay was the fungal culture with only 17 tissue samples (12%) yielding G. destructans in culture. The data suggested that the real-time PCR assay is highly promising for rapid, sensitive, and specific identification of G. destructans. Further trials and inter-laboratory comparisons of this novel assay are recommended to improve the diagnosis of bat geomycosis.

ORAL RABIES VACCINATION STRATEGIES TOWARD RACCOON (PROCYON LOTOR) RABIES ELIMINATION ON SUBURBAN LONG ISLAND, NEW YORK, USA.

Approximately 1.86 million baits containing a vaccinia-rabies glycoprotein recombinant vaccine were distributed with helicopters, vehicles, and bait stations during 2006-10. A bait density of 250 baits/km2 effectively controlled rabies cases in enzootic and preepizootic areas. However, a cluster of 11 rabid raccoons at the eastern edge of infection resulted in the initiation of semiannual, high-density (500 baits/km2) vaccination campaigns in approximately 20% of the oral rabies vaccination zone during July and September (2007-09). Bait success (i.e., chewed sachets or removed baits) at bait stations was negatively associated with station distances from water. Conversely, bait success improved with increasing distances from roads. Bait stations deployed significantly more baits in developed open space when compared to low- and medium- to high-intensity developed areas. However, a difference was not detected between developed open space and forest habitats. Rabies was confined to 86 raccoons within 317 km2 (10%) of a 3,133 km2 suburban landscape, with a disproportionate number of rabid raccoons (n=74) in developed areas, when compared to 10 cases in forest-wetland habitats. Two rabid raccoons did not fall within either general land-use classification. Rabies advanced 15.1 km eastward at a rate of 6.4 km/yr during a 28-mo interval (2004-06).

VIRUS NEUTRALIZING ANTIBODY FOLLOWING ORAL RABIES VACCINATION OF RACCOONS (PROCYON LOTOR) ON SUBURBAN LONG ISLAND, NEW YORK, USA.

Vaccine-laden baits were distributed to interrupt and halt raccoon (Procyon lotor) rabies transmission in suburban Nassau and Suffolk counties on Long Island, New York, US. Fishmeal polymer baits containing the RABORAL V-RG® vaccine were deployed with helicopters, bait stations, and vehicles at a target density of 250 baits/km2 during annual September campaigns (2006-10). Semiannual campaigns (500 baits/km2) were also initiated in a portion of the treatment zone (2007-09) in response to a persistent focus of rabid raccoons. The last enzootic case was reported in January 2009. The final vaccination campaign was completed in 2010. The raccoon variant of rabies virus is no longer circulating in Nassau or Suffolk counties. Significantly greater probabilities of raccoon seroconversion were observed in helicopter-deployed bait zones. The lowest probabilities of seroconversion were identified in vehicle and bait station-deployment bait zones, with a marginal advantage associated with bait-station deployment. Seroconversion was negatively associated with developed, medium-intensity areas and increasing human population density. Significantly higher rabies virus neutralizing antibody endpoint titrations were detected in helicopter and bait station-deployment zones.

Detection of rabies viral neutralizing antibodies in the Puerto Rican Brachyphylla cavernarum.

The purpose of this study was to determine if Puerto Rican bats had previous exposure to rabies virus based on viral neutralizing antibodies. Our results demonstrate that 6.5% of the bats in this study had some exposure to rabies virus. The route of exposure is unknown but may have occurred following interaction with a rabid terrestrial animal or an unidentified bat rabies virus.

Cost and Relative Value of Road Kill Surveys for Enhanced Rabies Surveillance in Raccoon Rabies Management.

Oral rabies vaccination (ORV) requires knowledge of the spatial-temporal distribution of rabies virus variants targeted for control. Rabies-exposure based public health surveillance alone may not provide a sound basis for ORV decisions. The value and cost of road kill surveys was evaluated for the late spring⁻early fall 2005⁻2007 as a part of enhanced rabies surveillance in northern New York, where raccoon rabies is enzootic and ORV has occurred since the late 1990s. Structured surveys were conducted to collect raccoons and other meso-carnivores for rabies testing at the New York State Rabies Laboratory. Of the 209 meso-carnivore heads collected and submitted for testing, 175 were testable by direct fluorescent antibody; none was rabid. Rabies was also not reported through public health surveillance in survey zones during 2005⁻2007. Overall, survey costs were $37,118 (2016 USD). Salaries and benefits accounted for 61% of costs, followed by fuel (22%), vehicle depreciation (14%), and sample shipping (3%). Mean daily distance driven was 303 ± 37 km and 381 ± 28 km for total road kills and raccoons, respectively. Costs/road kill collected and submitted was $176/all species and $224/raccoon. This study provides costs for planning road kill surveys and underscores the need to continually improve enhanced rabies surveillance approaches to support ORV decision making.

Identification of secreted and membrane-bound bat immunoglobulin using a Microchiropteran-specific mouse monoclonal antibody.

Bat immunity has received increasing attention because some bat species are being decimated by the fungal disease, White Nose Syndrome, while other species are potential reservoirs of zoonotic viruses. Identifying specific immune processes requires new specific tools and reagents. In this study, we describe a new mouse monoclonal antibody (mAb) reactive with Eptesicus fuscus immunoglobulins. The epitope recognized by mAb BT1-4F10 was localized to immunoglobulin light (lambda) chains; hence, the mAb recognized serum immunoglobulins and B lymphocytes. The BT1-4F10 epitope appeared to be restricted to Microchiropteran immunoglobulins and absent from Megachiropteran immunoglobulins. Analyses of sera and other E. fuscus fluids showed that most, if not all, secreted immunoglobulins utilized lambda light chains. Finally, mAb BT1-4F10 permitted the identification of B cell follicles in splenic white pulp. This Microchiropteran-specific mAb has potential utility in seroassays; hence, this reagent may have both basic and practical applications for studying immune process.

The Effects of Cutaneous Fatty Acids on the Growth of Pseudogymnoascus destructans, the Etiological Agent of White-Nose Syndrome (WNS).

White Nose Syndrome (WNS) greatly increases the over-winter mortality of little brown (Myotis lucifugus), Indiana (Myotis sodalis), northern (Myotis septentrionalis), and tricolored (Perimyotis subflavus) bats. It is caused by a cutaneous infection with the fungus Pseudogymnoascus destructans (Pd). Big brown bats (Eptesicus fuscus) are much more resistant to cutaneous infection with Pd, however. We thus conducted analyses of wing epidermis from hibernating E. fuscus and M. lucifugus to determine their fatty acid compositions, and laboratory Pd culture experiments at 4.0-13.4°C to determine the effects of these fatty acids on Pd growth. Our analyses revealed that the epidermis of both bat species contain the same 7 fatty acid types (14:0, 15:0, 16:0. 16:1, 18:0, 18:1, & 18:2), but the epidermis of M. lucifugus contains: a) more stearic (18:0) acid, b) less palmitoleic (16:1) acid, c) less myristic (14:0) acid, and, d) less oleic (18:1) acid than that of E. fuscus. The growth of Pd was inhibited by: a) myristic and stearic acids at 10.5-13.4°C, but not at 4.0-5.0°C, b) oleic acid at 5.0-10.6°C, c) palmitoleic acid, and, d) linoleic (18:2) acid at 5.0-10.6°C. One set of factors that enables E. fuscus to better resist cutaneous P. destructans infections (and thus WNS) therefore appears to be the relatively higher myristic, palmitoleic, and oleic acid contents of the epidermis.

Overwintering of Rabies Virus in Silver Haired Bats (Lasionycteris noctivagans).

Silver-haired bats, (Lasionycteris noctivagans) are semi-colonial, migratory tree bats that have infrequent contact with humans. Despite the species rarity, the L. noctivagans rabies variant is the most commonly reported rabies virus variant (RABV) in domestically acquired human rabies cases in the US. Unlike big brown bats (Eptesicus fuscus) and little brown bats (Myotis lucifugus), L. noctivagans are not considered true hibernators. It is unknown if RABV can overwinter in hibernating L. noctivagans or is only maintained in members of this taxa that migrate to warmer climates. To better understand RABV overwintering in this species, L. noctivagans were inoculated intramuscularly with either a homologous RABV (L. noctivagans Virus 1) or one of two heterologous RABV (Eptesicus fuscus Virus 2 and Myotis lucifugus Virus 1). Five days following inoculation, L. noctivagans were placed in a hibernation chamber for 6 weeks. Our results demonstrate that rabies virus can overwinter in L. noctivagans yet the incubation period was extended 6 weeks when compared to bats maintained at ambient temperatures. Additionally, we found that the longer the incubation period, the greater the viral dissemination to the salivary glands. Similar to our previous studies, L. noctivagans were most susceptible to a homologous variant. In summary, we found that RABV incubation is extended following a subcutaneous exposure or maintenance in hibernation and longer incubation times increase dissemination and potential for transmission.

A need for standardized rabies-virus diagnostic procedures: effect of cover-glass mountant on the reliability of antigen detection by the fluorescent antibody test.

The direct fluorescent antibody test is a sensitive and specific procedure used in the routine diagnosis of rabies. However, given the critical role of the rabies diagnostic laboratory in patient management and public health decision-making, the use of a standardized national rabies diagnostic procedure is highly recommended. Seemingly small variations in test procedures may have dramatic effects on sensitivity. For example, two independent reports of diminished staining performance of two lots of a commercial anti-rabies conjugate were investigated in this study. The diminished staining occurred only with a single rabies-virus variant, associated with big brown bats, Eptesicus fuscus, in the southwestern United States. Similarly diluted and prepared diagnostic reagents provided bright staining on all other variants of rabies-virus tested. Subsequent evaluation disclosed that the phenomenon was associated with the relative concentrations of glycerol used in the mounting media by the reporting laboratories. These findings, related to the proper selection of an optimal cover-glass mountant for use in the immunofluorescence procedure, demonstrate the potential for erroneous results with severe implications for patient health, when uncontrolled variations in protocol occur. This paper underscores the necessity for all rabies diagnostic laboratories to follow one standard protocol. Such a protocol has been placed on the websites maintained by the Centers for Disease Control and Prevention: .

Rabies virus infection in a pet guinea pig and seven pet rabbits.

Raccoon-variant rabies was confirmed in 7 pet rabbits and 1 pet guinea pig in New York State, and postexposure treatment was required in several adults and children. To prevent rabies virus infection, domestic rabbits and pet rodents should be protected from contact with wild animals, including double-cage housing when housed outside. Pet rabbits or rodents with any possible contact with a wild animal, particularly if the rabbit or rodent had wounds of unknown origin, should be quarantined for 6 months for observation, to prevent escape, and to avoid contact with humans, who will require treatment if the rabbit or rodent develops rabies. Bites and scratches to humans from rodents and lagomorphs should be evaluated for potential rabies exposure on an individual basis, with consideration of whether the animal was caged outside or permitted outdoors unsupervised.

Antigen detection, rabies virus isolation, and Q-PCR in the quantification of viral load in a natural infection of the North American beaver (Castor canadensis).

All mammals are believed susceptible to rabies virus infection, yet transmission from nonreservoir hosts to humans is uncommon. However, interactions between nonreservoir hosts and humans occur frequently and risk of exposure increases where rabies is enzootic. We describe rabies and apparent pantropism of rabies virus in a beaver (Castor canadensis).

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

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.

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/km(2). 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.

Rabies virus infection in Eptesicus fuscus bats born in captivity (naïve bats).

The study of rabies virus infection in bats can be challenging due to quarantine requirements, husbandry concerns, genetic differences among animals, and lack of medical history. To date, all rabies virus (RABV) studies in bats have been performed in wild caught animals. Determining the RABV exposure history of a wild caught bat based on the presence or absence of viral neutralizing antibodies (VNA) may be misleading. Previous studies have demonstrated that the presence of VNA following natural or experimental inoculation is often ephemeral. With this knowledge, it is difficult to determine if a seronegative, wild caught bat has been previously exposed to RABV. The influence of prior rabies exposure in healthy, wild caught bats is unknown. To investigate the pathogenesis of RABV infection in bats born in captivity (naïve bats), naïve bats were inoculated intramuscularly with one of two Eptesicus fuscus rabies virus variants, EfV1 or EfV2. To determine the host response to a heterologous RABV, a separate group of naïve bats were inoculated with a Lasionycteris noctivagans RABV (LnV1). Six months following the first inoculation, all bats were challenged with EfV2. Our results indicate that naïve bats may have some level of innate resistance to intramuscular RABV inoculation. Additionally, naïve bats inoculated with the LnV demonstrated the lowest clinical infection rate of all groups. However, primary inoculation with EfV1 or LnV did not appear to be protective against a challenge with the more pathogenic EfV2.

Structuring targeted surveillance for monitoring disease emergence by mapping observational data onto ecological process.

An efficient surveillance system is a crucial factor in identifying, monitoring and tackling outbreaks of infectious diseases. Scarcity of data and limited amounts of economic resources require a targeted effort from public health authorities. In this paper, we propose a mathematical method to identify areas where surveillance is critical and low reporting rates might leave epidemics undetected. Our approach combines the use of reference-based susceptible-exposed-infectious models and observed reporting data; We propose two different specifications, for constant and time-varying surveillance, respectively. Our case study is centred around the spread of the raccoon rabies epidemic in the state of New York, using data collected between 1990 and 2007. Both methods offer a feasible solution to analyse and identify areas of intervention.

Extended incubation period of rabies virus in a captive big brown bat (Eptesicus fuscus).

Rabies virus incubation in bats is typically less than 180 days, yet longer incubation periods have been described. We report a 267-day incubation in a big brown bat (Eptesicus fuscus) exposed to rabies virus before entering our captive colony.