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.

Detection of Rabies IgG and IgM Antibodies Using the Rabies Indirect Fluorescent Antibody Test.

The rabies indirect fluorescent antibody (IFA) test was developed to detect various rabies-specific antibody isotypes in sera or cerebral spinal fluid. This test provides rapid results and can be used to detect rabies antibodies in several different scenarios. The rabies IFA test is especially useful for the quick and early detection of antibodies to evaluate the immune response in a patient who has developed rabies. Although other methods for antemortem rabies diagnosis take precedence, this test may be utilized to demonstrate recent rabies virus exposure through antibody detection. The IFA test does not provide a virus-neutralizing antibody (VNA) titer, but the pre-exposure prophylaxis (PrEP) response can be evaluated through positive or negative antibody presence. This test can be utilized in various situations and can provide results for a number of different targets. In this study, we used several paired serum samples from individuals who received PrEP and demonstrated their rabies antibody presence over time using the IFA test.

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.

Clarifying Indeterminate Results on the Rabies Direct Fluorescent Antibody Test Using Real-Time Reverse Transcriptase Polymerase Chain Reaction.

OBJECTIVES: Each year, rabies virus infection results in the death of more than 50 000 persons worldwide. In the United States, the Centers for Disease Control and Prevention (CDC) reported 23 human rabies cases from May 1, 2008, through October 1, 2017. Although rabies testing in the United States is highly reliable, some specimens submitted to rabies laboratories do not have adequate tissues or may be substantially decomposed. In these instances, the specimen may be considered unsatisfactory for testing or produce indeterminate results using the gold standard direct fluorescent antibody test. The objective of this study was to evaluate the number of unsatisfactory samples or samples with indeterminate results that were positive for rabies virus after additional testing using real-time reverse transcriptase polymerase chain reaction (RT-PCR). METHODS: In 2016, we retested all unsatisfactory specimens or specimens with indeterminate results using real-time RT-PCR. We further typed any sample that was real-time RT-PCR positive to identify the infecting rabies virus variant. RESULTS: Of 210 retested unsatisfactory specimens or specimens with indeterminate results, 9 (4.3%) were positive for rabies. In each case, the animal was infected with a homologous rabies virus variant. CONCLUSION: These results confirm the recommendation by CDC and state public health laboratories that indeterminate results should be considered positive and justify the prompt treatment of exposed persons through an animal that is suspected to have rabies.

Rabies Necropsy Techniques in Large and Small Animals.

The New York State Department of Health (NYSDOH) Rabies Laboratory receives between 6,000 to 9,000 specimens annually and performs rabies testing for the entire state, with the exception of New York City. The Rabies laboratory necropsies a variety of animals ranging in size from bats to bovids. Most of these specimens are animals exhibiting neurological signs, however, less than 10% actually test positive for rabies; implying trauma, lesions or other infectious agents as the cause of these symptoms. Due to the risk of aerosolizing undiagnosed infectious agents, the Rabies Laboratory does not use power tools or saws. Three necropsy techniques will be presented for animals whose skulls are impenetrable with scissors. The laboratory has implemented these techniques to decrease potential exposure to infectious agents, eliminate unnecessary manipulation of the specimen and reduce processing time. The advantages of a preferred technique opposed to another is subject to the trained individual processing the specimen.

HEALTH SURVEY OF FREE-RANGING RACCOONS (PROCYON LOTOR) IN CENTRAL PARK, NEW YORK, NEW YORK, USA: IMPLICATIONS FOR HUMAN AND DOMESTIC ANIMAL HEALTH.

We conducted health assessments on 113 free-ranging raccoons ( Procyon lotor ) in Central Park, New York City, US, in February 2010, September 2010, and November 2011 in conjunction with a trap-vaccinate-release program to control a raccoon rabies epizootic. Five individuals were sampled at two time points for 118 raccoon examinations in total. We tested 13 of 13 and 8 of 13 euthanized raccoons for rabies and canine distemper virus (CDV), respectively, by antigen testing on brain tissue; all were negative for both viruses. Endoparasitism was the most common necropsy finding, with definitive identification of Baylisascaris procyonis in six of eight (75%) necropsied raccoons. Multiple intestinal parasites were detected in feces of living raccoons, including ascarid-type ova in 25 of 80 (31%) raccoons, with B. procyonis confirmed in one sample. Median blood lead level was 7.3 µg/dL (n=104). Rabies virus neutralizing antibody titer was ≥0.5 IU/mL in 9 of 88 (10%) raccoons naive to rabies vaccination and in 13 of 20 (65%) previously vaccinated raccoons. The majority of raccoons we tested were seropositive for canine parvovirus-2 (54/59, 92%) and Toxoplasma gondii (39/60, 65%). Fewer were seropositive for Rickettsia rickettsii (3/30, 10%). None were seropositive for CDV (n=108), canine adenovirus-1 (n=60), or Borrelia burgdorferi (n=30). Ectoparasites found during 16 of 118 (13.6%) physical examinations included Ixodes texanus ticks (15/118, 12.7%) and Trichodectes octomaculatus lice (1/118, 0.8%). We detected Campylobacter jejuni in 5 of 79 (6%) fecal samples. We detected 11 Salmonella enterica serotypes in 70 of 111 (63.1%) enteric cultures, the most common of which were Salmonella Newport (20/70, 29%) and Salmonella Oranienburg (20/70, 29%). These results indicate that raccoons in Central Park likely are involved in the environmental occurrence and potential disease transmission of a variety of infectious and noninfectious diseases of concern for human, wildlife, and domestic animal health.

Rabies direct fluorescent antibody test does not inactivate rabies or eastern equine encephalitis viruses.

An examination using the routine rabies direct fluorescent antibody test was performed on rabies or Eastern equine encephalitis positive mammalian brain tissue to assess inactivation of the virus. Neither virus was inactivated with acetone fixation nor the routine test, thus laboratory employees should treat all samples as rabies and when appropriate Eastern equine encephalitis positive throughout the whole procedure.

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.

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.

Naturally acquired rabies virus infections in wild-caught bats.

The study of a zoonotic disease requires an understanding of the disease incidence in animal reservoirs. Rabies incidence in bats submitted to diagnostic laboratories does not accurately reflect the true incidence in wild bat populations as a bias exists for testing bats that have been in contact with humans or pets. This article details the rabies incidence in two species of bats collected from natural settings without such bias. In this study, brain smears from 0.6% and 2.5% of wild-caught and apparently healthy Tadarida brasiliensis and Eptesicus fuscus, respectively, were positive for rabies virus (RV) antigen. Conversely, 92% of the grounded T. brasiliensis were positive for RV. Serology performed on captive colony and sick bats reveal an immune response to rabies. This work illustrates the complex interplay between immunity, disease state, and the conundrum of RV maintenance in bats.