Virally-vectored vaccine candidates against white-nose syndrome induce anti-fungal immune response in little brown bats (Myotis lucifugus).

White-nose syndrome (WNS) caused by the fungus, Pseudogymnoascus destructans (Pd) has killed millions of North American hibernating bats. Currently, methods to prevent the disease are limited. We conducted two trials to assess potential WNS vaccine candidates in wild-caught Myotis lucifugus. In a pilot study, we immunized bats with one of four vaccine treatments or phosphate-buffered saline (PBS) as a control and challenged them with Pd upon transfer into hibernation chambers. Bats in one vaccine-treated group, that received raccoon poxviruses (RCN) expressing Pd calnexin (CAL) and serine protease (SP), developed WNS at a lower rate (1/10) than other treatments combined (14/23), although samples sizes were small. The results of a second similar trial provided additional support for this observation. Bats vaccinated orally or by injection with RCN-CAL and RCN-SP survived Pd challenge at a significantly higher rate (P = 0.01) than controls. Using RT-PCR and flow cytometry, combined with fluorescent in situ hybridization, we determined that expression of IFN-γ transcripts and the number of CD4 + T-helper cells transcribing this gene were elevated (P < 0.10) in stimulated lymphocytes from surviving vaccinees (n = 15) compared to controls (n = 3). We conclude that vaccination with virally-vectored Pd antigens induced antifungal immunity that could potentially protect bats against WNS.

Protection of bats (Eptesicus fuscus) against rabies following topical or oronasal exposure to a recombinant raccoon poxvirus vaccine.

Rabies is an ancient neglected tropical disease that causes tens of thousands of human deaths and millions of cattle deaths annually. In order to develop a new vaccine for potential use in bats, a reservoir of rabies infection for humans and animals alike, an in silico antigen designer tool was used to create a mosaic glycoprotein (MoG) gene using available sequences from the rabies Phylogroup I glycoprotein. This sequence, which represents strains more likely to occur in bats, was cloned into raccoonpox virus (RCN) and the efficacy of this novel RCN-MoG vaccine was compared to RCN-G that expresses the glycoprotein gene from CVS-11 rabies or luciferase (RCN-luc, negative control) in mice and big brown bats (Eptesicus fuscus). Mice vaccinated and boosted intradermally with 1 x 107 plaque forming units (PFU) of each RCN-rabies vaccine construct developed neutralizing antibodies and survived at significantly higher rates than controls. No significant difference in antibody titers or survival was noted between rabies-vaccinated groups. Bats were vaccinated either oronasally (RCN-G, RCN-MoG) with 5x107 PFU or by topical application in glycerin jelly (RCN-MoG, dose 2x108 PFU), boosted (same dose and route) at 46 days post vaccination (dpv), and then challenged with wild-type big brown variant RABV at 65 dpv. Prior to challenge, 90% of RCN-G and 75% of RCN-MoG oronasally vaccinated bats had detectable levels of serum rabies neutralizing antibodies. Bats from the RCN-luc and topically vaccinated RCN-MoG groups did not have measurable antibody responses. The RCN-rabies constructs were highly protective and not significantly different from each other. RCN-MoG provided 100% protection (n = 9) when delivered oronasally and 83% protection (n = 6) when delivered topically; protection provided by the RCN-G construct was 70% (n = 10). All rabies-vaccinated bats survived at a significantly (P ≤ 0.02) higher rate than control bats (12%; n = 8). We have demonstrated the efficacy of a novel, in silico designed rabies MoG antigen that conferred protection from rabies challenge in mice and big brown bats in laboratory studies. With further development, topical or oronasal administration of the RCN-MoG vaccine could potentially mitigate rabies in wild bat populations, reducing spillover of this deadly disease into humans, domestic mammals, and other wildlife.

Infectivity of attenuated poxvirus vaccine vectors and immunogenicity of a raccoonpox vectored rabies vaccine in the Brazilian Free-tailed bat (Tadarida brasiliensis).

Bats (Order Chiroptera) are an abundant group of mammals with tremendous ecological value as insectivores and plant dispersers, but their role as reservoirs of zoonotic diseases has received more attention in the last decade. With the goal of managing disease in free-ranging bats, we tested modified vaccinia Ankara (MVA) and raccoon poxvirus (RCN) as potential vaccine vectors in the Brazilian Free-tailed bat (Tadarida brasiliensis), using biophotonic in vivo imaging and immunogenicity studies. Animals were administered recombinant poxviral vectors expressing the luciferase gene (MVA-luc, RCN-luc) through oronasal (ON) or intramuscular (IM) routes and subsequently monitored for bioluminescent signal indicative of viral infection. No clinical illness was noted after exposure to any of the vectors, and limited luciferase expression was observed. Higher and longer levels of expression were observed with the RCN-luc construct. When given IM, luciferase expression was limited to the site of injection, while ON exposure led to initial expression in the oral cavity, often followed by secondary replication at another location, likely the gastric mucosa or gastric associated lymphatic tissue. Viral DNA was detected in oral swabs up to 7 and 9 days post infection (dpi) for MVA and RCN, respectively. While no live virus was detected in oral swabs from MVA-infected bats, titers up to 3.88 x 104 PFU/ml were recovered from oral swabs of RCN-infected bats. Viral DNA was also detected in fecal samples from two bats inoculated IM with RCN, but no live virus was recovered. Finally, we examined the immunogenicity of a RCN based rabies vaccine (RCN-G) following ON administration. Significant rabies neutralizing antibody titers were detected in the serum of immunized bats using the rapid fluorescence focus inhibition test (RFFIT). These studies highlight the safety and immunogenicity of attenuated poxviruses and their potential use as vaccine vectors in bats.

Analgesic effects of carprofen and liposome-encapsulated butorphanol tartrate in Hispaniolan parrots (Amazona ventralis) with experimentally induced arthritis.

OBJECTIVE: To evaluate the microcrystalline sodium urate (MSU) method for inducing arthritis in parrots and to compare the analgesic efficacy of long-acting liposome-encapsulated butorphanol (LEBT), carprofen, or a combination of both. ANIMALS: 20 Hispaniolan parrots. PROCEDURES: MSU was injected into a tibiotarsal-tarsometatarsal (intertarsal) joint to induce arthritis (time 0). Four treatments were compared (LEBT [15 mg/kg, SC] administered once at time 0; injections of carprofen [3 mg/kg, IM, q 12 h] starting at time 0; administration of LEBT plus carprofen; and a control treatment of saline [0.9% NaCl] solution). Weight load testing and behavioral scoring were conducted at 0, 2, 6, 26, and 30 hours. RESULTS: Injection of MSU into the intertarsal joint induced arthritis, which resolved within 30 hours. Treatment with LEBT or LEBT plus carprofen resulted in significantly greater weight-bearing load on the limb with induced arthritis, compared with the control treatment. Treatment with carprofen alone caused a slight but nonsignificant improvement in weight-bearing load on the arthritic limb, compared with the control treatment. Behaviors associated with motor activity and weight bearing differed between the control and analgesic treatments. CONCLUSIONS AND CLINICAL RELEVANCE: Butorphanol was an effective treatment for pain associated with arthritis, but carprofen administered every 12 hours was insufficient. Injection of MSU to induce arthritis in a single joint was a good method for evaluating tonic pain in parrots, and measurement of the weight-bearing load was accurate for assessment of arthritic pain; however, behavioral changes associated with pain were subtle.