Introduction: Rabies is a fatal disease caused by rabies virus (RABV) of the family Rhabdoviridae and the genus Lyssavirus. RABV kills warm-blooded animals including dogs and cattle. Pets and domestic animals, particularly dogs, are at increased risk if they live in regions populated by raccoon dogs (Nyctereutes procyonoises koreensis), which is a known reservoir of RABV and has played a key role in rabies transmission in South Korea. A total of 486 animals infected by rabid raccoons have been identified since 1993; these include 221 cattle, 184 dogs, 76 other raccoons, 4 feral cats, and 1 deer (www.kahis.go.kr). Prevention of rabies in animals is essential to reduce human rabies; more than 95% of all human cases are caused by dog bites. Both vaccination and the use of a bait vaccine (vaccinia-rabies glycoprotein [V-RG]) have been used successfully in South Korea; no case of animal rabies was recorded during 2013~2016. We previously constructed the recombinant rabies vaccine strain ERAGS with two mutations in the glycoprotein gene using reverse genetics. The strain was immunogenic in both mice and raccoon dogs when given either IM or orally. In the present study, we evaluated the safety and immunogenicity of the vaccine in dogs and cattle. Materials and Methods: Virus: ERAGS, which has Asn194Ser and Arg333Glu substitutions in the glycoprotein, was constructed using site-directed mutagenesis. CVS11, which was a fixed rabies strain, was used for fluorescent antibody virus neutralization (FAVN) tests. Safety and immunogenicity of ERAGS strain in animals: Four-month-old dogs, seronegative against rabies virus (RABV), were divided into 10 groups consisting of four dogs. Each group of dogs was inoculated with the ERAGS strain (10^7.0 TCID₅₀/mL to 10^2.0 TCID₅₀/mL) via IM or SC route. Cattle were also divided into six groups comprising five heads. Each group was inoculated with the same dose via IM route. Control animals received no treatment. Following inoculation, at 0, 4, and 8 weeks, blood was collected from all animals to measure virus neutralizing antibodies (VNA) against rabies virus. All animals were monitored daily for adverse effects, including anorexia, prostration, anxiety, agitation, aggression, and paralysis. FAVN test: rabies virus (CVS-11 strain) containing about 100 FAID₅₀/50 μl was added to each well. A 50 μl aliquot of BHK-21 cells containing 4 × 10⁵ cells/ mL was added to each well after 60 min of incubation at 37℃, and the microplates were incubated in a humidified incubator with 5% CO₂ at 37℃ for 72 h. The microplates were then fixed in cold acetone for 20 min. After staining with fluorescence isothiocyanate conjugated goat-anti mouse IgG + IgM, the microplates were air-dried and examined at 400× under a fluorescence microscope. Results: No clinical sign of rabies was noted after inoculation via the IM or SC route. Unvaccinated controls also remained normal through 8 WPI. All dogs in group 1 inoculated with 10^7.0 FAID₅₀/mL vaccine via the IM route developed high VNA titers (7.9~23.9 IU/mL [mean, 15.9 IU/mL]) by 4 WPI. Dogs in groups 2-4 inoculated with 10^5.0~10^3.0 FAID₅₀/mL vaccine via the IM route developed mean VNA titers of 4.56~0.61 IU/mL by 4 WPI. However, moderate decreases in mean VNA titers were evident in all groups by 8 WPI. Dogs in groups 6~8 inoculated with 10^5.0~10^3.0 FAID₅₀/mL vaccine via the SC route exhibited a slightly lower mean VNA titer (1.44~0.1 IU/mL) at 4 WPI. Dogs in groups 5 and 9 inoculated with 10^2.0 FAID₅₀/mL vaccine via the IM and SC routes, respectively did not develop VNAs by 4 or 8 WPI. All dog sera were subjected to the Platelia Rabies ELISA to detect anti-RABV antibodies. Cattle in group 11 inoculated with 10^7.0 FAID₅₀/mL vaccine via the IM route developed a moderate mean VNA titer of 3.05 IU/mL by 4 WPI. Cattle in groups 12~14 inoculated with diluted vaccine at 10^5.0~10^3.0 FAID₅₀/mL via the IM route developed mean VNA titers of 0.97~0.13 IU/mL by 4 WPI. However, moderate decreases in the mean VNA titers were apparent by 8 WPI. Cattle in group 15 inoculated with 10^2.0 FAID₅₀/mL vaccine via the IM route did not develop VNA Conclusions: Our findings indicate that the new rabies vaccine for pet and domestic animals is safe and immunogenic in dogs and cattle. References 1. Yang DK, Kim HH, Choi SS, Kim JT, Lee KB, Lee SH, Cho IS. Safety and immunogenicity of recombinant rabies virus (ERAGS) in mice and raccoon dogs. Clin Exp Vaccine Res. 2016 Jul;5(2):159-168. 2. Hanlon CA, Niezgoda M, Morrill P, Rupprecht CE. Oral efficacy of an attenuated rabies virus vaccine in skunks and raccoons. J Wildl Dis 2002;38:420-7.