DOI: 10.7589/2013-01-020
Journal of Wildlife Diseases, 50(2), 2014, pp. 374–377 # Wildlife Disease Association 2014
Experimental Oral Immunization of Ferret Badgers (Melogale moschata) with a Recombinant Canine Adenovirus Vaccine CAV-2-E3D-RGP and an Attenuated Rabies Virus SRV9 Jinghui Zhao,1,2 Ye Liu,1,2 Shoufeng Zhang,1,2 Lijun Fang,1 Fei Zhang,1 and Rongliang Hu1,3 1Laboratory of Epidemiology, Key Laboratory of Jilin Province for Zoonosis Prevention and Control, Military Veterinary Research Institute, Academy of Military Medical Sciences, 666 Liuying West Road, Changchun 130122, People’s Republic of China; 2These authors contributed equally to the manuscript; 3Corresponding author (email: [email protected])
suggested that FB rabies has likely formed an independent enzootic transmission cycle (Zhang et al. 2009). Recently, there was an outbreak of FB rabies in Taiwan after more than 50 yr rabies-free status since 1961, in which 150 FBs, one house shrew (Suncus murinus), and one dog were confirmed to have contracted rabies as of 30 September 2013 (Taiwan Central Epidemic Command Center 2013). This situation suggests the threat of FB rabies transmission to humans. Prevention of FB rabies may be an effective way to interrupt rabies virus transmission in this species. Oral vaccination has been demonstrated to be a practicable way to control wildlife rabies (Rupprecht et al. 2006). Attenuated rabies viruses (ERA/ SAD/SAG), recombinant vaccinia virus (V-RG), and recombinant human adenovirus (AdRG1.3) have all been used successfully in oral vaccination field programs to limit the spread of rabies in coyotes (Canis latrans), red foxes (Vulpes vulpes), raccoon dogs (Nyctereutes procyonoides), and raccoons (Procyon lotor) in North America and Western Europe (Rupprecht et al. 2004; Brown et al. 2012; Robardet et al. 2012). Previously, we constructed a recombinant canine adenovirus, CAV-2-E3DRGP, expressing the rabies virus glycoprotein (RGP). This recombinant vaccine stimulates an immunologic response in domestic dogs, domestic cats (Felis catus), and swine (Sus scrofa; Hu et al. 2006, 2007; Liu et al. 2008). Others have also demonstrated the efficacy of CAV-RGP recombinants in several animal species (Li et al. 2006; Henderson et al. 2009). The SRV9 virus (GenBank AF499686), an experimental
Ferret badgers (Melogale moschata) are a major reservoir of rabies virus in southeastern China. Oral immunization has been shown to be a practical method for wildlife rabies management in Europe and North America. Two groups of 20 ferret badgers were given a single oral dose of a recombinant canine adenovirus-rabies vaccine, CAV-2-E3D-RGP, or an experimental attenuated rabies virus vaccine, SRV9. At 21 days, all ferret badgers had seroconverted, with serum virus-neutralizing antibodies ranging from 0.1 to 4.5 IU/mL. Titers were .0.50 IU/mL (an acceptable level) in 17/20 and 16/20 animals receiving CAV-2-E3D-RGP or SRV9, respectively. The serologic results indicate that the recombinant CAV-2-E3D-RGP is at least as effective as the attenuated rabies virus vaccine. Both may be considered for additional research as oral rabies vaccine candidates for ferret badgers. Key words: Ferret badgers, oral immunization, recombinant rabies–canine adenovirus vaccine.
ABSTRACT:
Rabies is an acute encephalomyelitis caused by rabies virus or other lyssaviruses. Although dogs (Canis lupus domesticus) are the main reservoir worldwide, all mammals are believed to be susceptible and can transmit rabies virus to humans (Rupprecht et al. 2002). Human rabies cases associated with ferret badger (FB, Melogale moschata) have been reported in southeastern China since 1994, with 17 of 22 human rabies cases in Hangzhou, and 12 of 20 in Huzhou between 1994 and 2004 (Zhang et al. 2009). Rabies in FBs is an increasing public health threat to humans in southeastern China (including northern Jiangxi, central to western Zhejiang, and eastern Anhui provinces; Gong et al. 2007; Hu et al. 2009). Epidemiologic studies have 374
SHORT COMMUNICATIONS
FIGURE 1. Trap used for catching ferret badgers (Melogale moschata) in near Poyang Lake, Jiangxi province, China, March, 2011.
attenuated rabies virus vaccine, is a plaquecloned vaccine strain of SAD, adapted to BHK-21 cells, and has been maintained in our laboratory for .20 yr (Hu et al. 2006). Our objective was to determine if the CAV2-E3D-RGP vaccine and SRV9, when given by direct instillation into the oral cavity, could stimulate an effective immune response in FBs. We used FBs, of mixed ages and sexes. The animals were captured around Poyang Lake (approximately 29u059N, 116u179E) in Jiangxi province in March 2011, using specially designed live traps (Fig. 1). Live fish bait was attached to the door by a cord with a hook at the end, so that when the bait was disturbed, the door would close. The experiment was carried out in captivity after an acclimatization period of at least 5 days immediately after the capture in an isolated yard in Nanchang, Jiangxi. All animal experiments were conducted according to the Guideline on the Humane Treatment of Laboratory Animals (Ministry of Science and Technology of the People’s Republic of China 2006) and approved by the Animal Welfare Committee of Military Veterinary Research Institute, Changchun, China. All animals were housed individually in stainless steel cages and fed fresh meat (fish, chicken, or beef), ham, fruits, and commercial dog food; water was provided ad libitum. They were randomly divided into
375
three groups. Group 1 (n520) was given the CAV-2-E3D-RGP, and group 2 (n520), the attenuated rabies virus SRV9; group 3 (n510) was an unvaccinated control. For oral vaccination, 1 mL of vaccine containing 107.5 plaque-forming units of the viruses (CAV-2-E3D-RGP or SRV9) was administered to nonsedated animals (groups 1 and 2) by placing on the tongue, using a needleless syringe. All animals were bled by venipuncture before oral vaccination, then at 14 and 21 days thereafter. Sera were analyzed for rabies virus-specific neutralizing antibodies (VNA) using the fluorescent antibody virus neutralization test with BHK-21 cells (Cliquet et al. 1998). The data are summarized in Table 1. All animals were antibody negative (VNA50) for rabies VNA before vaccination. By day 14, 18 of 20 group 1 animals had developed VNA, and by day 21, all had seroconverted (range, 0.1–2.6 IU/mL), 17 with VNA titers .0.5 IU/mL (the minimum defined acceptable threshold as recommended by the World Health Organization [WHO] for basic human immune response comparisons). In group 2, 17 of 20 animals had detectable VNA on day 14, with all seroconverting by day 21 (range, 0.1–4.5 IU/mL), and with 16 having VNA titers . 0.5 IU/mL. Group 3 animals remained antibody negative throughout the experiment. The observed intergroup differences of the VNA titer were analyzed via the rank sum test using the Statistical Package for Social Sciences (SPSS Inc., Chicago, Illinois, USA), version 13.0. There was no statistically significant difference between the two groups. Street rabies virus challenge could not be performed due to the continuing death of FBs 21 days post vaccination in all three groups. The death of FBs was apparently not caused by rabies because the brains of all the dead FBs were negative by fluorescent antibody test; mortality was probably due to poor adaptation to captivity or other unknown disease.
376
JOURNAL OF WILDLIFE DISEASES, VOL. 50, NO. 2, APRIL 2014
TABLE 1. Rabies virus neutralizing antibody titers (IU/mL)a in ferret badgers (Melogale moschata) vaccinated with CAV-2-E3D-RGP and SRV9 in Nanchang, Jiangxi Province, China, 2011. Animal grouping and number
Group 1 (CAV-2E3D-RGP)
Mean6SD Group 2 (SRV9)
Mean6SD a
n
Day 0
20
,0.1 ,0.1 ,0.1 ,0.1 ,0.1 ,0.1 ,0.1 ,0.1 ,0.1 ,0.1 ,0.1 ,0.1 ,0.1 ,0.1 ,0.1 ,0.1 ,0.1 ,0.1 ,0.1 ,0.1 — ,0.1 ,0.1 ,0.1 ,0.1 ,0.1 ,0.1 ,0.1 ,0.1 ,0.1 ,0.1 ,0.1 ,0.1 ,0.1 ,0.1 ,0.1 ,0.1 ,0.1 ,0.1 ,0.1 ,0.1 —
20
Day 14
Day 21
1.4 2. 0 0.3 0.2 0.2 0.3 0.5 0.7 0.5 0.5 1.5 1.4 0.3 0.9 0.9 0.7 ,0.1 0.2 1.1 1.4 0.9 1.1 0.9 0.9 0.5 0.5 2.6 2.6 1.1 2.0 0.7 0.9 1.5 1.5 ,0.1 0.7 1.4 1.1 0.7 0.7 0.860.6 1.060.6 0.1 1.5 ,0.1 0.5 0.2 0.2 ,0.1 0.1 0.2 4.5 0.3 2.6 2.6 2.6 0.4 0.4 0.9 1.1 2.0 1.4 0.7 0.7 0.9 3.4 0.3 2.6 0.23 0.7 2.0 2.0 2.6 3.4 ,0.1 0.1 0.7 0.9 1.1 1.1 2.6 2.6 0.960.9 1.661.3
VNA titers .0.1 IU/mL are considered seroconverted; .0.5 IU/mL are considered to be the minimum defined acceptable threshold.
Consequently, longer term studies were not possible. However, our preliminary results showed that all animals, given a single oral dose of either the recombinant
canine adenovirus or an attenuated rabies virus vaccine, seroconverted, with the majority (80–85%) producing VNA titers .0.50 IU/mL, indicative of levels above a minimum threshold (WHO 2013). The recombinant CAV-2-E3D-RGP was at least as effective as the attenuated live vaccine with regard to comparative immunogenicity. The recombinant CAV-2-E3DRGP may have greater potential for practical use than the experimental SRV9, because the recombinant utilizes an attenuated canine adenovirus strain as a vector and no live rabies virus is involved; therefore it is potentially safer from a public health and agricultural perspective. The vaccines were well tolerated. No adverse clinical signs were recorded during the 21-day observation period in the test groups, compared to the control group. Although some reports have shown that the preexisting immunity against CAV apparently hinders the replication of the recombinant CAV-2 (Sumner et al. 1988; Hamir et al. 1992), our previous tests had shown that there was no CAV-2–specific neutralizing antibody in FBs and the hemagglutination inhibition (HI) test on the serum samples before vaccination also were HI antibody negative. Therefore, alternative use of the recombinant vaccine compared to the attenuated SRV9 rabies virus may be a practical way in avoiding existing antibodies against the vector. Due to continuing deaths, all remaining animals were euthanized by overdose of pentobarbital sodium 25 days after oral vaccination. Following euthanasia, no rabies virus was detected in the brains of the FBs including the controls by the direct immunofluorescence assay (Dean et al. 1996). In China, no practical rabies vaccine has been developed for wildlife, and no antirabies oral vaccine has been registered. This study represents the first use of a recombinant canine adenovirus-rabies vaccine (CAV-2-E3D-RGP) and an attenuated rabies virus vaccine SRV9 in FBs and provides evidence of their promise as
SHORT COMMUNICATIONS
candidates for oral rabies vaccination. Future work should include the development of attractive baits for delivery and improved husbandry and management of FB in captivity to further test the safety and efficacy of delivery of prospective vaccine laden baits. This investigation was funded by the National Natural Science Foundation of China (30630049, 30972199) and the China National ‘‘973’’ Program (2005CB52300, 2011CB500705). LITERATURE CITED Brown LJ, Rosatte RC, Fehlner-Gardiner C, Taylor JS, Davies JC, Donovan D. 2012. Immune response and protection in raccoons (Procyon lotor) following consumption of baits containing ONRABH, a human adenovirus rabies glycoprotein recombinant vaccine. J Wildl Dis 48:1010– 1020. Cliquet F, Aubert M, Sagne L. 1998. Development of a fluorescent antibody virus neutralisation test (FAVN test) for the quantitation of rabiesneutralising antibody. J Immunol Methods 212:79–87. Dean DJ, Abelseth MK, Atanasiu P. 1996. The fluorescent antibody test. In: Laboratory techniques in rabies, 4th Ed., Meslin FX, Kaplan MM, Koprowski H, editors. World Health Organization, Geneva, Switzerland, pp. 88–93. Gong Z, Wang Z, Chen E, He F, Lin J, Li Y, Ding G, Fontaine RE. 2007. Human rabies cluster following badger bites, People’s Republic of China. Emerg Infect Dis 13:1956–1957. Hamir AN, Raju N, Rupprecht CE. 1992. Experimental oral administration of canine adenovirus (type 2) to raccoons (Procyon lotor). Vet Pathol 29:509–513. Henderson H, Jackson F, Bean K, Panasuk B, Niezgoda M, Slate D, Li J, Dietzschold B, Mattis J, Rupprecht CE. 2009. Oral immunization of raccoons and skunks with a canine adenovirus recombinant rabies vaccine. Vaccine 27:7194–7197. Hu R, Liu Y, Zhang S, Zhang F, Fooks AR. 2007. Experimental immunization of cats with a recombinant rabies-canine adenovirus vaccine elicits a long-lasting neutralizing antibody response against rabies. Vaccine 25:5301–5307. Hu R, Tang Q, Tang J, Fooks AR. 2009. Rabies in China: An update. Vector Borne Zoonotic Dis 9:1–12.
377
Hu R, Zhang S, Fooks AR, Yuan H, Liu Y, Li H, Tu C, Xia X, Xiao Y. 2006. Prevention of rabies virus infection in dogs by a recombinant canine adenovirus type-2 encoding the rabies virus glycoprotein. Microbes Infect 8:1090–1097. Li J, Faber M, Papaneri A, Faber ML, McGettigan JP, Schnell MJ, Dietzschold B. 2006. A single immunization with a recombinant canine adenovirus expressing the rabies virus G protein confers protective immunity against rabies in mice. Virology 356:147–154. Liu Y, Zhang S, Ma G, Zhang F, Hu R. 2008. Efficacy and safety of a live canine adenovirusvectored rabies virus vaccine in swine. Vaccine 26:5368–5372. Ministry of Science and Technology of the People’s Republic of China (MOST). 2006. Guideline on humane treatment of laboratory animals, www. most.gov.cn/fggw/zfwj/zfwj2006/zf06wj/zf06bfw/ 200609/t20060930_54196.htm. Accessed February 2011. Robardet E, Demerson JM, Andrieu S, Cliquet F. 2012. First European interlaboratory comparison of tetracycline and age determination with red fox teeth following oral rabies vaccination programs. J Wildl Dis 48:858–868. Rupprecht CE, Hanlon CA, Hemachudha T. 2002. Rabies re-examined. Lancet Infect Dis 2:327–343. Rupprecht CE, Hanlon CA, Slate D. 2004. Oral vaccination of wildlife against rabies: Opportunities and challenges in prevention and control. Dev Biol (Basel) 119:173–184. Rupprecht CE, Willoughby RR, Slate DD. 2006. Current and future trends in the prevention, treatment and control of rabies. Expert Rev Anti Infect Ther 4:1021–1038. Sumner JW, Shaddock JH, Wu GJ, Baer GM. 1988. Oral administration of an attenuated strain of canine adenovirus (type 2) to raccoons, foxes, skunk, and mongoose. Am J Vet Res 49:169–171. Taiwan Central Epidemic Command Center. 2013. Rabies real-time distribution, www.cdc.gov.tw/ professional/submenu.aspx?treeid5BEAC9C103 DF952C4&nowtreeid5B4B3A2CFD4480AA6. Accessed September 2013. World Health Organization. 2013. WHO Expert Consultation on Rabies. 2013. Second Report, WHO technical report series 982. World Health Organization, Geneva, Switzerland, 59 pp. Zhang S, Tang Q, Wu X, Liu Y, Zhang F, Rupprecht CE, Hu RL. 2009. Rabies in ferret badgers, southeastern China. Emerg Infect Dis 15:946–949. Submitted for publication 26 January 2013. Accepted 13 October 2013.
Journal of Wildlife Diseases, 50(2), 2014, pp. 374–377 # Wildlife Disease Association 2014
Experimental Oral Immunization of Ferret Badgers (Melogale moschata) with a Recombinant Canine Adenovirus Vaccine CAV-2-E3D-RGP and an Attenuated Rabies Virus SRV9 Jinghui Zhao,1,2 Ye Liu,1,2 Shoufeng Zhang,1,2 Lijun Fang,1 Fei Zhang,1 and Rongliang Hu1,3 1Laboratory of Epidemiology, Key Laboratory of Jilin Province for Zoonosis Prevention and Control, Military Veterinary Research Institute, Academy of Military Medical Sciences, 666 Liuying West Road, Changchun 130122, People’s Republic of China; 2These authors contributed equally to the manuscript; 3Corresponding author (email: [email protected])
suggested that FB rabies has likely formed an independent enzootic transmission cycle (Zhang et al. 2009). Recently, there was an outbreak of FB rabies in Taiwan after more than 50 yr rabies-free status since 1961, in which 150 FBs, one house shrew (Suncus murinus), and one dog were confirmed to have contracted rabies as of 30 September 2013 (Taiwan Central Epidemic Command Center 2013). This situation suggests the threat of FB rabies transmission to humans. Prevention of FB rabies may be an effective way to interrupt rabies virus transmission in this species. Oral vaccination has been demonstrated to be a practicable way to control wildlife rabies (Rupprecht et al. 2006). Attenuated rabies viruses (ERA/ SAD/SAG), recombinant vaccinia virus (V-RG), and recombinant human adenovirus (AdRG1.3) have all been used successfully in oral vaccination field programs to limit the spread of rabies in coyotes (Canis latrans), red foxes (Vulpes vulpes), raccoon dogs (Nyctereutes procyonoides), and raccoons (Procyon lotor) in North America and Western Europe (Rupprecht et al. 2004; Brown et al. 2012; Robardet et al. 2012). Previously, we constructed a recombinant canine adenovirus, CAV-2-E3DRGP, expressing the rabies virus glycoprotein (RGP). This recombinant vaccine stimulates an immunologic response in domestic dogs, domestic cats (Felis catus), and swine (Sus scrofa; Hu et al. 2006, 2007; Liu et al. 2008). Others have also demonstrated the efficacy of CAV-RGP recombinants in several animal species (Li et al. 2006; Henderson et al. 2009). The SRV9 virus (GenBank AF499686), an experimental
Ferret badgers (Melogale moschata) are a major reservoir of rabies virus in southeastern China. Oral immunization has been shown to be a practical method for wildlife rabies management in Europe and North America. Two groups of 20 ferret badgers were given a single oral dose of a recombinant canine adenovirus-rabies vaccine, CAV-2-E3D-RGP, or an experimental attenuated rabies virus vaccine, SRV9. At 21 days, all ferret badgers had seroconverted, with serum virus-neutralizing antibodies ranging from 0.1 to 4.5 IU/mL. Titers were .0.50 IU/mL (an acceptable level) in 17/20 and 16/20 animals receiving CAV-2-E3D-RGP or SRV9, respectively. The serologic results indicate that the recombinant CAV-2-E3D-RGP is at least as effective as the attenuated rabies virus vaccine. Both may be considered for additional research as oral rabies vaccine candidates for ferret badgers. Key words: Ferret badgers, oral immunization, recombinant rabies–canine adenovirus vaccine.
ABSTRACT:
Rabies is an acute encephalomyelitis caused by rabies virus or other lyssaviruses. Although dogs (Canis lupus domesticus) are the main reservoir worldwide, all mammals are believed to be susceptible and can transmit rabies virus to humans (Rupprecht et al. 2002). Human rabies cases associated with ferret badger (FB, Melogale moschata) have been reported in southeastern China since 1994, with 17 of 22 human rabies cases in Hangzhou, and 12 of 20 in Huzhou between 1994 and 2004 (Zhang et al. 2009). Rabies in FBs is an increasing public health threat to humans in southeastern China (including northern Jiangxi, central to western Zhejiang, and eastern Anhui provinces; Gong et al. 2007; Hu et al. 2009). Epidemiologic studies have 374
SHORT COMMUNICATIONS
FIGURE 1. Trap used for catching ferret badgers (Melogale moschata) in near Poyang Lake, Jiangxi province, China, March, 2011.
attenuated rabies virus vaccine, is a plaquecloned vaccine strain of SAD, adapted to BHK-21 cells, and has been maintained in our laboratory for .20 yr (Hu et al. 2006). Our objective was to determine if the CAV2-E3D-RGP vaccine and SRV9, when given by direct instillation into the oral cavity, could stimulate an effective immune response in FBs. We used FBs, of mixed ages and sexes. The animals were captured around Poyang Lake (approximately 29u059N, 116u179E) in Jiangxi province in March 2011, using specially designed live traps (Fig. 1). Live fish bait was attached to the door by a cord with a hook at the end, so that when the bait was disturbed, the door would close. The experiment was carried out in captivity after an acclimatization period of at least 5 days immediately after the capture in an isolated yard in Nanchang, Jiangxi. All animal experiments were conducted according to the Guideline on the Humane Treatment of Laboratory Animals (Ministry of Science and Technology of the People’s Republic of China 2006) and approved by the Animal Welfare Committee of Military Veterinary Research Institute, Changchun, China. All animals were housed individually in stainless steel cages and fed fresh meat (fish, chicken, or beef), ham, fruits, and commercial dog food; water was provided ad libitum. They were randomly divided into
375
three groups. Group 1 (n520) was given the CAV-2-E3D-RGP, and group 2 (n520), the attenuated rabies virus SRV9; group 3 (n510) was an unvaccinated control. For oral vaccination, 1 mL of vaccine containing 107.5 plaque-forming units of the viruses (CAV-2-E3D-RGP or SRV9) was administered to nonsedated animals (groups 1 and 2) by placing on the tongue, using a needleless syringe. All animals were bled by venipuncture before oral vaccination, then at 14 and 21 days thereafter. Sera were analyzed for rabies virus-specific neutralizing antibodies (VNA) using the fluorescent antibody virus neutralization test with BHK-21 cells (Cliquet et al. 1998). The data are summarized in Table 1. All animals were antibody negative (VNA50) for rabies VNA before vaccination. By day 14, 18 of 20 group 1 animals had developed VNA, and by day 21, all had seroconverted (range, 0.1–2.6 IU/mL), 17 with VNA titers .0.5 IU/mL (the minimum defined acceptable threshold as recommended by the World Health Organization [WHO] for basic human immune response comparisons). In group 2, 17 of 20 animals had detectable VNA on day 14, with all seroconverting by day 21 (range, 0.1–4.5 IU/mL), and with 16 having VNA titers . 0.5 IU/mL. Group 3 animals remained antibody negative throughout the experiment. The observed intergroup differences of the VNA titer were analyzed via the rank sum test using the Statistical Package for Social Sciences (SPSS Inc., Chicago, Illinois, USA), version 13.0. There was no statistically significant difference between the two groups. Street rabies virus challenge could not be performed due to the continuing death of FBs 21 days post vaccination in all three groups. The death of FBs was apparently not caused by rabies because the brains of all the dead FBs were negative by fluorescent antibody test; mortality was probably due to poor adaptation to captivity or other unknown disease.
376
JOURNAL OF WILDLIFE DISEASES, VOL. 50, NO. 2, APRIL 2014
TABLE 1. Rabies virus neutralizing antibody titers (IU/mL)a in ferret badgers (Melogale moschata) vaccinated with CAV-2-E3D-RGP and SRV9 in Nanchang, Jiangxi Province, China, 2011. Animal grouping and number
Group 1 (CAV-2E3D-RGP)
Mean6SD Group 2 (SRV9)
Mean6SD a
n
Day 0
20
,0.1 ,0.1 ,0.1 ,0.1 ,0.1 ,0.1 ,0.1 ,0.1 ,0.1 ,0.1 ,0.1 ,0.1 ,0.1 ,0.1 ,0.1 ,0.1 ,0.1 ,0.1 ,0.1 ,0.1 — ,0.1 ,0.1 ,0.1 ,0.1 ,0.1 ,0.1 ,0.1 ,0.1 ,0.1 ,0.1 ,0.1 ,0.1 ,0.1 ,0.1 ,0.1 ,0.1 ,0.1 ,0.1 ,0.1 ,0.1 —
20
Day 14
Day 21
1.4 2. 0 0.3 0.2 0.2 0.3 0.5 0.7 0.5 0.5 1.5 1.4 0.3 0.9 0.9 0.7 ,0.1 0.2 1.1 1.4 0.9 1.1 0.9 0.9 0.5 0.5 2.6 2.6 1.1 2.0 0.7 0.9 1.5 1.5 ,0.1 0.7 1.4 1.1 0.7 0.7 0.860.6 1.060.6 0.1 1.5 ,0.1 0.5 0.2 0.2 ,0.1 0.1 0.2 4.5 0.3 2.6 2.6 2.6 0.4 0.4 0.9 1.1 2.0 1.4 0.7 0.7 0.9 3.4 0.3 2.6 0.23 0.7 2.0 2.0 2.6 3.4 ,0.1 0.1 0.7 0.9 1.1 1.1 2.6 2.6 0.960.9 1.661.3
VNA titers .0.1 IU/mL are considered seroconverted; .0.5 IU/mL are considered to be the minimum defined acceptable threshold.
Consequently, longer term studies were not possible. However, our preliminary results showed that all animals, given a single oral dose of either the recombinant
canine adenovirus or an attenuated rabies virus vaccine, seroconverted, with the majority (80–85%) producing VNA titers .0.50 IU/mL, indicative of levels above a minimum threshold (WHO 2013). The recombinant CAV-2-E3D-RGP was at least as effective as the attenuated live vaccine with regard to comparative immunogenicity. The recombinant CAV-2-E3DRGP may have greater potential for practical use than the experimental SRV9, because the recombinant utilizes an attenuated canine adenovirus strain as a vector and no live rabies virus is involved; therefore it is potentially safer from a public health and agricultural perspective. The vaccines were well tolerated. No adverse clinical signs were recorded during the 21-day observation period in the test groups, compared to the control group. Although some reports have shown that the preexisting immunity against CAV apparently hinders the replication of the recombinant CAV-2 (Sumner et al. 1988; Hamir et al. 1992), our previous tests had shown that there was no CAV-2–specific neutralizing antibody in FBs and the hemagglutination inhibition (HI) test on the serum samples before vaccination also were HI antibody negative. Therefore, alternative use of the recombinant vaccine compared to the attenuated SRV9 rabies virus may be a practical way in avoiding existing antibodies against the vector. Due to continuing deaths, all remaining animals were euthanized by overdose of pentobarbital sodium 25 days after oral vaccination. Following euthanasia, no rabies virus was detected in the brains of the FBs including the controls by the direct immunofluorescence assay (Dean et al. 1996). In China, no practical rabies vaccine has been developed for wildlife, and no antirabies oral vaccine has been registered. This study represents the first use of a recombinant canine adenovirus-rabies vaccine (CAV-2-E3D-RGP) and an attenuated rabies virus vaccine SRV9 in FBs and provides evidence of their promise as
SHORT COMMUNICATIONS
candidates for oral rabies vaccination. Future work should include the development of attractive baits for delivery and improved husbandry and management of FB in captivity to further test the safety and efficacy of delivery of prospective vaccine laden baits. This investigation was funded by the National Natural Science Foundation of China (30630049, 30972199) and the China National ‘‘973’’ Program (2005CB52300, 2011CB500705). LITERATURE CITED Brown LJ, Rosatte RC, Fehlner-Gardiner C, Taylor JS, Davies JC, Donovan D. 2012. Immune response and protection in raccoons (Procyon lotor) following consumption of baits containing ONRABH, a human adenovirus rabies glycoprotein recombinant vaccine. J Wildl Dis 48:1010– 1020. Cliquet F, Aubert M, Sagne L. 1998. Development of a fluorescent antibody virus neutralisation test (FAVN test) for the quantitation of rabiesneutralising antibody. J Immunol Methods 212:79–87. Dean DJ, Abelseth MK, Atanasiu P. 1996. The fluorescent antibody test. In: Laboratory techniques in rabies, 4th Ed., Meslin FX, Kaplan MM, Koprowski H, editors. World Health Organization, Geneva, Switzerland, pp. 88–93. Gong Z, Wang Z, Chen E, He F, Lin J, Li Y, Ding G, Fontaine RE. 2007. Human rabies cluster following badger bites, People’s Republic of China. Emerg Infect Dis 13:1956–1957. Hamir AN, Raju N, Rupprecht CE. 1992. Experimental oral administration of canine adenovirus (type 2) to raccoons (Procyon lotor). Vet Pathol 29:509–513. Henderson H, Jackson F, Bean K, Panasuk B, Niezgoda M, Slate D, Li J, Dietzschold B, Mattis J, Rupprecht CE. 2009. Oral immunization of raccoons and skunks with a canine adenovirus recombinant rabies vaccine. Vaccine 27:7194–7197. Hu R, Liu Y, Zhang S, Zhang F, Fooks AR. 2007. Experimental immunization of cats with a recombinant rabies-canine adenovirus vaccine elicits a long-lasting neutralizing antibody response against rabies. Vaccine 25:5301–5307. Hu R, Tang Q, Tang J, Fooks AR. 2009. Rabies in China: An update. Vector Borne Zoonotic Dis 9:1–12.
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