Preliminary Evaluation of Raboral V-RG^® Oral Rabies Vaccine in Arctic Foxes (Vulpes lagopus)

Abstract

We tested the Raboral V-RG^® recombinant oral rabies vaccine for its response in Arctic foxes (Vulpes lagopus), the reservoir of rabies virus in the circumpolar North. The vaccine, which is currently the only licensed oral rabies vaccine in the United States, induced a strong antibody response and protected foxes against a challenge of 500,000 mouse intracerebral lethal dose 50% of an Arctic rabies virus variant. However, one unvaccinated control fox survived challenge with rabies virus, either indicating a high resistance of Arctic foxes to rabies infection or a previous exposure that induced immunity. This preliminary study suggested that Raboral V-RG vaccine may be efficacious in Arctic foxes.

Rabies is an important zoonotic disease that is endemic throughout the Arctic (Mork and Prestrud, 2004). Epizootics occur every 3–4 yr in these areas, with the most recent during the winter of 2006–2007 (Kuzmin et al., 2008). The Arctic fox is considered the primary rabies reservoir in Alaska, along the northern and western coast, and in the high Canadian Arctic (Mork and Prestrud, 2004). Although Arctic foxes maintain defended territories during the breeding period (March–August), they expand their range during winter (Pamperin, 2008; Pamperin et al., 2008). To secure food, they often congregate at dumpsters and landfills in areas of human development. Rabies epizootics occur during winter, as the probability for contact between animals is much higher than during summer when foxes avoid contacts outside the family unit. Although oral rabies vaccination programs have been conducted successfully in Europe and North America for red foxes (V. vulpes), raccoons (Procyon lotor), gray foxes (Urocyon cinereoargenteus), and coyotes (Canis latrans; Slate et al., 2009), no conclusive oral rabies vaccine (ORV) study has been conducted in the Arctic using oral rabies vaccine licensed in the United States in Arctic foxes. However, human habitations or industrial development sites, such as oil fields and other areas of fox congregation, provide feasible locations for targeted oral vaccination campaigns. Currently, Raboral V-RG^® is the only licensed ORV in the United States (NASPHV, 2008). Vaccine baits have been distributed and used successfully in ORV programs within the eastern and southwestern United States to control rabies (Slate et al., 2009).

Twelve Arctic foxes were trapped in the oilfield region of the North Slope of Alaska using baited Tomahawk live traps (Tomahawk live traps, Tomahawk, Wisconsin, USA). Serum from each animal was analyzed for rabies virus antibody using the rapid fluorescent focus inhibition test (RFFIT) at the Kansas State University Department of Veterinary Diagnosis. After 5 wk of quarantine, eight foxes were selected for vaccination and the remaining four served as unvaccinated controls. Foxes were anesthetized using 10 mg xylazine and 20 mg ketamine and 5–6 ml blood were drawn for a second RFFIT just before vaccination on day 0.

Eight sedated foxes were vaccinated by placing 2.0 ml (10^7.7 plaque forming units per ml) of Raboral V-RG into their oral cavities. Foxes were placed in sternal recumbency. Vaccine was delivered via a plastic syringe placed inside the animal’s mouth. The vaccinated foxes (no. 1–8) and three control foxes (no. 9, 10, 12) had blood drawn at 1, 2, 4, and 8 wk postvaccination to test for rabies virus neutralizing antibodies. One unvaccinated fox (no. 11) developed clinical signs of rabies infection after 40 days in captivity and was euthanized. Rabies virus was detected in brain tissue by direct fluorescent antibody test, indicating infection occurred prior to capture.

Sixteen weeks postvaccination, all vaccinates and control foxes (n=11) were anesthetized and serum was obtained for RFFIT. Following blood collection, and while under sedation, each fox was injected bilaterally in the masseter muscles with 1.0 ml of neural (brain) tissue containing rabies virus. This challenge virus consisted of an isolate of an Arctic rabies virus variant obtained from a rabid dog from western Alaska. The virus was titered in 19- to 21-day-old Ha/ICR-CDR mice by intracerebral inoculation. The mouse titration dose equaled 10^3.8/0.03 ml. Each fox received approximately a 500,000 mouse intracerebral lethal dose 50% (MICLD50) per dose of rabies virus.

Foxes were observed postinfection twice daily for clinical signs associated with rabies. Blood (5–6 ml) was obtained from vaccinated and control foxes prior to infection, on the day of challenge and 1, 2, 4 and 9 wk postchallenge for RFFIT. Any foxes beginning to show signs of rabies, such as anorexia, pelt scruffiness, and agitation, were observed several times per day. When posterior paralysis became evident, foxes were anesthetized for blood collection and euthanized with an overdose of barbiturates (390 mg) according to approved procedures (Institutional Animal Care and Use Committee, University of Alaska, Fairbanks). Control fox no. 10 was euthanized 14 days postchallenge and control fox no. 12 was euthanized 23 days postchallenge. Surviving foxes (eight vaccinated and one control) were euthanized 9 wk postchallenge. Neural tissue was collected and examined for rabies virus using the direct fluorescent antibody testing (Hanlon et al., 1999).

All vaccinated foxes developed a measurable rabies virus–neutralizing antibody response after a single oral vaccination (Table 1). At 14 days postvaccination, titers ranged from 1:163 to 1:6,000 for all vaccinated foxes. These foxes maintained titers over 1:85 for 18 wk. This titer corresponded to approximately 0.8 IU. The World Health Organization suggests booster vaccination for humans with titers below 0.5 IU (WHO, 2005). However the level of protective antibodies has not been defined for any species. Therefore no definite conclusion on protection can be drawn from rabies virus antibody titers alone.

TABLE 1.

Rabies virus–neutralizing antibody titers of vaccinated and control foxes. Arctic foxes (Vulpes lagopus) were vaccinated against rabies with Raboral V-RG^® recombinant oral rabies vaccine in week 0 and challenged after 18 wk. Fox no. 11 died of rabies prior to challenge and foxes no. 10 and no. 12 died of rabies in the last week with reported antibody titers.

Fox no.	Week
	−5	0	1	2	4	8	13	18	19	20	22	27
Vaccinated
1	<5	<5	50	1,200	1,100	1,300	1,200	1,800	>7,000	>7,000	4,100	2,400
2	<5	<5	125	480	1,200	1,000	3,600	3,125	>7,000	>7,000	>7,000	6,700
3	<5	<5	13	163	700	270	180	95	>7,000	6,700	3,600	1,300
4	<5	<5	180	340	6,700	2,200	2,700	1,800	>7,000	>7,000	6,700	6,700
5	<5	<5	54	6,000	1,300	320	900	1,200	>7,000	>7,000	2,400	1,800
6	<5	<5	<5	110	270	230	215	95	>7,000	>7,000	>7,000	4,900
7	<5	<5	<5	245	110	125	95	85	>7,000	>7,000	625	1,100
8	<5	<5	50	370	800	300	270	400	>7,000	>7,000	6,000	1,300
Unvaccinateda
9	<5	<5	n/a	n/a	n/a	n/a	<5	<5	125	n/a	17	45
10	<5	<5	n/a	n/a	n/a	n/a	<5	<5	<5	<5	n/a	n/a
11	<5	<5	n/a	n/a	n/a	n/a	n/a	n/a	n/a	n/a	n/a	n/a
12	<5	<5	n/a	n/a	n/a	n/a	<5	<5	<5	n/a	700	n/a

^an/a=not tested.

All vaccinated foxes mounted a strong secondary response with rabies virus–neutralizing antibody titers above 1:7,000 1 wk postchallenge. Antibody titers above 1:1,000 were maintained until the end of the study at 9 wk postchallenge. No vaccinated foxes showed clinical signs of rabies. One nonvaccinated fox (no. 9) survived challenge and was euthanized with the surviving vaccinates. Rabies virus was detected in the neural tissue of the two control foxes that demonstrated signs of encephalitis after the rabies virus challenge infection, but not in vaccinated foxes or the control fox (no. 9) that survived the challenge.

Despite the limitations of this study due to the death of one control fox prior to challenge, and the survival of one control fox after virus challenge, these results suggest effectiveness of oral instillation of Raboral V-RG in Arctic foxes. In addition, rabies virus antibody titers were higher than those observed in previous vaccination trials of Arctic foxes receiving SAD-BHK21, SAG-1, and SAG-2 viruses by the oral route (Follmann et al., 1988, 1992, 2004). We used wild-caught foxes whose rabies exposure history prior to capture is unknown. Previous studies of infection of Arctic foxes have shown that a relatively high challenge dose of rabies virus (~500,000 MICLD50) is needed to induce 100% rabies mortality compared to other canids (Follmann et al., 1988, 2004). In our experiment, that dose also resulted in one survivor of three control foxes. In all these studies, previous exposure to rabies virus and a resulting immune response prior to capture in control animals cannot be excluded. We did not detect measurable rabies virus–neutralizing antibodies prior to experimental infection in the Arctic fox that survived the challenge infection. In previous studies no antibodies against rabies virus were detected in surviving foxes. An alternative explanation would be a very high inherent resistance of Arctic foxes to Arctic rabies virus variants. The observation that the surviving control fox had detectable antibody suggests that an infection occurred, resulting in an immune response. A third, less likely explanation would be that the challenge virus contained sufficient nonviable virus material to provide antigen for the immune system to mount a successful response. Controlled captive trials with Arctic foxes of known rabies exposure status are needed to resolve these alternative explanations for the suspected high resistance of Arctic foxes to rabies.

While this study suggests the effectiveness of the Raboral V-RG rabies vaccine in Arctic foxes, the unforeseen rabies virus infection of one control animal prior to challenge and the survival of one of the remaining three control animals limits the interpretation of these data. In addition, careful evaluation of bait systems for an Arctic environment targeting Arctic foxes must be considered prior to implementation of any practical vaccine efforts. Apart from immunologic effects, an ORV program of wildlife in the Arctic poses significant challenges. During the summer, Arctic foxes establish territories and are dispersed throughout a large remote landscape. In winter, Arctic foxes can travel great distances. These factors make successful ORV campaigns difficult. In winter, targeting areas around human settlements or industrial sites is more feasible. However, low temperatures will affect consistent delivery of ORV to target species. A frozen vaccine dose could be swallowed whole and be inactivated in the stomach. Such issues will need to be assessed in field studies.