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Oral vaccination and protection of red foxes (Vulpes vulpes) against rabies using ONRAB® , an adenovirus-rabies recombinant vaccine夽
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L.J. Brown a,∗ , R.C. Rosatte a , C. Fehlner-Gardiner b , P. Bachmann a , J.A. Ellison c , F.R. Jackson c , J.S. Taylor a , C. Davies a , D. Donovan a a Wildlife Research and Development Section, Ontario Ministry of Natural Resources, Trent University, DNA Building, 2140 East Bank Drive, Peterborough, Ontario K9J 7B8, Canada b Centre of Expertise for Rabies, Canadian Food Inspection Agency, 3851 Fallowfield Road, P.O. Box 11300, Station H, Ottawa, Ontario K2H 8P9, Canada c Division of High-Consequence Pathogens and Pathology, Centers for Disease Control and Prevention, 1600 Clifton Road NE, Mailstop G-33, Atlanta, GA 30329, USA
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Article history: Received 31 July 2013 Received in revised form 29 November 2013 Accepted 10 December 2013 Available online xxx
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Keywords: Human adenovirus serotype 5 Immune response ONRAB® Oral rabies vaccination Rabies control Rabies virus challenge Red fox Vulpes vulpes
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1. Introduction
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Twenty-seven red foxes (Vulpes vulpes) were each offered a bait containing ONRAB® , a recombinant oral rabies vaccine that uses a human adenovirus vector to express the immunogenic rabies virus glycoprotein; 10 controls received no vaccine baits. Serum samples collected from all foxes before treatment, and each week post-treatment for 16 weeks, were tested for the presence of rabies virus neutralizing antibody (RVNA). In the bait group, a fox was considered a responder to vaccination if serum samples from 3 or more consecutive weeks had RVNA ≥0.5 IU/ml. Using this criterion, 79% of adult foxes (11/14) and 46% of juveniles (6/13) responded to vaccination with ONRAB® . Serum RVNA of adults first tested positive (≥0.5 IU/ml) between weeks 1 and 3, about 4 weeks earlier than in juveniles. Adults also responded with higher levels of RVNA and these levels were maintained longer. Serum samples from juveniles tested positive for 1–4 consecutive weeks; in adults the range was 2–15 weeks, with almost half of adults maintaining titres above 0.5 IU/ml for 9 or more consecutive weeks. Based on the kinetics of the antibody response to ONRAB® , the best time to sample sera of wild adult foxes for evidence of vaccination is 7–11 weeks following bait distribution. Thirty-four foxes (25 ONRAB® , 9 controls) were challenged with vulpine street virus 547 days post-vaccination. All controls developed rabies whereas eight of 13 adult vaccinates (62%) and four of 12 juvenile vaccinates (33%) survived. All foxes classed as non-responders to vaccination developed rabies. Of foxes considered responders to vaccination, 80% of adults (8/10) and 67% of juveniles (4/6) survived challenge. The duration of immunity conferred to foxes would appear adequate for bi-annual and annual bait distribution schedules as vaccinates were challenged 1.5 years post-vaccination. © 2013 The Authors. Published by Elsevier Ltd. All rights reserved.
In many developed countries, wild animals are the primary vectors of the rabies virus (RV) and maintain independent cycles of infection [1]. Until recently, the red fox (Vulpes vulpes) was the main rabies reservoir in Ontario and the principle target species for rabies management strategies [2,3]. The fox epizootic in Ontario originated in the Canadian Arctic, where the key wildlife vector is the Arctic fox (Vulpes lagopus) [4]. As the disease spread into
夽 This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-No Derivative Works License, which permits non-commercial use, distribution, and reproduction in any medium, provided the original author and source are credited. ∗ Corresponding author. Tel.: +1 705 755 2285; fax: +1 705 755 1559. E-mail address: [email protected] (L.J. Brown).
southern Ontario, red foxes and striped skunks (Mephitis mephitis) became the main reservoirs and vectors of Arctic rabies virus variant (ARVV), accounting for 68% and 30% of the terrestrial wildlife rabies cases, respectively, between 1958 and 1989 [5]. More recently in Ontario (1999–2005), raccoons (Procyon lotor) and, to a lesser extent, striped skunks were the main species affected by raccoon rabies virus variant (RRVV) [6,7], a variant associated with raccoons throughout the US eastern seaboard [8]. Oral rabies vaccination (ORV) is a cost-effective, socially acceptable disease control method for wildlife reservoirs that can be applied over large geographic areas [9–11]. ORV requires a vaccine that is effective by the oral route and a vaccine delivery package suitable for the target species. In eastern Ontario, ARVV was eliminated from the red fox population through large-scale ORV using ERA-BHK21 , a modified-live RV vaccine [2,3]. Similar results were attained throughout Europe with related attenuated strains of RV (SADBern , SADB19 , SAG1, and SAG2) [12]. Although live-attenuated
0264-410X/$ – see front matter © 2013 The Authors. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.vaccine.2013.12.015
Please cite this article in press as: Brown LJ, et al. Oral vaccination and protection of red foxes (Vulpes vulpes) against rabies using ONRAB® , an adenovirus-rabies recombinant vaccine. Vaccine (2013), http://dx.doi.org/10.1016/j.vaccine.2013.12.015
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vaccines have proven successful for fox rabies control, their continued use in ORV remains controversial due to residual pathogenicity for a variety of rodent species [13–16], and vaccine-induced rabies in target and non-target animals [17–19]. Other considerations of note include sensitivity to heat [20] and ineffectiveness by the oral route in rabies reservoirs such as raccoons and skunks [17,21,22]. An alternative ORV strategy involves the use of recombinant vaccines constructed from heterologous virus vectors that express the RV glycoprotein. These vaccines reportedly offer improved safety and better heat stability [23–25]. In Europe, vaccinia virus recombinant vaccines (VVTGgRAB and V-RG) have proven effective by the oral route in red foxes [23,25,26] but efforts to immunize raccoons and skunks have proven less successful [6,9,27–30]. Recombinant vaccines that use adenoviruses as vectors have also been considered and show promise for wildlife ORV [31–37]. ONRAB® (AdRG1.3) is a recombinant oral rabies vaccine that uses a human adenovirus vector (serotype 5) to express the RV glycoprotein [33]. In early Ontario field trials (2006–2007), both raccoons and skunks demonstrated serologic evidence of an immune response to ONRAB® [38]. A high proportion of raccoons in captivity also responded to vaccination with ONRAB® and most responders survived rabies challenge almost 1 year postvaccination (PV) [39]. The current study was conducted to see if similar results could be attained in red foxes, as a single oral rabies vaccine that is immunogenic in multiple North American rabies vector species (especially raccoon, red fox, and striped skunk) offers the best chance of eventual virus containment and elimination. The objectives of this study were to (1) quantify the longitudinal humoral immune response of captive foxes following ORV with ONRAB® and (2) test the protection afforded by the vaccine against RV.
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2. Methods
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2.1. Study animals and housing
then every 15 min for the next 1.5 h. Ten controls (5 adults, 5 juveniles) received no vaccine baits. 2.3. Blood collection A blood sample (3–5 ml) was collected from controls and vaccinates 2 weeks before bait presentation, and then each week after treatment for 16 consecutive weeks. Animals were anesthetised and blood samples collected as described in [39]. Blood was refrigerated at 4 ◦ C for up to 48 h and then centrifuged at 1000 × g for 12 min at 4 ◦ C. Harvested serum was maintained at −20 ◦ C and then heat-inactivated at 56 ◦ C for 30 min prior to testing. 2.4. Rabies challenge Thirty-four foxes (9 controls, 25 vaccinates) from the serology study were subjected to rabies challenge on February 10, 2010. The challenge virus was isolated from a naturally infected rabid fox and identified as ARVV using a panel of anti-nucleocapsid monoclonal antibodies and sequence analysis [42] (CDC SM1515; GenBank Accession No. JQ685990). The RV was adapted to murine neuroblastoma cells with 3 serial passages. The supernatant was harvested and clarified by low speed centrifugation (800 × g) for 10 min then stored at −120 ◦ C before use. Five hundred and forty-seven days PV, foxes were anesthetised and a blood sample (3 ml) was collected from the jugular vein for baseline serology. Each fox was then inoculated bilaterally with 0.5 ml of RV (104.9 MICLD50 ) into the masseter muscle. Foxes were also bled 7 days post-inoculation (PI). Animals were observed twice daily for clinical signs of rabies (paralysis, ataxia, behavioural change, lack of appetite, hyper-salivation, vocalization, agitation, tremors, convulsions, aggression) and humanely euthanized when two or more signs appeared. All surviving animals were euthanized 60 days PI and submitted for rabies diagnosis using the direct fluorescent antibody test [43].
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Thirty-seven captive-reared foxes were purchased from an Ontario breeder. At treatment, juveniles were aged 14–17 weeks; adults were 1.3–7.3 years. Prior to treatment, foxes were wormed (Strongid*T; 50 mg/ml; Pfizer Animal Health, Quebec, Canada) and vaccinated against canine distemper (Galaxy*D; Schering-Plough Animal Health, Nebraska, USA). For the serology study, foxes were housed in separate units within outdoor enclosures. For the challenge study, foxes were housed in kennel runs in an Animal BioContainment Level-2 facility. Animals were provided a commercial feed once a day and water ad libitum. All animals were maintained in accordance with national guidelines (Canadian Council on Animal Care Guidelines [40] or Guide for the Care and Use of Laboratory Animals [41]) and protocols were approved by Institutional Animal Care and Use Committees (Ontario Ministry of Natural Resources [OMNR], Canada or Centers for Disease Control and Prevention [CDC], Georgia, USA).
A modified fluorescent antibody virus neutralization test [44] was used to detect rabies virus neutralizing antibody (RVNA) in sera from the humoral immune response study. Titres were calculated using the Spearman–Kärber formula [45] and expressed in international units (IU) per ml calibrated against the World Health Organization 2nd International Reference Standard (National Institute of Biological Standards and Controls, Potter’s Bar, UK). A rapid fluorescent focus inhibition test [46] with endpoint titres determined [47] was used to detect RVNA in sera from the challenge study. Titres were reported in IU/ml following normalization against the US Standard Rabies Immune Globulin (Laboratory of Standards and Testing, Food and Drug Administration, USA) diluted to 2 IU/ml. For both studies, serum samples were considered positive for RVNA if the titre was ≥0.5 IU/ml.
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2.2. Vaccination protocol
2.6. Statistical analyses
Ultra-lite baits containing 1.8 ml (±0.1 ml) of ONRAB® (1010 TCID50 /ml) were manufactured by Artemis Technologies Inc., Guelph, Ontario, Canada [38]. Foxes were randomly assigned to a control or vaccinate group with minor adjustments to ensure similar age-sex representation among groups. Twenty-seven foxes (14 adults, 13 juveniles) were each offered one ONRAB® bait on August 12, 2008 following a 24 h fasting period. Bait debris and vaccine spillage were collected on plastic sheets below each cage. The amount of bait ingested, vaccine loss during ingestion, and timing of ingestion were recorded every 10 min for the first 0.5 h
Fisher’s exact tests (FET) were used to examine associations between age and response to vaccination and between age and RV infection post-challenge. FET were also used to assess differences in RV infection among controls and vaccinates post-challenge and test the association between response to vaccination and survival. A Mann–Whitney test was used to determine whether the number of days until death differed among controls and vaccinates post-challenge [48]. For foxes considered responders to vaccination, Mann–Whitney tests were used to determine whether there was a difference between adults and juveniles in the week RVNA
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2.5. Serologic analyses
Please cite this article in press as: Brown LJ, et al. Oral vaccination and protection of red foxes (Vulpes vulpes) against rabies using ONRAB® , an adenovirus-rabies recombinant vaccine. Vaccine (2013), http://dx.doi.org/10.1016/j.vaccine.2013.12.015
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Table 1 Numbers of red foxes (Vulpes vulpes) in the control and ONRAB® bait groups that tested positive for rabies virus neutralizing antibody (RVNA) post-vaccination (PV), numbers that tested positive for RVNA on day 7 post-infection (7 d PI), and numbers that survived rabies challenge 547 days post-vaccination. RVNA+ PV (%)
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0/9 (0%) 8/13 (62%) 4/12 (33%)
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was first detected and the number of consecutive weeks an animal tested RVNA positive. STATISTICA© was used for all analyses (StatSoft, Tulsa, Oklahoma, USA).
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3.1. Bait consumption and vaccination
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Eighteen foxes consumed baits within 20 min; nine animals took 1–2 h. Unconsumed vaccine (0.15 ml) was recovered from only one cage (#823).
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3.2. Serologic response to ONRAB®
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No vaccine-induced morbidity or mortality was observed. Prevaccination sera for all animals, and sera for all control foxes throughout the 16 week study, had titres ≤0.03 IU/ml. A fox was considered a ‘responder’ to vaccination if RVNA was detected for 3 or more consecutive weeks. Using this criterion, 79% of adults (11/14) and 46% of juveniles (6/13) were classed as responders (Table 1). Although fewer juveniles responded to vaccination, the difference was not significant (P = 0.1201; FET). RVNA was detected earlier in adults than juveniles (U = 4.5, P = 0.0006) (Fig. 1). RVNA first appeared in one adult at week 1, in four at week 2, and in two at week 3. In the remaining adults, RVNA first occurred at weeks 4, 5, or 6. The earliest evidence of RVNA in juveniles was at week 5, the latest at week 9. Adults exhibited higher RVNA titres and more consecutive weeks with RVNA than juveniles (U = 17.5, P = 0.0125) (Fig. 1). Six adult foxes had RVNA detected for 9–15 consecutive weeks; five others, for 3–5 consecutive weeks. Two adults had RVNA for only 2 consecutive weeks, while one adult remained RVNA negative. One juvenile had RVNA detected for 4 consecutive weeks; five others for 3 consecutive weeks. Two juveniles had RVNA detected at 1 or 2 weeks; the remaining five juveniles tested RVNA-negative throughout the study (mode 0.03 IU/ml; range 0.01–0.42 IU/ml). At week 4 PV, the proportion of adult foxes with RVNA was 57% (Fig. 2). Seropositivity increased to 71% by weeks 5 and 6, reached a peak of 86% by week 7, then declined slightly through weeks 8 and 9 (79% and 71%, respectively). Forty-three percent to 57% of adults had RVNA detectable between weeks 10 and 16. The proportion of juveniles with RVNA was highest between weeks 7 and 9 PV (38–46%); peak at week 8 (Fig. 2).
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3.3. Resistance to rabies challenge
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All control foxes (n = 9) succumbed to RV infection as confirmed by the direct fluorescent antibody test (Table 1). Eight of 13 adults (62%) and four of 12 juveniles (33%) offered ONRAB® baits survived challenge (Table 1). Although survival was lower in juveniles than adults, the difference was not significant (P = 0.2377, FET). Of the 10 adult foxes classed as responders to vaccination, seven demonstrated RVNA on the day of challenge (range 0.64–12.88 IU/ml). All eight survivors in the adult group were classed as responders to vaccination and all mounted an anamnestic response PI (range
Survived challenge
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Fig. 1. Temporal development of rabies virus neutralizing antibody (RVNA) in sera collected from (a) adult (n = 11) and (b) juvenile (n = 6) red foxes (Vulpes vulpes) considered responders to vaccination following consumption of ONRAB® baits. Solid lines track median titres for adult and juvenile foxes at each week post-vaccination.
3.35–298.71 IU/ml). Two adult responders that did not exhibit RVNA on the day of challenge and showed no anamnestic response did not survive challenge. Three adults classed as non-responders did not exhibit RVNA on the day of challenge and did not survive challenge (Table 1). One juvenile responder had detectable RVNA on the day of challenge (0.94 IU/ml); two others had titres that fell just below the 0.5 IU/ml threshold (0.48 IU/ml). All four juvenile foxes that survived challenge were responders to vaccination and had RVNA levels suggestive of an anamnestic response (range 60.09–312.88 IU/ml). Two juvenile responders did not survive challenge (Table 1). Both animals had no RVNA on the day of challenge,
Please cite this article in press as: Brown LJ, et al. Oral vaccination and protection of red foxes (Vulpes vulpes) against rabies using ONRAB® , an adenovirus-rabies recombinant vaccine. Vaccine (2013), http://dx.doi.org/10.1016/j.vaccine.2013.12.015
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Fig. 2. Proportion of red foxes (Vulpes vulpes) that had rabies virus neutralizing antibody (RVNA) ≥0.5 IU/ml at each week post-vaccination. Vaccinates were each offered one ONRAB® bait. Closed circles represent adults (n = 14); open circles, juveniles (n = 13).
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although one juvenile did have RVNA seven days PI (1.99 IU/ml). All six juvenile non-responders to vaccination did not survive rabies challenge (Table 1), but two animals did have RVNA on day 7 PI (1.55 and 2.88 IU/ml). More foxes offered ONRAB® baits survived challenge with ARVV than controls (P = 0.0132, FET). Within the ONRAB® group, more foxes classed as responders to vaccination tested negative for rabies (P = 0.0005, FET). Among controls and vaccinates that succumbed to rabies, there was no significant difference in the number of days to death or humane euthanasia PI (median = 12 days; range 11–15 days) (U = 44.5, P = 0.3498).
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4. Discussion
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This is the first study to examine the longitudinal antibody response of red foxes to ONRAB® (AdRG1.3) presented in baits and results provide evidence of its promise as a candidate oral rabies vaccine for this species. Seventy-nine percent (11/14) of adult foxes offered ONRAB® baits seroconverted within 6 weeks. Similar results were achieved in a previous study where foxes aged 1–2 years were immunized with an earlier version of the vaccine (Ad5RG1) by direct instillation into the oral cavity; 87% (14/16) seroconverted within 9 weeks PV [32]. Our results also compare favourably with similar studies where 50–100% of foxes produced RVNA in response to V-RG, a vaccinia-rabies recombinant vaccine, in different baits [23,49–51]. Although a variety of live-attenuated oral rabies vaccines have also proven efficacious in red foxes [52–54], safety and stability concerns make them less desirable for field applications and they are ineffective in other important rabies vector species such as raccoons and skunks [17,21,22]. During this study and two other ONRAB® efficacy trials, no vaccine-induced morbidity or mortality was observed in foxes, raccoons or skunks [39,55]. Field results also support that ONRAB® is safe for foxes as well as other wildlife species. ONRAB® was quickly excreted by target and non-target animals that contacted vaccine baits and viral replication following vaccine ingestion was short lived, suggesting a low risk of virus transmission in the natural environment [32,44,56,57]. Juveniles as a group did not respond to vaccination as well as adults, but similar results were also seen in other laboratory and field trials [13,52,58–62]. In our study, 46% (6/13) of juvenile foxes offered ONRAB® baits seroconverted within 9 weeks. Although our
study was not designed to evaluate seroconversion in juveniles of different ages, older juveniles did respond better to vaccination. All four juveniles less than 15 weeks did not respond to vaccination, whereas only three of nine juveniles aged 16–17 weeks were nonresponders. During fall ORV campaigns, juvenile foxes would be several weeks older than those used in our captive study; thus, it is possible that at baiting time the immunization rate of matured young foxes would approach that of adults. RVNA was first detected in some adult foxes as early as 14 days after presentation of ONRAB® baits. This is similar to other studies in foxes with V-RG, SAG2, and ERA vaccines [50–52,63]. Adult foxes demonstrated RVNA earlier, by about 4 weeks, and maintained RVNA longer than juveniles. Based on the kinetics of the antibody response of foxes to ONRAB® , and assuming most foxes consume baits within 2 weeks of field distribution [64,65], the best time to collect sera from adult foxes for RVNA evaluation is 7–11 weeks post-bait distribution. For maximal detection of RVNA in juveniles, field sampling should be conducted between weeks 9 and 11. In the original study design, rabies challenge was scheduled to occur 90 days PV; however, various logistical factors delayed the challenge more than 1 year, providing a unique opportunity to test the long-term protective potential of ONRAB® in red foxes. All nine controls developed rabies whereas eight of 13 adult vaccinates (62%) and four of 12 juvenile vaccinates (33%) survived rabies challenge 1.5 years PV. Survival of adult foxes was higher after consumption of ERA baits (100%) [13,16,53,66,67] but protection provided by ONRAB® was within the range of that provided by V-RG in different baits (50–100%) [23,49–51,68]. In ERA trials, foxes were challenged 28 days to 7 years PV; V-RG challenge trials all occurred within 91 days PV. For successful fox rabies control, duration of immunity should extend through to the next baiting campaign [53]. Although bi-annual baiting schedules (spring and fall) have been applied in Europe [60], annual fall campaigns were preferred in Ontario [2]. The duration of immunity conferred to foxes in this study would appear adequate for bi-annual and annual bait distribution schedules as vaccinates were challenged 1.5 years PV. In this study, and in many others, the correlation between the production of RVNA and protection of foxes against rabies was relatively strong. In our study, all foxes classed as non-responders to vaccination developed rabies. Of foxes considered responders to vaccination, 80% of adults (8/10) and 67% of juveniles (4/6) survived challenge. In several vaccine efficacy trials, seropositivity equated to protection [16,49,50,53,54,67,69]. In others, survival rate was higher than seropositivity [13,49,50] or, as in our study, it was slightly lower [23,53]. Four animals in our study with RVNA for three or more consecutive weeks did not survive challenge. None of these individuals had detectable rabies antibody prior to challenge and only one had a positive titre at 7 days PI. These data suggest that despite stimulating a primary humoral immune response, immunological memory was not established in these four animals. Similar results were also seen in captive raccoons. Of raccoons considered responders to oral vaccination after consumption of ONRAB® baits, 67% (10/15) survived rabies challenge [39]; one nonresponder also survived. Interestingly, in captive skunks, survival rates following oral vaccination with ONRAB® surpassed vaccination estimates [55] which would suggest that immunity factors in addition to those measured by the presence of antibodies likely contributed to protection in this species. In summary, we have demonstrated that ONRAB® , an adenovirus-rabies recombinant vaccine, is innocuous in the red fox and, when presented in baits, elicited RVNA at levels comparable to those observed with live-attenuated and vaccinia-recombinant rabies vaccines, with protection exceeding 1 year in duration. These results, together with safety and effectiveness in additional rabies
Please cite this article in press as: Brown LJ, et al. Oral vaccination and protection of red foxes (Vulpes vulpes) against rabies using ONRAB® , an adenovirus-rabies recombinant vaccine. Vaccine (2013), http://dx.doi.org/10.1016/j.vaccine.2013.12.015
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reservoirs, make ONRAB® an attractive alternative to oral rabies vaccines used in the past for fox rabies control. Acknowledgements We would like to thank staff of the Ontario Ministry of Natural Resources and Canadian Food Inspection Agency for animal care, and field and laboratory technical assistance: Erin Scharf, Tara MacDonald, Andrea Clark, and Bharat Bongale. We would also like to acknowledge the contributions of CDC staff responsible for animal care and all aspects of the challenge portion of this study. Andrew Beresford and Al Beath at Artemis Technologies Inc. prepared the vaccine baits used in this study and Beverly Stevenson and Kim Bennett commented on an early draft of the manuscript. This project was supported by the Ontario Rabies Advisory Committee (Jim Broadfoot, Chair). References [1] Rosatte RC, Tinline RR, Johnston DH. Rabies control in wild carnivores. In: Jackson A, Wunner W, editors. Rabies. San Diego, CA: Academic Press; 2007. p. 595–634. [2] MacInnes CD, Smith SM, Tinline RR, Ayers Neil R, Bachmann P, Ball DG, et al. Elimination of rabies from red foxes in eastern Ontario. J Wildl Dis 2001;37:119–32. [3] Rosatte RC, Power MJ, Donovan D, Davies JC, Allan M, Bachmann P, et al. Elimination of arctic variant rabies in red foxes, metropolitan Toronto. Emerg Infect Dis 2007;13:25–7. [4] Tabel H, Corner AH, Webster WA, Casey CA. History and epizootiology of rabies in Canada. Can Vet J 1974;15:271–81. [5] Agriculture Canada Report of Rabies Diagnosed – 1958 to 1989, Ottawa; 1958–1989. [6] Rosatte R, Allan M, Bachmann P, Sobey K, Donovan D, Davies JC, et al. Prevalence of tetracycline and rabies virus antibody in raccoons, skunks, and foxes following aerial distribution of V-RG baits to control raccoon rabies in Ontario, Canada. J Wildl Dis 2008;44:946–64. [7] Rosatte RC, Donovan D, Allan M, Bruce L, Buchanan T, Sobey K, et al. The control of raccoon rabies in Ontario, Canada: proactive and reactive tactics, 1994–2007. J Wildl Dis 2009;45:772–84. [8] Wandeler AI. Raccoon rabies in eastern Ontario. Can J Vet Res 1999;40:731. [9] Slate D, Algeo TP, Nelson KM, Chipman RB, Donovan D, Blanton JD, et al. Oral rabies vaccination in North America: opportunities, complexities, and challenges. PLoS Negl Trop Dis 2009;3:e549. [10] Rosatte RC, Donovan D, Davies JC, Brown L, Allan M, von Zuben V, et al. Highdensity baiting with ONRAB® rabies vaccine baits to control Arctic variant rabies in striped skunks in Ontario, Canada. J Wildl Dis 2011;47:459–65. [11] Shwiff SA, Nunan CP, Kirkpatrick KN, Shwiff SS. A retrospective economic analysis of the Ontario red fox oral rabies vaccination programme. Zoonoses Public Health 2011;58:169–77. [12] Wandeler AI. Oral immunization against rabies: afterthoughts and foresight. Schweiz Arch Tierheilkd 2000;142:455–62. [13] Black JG, Lawson KF. The safety and efficacy of immunizing foxes (Vulpes vulpes) using bait containing attenuated rabies virus vaccine. Can J Comp Med 1980;44:169–76. [14] Artois M, Guittré C, Thomas I, Leblois H, Brochier B, Barrat J. Potential pathogenicity for rodents of vaccines intended for oral vaccination against rabies: a comparison. Vaccine 1992;10:524–8. [15] Vos A, Neubert A, Aylan O, Schuster P, Pommerening E, Muller T, et al. An update on safety studies of SAD B19 rabies virus vaccine in target and non-target species. Epidemiol Infect 1999;123:165–75. [16] Lawson KF, Hertler R, Charlton KM, Campbell JB, Rhodes AJ. Safety and immunogenicity of ERA strain of rabies virus propagated in a BHK-21 cell line. Can J Vet Res 1989;53:438–44. [17] Rupprecht CE, Charlton KM, Artois M, Casey GA, Webster WA, Campbell JB, et al. Ineffectiveness and comparative pathogenicity attenuated rabies virus vaccines for the striped skunk (Mephitis mephitis). J Wildl Dis 1990;26:99–102. [18] Fehlner-Gardiner C, Nadin-Davis S, Armstrong J, Muldoon F, Bachmann P, Wandeler A. Era vaccine-derived cases of rabies in wildlife and domestic animals in Ontario, Canada, 1989–2004. J Wildl Dis 2008;44:71–85. [19] Müller T, Bätza H-J, Beckert A, Bunzenthal C, Cox JH, Freuling CM, et al. Analysis of vaccine-virus-associated rabies cases in red foxes (Vulpes vulpes) after oral rabies vaccination campaigns in Germany and Austria. Arch Virol 2009;154:1081–91. [20] Winkler WG, McLean RG, Cowart JC. Vaccination of foxes against rabies using ingested baits. J Wildl Dis 1975;11:382–8. [21] Tolson ND, Charlton KM, Lawson KF, Campbell JB, Stewart RB. Studies of ERA BHK-21 rabies vaccine in skunks and mice. Can J Vet Res 1988;52:58–62. [22] Rupprecht CE, Dietzschold B, Cox JH, Schneider LG. Oral vaccination of raccoons (Procyon lotor) with an attenuated (SAD-B19) rabies virus vaccine. J Wildl Dis 1989;25:548–54.
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[48] Zar JH. Biostatistical analysis. 4th ed. Upper Saddle River, NJ: Prentice-Hall, Inc.; 1999. [49] Artois M, Masson E, Barrat J, Aubert MFA. Efficacy of three oral rabies vaccinebaits in the red fox: a comparison. Vet Microbiol 1993;38:167–72. [50] Cliquet F, Barrat J, Guiot AL, Caël N, Boutrand S, Maki J, et al. Efficacy and bait acceptance of vaccinia vectored rabies glycoprotein vaccine in captive foxes (Vulpes vulpes), raccoon dogs (Nyctereutes procyonoides) and dogs (Canis familiaris). Vaccine 2008;26:4627–38. [51] Tolson ND, Charlton KM, Casey GA, Knowles MK, Rupprecht CE, Lawson KF, et al. Immunization of foxes against rabies with a vaccinia recombinant virus expressing the rabies glycoprotein. Arch Virol 1988;102:297–301.
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Please cite this article in press as: Brown LJ, et al. Oral vaccination and protection of red foxes (Vulpes vulpes) against rabies using ONRAB® , an adenovirus-rabies recombinant vaccine. Vaccine (2013), http://dx.doi.org/10.1016/j.vaccine.2013.12.015
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Contents lists available at ScienceDirect
Vaccine journal homepage: www.elsevier.com/locate/vaccine
Oral vaccination and protection of red foxes (Vulpes vulpes) against rabies using ONRAB® , an adenovirus-rabies recombinant vaccine夽
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L.J. Brown a,∗ , R.C. Rosatte a , C. Fehlner-Gardiner b , P. Bachmann a , J.A. Ellison c , F.R. Jackson c , J.S. Taylor a , C. Davies a , D. Donovan a a Wildlife Research and Development Section, Ontario Ministry of Natural Resources, Trent University, DNA Building, 2140 East Bank Drive, Peterborough, Ontario K9J 7B8, Canada b Centre of Expertise for Rabies, Canadian Food Inspection Agency, 3851 Fallowfield Road, P.O. Box 11300, Station H, Ottawa, Ontario K2H 8P9, Canada c Division of High-Consequence Pathogens and Pathology, Centers for Disease Control and Prevention, 1600 Clifton Road NE, Mailstop G-33, Atlanta, GA 30329, USA
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Article history: Received 31 July 2013 Received in revised form 29 November 2013 Accepted 10 December 2013 Available online xxx
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Keywords: Human adenovirus serotype 5 Immune response ONRAB® Oral rabies vaccination Rabies control Rabies virus challenge Red fox Vulpes vulpes
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1. Introduction
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Twenty-seven red foxes (Vulpes vulpes) were each offered a bait containing ONRAB® , a recombinant oral rabies vaccine that uses a human adenovirus vector to express the immunogenic rabies virus glycoprotein; 10 controls received no vaccine baits. Serum samples collected from all foxes before treatment, and each week post-treatment for 16 weeks, were tested for the presence of rabies virus neutralizing antibody (RVNA). In the bait group, a fox was considered a responder to vaccination if serum samples from 3 or more consecutive weeks had RVNA ≥0.5 IU/ml. Using this criterion, 79% of adult foxes (11/14) and 46% of juveniles (6/13) responded to vaccination with ONRAB® . Serum RVNA of adults first tested positive (≥0.5 IU/ml) between weeks 1 and 3, about 4 weeks earlier than in juveniles. Adults also responded with higher levels of RVNA and these levels were maintained longer. Serum samples from juveniles tested positive for 1–4 consecutive weeks; in adults the range was 2–15 weeks, with almost half of adults maintaining titres above 0.5 IU/ml for 9 or more consecutive weeks. Based on the kinetics of the antibody response to ONRAB® , the best time to sample sera of wild adult foxes for evidence of vaccination is 7–11 weeks following bait distribution. Thirty-four foxes (25 ONRAB® , 9 controls) were challenged with vulpine street virus 547 days post-vaccination. All controls developed rabies whereas eight of 13 adult vaccinates (62%) and four of 12 juvenile vaccinates (33%) survived. All foxes classed as non-responders to vaccination developed rabies. Of foxes considered responders to vaccination, 80% of adults (8/10) and 67% of juveniles (4/6) survived challenge. The duration of immunity conferred to foxes would appear adequate for bi-annual and annual bait distribution schedules as vaccinates were challenged 1.5 years post-vaccination. © 2013 The Authors. Published by Elsevier Ltd. All rights reserved.
In many developed countries, wild animals are the primary vectors of the rabies virus (RV) and maintain independent cycles of infection [1]. Until recently, the red fox (Vulpes vulpes) was the main rabies reservoir in Ontario and the principle target species for rabies management strategies [2,3]. The fox epizootic in Ontario originated in the Canadian Arctic, where the key wildlife vector is the Arctic fox (Vulpes lagopus) [4]. As the disease spread into
夽 This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-No Derivative Works License, which permits non-commercial use, distribution, and reproduction in any medium, provided the original author and source are credited. ∗ Corresponding author. Tel.: +1 705 755 2285; fax: +1 705 755 1559. E-mail address: [email protected] (L.J. Brown).
southern Ontario, red foxes and striped skunks (Mephitis mephitis) became the main reservoirs and vectors of Arctic rabies virus variant (ARVV), accounting for 68% and 30% of the terrestrial wildlife rabies cases, respectively, between 1958 and 1989 [5]. More recently in Ontario (1999–2005), raccoons (Procyon lotor) and, to a lesser extent, striped skunks were the main species affected by raccoon rabies virus variant (RRVV) [6,7], a variant associated with raccoons throughout the US eastern seaboard [8]. Oral rabies vaccination (ORV) is a cost-effective, socially acceptable disease control method for wildlife reservoirs that can be applied over large geographic areas [9–11]. ORV requires a vaccine that is effective by the oral route and a vaccine delivery package suitable for the target species. In eastern Ontario, ARVV was eliminated from the red fox population through large-scale ORV using ERA-BHK21 , a modified-live RV vaccine [2,3]. Similar results were attained throughout Europe with related attenuated strains of RV (SADBern , SADB19 , SAG1, and SAG2) [12]. Although live-attenuated
0264-410X/$ – see front matter © 2013 The Authors. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.vaccine.2013.12.015
Please cite this article in press as: Brown LJ, et al. Oral vaccination and protection of red foxes (Vulpes vulpes) against rabies using ONRAB® , an adenovirus-rabies recombinant vaccine. Vaccine (2013), http://dx.doi.org/10.1016/j.vaccine.2013.12.015
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vaccines have proven successful for fox rabies control, their continued use in ORV remains controversial due to residual pathogenicity for a variety of rodent species [13–16], and vaccine-induced rabies in target and non-target animals [17–19]. Other considerations of note include sensitivity to heat [20] and ineffectiveness by the oral route in rabies reservoirs such as raccoons and skunks [17,21,22]. An alternative ORV strategy involves the use of recombinant vaccines constructed from heterologous virus vectors that express the RV glycoprotein. These vaccines reportedly offer improved safety and better heat stability [23–25]. In Europe, vaccinia virus recombinant vaccines (VVTGgRAB and V-RG) have proven effective by the oral route in red foxes [23,25,26] but efforts to immunize raccoons and skunks have proven less successful [6,9,27–30]. Recombinant vaccines that use adenoviruses as vectors have also been considered and show promise for wildlife ORV [31–37]. ONRAB® (AdRG1.3) is a recombinant oral rabies vaccine that uses a human adenovirus vector (serotype 5) to express the RV glycoprotein [33]. In early Ontario field trials (2006–2007), both raccoons and skunks demonstrated serologic evidence of an immune response to ONRAB® [38]. A high proportion of raccoons in captivity also responded to vaccination with ONRAB® and most responders survived rabies challenge almost 1 year postvaccination (PV) [39]. The current study was conducted to see if similar results could be attained in red foxes, as a single oral rabies vaccine that is immunogenic in multiple North American rabies vector species (especially raccoon, red fox, and striped skunk) offers the best chance of eventual virus containment and elimination. The objectives of this study were to (1) quantify the longitudinal humoral immune response of captive foxes following ORV with ONRAB® and (2) test the protection afforded by the vaccine against RV.
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then every 15 min for the next 1.5 h. Ten controls (5 adults, 5 juveniles) received no vaccine baits. 2.3. Blood collection A blood sample (3–5 ml) was collected from controls and vaccinates 2 weeks before bait presentation, and then each week after treatment for 16 consecutive weeks. Animals were anesthetised and blood samples collected as described in [39]. Blood was refrigerated at 4 ◦ C for up to 48 h and then centrifuged at 1000 × g for 12 min at 4 ◦ C. Harvested serum was maintained at −20 ◦ C and then heat-inactivated at 56 ◦ C for 30 min prior to testing. 2.4. Rabies challenge Thirty-four foxes (9 controls, 25 vaccinates) from the serology study were subjected to rabies challenge on February 10, 2010. The challenge virus was isolated from a naturally infected rabid fox and identified as ARVV using a panel of anti-nucleocapsid monoclonal antibodies and sequence analysis [42] (CDC SM1515; GenBank Accession No. JQ685990). The RV was adapted to murine neuroblastoma cells with 3 serial passages. The supernatant was harvested and clarified by low speed centrifugation (800 × g) for 10 min then stored at −120 ◦ C before use. Five hundred and forty-seven days PV, foxes were anesthetised and a blood sample (3 ml) was collected from the jugular vein for baseline serology. Each fox was then inoculated bilaterally with 0.5 ml of RV (104.9 MICLD50 ) into the masseter muscle. Foxes were also bled 7 days post-inoculation (PI). Animals were observed twice daily for clinical signs of rabies (paralysis, ataxia, behavioural change, lack of appetite, hyper-salivation, vocalization, agitation, tremors, convulsions, aggression) and humanely euthanized when two or more signs appeared. All surviving animals were euthanized 60 days PI and submitted for rabies diagnosis using the direct fluorescent antibody test [43].
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Thirty-seven captive-reared foxes were purchased from an Ontario breeder. At treatment, juveniles were aged 14–17 weeks; adults were 1.3–7.3 years. Prior to treatment, foxes were wormed (Strongid*T; 50 mg/ml; Pfizer Animal Health, Quebec, Canada) and vaccinated against canine distemper (Galaxy*D; Schering-Plough Animal Health, Nebraska, USA). For the serology study, foxes were housed in separate units within outdoor enclosures. For the challenge study, foxes were housed in kennel runs in an Animal BioContainment Level-2 facility. Animals were provided a commercial feed once a day and water ad libitum. All animals were maintained in accordance with national guidelines (Canadian Council on Animal Care Guidelines [40] or Guide for the Care and Use of Laboratory Animals [41]) and protocols were approved by Institutional Animal Care and Use Committees (Ontario Ministry of Natural Resources [OMNR], Canada or Centers for Disease Control and Prevention [CDC], Georgia, USA).
A modified fluorescent antibody virus neutralization test [44] was used to detect rabies virus neutralizing antibody (RVNA) in sera from the humoral immune response study. Titres were calculated using the Spearman–Kärber formula [45] and expressed in international units (IU) per ml calibrated against the World Health Organization 2nd International Reference Standard (National Institute of Biological Standards and Controls, Potter’s Bar, UK). A rapid fluorescent focus inhibition test [46] with endpoint titres determined [47] was used to detect RVNA in sera from the challenge study. Titres were reported in IU/ml following normalization against the US Standard Rabies Immune Globulin (Laboratory of Standards and Testing, Food and Drug Administration, USA) diluted to 2 IU/ml. For both studies, serum samples were considered positive for RVNA if the titre was ≥0.5 IU/ml.
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2.6. Statistical analyses
Ultra-lite baits containing 1.8 ml (±0.1 ml) of ONRAB® (1010 TCID50 /ml) were manufactured by Artemis Technologies Inc., Guelph, Ontario, Canada [38]. Foxes were randomly assigned to a control or vaccinate group with minor adjustments to ensure similar age-sex representation among groups. Twenty-seven foxes (14 adults, 13 juveniles) were each offered one ONRAB® bait on August 12, 2008 following a 24 h fasting period. Bait debris and vaccine spillage were collected on plastic sheets below each cage. The amount of bait ingested, vaccine loss during ingestion, and timing of ingestion were recorded every 10 min for the first 0.5 h
Fisher’s exact tests (FET) were used to examine associations between age and response to vaccination and between age and RV infection post-challenge. FET were also used to assess differences in RV infection among controls and vaccinates post-challenge and test the association between response to vaccination and survival. A Mann–Whitney test was used to determine whether the number of days until death differed among controls and vaccinates post-challenge [48]. For foxes considered responders to vaccination, Mann–Whitney tests were used to determine whether there was a difference between adults and juveniles in the week RVNA
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2.5. Serologic analyses
Please cite this article in press as: Brown LJ, et al. Oral vaccination and protection of red foxes (Vulpes vulpes) against rabies using ONRAB® , an adenovirus-rabies recombinant vaccine. Vaccine (2013), http://dx.doi.org/10.1016/j.vaccine.2013.12.015
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Table 1 Numbers of red foxes (Vulpes vulpes) in the control and ONRAB® bait groups that tested positive for rabies virus neutralizing antibody (RVNA) post-vaccination (PV), numbers that tested positive for RVNA on day 7 post-infection (7 d PI), and numbers that survived rabies challenge 547 days post-vaccination. RVNA+ PV (%)
Survival (%)
RVNA post-vaccination Positive n
Control Bait
All foxes Adults Juveniles
0/10 (0%) 11/14 (79%) 6/13 (46%)
0/9 (0%) 8/13 (62%) 4/12 (33%)
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Negative RVNA+ 7 d PI 8 5
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was first detected and the number of consecutive weeks an animal tested RVNA positive. STATISTICA© was used for all analyses (StatSoft, Tulsa, Oklahoma, USA).
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3.1. Bait consumption and vaccination
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Eighteen foxes consumed baits within 20 min; nine animals took 1–2 h. Unconsumed vaccine (0.15 ml) was recovered from only one cage (#823).
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3.2. Serologic response to ONRAB®
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No vaccine-induced morbidity or mortality was observed. Prevaccination sera for all animals, and sera for all control foxes throughout the 16 week study, had titres ≤0.03 IU/ml. A fox was considered a ‘responder’ to vaccination if RVNA was detected for 3 or more consecutive weeks. Using this criterion, 79% of adults (11/14) and 46% of juveniles (6/13) were classed as responders (Table 1). Although fewer juveniles responded to vaccination, the difference was not significant (P = 0.1201; FET). RVNA was detected earlier in adults than juveniles (U = 4.5, P = 0.0006) (Fig. 1). RVNA first appeared in one adult at week 1, in four at week 2, and in two at week 3. In the remaining adults, RVNA first occurred at weeks 4, 5, or 6. The earliest evidence of RVNA in juveniles was at week 5, the latest at week 9. Adults exhibited higher RVNA titres and more consecutive weeks with RVNA than juveniles (U = 17.5, P = 0.0125) (Fig. 1). Six adult foxes had RVNA detected for 9–15 consecutive weeks; five others, for 3–5 consecutive weeks. Two adults had RVNA for only 2 consecutive weeks, while one adult remained RVNA negative. One juvenile had RVNA detected for 4 consecutive weeks; five others for 3 consecutive weeks. Two juveniles had RVNA detected at 1 or 2 weeks; the remaining five juveniles tested RVNA-negative throughout the study (mode 0.03 IU/ml; range 0.01–0.42 IU/ml). At week 4 PV, the proportion of adult foxes with RVNA was 57% (Fig. 2). Seropositivity increased to 71% by weeks 5 and 6, reached a peak of 86% by week 7, then declined slightly through weeks 8 and 9 (79% and 71%, respectively). Forty-three percent to 57% of adults had RVNA detectable between weeks 10 and 16. The proportion of juveniles with RVNA was highest between weeks 7 and 9 PV (38–46%); peak at week 8 (Fig. 2).
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3.3. Resistance to rabies challenge
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All control foxes (n = 9) succumbed to RV infection as confirmed by the direct fluorescent antibody test (Table 1). Eight of 13 adults (62%) and four of 12 juveniles (33%) offered ONRAB® baits survived challenge (Table 1). Although survival was lower in juveniles than adults, the difference was not significant (P = 0.2377, FET). Of the 10 adult foxes classed as responders to vaccination, seven demonstrated RVNA on the day of challenge (range 0.64–12.88 IU/ml). All eight survivors in the adult group were classed as responders to vaccination and all mounted an anamnestic response PI (range
Survived challenge
n
RVNA+ 7 d PI
Survived challenge
8 4
9 3 6
1 1 2
0 0 0
Fig. 1. Temporal development of rabies virus neutralizing antibody (RVNA) in sera collected from (a) adult (n = 11) and (b) juvenile (n = 6) red foxes (Vulpes vulpes) considered responders to vaccination following consumption of ONRAB® baits. Solid lines track median titres for adult and juvenile foxes at each week post-vaccination.
3.35–298.71 IU/ml). Two adult responders that did not exhibit RVNA on the day of challenge and showed no anamnestic response did not survive challenge. Three adults classed as non-responders did not exhibit RVNA on the day of challenge and did not survive challenge (Table 1). One juvenile responder had detectable RVNA on the day of challenge (0.94 IU/ml); two others had titres that fell just below the 0.5 IU/ml threshold (0.48 IU/ml). All four juvenile foxes that survived challenge were responders to vaccination and had RVNA levels suggestive of an anamnestic response (range 60.09–312.88 IU/ml). Two juvenile responders did not survive challenge (Table 1). Both animals had no RVNA on the day of challenge,
Please cite this article in press as: Brown LJ, et al. Oral vaccination and protection of red foxes (Vulpes vulpes) against rabies using ONRAB® , an adenovirus-rabies recombinant vaccine. Vaccine (2013), http://dx.doi.org/10.1016/j.vaccine.2013.12.015
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Fig. 2. Proportion of red foxes (Vulpes vulpes) that had rabies virus neutralizing antibody (RVNA) ≥0.5 IU/ml at each week post-vaccination. Vaccinates were each offered one ONRAB® bait. Closed circles represent adults (n = 14); open circles, juveniles (n = 13).
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although one juvenile did have RVNA seven days PI (1.99 IU/ml). All six juvenile non-responders to vaccination did not survive rabies challenge (Table 1), but two animals did have RVNA on day 7 PI (1.55 and 2.88 IU/ml). More foxes offered ONRAB® baits survived challenge with ARVV than controls (P = 0.0132, FET). Within the ONRAB® group, more foxes classed as responders to vaccination tested negative for rabies (P = 0.0005, FET). Among controls and vaccinates that succumbed to rabies, there was no significant difference in the number of days to death or humane euthanasia PI (median = 12 days; range 11–15 days) (U = 44.5, P = 0.3498).
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4. Discussion
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This is the first study to examine the longitudinal antibody response of red foxes to ONRAB® (AdRG1.3) presented in baits and results provide evidence of its promise as a candidate oral rabies vaccine for this species. Seventy-nine percent (11/14) of adult foxes offered ONRAB® baits seroconverted within 6 weeks. Similar results were achieved in a previous study where foxes aged 1–2 years were immunized with an earlier version of the vaccine (Ad5RG1) by direct instillation into the oral cavity; 87% (14/16) seroconverted within 9 weeks PV [32]. Our results also compare favourably with similar studies where 50–100% of foxes produced RVNA in response to V-RG, a vaccinia-rabies recombinant vaccine, in different baits [23,49–51]. Although a variety of live-attenuated oral rabies vaccines have also proven efficacious in red foxes [52–54], safety and stability concerns make them less desirable for field applications and they are ineffective in other important rabies vector species such as raccoons and skunks [17,21,22]. During this study and two other ONRAB® efficacy trials, no vaccine-induced morbidity or mortality was observed in foxes, raccoons or skunks [39,55]. Field results also support that ONRAB® is safe for foxes as well as other wildlife species. ONRAB® was quickly excreted by target and non-target animals that contacted vaccine baits and viral replication following vaccine ingestion was short lived, suggesting a low risk of virus transmission in the natural environment [32,44,56,57]. Juveniles as a group did not respond to vaccination as well as adults, but similar results were also seen in other laboratory and field trials [13,52,58–62]. In our study, 46% (6/13) of juvenile foxes offered ONRAB® baits seroconverted within 9 weeks. Although our
study was not designed to evaluate seroconversion in juveniles of different ages, older juveniles did respond better to vaccination. All four juveniles less than 15 weeks did not respond to vaccination, whereas only three of nine juveniles aged 16–17 weeks were nonresponders. During fall ORV campaigns, juvenile foxes would be several weeks older than those used in our captive study; thus, it is possible that at baiting time the immunization rate of matured young foxes would approach that of adults. RVNA was first detected in some adult foxes as early as 14 days after presentation of ONRAB® baits. This is similar to other studies in foxes with V-RG, SAG2, and ERA vaccines [50–52,63]. Adult foxes demonstrated RVNA earlier, by about 4 weeks, and maintained RVNA longer than juveniles. Based on the kinetics of the antibody response of foxes to ONRAB® , and assuming most foxes consume baits within 2 weeks of field distribution [64,65], the best time to collect sera from adult foxes for RVNA evaluation is 7–11 weeks post-bait distribution. For maximal detection of RVNA in juveniles, field sampling should be conducted between weeks 9 and 11. In the original study design, rabies challenge was scheduled to occur 90 days PV; however, various logistical factors delayed the challenge more than 1 year, providing a unique opportunity to test the long-term protective potential of ONRAB® in red foxes. All nine controls developed rabies whereas eight of 13 adult vaccinates (62%) and four of 12 juvenile vaccinates (33%) survived rabies challenge 1.5 years PV. Survival of adult foxes was higher after consumption of ERA baits (100%) [13,16,53,66,67] but protection provided by ONRAB® was within the range of that provided by V-RG in different baits (50–100%) [23,49–51,68]. In ERA trials, foxes were challenged 28 days to 7 years PV; V-RG challenge trials all occurred within 91 days PV. For successful fox rabies control, duration of immunity should extend through to the next baiting campaign [53]. Although bi-annual baiting schedules (spring and fall) have been applied in Europe [60], annual fall campaigns were preferred in Ontario [2]. The duration of immunity conferred to foxes in this study would appear adequate for bi-annual and annual bait distribution schedules as vaccinates were challenged 1.5 years PV. In this study, and in many others, the correlation between the production of RVNA and protection of foxes against rabies was relatively strong. In our study, all foxes classed as non-responders to vaccination developed rabies. Of foxes considered responders to vaccination, 80% of adults (8/10) and 67% of juveniles (4/6) survived challenge. In several vaccine efficacy trials, seropositivity equated to protection [16,49,50,53,54,67,69]. In others, survival rate was higher than seropositivity [13,49,50] or, as in our study, it was slightly lower [23,53]. Four animals in our study with RVNA for three or more consecutive weeks did not survive challenge. None of these individuals had detectable rabies antibody prior to challenge and only one had a positive titre at 7 days PI. These data suggest that despite stimulating a primary humoral immune response, immunological memory was not established in these four animals. Similar results were also seen in captive raccoons. Of raccoons considered responders to oral vaccination after consumption of ONRAB® baits, 67% (10/15) survived rabies challenge [39]; one nonresponder also survived. Interestingly, in captive skunks, survival rates following oral vaccination with ONRAB® surpassed vaccination estimates [55] which would suggest that immunity factors in addition to those measured by the presence of antibodies likely contributed to protection in this species. In summary, we have demonstrated that ONRAB® , an adenovirus-rabies recombinant vaccine, is innocuous in the red fox and, when presented in baits, elicited RVNA at levels comparable to those observed with live-attenuated and vaccinia-recombinant rabies vaccines, with protection exceeding 1 year in duration. These results, together with safety and effectiveness in additional rabies
Please cite this article in press as: Brown LJ, et al. Oral vaccination and protection of red foxes (Vulpes vulpes) against rabies using ONRAB® , an adenovirus-rabies recombinant vaccine. Vaccine (2013), http://dx.doi.org/10.1016/j.vaccine.2013.12.015
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reservoirs, make ONRAB® an attractive alternative to oral rabies vaccines used in the past for fox rabies control. Acknowledgements We would like to thank staff of the Ontario Ministry of Natural Resources and Canadian Food Inspection Agency for animal care, and field and laboratory technical assistance: Erin Scharf, Tara MacDonald, Andrea Clark, and Bharat Bongale. We would also like to acknowledge the contributions of CDC staff responsible for animal care and all aspects of the challenge portion of this study. Andrew Beresford and Al Beath at Artemis Technologies Inc. prepared the vaccine baits used in this study and Beverly Stevenson and Kim Bennett commented on an early draft of the manuscript. This project was supported by the Ontario Rabies Advisory Committee (Jim Broadfoot, Chair). References [1] Rosatte RC, Tinline RR, Johnston DH. Rabies control in wild carnivores. In: Jackson A, Wunner W, editors. Rabies. San Diego, CA: Academic Press; 2007. p. 595–634. [2] MacInnes CD, Smith SM, Tinline RR, Ayers Neil R, Bachmann P, Ball DG, et al. Elimination of rabies from red foxes in eastern Ontario. J Wildl Dis 2001;37:119–32. [3] Rosatte RC, Power MJ, Donovan D, Davies JC, Allan M, Bachmann P, et al. Elimination of arctic variant rabies in red foxes, metropolitan Toronto. Emerg Infect Dis 2007;13:25–7. [4] Tabel H, Corner AH, Webster WA, Casey CA. History and epizootiology of rabies in Canada. Can Vet J 1974;15:271–81. [5] Agriculture Canada Report of Rabies Diagnosed – 1958 to 1989, Ottawa; 1958–1989. [6] Rosatte R, Allan M, Bachmann P, Sobey K, Donovan D, Davies JC, et al. Prevalence of tetracycline and rabies virus antibody in raccoons, skunks, and foxes following aerial distribution of V-RG baits to control raccoon rabies in Ontario, Canada. J Wildl Dis 2008;44:946–64. [7] Rosatte RC, Donovan D, Allan M, Bruce L, Buchanan T, Sobey K, et al. The control of raccoon rabies in Ontario, Canada: proactive and reactive tactics, 1994–2007. J Wildl Dis 2009;45:772–84. [8] Wandeler AI. Raccoon rabies in eastern Ontario. Can J Vet Res 1999;40:731. [9] Slate D, Algeo TP, Nelson KM, Chipman RB, Donovan D, Blanton JD, et al. Oral rabies vaccination in North America: opportunities, complexities, and challenges. PLoS Negl Trop Dis 2009;3:e549. [10] Rosatte RC, Donovan D, Davies JC, Brown L, Allan M, von Zuben V, et al. Highdensity baiting with ONRAB® rabies vaccine baits to control Arctic variant rabies in striped skunks in Ontario, Canada. J Wildl Dis 2011;47:459–65. [11] Shwiff SA, Nunan CP, Kirkpatrick KN, Shwiff SS. A retrospective economic analysis of the Ontario red fox oral rabies vaccination programme. Zoonoses Public Health 2011;58:169–77. [12] Wandeler AI. Oral immunization against rabies: afterthoughts and foresight. Schweiz Arch Tierheilkd 2000;142:455–62. [13] Black JG, Lawson KF. The safety and efficacy of immunizing foxes (Vulpes vulpes) using bait containing attenuated rabies virus vaccine. Can J Comp Med 1980;44:169–76. [14] Artois M, Guittré C, Thomas I, Leblois H, Brochier B, Barrat J. Potential pathogenicity for rodents of vaccines intended for oral vaccination against rabies: a comparison. Vaccine 1992;10:524–8. [15] Vos A, Neubert A, Aylan O, Schuster P, Pommerening E, Muller T, et al. An update on safety studies of SAD B19 rabies virus vaccine in target and non-target species. Epidemiol Infect 1999;123:165–75. [16] Lawson KF, Hertler R, Charlton KM, Campbell JB, Rhodes AJ. Safety and immunogenicity of ERA strain of rabies virus propagated in a BHK-21 cell line. Can J Vet Res 1989;53:438–44. [17] Rupprecht CE, Charlton KM, Artois M, Casey GA, Webster WA, Campbell JB, et al. Ineffectiveness and comparative pathogenicity attenuated rabies virus vaccines for the striped skunk (Mephitis mephitis). J Wildl Dis 1990;26:99–102. [18] Fehlner-Gardiner C, Nadin-Davis S, Armstrong J, Muldoon F, Bachmann P, Wandeler A. Era vaccine-derived cases of rabies in wildlife and domestic animals in Ontario, Canada, 1989–2004. J Wildl Dis 2008;44:71–85. [19] Müller T, Bätza H-J, Beckert A, Bunzenthal C, Cox JH, Freuling CM, et al. Analysis of vaccine-virus-associated rabies cases in red foxes (Vulpes vulpes) after oral rabies vaccination campaigns in Germany and Austria. Arch Virol 2009;154:1081–91. [20] Winkler WG, McLean RG, Cowart JC. Vaccination of foxes against rabies using ingested baits. J Wildl Dis 1975;11:382–8. [21] Tolson ND, Charlton KM, Lawson KF, Campbell JB, Stewart RB. Studies of ERA BHK-21 rabies vaccine in skunks and mice. Can J Vet Res 1988;52:58–62. [22] Rupprecht CE, Dietzschold B, Cox JH, Schneider LG. Oral vaccination of raccoons (Procyon lotor) with an attenuated (SAD-B19) rabies virus vaccine. J Wildl Dis 1989;25:548–54.
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Please cite this article in press as: Brown LJ, et al. Oral vaccination and protection of red foxes (Vulpes vulpes) against rabies using ONRAB® , an adenovirus-rabies recombinant vaccine. Vaccine (2013), http://dx.doi.org/10.1016/j.vaccine.2013.12.015
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