COMPARISON OF ANTIBODY RESPONSE TO A NONADJUVANTED, LIVE CANARYPOX-VECTORED RECOMBINANT RABIES VACCINE AND A KILLED, ADJUVANTED RABIES VACCINE IN ELD'S DEER (RUCERVUS ELDI THAMIN) Author(s): Judilee C. Marrow, D.V.M., Luis R. Padilla, D.V.M., Dipl. A.C.Z.M., Lee-Ann C. Hayek, Ph.D., Mitch Bush, D.V.M., Dipl. A.C.Z.M., and Suzan Murray, D.V.M., Dipl. A.C.Z.M. Source: Journal of Zoo and Wildlife Medicine, 45(2):315-320. 2014. Published By: American Association of Zoo Veterinarians DOI: http://dx.doi.org/10.1638/2013-0171R1.1 URL: http://www.bioone.org/doi/full/10.1638/2013-0171R1.1

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Journal of Zoo and Wildlife Medicine 45(2): 315–320, 2014 Copyright 2014 by American Association of Zoo Veterinarians

COMPARISON OF ANTIBODY RESPONSE TO A NONADJUVANTED, LIVE CANARYPOX-VECTORED RECOMBINANT RABIES VACCINE AND A KILLED, ADJUVANTED RABIES VACCINE IN ELD’S DEER (RUCERVUS ELDI THAMIN) Judilee C. Marrow, D.V.M., Luis R. Padilla, D.V.M., Dipl. A.C.Z.M., Lee-Ann C. Hayek, Ph.D., Mitch Bush, D.V.M., Dipl. A.C.Z.M., and Suzan Murray, D.V.M., Dipl. A.C.Z.M.

Abstract: Captive Eld’s deer (Rucervus eldi thamin) were evaluated for the presence of rabies virus–neutralizing antibodies using a rapid fluorescent focus inhibition after vaccination with either a live canarypox-vectored recombinant rabies vaccine or a killed monovalent rabies vaccine. Twelve deer were vaccinated with 1.0 ml of killed, adjuvanted, monovalent rabies vaccine at 5–33 mo of age then annually thereafter, and 14 deer were vaccinated with 1.0 ml nonadjuvanted, live canarypox-vectored rabies vaccine at 3–15 mo of age then annually thereafter. Banked serum was available or collected prospectively from deer at 6 mo and 1 yr after initial vaccination, then collected annually. Rabies virus–neutralizing antibodies considered adequate (.0.5 IU/ml) were present in 20/34 samples vaccinated with canarypox-vectored rabies vaccine and in 12/14 samples vaccinated with killed adjuvanted rabies vaccine. Poor seroconversion was noted in deer less than 6 mo of age vaccinated with the canarypox-vectored rabies vaccine. Key words: Cervid, Eld’s deer, rabies, Rucervus eldi thamin, vaccination.

INTRODUCTION Rabies has a worldwide geographic distribution and remains an important disease of animals and humans. Rabies virus (family Rhabdoviridae, genus Lyssavirus) causes acute fatal encephalitis, most commonly after the bite of an infected animal.2,4 Development of effective vaccines and widespread vaccination has greatly reduced cases of rabies in domestic animals and humans worldwide.2 There are currently no approved parenteral vaccines for use in captive wildlife or exotic mammals, although these animals have been routinely vaccinated using killed monovalent rabies vaccine products for decades.3 Killed monovalent rabies vaccines have been deemed safe when used in most species, as they cannot cause infection or spread the virus, and efficacy From the Smithsonian Conservation Biology Institute, Species Survival Center, 1500 Remount Road, Front Royal, Virginia 22630, USA (Marrow, Padilla, Bush); Smithsonian Institution National Zoological Park, Smithsonian Conservation Biology Institute, Wildlife Health Sciences, 3001 Connecticut Avenue, Washington, D.C. 20008, USA (Marrow, Murray); and Smithsonian Institution, MRC 121, P.O. Box 7012, Washington, D.C. 20013, USA (Hayek). Present addresses (Marrow): Binder Park Zoo, 7400 Division Drive, Battle Creek, Michigan 49014, USA; (Padilla): Saint Louis Zoo, One Government Drive, St. Louis, Missouri 63110, USA. Correspondence should be directed to Dr. Marrow (judileemarrowdvm@ gmail.com).

has been established in a wide variety of domestic species.2,4 Commercially available killed vaccines often add adjuvants to boost a host’s immune response. The use of adjuvants has increasingly been associated with allergic-type reactions.1,6,12 With the development of recombinant vaccine products, such as canarypox-vectored vaccines, a strong antibody response can be achieved without the use of adjuvants or the ability of the vaccine virus strain to revert to live virus strain as with more traditional live vaccines.15,16 The Smithsonian Conservation Biology Institute’s (SCBI’s) Front Royal Facility maintains a herd of the endangered Eld’s deer (Rucervus eldi thamin) and participates in the Species Survival Plan for this species. Historically, a killed rabies vaccine was used for routine vaccination as this was the only vaccine commercially available. In 2006, two individuals developed suspected vaccine-related adverse reactions following routine booster vaccination 1 yr after initial vaccination. Clinical signs included facial swelling and difficulty breathing; both animals responded to supportive care with antihistamines and steroids. The preventative medicine program was adjusted and the herd was subsequently vaccinated with a canarypox-vectored recombinant rabies vaccine. The purpose of the study is to evaluate the humoral antibody response of Eld’s deer when vaccinated with a live canarypox-vectored recombinant vaccine and to compare the humoral

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antibody response to deer vaccinated with a killed vaccine product.

MATERIALS AND METHODS Twenty-six Eld’s deer (7 males and 19 females) were included in this study; prospectively collected samples were approved by the animal care and use committee. Deer were housed in compatible groups of females and singly housed males in indoor–outdoor facilities at SCBI in Front Royal, Virginia. All animals were deemed healthy based on routine physical examinations and daily observation by animal care staff throughout the duration of the study. Prior to 2006, animals were vaccinated with a killed, adjuvanted rabies (KAR) vaccine product (IMRABt large animal, Merial, Duluth, Georgia 30096, USA; 1.0 ml s.c. or i.m.) at 5–33 mo of age and received booster vaccines annually, as part of the routine preventative medicine protocol at SCBI. Fourteen banked serum samples were available from 12 deer (three males, nine females) vaccinated with KAR vaccine. Samples were stored at 708C prior to analysis. After 2006, following commercial availability, deer were vaccinated with a nonadjuvanted canarypox-vectored rabies (CPVR) vaccine (Purevax, Merial; 1.0 ml s.c.) at 3–15 mo of age and received booster vaccination annually as part of the preventative medicine protocol at SBCI. Prospectively, blood was obtained under conditioned restraint, which is part of the management of this herd. Blood was collected opportunistically at 6 6 2 mo and 12 6 2 mo after initial vaccination, and then collected annually at the time of booster vaccination each subsequent year. Whole blood was collected in serum tubes and centrifuged; serum was removed and stored at 708C for batch analysis. Thirty-four serum samples were collected from 14 deer (4 males, 10 females) between 2007 and 2012 for batch evaluation. Rabies virus–neutralizing antibodies (RVNA) were quantified via rapid fluorescent focus inhibition test (RFFIT) at Kansas State University.11 The low end of the limit was reported at 0.1 IU/ ml and the high end of the detectable titer was 15 IU/ml. As no challenge studies are available for nondomestic cervids, a titer of 0.5 IU/ml was considered adequate based on previously published reports.9,10 Data were collected and recorded as binary; the outcomes were recorded as either an adequate titer or not. Fisher’s exact tests on counts were used for all two-by-two tables to determine if

there was an association between the variables in rows and columns for each test, and exact probabilities for tests of larger numbers of proportions were calculated with commercially available software (Mathcad 15t 2012 for Windows PTC, Needham, Massachusetts 02494, USA).

RESULTS Rabies was not suspected or diagnosed in any of the animals in the SCBI collection or in any wild animals on site, including the individuals in the Eld’s deer herd, during the study period. Additionally, vaccine reactions or other adverse effects from vaccination with the CPVR vaccine were not documented. Although not included in this study, the two animals with previously documented vaccine reactions to the KAR vaccine did not demonstrate adverse reactions after being vaccinated for consecutive years with the CPVR vaccine. Between 1996 and 2012, 48 serum samples from 26 Eld’s deer were available for evaluation of RVNA titer by RFFIT (Table 1). Thirty-seven of the 48 samples had detectable RVNA titers. In deer vaccinated with the CPVR vaccine, 23/34 available samples elicited a detectable antibody response, but only 20/34 (58.8%) demonstrated adequate titers (Table 2). In deer vaccinated with the KAR vaccine, all serum samples had detectable RVNA titers and 12/14 (85.7%) demonstrated adequate titers. Deer vaccinated with KAR vaccine product were significantly more likely to elicit a detectable titer in Eld’s deer (P ¼ 0.021). When a detectable titer was present, the KAR and CPVR groups were not statistically different in the ability to develop an adequate titer after vaccination (P ¼ 0.098). Titers ranged between 0.1 and .15 IU/ml in both vaccine groups. Poor seroconversion of adequate RVNA titers was detected in deer vaccinated with CPVR vaccine under 6 mo of age (Table 3). Comparison of deer under 6 mo of age vaccinated with KAR versus CPVR vaccine products was not possible because serum samples were not available for deer vaccinated with KAR vaccine product at 12 mo following initial vaccination. In deer vaccinated at 6 mo of age or greater, neither KAR nor CPVR vaccine groups demonstrated any difference in proportion of adequate titers (P ¼ 0.098). Within the vaccine groups, no statistical difference between titer responses in males and females could be determined (Table 3).

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MARROW ET AL.—SERUM RABIES TITERS IN ELD’S DEER

Table 1. Serum rabies virus–neutralizing antibody titers in captive Eld’s deer (Rucervus eldi thamin) vaccinated with monovalent killed, adjuvanted rabies vaccine or live canarypox-vectored rabies vaccine products as determined by rapid fluorescent focus inhibition test. Serum virus-neutralizing antibody titer

CPVRa 1 2 3 4 5 6 7 8 9 10 11 12 13 14 KAR 15 16 17 18 19 20 21 22 23 24 25 26

Sex

Age atinitial vaccination (mo)

6 mo post initial vaccination

12 mo post initial vaccination

12 mo post first booster

12 mo post second booster

12 mo post third booster

M F F F F M F M F F M F F F

10 4 5 15 6 11 14 4 4 4 4 4 3 3

— — — A — A A N N N N — — —

— A D A — A A N N N N N N N

— — — A D A A A A D A — — —

— — — — A — — — — — — — — —

A A A — — — — — — — — — — —

F F F F F F F F M F M M

7 5 8 8 32 7 9 8 18 33 7 17

— — — — — D D — — — A —

— — — A A — — — A A — —

— — — A — — — A — — — A

— — — — — — — — — — — —

A A A — — — — A — — — —

a Abbreviations: CPVR, canarypox-vectored rabies vaccine; M, male; F, female; A, Adequate RVNA titer (.0.5 IU/ml); D, detectable RVNA titer (0.1–0.49 IU/ml); N, no detectable RVNA titer (,0.1 IU/ml); KAR, killed, adjuvanted rabies vaccine.

DISCUSSION Rabies has been rarely reported in captive and wild deer.8,10,13,14,17 There are no approved rabies vaccines for use in wildlife species; however, many institutions housing captive animals and many farms choose to vaccinate deer to help protect valuable animals and decrease risk for human exposure.3,4,9,10 Deer vaccinated with the KAR vaccine product were significantly more likely to develop detectable RVNA titers as compared to those deer vaccinated with the CPVR vaccine. When evaluating the development of titers considered to be adequate no statistical difference could be determined between KAR and CPV vaccine groups. Two deer (animals 5 and 10) demonstrated suspected vaccine failures after vaccination with the CPVR vaccine. Deer 10 did not have a detectable RVNA titer at 6 or 12 mo

after initial vaccination, or at 12 mo after first annual booster, but did eventually demonstrate an adequate titer at 12 mo after second annual booster vaccine. Deer 5 did not have serum

Table 2. Serum virus-neutralizing antibody (RVNA) titers to rabies vaccination in captive Eld’s deer (Rucervus eldi thamin) vaccinated with monovalent killed, adjuvanted rabies vaccine (KAR) or live canarypox-vectored rabies vaccine (CPVR) products as determined by rapid fluorescent focus inhibition test. Deer receiving KAR vaccine were significantly more likely to develop a detectable RVNA titer than deer receiving CPVR vaccine (P ¼ 0.021).

Vaccine group

Detectable titer, .0.1 IU/ml (%)

Adequate titer, .0.5 IU/ml (%)

KAR CPVR

14/14 (100) 23/34 (67.6)

12/14 (85.7) 20/34 (58.8)

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Table 3. Comparison of the development of adequate serum rabies virus–neutralizing antibody (RVNA) titer (.0.5 IU/ml) in captive Eld’s deer (Rucervus eldi thamin) vaccinated before 6 mo of age or after 6 mo of age when vaccinated with monovalent killed, adjuvanted rabies vaccine (KAR) or live canarypox-vectored rabies vaccine (CPVR) as determined by rapid fluorescent focus inhibition test (RFFIT). Serum samples obtained 12 mo after initial vaccination from available serum samples. No. of animals with adequate RVNA titer (.0.5 IU/ml) Vaccine group

,6 mo at initial vaccination

.6 mo at initial vaccination

Males

Females

KAR CPVR

— 1/8

4/4 2/3

1/1 1/3

3/3 3/9

samples at 6 or 12 mo after initial vaccination and failed to produce an adequate titer at 12 mo after first annual booster before developing an adequate titer at 12 mo after second annual booster vaccination. Although many of the deer vaccinated with CPVR vaccines demonstrate an RVNA titer after multiple vaccinations, more frequent titer screening or vaccination may be necessary to ensure adequate humoral protection in Eld’s deer vaccinated with the CPVR vaccine. During the study period, no adverse reactions were documented in any of the deer vaccinated with the CPVR vaccine. In the two deer with previous reactions, no subsequent reactions were noted when vaccinated with the CPVR vaccine product. These deer also demonstrated protective titers after boosters with the CPVR vaccine (data not shown). In a limited sample size, CPVR vaccine appears safe when used to vaccinate Eld’s deer, even in animals with previous reactions to other rabies vaccines. Since KAR vaccines are more likely to produce a detectable RVNA titer in Eld’s deer, it may be advisable to initially vaccinate with a KAR vaccine, then follow with annual boosters of a CPVR vaccine to minimize the risk for allergic vaccination reactions. Adverse reactions after vaccination, thought to be mediated by the immune response, have been associated with the use of adjuvants.1,12 The occurrence of adjuvant-related immunotoxicity, including allergies, hypersensitivity reactions, and the trigger of autoimmune disease syndromes is likely the result of an exaggerated activation of the immune and inflammatory response pathways deemed essential for normal function of both the innate and adaptive immune response.1 Rabies virus antibody titers are indicative of a response to vaccination or exposure to live virus, but do not directly correlate with protection against rabies infection.2 There are no studies that have evaluated rabies challenge in cervids, but 0.5 IU/ml has been used as a measure of an adequate vaccination response and cervids are

thought to have similar vaccination responses as other domestic and nondomestic ruminants.8,10 In this study, the route of vaccine administration may have an effect on the desired immune response to vaccination. Contact with different cell types at the injection site may dictate the immediate, local inflammatory reaction, as well as the subsequent humoral response. In this population of deer, the KAR vaccine was administered either intramuscularly or subcutaneously, whereas the CPVR vaccine was always administered subcutaneously. Differences in the route of vaccine administration may account for some of the attenuated responses seen, as well as variability in strength of the titer response. Since the injection site was not standardized or always recorded in this herd, further statistical analysis of these data set was deemed inappropriate for drawing conclusions, but it is an area worth exploring in further trials of vaccine response. Deer vaccinated with CPVR vaccine under 6 mo of age demonstrated poor seroconversion when compared to animals vaccinated with CPVR or KAR vaccine at or after 6 mo of age. The observed difference in response could be attributed to interference with maternally derived antibodies present at the time of vaccination, or may simply indicate individual variation in host response or in maturation of the immune system. Relatively little has been published evaluating the persistence of passively acquired, colostrum-derived, maternal antibodies in cervids and this has not been evaluated in Eld’s deer.5,7 Three deer (animals 8, 9, and 11) vaccinated under 6 mo of age failed to develop an adequate titer at 6 or 12 mo after initial vaccination but demonstrated an adequate RVNA titer 12 mo after the first annual booster vaccination. Maternal antibodies would be expected to have dissipated by the time of booster vaccination at 1 yr of age. The only animal (deer 2) vaccinated under 6 mo of age that developed a protective titer after initial vaccination had adequate colostral transfer. Decreased interaction of

MARROW ET AL.—SERUM RABIES TITERS IN ELD’S DEER

maternally derived antibodies was considered unlikely in this case. Interestingly, two deer (animals 20 and 21) vaccinated with KAR at 7 and 9 mo of age also failed to develop an adequate titer 6 mo after initial vaccination. Serum samples were not available at later time points for further comparison. Although not evaluated in this study, vaccination in the face of maternally derived antibodies can induce a cellular immune response.18 Further investigation is needed to determine if vaccination with CPVR vaccine can also provide a protective cell-mediated vaccine response in Eld’s deer. Changes to the preventative medicine plan for this herd at the time of vaccination and a relatively small sample size make further analysis challenging and additional data would be needed to determine if vaccination with CPVR vaccine after 6 mo of age can result in protective RVNA titers comparable to those documented in the KAR vaccine group. In cervids vaccinated against rabies with monovalent killed, adjuvanted vaccine products, reported titers have been relatively short-lived, requiring frequent boosters to maintain a protective level.10 In this study, Eld’s deer vaccinated with CPVR or KAR vaccine products were capable of demonstrating protective titers as long as 12 mo after vaccination and developed higher titer levels with subsequent booster vaccinations. When CPVR vaccine products are used in Eld’s deer, vaccination after 6 mo of age or increased frequency of booster vaccination may be required to develop a protective humoral immune response.

CONCLUSION In Eld’s deer, canarypox-vectored rabies vaccination is considered safe and can produce sustained protective titers against rabies. Animals vaccinated with monovalent killed rabies vaccines in this study were more likely to develop detectable rabies-neutralizing antibodies. Cell-mediated immunity after CPVR vaccination needs to be further evaluated in this species. If younger than 6 mo, deer vaccinated with the CPVR product are less likely to seroconvert. When using the CPVR vaccine, deer should be vaccinated at 6 mo of age or older and subsequently boostered, or RVNA titers should be performed to ensure the development of adequate rabies titers. Acknowledgements: The authors would like to thank the research, veterinary, and animal care staff from the Smithsonian Conservation Biology Institute for their care for the animals included in

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this study, with special attention to Lisa H. Ware and Dolores Reed for invaluable support in collecting, processing, and preparing all the samples used in this study.

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17. Wilson JE. Occurrence of rabies in deer (Odocoileus virginianus). J Mammal. 1949;30:203. 18. Zimmerman AD, Boots RE, Valli JL, Chase CCL. Evaluation of protection against virulent bovine viral diarrhea virus type 2 in caves that had maternal antibodies and were vaccinated with a modified-live vaccine. J Am Vet Med Assoc. 2006;228:1757–1761. Received for publication 21 July 2013