Journal of Wildlife Diseases, 47(1), 2011, pp. 182–194 # Wildlife Disease Association 2011
IMMUNOGENICITY AND EFFICACY OF TWO RABIES VACCINES IN WILD-CAUGHT, CAPTIVE RACCOONS L. J. Brown,1,3 R. C. Rosatte,1 C. Fehlner-Gardiner,2 M. K. Knowles,2 P. Bachmann,1 J. C. Davies,1 A. Wandeler,2 K. Sobey,1 and D. Donovan1 1 Ontario Ministry of Natural Resources, Wildlife Research and Development Section, Trent University, DNA Building, 2140 East Bank Drive, Peterborough, Ontario K9J 7B8, Canada 2 Canadian Food Inspection Agency, Ottawa Laboratory Fallowfield, PO Box 11300, Station H, Ottawa, Ontario K2H 8P9, Canada 3 Corresponding author (email:
[email protected])
The immunogenicity and efficacy of two rabies vaccines in wild-caught, captive raccoons (Procyon lotor) were investigated. Raccoons were fed Ontario Slim (OS) baits containing a recombinant vaccinia virus-rabies glycoprotein (VRG) oral rabies vaccine, or they were given an intramuscular (IM) injection of IMRABH 3 rabies vaccine. Blood samples collected before treatment and from weeks 1 to 16 posttreatment were assessed for the presence of rabies virus antibody (RVA). There were significantly more positive responders in the group that received an IM injection of IMRAB 3 (18/27) than in the group that consumed VRG in OS baits (VRG-OS; 4/ 26). There were no significant associations among age, sex, and seroconversion. Of those animals that mounted a humoral immune response to vaccination, RVA was first detected between weeks 1 and 5, with the majority of initial seroconversions detectable at week 2. A subsample of 50 raccoons (19 VRG-OS, 18 IMRAB 3, and 13 controls) from the longitudinal serology study was challenged with live raccoon variant rabies virus 442 days after initial treatment. There were significantly more survivors in the group that received IMRAB 3 (13/18) than in the VRG-OS (5/19) or control (2/13) groups. All 15 raccoons that demonstrated a serologic response survived challenge regardless of treatment. Of the 35 raccoons with no detectable serologic response, 30 (86%) succumbed to rabies virus infection (14/15 VRG-OS, 5/7 IMRAB 3, and 11/13 controls). Key words: Immune response, IMRAB 3, Procyon lotor, rabies virus challenge, raccoon, raccoon rabies, vaccinia virus-rabies glycoprotein. ABSTRACT:
In eastern Ontario, a variety of tactics were used between 1999 and 2006 to contain, control, and eventually eliminate raccoon rabies. These tactics included population reduction, trap-vaccinate-release (TVR), and oral rabies vaccination (ORV; Rosatte et al., 2001, 2007a, 2008). Between 2000 and 2006, the Ontario Ministry of Natural Resources (OMNR) vaccinated 24,544 raccoons and 2,863 skunks with IMRABH 3 rabies vaccine (Merial Limited, Athens, Georgia, USA) and distributed approximately 3.6 million Ontario Slim (OS) baits (Artemis Technologies Inc., Guelph, Ontario, Canada) containing a recombinant vaccinia virusrabies glycoprotein (VRG) oral rabies vaccine (Rosatte et al., 2008, 2009). The vaccine-bait combination used (VRG-OS) was experimental in that the vaccine was purchased in bulk from the manufacturer (Merial) and then incorporated into OS baits in Ontario (Rosatte et al., 2008).
INTRODUCTION
Raccoons (Procyon lotor) are major reservoirs of rabies virus in the eastern United States and carry a distinct variant of the virus. Translocation of raccoons in the late 1970s from the southeastern United States where the disease was enzootic resulted in a rabies outbreak on the Virginia-West Virginia border (Nettles et al., 1979; Jenkins et al., 1988). This epizootic progressively spread north through populations of raccoons and striped skunks (Mephitis mephitis) in the mid-Atlantic states (Centers for Disease Control and Prevention, 2000) and eventually crossed the international border into Canada: Ontario in 1999 (Wandeler and Salsberg, 1999; Rosatte et al., 2006), New Brunswick in 2000, and Quebec in 2006. The rabies virus variant in these regions is commonly referred to as raccoon variant. 182
BROWN ET AL.—VACCINATION AND PROTECTION AGAINST RABIES
Field surveillance was carried out each year 5–6 wk postbaiting to examine bait uptake by raccoons, assessed by detection of a tetracycline biomarker in teeth (second premolars), and seroconversion, as evidenced by the presence of rabies virus antibodies (RVAs) in sera. Between 2000 and 2002, bait uptake ranged from 51% to 83% depending on bait density (75 or 150 baits/km2). In contrast, serology indicated that only 11% of sampled raccoons had developed RVA (Rosatte et al., 2008). These results raised concern that VRG at a titer of 107.7 tissue culture infective dose (TCID50)/ dose incorporated into OS baits was not immunizing a large enough proportion of the raccoon population to stop the spread of disease. In the same assessment period, 72– 79% of the estimated raccoon population was vaccinated with IMRAB 3 (population density estimated using TVR capture-markrecapture data). Although high costs associated with this labor-intensive method meant TVR areas were much smaller than ORV areas (400–850 km2 and 4,000– 9,700 km2, respectively), the antibody prevalence achieved probably contributed significantly to stopping the spread of disease (Rosatte et al., 2007b, 2009). It is possible that blood samples were collected from raccoons outside of the peak serologic response period and that raccoons in the vaccination zone still would have been protected from disease if exposed. However, without detailed knowledge of the timing of serologic response in raccoons and a definitive test of protection using lethal virus, the effectiveness of VRG-OS as a tool to stop the spread of rabies in the field would remain questionable. In 2003, a decision was made to examine the longitudinal humoral immune response of raccoons to both VRG (in OS baits) and IMRAB 3 rabies vaccines under controlled conditions. The relationship between serologic response and protection against disease also was investigated by challenging raccoons with field isolates of the raccoon rabies virus 442 days postvaccination.
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MATERIALS AND METHODS Vaccines
IMRAB 3 was used off-label as the injectable rabies vaccine and was the same lot used in OMNR 2003 TVR field operations. For this study, and 2003 ORV field campaigns, VRG oral rabies vaccine was purchased in bulk from Merial (Rabies Vaccine, Live Vaccinia Vector– Bulk Product for Further Manufacture), shipped to Ontario, and incorporated into OS baits at Artemis Technologies. OS baits consisted of an outer oleo-wax matrix surrounding a polystyrene blister pack containing 1.860.1 ml VRG rabies vaccine (Rosatte et al., 2008). The titer of the vaccine after incorporation into OS baits was 107.4 TCID50/ml, giving a total dose of 107.7 TCID50 in each bait. This oral rabies vaccine-bait combination (VRG-OS, lot 2003-01) was considered experimental because the OS bait was not a component of the original RABORAL V-RG package (Merial). IMRAB 3 was refrigerated and vaccine baits were frozen until the morning of treatment administration. Study animals and housing
For the longitudinal immune response study, 80 wild raccoons were trapped in fall 2003 in a 2,000-km2 area approximately 32 km south and 80 km west of London, Ontario, Canada (42u599N, 81u159W). Approximately equal numbers of raccoons within each sex and age class were retained. There had never been a confirmed case of raccoon variant rabies in the area, and the last ORV campaign to vaccinate red foxes (Vulpes vulpes) against Arctic variant rabies (using baits containing ERA-BHK21 oral rabies vaccine) occurred in 1999 (OMNR, unpubl. data). Raccoons were housed in separate units within outdoor enclosures at the OMNR Wildlife Research Facility in Codrington, Ontario, Canada (44u109N, 77u489W). Each unit consisted of a covered plywood nest box for privacy and sleeping and an open wire cage used for feeding, exercise, and enrichment. Treatments
Each raccoon was randomly assigned to one of three groups, with minor adjustments to ensure similar age-sex representation among the groups. Treatments occurred over 3 days (6–8 October 2003) and were administered early in the morning before regular feeding. Thirty-one raccoons were offered VRG-OS from the same bait production lot used in the field in 2003. Pliable plastic sheets were
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placed below each cage to collect bait debris and vaccine spillage. The amount of bait ingested, vaccine loss during ingestion, and timing of ingestion were recorded for each raccoon. Thirty raccoons were given an intramuscular (IM) injection of 1 ml of IMRAB 3 rabies vaccine into a hind leg. Raccoons were restrained in their nest boxes, and the vaccine was administered without anesthesia. Nineteen control raccoons received no treatment. Blood collection
A blood sample (3–5 ml) was collected from each raccoon before treatment and then each week after treatment for 16 wk, with the exception of weeks 11 and 12. Animals were anesthetized by IM injection of a 10:1 mixture of ketamine hydrochloride (HCl; 100 mg/ml; Pfizer Canada Inc., London, Ontario, Canada) and xylazine HCl (100 mg/ml; Novopharm, Toronto, Ontario, Canada) into a hind leg at a dose of 20 mg ketamine HCl/kg. Blood samples were collected from the subclavian or brachiocephalic veins into 10-ml VacutainerH tubes (red stopper, no additive, silicone coating; BD Biosciences, Franklin Lakes, New Jersey, USA) after which a weight-dependant dose (0.2 mg/kg) of yohimbine (2.0 mg/ml; Novopharm) was administered IM to reverse the sedation effects of xylazine. Blood samples were refrigerated at 4 C for up to 48 hr and centrifuged at 1,000 3 G for 12 min at 4 C. Serum (1–2 ml) was collected and frozen at 220 C until testing. Before testing, serum was thawed and heat inactivated at 56 C for 30 min. Serologic analyses
To facilitate the testing of the large number of serum samples generated in this and a companion study (.1,800), a competitive enzyme-linked immunosorbent assay (CELISA) as described by Elmgren and Wandeler (1996) was used to detect the presence of RVA in sera. The optimal positive cut-off value for the C-ELISA was determined by receiver operating characteristics (ROC) analysis (Zweig and Campbell, 1993) of 1,289 paired samples, by using neutralizing titer as the reference standard. Neutralizing titers were determined as described in Knowles et al. (2009). Serum samples were classified as antibody-positive if the neutralizing titer was $0.5 IU/ml. For the set of sera obtained from raccoons trapped in the London area in 2003, ROC analysis using the MEDCALC program (Medcalc Software, Mariakerke, Belgium) established that a C-ELISA value of $28% inhibition (I) generated the lowest number of
false positives and false negatives compared with the neutralizing titer positive cut-off. At this cut-off value, the C-ELISA had a sensitivity of 95% and a specificity of 93% compared with the virus neutralization assay. Based on these data, sera were considered positive for the presence of RVA if an inhibition value was $28%. Challenge virus preparation
Salivary glands were removed from wild raccoons diagnosed as infected with the raccoon rabies virus variant by the Canadian Food Inspection Agency (CFIA, Ottawa, Ontario, Canada) Centre of Expertise for Rabies and frozen at 280 C until use. A 20% suspension of salivary glands was prepared in 0.01 M phosphate-buffered saline, pH 7.4, containing 10% fetal bovine serum, 13 antibiotic-antimycotic (100 units/ml penicillin, 0.1 mg/ml streptomycin, 0.25 mg/ml amphotericin B; Sigma, Oakville, Ontario, Canada), 0.1 mg/ml gentamycin (Sigma), and 0.1 mg/ml neomycin (Sigma). Each individual salivary gland suspension was titrated on MNA cells, and those suspensions with sufficient titer were pooled and dispensed into 10-ml aliquots. The pooled suspension was retitrated in triplicate on MNA cells and was found to have a geometric mean titer of 106.7 TCID50/ml. On the day of challenge, vials were thawed, combined, and dispensed into syringes. Challenge of raccoons with rabies virus
A subsample of 50 raccoons from the longitudinal immune response study was selected for rabies challenge based on serology results at weeks 3 and 6 (19 VRG-OS, 18 IMRAB 3, and 13 controls). Animals for challenge were selected to give an equal agesex representation among treatment groups and included individuals whose serum samples were considered positive for the presence of RVA (C-ELISA $28% I) at weeks 3 and 6, and raccoons whose sera were negative (CELISA ,28% I) at the same weeks. Raccoons were housed in a level-3 biocontainment facility (CFIA) in individual stainless steel cages with nest boxes and environmental enrichment. Food and water were provided ad libitum. Animals in both studies were maintained in accordance with the Canadian Council on Animal Care (CCAC) guidelines (CCAC, 2003), and all protocols were approved by institutional Animal Care Committees of both OMNR and CFIA. At 442 days posttreatment, raccoons selected for challenge were anesthetized by IM injection of 1–3 ml of a 10:1 mixture of
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ketamine HCl (100 mg/ml; CDMV, SainteHyacinthe, Que´bec, Canada) and acepromazine maleate (25 mg/ml; CDMV) into a hind leg. A 3–5-ml blood sample was collected from the jugular vein, and the raccoon was injected with 1 ml of the challenge virus preparation into the masseter muscle. Animals were observed daily by two independent observers for clinical signs of rabies (e.g., a combination of lack of food consumption, hypersalivation, vocalization, agitation, tremors, convulsions, paralysis, and unprovoked aggression, among others). A score or range of scores was attached to each clinical sign, and raccoons were humanely euthanized by an intercardiac injection of 1–2 ml of sodium pentobarbital (540 mg/ml, BimedaH, MTC Animal Health Inc., Cambridge, Ontario, Canada) when a score of 15 was reached. Surviving animals were humanely euthanized 60 days postchallenge. A blood sample was collected at death or after euthanasia. Brains of all animals were tested for the presence of rabies virus antigen by the fluorescent antibody test (FAT) as described by Dean et al. (1996). Serum samples collected before challenge and at death were assayed for the presence of RVA as described above.
plastic blister pack. One raccoon consumed 90% of the bait matrix, and 0.1 ml of vaccine was recovered from the chewed blister pack. One other raccoon consumed only 10% of the bait matrix, and 1.1 ml of vaccine was collected from the plastic sheet below the wire cage; this animal was excluded from the VRG-OS group. Ten raccoons consumed their baits within 30 min; nine within 1 hr; eight within 2.5 hr; and in three cases, vaccine baits had to be left overnight, but they were completely consumed by the next morning. Several raccoons in each treatment group (three VRG-OS, four IMRAB 3, and five controls) died before the end of the longitudinal immune response study due to canine distemper virus (CDV) or parvovirus enteritis (confirmed by postmortem diagnosis at the Canadian Cooperative Wildlife Health Centre, Guelph, Ontario, Canada). Data from these animals are not included in this report.
Statistical analyses
Antibody development in response to VRG-OS and IMRAB 3 rabies vaccines
A log-linear analysis was used to test for main and interaction effects among the categoric variables: treatment (VRG-0S or IMRAB 3), age (adult-juvenile), sex (malefemale), and overall serologic response (positive or negative) simultaneously using STATISTICA# (StatSoft, Tulsa, Oklahoma, USA). Overall serologic response was considered positive if there were four or more consecutive sampling dates where C-ELISA was $28% I between weeks 1 and 16. Any significant two-way associations were further examined using chi-square tests of independence. A chi-square analysis also was used to test the association between overall serologic response and survival after rabies challenge. A Kruskal-Wallis test was used to test the null hypothesis that there was no difference in the number of days until death among the control and two vaccination groups after rabies virus infection. RESULTS Bait consumption by raccoons
Of the 31 raccoons presented VRG-OS, 29 consumed 100% of the matrix around the bait and left only an empty, chewed
All serum samples collected from raccoons before vaccine administration were negative for RVA. No raccoon in the control group seroconverted at any time during the longitudinal serology study (Fig. 1a). Several raccoons vaccinated parenterally with IMRAB 3 (Fig. 1b) or orally by consumption of VRG-OS (Fig. 1c) showed evidence of seroconversion between weeks 1 and 16 posttreatment. Regardless of rabies vaccine or method of administration, individual raccoons showed either an overall positive serologic response (C-ELISA $28% I for at least four consecutive weeks) or they did not respond. One raccoon in the IMRAB 3 group had a C-ELISA value equal to 28% I at week 2 and 30% I at week 5, but values at all other weeks fell below the 28% I cut-off; this animal was considered a nonresponder. The model that best fit the multiway frequency table was comprised of only one significant two-way association, treatment 3
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FIGURE 1. Longitudinal humoral immune response of raccoons in (a) the control group, (b) the group vaccinated parenterally with IMRAB 3, and (c) the group vaccinated orally via consumption of Ontario Slim baits containing a recombinant vaccinia virus-rabies glycoprotein oral rabies vaccine. Each line represents one raccoon. The hatched line at competitive enzyme-linked immunosorbent assay equal to 28% inhibition represents the cut-off value above which sera were considered positive for the presence of rabies virus antibodies.
serologic response (n553; x256.693; P50.877). Further examination of this result using a chi-square test of independence revealed more responders in the group that
received an IM injection of IMRAB 3 rabies vaccine than in the group fed VRG-OS (n553; x2516.154; P50.0001). Eighteen of 26 raccoons (69%) in the IMRAB 3 group showed a serologic response to vaccination compared with only four of 27 (15%) in the group that consumed VRG-OS. A measurable antibody response to IMRAB 3 was first detected in one raccoon at week 1, but in most animals (14/18) the first measurable response occurred at week 2 (Fig. 1b). In these individuals, the immune response was maintained through week 5, with several animals exhibiting a sustained response through week 16. Two raccoons showed initial evidence of seroconversion at week 3 and one raccoon at week 5, but the response remained strong in all three animals through week 16. Although the proportion of raccoons exhibiting an antibody response was significantly lower in the VRG-OS group, the kinetics of the response was similar (Fig. 1c). Three of four raccoons exhibited an initial serologic response at week 2; in the fourth animal, it occurred at week 4. Two of the four raccoons that responded to VRG-OS exhibited a sustained serologic response through week 16; in the others, it was maintained through weeks 9 and 13. The kinetics of the humoral immune response by adults and juveniles within each vaccination group is shown in Fig. 2. Both adult and juvenile raccoons in the IMRAB 3 group showed a strong serologic response between weeks 2 and 5 (60–80% antibody-positive). The percentage of antibody-positive adults dropped to 38% by week 7 and continued to decline through week 16, whereas that of juveniles remained high (70–80%) through week 10, dropping off to 50–60% only in the last 4 wk of the study. Juveniles made up the majority of positive responders in the VRG-OS group (3/4) and the response in this age group was maintained through weeks 13 (one raccoon) and 16 (two raccoons). The one adult raccoon that responded to VRG-OS maintained a strong serologic response through week 9.
BROWN ET AL.—VACCINATION AND PROTECTION AGAINST RABIES
FIGURE 2. Kinetics of the humoral immune response by adult (open symbols) and juvenile (closed symbols) raccoons after parenteral vaccination with IMRAB 3 (squares) or consumption of Ontario Slim baits containing a recombinant vaccinia virus-rabies glycoprotein oral rabies vaccine (triangles). Seropositive result 5 competitive enzymelinked immunosorbent assay $28% inhibition. Control animals did not test positive for seroconversion at any time during the longitudinal immune response study. Challenge of study animals with raccoon variant rabies virus
Eleven of 13 control animals (85%) succumbed to rabies challenge as confirmed by FAT (Table 1). Two raccoons (one adult and one juvenile) survived challenge with raccoon variant rabies virus despite the absence of detectable RVA throughout the longitudinal serology study. These animals also did not show evidence of an immune response to rabies virus infection at the end of the challenge experiment. In the VRG-OS group, 14/19 (74%) animals succumbed to rabies virus infection, whereas only 5/18 (28%) died in the IMRAB 3 group (Table 1). There was no significant difference among the three groups in the number of days to death or humane euthanasia after rabies virus infection (n530; H52.09; P50.352). The median number of days to death or exhibition of sufficient clinical signs to warrant humane euthanasia was 18 (range 14–35). All 15 vaccinates that showed a serologic response in the longitudinal study survived when challenged with the rabies virus (Table 2). Only two of seven nonre-
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sponders survived in the IMRAB 3 group and only one of 15 survived in the VRGOS group, for a combined survival rate of 14%, similar to that of the control group (15%). All three surviving raccoons were adults. Combining results from all treatment groups, there was a significant association between serologic response and response to challenge with raccoon variant rabies virus (n550; x2532.14; P50.000), with higher survival associated with an overall positive serologic response. DISCUSSION
This study was conducted to examine under controlled conditions the longitudinal humoral immune response of raccoons to two rabies vaccines used extensively during rabies control field operations in Ontario and to assess the protective effect conferred to those animals after vaccination. The large serology dataset generated in this study also presented a unique opportunity to examine the relationship between antibody response and protection from rabies virus infection. In this study, most raccoons that responded to vaccination with IMRAB 3 or VRG in OS baits demonstrated an initial measureable antibody response at week 2 and maintained the response through at least week 5. In several raccoons, RVA was detectable through the end of the 16-wk study. These results are consistent with those observed in the first field release of VRG in North America, where raccoons sequentially trapped after bait distribution exhibited the highest geometric mean neutralizing titers between weeks 4 and 6 (Hanlon et al., 1998). Similarly, Sattler et al. (2009) observed that the overall proportion of antibody-positive raccoons was highest at 5 weeks postbaiting after a spring ORV campaign in Ohio. Because 80–100% of baits distributed during ORV campaigns in Ontario, Ohio, and New Jersey were consumed within 1–2 wk (Roscoe et al., 1998; Rosatte and Lawson, 2001; Black-
8 15 7 10 2 2 2 3 16 5 3 3 5
NS 41 77 20 5 19 NS 41 77 15 71 20 22
D
D D D D D
D D D D D
(21) (18) (27) (16) (16) S (16) (18) (35) (14) (21) S (19)
14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32
Rac
8 0 0 0 0 0 0 0 0 10 0 13c 0 0 11 0 0 0 0
19 2 5 18 8 9 2 0 16 24 6 31 16 3 20 1 0 3 9
#W $28% I C0 (% I)
VRG-OS
87 NS 58 27 9 28 44 45 72 38 13 78 35 18 89 54 9 40 NS
CT (% I)
S D (17) D (19) D (18) S D (30) D (15) D (16) D (26) S D (16) S D (15) D (16) S D (16) D (18) D (14) D (18)
Challenge result
33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50
Rac
5 6 0 1 13 9 0 0 0 13 13 10 4 0 13 10 0 8
15 19 21 19 13 18 0 6 15 29 54 14 10 20 74 20 13 18
#W $28% I C0 (% I)
IMRAB 3
48 45 63 84 95 34 17 74 57 70 59 12 27 85 94 90 51 59
CT (% I)
D
D
D
D D
S S (17) (21) S S S (16) S S S S S (21) S S (20) S
Challenge result
Day animal was humanely euthanized or found dead is shown in brackets.
C-ELISA values were from 29% I to 50% I between weeks 2 and 16 except at week 7 where the value was 27% I, just below the 28% I positive cut-off.
c
Abbreviations: Rac 5 raccoon identification; #W $28% I 5 number of consecutive weeks with competitive enzyme-linked immunosorbent assay (C-ELISA) $28% inhibition; C0 5 C-ELISA value at challenge; CT 5 C-ELISA value at death or challenge termination; NS 5 no sample; D 5 died; S 5 survived.
0 0 0 0 0 0 0 0 0 0 0 0 0
CT (% I)
Challenge resultb
b
a
1 2 3 4 5 6 7 8 9 10 11 12 13
C0 (% I)
Control
Treatment group
Response of individual raccoons to rabies vaccination and virulent raccoon rabies challenge.
Raca #W $28% I
TABLE 1.
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TABLE 2. Survival of control and vaccinated raccoons challenged with raccoon variant rabies virus 442 days posttreatment. Serologic response was considered positive if C-ELISA values were $28% inhibition for four or more consecutive weeks between weeks 1 and 16. Serologic response Positive
Negative
Group
Total no. raccoons
n
Survived challenge (%)
n
Control IMRAB 3 VRG-OS All groups
13 18 19 50
0 11 4 15
— 11 (100) 4 (100) 15 (100)
13 7 15 35
well et al., 2004), the current field sampling regime targeted at 5–6 wk postORV, seems adequate to detect seroconversion in response to VRG-OS. Our data suggest the sampling period could even be extended by several weeks without loss of sensitivity. Thus, the low seroconversion rates observed in Ontario after the distribution of VRG-OS between 2000 and 2006 (Rosatte et al., 2008) do not seem to be the result of inappropriate timing of the post-ORV assessments. In TVR operations conducted in the St. Lawrence and Niagara regions of southern Ontario in 2003, seroconversion rates for raccoons treated with IMRAB 3 ranged from 88% to 100% in adults tested 3–4 wk postvaccination (Sobey et al., 2010). Based on those results, the seroconversion rate of 69% for IMRAB 3 animals in our study was lower than expected. Similarly, the 15% seroconversion rate for raccoons in the VRG-OS group was much lower than rates obtained in early laboratory and field trials with VRG (Rupprecht et al., 1986, 1988; Hanlon et al., 1998; Roscoe et al., 1998). Several experimental animals died during the present study due to CDV or parvovirus enteritis. Because infections with morbilliviruses such as CDV are known to cause immune suppression in some species (Schneider-Schaulies and Schneider-Schaulies, 2008), the occurrence of CDV in the raccoon colony may have contributed to the reduced seroconversion rates observed in the IMRAB 3 and VRG-OS treatment groups. However, the seroconversion rate of 15% in the
Survived challenge (%)
2 2 1 5
(15) (29) (7) (14)
VRG-OS group was consistent with rates obtained in eastern Ontario (10–23%) after ORV campaigns in 2003 that used the same vaccine-bait combination (Rosatte et al., 2008). The dose of VRG used in OS baits was slightly lower than the 108.0 plaque-forming units reported in early laboratory and field studies of VRG efficacy (Rupprecht et al., 1986, 1988; Hanlon et al., 1998; Roscoe et al., 1998) and may have contributed to the lower-than-expected seroconversion rates in the field (Rosatte et al., 2008) and in this study. However, the vaccine dose used in OS baits was well above the reported minimum protective titer of 107.0 TCID50 /ml required for licensure of RABORAL V-RG for use in raccoons in the USA (Rosatte et al., 2008), and it was consistent with reported field doses of 107.7 TCID50 for raccoons (as referenced in Grosenbaugh et al., 2007) and for gray foxes (Urocyon cinereoargenteus) and coyotes (Canis latrans; Sidwa et al., 2005). It is difficult to directly compare seroconversion rates among vaccine studies due to differences in bait size, shape, and composition, and differences in serologic analyses and cut-off values used to determine antibody positivity. Different study environments (laboratory and field), experimental designs, and vaccine presentation methods (bait vs. oral) are also complicating factors. Laboratory studies of VRG immunogenicity reporting seroconversion rates approaching 100% involved either direct instillation of the vaccine into the oral cavity (Blanton et al., 2007) or
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ingestion of vaccine via polyurethane sponge baits (Rupprecht et al., 1986, 1988). Seroconversion rates of 60–70% were reported in field studies when VRG was dispensed in a wax ampule inserted into a fishmeal polymer (FP) bait (Hanlon et al., 1998; Roscoe et al., 1998). Recent field assessments of the immunogenicity of RABORAL V-RG in FP and CS baits (where the vaccine was contained in a polyethylene sachet) are more consistent with the seroconversion rates in the present study using the OS bait. Using a neutralizing positive titer threshold of 0.05 IU/ml, the average seroconversion rate for raccoons in regions of the USA baited with RABORAL V-RG between 1997 and 2007 was 30% (range approximately 20–42%; Slate et al., 2009). PostORV antibody prevalence in northeastern Ohio, where FP baits were distributed at 75–150 baits/km2, ranged from 4% to 18% using neutralization titer thresholds of $0.25 IU/ml (Ramey et al., 2008) and .1:12 (Sattler et al., 2009). Rosatte et al. (2008) observed similar seroconversion rates of 14–27% in Ontario field trials where RABORAL V-RG FP and CS baits were distributed at 70–150 baits/km2 (using a C-ELISA cut-off that corresponded with a neutralizing titer positive threshold of 0.5 IU/ml). Seroconversion rates reported from some field studies may have been influenced by bait density. As bait density increases, so does the opportunity for raccoons to consume more than one bait. In the field study by Hanlon et al. (1998), baits were distributed at a density of 1,000 baits/km2, significantly higher than the 75–150 baits/km2 used during raccoon rabies ORV in Ontario (Rosatte et al., 2008). At such a high bait density, an enhancing effect of multiple vaccine doses on immune response cannot be discounted unless bait consumption is assessed or controlled as in the present study. Neither vaccine conferred complete protection against rabies virus infection; however, there were significantly more survivors in the IMRAB 3 group (72%)
than in either the VRG-OS (26%) or the control (15%) groups. In both vaccinated groups, a sustained antibody response postvaccination was highly predictive of protection from lethal rabies virus challenge, and a lack of detectable RVA was correlated with a high susceptibility to virus infection. Two raccoons in the IMRAB 3 group and one in the VRG-OS group also survived challenge despite lack of detectable RVA throughout the 16-wk study period. Although we cannot rule out the contribution of other factors, such as cell-mediated immune responses (Nathanson and Gonzalez-Scarano, 1991; Lambot et al., 2001), to survival of some animals in the absence of a strong humoral immune response, the fact that a similar proportion of control animals also survived suggests that survival of these vaccinates was not due to immunization. The challenge virus dose was chosen to result in a minimum of 80% mortality in the nonvaccinated group as per Title 9, Part 113 of the Code of Federal Regulations (CFR, 2010); therefore, the presence of survivors in the control group was not unexpected and also has been reported in other studies (Black and Lawson, 1970; Rupprecht et al., 1988, 1993; Se´tien et al., 1998; Blanton et al., 2007; Henderson et al., 2009; Mu¨ller et al., 2009). Despite clear differences between the vaccines in composition and route of administration, the effect of the humoral immune response, once initiated, was similar with respect to protection from disease. This also has been observed in foxes orally vaccinated with the attenuated live rabies vaccine SADB19 (Mu¨ller et al., 2001) and in raccoons (Blanton et al., 2007) and skunks (Grosenbaugh et al., 2007) administered VRG by direct oral instillation. In contrast, in studies of raccoons (Rupprecht et al., 1986, 1988) and skunks (Tolson et al., 1987) orally vaccinated with VRG in baits, some animals did not survive challenge despite demonstrating good antibody responses; and, in other studies, protection in the
BROWN ET AL.—VACCINATION AND PROTECTION AGAINST RABIES
absence of demonstrable or very low antibody levels has been reported in some foxes (Blancou et al., 1986; Brochier et al., 1990; Pastoret et al., 1992), skunks (Grosenbaugh et al., 2007), and dogs (Canis familiaris; Cliquet et al., 2008). It is possible that we found a strong correlation between antibody detection and protection from disease because an animal was considered a positive responder only if RVA was detectable by C-ELISA over a period of consecutive weeks, rather than at one point in time, or over collection periods separated by weeks or months. The neutralizing titer cut-off of 0.5 IU/ml used in the ROC analysis to establish the C-ELISA positive cut-off also may have contributed to the strong correlation between antibody response and protection. It is expected that the nature of a vaccine (live attenuated, killed, live recombinant); route of administration (oral instillation, IM, and bait); challenge virus used (dose, site of inoculation, and variant); time interval between vaccination and challenge; species; and age, health, and nutritional status will all influence the outcome of vaccination and challenge. Thus, when new products (vaccines or vaccine-bait combinations), or new applications of old products, are being evaluated for licensing, they should be administered via the planned route to the intended target species and evaluation of efficacy should be based on serology combined with challenge data (WHO, 2004). We used sample sizes of adequate statistical power, and the immunogenicity and efficacy of IMRAB 3 administered IM, and VRG presented in OS baits, were assessed in raccoons through the collection of extensive serologic data followed by exposure to field isolates of the rabies virus most likely to be encountered by raccoons in the wild. Although many of the raccoons had sustained antibody levels, some for the entire 16-wk study, few had detectable antibody at challenge. Survival after rabies challenge suggests both IMRAB 3 and
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VRG-OS rabies vaccines stimulated lasting immunologic memory in raccoons. These data reinforce the importance of selecting wild raccoons for vaccine trials from geographic areas in which vaccination campaigns have not been carried out for several years to ensure a naive study population. Previous laboratory studies have demonstrated that VRG could provide protective immunity in raccoons up to 6 mo postimmunization (Rupprecht et al., 1986, 1988). We have extended these studies by showing that immunity induced by VRG-OS and IMRAB 3 rabies vaccines lasts at least 14 mo. In Ontario, TVR campaigns to control raccoon rabies were carried out annually (Rosatte et al., 2001, 2008, 2009), and results of our study suggest this frequency is adequate because protective immunity should last at least until the next year. Integral to the success of any wildlife rabies control program is the ability to immunize a large enough proportion of the target population to prevent epizootic spread of the disease, that is, to reach the herd immunity threshold. For domestic dog populations, it is estimated that an immunization rate of 70% is required to control the spread of rabies (WHO, 1992). It is generally assumed that similar levels of vaccine coverage may be required for free-ranging raccoons, as well as other wildlife, although results of recent modeling studies for red fox ORV in Europe suggest lower vaccination thresholds may provide disease control (Thulke and Eisinger, 2008). Although the serologic response of raccoons to VRG-OS in this study was slightly higher than that observed in the field after aerial distribution of similar vaccine baits between 2000 and 2002 (Rosatte et al., 2008), the response rate of 15% is still well below that assumed sufficient for disease control. Results of both studies suggest that VRG incorporated into OS baits at a dose of 107.7 TCID50, and distributed during rabies control operations in eastern Ontario during 2000–2006, may not have
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resulted in sufficient vaccination coverage to contain the outbreak of raccoon rabies. Rather, intensive population reduction and TVR with IMRAB and IMRAB 3 were probably instrumental in eliminating the disease from the province (Rosatte et al., 2001, 2008, 2009). The IMRAB and IMRAB 3 rabies vaccines are known to induce RVA and protective immunity in multiple domestic animal species and are registered for such use in Canada and the USA (CFIA, 2010; USDA, 2009). Their ability to induce RVA after parenteral administration in raccoons has been demonstrated in this study and previously (Rosatte et al., 1990; Sobey et al., 2010), and we have shown here that the antibody response to IMRAB 3 in raccoons is protective. During several years of intensive TVR campaigns after confirmation of raccoon rabies in eastern Ontario, an estimated 72–79% of the raccoon population received parenteral IMRAB 3 (Rosatte et al., 2009). Similar vaccination coverage (54–78%) was attained with IMRAB 3 in the Niagara region of southern Ontario, and since 1994 raccoon rabies has not been detected in that area (OMNR, unpubl. data) despite its presence in neighboring New York state (Wadsworth Center, 2010). These data from field and laboratory studies suggest that appropriate levels of vaccine coverage can be attained with parenteral administration of IMRAB 3 and support its use as one strategy for raccoon rabies control. Unfortunately, the costs of such programs preclude their use over large areas (Rosatte et al., 1990, 2001, 2009). The ultimate challenge of raccoon rabies control is the development of an effective rabies vaccine combined with a proven and cost-effective delivery method that results in sufficient herd immunity to prevent spread of the disease. ACKNOWLEDGMENTS
We thank Jan Armstrong, Andrea Clark, Frances Muldoon, Maria Harding, and Ruth Beylea of the Centre of Expertise for Rabies,
Canadian Food Inspection Agency; Bob Davis, Leith Kealey, Richard Legault, Sandra McIntosh, and Bill Trip, Animal Care Section, Canadian Food Inspection Agency; and Scott Taylor, Vaughn Jaimeson, Mark Gibson, Kimberly Laframboise, and Beverly Stevenson, Rabies Research and Development Unit, Ontario Ministry of Natural Resources, for the field and technical assistance essential to this research. This work was funded through a Collaborative Research Agreement between the Ontario Ministry of Natural Resources and the Canadian Food Inspection Agency. LITERATURE CITED BLACK, J. G., AND K. F. LAWSON. 1970. Sylvatic rabies studies in the silver fox (Vulpes vulpes). Susceptibility and immune response. Canadian Journal of Comparative Medicine 34: 309–311. BLACKWELL, B. F., T. W. SEAMANS, R. J. WHITE, Z. J. PATTON, R. M. BUSH, AND J. D. CEPEK. 2004. Exposure time of oral rabies vaccine baits relative to baiting density and raccoon population density. Journal of Wildlife Diseases 40: 222–229. BLANCOU, J., M. P. KIENY, R. LATHE, J. P. LECOCQ, P. P. PASTORET, J. P. SOULEBOT, AND P. DESMETTRE. 1986. Oral vaccination of the fox against rabies using a live recombinant vaccinia virus. Nature 322: 373–375. BLANTON, J. D., J. SELF, M. NIEZGODA, M. L. FABER, B. DIETZSCHOLD, AND C. RUPPRECHT. 2007. Oral vaccination of raccoons (Procyon lotor) with genetically modified rabies virus vaccines. Vaccine 16: 7296–7300. BROCHIER, B. M., B. LANGUET, M. ARTOIS, S. ZANKER, C. GUITTRE, J. BLANCOU, G. CHAPPUIS, P. DESMETTRE, AND P. P. PASTORET. 1990. Efficacy of a baiting system for vaccinating foxes against rabies with vaccinia-rabies recombinant virus. Veterinary Record 18: 165–167. CANADIAN COUNCIL ON ANIMAL CARE (CCAC). 2003. Guidelines on the care and use of wildlife. Canadian Council on Animal Care, Ottawa, Ontario, Canada, 66 pp. CENTERS FOR DISEASE CONTROL AND PREVENTION. 2000. Update: Raccoon rabies epizootic–United States and Canada, 1999. Morbidity and Mortality Weekly Report 49: 31–35. CANADIAN FOOD INSPECTION AGENCY (CFIA). 2010. Veterinary Biological Products Licensed in Canada, http://active.inspection.gc.ca/eng/anima/ vetbio/vetbio_dbe.asp. Accessed June 2010. CLIQUET, F., J. BARRAT, A. L. GUIOT, N. CAE¨L, S. BOUTRAND, J. MAKI, AND C. L. SCHUMACHER. 2008. Efficacy and bait acceptance of vaccine vectored rabies glycoprotein vaccine in captive foxes (Vulpes vulpes), raccoon dogs (Nyctereutes procyonoides) and dogs (Canis familaris). Vaccine 26: 4627–4638.
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