Rabies challenge of captive striped skunks (Mephitis mephitis) following oral administration of a live vaccinia-vectored rabies vaccine

Twenty-four adult striped skunks (Mephitis mephitis) were administered the raccoon product formulation of Rabies Vaccine, Live Vaccinia-Vectored (Raboral V-RG, Merial Limited, Athens, Georgia, USA), either by oral instillation or in vaccine-filled coated sachets either as single or multiple doses. A control group remained unvaccinated. Twenty-three of the skunks were challenged 116 days postvaccination with rabies virus (skunk isolate). Six of six naive skunks succumbed to challenge. Four of six skunks that received the vaccine by oral instillation survived challenge. The skunks that did not survive failed to seroconvert following vaccination. None of the skunks that accepted multiple doses of the vaccine offered in coated sachets survived challenge, nor were rabies virus-neutralizing antibodies (VNAs) detected in the sera. Likewise, none of the five skunks ingesting a single sachet developed VNA against rabies. However, in this group one skunk did survive rabies challenge. This preliminary study showed that the vaccinia-vectored oral rabies vaccine Raboral V-RG, as formulated for use in raccoons, is capable of protecting a percentage of skunks against rabies. However, although the fishmeal-coated sachets were readily consumed, subsequent challenge of these animals revealed poor vaccine delivery efficiency.     

Comparing ONRAB® AND RABORAL V-RG® oral rabies vaccine field performance in raccoons and striped skunks, New Brunswick, Canada, and Maine, USA

Control of rabies in mesocarnivore reservoirs through oral rabies vaccination (ORV) requires an effective vaccine bait. Oral rabies vaccine performance in the field may be affected by a variety of factors, including vaccine bait density and distribution pattern, habitat, target species population density, and the availability of competing foods. A field study in which these covariates were restricted as much as possible was conducted along the international border of the state of Maine (ME), USA, and the province of New Brunswick (NB), Canada, to compare the performance of two oral rabies vaccines in raccoons (Procyon lotor) and striped skunks (Mephitis mephitis). RABORAL V-RG(?) (vaccinia-rabies glycoprotein recombinant oral vaccine in fishmeal-coated sachet) or ONRAB(?) (adenovirus-rabies glycoprotein recombinant oral vaccine in Ultralite bait matrix) were distributed in ME and NB, respectively, by fixed-wing aircraft at a density of 75 baits/km(2) along parallel flight lines spaced 1.0 km apart. Sera were collected from live-trapped raccoons and skunks 5-7 wk post-ORV and assayed to determine antibody prevalence in each area. Duplicate serum samples were provided blind to two different laboratories for analyses by rabies virus serum neutralization assays (at both laboratories) and a competitive enzyme-linked immunosorbent assay (at one laboratory). There was no significant difference in the proportion of antibody-positive animals determined by the three serologic methods, nor was there a significant difference between ONRAB and RABORAL V-RG in the proportion of antibody-positive striped skunks observed post-ORV. In contrast, the proportion of antibody-positive raccoons was significantly higher in the ONRAB- versus the RABORAL V-RG-baited areas (74% vs. 30%; χ(2)=89.977, df=5, P<0.0001). These data support that ONRAB may serve as an effective tool for raccoon rabies control.     

Consideration of inactivated rabies vaccines as oral immunogens of wild carnivores.

An experimental beta-propiolactone (BPL)-inactivated rabies virus vaccine was evaluated for the oral immunization of captive raccoons (Procyon lotor) and red foxes (Vulpes vulpes). None of 10 red foxes administered a single 1.0 ml dose of BPL-inactivated rabies virus vaccine (PM strain; 100 or 500 micrograms protein) per os developed detectable anti-rabies virus-neutralizing antibodies (VNA) at any time over 8 wk of observation. Foxes were excluded from further study. In two different groups of five to six raccoons, each administered a single 1.0 ml dose of BPL-inactivated rabies virus vaccine (ERA strain) per os, at concentrations of 100 or 400 micrograms protein, only a single animal in each group demonstrated evidence of seroconversion within 4 wk. In contrast, instillation of a single dose (500 micrograms protein) of BPL-inactivated rabies virus vaccine (ERA strain), directly into the small intestine via fiberoptic endoscope, or ERA vaccine (800 micrograms protein) instillation to the buccal cavity by needle-less syringe, resulted in the production of rabies-specific VNA and protection against lethal rabies infection in three of six, and in four of six raccoons, respectively; all seven control raccoons succumbed to street virus challenge. These preliminary challenge studies, while somewhat encouraging, demonstrate that considerable quantities of purified viral antigen are required for even minimal oral efficacy against lethal rabies infection. At the present time, therefore, potent, self-replicating, attenuated, or recombinant viruses offer the most versatile, economic, efficacious, and safe solutions to terrestrial rabies control of free-ranging carnivores.     

Oral vaccination of raccoons (Procyon lotor) with an attenuated (SAD-B19) rabies virus vaccine

Unlike previous reports to the contrary, raccoons (Procyon lotor) were successfully vaccinated against rabies with a liquid SAD-B19 attenuated virus vaccine administered per os and given in vaccine-laden baits. There was neither evidence of vaccine-induced rabies in raccoons nor in a limited safety trial with opossums (Didelphis virginiana) given SAD-B19. Protection from lethal street rabies virus infection was not absolute: only three of nine raccoons given 1 x 10(6.0) TCID/ml were protected versus five of 10 raccoons given 1 x 10(7.0) TCID/ml of SAD-B19 and challenged 4 mo after consumption of vaccine-laden baits. Six of eight raccoons consuming 1 x 10(8.8) TCID/ml of SAD-B19 vaccine in baits survived street rabies virus challenge 2 mo postvaccination. Raccoon survivorship was not wholly dependent upon rabies virus-neutralizing antibody titer on the day of challenge. Vaccinated raccoons demonstrated a prominent anamnestic response within 1 wk following challenge. Surviving raccoons were observed for a minimum of 3 mo following street rabies virus challenge with neither clinical nor pathologic evidence of rabies. The SAD-B19 rabies vaccine administered within baits in captivity appears less effective for raccoons than for its demonstrated efficacy in the immunization of free-ranging foxes (Vulpes vulpes) in Europe.     

Intramuscular vaccination of skunks and raccoons against rabies

Live-captured striped skunks (Mephitis mephitis) and raccoons (Procyon lotor) were immunized with inactivated rabies vaccine by intramuscular injection and released at the point of capture during a rabies control program in Metropolitan Toronto (Ontario, Canada). Serum samples collected prior to and following vaccination revealed that 100% of the skunks and 98% of the raccoons seroconverted. Rabies antibody was still detectable 314 to 757 days postvaccination. Five of six skunks vaccinated in the laboratory survived challenge with rabies virus 90 days postvaccination. To our knowledge, this is the first documentation of the successful seroconversion of skunks and raccoons vaccinated against rabies in the field.     

Safety studies of the oral rabies vaccine SAD B19 in striped skunk (Mephitis mephitis)

Safety of the modified live rabies virus vaccine, SAD B19, was studied in striped skunks (Mephitis mephitis). Seven skunks received 10(7.9) foci formatting units by direct oral administration. In four cages, a vaccinated animal was placed with a control animal, the other three vaccinated skunks were housed individually. Saliva and nasal swabs were collected 1, 2, 4, 24, 48, and 72 hr post-vaccination. From all vaccinated and control animals (n = 11) blood samples were collected 0, 28, 56, 84, and 296 days post-vaccination. Three of seven vaccinated skunks seroconverted. None of the control animals had detectable levels of rabies virus neutralizing antibodies. Also no vaccine virus was isolated from the nasal and saliva swabs collected from any animal. Thus, SAD B19 was innocuous for skunks in our study after direct oral administration at field concentration.     

Oral rabies vaccine (ORV) bait uptake by captive striped skunks

Aerial delivery of oral rabies vaccine (ORV) baits has proven effective in large-scale efforts to immunize wildlife against rabies, and in North America this strategy currently is being used to immunize foxes (Urocyon cinereoargenteus and Vulpes vulpes), raccoons (Procyon lotor), and coyotes (Canis latrans). Skunks are also a major reservoir and vector of rabies, but at present oral vaccines for use in skunks are not licensed. Furthermore, given differences in morphology (smaller jaws) and behavior (food handling and consumption), it is unknown if baits currently used in ORV campaigns would be effective for skunks. Because oral vaccine delivery is contingent upon puncture of the vaccine container (VC), baits need to be sufficiently attractive to elicit selection and consumption. Manipulation of the bait to facilitate vaccine ingestion by the target species is a critical element for an effective ORV bait. The objectives of this study were to assess manipulation and consumption of current ORV baits by striped skunks (Mephitis mephitis). We conducted four independent trials with penned animals and various baits to assess bait selection frequency, VC puncture frequency, and consumption. Video recorded trials were used to assess attractiveness of baits and consumption behavior of skunks. Bait characteristics, such as texture, size, and flavor influenced selection and consumption. Fish and chicken flavors were preferred and vaccine containers within selected baits were likely to be punctured. Vaccine ingestion seemed more likely if VCs were directly coated with the bait matrix. To make baits attractive to skunks and to ensure puncture of the VC, modifications to current baits should consider a smaller size, a meat-flavored matrix, a slightly pressurized VC, and a direct coating of matrix on the VC.     

Vaccination of small Asian mongoose (Herpestes javanicus) against rabies

Oral vaccination of free-ranging wildlife is a promising technique in rabies control. The small Asian mongoose (Herpestes javanicus) is an important reservoir of rabies on several Caribbean islands, but no vaccines have been evaluated for this species. Captive mongooses were used to test the safety and efficacy of the commercially licensed vaccinia-rabies glycoprotein (V-RG) recombinant vaccine and a newly developed genetically engineered oral rabies virus vaccine (SPBNGA-S). In one study using V-RG, no vaccinated animals developed detectable rabies virus-neutralizing antibodies, and all but one died after experimental challenge with rabies virus. In contrast, all animals given SPBNGA-S demonstrated seroconversion within 7 to 14 days after vaccination and survived rabies virus challenge. On the basis of these preliminary results indicating the greater efficacy of SPBNGA-S vs. V-RG vaccine, additional investigations will be necessary to determine the optimal dose and duration of vaccination, as well as incorporation of the SPBNGA-S vaccine into edible bait.     

Immunogenicity and efficacy of two rabies vaccines in wild-caught, captive raccoons

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 IMRAB(?) 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).     

Experimental inoculation of raccoons (Procyon lotor) with rabies virus of skunk origin.

To determine raccoon (Procyon lotor) susceptibility and serum neutralizing antibody response to a skunk salivary gland rabies virus, raccoons were inoculated with a rabies virus isolated from a naturally-infected striped skunk (Mephitis mephitis). Raccoons were divided into four groups of three animals each. A dilution of the rabies virus suspension, 10(2.4), 10(3.4), or 10(4.8), mouse intracerebral lethal dose50 (MICLD50), was administered into the masseter muscles of each animal. Three negative control animals received only diluent. Saliva and sera were collected on post-inoculation days 35, 63 and 92 for virus isolation and determination of serum neutralizing antibody titer. All animals survived the 92 day observation period and none exhibited the behavioral changes classically associated with clinical rabies virus infections. Rabies virus was not detected in the saliva of any raccoon and two of the three animals receiving the highest inoculum developed serum neutralizing antibodies (SNA). On day 92, a challenge suspension of New York City/Georgia (NYC/GA) strain rabies virus in fox salivary glands (10(3.2) MICLD50) was administered to all 12 raccoons. All animals succumbed to rabies virus except the two animals that had earlier developed SNA. The results of this study provided evidence about the susceptibility of raccoons to a skunk rabies virus and demonstrated that exposed raccoons could survive for at least 92 days following exposure. Furthermore, animals developing SNA under such circumstances were capable of withstanding challenge with rabies virus that was fatal for seronegative raccoons.     

Vaccination of free-ranging Pennsylvania raccoons (Procyon lotor) with inactivated rabies vaccine

Thirty-nine free-ranging raccoons (Procyon lotor) in an endemic rabies area of Pennsylvania (USA) were vaccinated with a single intramuscular inoculation of commercial inactivated rabies virus vaccine, 17 June to 23 August 1987. Paired serum samples, pre- and postvaccination, were obtained from eight raccoons and were analyzed in vitro for rabies virus neutralizing antibody using a modified rapid fluorescent focus inhibition test. Seven of eight (88%) recaptured raccoons demonstrated seroconversion within 15 to 26 days of vaccination. At 1 yr postvaccination, three vaccinated raccoons were recaptured and challenged in captivity with street rabies virus, resulting in the death of two of three vaccinates and five of five unvaccinated control raccoons.     

High-density baiting with ONRAB® rabies vaccine baits to control Arctic-variant rabies in striped skunks in Ontario, Canada

The Arctic variant of rabies virus has been maintained in striped skunks in small foci in southwestern Ontario, Canada, despite the control of the disease in red foxes. To control the disease in skunks, high-density baiting with ONRAB(?) oral rabies vaccine baits was conducted by air and by hand distribution of baits in the vicinity of skunk cases. During 2009, antibody prevalences in skunks were higher in areas baited at a density of 300 baits/km(2) and flight-line spacing of 0.25 km than at 0.5-km spacing. Once an area containing Arctic-variant cases was treated with high densities of ONRAB baits, the disease did not reoccur in skunks in those areas. During 2009, only eight skunks were diagnosed with the Arctic variant of rabies virus in Ontario.     

Predictive spatial dynamics and strategic planning for raccoon rabies emergence in Ohio

Rabies is an important public health concern in North America because of recent epidemics of a rabies virus variant associated with raccoons. The costs associated with surveillance, diagnostic testing, and post-exposure treatment of humans exposed to rabies have fostered coordinated efforts to control rabies spread by distributing an oral rabies vaccine to wild raccoons. Authorities have tried to contain westward expansion of the epidemic front of raccoon-associated rabies via a vaccine corridor established in counties of eastern Ohio, western Pennsylvania, and West Virginia. Although sporadic cases of rabies have been identified in Ohio since oral rabies vaccine distribution in 1998, the first evidence of a significant breach in this vaccine corridor was not detected until 2004 in Lake County, Ohio. Herein, we forecast the spatial spread of rabies in Ohio from this breach using a stochastic spatial model that was first developed for exploratory data analysis in Connecticut and next used to successfully hind-cast wave-front dynamics of rabies spread across New York. The projections, based on expansion from the Lake County breach, are strongly affected by the spread of rabies by rare, but unpredictable long-distance translocation of rabid raccoons; rabies may traverse central Ohio at a rate 2.5-fold greater than previously analyzed wildlife epidemics. Using prior estimates of the impact of local heterogeneities on wave-front propagation and of the time lag between surveillance-based detection of an initial rabies case to full-blown epidemic, specific regions within the state are identified for vaccine delivery and expanded surveillance effort.     

Era vaccine-derived cases of rabies in wildlife and domestic animals in Ontario, Canada, 1989-2004

A vaccination program for the control of terrestrial rabies in the province of Ontario, Canada, began in 1989. During the period between 1989 and 2004, over 13 million baits containing the live, attenuated rabies virus ERA-BHK21 were distributed across the province, with the aim of immunizing foxes by the oral route. Animals recovered from bait distribution areas were assayed by fluorescent antibody test for rabies virus infection. Immunoreactivity with a panel of monoclonal antibodies that discriminate between ERA and rabies virus variants known to circulate in Ontario, and molecular genetic analyses were used to identify animals infected with ERA. Nine cases of ERA variant rabies were identified over the 16-yr period of study; these did not appear to be stratified by species, year of discovery, or location of capture. The ERA-positive animals were found across the province in eight counties, all of which had been baited in the year of case discovery. The nine ERA-positive cases included four red foxes (Vulpes vulpes), two raccoons (Procyon lotor), two striped skunks (Mephitis mephitis), and one bovine calf (Bos taurus). Molecular phylogenetic analyses of the partial N gene sequences generated from these isolates indicated that these nine cases were due to infection with the ERA variant. The glycoprotein sequences were predicted from G gene sequencing of all nine field isolates and two laboratory stock ERA viruses. This revealed some heterogeneity at residue 120 (either arginine or histidine) in both field and laboratory stocks as well as a few other mutations in field isolates. The significance of this heterogeneity remains unclear. Our data demonstrate that the ERA vaccine distributed in Ontario carried residual pathogenicity; however, there does not appear to be any evidence of ERA establishment in wildlife populations over the 16-yr period. These results are consistent with previous reports of the rare detection of ERA vaccine-induced rabies and with laboratory studies of ERA pathogenicity.     

Rabies detection in road-killed skunks (Mephitis mephitis)

Three diagnostic techniques (1) microscopic examination for Negri bodies, (2) mouse inoculation and (3) fluorescent antibody tagging were compared as to their ability to detect rabies virus in 14 experimentally infected striped skunks (Mephitis mephitis) exposed to normal summer weather conditions after death. The fluorescent antibody technique correctly identified rabies virus longer than either of the other methods. Rabies incidence in 61 road-killed skunks collected on the day following death in southeastern North Dakota was 26 per cent.     

Emergency response to raccoon rabies introduction into Ontario

During 15 July to 4 October, 1999, rabies control programs were implemented with the objective being to contain the first three confirmed cases of raccoon rabies in Canada. The strategy, called point infection control (PIC) involved the use of three tactics: population reduction (PR), trap-vaccinate-release (TVR) and oral rabies vaccination with baits (ORV), to control the spread of raccoon rabies. A total of 1,202 raccoons (Procyon lotor) and 337 skunks (Mephitis mephitis) were captured and euthanized using 24,719 trap-nights in the three PR zones around the location of the three rabies cases, near Brockville, Ontario. That represented an 83% to 91% reduction in the raccoon populations in an approximate 225 km2 area around the three rabies cases. Raccoon density in the PR zones declined from 5.1-7.1/km2 to 0.6-1.1/km2 following control. All tested specimens were negative for rabies by the fluorescent antibody test (FAT). In addition, 1,759 raccoons and 377 skunks were intramuscularly vaccinated against rabies and released using 27,956 trap-nights in an approximate 485 km2 TVR zone implemented outside of the PR zones. A total of 856 cats from both PR and TVR areas were also captured, vaccinated and released. Cost for the three PIC operations was $363,000.00 Cdn or about $500.00 Cdn/km2. To further contain the outbreak, about 81,300 baits containing Raboral V-RG oral rabies vaccine were aerially distributed on 8 and 27 September 1999, to create an 8 to 15 km wide buffer zone (1,200 km2 area) of vaccinated raccoons immediately beyond the PR and TVR zones. This was the first time that V-RG was used in Canada to orally vaccinate free ranging raccoons against rabies. Baiting costs were $241,000.00 Cdn or about $200.00 Cdn/km2 including post baiting assessment costs. As of 31 August, 2000, thirty-five additional cases (38 in total) of raccoon rabies have occurred in the control and vaccination zones. This number is far below the level of rabies prevalence in USA jurisdictions where raccoon rabies was epizootic. In the future, PIC methodologies will continue to be used in Ontario to contain isolated cases of raccoon rabies.     

Formulation and evaluation of baits for oral rabies vaccination of raccoons (Procyon lotor)

Captive raccoons were offered a variety of vaccine containers and bait components in a series of three-choice tests. Paraffin wax ampules were the most readily accepted vaccine container. Preferred bait components included corn and shellfish oils, deep fried corn meal batter, and egg, apple and buttermilk flavorings. These results, together with factors including ease of bait formulation, cost, and suitability for field use, were used to develop an experimental delivery system for an oral rabies vaccine. The developed system was composed of a polyurethane sleeve (1.5 x 5.5 cm) dipped in a commercial food batter mix together with corn meal, milk and egg. The sleeve was deep fried in corn oil and a 2.0 ml ampule containing a recombinant rabies vaccine was then inserted into the sleeve bait. These baits were presented to 10 captive raccoons. Nine of the 10 animals developed high levels of rabies virus neutralizing antibodies. Field tests are needed to determine if the delivery system developed also is effective for wild raccoons.     

Measurement of rabies-specific antibodies in carnivores by an enzyme-linked immunosorbent assay

We describe an indirect enzyme-linked immunosorbent assay (ELISA) that utilizes anticanine immunoglobulin for the measurement of rabies-specific antibody in the sera of the major domestic and wildlife reservoirs of rabies in North America. Sufficient cross-reactivity was found to exist between anticanine IgG and serum antibody from all carnivores tested, including dogs, cats, foxes (Vulpes vulpes), skunks (Mephitis sp.) and raccoons (Procyon lotor). With sera of most species, good correlation was observed between results obtained with the ELISA and with the fluorescence inhibition microtest (FIMT). Some wildlife specimens, particularly of skunk and raccoon origin, were cytotoxic in the FIMT, resulting in possible false-positive reactions. In view of this, and since the ELISA is rapid, economical and reproducible (coefficient of variation less than 13%), we consider it to be a favorable alternative to the fluorescence inhibition test for assay of wildlife sera.     

Evaluation of rabies virus neutralizing antibody titres induced by intramuscular inoculation of rabies DNA vaccine in mice and Bonnet monkeys (Macaca radiata)

A rabies DNA vaccine consisting of plasmid DNA expressing the rabies virus surface glycoprotein was injected (im) twice at two week interval to outbred swiss mice or Bonnet monkeys (Macaca radiata) and the levels of rabies virus neutralizing antibody (VNA) titres were examined over a one year period. In mice, the VNA titre was maintained above the minimum protective level (0.5 I.U./ml) up to 10 months after primary immunization, while in monkeys, the titre dropped below the protective level by 6 months. An anamnestic B cell response was seen in both mice and monkeys following the administration of a booster dose, 10 and 6 months after the primary immunization, respectively. These results indicate that im injection of rabies DNA vaccine induces VNA in nonhuman primates and mice unlike intradermal (id) immunization, which was shown to induce VNA only in mice but not in monkeys. This is the first report on the induction of VNA in nonhuman primates by im inoculation of rabies DNA vaccine.     

Ineffectiveness and comparative pathogenicity of attenuated rabies virus vaccines for the striped skunk (Mephitis mephitis)

Three attenuated rabies virus vaccines (SAD-B19, ERA/BHK-21, AZA 2) were compared for efficacy and safety in the striped skunk (Mephitis mephitis) by the oral and intranasal routes. The SAD-B19 and ERA/BHK-21 vaccines were given orally; all three vaccines were given intranasally. Oral administration of SAD-B19 and ERA/BHK-21 vaccines induced neither seroconversion nor significant protection against rabies challenge. One skunk which consumed a SAD-B19 vaccine-laden bait succumbed to vaccine-induced rabies. Intranasal instillation of the three vaccines resulted in the deaths of two of six (AZA 2), three of six (ERA/BHK-21) and six of six (SAD-B19) skunks.