A field trial in Belgium to control fox rabies by oral immunisation

Campaigns of fox vaccination against rabies were carried out in Belgium in September 1986 and June and September 1987. The SAD B19 attenuated strain of rabies virus was inserted into baits which were distributed over an area of 2100 km2 at a density of 11 baits/km2. As recommended by the World Health Organisation, the efficacy and the innocuity of the method were controlled in the field and in the laboratory. Samples of blood and brain and jaw were taken from foxes which were shot or found dead in the vaccination area, for the diagnosis of rabies, the titration of antirabies antibody and the detection of tetracycline marker. In rabid animals, the virus strain was characterised by immunofluorescence using monoclonal antibodies. In September 1987, the uptake of the baits had reached 72 per cent by 14 days after distribution. Several wild species competed with foxes in taking the baits. After the last campaign, tetracycline was found in 65 per cent of the healthy foxes collected and rabies virus neutralising antibodies were detected in 77 per cent of them. In 1987, the incidence of rabies decreased markedly in the vaccination area compared with the untreated areas. No vaccine virus was isolated either from rabid animals or from 228 small mammals trapped in the vaccination area.     

Challenges to controlling rabies in skunk populations using oral rabies vaccination: A review

Controlling rabies in skunk populations is an important public health concern in many parts of the United States due to the potential for skunk rabies outbreaks in urban centres and the possible role for skunks in raccoon rabies variant circulation. Oral rabies vaccination (ORV) programmes have supported wildlife rabies control efforts globally but using ORV to control rabies in skunk populations has proven more challenging than with other target species, like foxes, coyotes and raccoons. A review of published studies found that some ORV constructs are immunogenic in skunks and protect against virulent rabies virus challenges, especially when delivered by direct installation into the oral cavity. However, in field ORV programmes using currently available vaccine‐bait formats and distribution methods targeting other rabies reservoir species, skunks often fail to seroconvert. Field effectiveness of ORV in skunks appears to be limited by poor bait uptake or inadequate ingestion of vaccine rather than from poor vaccine efficacy. Observations of captive skunks revealed vaccine spillage when handling and biting into baits such that modification of bait formats might improve field effectiveness. In addition, a dose–response relationship between bait distribution density and post‐baiting seroconversion among skunks was observed across the limited number of field studies. Additional research is needed to identify opportunities to modify ORV baits and distribution strategies to improve the viability of ORV as a rabies control strategy in skunks.     

Pathogenesis studies with Australian bat lyssavirus in grey-headed flying foxes (Pteropus poliocephalus)

OBJECTIVE: To examine the susceptibility of the grey-headed flying fox (Pteropus poliocephalus) to Australian bat lyssavirus (ABL), and to provide preliminary observations on the pathogenesis of the disease in flying foxes. PROCEDURE: Ten flying foxes were inoculated intramuscularly with ABL, and four with a bat-associated rabies virus. Inoculated animals were observed daily, and clinical samples collected every 9 to 14 days. Animals with abnormal clinical signs were euthanased, and samples collected for histological, serological, virological and immunohistological examinations. At 3 months post inoculation (PI), all survivors were euthanased, and each submitted to a similar examination. RESULTS: Three ABL-inoculated flying foxes, and two rabies-inoculated animals developed abnormal clinical signs between 15 and 24 days PI. All three ABL-inoculated animals had histological lesions consistent with a lyssavirus infection, and lyssaviral antigen was identified in the central nervous system (CNS) of each. Virus was isolated from the brain of two affected animals. Of the rabies-inoculated flying foxes, both had histological lesions and viral antigen in the CNS. Virus was recovered from the brain of only one. None of the five affected flying foxes developed anti-lyssavirus antibodies, but, by 3 months PI, five of the seven ABL-inoculated survivors, and one of the two rabies virus-inoculated survivors, had seroconverted. The dynamics of the immune responses were quite variable. CONCLUSIONS: The response of flying foxes to ABL, administered by a peripheral route of inoculation, was similar to that of bats inoculated peripherally with bat-derived rabies viruses.     

Immunization of foxes Vulpes vulpes by the oral and intramuscular routes with inactivated rabies vaccines.

Inactivated rabies vaccines prepared from common vaccine strains of virus were inoculated into foxes by the intramuscular and intestinal route. There were differences among the vaccines in the duration of antibody produced after intramuscular administration. Inactivated vaccines deposited directly into the lumen of the duodenum by means of a fiberscope caused seroconversion in some foxes, especially following a booster dose, but the antibodies produced were for the most part of short duration. The ERA modified live virus vaccine, in contrast, produced a satisfactory and long lasting antibody after intestinal instillation.     

Safety and immunogenicity of ERA strain of rabies virus propagated in a BHK-21 cell line.

The ERA strain of rabies virus was propagated in a baby hamster kidney cell line (BHK-21/C13). The viral titer was 10(1.8) tissue culture infective doses (TCID) higher than that of commercial ERA vaccine. The ERA/BHK-21 vaccine in baits retained titers of 10(6.3) to 10(6.4), TCID when subjected to daily temperature fluctuations from 9 degrees C to 24 degrees C for 21 days. This titer, according to a dose response in laboratory foxes, was still capable of immunizing up to 100% of foxes consuming a bait. The ERA/BHK-21 vaccine, when presented in baits, produced antibodies in 80 to 100% of dogs consuming more than one bait. Duration of immunity in foxes, from feeding the ERA strain rabies virus in baits, as determined by resistance to challenge with virulent virus, was at least 48 months. The vaccine strain retained some pathogenicity for nontarget species. In tests carried out on foxes, raccoons, dogs, cats and cattle, the vaccine did not cause vaccine-induced rabies. One of 14 skunks which consumed four baits developed vaccine-induced rabies, but virus could not be isolated from the salivary glands of this animal. The vaccine, when presented in baits, caused vaccine-induced rabies in 37% of laboratory mice, 3.4% of Microtus and 2.6% of Peromyscus species. Rabies virus could not be isolated from the salivary glands of rodents with vaccine-induced rabies. It was concluded that ERA virus propagated in BHK-21/C13 cells and incorporated in an acceptable bait produced a high titer, stable, immunogenic and safe vaccine for foxes.     

Immunogenicity and efficacy of the oral rabies vaccine SAD B19 in foxes

Studies on the immunogenicity and efficacy of SAD B19 attenuated rabies virus vaccine in foxes under laboratory conditions were conducted. Twenty-seven foxes (Vulpes vulpes) were offered a vaccine bait containing 10(6.3) FFU/ml SAD B19. Blood samples were collected 60, 110 and 190 days post-vaccination. On day 190 post vaccination the animals and 14 controls were challenged with a canid street rabies virus. Twenty-four of the 26 vaccinated foxes (92.3%) survived the challenge, whereas all the controls died from rabies. The two vaccinated foxes that did not survive the challenge did not show any detectable level of rabies neutralizing antibodies at any time after vaccination. The geometric mean titres (GMT) of foxes that seroconverted after vaccination were 43.5, 33.9 and 43.5 IU/ml 60, 110 and 190 days post-vaccination, respectively. Furthermore, to test the vaccine virus under sub-optimal conditions five naive and nine previously vaccinated vixens received 2 ml SAD B19 (10(6.7) FFU/ml) by direct administration of the vaccine virus into the oral cavity shortly before or during pregnancy. All vixens seroconverted above the threshold of 0.5 IU/ml. No booster effect was observed in the immune response of the previously vaccinated animals.     

An epidemic of sylvatic rabies in Finland--descriptive epidemiology and results of oral vaccination.

When rabies reappeared in Finland in April 1988, the country had been rabies free since 1959. Soon a picture of sylvatic rabies become evident, its main vector and victim being the raccoon dog (Nyctereutes procyonoides). Between 8 April 1988 and 16 February 1989, 66 virologically verified cases were recorded (48 raccoon dogs, 12 red foxes, 2 badgers, 2 cats, 1 dog and 1 dairy bull) in an area estimated at 1700 km2 in south-eastern Finland. The greatest distance between recorded cases was 67 km. A positive reaction with monoclonal antibody p-41 indicated that the virus was an arctic-type strain. A field trial on oral immunization of small predators was initiated in September 1988 using Tübingen fox baits according to the Bavarian model of bait distribution. Each bait contained 5*10(7) TCID50/ml modified live rabies virus (SAD-B19). The 6 months' surveillance indicate a seroconversion rate of 72% (N = 126) in the raccoon dog population, 67% (N = 56) in the red foxes and 13% (N = 16) in the badgers, when titers greater than or equal to 1.0 IU/ml are considered seropositive. In the whole follow-up period, no statistically significant difference could be detected between the raccoon dogs and red foxes in the rate of seroconversion or in the uptake of tetracycline from the baits. Notably high antibody levels were recorded in both raccoon dogs and red foxes within 4-5 months after vaccination. Of the seropositive animals, the proportion of animals with titers 3.0 IU/ml or greater was higher in raccoon dogs (73%) than in red foxes (51%) (x2 = 5.29, p less than 0.05). The trial shows that raccoon dogs can be immunized against rabies in the field with vaccine baits originally developed for controlling sylvatic rabies in foxes.     

The safety and efficacy of immunizing foxes (Vulpes vulpes) using bait containing attenuated rabies virus vaccine.

Foxes given ERA rabies vaccine baits were challenged at one, six, 12 and 24 months later and showed a resistance to challenge in 80%, 78%, 60% and 44% of individuals respectively. All animals showing seroconversion following vaccination, resisted challenge at 24 months, suggesting that successful vaccination by the oral route could confer a relatively long term duration of immunity. The trials showed that fox pups did not immunize as easily as adult foxes using ERA rabies vaccine baits. Back-passage studies and the consumption of ERA injected mice by foxes failed to show any reversion of the vaccine virus to a virulent state. The fox and mouse are shown to be highly susceptible to rabies street virus, while the domestic species tested are consisderably more resistant. Monkeys were found to be intermediate in susceptibility to the virus. Safety tests carried out on various species of wildlife showed only the mouse to be susceptible to infection from ingesting the vaccine in the form of a bait. ERA rabies vaccine was shown to be safe in monkeys even when high titred virus was administered by the oral route.     

Stability of attenuated live virus rabies vaccine in baits targeted to wild foxes under operational conditions

The viability of an attenuated live virus rabies vaccine in a bait targeted to red foxes was examined under various operational conditions in a series of experiments in Ontario. The virus was relatively stable over a 28-day period in the field, losing a mean 0.5, s = 0.2 log10 of virus titer. The micro-environment into which the bait was placed (open cultivated field, grassy meadow, wooded grove, sun or shade) did not make an appreciable difference in the viability of the virus. There was no significant difference (P < or = 0.05) between mean ambient temperatures and the temperature of fluids in blister packs of baits placed in sun or shade. Sixty-three percent of foxes fed baits exposed to sun and shade conditions for 21 days (titer 10(6.2) tissue culture infective doses per 1 mL) developed rabies virus-neutralizing antibodies. Storage of vaccine baits at -30 degrees C prior to bait distribution was important in maintaining virus viability.     

Oral vaccination against rabies and the behavioural ecology of the red fox (Vulpes vulpes)

As a result of oral vaccination of foxes (Vulpes vulpes) against rabies, this virus disease has almost been completely eradicated from West- and Central Europe. In most countries, vaccine baits were distributed twice a year: during spring (March to May) and autumn (September to October). This strategy has shown to be able to control and eventually eradicate rabies. However, it remains to be clarified if this is the most cost-effective strategy. Astonishingly, the behavioural ecology of the target species, the red fox, did receive only limited attention selecting the periods when baits should be distributed. Considering the behavioural ecology and rabies epidemiology of foxes, territory owners seem to play a key role in the spread and maintenance of rabies. Thus, oral vaccination campaigns should be targeted primarily at these animals. It is suggested that the optimal timing for bait distribution in Europe is late autumn (November) or early winter (December), depending on the prevailing climatic conditions. Additional campaigns, when financially feasible, can be implemented in order to maintain a high vaccination coverage during the remaining year. Furthermore, different baiting strategies can be selected in case of re-infection or persistent residual foci.     

Pathogenesis of Rabies in the Rat

Three strains of rabies virus, one fixed and two street, were compared for invasiveness in neurectomized rats following foot pad inoculation. Approximately one-fifth of test rats studied revealed that the virus bypassed the severed sciatic and saphenous nerves and reached the brain.

All rats dying within 20 days were positive for rabies upon fluorescent antibody and mouse mortality tests.

Viremia was demonstrated in 20 per cent of both neurectomized or non-neurectomized rats one hour after inoculation of rabies virus, both fixed and street strains, into the foot pad.

     

Raccoon dog rabies surveillance and post-vaccination monitoring in Lithuania 2006 to 2010

Background

Oral rabies vaccination (ORV) in rabies infected regions should target the primary rabies vector species, which in Lithuania includes raccoon dogs as well as red foxes. Specific investigations on ORV in raccoon dogs are needed e.g. evaluation of vaccine effectiveness under field conditions. The objective of the current study was to investigate the efficacy of the ORV programme 2006-2010 in Lithuania by examining the number of rabies cases and estimating the prevalences of a tetracycline biomarker (TTC) and rabies virus antibodies in raccoon dogs.

Methods

From 2006 to 2010, 12.5 million rabies vaccine-baits were distributed by aircraft. Baiting occurred twice per year (spring and autumn), targeting raccoon dogs and red foxes in a 63,000 km² area of Lithuania. The mandibles of raccoon dogs found dead or killed in the vaccination area were analyzed by fluorescence microscopy for the presence of the TTC. Rabies virus sera neutralizing anti-glycoprotein antibody titres were determined using an indirect ELISA method and seroconversion (> 0.5 EU/ml) rates were estimated.

Results

During the study period, 51.5% of raccoon dog mandibles were positive for TTC. 1688 of 3260 tested adults and 69 of 175 tested cubs were TTC positive. Forty-seven percent of raccoon dog serum samples were positive for rabies virus antibodies. 302 of 621 investigated adults and 33 of 95 investigated cubs were seropositive. In the same time 302 of 684 and 43 of 124 tested samples were TTC and ELISA positive in spring; whereas 1455 of 2751 and 292 of 592 tested samples were TTC and ELISA positive in autumn. There was a positive correlation between the number of TTC and antibody positive animals for both adult and cub groups.

Conclusions

ORV was effective in reducing the prevalence of rabies in the raccoon dog population in Lithuania. The prevalence of rabies cases in raccoon dogs in Lithuania decreased from 60.7% in 2006-2007 to 6.5% in 2009-2010.     

First Report on the Efficiency of Oral Vaccination of Foxes against Rabies in Serbia

Rabies is one of the oldest known zoonotic diseases that has significant impact on public health, but still remains neglected in Serbia. Rabies virus can infect humans and other mammals and causes inflammation of the brain associated with encephalomyelitis and neurological symptoms. In 2010, Veterinary Directorate (national Competent Authority for animal health in Serbia) has started multi‐annual project of oral rabies vaccination of foxes and other wild carnivores (e.g. jackals), as support of long‐term programme of eradication of rabies in Serbia, co‐funded by EU (financed by Instrument for Pre‐Accession Assistance). Monitoring of the effectiveness of oral vaccination campaigns has been carried out in continuation from 2011 and was based on: (i) post‐mortem laboratory examination of brain tissue of target animals (foxes, jackals and other carnivores) by fluorescent antibody test (FAT), (ii) detection of antibodies against rabies virus in serum samples by ELISA and (iii) detection of tetracycline biomarker in the mandibles for the evaluation of vaccine bait uptake. From September 2011 to May 2014, the total number of 4943 brain tissue samples, 4241 sera and 4971 mandibles were analysed. Confirmed rabies‐positive brains decreased from 10 in 2011/2012 to 6 in 2012/2013 and eventually to 1 positive case in 2013/2014. The seroconversion rate increased from 10.48% (133/1269) in 2011/2012 to 20.11% (362/1800) in 2012/2013 and 42.23% (495/1172) in 2013/2014. Along with the seroconversion, the number of detected tetracycline‐positive mandibles demonstrated an increasing tendency in the same period, being 49.67% (682/1373) in 2011/2012, 62.60% (1294/2067) in 2012/2013 and 90.33% (1383/1531) in the monitoring programme carried out in 2013/2014. Presented results confirmed that ORV of foxes and other wildlife in Serbia against rabies was successful and characterized by steady increase of vaccine baits uptake and immunization of animals.     

Comparative immunogenicity and efficacy studies with oral rabies virus vaccine SAD P5/88 in raccoon dogs and red foxes.

A comparative study of immunogenicity and efficacy of the oral rabies virus vaccine SAD P5/88 in raccoon dogs and foxes was conducted. The raccoon dogs received 10(6.9) (n = 6), 10(6.3) (n = 6) or 10(5.7) FFU SAD P5/88 (n = 5) by direct oral application, and subsequently all animals seroconverted. The foxes received 10(7.2) (n = 4), 10(6.2) (n = 4), 10(5.2) (n = 4) and 10(4.2) FFU SAD P5/88 (n = 5) by the same route. On days 106 and 196 post vaccination 10 raccoon dogs and 16 foxes were challenged with a relevant street virus, respectively. All 10 raccoon dogs vaccinated with 10(6.3) (n = 5) or 10(5.7) FFU SAD P5/88 (n = 5) survived the challenge, whereas all control animals (n = 5) died of rabies. Two foxes vaccinated with 10(4.2) FFU and one fox vaccinated with 10(5.2) FFU died of rabies on day 7, 17 and 12 post infection, respectively. Also all control foxes succumbed to rabies. Our findings demonstrate that SAD P5/88 is not only an effective vaccine for oral vaccination of foxes but also for that of raccoon dogs.     

Experimental infection of European red foxes (Vulpes vulpes) with canine herpesvirus

We report on the pathogenicity of canine herpesvirus (CHV) for European red foxes. In the first experiment, we inoculated 10 adult foxes intravenously with a canine isolate of CHV. All foxes became infected and shed CHV in saliva and genital secretions for up to 14 days post-inoculation (p.i.) as evaluated by PCR and/or by virus isolation. All foxes developed clinical signs such as fever, lethargy and evidence of respiratory tract disease. Two foxes died on day 6 p.i., one on day 7 p.i., and one fox was euthanased on day 6 p.i. Tissues taken from the four dead foxes were positive for CHV by PCR. The remaining six foxes recovered after approximately 14 days p.i. Virus particles with morphology typical of herpesviruses were found by electron microscopy in the liver of an infected animal. All surviving foxes developed serum anti-CHV antibodies. In a second experiment, six foxes were dosed perorally with CHV and paired with six untreated controls. Neither the perorally dosed nor the in-contact control foxes developed clinical signs of disease. Infectious CHV was not isolated from any of the dosed or the in-contact foxes but all perorally-infected foxes and one of the in-contact foxes tested PCR-positive for CHV on several occasions p.i. All perorally-infected foxes, but none of the in-contact foxes, seroconverted. In summary, intravenous CHV inoculation caused a clinical disease in adult foxes much more severe than observed in experimentally-infected adult dogs. No clinical disease or virus spread was observed after peroral dosing although viral infection occurred as evidenced by seroconversion.     

Detection of rabies viral antigens in non-autolysed and autolysed tissues by using an immunoperoxidase technique

The objectives of this study were to determine the potential of an immunoperoxidase technique involving the avidin-biotin complex (ABC) stain for the diagnosis of rabies in fresh tissues and compare it with other standard methods, including the fluorescent antibody test (FAT), haematoxylin and eosin and Seller's stain, and to investigate its capacity to detect rabies antigen in autolysed tissues. Samples of non-autolysed brain from 81 domestic and wild animals suspected of having rabies were examined. Rabies antigen was detected by FAT in 41 of these samples and Negri bodies were detected in 40 (97.6 per cent) of them by the immunoperoxidase technique, in 25 by haematoxylin and eosin and in 22 by Seller's stain. The sensitivity of the immunoperoxidase technique decreased as the tissues were left to autolyse; after two days it was 91.2 per cent, after four days 70.6 per cent, and after seven days 11.8 per cent.     

Serological responses of adult dogs to revaccination against distemper, parvovirus and rabies

Serum antibody titres to canine distemper virus (CDV), canine parvovirus (CPV) and rabies were measured in dogs that had not been revaccinated annually and compared with the titres in a control group of regularly vaccinated animals; 83 per cent (171 of 207) of the dogs vaccinated against CDV one or more years earlier had serum neutralising antibody titres equal to or greater than 16; 64 per cent (136 of 213) of the dogs vaccinated against CPV one or more years earlier had haemagglutination inhibiting titres equal to or greater than 80; and 59 per cent (46 of 78) of the dogs vaccinated against rabies two or more years earlier had serum neutralising antibody titres equal to or greater than 0.5 iu/ml. Three weeks after a single booster vaccination the dogs' antibody titres against CDV had increased above the threshold level in 94 per cent of the dogs, against CPV in 68 per cent, and against rabies in 100 per cent.     

Use of recombinant vaccinia-rabies virus for oral vaccination of fox cubs (Vulpes vulpes, L) against rabies

Thirteen fox cubs were orally administered 10(7.2) plaque-forming units of live vaccinia-rabies glycoprotein recombinant virus. On Day 28 post-vaccination, all but 1 cub had produced rabies virus antibodies. Twelve animals were intramuscularly inoculated with 10(3.2) mouse intracerebral LD50 of rabies virus suspension on Days 33 (5 foxes), 180 (4 foxes) or 360 (3 foxes) after vaccination. Eleven of them resisted rabies challenge. Unvaccinated foxes, either put in contact with 1 vaccinated animal or used as controls, died after challenge applied on Day 33. The absence of horizontal transmission of this vaccine strain and its innocuity to cubs were also demonstrated.     

Oral immunization of foxes with avirulent rabies virus mutants

SAG1, a rabies virus strain bearing one mutation which abolishes virulence for adult animals, was constructed from the SADBern strain of rabies virus which has previously been used as live vaccine for oral immunization of foxes. SAG1 also bears an antigenic mutation which serves as an additional marker of the strain. Studies on mice and four species of wild rodents showed that SAG1 is totally avirulent whereas SADBern is still pathogenic after intracerebral, intramuscular or oral inoculation and thus could cause cases of rabies. Trials of oral vaccination performed on foxes with SAG1 indicate that it is as effective as SADBern. The SAG1 strain represents a significant progress in the search for an efficient and safe live rabies for the oral immunization of wild animals.