The development of strategies for the field application of oral immunization of foxes against rabies

Preconditions for successful field application of the idea to orally immunize foxes against rabies were a) the development of an efficacious and safe vaccine and an attractive bait and b) a suitable strategy to reach a high proportion of the vector population with vaccine-ladden baits. A geographical and epidemiological concept on how to implement the vaccination strategy and how to achieve a sufficiently high level of surveillance were further cornerstones contributing to the success of the method. Finally, political decisions and a legal basis for the application of the method of oral immunization were required to eradicate fox rabies in Switzerland.     

The development of rabies in Switzerland--landscape determines the spread of a wildlife epidemic

The rabies epidemic that reached Switzerland in 1967 developed in response to landscape factors as long as no efficient control strategies were available. The landscape acted either as barrier to the spread of rabies, or it influenced the density of red foxes and thus the habitat of the epidemic. Following the first cases in the canton of Schaffhausen, the whole northwestern Switzerland was infected followed by the eastern Alps, large parts of the Plateau and the Jura mountains. In 1978, in the canton of Valais, the first campaigns of oral immunization of foxes against rabies started. The design of vaccination campaigns during the next two decades was always closely linked to landscape features. Thus, it was possible to free first the Alps and then the Plateau from rabies and finally, at the end of the 1990s, to eliminate it completely within the country. We describe the entire development of the epidemic within the period of 30 years from the first infection up to the last registered case and the final vaccination campaign.     

The final stage of rabies in Switzerland

Since summer 1989, rabies in Switzerland has been restricted to the Jura Mountains in the north-west of the country. Even there, the last endemic focus disappeared in 1990, but a re-infection in the same year caused a new flare-up of the epizootic. Until 1994, the number of rabies cases increased again to 225. Control measures were intensified with doubled vaccination campaigns, increased bait densities, and additional vaccination campaigns to immunize young foxes at the den. As a consequence, the number of cases dropped to 25 in 1995 and to 6 in 1996. On December 21, 1996, the last endemic case of rabies in Switzerland was registered. After two years of continuing vaccination campaigns and surveillance, Switzerland became officially rabies-free at the beginning of 1999. In the present paper, we analyse the final stage of the epizootic. The re-infection in 1990 was caused by infected foxes immigrating from France, but as the immunization of the fox population in Switzerland was insufficient, the disease became again endemic immediately. The lacking herd immunity was partly a consequence of problems related to the vaccination system and even more of the rapid increase of the fox population.     

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.     

Oral vaccination of foxes against rabies in Turkey between 2008 and 2010

Following a sustained spill-over event from dogs to foxes, fox rabies spread rapidly in the Aegean region, Turkey. In order to control the outbreak a program of oral vaccination of foxes against rabies was introduced. In the selected vaccination area three annual campaigns between 2008 and 2010 were undertaken during the winter months whereby the vaccine baits were distributed exclusively by plane using a density of 18 baits per km2. Subsequently, fox rabies cases were reported only from locations bordering the non-vaccinated areas. Hence, it was shown that fox rabies control by means of oral rabies vaccination is feasible in Turkey. However, for the progress towards the elimination of fox-mediated rabies in Turkey to be maintained, it is necessary that political and financial support is secured to extend oral vaccination where infected foxes remain.     

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.     

Efficacy of oral vaccination in the final stage of fox rabies elimination in Switzerland

Subsequent to rabies vaccination campaigns, two well-established methods for the determination of the proportion of vaccinated foxes--the detection of tetracycline (TC) in bones and the detection of virus neutralizing antibodies (VNA) in thoracic fluids--were used and compared. Special emphasis was given to the effect of a new method of bait distribution at the den, which is primarily targeted at young foxes. The overall proportion of vaccinated animals estimated by TC was 60% as compared to 50% by VNA. In young foxes overall, significantly lower proportions of vaccinated animals (58% by TC and 40% by VNA) than in adult foxes (75 and 59%) were estimated with both methods. Low proportions of vaccinated young animals were found after spring (39 and 18%), but also after autumn vaccination (56 and 35%). In contrast, after den vaccination the level of vaccination of young foxes reached that of adult foxes. The theoretical implication of the successful elimination of fox rabies in Switzerland in spite of a relatively low overall proportion of VNA-positive animals is discussed.     

Clinical experiences with rabies in cattle in Hungary

Over the past years, bovine rabies has become a significant problem in Hungary. Not only do foxes play and important role in the transmission of the virus to cattle, but also cats have recently been recognized as an important source of infection in cattle. In the investigated regions, seasonal peaks of transmission could be observed. The first cases of the year occurred in June, but the most cases were diagnosed in autumn and early winter. The average clinical course of rabies in infected animals extended over a period of 2-6 days, occasionally shorter and longer courses were presented. A short clinical course was usually related to an early onset of prominent nervous symptoms, while prolonged cases expressed fewer nervous signs. Prevention and control of the disease can be best achieved by regional oral vaccination of foxes as the positive results demonstrate in the Transdanubian region of Hungary.     

Re-emergence of rabies in Mazowieckie Voivodeship,Poland, 2021

Due to the oral vaccination of foxes against rabies most of the territory of Poland was freed from rabies of non-flying mammals. In January 2021, rabies was diagnosed in fox in the central part of Mazowieckie Voivodeship where rabies has not been detected since last 17 years. Subsequently, in the following months the rabies virus infection spread southward reaching the voivodeship of Świętokrzyskie in November 2021. Emergency actions were implemented aiming at rapid rabies elimination.     

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.     

Elimination of terrestrial rabies in Germany using oral vaccination of foxes

Oral rabies vaccination (ORV) has become the method of choice in fox rabies control in Europe. During the past three decades fox-mediated rabies virtually disappeared from Western and Central Europe. Following Switzerland, Germany was the second European country to launch ORV field trials on its territory in 1983. This paper provides a historical overview on the emergence of fox rabies in Germany; describing the basic principles and milestones of the German rabies eradication programme and presenting results of two decades of efforts to control the disease in foxes. Also, setbacks as well as country-specific differences and particularities on Germany's long way to rabies elimination in comparison to other European countries are addressed. Since the first field trials in Germany the number of rabies cases steadily decreased from 10 484 in 1983 to three cases recorded in 2006. On February 3rd 2006 the last case of terrestrial rabies in Germany was detected in a fox near the town of Mainz, Rhineland-Palatinate. In 2008, ORV ceased after 25 years and Germany was officially declared as free from terrestrial rabies. The German rabies eradication programme did cost approximately 100 million euro of which 37 million euro were covered by the EU. For the future, efforts should focus on maintaining a rabies free status by implementing measures to prevent reintroduction of terrestrial rabies from endemic countries.     

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.     

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.     

Rabies oral vaccination of foxes during the summer with the VRG vaccine bait

The vaccination of foxes by distributing vaccine baits in the environment was initiated in France in 1986. Two campaigns per year were carried out: one in the spring and one in the autumn. After the spring campaigns, only 22-52% of fox cubs consumed vaccine baits compared to 75% of the adults and 70-80% of the adults or fox cubs after autumn campaigns. In order to reduce the period of time during which fox cubs do not have access to baits and are not immunised, a vaccination campaign was organised during the summer of 1992 over a contaminated area of 25,748 km2 where vaccines had never previously been given. Vaccine bait stability was assessed during the same summer in the field and their appetence tested on captive foxes. The efficacy of the campaign was evaluated by the relative decrease in rabies incidence and the rate of bait uptake by foxes compared to those from neighbouring areas vaccinated for the first time with the same vaccine during the spring or autumn. Summer vaccination significantly increased (P < 0.01) bait uptake by fox cubs (71%) compared with spring vaccination (39%), but no significant difference was observed for adult foxes. Moreover, the decrease in rabies incidence, measured during the 6-month period following the campaigns was less pronounced after summer vaccination (49% decrease) than when the first vaccination was carried out during the spring or autumn (79 and 72% decrease, respectively). Three campaigns led to an apparent elimination of rabies when the first campaign was performed in the spring or autumn, but only to a 76% decrease in rabies incidence density index when the first campaign was performed during the summer. The high thermostability of the Raboral VRG bait permits its use during the summer for an emergency campaign. For routine vaccination plans, however, the classical calendar of spring and autumn vaccination campaigns should continue to be preferred.     

Experiences with follow-up investigations of oral vaccination campaigns against rabies in foxes in Saxony with special emphasis on a standardised serology

An 8-year experience with organisation and standardisation of follow-up investigations within oral vaccination campaigns against rabies in foxes (OVF) in Saxony is summarised. With respect to OVF, the number of diagnostic tests performed during the years 1992-2000 on foxes amounts to a total of 52,226 Fluorescence antibody-(FAT), 7,551 marker-(TC) and 11,645 serological tests. The mean bait-uptake and the mean immunisation rate in foxes ranged between 78-86% and 60-89%, respectively. Based on the seroconversion rates of the years 1997-2000 observed in vaccination areas and in areas where vaccination was already finished, experience with a standardised serology under routine conditions is presented and discussed. Furthermore, recommendations concerning organisation and logistics of sampling are given.     

Pyrosequencing of the rabies virus glycoprotein gene to demonstrate absence of vaccine-associated rabies cases following oral vaccination

Replication competent vaccines have been used successfully for the control of terrestrial rabies, mainly in wildlife; however, these vaccine strains occasionally may induce rabies. In this study, a pyrosequencing protocol for the rapid identification of vaccine-associated rabies viruses was applied to the 2008–2011 Italian epidemic. There was no evidence of vaccine-associated rabies cases following oral vaccination of foxes with the SAG2 and SADB19 vaccine strains.     

Duration of immunity in foxes vaccinated orally with ERA vaccine in a bait.

Red foxes (Vulpes vulpes) vaccinated orally with the ERA strain of rabies vaccine in a bait were challenged after 83 mo. Ten of 11 foxes that had seroconverted following vaccination resisted challenge with a virulent rabies virus which produced clinical signs of rabies in 6 of 6 unvaccinated foxes. Five of 11 vaccinated animals retained titers of rabies virus neutralizing antibody throughout the period. Although 6 of 11 had no detectable antibody at the time of challenge, 5 of these 6 resisted challenge and had an anamnestic response, as indicated by elevated titers of antibody when measured at day 77 postchallenge. These results show that foxes can be immunized successfully with a single oral dose of ERA vaccine, probably with protection against a lethal rabies challenge, for at least 7 y.     

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.     

Twenty year experience of the oral rabies vaccine SAG2 in wildlife: a global review

The SAG2 vaccine (RABIGEN® SAG2) is a modified live attenuated rabies virus vaccine, selected from the SAD Bern strain in a two-step process of amino acid mutation using neutralizing monoclonal antibodies. The strain is genetically stable and does not spread in vivo or induce a persistent infection. Its absence of residual pathogenicity was extensively demonstrated in multiple target and non target species (such as wild carnivores and rodent species), including non-human primates. The efficacy of SAG2 baits was demonstrated according to the EU requirements for the red fox and raccoon dog. The use of safe and potent rabies vaccines such as SAG2 largely contributed to the elimination of rabies in Estonia, France, Italy and Switzerland. Importantly, these countries were declared free of rabies after few years of oral vaccination campaigns with SAG2 baits distributed with an appropriate strategy. The excellent tolerance of the SAG2 vaccine has been confirmed in the field since its first use in 1993. No safety issues have been reported, and in particular no vaccine-induced rabies cases were diagnosed, after the distribution of more than 20 million SAG2 baits in Europe.     

The economic implications of sylvatic rabies eradication in Italy

After more than 10 years of absence, sylvatic rabies re‐appeared in Italy in 2008. To prevent disease spread, three oral rabies vaccination (ORV) campaigns targeting red foxes were performed through manual distribution of vaccine baits between January and September 2009. As these campaigns proved unsuccessful, at the end of December 2009, baits started being distributed using helicopters, allowing uniform coverage of larger areas in a shorter time period. From winter 2009 to autumn 2016, a total of 15 ORV campaigns (four emergency, four regular and seven preventive ORV) were implemented through aerial distribution of baits. In this study, we assessed the costs of the aerial ORV campaigns, which were aimed at eradicating the disease and reobtaining the rabies‐free status. Cumulative costs per km² were estimated at €59.45 during emergency campaigns and ranged between €51.94 and €65.67 in the regular vaccinations. The main portion of costs for ORV programmes were related to baits supply and distribution: €49.24 (82.83%) in emergency campaigns and from €40.33 to € 43.35 in regular ORVs (71.97% and 66.02%, respectively). At the end of each ORV campaign, the efficacy of vaccination activities was estimated by assessing the proportion of foxes testing positive for tetracycline biomarker in jawbone, indicating bait intake. Results revealed that the proportion of foxes that ingested baits varied between 70.97% and 95.51%. Statistical analysis indicated that reducing the density of dropped baits could potentially lead to a cost‐saving of 22.81%, still maintaining a satisfactory level of bait intake by the fox population.