Introduction

Terrestrial rabies in carnivores is caused by negative-strand RNA viruses of the order Mononegavirales, family Rhabdoviridae, genus Lyssavirus, species Rabies virus, genotype 1. The latest sylvatic rabies outbreak among red foxes (Vulpes vulpes) in Europe started south of Gdansk in Poland in 1939. It reached Germany and Austria in 1950 and 1966, respectively [32]. In the following years, it spread rapidly over the entire territory of these countries, reaching peak case numbers in Germany (10,484 cases) and in Austria (2,514 cases) in 1983 and 1990, respectively.Footnote 1 Traditional control methods based on killing of foxes by various means did not decrease the number of rabies cases and/or slow its spread. It has actually been suggested that these measures increased transmission possibilities by de-stabilizing the social structure of the fox population [32]. After the first field studies in Switzerland at the end of the 1970s demonstrated that oral immunization with vaccine baits represents a highly effective control method [42], Germany and Austria started large-scale bait vaccination in 1983 and 1986, respectively. Since 2006 in Austria and 2007 in Germany, no terrestrial rabies cases have been reported from either country (source: Rabies Bulletin Europe). This represents an important success in controlling an infectious disease in wildlife.

Four different rabies vaccine viruses were used, which, like all other commercially available oral rabies vaccines, are based on replication-competent, attenuated viruses that must infect host tissues to elicit an immune response. It is well known that live modified virus vaccines can induce disease under certain circumstances which, e.g., has been shown for oral polio virus vaccines in humans and canine distemper vaccines in pets and wildlife [7–9, 17, 18, 25, 30, 36]. Rabies vaccines are no exception, and vaccine-associated rabies cases associated with the ERA-BHK21 and SAD Berne rabies vaccine strains have been reported in wildlife [22, 47]. All four vaccine strains used in Germany and Austria are claimed to be direct derivatives of the SAD Berne. Although they are more attenuated than the SAD Bern strain and non-pathogenic for the target species, residual pathogenicity for rodents has been observed under experimental conditions. Also, in immunocompromised hosts, these vaccine viruses can cause disease [20, 45]. Hence, the uptake of vaccine baits containing these viruses could result in vaccine-virus-induced rabies in target and non-target species, including humans. To quantify these potential risks, foxes and other animals that tested rabies-positive during active surveillance programs established as part of the oral rabies vaccination (ORV) campaigns and/or routine rabies diagnosis were examined using a panel of monoclonal antibodies (MAb) for possible vaccine-virus-associated rabies cases. More recently, molecular technologies (RT-PCR) have been used. This paper summarizes the results of the post-vaccination surveillance in Germany and Austria. In total, 6 possible vaccine-virus-associated rabies cases have been identified after the distribution of almost 97 million vaccine baits.

Methods

Vaccine virus

The oral rabies vaccine viruses used in Germany and Austria were SAD B19, SAD VA1, SAD P5/88 and SAG1. All of them have been reported to be derived from rabies virus strain SAD (Street Alabama Dufferin), which was isolated from a dog in 1935 [5], although recent studies have questioned the link of some of them to the ancestor SAD Bern [26]. After adaptation to cell culture, SAD was referred to as ERA (Evelyn Rokitniki Abelseth) [23]. ERA was further propagated in BHK21-cells [22] and used to develop the first oral rabies vaccine virus used in the field, SAD Berne [42].

The most widely used oral rabies vaccine virus, SAD B19, was selected for its improved thermostability and its reduced residual pathogenicity for rodents in comparison with its parental strain, SAD Berne [38]. The SAD P5/88 vaccine virus was developed by adaptation of SAD Berne to the high-producer cell line BHK-21 clone BSR P5/88 [40]. SAG1 and SAD VA1 were selected from SAD Berne in the presence of anti-rabies glycoprotein monoclonal antibodies and contain a mutation at arginine 333 of the glycoprotein [2, 26, 31].

Animal specimens

Animals were submitted for routine rabies diagnosis and during active surveillance programs following oral rabies campaigns as recommended by the EU [20]. In Germany, animals for rabies diagnosis are submitted to the responsible regional veterinary laboratories. For the cases described in this report, the following laboratories were involved: Krefeld (North Rhine Westphalia), Giessen (Hessen) and Koblenz (Rhineland-Palatinate). In Austria, animals were submitted directly to the national reference laboratory for rabies at the Agency for Food and Health Safety GmbH (AGES), Mödling. Initial diagnosis, e.g. fluorescence antibody test (FAT), virus isolation, immunohistochemistry (IHC), and to some extent, RT-PCR and partial sequencing (Austria), was performed there. For further characterization, original brain material, mouse-brain-passaged material, cell culture supernatant and/or RNA was sent to the WHO Collaborating Centre for Rabies Surveillance and Research at the Friedrich-Loeffler-Institut (FLI), Wusterhausen, Germany, where antigenic typing using MAbs, as well as partial and full-genome sequencing, was performed. Partial sequencing results were independently confirmed at the Veterinary Laboratory Agency (VLA), Weybridge, UK, using blinded samples.

Rabies diagnosis

Brain tissues were tested for the presence of rabies virus by FAT [15].Viruses were isolated in murine neuroblastoma cells in the rabies tissue culture infection test (RTCIT) [48]. For antigenic typing, an indirect FAT was applied in cell culture using a reduced panel of anti-nucleocapsid MAbs (W239.17, W187.5, MW187.6.1) that had previously been shown to discriminate between field rabies and SAD vaccine virus strains [14, 37]. Staining patterns for each specimen were assessed by fluorescence microscopy and used to determine the presence of the variant type. Since 2000 (Germany) and 2004 (Austria), PCR has been available as an additional tool to detect rabies after RNA extraction of brain tissue of selected animals or from virus isolates.

Immunohistochemistry

One German specimen was analyzed by immunohistochemistry (IHC) after fixation of brain tissue in 10% buffered formalin and embedding in paraffin wax. IHC detection of lyssavirus nucleocapsid was performed as described [29], using an anti-rabies nucleocapsid monoclonal antibody (HAM; obtained from the Swiss Rabies Centre, Bern, Switzerland). Sections were counterstained with Meyer’s haematoxylin-eosin and mounted.

Partial sequencing

To identify the virus isolates, RNA was extracted (RNeasy Mini Kit, Qiagen, Hilden, Germany) directly from brain tissue and amplified, and 580-bp (position 55–641), 380-bp (position 1198–1579) and 769-bp (position 4692–5461) fragments of the N gene, the N–P intergenic region [19, 27, 28] and the G-gene, respectively, were sequenced on both strands. For the last fragment, the following primers were used: (1) G4243Fwd (5′-CAT GAC AAC CAA GTC AGT GAG-3′) and G4484Rev (5′-ATT GCA TCT CTG GGA TTA AGA C-3′). Briefly, the G-gene-specific fragment was amplified with Invitrogen SuperScript III OneStep (Invitrogen, Karlsruhe, Germany) at 94°C for 2 min, 40 cycles of 94°C for 15 s, 56°C for 30 s and 68°C for 60 s. A 5-μl aliquot taken directly from the amplification reaction was used for sequencing. Molecular phylogenetic analysis of the sequences was conducted as described [28].

Full-genome sequencing

To identify all alterations, complete genome sequences were obtained for each presumptive SAD-vaccine-associated case. The isolates were propagated in murine neuroblastoma cells for up to two passages until 100% of the monolayer was infected. Subsequently, RNA was extracted from the infected cell supernatant using a commercially available RNA extraction kit (RNeasy Mini Kit, Qiagen, Hilden, Germany). RT-PCR as well as forward and reverse sequencing of the PCR products by direct cycle sequencing was performed essentially as described [26]. Presumptive SAD isolates were sequenced from nucleotide position 10 to 11904 of the SAD B19 genome [12]. All PCR products were independently sequenced at least three to six times in each direction using an automatic LI-COR DNA Sequencer 4200 (LI-COR GmbH, Bad Homburg, Germany).

Sequence analysis

Sequence analysis was performed using the Lasergene DNAStar program 7.0 package (DNAstar Inc., Madison, USA). Pairwise and multiple alignments of DNA and deduced amino acid sequences were done using the Megalign tool (DNAStar). Sequences were compared to complete genome sequences of commercially available SAD derivatives (accession numbers EF206708, EF206709, EF206715, EF206719, EF206720) from GenBank [26]. Complete genome sequences generated in this study have been submitted to GenBank and assigned the following accession numbers: EU886631—isolate Germany 2001 (Leverkusen), EU886632—isolate Germany 2002 (Limburg), EU886633—isolate Austria 2004 (Völkermarkt), EU886634—isolate Germany 2004 (Mettmann), EU886635 isolate Germany 2005 (Kusel) and EU886636—isolate Austria 2006 (Oberloisdorf).

Experimental studies

To test for pathogenicity of the SAD vaccine virus isolates in foxes, virus was grown to high titers in neuroblastoma cells to obtain inocula that were clearly above the minimum effective dose for both vaccine strains. Two farm-bred fox cubs each were inoculated intramuscularly (i.m.) in the masseter muscle with 1 ml containing 106.8 and 106.3 FFU of the isolates from the foxes from Limburg (no. 2) and Völkermarkt (no. 3). For comparison, one fox each was vaccinated i.m. with working seed virus (WSV) SAD P5/88 (107.0 FFU/ml) and WSV SAD B19 (106.5 FFU/ml), used for the production of the vaccine bait batches distributed. In addition, two foxes were inoculated with 105.7 FFU of a rabies virus isolate from a dog originating in Azerbaijan that had been imported into Germany in 2002. All foxes were serologically negative for rabies neutralising antibodies prior to this study. Animals were observed for 110 days after infection. Blood samples were collected at 0, 14, 29, 64 and 110 days postinfection and saliva swabs were collected at 14, 21, 29, 35, 42, 64 and 110 days postinfection. Brain samples of all animals that had been euthanized at the end of the experiment or which had died during the observation period were examined for the presence of viral antigen by FAT [15]. Saliva swabs and salivary glands were tested for the presence of rabies-specific RNA using PCR [19]. Virus-neutralising antibody (VNA) titres were determined using a rapid fluorescence focus inhibition test (RFFIT) [41], which was modified as described [13]. The international standard immunoglobulin (second human rabies immunoglobulin preparation, UK) adjusted to 0.5 IU/ml served as a positive control. VNA titres were converted to International Units (IU/ml). All handling and invasive procedures were conducted in compliance with the German Animal Welfare Act 2 and 2a and recommendations of the GV-SOLAS (Society for Laboratory Animal Science).

Results

Since the beginning of oral rabies vaccination in Germany (1983) and Austria (1986), approximately 83.1 and 13.8 million vaccine baits have been distributed, respectively (Table 1). The most widely used vaccine viruses were SAD B19 (51.6 million; 53.2%) and SAD P5/88 (43.1 million; 44.5%). Vaccine baits containing SAD VA1 (0.5 million; 0.4%) and SAG1 (1.8 million; 1.9%) were used to a lesser extent. The bait density in both countries varied between 15 and 30 baits/km2 at the beginning of the campaigns and in the final stage of rabies elimination, respectively, resulting in an average of 20–25 baits/km2. In both countries, the number of rabies cases could be drastically reduced, and terrestrial wildlife rabies was eventually eliminated. As part of these campaigns, all rabies-positive cases from vaccination areas in Germany were further examined to identify possible vaccine-induced cases by virus typing using the abovementioned panel of MAbs. At least 7,273 foxes and 2,509 other animal species were analyzed between 1983 and 2007. The exact number of examinations from 1998 to 2007 for the three federal states in Germany from which the vaccine-associated cases have been reported is shown in Table 2. Since the first campaigns in 1983 (Germany) and 1986 (Austria), a total of four and two vaccine-associated rabies cases, respectively, have been detected exclusively in red foxes (Table 3). In contrast to the cases in Germany, the Austrian cases 3 and 6 were reportedly clinically inconspicuous.

Table 1 Number of vaccine baits distributed in Germany and Austria (1983–2008) and number of reported rabies cases
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Table 2 Rabies surveillance in the three German federal states from which vaccine-virus-associated cases have been reported (1998–2007)
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Table 3 Oral-vaccine-associated rabies cases from Germany and Austria
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The first case (Leverkusen) involved a young fox that was found underneath a car and did not leave the spot even after the car was moved. Due to this behaviour, a hunter was called to kill the animal, and subsequently, the animal was submitted for rabies testing. The two nearest foci of field rabies infection were 150 km away. The last vaccination campaign with SAD P5/88 virus in the area where the animal had been found had taken place approximately 3 months earlier. Two other vaccine viruses, SAD VA1 and SAD B19, had been distributed during the same period in areas located more than 20 and 45 km away. The second case (Limburg) was an approximately 6-month-old juvenile fox that was observed on the street at the edge of the city. The emaciated animal with a slight paralysis of the hind legs showed no timidity and was shot. Pathological examinations revealed severe cachexia and double-sided profound interstitial bacterial pneumonia. The nearest rabies focus was about 55 km away, but SAD B19-loaded vaccine baits had been distributed in the immediate vicinity 1 month earlier. Baits containing SAD VA1 and SAD P5/88 had been distributed more recently (1–2 weeks earlier) in areas located approximately 55 km away.

The first Austrian case (no. 3, “Völkermarkt”) was a young fox showing no typical signs of rabies; approximately 1 month earlier SAD P5/88 vaccine baits had been distributed in this area. The fourth fox (Mettmann) also showed paralysis of the hind quarters and moved in circles before being run over by a tractor. More than 2 months before, SAD B19-containing vaccine baits had been distributed in the area during the annual spring campaign. The nearest recent reported rabies case involved a badger more than 150 km away. The fifth case (Kusel) concerns a lactating vixen that did not show normal shy behaviour, whereupon it was killed and submitted for rabies diagnosis. A rabies focus caused by a recent outbreak was located about 20 km away and approximately 1 month before SAD B19 vaccine baits had been distributed in the area.

The Austrian case no. 6 was an adult fox that was shot during a regular hunting trip close to the border with Hungary. No vaccine bait had been distributed in this area for at least 3–4 months. Furthermore, no other rabies case had been reported in this area.

Rabies diagnostic tests performed at the veterinary laboratories and at FLI are summarized in Table 4. Three animals tested negative for rabies using the standard FAT but were positive in RTCIT after two passages. With the panel of anti-nucleocapsid MAbs, the isolates were characterised in five cases as atypical RABV strains and not vaccine virus; no typing was done with no. 6 (Table 4). Although case no. 5 (Kusel) tested negative for rabies in FAT, histological examinations of the brain tissue revealed a profound mononuclear perivasculitis with infiltration of lymphocytes and histiocytes accompanied by the appearance of singular neutrophile granulocytes (Fig. 1a). IHC showed granular rabies-specific reactions in the cytoplasm of singular ganglia cells in the cortex, brain stem and Pukinje cells of the cerebellum but not in the Ammons’ horn (Fig. 1b).

Table 4 Rabies cases associated with oral rabies vaccine virus: results of the different rabies diagnosis tests applied
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Fig. 1
figure 1

Histopathological findings at high magnification (×40) (a) and rabies-antigen-specific IHC staining (dark red pigment) (b) in the brain tissue of the fifth reported vaccine-associated fox rabies case (Kusel)

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In two (no. 1 and 6) and three cases (no. 2, 4, and 5), respectively, partial sequence analysis of the 586-, 367- and 216-bp fragments of the N gene, the N–P intergenic region and the G gene revealed a 100% sequence identity to the vaccine virus strains SAD P5/88 and SAD B19 distributed in the area, when compared to sequences of commercially available SAD derivatives. In one case (no. 3), molecular characterisation of the SAD isolate revealed identity to a vaccine strain that was different from the one reportedly distributed in the area in 2004 (Table 5).

Table 5 Results of partial sequencing of the 6 vaccine-virus-associated rabies cases from Germany and Austria
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Complete genome sequences of the two SAD P5/88 virus isolates showed one identical nucleotide exchange in the L gene that did not alter the deduced amino acid sequence when compared to the SAD P5/88 virus (EF206715). In contrast, 3 of the 4 SAD B19 virus isolates had between 1 and 5 nucleotide alterations in different genes, of which 4 resulted in an exchange of amino acids in comparison with SAD B19 (EF206709). Nucleotide alterations mostly occurred in the highly conserved blocks (I–IV) and in block V of the L gene. Only one isolate (no. 4, EU886634) was 100% identical to the original vaccine virus (Table 6).

Table 6 Differences in complete genome sequence between the SAD isolates and commercial batches of SAD P5/88 and SAD B19 [26]
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Fox cubs experimentally inoculated with high doses of isolates no. 2 and 3 and with WSV of both vaccine viruses did not succumb to rabies and survived a 110-day observation period. No rabies-specific RNA was found in any of the saliva swabs tested. All animals, except two foxes that had been inoculated with a rabies virus isolate of dog origin as a positive control, developed high levels of VNA. The two control animals died 7–8 days post inoculation, and the presence of rabies was confirmed by FAT (Table 7).

Table 7 Virological and serological results for foxes inoculated with two vaccine-virus-associated isolates, original vaccine viruses and a RABV of dog origin
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Discussion

Despite extensive safety studies required for oral rabies vaccine viruses [49, 50], vaccination with vaccines containing replication-competent attenuated viruses can induce disease under certain circumstances. Hence, the six SAD B19- and SAD P5/88-associated rabies cases described here parallel similar findings with other oral rabies vaccine viruses such as ERA and SAD Berne [22, 47]. The results obtained in this study provide a basis for a detailed risk assessment for the observed vaccine-virus-associated rabies cases in red foxes after oral rabies vaccination campaigns in Germany and Austria.

Three of the six cases (no. 1, 4, and 5) tested negative for rabies using FAT. However, histological and histochemical examination of the brain of the FAT-negative animal no. 5 clearly revealed changes that are typical for a rabies infection. Although the standard procedure for the FAT requires testing of brain tissue from different areas (ammonshorn, cerebellum, brain stem) within the CNS [15], the reason for the failure of the FAT to detect rabies infection in these samples is unknown. Presumably, vaccine virus was present below the threshold of detection, which is supported by the fact that serial passaging in cell culture was necessary to isolate virus in RTCIT. The occurrence of such a diagnostic result, FAT-negative but RTCIT-positive, is very rare; only 0.05% of approximately 21,700 samples in a German database tested RTCIT-positive after the FAT gave a negative or inconclusive result (Müller, unpublished data). Apparently, the high incidence of such results in the cases described here is not caused by certain properties of the vaccine virus. During experimental studies it was shown that when small rodents succumbed to rabies after oral administration with SAD B19, the animal tested positive for rabies using FAT [45]. On the other hand, extensive safety studies indicated that after intracerebral inoculation into foxes, SAD B19 did not induce disease and was rapidly cleared from the brain [45]. Thus, at the time the animals were examined, the vaccine virus may have almost vanished. Under routine rabies surveillance, no further tests would have to be performed on the foxes because the FAT was negative and no direct human contact had occurred [15]. These animals, however, were reported to have shown atypical behaviour. This could also have been due to an infection with canine distemper virus (CDV), which is known to occur in Central Europe and induces symptoms that look very similar to rabies [24]. A differential diagnosis for CDV or any other agents causing neurological disorders was not conducted as a matter of routine; however, confirmatory tests for rabies (RTCIT) were conducted, and eventually, rabies infections were diagnosed.

All cases except no. 5 were found at great distances from the residual rabies foci in the respective areas, prompting characterization of the isolates to investigate their origin. Examination with the panel of anti-rabies-nucleoprotein MAbs gave questionable results, and neither of the isolates could be differentiated initially as vaccine virus, but were identified as atypical RABV strains [14, 37]. Since the atypical fox rabies strain known to circulate in northeastern Europe, including the eastern parts of Germany (Table 4; [43]), had never been reported from West Germany, where all the German cases (no. 1, 2, 4, and 5) were located, further investigations were needed. The observed variance in the typing with MAb MW 187.6.1 may be one explanation for why vaccine-associated cases were not reported using this MAb panel, especially in countries where atypical RABV variants are known to circulate [33]. Hence, the validity of the reduced MAb panel needs reassessment. Also, a comparative study on the reactivity of this particular MAb with oral rabies vaccines other than SAD-B19 and SAD P5/88 has not been undertaken yet. For example, since 1992, approximately 23 million SAD Berne vaccine baits have been distributed, and all RABV isolates from vaccination areas were identified as field strains using exactly the same panel of MAbs [33]. However, using only a fraction of these numbers of baits, three SAD Berne-associated rabies cases were reported from Switzerland [47].

Partial sequence analysis of the N and G genes, and the N–P intergenic region clearly identified the isolates as being similar to the vaccine virus distributed in the area, except for no. 3, which showed 100% identity with SAD B19 (Table 5). Vaccine baits containing SAD B19 were used for vaccination campaigns in Austria for the last time in 1994 (Table 1). Also, the baits distributed in 2004 and 2006 contained SAD P5/88, as confirmed by sequence analysis at the national reference laboratory (data not shown). It seems unlikely that the animal may have contracted the virus from SAD B19 baits distributed in neighbouring Slovenia, which is separated from Austria by a high Alpine mountain range, or translocation of wildlife [10]. Thus, its origin remains unclear.

Early or late death phenomena as consequences of interactions between oral vaccination and rabies infection [4, 6] can also be ruled out considering the fact that five of six cases occurred in areas that were free of terrestrial rabies, with the nearest rabies foci 60–150 km away. Data on the ranging behaviour of rabid foxes suggest that infected animals are normally found within or at the borders of their home ranges [1].

The most likely explanation for these cases is reversion to virulence or residual pathogenicity of the vaccine virus. However, although there is a low residual pathogenicity in rodents for both vaccine viruses after experimental inoculation, during extensive safety and immunogenicity studies, no adverse reactions were observed in the target species (n > 200), including pregnant vixens and cubs [34, 35, 39]. Foxes, including young animals, were also exposed to extremely high doses of up to 100 times the minimum effective dose without causing signs of disease [45]. However, the immune status of the animals involved may differ, with serious effects on the outcome of vaccination. In not-yet-immunocompetent (no. 1–4) and/or immunocompromised animals (no. 2 had been diagnosed with a severe pneumonia), this could lead to lethal infection resulting in clinical disease. However, the vaccine virus associated with this latter case, SAD B19, did not induce disease, even in animals that has been experimentally treated with corticosteroids to mediate immunosuppression [11].

With respect to reversion to virulence, RNA viruses like rabies are known to have high mutation rates, resulting from the lack of proofreading by RNA polymerases. Several parts of the rabies genome are known to affect virus virulence, like residues 333 and 194 of the rabies virus glycoprotein [16, 21]. However, a high genetic stability of SAD B19 after serial passages in vivo has been demonstrated [3]. Sequence analyses of the virus isolates described here confirm these findings, as the great majority of genetic alterations in the full-genome sequences of 5 individual isolates were silent and mainly found in the L gene (Table 6). Mutations due to the manufacturing process, resulting in possible reversion of the vaccine viruses can be ruled out, as studies showed 100% identity in the complete genome of a recent SAD B19 batch compared to the original strain [12, 26]. Therefore, the observed mutations in the genomes of the isolates from the individual cases occurred most likely after bait uptake. Whereas three isolates (no. 1, 4, and 6) either had no or only a single silent nucleotide alteration, single amino acid exchanges were found in viruses isolated from three animals (no. 2, 3, and 5) but not in regions known to play a role in pathogenicity. This is supported by the animal experiments conducted with material prepared from the isolates from two of the vaccine-associated cases (no. 2, and 3). Inoculation of the animals with high doses of the isolated vaccine viruses did not lead to any signs of disease, histopathological alterations or virus excretion in saliva.

Certain aspects of the epidemiology of the vaccine-associated rabies cases described here, however, remain inconclusive. In cases 1 and 6, the last vaccination campaign in the area had taken place many months earlier, indicating an unusually long incubation period for attenuated rabies viruses. However, similar prolonged incubation periods have been reported for rabies cases associated with the ERA strain [22]. Also, in three cases (no. 1, 3, and 4) the infected foxes were either not yet born or had just been born at the time of bait distribution in spring. Direct consumption of the bait by the young animals seems unlikely, because they would have been at most 3 weeks old and hardly able to feed on the solid bait. Also, in the rare cases in which vaccine baits were located and consumed 1 month or more after distribution in the field [46], the thermally induced loss of infectivity would have reached a level at which it would be unlikely that the vaccine virus could actively replicate, much less infect and induce disease [44]. Unfortunately, it was not possible to determine if the animals had indeed consumed a vaccine bait because since the end of the 1990s, vaccine baits used in Germany were not allowed to contain the biomarker tetracycline. However, it cannot be excluded that the young foxes actually had direct contact with the baits. Another possibility is that the vixen could have consumed a bait and licked (grooming behaviour) her offspring immediately afterwards, transferring the vaccine virus to her not yet immunocompetent cub(s). However, horizontal or vertical transmission seems unlikely, since intensive studies in young foxes born to recently vaccinated vixens never showed transmission of the vaccine virus from mother to offspring or from vaccinated cubs to non-vaccinated littermates [34]. More convincing evidence against establishment of the vaccine virus in the fox population is given by the fact that no further cases were reported from the affected areas. For the sake of completeness, human error during diagnosis must be mentioned as a possibility as well. For example, recently, two unrelated suspected rabies cases from non-vaccinated areas in Germany were identified as false positives during routine surveillance (unpublished results).

In summary, we describe six vaccine-associated cases of rabies in red foxes, detected when the vaccination campaigns were nearing their ends, i.e. when rabies cases were rare, and these cases were therefore investigated more thoroughly than those in the early-years of oral vaccination. After the first detection of possible vaccine-associated cases, awareness of such incidents increased. All of these cases were spatially and temporarily isolated and did not spread or become established within the target species population. Therefore, these cases have no epidemiological relevance [22].

According to recommendations, every rabies case from vaccination areas should be subjected to further characterization to distinguish field strains from oral vaccine virus [20]. To address this recommendation objectively, a comparison of the number of vaccine doses of different oral rabies vaccines used for oral vaccination campaigns throughout Europe and the number of RABV isolates characterised from areas where those vaccines have been distributed would be needed. However, for many European countries, data are not available. Since the large-scale implementation of ORV in the 1980s, SAD B19 and SAD P5/88 have been the most widely used vaccine virus strains throughout Europe [26]. Since then, more than 10,200 rabies cases from vaccination areas have been characterized, and an involvement of SAD vaccine virus was suggested in only 6 cases.

Using standard rabies diagnostic tools (FAT), only 3 of 6 cases would have been identified as rabies cases. Furthermore, only after intensified examination were these cases linked to the oral rabies vaccine viruses, emphasizing the intensive screening required to detect these cases. Therefore, there is reason to believe that the occurrences of similar cases have been underreported in Europe. Hence, it seems that the number of reported vaccine virus-associated cases is determined more by the intensity of the surveillance efforts than by differences among the vaccine viruses.