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Background: Domestic carnivores can introduce rabies into disease-free countries or areas if they are incubating the disease and transported during the pre-symptomatic period. For pets moved into the European Union, the European Commission decided to establish a system of community approval of laboratories willing to carry out the rabies serological controls to guarantee an effective control system. As the specific institute to coordinate the approval of the laboratories, designated by the European Commission in 2000, our laboratory organizes annual proficiency tests (PT) for laboratories already agreed or willing to be agreed to perform rabies serological controls (by detecting rabies virus neutralizing antibodies only) in the frame of international trade., Methodology/principal Findings: The assessment criteria of this PT rely on the analysis of the specificity and the intra-laboratory consistency. The approach used to evaluate the degree of laboratory consistency is based on the use of compiled data obtained from previous PT campaigns, and is measured by the quality of a regression model. By using historical data for calculating assigned values and associated standard deviations, instead of values obtained from only one campaign, they became robust without any additional statistical treatment. In the present paper, more than 800 historical values were compiled for each of the regression parameters., Conclusions/significance: Since the beginning of these PT schemes in 1999, the overall percentage of failing laboratories remained stable over the years (4.1%) while the number of participants increased to 79 in 2018. This highlighted the robustness and the consistency of the statistical analyses used to assess the laboratory's performance over the years. The improvements carried out and the consistency of our statistical analyses have resulted in the compliance of the rabies serology PT with the ISO/IEC 17043 and ISO 13528:2015 International Standards., Competing Interests: The authors have declared that no competing interests exist.

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.

Regulations governing international movements of domestic carnivores from rabies-infected to rabies-free countries have recently been loosened, with the adoption of a system that combines vaccination against rabies and serological surveillance (neutralising antibody titration test with a threshold of 0.5 UI/ml). Since 1993, the Research Laboratory for Rabies and Wild Animal Pathology in Nancy, France, has analysed over 25,000 sera from dogs and cats using a viral seroneutralisation technique. The statistical analyses performed during this time show that cats respond better than dogs. Although no significant difference in titres was observed between primovaccinated and repeat-vaccinated cats, repeat-vaccinated dogs had titres above 0.5 IU/ml more frequently. In primovaccinated dogs, monovalent vaccines offered a better serological conversion rate than multivalent ones. Finally, the results of these analyses showed a strong correlation between antibody counts and the time that elapsed between the last vaccination and the blood sampling.

Humoral and cell-mediated immune responses of 36 captive foxes to two oral vaccines against rabies currently used for foxes in Europe were studied. The Street Alabama Dufferin (SAD) mutant Gif (SAG2) vaccine has been selected by double mutation from the SAD virus. The vaccinia recombinant virus (V-RG) expresses the rabies glycoprotein. Both vaccines induce similar humoral and cell-mediated responses after primary and secondary oral administration. We observed a typical anamnestic response, although of a limited duration, after the booster vaccination. Therefore, our results suggested that two successive oral vaccination campaigns should not significantly improve the immunisation of foxes. Lymphocyte in vitro proliferative response to the SAD antigen highlighted the presence in blood of a T-cell specific memory 6 months after vaccination. The synthesis of several vulpine cytokines was detected in peripheral blood mononuclear cells (PBMC) stimulated by SAD antigen via reverse transcription polymerase chain amplification. The data showed a concomitant expression of interleukin (IL)-4 and interferon-gamma in PBMC of vaccinated foxes. No change was detected in the level of IL-2, IL-10 and IL-12 synthesis, whereas the pro-inflammatory cytokine tumour necrosis factor-alpha seemed involved in the activation of naive T lymphocytes.

The safety of the SAG2 rabies virus, a highly attenuated mutant of the SAD strain intended to vaccinate dogs by the oral route, was evaluated in local Tunisian dogs and in five other local species likely to consume vaccine baits. These species were the domestic cat (Felis catus), the jackal (Canis aureus), the jerboa (Jaculus orientalis), the merion (Meriones sp.) and the gerbil (Gerbillus campestris). The vaccine was administered orally to 21 dogs, 11 cats and eight jackals and orally or intramuscularly to 62 wild rodents of the above-mentioned species. Seven dogs, one cat, five jackals all juvenile and with poor health status) and two rodents died for intercurrent causes. The others were observed for 60-180 days. No animal showed any rabies symptom. Seroneutralizing antibodies were observed in all experimental groups, only after vaccination, with the highest rate being observed in jackals and rodents. The rabies virus was detected in the oral cavity of three cats 6 h after oral instillation, but was not isolated later either in saliva or in salivary glands. Tissue samples (brain and salivary glands) from dead or euthanized animals were examined for the rabies virus antigen by a fluorescent antibody test. No rabies antigen was detected. These trials confirm the safety of the SAG2 strain on the Tunisian species already demonstrated by other authors on many other target and non target species.

The rapid fluorescent focus inhibition test (RFFIT) and the fluorescent antibody virus neutralization test (FAVNT) are both diagnostic tests for determining levels of rabies neutralizing antibodies. An automated method for determining fluorescence has been implemented to reduce the work time required for fluorescent visual microscopic observations. The automated method offers several advantages over conventional visual observation, such as the ability to rapidly test many samples. The antibody titers obtained with automated techniques were similar to those obtained with both the RFFIT (n = 165, r = 0.93, P < 0.001) and the FAVNT (n = 52, r = 0.99, P < 0.001).

Two bait delivery systems for the oral immunization of dogs against rabies were tested in small scale field trials in a semi-rural area in Tunisia: bait delivery to owned dogs during door to door visits of households (door to door baiting) and distribution of baits on transect lines (transect line baiting). A prototype bait (DBL2) configured for industrial production and containing either sulfadimethoxine (SDM) as a systemic marker or Rhodamine B as a topical marker was used. The overall proportion of dogs which took a bait and presented topical marker staining after door to door baiting was 59.1%. The total time and costs spent per bait accepting dog averaged 34 person minutes and US$4, respectively. Unconsummated baits were readily recovered. No unprotected human contacts with baits were recorded. Door to door baiting is a very specific but time-consuming method that enables a safe administration of vaccine baits to owned dogs. For transect line baiting, baits were distributed at a density of ca 3000 baits per km2 along double transect lines. Baits were recovered after 20 h. According to the proportion of SDM positive serum samples, 24.1% of owned dogs in the baiting area had consumed baits. Of all owned and ownerless dogs, presumably free-roaming during transect line baiting, > 40% had consumed baits. The total time and costs spent per bait accepting dog averaged 48 person minutes and ca US$20, respectively. The household census revealed 32 direct human contacts with the bait matrix which corresponds to 1.4% of inhabitants. Placing baits on transect lines gives the possibility to vaccinate dogs not accessible by vaccination systems which base on dog owner participation. However, the method is not specific, less safe than other systems, not easily accepted by the human population, and costly.

A microtest named the FAVN test (fluorescent antibody virus neutralisation test), which is an adaptation of the original rapid fluorescent focus inhibition test (RFFIT) has been developed and evaluated. One hundred percent specificity was estimated using 414 sera from dogs sampled in rabies-free areas or from non-vaccinated animals. The accuracy as determined by the agreement between observed and expected values using sera of known titres was satisfactory. Serum samples from unvaccinated and vaccinated dogs (using sera with titres near 0.5 IU/ml) were assayed for rabies antibody by the FAVN test, the RFFIT and the mouse neutralisation test (MNT): comparative results obtained on the same sera with the three tests showed good agreement. Furthermore, distinguishing negative sera from positive sera with low titres is much easier with the FAVN test than with the RFFIT.

The safety of the SAG2 virus, a low virulence mutant of the SAD strain, was investigated in ten species of mammals and seven species of birds liable to consume vaccine baits. These species are the western hedgehog (Erinaceus europaeus), the meadow vole (Microtus arvalis), the bank vole (Clethrionomys glareolus), the water vole (Arvicola terrestris), the field mouse (Apodemus flavicollis or A. sylvaticus), the Norway rat (Rattus norvegicus), the european badger (Meles meles), the domestic ferret (Mustela putorius furo), the wild boar (Sus scrofa), the domestic goat (Capra hircus), the carrion crow (Corvus corone), the rook (Corvus frugilegus), the buzzard (Buteo buteo), the red kite (Milvus milvus), the tawny owl (Strix aluco), the long-eared owl (Asio otus) and the barn owl (Tyto alba). The vaccine was administered orally to each species, by an intramuscular (i.m.) route to the rodents and ferret, and by an intracerebral route to the field mouse. No pathogenicity was observed in the 169 animals vaccinated throughout an observation period of over 30 days. After euthanasia, no rabies virus could be detected either in the brain or in the salivary glands of any of the animals. The SAG2 virus administered orally, triggered a specific seroconversion in the field mouse, wild boar, ferret and most of the raptors. Following administration by the i.m. route, specific antibody titres were observed in most of the rodents, as well as in the ferrets.

Doubt has sometimes been cast upon the protective effect of rabies antibodies in serum. Animals and humans suffering from fatal rabies often produce high antibody titres, while rabies cases are also observed in vaccinated animals. Cellular immunity is also largely involved in protection. Nevertheless, a large number of laboratory experiments and field observations clearly demonstrate that cats and dogs which develop antibodies after vaccination and before challenge have a very high probability of surviving any challenge, no matter how strong the dose and which virus strain was used. Rabies antibody titration can, therefore, afford a strong additional guarantee to the vaccination certificates accompanying domestic carnivores during transportation between countries. Quarantine rules should also be adapted to the epidemiological features in the exporting country, e.g. statistics of vaccination failure in cats and dogs and host-virus adaptation of the rabies strains circulating in these countries.

Two rabies virus strains collected from naturally infected foxes in France in 1976 and 1986 were inoculated in 2 groups each consisting of 10 foxes (approximately 50 lethal doses50 mouse intracerebral per fox). Another 20 healthy foxes were kept in the same cages as the inoculated animals in order to study the transmission of both strains. All the inoculated foxes became rabid and transmitted rabies to their cage companion. The intervals between death of the inoculated foxes and death of the cage companion were significantly more variable (P less than 0.001) with the earlier strain, from 1 to 33 days compared to the range obtained with the later strain, 10-14 days. However, the mean intervals did not differ. Epidemiological consequences of such a decrease in variability, if confirmed in the field could decrease the epidemiological extent of rabies. Viremia was demonstrated in 2 foxes inoculated with the later strain but we cannot assume this to be a characteristic of this strain compared with the earlier strain. Rabies virus (both strains) was isolated from the saliva of foxes 29 days or less before the onset of clinical symptoms. This last result raises the question of the time limit as regards security before onset of clinical symptoms in animals involved in human contamination.

Four different serological techniques were used for the determination of antibody levels against rabies virus in 55 vaccinated street dogs: mouse neutralization test (reference), rapid fluorescent focus inhibition test, plaque reduction test and immunoenzymatic test (protein A). The results obtained with each of the last three methods were compared with those obtained with the reference test: correlation coefficients were, respectively, 0.810, 0.812 and 0.682, each being significantly correlated with the others. Therefore the three techniques may each be recommended as an alternative to the mouse neutralization test for routine titrations; the immunoenzymatic method, which titrates not only neutralizing antibodies being, nevertheless, better used as a screening test.

Rabies virus from the submandibular salivary gland of a naturally infected fox was adapted to growth in BHK-21 cells. The pathogenicity of the original isolate and the cell culture adapted virus were compared by the intramuscular and oral routes in mice and foxes. Animals surviving exposure were tested for serum rabies antibodies (immunogenic efficiency) and for their ability to survive a second challenge with rabies virus (protective efficiency). In mice, ratios between lethal and protective doses of the two strains via the oral or muscular route were similar. In foxes, however, pathogenic efficiency was modified after cell culture adaptation, although good immunogenic and protective efficiency was maintained. Inoculation via the oral route resulted in lethal infection with the wild-type strain and survival (with some subsequent immunity) with the cell-adapted strain. Via the intramuscular route, foxes given high doses of cell culture adapted virus survived whereas several foxes given lower doses died and virus could not be reisolated. It is concluded that safety testing of modified strains should be done with different dilutions of virus in the target species. "Autosterilizing" infections may result in erroneous diagnosis.

Antigenic differences between several strains of rabies virus, namely CVS, SAD and Flury (LEP) strains, were studied in cross-challenge experiments or cross-neutralization tests performed on sera of mice immunized with vaccines containing each strain. A typical wild fox virus strain was also included as challenge virus. The strain differences affected the relative potencies of the three vaccines in the European Pharmacopoeia mouse protection test for veterinary rabies vaccines, in that higher antigenic values were obtained when the vaccine strain was homologous to the challenge virus. This observation was confirmed by neutralizing antibody induction in mice.