Florence Cliquet

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.

International interlaboratory trials on rabies diagnosis: An overview of results and variation in reference diagnosis techniques (fluorescent antibody test, rabies tissue culture infection test, mouse inoculation test) and molecular biology techniques

Interlaboratory trials on rabies diagnosis were organised in 2009 and in 2010 by the European Union Reference Laboratory (EURL) for rabies. In 2009, two panels of virus samples were sent to participating laboratories to compare results on reference diagnosis techniques and on RT-PCR. A single panel was sent in 2010 to test FAT (fluorescent antibody test), RTCIT (rabies tissue culture infection test) and RT-PCR techniques. The virus panels included the RABV, EBLV-1, EBLV-2 and ABLV strains. Results revealed that laboratories produced the highest proportion of concordant results using RT-PCR (90.5%) and FAT (87.1%), followed by RTCIT (70.0%) and MIT (35.0%) in 2009 and in FAT (85.0%) and RT-PCR (80.6%) followed by RTCIT (77.3%) in 2010. Errors were only observed in bat strains (i.e. none in the RABV strain) for the RT-PCR or FAT techniques, highlighting the need to improve diagnosis most specifically in such strains. RT-PCR was the technique showing the lowest rate of false negative results in either trial year, while RTCIT and MIT (performed in 2009 only) were the techniques with the lowest proportion of false positive results. Nevertheless, the FAT technique represented a good compromise with both satisfactory sensitivity and specificity, as only a few false positive (1.6% in 2009, 5.8% in 2010) and false negative results (1.6% in both 2009 and 2010) were detected. The analysis of technical questionnaires describing the protocols used by participating laboratories revealed variation in the methods used that may induce inconsistencies in the results. In this study, the number of readers for FAT slide examination was identified as a factor affecting significantly the results of laboratories, suggesting that two independent readers are necessary for routine rabies diagnosis. Our findings highlight the need for all rabies diagnostic laboratories to improve harmonisation of procedures.

Evaluation of ELISA for detection of rabies antibodies in domestic carnivores.

Serological tests of pets have increased as many rabies-free countries have amended their quarantine measures and adopted a scheme requiring rabies vaccination followed by a serological test. A European directive requires the measurement of neutralising antibodies as proof of protection to allow the free movement of pets within the European Union and between third countries non listed in the list C of regulation 998/2003 and European countries. At present, the recommended neutralisation tests (FAVN test or RFFIT) are time-consuming, expensive and require highly trained technicians as well as special laboratory facilities. The rabies ELISA designed by BioPro was developed initially for use for field samples from foxes to check the efficacy of oral vaccination campaigns in Europe. In this study, the specificity, sensitivity and reliability of this commercial rabies ELISA was evaluated for testing sera from dogs and cats involved in international trade. The specificity evaluated in 315 unvaccinated animals was 100%. Concordance of 86.2% was obtained when comparing BioPro ELISA to the gold standard FAVN test in 701 samples from vaccinated dogs and cats. The rabies ELISA developed recently can be considered a valuable method for the assessment of rabies antibodies in vaccinated domestic carnivores in combination with neutralisation tests.

Evaluation of a rapid immunochromatographic diagnostic test for the detection of rabies from brain material of European mammals

The surveillance of rabies relies on investigations conducted on dead suspected animals or animals showing clinical signs suggestive of rabies. An immunochromatographic method based on lateral flow principle has been evaluated against a collection of brain samples mainly of European mammals including bats. The performance of this new test has been compared to the conventional gold standard methods: the fluorescent Antibody Test (FAT) and the Rapid Tissue Culture Infection Test (RTCIT). This test enabled the detection of various rabies strains belonging to rabies species 1, 5, 6 and 7 and demonstrated an overall specificity of 100% and a sensitivity of more than 88% when compared to FAT and RTCIT. A total agreement between the Rapid Immunochromatographic Diagnostic Test and conventional technique results have been obtained for European bat samples.

Active surveillance of bat rabies in France: A 5-year study (2004–2009)

Active surveillance of bats in France started in 2004 with an analysis of 18 of the 45 bat species reported in Europe. Rabies antibodies were detected in six indigenous species, mainly in Eptesicus serotinus and Myotis myotis, suggesting previous contact with the EBLV-1 rabies virus. Nineteen of the 177 tested bats were shown serologically positive in seven sites, particularly in central and south-western France. Neither infectious viral particles nor viral genomes were detected in 173 and 308 tested oral swabs, respectively. The presence of neutralising antibodies in female bats (18.6%) was significantly higher than in males (5.6%).

Rabies in Europe: what are the risks?

Rabies remains a serious endemic disease in animal populations in many European countries. Oral vaccination by use of rabies vaccine baits has proved to be durably efficient for controlling and eliminating terrestrial rabies. However, the recurrence of rabies in some countries highlights the fragility of rabies-free country status and the need for continuous surveillance. In Eastern and Southern countries, the rabies control programmes for foxes should be accompanied by stray dog management measures in view of the high populations of strays in certain areas. Alerts of rabies in pets imported from enzootic countries are regularly reported in Europe, threatening the rabies-free status of terrestrial animals. New variants of rabies virus have been recently discovered in autochthonous bats, implying research studies to assess the efficacy of the current vaccines against those strains and the possible crossing of the species barrier in terrestrial mammals. The incidence of the disease in humans is very low, with cases contracted in Europe or in enzootic countries. Sustainable strategies of vaccination programmes in animals and improvement of public awareness, particularly for travelers, regarding rabies risks and legislation for pet movements would render accessible the elimination of rabies in Europe.

Evaluation of an ELISA to detect rabies antibodies in orally vaccinated foxes and raccoon dogs sampled in the field

The assessment of the efficacy of oral vaccination in wildlife is based on detection in the teeth of a biomarker (tetracycline) which is incorporated in the vaccine bait, and the quantification of rabies antibodies. A blocking ELISA was evaluated and compared with the FAVN test and a validated in-house ELISA, using sera from foxes and raccoon dogs collected following oral vaccination campaigns in France and Estonia. Specificity reached 100% in sera from naïve animals. A high concordance (95%) was observed between the BioPro ELISA and the FAVN test, which was similar in sera from red foxes and raccoon dogs. Concordance between the BioPro ELISA and the in-house ELISA reached 96.5% for sera from red foxes. The agreement with tetracycline results was excellent in the fox for both the BioPro ELISA (95.9%) and the FAVN test (91.8%). Concordance was slightly lower in the raccoon dog, with a value of 82.8% for the BioPro ELISA and 78.4% for the FAVN test. Rabies antibodies were detected with the BioPro ELISA in animals vaccinated with different types of vaccines and in highly haemolysed sera. The BioPro ELISA is a valuable test to assess the efficacy of oral vaccination in foxes and raccoon dogs.

Bat Rabies in France: A 24-Year Retrospective Epidemiological Study

Since bat rabies surveillance was first implemented in France in 1989, 48 autochthonous rabies cases without human contamination have been reported using routine diagnosis methods. In this retrospective study, data on bats submitted for rabies testing were analysed in order to better understand the epidemiology of EBLV-1 in bats in France and to investigate some epidemiological trends. Of the 3176 bats submitted for rabies diagnosis from 1989 to 2013, 1.96% (48/2447 analysed) were diagnosed positive. Among the twelve recognised virus species within the Lyssavirus genus, two species were isolated in France. 47 positive bats were morphologically identified as Eptesicus serotinus and were shown to be infected by both the EBLV-1a and the EBLV-1b lineages. Isolation of BBLV in Myotis nattereri was reported once in the north-east of France in 2012. The phylogenetic characterisation of all 47 French EBLV-1 isolates sampled between 1989 and 2013 and the French BBLV sample against 21 referenced partial nucleoprotein sequences confirmed the low genetic diversity of EBLV-1 despite its extensive geographical range. Statistical analysis performed on the serotine bat data collected from 1989 to 2013 showed seasonal variation of rabies occurrence with a significantly higher proportion of positive samples detected during the autumn compared to the spring and the summer period (34% of positive bats detected in autumn, 15% in summer, 13% in spring and 12% in winter). In this study, we have provided the details of the geographical distribution of EBLV-1a in the south-west of France and the north-south division of EBLV-1b with its subdivisions into three phylogenetic groups: group B1 in the north-west, group B2 in the centre and group B3 in the north-east of France.

Genetic strain modification of a live rabies virus vaccine widely used in Europe for wildlife oral vaccination

In Europe, the main reservoir and vector of rabies has been the red fox (Vulpes vulpes). Oral immunization of foxes with live vaccines, using attenuated rabies strains (SAD B19, SAD Bern), apathogenic mutants of an attenuated strain (SAG2) and the vaccinia-rabies glycoprotein recombinant virus vaccine (V-RG), has been shown to be the most effective method for the control and elimination of rabies. Among all vaccines currently used for wildlife oral vaccination, one vaccine (marketed as SAD Bern strain) has been widely used in Europe since 1992 with the distribution of 17million of baits in 2011. Because of the potential environmental safety risk of a live virus which could revert to virulence, the full genome sequencing of this vaccine was undertaken and the sequence was characterized and compared with those of referenced rabies viruses. The vaccine showed higher similarity to the strains belonging to the SAD B19 vaccine virus strains than to the SAD Bern vaccines. This study is the first one reporting on virus strain identity changes in this attenuated vaccine.

Comparative assay of fluorescent antibody test results among twelve European National Reference Laboratories using various anti-rabies conjugates

Twelve National Reference Laboratories (NRLs) for rabies have undertaken a comparative assay to assess the comparison of fluorescent antibody test (FAT) results using five coded commercial anti-rabies conjugates (Biorad, Bioveta, Fujirebio, Millipore, and SIFIN conjugates). Homogenized positive brain tissues infected with various lyssavirus species as well as negative samples were analyzed blindly using a standardized FAT procedure. Conjugates B, C, D, and E were found to be significantly more effective than conjugate A for GS7 (French RABV) diluted samples (1/8 and 1/100) while the frequency of concordant results of conjugates C and D differ significantly from conjugates A, B and E for CVS 27. For detection of EBLV-1 strains, conjugates C and D also presented a significantly lower frequency of discordant results compared to conjugates A, B and E. Conjugates B, C and D were found to be significantly more effective than conjugates E and A for EBLV-2 and ABLV samples. In view of these results, conjugates C and D set themselves apart from the others and appeared as the most effective of this 5-panel conjugates. This study clearly demonstrates that the variability of conjugates used by National Reference Laboratories can potentially lead to discordant results and influence assay sensitivity. In case of false negative results this could have a dramatic impact if the animal under investigation is responsible for human exposure. To avoid such situations, confirmatory tests should be implemented.