1. Towards the elimination of rabies in Eurasia, Paris, 27-30 May 2007 1 Efficacy of a square presentation of V-RG vaccine baits in red fox, domestic dog and raccoon dogs F. Cliquet 1, A.L. Guiot 2, C. Schumacher 3, J. Maki 3 and J. Barrat 1 1 WHO Collaborating Centre for Research and Management in Zoonoses Control, OIE Reference Laboratory for Rabies, Community Reference Laboratory for Rabies Serology, AFSSA Malzéville, France 2 Conseils en Pharmacie et Biologie Santé, Lyon, France 3 Merial, Lyon (France) and Athens (USA)

2. Towards the elimination of rabies in Eurasia, Paris, 27-30 May 2007 2 V-RG rabies vaccine * V-RG vaccine (Raboral ® V-RG) is a vaccinia virus recombinant coding for the rabies glycoprotein gene. * V-RG vaccine baits have been extensively used in France and in other European countries which are all rabies free (France, 2000 ; Belgium and Luxembourg, 2001) and is currently used in US and also in Ukraine. * This vaccine is one of the two vaccines recognised and recommended by the European Commission (2002) and WHO (2005) for oral vaccination of foxes against rabies. Rectangular Fishmeal polymer bait (France) for red foxes and raccoon dogs (Merial) Square Fishmeal or Chickenmeal Polymer bait (USA) for raccoons, coyotes and gray foxes (Merial)

3. Towards the elimination of rabies in Eurasia, Paris, 27-30 May 2007 3 To evaluate in captive animals (domestic dog, red fox and raccoon dog) the efficacy of a square V-RG bait produced in USA in captive animals Evaluation of : Attractiveness of the baits Induction of neutralising antibodies Protection after rabies challenge Objective

4. Towards the elimination of rabies in Eurasia, Paris, 27-30 May 2007 4 Experimental design Raccoon dogs vaccinated controls vaccinated controls vaccinated controls 9393 10 9 square VRG placebo bait square VRG placebo bait square VRG NA 10 8.4 TCID 50 /dose - 10 8.4 TCID 50 /dose - 10 8.8 TCID 50 /dose - D30 New York rabies strain D91 Salivary gland homogenate from rabid foxes, 400 MIC LD 50 D214 Salivary gland homogenate from a naturally infected coyote (CDC), 1600 MIC LD 50 Dogs Foxes NA : not administered All animals daily observed and kept at least for 90 days after challenge Number of animals DO : VaccinationVaccine titreChallenge

5. Towards the elimination of rabies in Eurasia, Paris, 27-30 May 2007 5 Results - Bait acceptability Species Dogs Foxes Raccoon dogs Bait consumption in vaccinatesplacebo Bait uptake time 8/9 1/9 10/10 1/3 2/3 10/10* - less than 1 hour less than 24 hours mean time of disappearance 4.2 hours (SD = 7 hours) * Doubtful for two foxes

6. Towards the elimination of rabies in Eurasia, Paris, 27-30 May 2007 6 Results Dogs : rabies virus neutralising antibodies and resistance to challenge S : survive D : die Only one dog out of 9 vaccinates seroconverted All three controls died between D14 and D17 One vaccinate died at D15

7. Towards the elimination of rabies in Eurasia, Paris, 27-30 May 2007 7 Results Foxes : rabies virus neutralising antibodies 2 vaccinated foxes did not seroconvert

8. Towards the elimination of rabies in Eurasia, Paris, 27-30 May 2007 8 Results Foxes : resistance to challenge Vaccinated Controls Mean mortality delay (days) 14 14,40 Total 10 Rabies diagnosis (FAT + cell culture) 2 pos, 8 neg 10 pos Dead 2 10 Group

9. Towards the elimination of rabies in Eurasia, Paris, 27-30 May 2007 9 Results Raccoon dogs : rabies virus neutralising antibodies

10. Towards the elimination of rabies in Eurasia, Paris, 27-30 May 2007 10 Vaccinated Controls Mean mortality delay (days) - 18,90 Total 10 9 Rabies diagnosis (FAT + cell culture) 10 neg 9 pos Dead 0 9 Group Results Raccoon dogs : resistance to challenge

11. Towards the elimination of rabies in Eurasia, Paris, 27-30 May 2007 11 Discussion - The square fishmeal polymer bait V-RG is well accepted by dogs, foxes and raccoon dogs. - The rate of seroconversion following bait uptake as well as the magnitude of antibody titres are different in the 3 species : 5.38 (4.23) Neutralising antibodies at D28 Dogs0.07 0.03 1.51 3.46 41.59 13.77 0.24 (1.09) 2.95 (13.27) 1/9 7/9 10/10Raccoon dogs Foxes min (IU/ml) max (IU/ml) GMT (S.D.) positive/ total The high levels of rabies antibodies observed in raccoon dogs have been also observed when vaccinated with an attenuated oral vaccine (Cliquet and al, 2006)

12. Towards the elimination of rabies in Eurasia, Paris, 27-30 May 2007 12 Discussion - The challenge was validated since all controls died from rabies (typical rabies signs were observed and diagnostic tests positive). - Protection after challenge was satisfactory in the three species tested. * Dogs : protection (8/9 dog) despite absence of seroconversion already largely documented. * Foxes : protection in 8/10 foxes ; two vaccinated foxes did not seroconvert and succumbed to rabies challenge. * Raccoon dogs : protection in all vaccinated raccoon dogs.

13. Towards the elimination of rabies in Eurasia, Paris, 27-30 May 2007 13 Conclusion - The square fishmeal polymer bait V-RG tested in domestic dogs, foxes and raccoon dogs was attractive, immunogenic and efficient. - The efficacy data support the idea of a potential sourcing of this oral rabies vaccine : for oral vaccination campaigns in those European countries where both foxes and raccoon dogs represent major wildlife species affected by rabies, for oral vaccination of dogs as a complement to other means (parenteral vaccination and ABC programs) in countries where canine rabies is endemic.

14. Towards the elimination of rabies in Eurasia, Paris, 27-30 May 2007 14 ACKNOWLEDGMENTS - Chuck Rupprecht (CDC) - Jacqueline Bailly, Michel Munier, Nicolas Penel, Josiane Ambert, Jean-Michel Bousset, Audrey Hamen, Michel Ritleng, Chantal Patron, Patricia Grosgeorge, Anouck Labadie, Estelle Litaize and Sébastien Kempff and Jean Luc Schereffer (AFSSA Nancy)