Efficacy of a square presentation of V-RG vaccine baits in red fox, domestic dog and raccoon dogs

1. Efficacy of a square presentation of V-RG vaccine baits in red fox, domestic dog and raccoon dogs F. Cliquet1, A.L. Guiot2, C. Schumacher3, J. Maki3 and J. Barrat1 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. 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) 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.

3. Objective 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

4. Experimental design Number of animals DO : Vaccination Vaccine titre Challenge vaccinated controls 9 3 square VRG placebo bait 108.4 TCID50/dose - D30 New York rabies strain Dogs D91 Salivary gland homogenate from rabid foxes, 400 MIC LD50 vaccinated controls 10 10 square VRG placebo bait 108.4 TCID50/dose - Foxes D214 Salivary gland homogenate from a naturally infected coyote (CDC), 1600 MIC LD50 vaccinated controls 10 9 square VRG NA 108.8 TCID50/dose - Raccoon dogs NA : not administered All animals daily observed and kept at least for 90 days after challenge

5. Results - Bait acceptability Bait uptake time Bait consumption in Species vaccinates placebo 8/9 1/9 1/3 2/3 less than 1 hour less than 24 hours Dogs Foxes 10/10 10/10* less than 24 hours mean time of disappearance 4.2 hours (SD = 7 hours) Raccoon dogs 10/10 - *Doubtful for two foxes

6. Rabies virus neutralising antibodies (IU/ml) Day 28 Day 120 Group Animal No. Day -12 Day 7 Day 14 Day 21 Response to challenge Challenge Euthanasia 16 0,29 0,06 0,03 0,10 0,29 D (15) 17 < 0,07 0,06 0,29 1,99 3,46 0,87 S Vaccinated 18 < 0,06 0,03 0,06 0,10 0,22 0,50 S 19 < 0,03 0,03 0,03 0,03 0,17 0,10 S 20 < 0,04 0,03 0,03 0,10 0,13 0,38 S 21 < 0,06 0,03 0,04 0,17 0,38 0,87 S 22 > 0,87 0,04 0,04 0,06 0,10 0,22 S 23 < 0,06 0,03 0,03 0,07 0,07 0,66 S 24 0,13 0,04 0,04 0,13 0,22 0,87 S < GMT 0,09 0,04 0,05 0,12 0,24 0,45 SD 0,27 0,01 0,08 0,63 1,09 0,31 4 0,17 0,07 0,04 0,17 0,87 0,87 D (17) 5 0,10 0,04 0,03 0,04 0,07 10,45 D (15) Controls 6 < 0,03 0,04 0,03 0,04 0,07 D (14) < GMT 0,08 0,05 0,03 0,06 0,16 3,02 SD 0,07 0,02 0,01 0,08 0,46 6,77 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. Results Foxes : rabies virus neutralising antibodies 2 vaccinated foxes did not seroconvert

8. Group Mean mortality delay (days) Rabies diagnosis (FAT + cell culture) Total Dead Vaccinated 10 2 14 2 pos, 8 neg Controls 10 14,40 10 pos 10 Results Foxes : resistance to challenge

9. Results Raccoon dogs : rabies virus neutralising antibodies

10. Mean mortality delay (days) Rabies diagnosis (FAT + cell culture) Total Dead 10 0 - 10 neg 9 18,90 9 pos 9 Results Raccoon dogs : resistance to challenge Group Vaccinated Controls

11. Neutralising antibodies at D28 max (IU/ml) GMT (S.D.) positive/ total min (IU/ml) Dogs 0.07 3.46 0.24 (1.09) 1/9 Foxes 0.03 41.59 2.95 (13.27) 7/9 Raccoon dogs 1.51 13.77 10/10 5.38 (4.23) 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 : 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. 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. 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. 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)