Effectivity of oral vaccination of foxes against rabies with Flury HEP strain 675

Comp. lmmun. Microbiol. inlet! Dis., Vol. 5, Nos. I-3, pp. 181-184. 1982. Printed in Great Britain.

0147 9571/82/010181 04 $03.00/0 .~ 1982 Pergamon Press Ltd.

EFFECTIVITY OF ORAL VACCINATION OF FOXES AGAINST RABIES WITH F L U R Y HEP STRAIN 675 J. W. FROST, H. FRIEDRICH a n d G. WACHENDORFER Staatliches Veterin~iruntersuchungsamt, DeutschordenstraBe 48, D-6000 Frankfurt/Main 71, Federal Republic of Germany A ~ t r a c t - - A group of 14 adult foxes was orally vaccinated with 2 ml Flury HEP strain 675, titre

108.2TCIDso. Each fox seroconverted neutralizing antibody titres ranging between 1:40 and 1:640. These foxes survived an intramuscular challenge with 8000 mouse LDs0 pathogenic rabies virus, whereas six out of seven unprotected control foxes died within 28 days. This study was accompanied by virus isolation, fluorescence microscopic examination and neutralization tests. Key words: Rabies, oral vaccination, seroconversion, protective effect in foxes, Flury HEP strain 675 EFFICN(TITE DE LA VACCINATION RABIQUE DES RENARDS P A R V O l E O R A L E A V E C S O U C H E F L U R Y H E P 675 Resum~-Un groupe de 14 renards adultes a et+ vaccin6 par voie orale avec 2 ml de la souche Flury HEP 675 titrant 108.2 DICTso. Tousles renards ont present6 une s+roconversion, les titres en anticorps neutralisants variant de 1:40 fi 1 : 640. Ces renards ont surv6cu fi une 6preuve par voie musculaire avec un virus rabique pathog+ne titrant 8000 DLsosouris alors que 6 renards t6moins sur 7 mouraient dans les 28 jours. Cette 6tude comportait l'isolement du virus, l'examen en microscopie par immunofluorescence et les tests de neutralisation. Mots-clefs: Rage, vaccination par voie orale, seroconversion, immunisation des renards, souche Flury Hep 675

INTRODUCTION A very p r o m i s i n g alternative to the gassing o f fox dens was d e m o n s t r a t e d by Black a n d L a w s o n [1] by orally v a c c i n a t i n g foxes a g a i n s t rabies. In the p a s t few years research g r o u p s in C a n a d a , the U.S.A., F r a n c e , Switzerland a n d the F e d e r a l R e p u b l i c o f G e r m a n y investigated this subject. W i t h the S A D strain p a s s a g e d in BHK-21 cells a b o u t 80~o o f the foxes c o u l d be i m m u n i z e d via baits [2, 3]. D u e to the residual p a t h o g e n i c i t y o f this v a r i a n t o f the S A D strain [3-5], it was decided to t u r n to a cloned F l u r y H E P strain 675, offered by Prof. Bijlenga, Lyon. P r e l i m i n a r y a n d c o n t r a d i c t o r y i m m u n i z a t i o n results with this strain were r e p o r t e d in 1977 in N a n c y [6]. A f t e r a s a t i s f a c t o r y o u t c o m e o f i n n o c u i t y tests with strain 675 [7, 8], the protective effect o f this strain in foxes was tested. M A T E R I A L S A N D METHODS Y o u n g foxes, dug out from their dens, were i m m u n i z e d a g a i n s t distemper, hepatitis, l e p t o s p i r o s e s a n d fox encephalitis after a n t h e l m i n t i c t r e a t m e n t . T h e y were k e p t in g r o u p s o f five to six adults. F o r the v a c c i n a t i o n trial the foxes were h o u s e d singly in cages. B l o o d samples were t a k e n from each individual. Strain 675 was o b t a i n e d directly from Prof. Bijlenga, L y o n , on d r y ice a n d was s t o r e d in 181

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J . W . FROST, H. FRIEDRICH and G. WACHENI~RFER

Table 1. Antibody formation and protective effect of Flury HEP strain 675 in foxes after oral application Fox No.

Neutralizing antibodies (on days) 0 63 133

1

0

1:640

2 3 4 5 6 7 8 9 10 11 12 13 14 C1 C2 C3 C4 C5 C6 C7

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

1:640 1:160 1:80 1:640 1:80 1:320 1:320 1 : 160 1:640 1:160 1:80 1:80 1:40 0 0 0 0 0 0 0

1:1280 1:640 1:640 1:640 1:1280 1:320 1:640 1:320 1:640 1:1280 1:1280 1:320 1:40 1:320

Died Killed (days after challenge) --------------12 12 13 13 20 28 --

70 70 70 70 70 70 70 70 70 70 70 70 70 ------70

o u r l a b o r a t o r y at - 7 5 ' C . * F o r use in t h e foxes it was r a p i d l y t h a w e d a n d m i x e d w i t h D E A E - d e x t r a n to a final c o n c e n t r a t i o n o f 5 0 0 / t g / m l . W i t h the aid o f an a u t o m a t i c d o s e r 14 foxes r e c e i v e d 2 ml o f the v a c c i n e i n t o the b u c c a l cavity. S e v e n foxes s e r v e d as c o n t r o l s . F o r 9 w e e k s t h e a n i m a l s w e r e o b s e r v e d daily. O n c e a g a i n b l o o d s a m p l e s w e r e d r a w n a n d t h e v i r u l e n t c h a l l e n g e d o n e by i n j e c t i o n o f 8000 m o u s e LDso o f t h e N Y C strain, 7th m o u s e p a s s a g e , i n t o the m u s c l e s o f t h e neck. B r a i n s f r o m all foxes d y i n g d u r i n g the e x p e r i m e n t w e r e e x a m i n e d for rabies virus a n t i g e n by the f l u o r e s c e n t a n t i b i o d y m e t h o d . T h e s u r v i v i n g a n i m a l s w e r e sacrified 70 d a y s a f t e r the v i r u l e n t c h a l l e n g e , b l o o d s a m p l e s w e r e c o l l e c t e d a n d the b r a i n s e x a m i n e d by i m m u n o f l u o r e s c e n c e . V i r u s i s o l a t i o n was a t t e m p t e d by i n o c u l a t i o n o f h o m o g e n i z e d b r a i n m a t e r i a l i n t o m i c e a n d B H K - 2 1 cell cultures. S e r a w e r e c h e c k e d for n e u t r a l i z i n g a n t i b o d i e s in B H K - 2 1 cells c u l t u r e s as a l r e a d y d e s c r i b e d [9].

RESULTS A f t e r a d m i n i s t r a t i o n o f F l u r y H E P strain 675 n o a d v e r s e effects c o u l d be seen in the foxes w i t h i n n i n e weeks. A t t h a t d a t e all 14 v a c c i n a t e d foxes h a d s e r o c o n v e r t e d w i t h titres b e t w e e n 1:40 a n d 1:640 m e a s u r e d a g a i n s t 100 T C I D s 0 o f the h o m o l o g o u s virus strain. F r o m t h e c o n t r o l g r o u p six o u t o f seven foxes d i e d d u r i n g the first 28 d a y s p o s t * The indicated titre was 2 × l0 s TCIDs0, confirmed by the authors.

Oral vaccination against rabies

183

Table 2. Comparison of neutralizing activity of selected fox sera after oral application of Flury HEP strain 675 Fox No

2 3 4 10 14

Neutralizing antibody titre against CVS

Flury HEP 675

ERA

1:640 1:80 1:80 1:320 1:160

1:640 1:160 1:80 1:640 1:40

1:160 1:80 1:5 1:80 negative

challenge. O n e control fox a n d the 14 vaccinated remained healthy for the rest o f the o b s e r v a t i o n period. O n day 70 b l o o d samples were taken a n d the foxes killed by intracardial injection of T 61. The e x a m i n a t i o n of the brains for rabies virus antigen was negative a n d n o infectivity could be recovered by m o u s e i n o c u l a t i o n a n d tissue culture. The serological results are s u m m a r i z e d in Table 1. Selected sera were also tested by the same m e t h o d against CVS a n d E R A strains (Table 2).

DISCUSSION These results emphasize the necessity to use the h o m o l o g o u s virus strain when titrating antisera against a vaccinal rabies virus a n d also d e m o n s t r a t e the differing antigenic c o m p o s i t i o n of the strain used. O u r findings are in fine accordance with an analysis of the structural c o m p o n e n t s o f several rabies virus strains [10]. F l u r y H E P strain 675 has shown satisfactory i m m u n o g e n i c activity by the oral route as d e m o n s t r a t e d by seroconversion a n d virulent challenge. A t present the application o f this strain by bait in a larger g r o u p of foxes is studied.

Acknowledgement--These studies were supported by grants from the Federal Ministry of Nutrition, Agriculture

and Forestry and the German Association for Game Protection. We wish to thank Mrs. U. Kretschmer for expert technical assistance. REFERENCES 1. Black, J. G. and Lawson, K. F., Sylvatic rabies in the silver fox (Vulpes vulpes). Susceptibilityand immune response, Can. J. comp. Med. 34, 309-311 (1970). 2. Manz, D., Experimentelleund praktische untersuchungen zur markierung des rotfuchses (Vulpes vulpes L.) als vorbedingung zur beantwortung der frage nach seiner erreichbarkeit fiber k6der ffir staatliche tierseuchenbek/impfungsmassnahmemHabilitationsschrift, Giessen, 215 p 0976), Anhang 1978. 3. Winkler, W. G., Shaddock, J. H. and Williams, L. W., Oral rabies vaccine: evaluation of its infectivity in three species of rodents, Am. J. EpidemioL 104, 294-298 (1976). 4. FSrster, U., WachendSrfer, G., Schnettler, R. und Webber, J., Unsch/idlichkeitsprfifungenvon TollwutLebendvakzinen an wildlebenden S~iugern,Adv. Vet. Med. 257 262 (1976). 5. Wachend6rfer, G., Farrenkopf, R., Lohrbach, W., F6rster, U., Frost, J. W. und Valder, W. A., Passageversuche mit einer varianten des toUwut-impfstammes ERA bei wildlebenden species (Ondatra zibethica und Rattus norvegicus), Dtsch. TieriirztL Wschr. 85, 273 285 (1978). 6. Report of consultations on oral vaccination of foxes against rabies, Malzeville, Nancy, 12 13 November 1977.

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7. Frost, J. W. und Kiefert, C., Zur oralen immunisierung des fuchses gegen tollwut, Z. Siiugetierkunde 44, 52-55 (1979). 8. Wachend6rfer, G., Kiefert, C. and Frost, J. W., Safety tests with Flury HEP strain n 675 in wild living European mammals, Presented at the Nancy meeting, 3-5 June 1981. 9. Frost, J. W., Neutralization and immunofluorescence test (NIF) for the demonstration of antibodies against rabies virus, Zbl. Vet. Med. B 25, 338-340 (1978). 10. Dietzschold, B., Cox, J. H. and Schneider, L. G., Rabies virus: a comparison study by polypeptide analysis of vaccine strains with different pathogenic patterns, Virology 98, 63 75 (1979).