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Immunogenicity, efficacy and safety of an oral rabies vaccine (SAG-2) in dogs
Elsevier 0264-410X(95)00244-8
ELSEVIER
Vaccine, Vol. 14, No. 6, pp. 465-468, 1996 Copyright 0 1996 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0264-410X/96 $15+0.00
Immunogenicity, efficacy and safety of an oral rabies vaccine (SAG-2) in dogs M. Fekadu*§, S.B. Linhartl
S.L. Nesby*, J.H. Shaddock*, and D.W. Sanderlin*
C.L. Schumacher-f,
A study of immunogenicity and eficacy of Street Alabama Gtf (SAG-2) attenuated rabies virus vaccine in laboratory beagles was conducted. Four groups of ten dogs each received either 1.0 ml of SAG-2 orally on the tongue or 1.5 ml in baits. On day 180postvaccination, all dogs were challenged with a street rabies virus. The antibody response in groups that received the vaccine directly on the tongue was higher than in those vaccinated with baits, but the dtflerence between groups was not statistically signtficant. All vaccinated dogs survived, whereas 80% of controls died of rabies. Our findings demonstrate that the SAG-2 is a safe and eflective vaccine for oral immunization of canines. Copyright 0 1996 Elsevier Science-L&I. Keywords:
Rabies; vaccine; oral; dogs; carnivore; SAG-2
Rabies remains endemic in most developing countries and continues to exact a tremendous toll on the health of the population and the economic resources of these countries. The incidence of human rabies closely parallels that of the disease among domestic animals; ~90% of human cases are believed to be due to dogs, and much of the remainder to other domestic animals, especially cats’. Parenteral rabies vaccination of domestic animals, particularly do4s+ has been used to control rabies in many countries . The development of safe and effective oral rabies vaccines for free-ranging dogs that cannot be reached or contained for parenteral vaccination would facilitate the vaccination of a greater proportion of these animals. A number of candidate oral rabies vaccines are currently being evaluated for efficac . and safety testing in dogs and nontarget animal species CT One of these vaccines, an attenuated live rabies Street Alabama Dufferin (SAD) Bern strain, which has some residual pathogenicity for nontarget species, has been used for the oral vaccination of foxes in some European countries and Canada 9- l4 . Under the pressure of specific monoclonal antibodies (Mabs) on the SAD Bern strain, an avirulent antigenic mutant, Street Alabama Gif (SAG-l), was selected. This mutant has a I-nucleotide substitution in amino-acid position 333 of the glycoprotein15 and when tested for its pathogenicity in adult mice was shown to be almost apathogenic for adult
mice inoculated by the intracerebral route’“‘8. Further attenuation from SAD Bern was accomplished with the production of a double mutation at amino-acid 333 of the glycoprotein. This virus strain, called SAG-2, has a glutamate coded by GAA at position 333 instead of an arginine. This new codon differs from all the arginine triplets by two nucleotides”. SAG-2 is avirulent in adult mice by intercerebral and intermuscular routes. We have evaluated the immunogenicity of SAG-2 administered with or without bait in laboratory beagles. The results of this study are presented in this report.
MATERIALS
AND METHODS
Animals
Four groups of ten 1.5- to 2.5-year-old, purpose-bred beagles each received either 1.O ml of SAG-2 (titer: 1O8.5 or 107.5suckling SMICLD,, ml - ‘) orally on the tongue or 1.5 ml of SAG-2 (1O8-5or 107.5 SMICLD,, ml- ‘) in baits. A fifth group of ten dogs was retained as controls (Table I). All dogs were challenged with a street rabies virus 6 months after vaccination and observed for at least 3 months following challenge. Five to 6 months after challenge all dogs were euthanized by intravenous injection of barbiturate. Table 1 Groups of dogs administered SAG-2 vaccine orally with (w) or without (w/o) baits
*Division of Viral and Rickettsial Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Public Health Service, US Department of Health and Human Services, Atlanta, GA 30333, USA. tVlRBAC Laboratories, B. P. 27-06511 Carros Cedex, France. fsoutheastern Cooperative Wildlife Disease Study, College of Veterinary Medicine, University of Georgia, Athens, GA 30602, USA. $To whom correspondence should be addressed. (Received 23 June 1995; revised 13 November 1995; accepted 13 November 1995)
Dogs (10 per group) Group Group Group Group Group
I
w Bait
II Ill IV V
w Bait w/o Bait w/o Bait
Vaccine/dose 107.5 SMICLD 10e.5 SMICLD: 1O7.5SMICLD 1O’=’ SMICLD; Control
e SAG-2 SAG-2 SAG-2 SAG-2
5uckling mouse intracranial lethal dose 50%
Vaccine
1996 Volume
14 Number
6
465
Oral rabies vaccine: M. Fekadu et al. Table 2
Number of dogs in each vaccination
group with titers of tl:5a
Vaccine/dose SAG-2 SAG-2 SAG-2 SAG-2
lo= SMICLD 50 1O’=’ SMICLD 50 1 O= SMICLD 1O8.5 SMICLD5’50
%omplete
neutralization
w Bait w Bait w/o Bait w/o Bait at 1:5, demonstrates
Seroconversion 14
(>1:5) days postvaccination 28 42
4 6 5 9
6 :
A SAG-2 vaccine, batch S2/-1 grown in BHK cells was obtained from VIRBAC Laboratories (B.P. 27, 06511 Carros, Cedex, France) and stored in liquid nitrogen until use. The vaccine was quick-thawed and titrated both in suckling mice and tissue culture before use in vaccination. Vaccine that was left over after the vaccination of dogs was retitrated in tissue culture to determine possible titer loss. Baits
The baits used to administer the vaccine were ca. 2 cm in diameter and 5 cm in length, and cylindrical custom made (DuPont Nemours, Orange, TX, USA) for this experiment. They consisted of 57% dog biscuit meal, 20% meat and bone meal, 13% aquabind (EVA polymer), as a binder and 10% soybean oil as lubricant. A 1.5 ml virus suspension was deposited into 2-ml cylinder wax ampules which was sealed with paraffin and then inserted into the bait and offered to individual dogs. Dogs were denied the morning ration of feed on the day of vaccination. To determine if the baits were accepted, we monitored the dogs for at least 1 hour after the administration of the vaccine. antibody determination
Blood specimens were collected after vaccination and after challenge virus to monitor humoral antibody body titer was determined lox the focus inhibition test (RFFIT) .
at weekly intervals with a street rabies response. The antirapid fluorescence
Challenge virus
The virus strain used for challenging all dogs in the study was a dose of 106.5 MICLD,, street rabies virus strain isolated from a naturally infected dog at the Texas-Mexico border, as previously reported21. Virus isolation attempts from saliva swabs
The oral cavity of all vaccinated dogs was swabbed with a cotton swab dipped in Eagle’s media: Minimum Essential Modified (MEM) virus growth media with 10% Fetal Bovine Serum (FBS) at 1, 7, and 24 h after vaccination to determine the presence and the amount of residual virus in the mouth. The virus isolation attempts from the swabs were made by tissue culture and by suckling mouse inoculation. Virus isolation attempts from tissue specimens
All dogs that survived rabies challenge were euthanized 5-6 months after challenge by intravenous
466
10
180
186
8 10 8 10
8 10 9 10
10 10 10 10
sero-conversion
Vaccine
Rabies neutralizing
7 10 8 10
108
Vaccine 1996 Volume 14 Number 6
injection of barbiturate. Tissue specimens from the brain, spinal cord, tonsil, tongue, salivary gland, adrenal glands, and bladder were collected at necropsy. These tissue specimens were examined for viral antigen by fluorescence antibody staining (FA) of frozen tissue sections and for viable virus by inoculation of mouse neuroblastoma cells. RESULTS Attempts to isolate virus from saliva swabs collected at 1, 7, and 24 h after vaccine administration showed no virus present in any swab. Serum specimens collected at weekly intervals from dogs vaccinated with a dose of lo’.’ SMICD,, of SAG-2 directly on the tongue showed seroconversion in 5, 8, 8, and 8 of dogs on 14, 28, 42, and 108 days postvaccination, respectively (Table 2). Of the 10 dogs vaccinated with 1O8.5SMICLD,, directly on the tongue, 9 and 10 showed evidence of seroconversion on days 14 and 28 postvaccination, respectively (Table 2). Of the dogs vaccinated with a dose of 1O7.5SMICD,, of SAG-2 in baits, however, 4, 6, 7, and 8 dogs seroconverted on 14, 28, 42, and 180 days, respectively (Table 2). Six, 9, and 10 of 10 dogs vaccinated with 1Os.5 SMICD,, seroconverted 14, 28, and 42 days postvaccination, respectively (Table 2). By day 180, when the dogs were challenged with 106.5 MICLD,, street rabies virus, Mexican dog strain the geometric mean titer (GMT) of rabies neutralizing antibody had risen steadily to 2.8 IU ml ~ ’ in dogs vaccinated with 1Os.5SMICLD,, directly on the tongue and to 1.5 IU ml - ’ in dogs that were vaccinated with 108.5SMICLD,, in baits (Table 3). Six days after challenge, all dogs (including those which had failed to seroconvert) had high anamnestic responses with titers in excess of 0.5 IU ml - ’ (Table 3). All vaccinated dogs survived challenge, whereas eight of 10 unvaccinated control dogs died of rabies (Table 3). No viable virus was isolated in tissue specimens (i.e. brain, spinal cord, tonsils, tongue, salivary glands, adrenal glands, and bladder) collected at necropsy cu. 1 year after vaccination and 5-6 months after virus challenge. A dose of 1O7.5SMICLD,, ml ~ ’ of SAG-2 was as immunogenic as 108.5 SMICLDso ml ~ ’ when administered with or without bait (Table 2). The antibody response in groups that received the vaccine directly on the tongue was higher than those vaccinated with baits, but the difference between groups was not statistically significant. DISCUSSION In many countries rabies has been reduced from an epidemic to an endemic disease, and some countries
Oral rabies vaccine: M. Fekadu et al. Table 3 Number of dogs in each vaccination after challenge Vaccine SAG-2 SAG-2 SAG-2 SAG-2 Control
Dose 1O= 1O= 1O75 1 O=
SMCILD,, SMICLD, SMICLD,, SMICLD,,
aDate of challenge;
group with titers of 20.50 IU ml-’
Pre-challenge w Bait w Bait w/o Bait w/o bait
bGMT=Geometric
0.5 IU ml-’
<0.1-4.1 0.2-4.1 0.1-4.1 0.6-l 9.1
mean value. “Survivors;
180 days? after vaccination,
GMTb
Post-challenge
0.7 1.5 0.9 2.8
1.0>20.6 4.1220.6 1.0220.6 2.8220.6
6 days after challenge and survival
0.5 IU ml-’
GMT 6.8 13.4 7.9 12.2
SUN”
10/lod lo/lo
lo/lo lo/lo 2110
“number of survivors/inoculated
have eliminated rabies by parenteral immunization of domestic animals, mainly dogsZm7. In a number of those countries that managed to control dog rabies, wildlife subsequently emerged as a new rabies host11,22. The human and domestic animal rabies cases reported from those countries are mainly due to wildlife exposure. Between 1978 and 1982, the repetition of field trials in Alpine valleys resulted in the elimination of rabies in several regions of the Swiss Alps12. Several European countries and Canada have used oral vaccination as a means of controlling rabies in wild carnivoresx*9.“. The vaccine strain used in those field trials was the SAD strain known to immunize foxes and to rotect against ,B street rabies challenge when given orally . However, the data obtained from orally vaccinated domestic dogs were inconclusive”. The SAD strain has some residual pathogenicity for a variety of rodent species, and occasionally for domestic animals and wild carnivores with an impaired immune response13. In order to select avirulent antigenic escape mutants the SAD strain was incubated with Mabs that possessed binding properties for the antigenic site III (amino acid 330-338) of the viral glycoprotein. Arginine at position 333 has been linked to pathogenicity in several rabies virus strains15. During the selection process, under the pressure of site III specific binding mabs, only avirulent SAG-l variants of SAD Bern escaped neutralization and were able to replicate in cell culture. The substitution of the arginine at position 333 of site III of the rabies glycoprotein decreased or almost eliminated the pathogenicity of several clones for adult laboratory mice15. Subsequently, another escape variant was selected from SAD Bern in two steps in the presence of two different anti-glycoprotein Mabs. Both of the first two nucleotides in the codon for the amino acid 333 were affected, resulting in the incorporation of glutamine instead of arginine at this position. The resulting double mutant, SAG-2, showed no signs of the residual pathogenicity that characterized its parent (SAD Bern). The safety of SAG-2 has been demonstrated in the laboratory mouse, which serves as a sensitive animal model for the detection of residual pathogenicity of modified rabies vaccines”. SAG-2 has thus far been shown to be avirulent to most immunocompetent adult animals. Because of the replacement of the first two nucleotides in the codon of amino acid 333 of the glycoprotein, SAG-2 cannot revert to the pathogenic phenotype of the parent strain, SAD Bern, bi a single mutational event, as may happen with SAG-l ’ _ Because of these unique properties of SAG-2, we wanted to test the immunogenicity, efficacy, duration of immunity and safety of the SAG-2 vaccine strain in laboratory beagles. Our results show that SAG-2 vaccine administered directly on the tongue or in baits
induces an adequate antibody response in most dogs as early as 14 days after vaccination. Dogs that had no detectable humoral antibody titer were also protected against challenge with a street rabies virus 6 months after vaccination; these dogs developed a high anamnestic response indicating that the vaccine had induced a cellular immune response. Induction of immunity and protection were not dose dependent in the range of 1O7.5 SMCIDso to 1O8.5 SMICD,, when administered with or without bait. We did not establish the lower limit of the effective dose. The antibody response in groups that received the vaccine directly on the tongue was higher than that for groups vaccinated with baits, but the difference between groups was not statistically significant. The protecting capability of this system in laboratory trials seems not to be dependent on whether or not the dogs developed a neutralizing antibody response. The safety of the vaccine was tested by monitoring for residual virus in the oral cavity of dogs given vaccine with or without bait, at times ranging between one and 24 h after vaccination, but no virus was isolated from any of the swabs. These findings provide clear evidence that the SAG-2 vaccine is an ideal candidate for the oral immunization of dogs and possibly other canids.
REFERENCES 1 2
3
4
8
9
10
11
WHO. World Survey of Rabies, 1994, World Health Organization, Geneva, p. 210 Tierkel, E.S. Inauguration of rabies control studies by the U.S. Public Health Service. J. Am. Vet Med. Assoc. 1948, 112, 18-24 Tierkel, ES., Graves, L.M. and Wadley, S.L. Effective control of an outbreak of rabies in Memphis and Shelby County Tennessee. Am. J. Pub/. Hlth 1950, 40, 1084-l 088 Chomel, B., Chappuis, G., Bullon, F. et al. Mass vaccination campaign against rabies: are dogs correctly protected? The Peruvian experience. Rev. Infect. Dis. 1988, 10 (Suppl. 4), S697-S702 Belotto, A.J. Organization of mass vaccination for dog rabies in Brazil. Rev. infect Dis. 1988, 10 (Suppl. 4), S693-S696 Beran, G.W. and Frith, M. Domestic animal rabies control: an overview. Rev. Infect. Dis. 1988, 10 (Suppl. 4), S672-S677 Beran, G.W., Nocete, A.P., Elvina, 0. et al. Epidemiological and control studies on rabies in the Philippines Southeast. Asian. J. Trop. Med. Pub/. Hlfh 1972, 3, 433-445 Chappuis, G., Languet, B., Duret, C. and Desmettre, P.H. The use of recombinant poxviruses by oral and parenteral route. Proceedings of the Symposium on Rabies Control in Asia. Jakarta, Indonesia 1993, pp. 125-137 Rosatte, R.C., Maclnnes, CD., Power, M.J. eta/. Tactics for the control of wildlife rabies in Ontario (Canada). Rev. Sci. Tech. 1993,12,95-98 Le Blois, H., Tuffereau, C., Blancou, J., Artois, M., Aubert, A. and Flamand, A. Oral immunization of foxes with avirulent rabies virus mutants. Vet. Microbial. 1990, 23, 259-266 Blancou, J., Pastoret, P.P., Brouchier, B., Thomas, I. and Bogel, K. Vaccinating wild animals against rabies. Rev. Scient. Techn. /‘Office Int. Epizooties 1988, 7, 1005-1013
Vaccine
1996
Volume
14 Number
6
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Oral rabies vaccine: M. Fekadu et al. 12
13
14
15
16
468
Steck, F., Wandeler, A., Bichsel, P., Capt, S. and Schneider, L. Oral immunization of foxes against rabies. A field study. Zentralbl. Veterinarmed. (B). 1982, 29, 372-396 Bingham, J., Foggin, CM., Gerber, H. et al. Pathogenicity of SAD rabies vaccine given orally in chacma baboons (fapio ursinus). Vet. Rec. 1992, 131, 55-56 Wandeler, AL, Bauder, W., Prochaska, S. and Steck, F. Small mammal studies in a SAD baiting area. Camp. Immunol. Microbial. Infect. Dis. 1982, 5, 173-l 76 Tuffereau, C., Leblois, H., Benejean, J., Coulon, P., Lafay, F. and Flamand, A. Arginine or lysine in position 333 of ERA and CVS glycoprotein is necessary for rabies virulence in adult mice. Virology 1989, 172, 206-212 Seif, I., Coulon, P., Rollin, P.E. and Flamand, A. Rabies virulence: effect on pathogenicity and sequence characterization of rabies virus mutations affecting antigenic site Ill of the glycoprotein. J. Viral. 1985, 53, 926-934
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17
18
19 20
21
22
Coulon, P., Rollin, P.E. and Flamand, A. Molecular basis of rabies virus virulence. II. Identification of a site on the CVS glycoprotein associated with virulence. J. Gen. Viral. 1983, 64. 693-696 Flamand, A., Coulon, P., Lafay, F. and Tuffereau, C. Avirulent mutants of rabies virus and their use as live vaccine. Trends Microbial. 1993, 1, 317-320 Schumacher, C.L., Coulon, P., Lafay, F. et al. SAG-2 oral rabies vaccine Onderstepoort. J. Vet. Res. 1993, 60, 459-462 Smith, J.S., Yager, P.A. and Baer, G.M. A rapid tissue culture test for determining rabies neutralizing antibody. WHO. Monogr. Ser. 1973, XX, 354-357 Fekadu, M. and Shaddock, J.H. Peripheral distribution of virus in dogs inoculated with two strains of rabies virus. Am. J. Vet. Res. 1984, 45, 724-729 Krebs, J.W., Strine, T.W. and Childs, J.E. Rabies surveillance in the United States during 1992. J. Am. Vet. Med. Assoc. 1993, 203, 1718-1731
ELSEVIER
Vaccine, Vol. 14, No. 6, pp. 465-468, 1996 Copyright 0 1996 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0264-410X/96 $15+0.00
Immunogenicity, efficacy and safety of an oral rabies vaccine (SAG-2) in dogs M. Fekadu*§, S.B. Linhartl
S.L. Nesby*, J.H. Shaddock*, and D.W. Sanderlin*
C.L. Schumacher-f,
A study of immunogenicity and eficacy of Street Alabama Gtf (SAG-2) attenuated rabies virus vaccine in laboratory beagles was conducted. Four groups of ten dogs each received either 1.0 ml of SAG-2 orally on the tongue or 1.5 ml in baits. On day 180postvaccination, all dogs were challenged with a street rabies virus. The antibody response in groups that received the vaccine directly on the tongue was higher than in those vaccinated with baits, but the dtflerence between groups was not statistically signtficant. All vaccinated dogs survived, whereas 80% of controls died of rabies. Our findings demonstrate that the SAG-2 is a safe and eflective vaccine for oral immunization of canines. Copyright 0 1996 Elsevier Science-L&I. Keywords:
Rabies; vaccine; oral; dogs; carnivore; SAG-2
Rabies remains endemic in most developing countries and continues to exact a tremendous toll on the health of the population and the economic resources of these countries. The incidence of human rabies closely parallels that of the disease among domestic animals; ~90% of human cases are believed to be due to dogs, and much of the remainder to other domestic animals, especially cats’. Parenteral rabies vaccination of domestic animals, particularly do4s+ has been used to control rabies in many countries . The development of safe and effective oral rabies vaccines for free-ranging dogs that cannot be reached or contained for parenteral vaccination would facilitate the vaccination of a greater proportion of these animals. A number of candidate oral rabies vaccines are currently being evaluated for efficac . and safety testing in dogs and nontarget animal species CT One of these vaccines, an attenuated live rabies Street Alabama Dufferin (SAD) Bern strain, which has some residual pathogenicity for nontarget species, has been used for the oral vaccination of foxes in some European countries and Canada 9- l4 . Under the pressure of specific monoclonal antibodies (Mabs) on the SAD Bern strain, an avirulent antigenic mutant, Street Alabama Gif (SAG-l), was selected. This mutant has a I-nucleotide substitution in amino-acid position 333 of the glycoprotein15 and when tested for its pathogenicity in adult mice was shown to be almost apathogenic for adult
mice inoculated by the intracerebral route’“‘8. Further attenuation from SAD Bern was accomplished with the production of a double mutation at amino-acid 333 of the glycoprotein. This virus strain, called SAG-2, has a glutamate coded by GAA at position 333 instead of an arginine. This new codon differs from all the arginine triplets by two nucleotides”. SAG-2 is avirulent in adult mice by intercerebral and intermuscular routes. We have evaluated the immunogenicity of SAG-2 administered with or without bait in laboratory beagles. The results of this study are presented in this report.
MATERIALS
AND METHODS
Animals
Four groups of ten 1.5- to 2.5-year-old, purpose-bred beagles each received either 1.O ml of SAG-2 (titer: 1O8.5 or 107.5suckling SMICLD,, ml - ‘) orally on the tongue or 1.5 ml of SAG-2 (1O8-5or 107.5 SMICLD,, ml- ‘) in baits. A fifth group of ten dogs was retained as controls (Table I). All dogs were challenged with a street rabies virus 6 months after vaccination and observed for at least 3 months following challenge. Five to 6 months after challenge all dogs were euthanized by intravenous injection of barbiturate. Table 1 Groups of dogs administered SAG-2 vaccine orally with (w) or without (w/o) baits
*Division of Viral and Rickettsial Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Public Health Service, US Department of Health and Human Services, Atlanta, GA 30333, USA. tVlRBAC Laboratories, B. P. 27-06511 Carros Cedex, France. fsoutheastern Cooperative Wildlife Disease Study, College of Veterinary Medicine, University of Georgia, Athens, GA 30602, USA. $To whom correspondence should be addressed. (Received 23 June 1995; revised 13 November 1995; accepted 13 November 1995)
Dogs (10 per group) Group Group Group Group Group
I
w Bait
II Ill IV V
w Bait w/o Bait w/o Bait
Vaccine/dose 107.5 SMICLD 10e.5 SMICLD: 1O7.5SMICLD 1O’=’ SMICLD; Control
e SAG-2 SAG-2 SAG-2 SAG-2
5uckling mouse intracranial lethal dose 50%
Vaccine
1996 Volume
14 Number
6
465
Oral rabies vaccine: M. Fekadu et al. Table 2
Number of dogs in each vaccination
group with titers of tl:5a
Vaccine/dose SAG-2 SAG-2 SAG-2 SAG-2
lo= SMICLD 50 1O’=’ SMICLD 50 1 O= SMICLD 1O8.5 SMICLD5’50
%omplete
neutralization
w Bait w Bait w/o Bait w/o Bait at 1:5, demonstrates
Seroconversion 14
(>1:5) days postvaccination 28 42
4 6 5 9
6 :
A SAG-2 vaccine, batch S2/-1 grown in BHK cells was obtained from VIRBAC Laboratories (B.P. 27, 06511 Carros, Cedex, France) and stored in liquid nitrogen until use. The vaccine was quick-thawed and titrated both in suckling mice and tissue culture before use in vaccination. Vaccine that was left over after the vaccination of dogs was retitrated in tissue culture to determine possible titer loss. Baits
The baits used to administer the vaccine were ca. 2 cm in diameter and 5 cm in length, and cylindrical custom made (DuPont Nemours, Orange, TX, USA) for this experiment. They consisted of 57% dog biscuit meal, 20% meat and bone meal, 13% aquabind (EVA polymer), as a binder and 10% soybean oil as lubricant. A 1.5 ml virus suspension was deposited into 2-ml cylinder wax ampules which was sealed with paraffin and then inserted into the bait and offered to individual dogs. Dogs were denied the morning ration of feed on the day of vaccination. To determine if the baits were accepted, we monitored the dogs for at least 1 hour after the administration of the vaccine. antibody determination
Blood specimens were collected after vaccination and after challenge virus to monitor humoral antibody body titer was determined lox the focus inhibition test (RFFIT) .
at weekly intervals with a street rabies response. The antirapid fluorescence
Challenge virus
The virus strain used for challenging all dogs in the study was a dose of 106.5 MICLD,, street rabies virus strain isolated from a naturally infected dog at the Texas-Mexico border, as previously reported21. Virus isolation attempts from saliva swabs
The oral cavity of all vaccinated dogs was swabbed with a cotton swab dipped in Eagle’s media: Minimum Essential Modified (MEM) virus growth media with 10% Fetal Bovine Serum (FBS) at 1, 7, and 24 h after vaccination to determine the presence and the amount of residual virus in the mouth. The virus isolation attempts from the swabs were made by tissue culture and by suckling mouse inoculation. Virus isolation attempts from tissue specimens
All dogs that survived rabies challenge were euthanized 5-6 months after challenge by intravenous
466
10
180
186
8 10 8 10
8 10 9 10
10 10 10 10
sero-conversion
Vaccine
Rabies neutralizing
7 10 8 10
108
Vaccine 1996 Volume 14 Number 6
injection of barbiturate. Tissue specimens from the brain, spinal cord, tonsil, tongue, salivary gland, adrenal glands, and bladder were collected at necropsy. These tissue specimens were examined for viral antigen by fluorescence antibody staining (FA) of frozen tissue sections and for viable virus by inoculation of mouse neuroblastoma cells. RESULTS Attempts to isolate virus from saliva swabs collected at 1, 7, and 24 h after vaccine administration showed no virus present in any swab. Serum specimens collected at weekly intervals from dogs vaccinated with a dose of lo’.’ SMICD,, of SAG-2 directly on the tongue showed seroconversion in 5, 8, 8, and 8 of dogs on 14, 28, 42, and 108 days postvaccination, respectively (Table 2). Of the 10 dogs vaccinated with 1O8.5SMICLD,, directly on the tongue, 9 and 10 showed evidence of seroconversion on days 14 and 28 postvaccination, respectively (Table 2). Of the dogs vaccinated with a dose of 1O7.5SMICD,, of SAG-2 in baits, however, 4, 6, 7, and 8 dogs seroconverted on 14, 28, 42, and 180 days, respectively (Table 2). Six, 9, and 10 of 10 dogs vaccinated with 1Os.5 SMICD,, seroconverted 14, 28, and 42 days postvaccination, respectively (Table 2). By day 180, when the dogs were challenged with 106.5 MICLD,, street rabies virus, Mexican dog strain the geometric mean titer (GMT) of rabies neutralizing antibody had risen steadily to 2.8 IU ml ~ ’ in dogs vaccinated with 1Os.5SMICLD,, directly on the tongue and to 1.5 IU ml - ’ in dogs that were vaccinated with 108.5SMICLD,, in baits (Table 3). Six days after challenge, all dogs (including those which had failed to seroconvert) had high anamnestic responses with titers in excess of 0.5 IU ml - ’ (Table 3). All vaccinated dogs survived challenge, whereas eight of 10 unvaccinated control dogs died of rabies (Table 3). No viable virus was isolated in tissue specimens (i.e. brain, spinal cord, tonsils, tongue, salivary glands, adrenal glands, and bladder) collected at necropsy cu. 1 year after vaccination and 5-6 months after virus challenge. A dose of 1O7.5SMICLD,, ml ~ ’ of SAG-2 was as immunogenic as 108.5 SMICLDso ml ~ ’ when administered with or without bait (Table 2). The antibody response in groups that received the vaccine directly on the tongue was higher than those vaccinated with baits, but the difference between groups was not statistically significant. DISCUSSION In many countries rabies has been reduced from an epidemic to an endemic disease, and some countries
Oral rabies vaccine: M. Fekadu et al. Table 3 Number of dogs in each vaccination after challenge Vaccine SAG-2 SAG-2 SAG-2 SAG-2 Control
Dose 1O= 1O= 1O75 1 O=
SMCILD,, SMICLD, SMICLD,, SMICLD,,
aDate of challenge;
group with titers of 20.50 IU ml-’
Pre-challenge w Bait w Bait w/o Bait w/o bait
bGMT=Geometric
0.5 IU ml-’
<0.1-4.1 0.2-4.1 0.1-4.1 0.6-l 9.1
mean value. “Survivors;
180 days? after vaccination,
GMTb
Post-challenge
0.7 1.5 0.9 2.8
1.0>20.6 4.1220.6 1.0220.6 2.8220.6
6 days after challenge and survival
0.5 IU ml-’
GMT 6.8 13.4 7.9 12.2
SUN”
10/lod lo/lo
lo/lo lo/lo 2110
“number of survivors/inoculated
have eliminated rabies by parenteral immunization of domestic animals, mainly dogsZm7. In a number of those countries that managed to control dog rabies, wildlife subsequently emerged as a new rabies host11,22. The human and domestic animal rabies cases reported from those countries are mainly due to wildlife exposure. Between 1978 and 1982, the repetition of field trials in Alpine valleys resulted in the elimination of rabies in several regions of the Swiss Alps12. Several European countries and Canada have used oral vaccination as a means of controlling rabies in wild carnivoresx*9.“. The vaccine strain used in those field trials was the SAD strain known to immunize foxes and to rotect against ,B street rabies challenge when given orally . However, the data obtained from orally vaccinated domestic dogs were inconclusive”. The SAD strain has some residual pathogenicity for a variety of rodent species, and occasionally for domestic animals and wild carnivores with an impaired immune response13. In order to select avirulent antigenic escape mutants the SAD strain was incubated with Mabs that possessed binding properties for the antigenic site III (amino acid 330-338) of the viral glycoprotein. Arginine at position 333 has been linked to pathogenicity in several rabies virus strains15. During the selection process, under the pressure of site III specific binding mabs, only avirulent SAG-l variants of SAD Bern escaped neutralization and were able to replicate in cell culture. The substitution of the arginine at position 333 of site III of the rabies glycoprotein decreased or almost eliminated the pathogenicity of several clones for adult laboratory mice15. Subsequently, another escape variant was selected from SAD Bern in two steps in the presence of two different anti-glycoprotein Mabs. Both of the first two nucleotides in the codon for the amino acid 333 were affected, resulting in the incorporation of glutamine instead of arginine at this position. The resulting double mutant, SAG-2, showed no signs of the residual pathogenicity that characterized its parent (SAD Bern). The safety of SAG-2 has been demonstrated in the laboratory mouse, which serves as a sensitive animal model for the detection of residual pathogenicity of modified rabies vaccines”. SAG-2 has thus far been shown to be avirulent to most immunocompetent adult animals. Because of the replacement of the first two nucleotides in the codon of amino acid 333 of the glycoprotein, SAG-2 cannot revert to the pathogenic phenotype of the parent strain, SAD Bern, bi a single mutational event, as may happen with SAG-l ’ _ Because of these unique properties of SAG-2, we wanted to test the immunogenicity, efficacy, duration of immunity and safety of the SAG-2 vaccine strain in laboratory beagles. Our results show that SAG-2 vaccine administered directly on the tongue or in baits
induces an adequate antibody response in most dogs as early as 14 days after vaccination. Dogs that had no detectable humoral antibody titer were also protected against challenge with a street rabies virus 6 months after vaccination; these dogs developed a high anamnestic response indicating that the vaccine had induced a cellular immune response. Induction of immunity and protection were not dose dependent in the range of 1O7.5 SMCIDso to 1O8.5 SMICD,, when administered with or without bait. We did not establish the lower limit of the effective dose. The antibody response in groups that received the vaccine directly on the tongue was higher than that for groups vaccinated with baits, but the difference between groups was not statistically significant. The protecting capability of this system in laboratory trials seems not to be dependent on whether or not the dogs developed a neutralizing antibody response. The safety of the vaccine was tested by monitoring for residual virus in the oral cavity of dogs given vaccine with or without bait, at times ranging between one and 24 h after vaccination, but no virus was isolated from any of the swabs. These findings provide clear evidence that the SAG-2 vaccine is an ideal candidate for the oral immunization of dogs and possibly other canids.
REFERENCES 1 2
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WHO. World Survey of Rabies, 1994, World Health Organization, Geneva, p. 210 Tierkel, E.S. Inauguration of rabies control studies by the U.S. Public Health Service. J. Am. Vet Med. Assoc. 1948, 112, 18-24 Tierkel, ES., Graves, L.M. and Wadley, S.L. Effective control of an outbreak of rabies in Memphis and Shelby County Tennessee. Am. J. Pub/. Hlth 1950, 40, 1084-l 088 Chomel, B., Chappuis, G., Bullon, F. et al. Mass vaccination campaign against rabies: are dogs correctly protected? The Peruvian experience. Rev. Infect. Dis. 1988, 10 (Suppl. 4), S697-S702 Belotto, A.J. Organization of mass vaccination for dog rabies in Brazil. Rev. infect Dis. 1988, 10 (Suppl. 4), S693-S696 Beran, G.W. and Frith, M. Domestic animal rabies control: an overview. Rev. Infect. Dis. 1988, 10 (Suppl. 4), S672-S677 Beran, G.W., Nocete, A.P., Elvina, 0. et al. Epidemiological and control studies on rabies in the Philippines Southeast. Asian. J. Trop. Med. Pub/. Hlfh 1972, 3, 433-445 Chappuis, G., Languet, B., Duret, C. and Desmettre, P.H. The use of recombinant poxviruses by oral and parenteral route. Proceedings of the Symposium on Rabies Control in Asia. Jakarta, Indonesia 1993, pp. 125-137 Rosatte, R.C., Maclnnes, CD., Power, M.J. eta/. Tactics for the control of wildlife rabies in Ontario (Canada). Rev. Sci. Tech. 1993,12,95-98 Le Blois, H., Tuffereau, C., Blancou, J., Artois, M., Aubert, A. and Flamand, A. Oral immunization of foxes with avirulent rabies virus mutants. Vet. Microbial. 1990, 23, 259-266 Blancou, J., Pastoret, P.P., Brouchier, B., Thomas, I. and Bogel, K. Vaccinating wild animals against rabies. Rev. Scient. Techn. /‘Office Int. Epizooties 1988, 7, 1005-1013
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Steck, F., Wandeler, A., Bichsel, P., Capt, S. and Schneider, L. Oral immunization of foxes against rabies. A field study. Zentralbl. Veterinarmed. (B). 1982, 29, 372-396 Bingham, J., Foggin, CM., Gerber, H. et al. Pathogenicity of SAD rabies vaccine given orally in chacma baboons (fapio ursinus). Vet. Rec. 1992, 131, 55-56 Wandeler, AL, Bauder, W., Prochaska, S. and Steck, F. Small mammal studies in a SAD baiting area. Camp. Immunol. Microbial. Infect. Dis. 1982, 5, 173-l 76 Tuffereau, C., Leblois, H., Benejean, J., Coulon, P., Lafay, F. and Flamand, A. Arginine or lysine in position 333 of ERA and CVS glycoprotein is necessary for rabies virulence in adult mice. Virology 1989, 172, 206-212 Seif, I., Coulon, P., Rollin, P.E. and Flamand, A. Rabies virulence: effect on pathogenicity and sequence characterization of rabies virus mutations affecting antigenic site Ill of the glycoprotein. J. Viral. 1985, 53, 926-934
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Coulon, P., Rollin, P.E. and Flamand, A. Molecular basis of rabies virus virulence. II. Identification of a site on the CVS glycoprotein associated with virulence. J. Gen. Viral. 1983, 64. 693-696 Flamand, A., Coulon, P., Lafay, F. and Tuffereau, C. Avirulent mutants of rabies virus and their use as live vaccine. Trends Microbial. 1993, 1, 317-320 Schumacher, C.L., Coulon, P., Lafay, F. et al. SAG-2 oral rabies vaccine Onderstepoort. J. Vet. Res. 1993, 60, 459-462 Smith, J.S., Yager, P.A. and Baer, G.M. A rapid tissue culture test for determining rabies neutralizing antibody. WHO. Monogr. Ser. 1973, XX, 354-357 Fekadu, M. and Shaddock, J.H. Peripheral distribution of virus in dogs inoculated with two strains of rabies virus. Am. J. Vet. Res. 1984, 45, 724-729 Krebs, J.W., Strine, T.W. and Childs, J.E. Rabies surveillance in the United States during 1992. J. Am. Vet. Med. Assoc. 1993, 203, 1718-1731