TITLE: ORAL RABIES VACCINATION OF DOGS
AUTHORS: C.E. Rupprecht*, J.S. Shaddock*, D.W. Sanderlin*, C.A. Hanlon*, M.
Niezgoda* and C.L. Schumacher*
*. Centers for Disease Control and Prevention, Atlanta, GA, 30333, USA,
**. Virbac Laboratories, Carros, 06517, France
Summary
Single administration of three graded doses of SAG2 oral rabies vaccine were given
in baits to 26 beagles in order to demonstrate its safety and efficacy. Adverse signs
were not observed throughout 3160 dog days of observation. At 6 weeks, the vaccine
protected 73% of vaccinated dogs against lethal challenge while all the control dogs
succumbed.
Introduction
Canine rabies is geographically widespread and continues to represent a significant
public health threat, particularly in developing countries, with an estimated annual
50,000 human fatalities (1). Human rabies prevention by the application of parenteral
rabies vaccination of domestic dogs en masse has been a traditional method of disease
control, leading to the virtual elimination of canine rabies in developed regions (2).
However, routine creation of adequate herd immunity among dog populations in
many countries has been inadequate (3). Low dog accessibility to individual
vaccination was frequently reported to be a major factor in program failure. The
concept of oral rabies vaccination, as it was originally applied to wildlife (4,5), offers
a potential solution to this problem. In collaboration with multiple investigators, the
promotion and coordination of research on oral canine vaccination towards the
development of safe and effective vaccines and baits has been supported by the World
Health Organization over the past decade (6-11). Examples of efficacious orallyadministered vaccines in baits include the modified-live virus SAD B19 (12) and the
vaccinia-rabies glycoprotein recombinant virus (13). Another candidate is the
attenuated rabies virus SAG2, isolated as an escape mutant by selection with
neutralizing monoclonal antibodies from the modified-live rabies vaccine strain SAD
(14). The SAG2 virus is avirulent when inoculated intracerebrally in adult immunocompetent mice, as well as by other experimental exposure. To date, SAG2 vaccine
has been tested safely in over 30 species in captivity and in the field, including
domestic animals, wildlife, rodents and non-human primates (15-19). Significantly,
SAG2 vaccine conferred long-term protection for dogs when administered per os
either in the form of a liquid suspension or as an experimental bait in laboratory trials
(20).
These preliminary results suggested that SAG2 could be an effective oral canine
vaccine. Moreover, considering the hot climate of many tropical and sub-tropical
areas where canine rabies is a problem, the issue of viral thermal stability was
addressed by the incorporation of lyophilized vaccine in the bait. A limited trial in
laboratory beagles using this prototype bait
(DBL2) conferred protection in 3 of 4 dogs against a virulent challenge in which all
the controls succumbed (21). This promising result suggested that a further
investigation was needed using commercially produced lots of vaccine and bait. An
additional efficacy and safety was then performed in laboratory dogs with different
concentrations of SAG2 vaccine in liquid or lyophilized bait form, as presented
below.ELJOURNAOFVETERINARMEDICISRAELJOURNALOFETERINARYM
EDICINE
Materials and Methods
A group of 31 healthy adult Beagles was obtained from a commercial supplier. The dogs were not
rabies vaccinated. On arrival at CDC, dogs were individually identified by tattoo, examined by a staff
veterinarian, and placed under a 30 day period of quarantine for observation. Dogs were housed
individually in indoor-outdoor runs, provided with water ad libitum, and fed commercial dog food
daily. Venous blood samples were taken before the beginning of the experiment. Blood was allowed to
clot at room temperature prior to low speed centrifugation, and the separated sera were stored at -20°C
until testing. Sera were tested for detection of rabies virus neutralizing antibodies (VNA) by the RFFIT
technique (22).
The dogs were randomly divided into 3 groups of 13, 13, and 5 (controls) individuals. Two of the
groups received a vaccine-laden bait, while controls did not. Two different baits were used, one
consisting of approximately 1.8 ml of liquid SAG2 vaccine contained within a blister sachet coated
with a flavored paraffin matrix bait (Rabigen Oral), while the second bait (DBL2) consisted of
lyophilized SAG2 vaccine. The Rabigen blister sachets containing SAG2 vaccine (batch RS2002),
consisted of 3 different concentrations, with a titer of 10 7.4, 107.5, or 108.2 TCID50 per bait (Virbac
Laboratories, Carros-Cedex, France). These were shipped on dry ice to the CDC, where the vaccine
baits were received frozen and stored at -20°C until further use. On the day of the experiment, these
vaccine baits were thawed at 4°C and were placed in a cooler where they remained until presentation to
the dogs. The second type of bait DBL 2 was a specifically designed, proprietary lyophilized form of
the SAG2 vaccine within a bait, as previously described (23). Briefly, the DBL2 bait was composed of
a lyophilized central core containing the SAG2 vaccine and a thin layer of matrix. The matrix coating
was composed of animal origin, artificial taste enhancers, and a synthetic polymer responsible for
impermeability and mechanical stability. The approximate dimensions of the bait were 1.5x3x2.5 cm,
with an approximate mass of 10 g. Contact with small amounts of aqueous solution led to the rapid
rehydration of the vaccine core. These baits were packaged and stored at 4°C until refrigerated
transport to CDC, where they were kept at 4°C until the start of the experiment. These DBL2 baits
(batches LE4, LE5, and LE7) consisted of 3 different concentrations. The titer per bait determined
before shipment was 106.9, 107.2, or 108.3 TCID50. The DBL2 baits were stored at 4°C until
presentation to the dogs.
On day 0, all the dogs, excluding the controls, were provided with one bait in the evening. In the
morning, baits were checked for signs of consumption. A dog was considered vaccinated if no bait
could be found, or if bait fragments or a punctured blister container was observed. A vaccinated dog
was allowed to consume only one bait. Any untouched baits were removed and the cycle was repeated
every few days over the next two weeks, until there were 3 to 5 dogs per vaccine group and
concentration. Group A consisted of the 13 dogs that received the Rabigen blister bait (5 at the highest
concentration, 3 in the intermediate dilution, and 5 in the lowest concentration). Group B consisted of
the 13 dogs that received the DBL2 bait (5 at the highest concentration, 4 in the intermediate dilution,
and 4 in the lowest concentration). Group C consisted of the 5 unvaccinated control dogs. Over the next
several weeks, dogs were observed many times daily for the detection of any adverse clinical signs
associated with vaccine administration. No sharing of equipment, such as food bowls or supplies, was
allowed between individual dog runs. Latex examination gloves were changed regularly between the
handling of different baits, vaccinated and control dogs, to minimize cross contamination.
Blood samples were obtained from all the dogs by venipuncture for the RFFIT assay to determine
rabies specific VNA at approximately weekly intervals, and at 1 week following rabies virus challenge.
A minimum positive rabies VNA result was defined as the titer that resulted in complete neutralization
of CVS-11 rabies virus at a dilution of 1/5. Antibody titers were compared with a national reference
serum (lot R-3), obtained from the United States Bureau of Biologics, National Institutes of Health,
expressed in IU/ml.
Approximately 6 to 7 weeks after consumption of a single bait, all 31 (vaccinated and unvaccinated
control) dogs were sedated by an intramuscular mixture of tiletamine hydrochloride and zolazepam
hydrochloride at a dose of 0.5 - 1.0 mg/kg. Sedated dogs were then inoculated into the masseter muscle
with 0.5 ml of a street virus strain (Reference 2951 R), a canid rabies virus variant characteristic of
viruses in circulation among dogs and coyotes (Canis latrans) at the Texas-Mexico border (24). This
virus was originally isolated from the salivary gland of a naturally infected rabid coyote collected from
Texas.
The salivary gland was homogenized using 2% horse serum in sterile distilled water to a 10%
suspension, and was clarified by low speed centrifugation.
The challenge virus suspension was stored at -70°C, and had an original concentration of 107.0
MICLD50 per ml. On the day of challenge, the challenge virus was thawed under cold running water
and was diluted 1:1000 in 2% horse serum in sterile distilled water. The viral suspension was kept on
ice in a cooler throughout the experiment until all dogs were inoculated. Thereafter, dogs were
observed multiple times daily for typical clinical signs suggestive of rabies, such as lethargy,
inappetance, agitation, paresis, paralysis, cranial nerve deficits, or acute death.
Suspect ill dogs were examined, sedated, and euthanized by an intravenous administration of a
barbiturate overdose. After euthanasia, brain tissue was collected from each dog at necropsy, and
examined for rabies virus antigen by the direct fluorescent antibody test (25).
Surviving dogs were observed for a minimum of 90 days after challenge, after which they were
sedated, euthanized, and examined at necropsy, as above. SRAELJOURNALOF
VETERINARY MEDICINE
Results
None of the 26 dogs provided with the SAG2 vaccine in baits exhibited any overt
adverse effects or specific clinical signs indicative of rabies during the 6-7 week
period following vaccination. The sachets from the baits in Group A, when found,
were either chewed thoroughly by the dogs or were punctured by teeth, with no
remaining liquid observed in the sachets. In group B, the baits were completely eaten
by the dogs, with the sole exception of one dog (#QCI6) given the highest vaccine
concentration, which ate approximately one-half of the lyophilized DBL2 bait.
No dog had detectable levels of rabies VNA prior to inclusion in the study, nor any
suggestion of an anamnestic response in the ensuing week of serum collection after
bait consumption from any previous rabies exposure or vaccination, since no rabies
VNA was detected in the first 7 days following vaccination. However, within two
weeks of bait consumption, titers of rabies VNA were detectable in 14 of 26 (54%)
vaccinated dogs (GMT, 0. 3 IU/ml; range 0.1-0.5 IU/ml). There was no significant
difference in primary rabies VNA response by either GMT or total numbers
responding per group, with 7 individuals from group A (3 each from the lowest and
highest concentrations and a single dog at the intermediate dose) and 7 individuals
from Group B (3 each at the highest and intermediate concentration and a single dog
at the lowest dose). Rabies VNA titers in individual dogs remained stable or gradually
declined thereafter, regardless of the group. Over the period of observation, 16 of the
26 (62%) vaccinated dogs had evidence of rabies virus seroconversion at least once
after bait consumption (one additional dog each from groups A and B, both at the
highest vaccine concentration), but this declined
to only 12 of 26 (46%) dogs on the day of challenge, because rabies VNA titers in 4
of these previously seropositve dogs had fallen to pretrial negative values: two in
Group A and two in group B. Throughout the pre-challenge period, only 6 vaccinated
dogs (three in each group) developed a primary VNA response greater than or equal
to 0.5 IU/ml. In group A, 2 of these 3 dogs were from the highest concentration, with
the remaining animal from the intermediate dilution. In group B, all 3 dogs were from
the highest concentration, including the dog #QCI6 that did not fully consume the
DBL2 bait.
Seven days after challenge, 19 of the 26 (73%) vaccinated dogs, including 3
previously seronegative animals and the 4 previously seropositive dogs that had
declined to baseline serum levels, had evidence of an anamnestic response with
significant VNA titers. The other 7 vaccinates (4 in Group A, 2 from the lowest
vaccine concentration and 1 each per the other dilutions; 3 in Group B, all from the
lowest vaccine concentration), failed to seroconvert at any time before or after rabies
virus challenge, as did the 5 controls. Of those vaccinated dogs with evidence of a
booster response, 18 of 19 (95%) individuals had a VNA titer greater than or equal to
0.5 IU/ml. Within 2 weeks of challenge, 12 dogs displayed typical clinical signs of
rabies and were euthanized. These were all 5 controls and the 7 seronegative
vaccinates. In the vaccinated groups, all dogs with any detectable VNA titers
anywhere up to 7 days after the challenge survived (9 from group A; 10 from group
B), regardless of VNA temporal induction, its magnitude, or duration. The diagnosis
of rabies was confirmed in all euthanized dogs that displayed clinical signs of rabies
during the experiment by histopathology of CNS tissue. In contrast, the brains of all
the vaccinated survivors euthanized at the end of the experiment were free of rabies
virus antigen.
Discussion
These data corroborate previous preliminary findings of the efficacy and safety of
SAG2 vaccine when delivered in bait form for the oral immunization of dogs.
Significantly, 19 of 26 (73%) vaccinated beagles survived a virulent rabies infection,
regardless of SAG2 vaccine formulation. There was no significant difference in
overall survival between Groups A and B, with only a suggestion of dose response
according vaccine-virus concentration. For example, 5 of the 6 dogs with the most
pronounced primary VNA responses in both groups (greater than or equal to 0.5
IU/ml) also originated from the highest vaccine-virus concentration.
Moreover, in group B, consumption of a single DBL2 bait containing between 108.3
and 107.5 TCID50 of SAG2 vaccine led to the induction of, or priming for, rabies
VNA and resulted in complete protection in all 9 dogs in the two dosage groups (but
survival of only 1 dog out of 4 given the lowest concentration), approximately 6
weeks following vaccination and challenge with canine street virus in which all 5
controls succumbed. Given these promising results in adult Beagles, future research
should focus on the efficacy and safety of SAG2 vaccine in indigenous, non-purpose
bred laboratory dogs, especially in puppies less than 3 months of age.
Although the SAG2 vaccine has convincing potency via the oral route, its specific
mode of action is unclear. The rabies virus glycoprotein structural protein induces
specific rabies VNA, which is the primary effector mechanism against lethal virus
infection (26, 27). Whereas only 16 of 26 vaccinated dogs developed detectable rabies
VNA titers at some point before the day of challenge, 19 dogs survived lethal
infection which killed all unvaccinated controls. In addition, although rabies VNA is
one important defense mechanism and is generally correlated with protection,
emphasis on a minimum "protective titer" alone is misleading, such as the 0.5 IU/ml
level utilized in human rabies vaccination as evidence of basic seroconversion. This is
because only few of the orally vaccinated dogs achieved this level in the present
study. Rather, evidence of any seroconversion was a good predictor of vaccine
efficacy, and based on the humoral anamnestic response observed 1 week post
challenge, apparently 7 of the 19 survivors receiving SAG2 vaccine had been
immunologically primed by vaccine, because a VNA response was not detected
previously in 3 dogs, and another 4 dogs had declined to pretrial VNA levels prior to
challenge. Perhaps VNA titers were either too low or transient to be readily detected
by the methods used in this experiment, or the specific primary immune response may
not have essentially involved VNA. Immune protection without the apparent
measured presence of VNA before challenge infection has also been observed when
SAG2 vaccine was administered to dogs by the oral route (20), and the priming for
VNA by other rabies virus structural proteins, such as the nucleoprotein, although
incompletely understood, must also be considered (28,29). Additional mediators, such
as the induction of antibodies to internal viral proteins or the occurrence of cytokines
such as interferon, should therefore be examined after oral vaccination.
The public health aspects of canine oral rabies vaccination have been broadly
discussed, because of the close association of dogs and people, particularly children
(30). Obviously, no vaccine should cause overt illness in the recipient nor should be
readily excreted. Regardless of bait presentation or individual dog characteristics,
adverse clinical effects attributable to SAG2 vaccine were not observed in any of the
26 vaccinated dogs enrolled in this study, with over 3160 dog days of observation,
and confirmed previous results, in which viral suspensions of SAG2 or experimental
baits were used in dogs (20). In addition, attempts to isolate input SAG2 virus from
the mouths of 40 vaccinated dogs in that study in saliva swabs collected at 1, 7 and 24
hours after vaccine administration failed to detect virus in any swab, indicating that
residual input virus concentration was low, even immediately following oral
inoculation of viral suspension or bait consumption. More concentrated virus has been
used in at least one set of previous laboratory studies than what is required for
commercial vaccine production, so it is quite unlikely under actual field conditions
that significantly higher passive amounts of SAG2 virus should be present in their
mouth. Thus, the likely risk of transmission of SAG2 virus to subsequent contacts
from a newly immunized dog is minimal after vaccine consumption. Further
investigations for residual viral detection in dogs should include the selected dose of
the vaccine and formulation of the bait as will be deployed in the field, such as the
DBL bait, because the distribution or accumulation of SAG2 virus in the canine oral
cavity may not necessarily be the same in a lyophilized state as with a viral
suspension or a bait employing liquid vaccine in sachet form.
Use of a vaccine in oral rabies immunization requires biologic presentation that is
well accepted by the target species and is readily available for delivery to the oral
cavity without inactivation (5). The lyophilized DBL2 bait was specifically designed
for dogs, by consisting of a porous cavity in which the vaccine is homogeneously
dispersed, and an outer heat and water-resistant flavored coat. Bait acceptance as
observed in this trial had also been demonstrated for dogs in both the laboratory and
field (23, 31). This particular DBL2 bait is advantageous because it not only
maximizes vaccine reconstitution when in contact with the canine oral mucosa, but
also decreases the chances of non-target species uptake, such as human contamination
in the field, because it contains no permanent vaccine container and can be easily
discarded in the process of bait consumption. Eventual utilization of lyophilized
products, such as SAG2 in the DBL2 bait format, should set a new standard for other
oral rabies vaccines currently produced in the liquid phase only, and that may be
affected by vaccine container rejection from target species or may be inactivated by
adverse environmental conditions.
Although the SAG2 DBL2 bait was specifically designed for manual distribution to
dogs, focus on other products or techniques should consider compatibility for aerial
distribution to a variety of species.
Oral vaccination promises to become an important adjunct to traditional rabies control
measures, including parenteral vaccination of dogs, stray domestic animal control,
public education, and human rabies prophylaxis (32). Considering the infancy of the
oral rabies vaccination field for dogs, research with other biologics, such as
alternative recombinant (33) or DNA vaccines (34), should continue to emphasize
safety, efficacy and cost, and thus overcome the limitations of single existing product.
Anticipated field trials in Asia, Africa, and Latin America should generate critical
information as to the viability of such techniques in developing countries and their
application towards ultimate dog rabies elimination.
Acknowledgements
The authors wish to thank the staff in the Viral and Rickettsial Zoonoses Branch and the Animal
Resources Branch, CDC, and at Virbac Laboratories, for their valuable technical expertise, without
which this work would not have been possible.
References
1. World Healt Organization: World survey of rabies N÷32 for the year 1996. World
Health Organization Publication WHO/EMC/ZDI/98.4, Geneva, Switzerland. p 27,
1998.
2. Meslin, F.X, Fishbein, D.B. and Matter, H.C.: Rationale and prospects for rabies
elimination in developing countries. Curr. Top. Microbiol. Immunol. 187:1-26, 1994.
3. Meslin, F.X. and Stohr, K.: Prospects for immunisation against rabies in
developing countries. In: Dodet B. and Meslin, (eds.) Rabies Control in Asia.
Elsevier, Paris, pp. 15-18, 1997.
4. Baer, G.M.: Oral rabies vaccination: an overview. Rev. Infect. Dis.10(4), S644S648, 1988.
5. Wandeler, A.I.: Oral immunization of wildlife. In: Baer, G.M., ed.: The Natural
History of Rabies, 2nd edn.. CRC Press Inc., Boca Raton, Florida. pp.485-503, 1991
6. World Health Organization: Report of the WHO Consultation on Oral
Immunization of Dogs against Rabies, WHO/Rab.Res/88.26, Geneva, Switzerland.
1988.
7. World Health Organization: Report of the WHO Consultation on Requirements
and Criteria for Field Trials on Oral Rabies Vaccination of Dogs and Wild
Carnivores, WHO/Rab.Res/89.32, Geneva, Switzerland. 1989.
8. World Health Organization: Report of the Third Consultation on Oral
Immunization of Dogs against Rabies, WHO/Rab.Res/93.38, Geneva, Switzerland.
1992.
9. World Health Organization: Report of the Fourth Consultation on Oral
Immunization of Dogs against Rabies, WHO/Rab.Res/93.40, Geneva, Switzerland.
1993.
10. World Health Organization: Report of the Fifth Consultation on Oral
Immunization of Dogs against Rabies, WHO/Rab.Res/94.45, Geneva, Switzerland.
1994.
11. World Health Organization: Report of the Sixth Consultation on Oral
Immunization of Dogs against Rabies, WHO/Rab.Res/95.48, Geneva, Switzerland.
1995.
12. Aylan, O. and Vos, A.: Efficacy studies with SAD B19 in Turkish dogs. J. Etlik
Vet. Microbiol. 9: 93-101, 1998.
13. Chappuis, G., Languet, B., Duret, C. and Desmettre, P.: Dog rabies vaccination:
the use of recombinant poxviruses by oral and parenteral route. In: Proceedings of the
Symposium on Rabies Control in Asia. Fondation Marcel Merieux. Lyon. pp. 125138. 1993.
14. Lafay, F., Benejean, J., Tuffereau, C., Flamand, A. and Coulon, P.: Vaccination
against rabies: construction and characterization of SAG2, a double derivative of
SAD Bern. Vaccine 12:317-320, 1994.
15. Schumacher, C.L., Coulon, P., Lafay, F., Benejean, J., Aubert, M.F.A., Barrat, J.,
Aubert, A. and Flamand, A.: SAG2 oral rabies vaccine. Onderstepoort. J. Vet. Res.
60:459-462, 1993.
16. Follman, E.H., Ritter, D.G. and Baer, G.M.: Evaluation of the safety of two
attenuated oral rabies vaccines, SAG1 and SAG2, in six arctic mammals. Vaccine
14:270-273, 1996.
17. Masson, E., Cliquet, F., Aubert, M.F.A., Barrat, J., Aubert, A.,
Artois, M. and Schumacher, C.: Safety study of the SAG2 rabies
virus mutant in several non-target species with a view to its future
use for the immunization of foxes in Europe. Vaccine 14:15061510, 1996.
18. Masson, E., Aubert, M.F.A., Barrat, J. and Vuillaume, P.:
Comparison of the efficacy of the antirabies vaccines used for
foxes in France. Vet. Res. 27:255-266, 1996.
19. Bingham, J., Schumacher, C.L., Aubert, M.F.A., Hill, F.W.G.
and Aubert, A.: Innocuity studies of SAG2 oral rabies vaccine in
various Zimbabwean wild non-target species. Vaccine 15: 937-943,
1997.
20. Fekadu, M., Nesby, S.L., Shaddock, J.H., Schumacher, C.L.,
Linhart, S.B. and Sanderlin, D.W.: Immunogenicity, efficacy and
safety of oral rabies vaccine (SAG2) in dogs. Vaccine 14:465-468,
1996.
21. Schumacher, C.L. and Aubert, A.: The oral delivery of rabies
vaccines to dogs. In: Bingham J., Bishop, G. and King, (eds.)
Proceedings of the Third International Conference of the Southern
and Eastern African Rabies Group. Harare, Zimbabwe. pp 150-158,
1995.
22. Smith, J.S., Yager, P.A. and Baer, G.M.: A rapid fluorescent
focus inhibition test (RFFIT) for determining rabies virusneutralizing antibody. In: Meslin F.-X., Kaplan M.M. and
Koprowski, H., (eds.): Laboratory Techniques in Rabies, 4th edn.
World Health Organization, Geneva. pp. 181-192, 1996.
23. Matter, H.C., Schumacher, C.L., Kharmachi, H., Hammami, S.,
Tlatli, A., Jemli, J., Marbet, L., Meslin, F.X., Aubert, M.F.,
Neuenschwander, B.E. et al.: Field evaluation of two bait delivery
systems for the oral immunization of dogs against rabies in
Tunisia. Vaccine, 16(7), 657-665, 1998.
24. Krebs, J.W., Smith, J.S., Rupprecht, C.E. and Childs, J.E.:
Rabies surveillance in the United States during 1996. J. Am. Vet.
Med. Assoc. 211: 1525-1539, 1997.
25. Dean, D., Abelseth, M.K. and Atanasiu, P.: The fluorescent
antibody test. In: Meslin F.-X., Kaplan and Koprowski, H., (eds.)
Laboratory Techniques in Rabies, 4th edn. World Health
Organization, Geneva. pp. 88-95, 1996.
26. Wiktor, T.J., Gyoergy, E., Schlumberger, E. and Koprowski,
H.: Antigenic properties of rabies virus components. J. Immunol.
110: 269-276, 1973.
27. Wunner, W.H.: The chemical composition and molecular
structure of rabies viruses. In: Baer, G.M., (ed.) The Natural
History of Rabies, 2nd edn. CRC Press Inc., Boca Raton, Florida.
pp. 31-67. 1991.
28. Fu, Z.F., Wunner, W.H. and Dietzschold, B.:
Immunoprotection by rabies virus nucleoprotein. Curr. Top.
Microbiol. Immunol. 187:161-172, 1994.
29. Lafon, M.: Immunobiology of lyssaviruses: the basis for
immunoprotection. Curr. Top. Microbiol. Immunol. 187:145-160,
1994.
30. Blancou, J. and Meslin, F.X.: Modified live-rabies virus
vaccines for oral immunization of carnivores. In: Meslin F.-X.,
Kaplan M.M. and Koprowski, H., (eds.) Laboratory Techniques in
Rabies, 4th edn. World Health Organization, Geneva. pp. 324-337.
1996.
31. Ben Youssef, S., Matter, H., Schumacher, C.L., Kharmachi, H.,
Jemili, J., Mrabet, L., Gharbi, M., Hammami, S., El Hicheri, K.,
Aubert, M.F.A. and Meslin, F.X.: Field evaluation of a dog owner,
participation-based bait delivery system for the oral immunisation
of dogs against rabies in Tunisia. Am. J. Trop. Med. Hyg. 58:835845, 1998.
32. Centers for Disease Control and Prevention: Compendium of
animal rabies control, 1998. Morbid. Mortal. Wkly. Rep. 47:RR-9.
1998.
33. Campbell, J B.: Oral rabies immunization of wildlife and dogs:
challenges to the Americas. Curr. Top. Microbiol. Immunol.
187:245-266, 1994.
34. Lodmell, D.L., Ray, N.B., Parnell, M.J., Ewalt, L.C., Hanlon,
C.A., Shaddock, J.S., Sanderlin, D.S. and Rupprecht, C.E.: DNA
immunization protects nonhuman primates against rabies virus.
Nature Medicine 4:949-952, 1998.