Modified live-virus (MLV) and inactivated vaccines can be produced in cell culture, using either primary cells or continuous cell lines. The seed virus/cell systems vary considerably between different manufacturers. Increasing use of inactivated vaccines for animal immunization can be expected as a result of recent improvements in vaccine production techniques.

For mass canine vaccination campaigns, the use of inactivated rabies vaccine is recommended. The management of inactivated vaccine in the field is easier than that of live vaccine, since it is less sensitive to changes in temperature. Furthermore, accidents of self-inoculation do not represent any risk for the vaccinator.

At least 70% of the dog population in each community should be vaccinated within a month. In areas where the dog population turnover is rapid, it may be necessary to carry out a mass vaccination campaign each year. However, if the effective immune period of the vaccine is longer and the system for identifying vaccinated dogs can be trusted to last more than one year, the advantage of vaccinating only the dogs entering the population after the last campaign should be considered, with revaccination of dogs vaccinated during the last campaign at intervals of about 2 years.

Combined vaccines are already used for the immunization of dogs and cats. Several different antigens are incorporated in canine rabies vaccine, such as canine distemper, canine hepatitis, leptospirosis and canine parvovirus. Combined rabies vaccines for cats may include various other antigens such as feline panleukopenia virus, feline calicivirus and feline parvoviruses. A combined rabies and foot-and-mouth disease vaccine is available for use in cattle, sheep and goats.

Several modified-live (ML) rabies virus vaccine candidates have been developed for the oral immunization of animals in the past 35 years. In 1978 the SAD-Bern vaccine was used in Switzerland in the first large scale field trial for oral vaccination of foxes ever carried out. However, only a small number has proved suitable in terms of efficacy and safety for use in the field. All modified-live rabies virus vaccines currently in use are derivatives of the original SAD (Street Alabama Dufferin) virus. These ML rabies vaccines are used for vaccination of foxes (e.g. in Canada, western and central Europe), raccoons (e.g. in Finland and Estonia, Lithuania, Latvia as well as in Russia).

The liquid vaccine usually contained in a sachet or blister pack should be incorporated in a bait preferably adapted to the target species with regard to taste, size and texture. All attenuated vaccines currently used are derived from the original SAD (Street Alabama Dufferin) strain, with various levels of attenuation after passage in cell cultures. Most vaccines are attenuated by serial in-vitro selection on cloned baby hamster kidney cells or by passaging in mice in vivo. Modified-live rabies virus oral vaccines used in the field for wildlife may have residual pathogenicity, depending on the level of attenuation of the viral strain. SAD-related vaccines (e.g. ERA, SAD-Bern, SAD-B19, and Vnukovo-32) are pathogenic for adult mice by the intracerebral, intramuscular and oral routes, and for many other rodent species. SAG2 is a mutant vaccine developed by using rabies virus glycoprotein monoclonal antibodies to select a highly attenuated virus carrying two mutations in position 333. SAG 2 vaccine is apathogenic in mice (adult or suckling) as well as in any wild rodents tested by the oral, intramuscular or intracerebral routes.

SAD-related vaccines have been widely used in the field. Since 1983 millions of baits have been distributed in Europe (including Belgium, France, Germany, Italy, Luxembourg and a number of Eastern European countries) and since 1989 in Canada contributing to wildlife rabies elimination in Western and parts of Central and Eastern Europe as well as Ontario province, Canada. SAD related vaccine-induced rabies cases following ingestion of vaccine bait(s) have been described first in Switzerland and more recently in foxes and other wild carnivores in Austria, Germany, and Slovenia and in Canada. SAG2 has been used extensively in the field to eliminate fox rabies for example in France, Switzerland and more recently in Italy.

Vaccines should not induce any adverse signs in either target or non-target species. In non-target species their safety should be assessed in relevant wild rodents and other wild and domestic species that live in the area and may consume baits, as well as in non-human primates. Any rabies virus isolated from animals in the area of vaccination should be characterized with monoclonal antibodies or molecular techniques to ensure that no vaccine-induced rabies has occurred.

National control laboratories or government institutions involved in licensing vaccines or evaluating oral rabies vaccination programmes may verify the viral titre of all batches of vaccine bait before and during a campaign. Such tests should be conducted in qualified laboratories with documented, validated methods and appropriate standards. Use of rabies virus strains that can cause rabies in wildlife species is not recommended.

In the event of accidental human exposure to attenuated rabies virus vaccines, medical attention should be sought and post-exposure prophylaxis considered.

The development of recombinant DNA technology has initiated a new era in rabies control. Recombinant vaccines cannot exhibit residual pathogenicity caused by rabies because they contain only single non-virulent gene products. The majority of the safety requirements for modified live-virus vaccines are also applicable to recombinant vaccines.

Various recombinant constructs (e.g. animal poxviruses or human or canine adenovirus as vector) expressing the rabies glycoprotein were tested in different target and non-target wildlife species by the oral route. The only recombinant that is now produced and used in large quantities is a vaccinia based recombinant product expressing the rabies glycoprotein (VRG).

VRG shares many basic properties with parental vaccinia virus (Copenhagen strain) but differs in other ways which make the vector virus safer. The deletion of the thymidine kinase gene dramatically decreases the pathogenicity of the vaccine for mice when it is given by the intracerebral and intraperitoneal routes. In addition, no viral spread from currently known sites of viral replication has been observed, and oral vaccination of dozens of animal species, including wild animals, has not revealed any residual pathogenicity.

When administered orally (by direct instillation in the mouth or in a bait) to young and adult foxes or raccoons, a dose of 10 8 TCID 50 (median tissue-culture infective dose) of VRG elicits high titres of virus-neutralizing antibodies and confers protection against a severe rabies challenge.

Studies have shown that the VRG vaccine is not pathogenic in over 10 avian and 35 mammalian species, including the majority of rabies reservoir hosts. Regardless of the vaccine dose or route of administration, the vaccinated animals have remained clinically normal, with no overt gross or histopathological lesions. Following oral administration, the VRG vaccine is cleared relatively quickly (e.g. within 48 hours). No abortifacient, teratogenic, or oncogenic side-effects have been noted. Large-scale field trials in foxes, raccoons and coyotes have been reported to date in Europe and North America.

The eighth report of the WHO Expert Committee on Rabies suggested that inactivated veterinary vaccines with a potency of less than 1.0 IU per dose, as measured by the NIH test, should not be licensed or released unless an adequately designed experiment has demonstrated a duration of immunity of at least one year in the species for which the vaccine is to be used.

The potency of live and inactivated vaccines should be ascertained at intervals after they have been distributed. Inactivated vaccine, even in liquid form, and lyophilized modified live-virus vaccines are relatively stable when stored under proper conditions. To verify that storage conditions are adequate, it is recommended that samples from the field that are approaching their expiry date be tested using the methods applied to newly manufactured products.

Minimum potency requirements for oral vaccines for immunization of wild animals have not been generally established, although the median effective doses (ED50) of various modified live-virus and recombinant vaccines are known.

To test the efficacy of candidate vaccines for oral immunization, sufficient numbers of target animals should be maintained under captive conditions, given the vaccine and challenged with the virus. The potency of the vaccines should be standardized to quantifiable levels (e.g. plaque-forming units/ml, TCID50/ml). Once efficacy has been demonstrated in the target species under laboratory conditions, the vaccine should be administered in a bait identical to that to be used in field trials.

Serial dilutions of test vaccine will determine the ED50. Animals should then be held for a minimum of 6-12 months prior to a challenge with a field strain administered by the intramuscular route; the interval between vaccine administration and challenge depends on the turnover rate of the target species. Potency estimates should not be based entirely on the ability of the vaccine to induce virus-neutralizing antibodies in the target species; environmental stability tests are also necessary to demonstrate that vaccine potency is retained under field conditions.