ORAL RABIES IMMUNIZATION OF SWINE : USE OF VNUKOVO-32 / 107 VACCINATION STRAIN

Ondrejka R. , A. Ondrejková, ·. ·vrãek, Z. Bení‰ek, J . Süli , J . Závadová, M. Maìar, J . MojÏi‰ová: Oral Rabies Immunization of Swine: Use of Vnukovo-32/107 Vaccination Strain. Acta Vet. Brno 2001, 70: 333–338. The apathogenicity and antigenic activity of the live oral rabies vaccine prepared from the strain Vnukovo-32/107 were evaluated in two experiments carried out on non-target species – swine (domestic swine Sus scrofa domestica and wild boar Sus scrofa). The Vnukovo-32/107 vaccination strain has been used to produce oral rabies vaccine Kamark for immunization of free living carnivores. Wild boars compete with foxes for acceptance of baits distributed in the field. The tested vaccination strain for oral application (including very high doses) proved apathogenic to non-target species – swine. Detection and quantification of rabies antibodies were carried out by an immunoenzymatic assay – ELISA, rapid fluorescence focus inhibition test (RFFIT) and virus neutralization test (VNT) on mice on days 30 and 90 post-immunization. The values obtained were expressed in international units (IU/cm3); the value of 0,5 IU/cm3 was used as a positivity criterium. Antirabic antibodies were detected in more than 50 % of animals in all three groups of two animal sets on day 30 post-immunization. The results obtained show good antigenic activity of the live oral rabies vaccine prepared from the strain Vnukovo-32/107. Rabies, vaccination strain Vnukovo-32/107, apathogenicity; antigenic activity Comprehensive analysis of topical knowledge on rabies immunoprophylaxis indicates that oral vaccination, performed for the first time by Baer et al. in 1971, is the only prospective method of rabies prophylaxis and control in free living carnivores. Many researchers from different countries focused on selection of suitable rabies vaccination strains and determination of their safety, efficacy and stability (Black and Lawson 1970; Baer et al. 1971; Johnston and Voigt 1982; Steck et al. 1982; Wandeler et al. 1982; ·vrãek et al. 1994). The vaccine intended for oral immunization of free-living animals should comply with a range of requirements (WHO 1989; Wandeler 1991). Apart from inducing immunity, it should fulfil the second most important requirement, i.e. be apathogenic to target and nontarget animal species. The harmlessness of various oral rabies vaccines was experimentally proved on many free-living animals, captured and held in captivity (Brochier et al. 1989; Guit t re et al. 1992; Cliquet et al. 1995; ·vrãek et al. 1995 a, b). The first experiments were carried out with a SAD strain (Baer et al. 1971; 1975; Debbie et al. 1972). The SAD strain (Street Alabama Dufferin) was originally isolated from a dog in Alabama, the USA, in 1935 (Fenje 1960). Different variants, used for production of oral vaccines, were derived from that strain: SAD-Bern, SAD-B19, ERA, SAG, SAG2, Vnukovo-32. The safety and effectiveness of the strain Vnukovo-32 was confirmed by many experiments (Sel imov 1978; 1987). High ACTA VET. BRNO 2001, 70: 333–338 Address for correspondence: Doc. MVDr. R. Ondrejka, CSc. Department of Infectology and Tropical Veterinary Medicine University of veterinary Medicine Komenského 73, 041 81 Ko‰ice, Slovak Republic Phone: +421 55 633 4581 E-mail: suliova@uvm.sk http://www.vfu.cz/acta-vet/actavet.htm

Comprehensive analysis of topical knowledge on rabies immunoprophylaxis indicates that oral vaccination, performed for the first time by Baer et al. in 1971, is the only prospective method of rabies prophylaxis and control in free living carnivores.Many researchers from different countries focused on selection of suitable rabies vaccination strains and determination of their safety, efficacy and stability (Black and Lawson 1970;Baer et al. 1971;Johnston and Voigt 1982;St eck et al. 1982;Wandeler et al. 1982;•vrãek et al. 1994).
The vaccine intended for oral immunization of free-living animals should comply with a range of requirements (WHO 1989;Wandeler 1991).Apart from inducing immunity, it should fulfil the second most important requirement, i.e. be apathogenic to target and nontarget animal species.
The harmlessness of various oral rabies vaccines was experimentally proved on many free-living animals, captured and held in captivity (Brochier et al. 1989;Guittre et al. 1992;Cliquet et al. 1995;•vrãek et al. 1995 a, b).The first experiments were carried out with a SAD strain (Baer et al. 1971;1975;Debbie et al. 1972).The SAD strain (Street Alabama Dufferin) was originally isolated from a dog in Alabama, the USA, in 1935 (Fenje 1960).Different variants, used for production of oral vaccines, were derived from that strain: SAD-Bern, SAD-B19, ERA, SAG, SAG2, Vnukovo-32.The safety and effectiveness of the strain Vnukovo-32 was confirmed by many experiments (Selimov 1978;1987).High degree of attenuation and good immunogenic activity of the strain Vnukovo-32 on the level of 107 th serial passage was proved by many years experience with the field use of live rabies vaccines for wild and domestic animals.
The results of experiments investigating apathogenicity of Vnukovo-32/107 vaccination strain and the antigenic activity of the oral rabies vaccine in immunization of swine are published in this study.

Apathogenicity of Vnukovo-32/107 vaccination strain Experimental animals
Non-target animals-domestic pigs (Sus scrofa domestica) and wild boars (Sus scrofa), 3-month-old, were tested.Both species of animals came from the school farm in Zemplínska Teplica, and had not been previously vaccinated.

Vaccine
Live tissue rabies vaccine -infectious tissue culture medium of the vaccination strain Vnukovo-32/107, cultured in the BHK-21 cell line, was used for the immunization.This vaccine strain was selected for production of oral rabies vaccine by the producer MEVAK Nitra (Slovak Republic), commercial name Kamark.Before using the vaccine or the infectious cell culture medium for immunization experiments, the virus titre of the vaccination strain was determined in parallel by intracerebral (i.c.) inoculation test (Koprowski 1973) on mice weighing 6.0 g; the volume of inoculum was 0.03 cm 3 and its decimal dilutions were prepared.Five mice were immunized, each with different dilution, and observed for 21 d post-infection.The specificity of dying was checked by examination of brain imprints of transversal cuts on the level of hippocampus by direct immunofluorescence test (DIFT) (Dean and Abelseth 1973) for detection of rabies antigen.The results were processed by the cumulative method according to Reed and Muench (1938), the rabies tissue culture infectious test RTCIT (Wiktor 1973;Rudd and Trimarchi 1978, modified), on 8 Lab-Tek cell slides, in a BHK-21/138 cell culture, followed by detection of virus multiplication by DIFT.

Immunization of animals Experiments in domestic swine
Testing of the apathogenicity of the Vnukovo-32/107 vaccination strain was carried out in 3 groups of domestic swine, 8 animals in each.
Group I: animals were immunized orally by a vaccination bait Kamark; Group II: animals were immunized orally by a dose 5× higher than the presumed immunization dose (3 cm 3 , 5× concentrated infectious tissue culture medium with 10% addition of stabilizer).
Group III: animals were immunized orally by a dose 15× higher than the presumed immunization dose (3 cm 3 15× concentrated infectious tissue culture medium with 10% addition of stabilizer).

Experiments in wild boars
Animals were divided into 3 groups, 5 animals in each.Group I: animals were immunized orally by a vaccination bait Kamark; Group II: animals were immunized orally by a dose 5 x higher than the presumed immunization dose (3 cm 3 5 x concentrated infectious tissue culture medium with 10% addition of stabilizer).
Group III: animals were immunized orally by a dose 15x higher than the presumed immunization dose (3 cm 3 15x concentrated infectious tissue culture medium with 10% addition of a stabilizer).

Verification of the apathogenicity
Animals were observed clinically for 90 d.In the observation period, saliva (swabs from oral cavity mucosa) samples were taken at 7-day intervals (days 7-63).Pool samples (2 for each group of domestic swine, 1 for each group of wild boars) were examined by i.c.inoculation test (MICIT) on suckling mice.
On day 180, the experimental animals were killed and partial necropsy was performed to obtain parts of the central nervous system (CNS) and submandibular salivary glands.The material obtained was used to prepare imprints for both DIFT and MICIT.

Antigenic activity
Antigenic activity was determined in animals from the experiments described above (3 groups of domestic swine, 3 groups of wild boars).Blood samples were taken on days 30 and 90 post-immunization and rabies antibodies were detected and quantified simultaneously by 3 following methods: ELISA test (kit developed in our laboratory; Süliová et al. 1988;Bení‰ek et al. 1989); RFFIT (Wiktor 1973;Bourhy and Sureau 1991) using CVS-11 as a challenge virus; VNT in 12 g mice (Atanasiu 1973) using CVS-11 as a challenge strain at a dose of 50 MICLD 50 .
All sera were examined by ELISA and RFFIT tests.Pool samples (4 pool samples for each group of domestic swine, 2 pool samples for each group of wild boars) were prepared for VNT.
Student's t-test was used for statistical analysis.The results obtained from Group 1 immunized by vaccine Kamark served as a basis for comparison.

Results and Discussion
Apathogenicity of the vaccination strain Vnukovo-32/107 was verified out in two sets of experiments on non-target species, swine (Sus scrofa domestica and Sus scrofa).Pool samples were examined by MICIT on suckling mice.No rabies antigen was isolated either from brain imprints prepared from parts of CNS or from submandibular salivary glands of animals killed on day 180 post-immunization.
The results confirmed that the vaccination strain Vnukovo-32/107, when given orally, is apathogenic to the non-target species, swine (including extremely high doses).
The results of detection and quantification of rabies antibodies in experiments on swine are shown in Table 1 and 2. Blood sera from both species of animals were used to determine antigenic activity of the vaccines.Detection and quantification of antibodies was carried out on days 30 and 90 post-vaccination using ELISA, RFFIT and VNT tests on mice.The values obtained were expressed in international units (IU/cm 3 ), in comparison with international reference serum.The value ≥ 0.5 IU/cm 3 was used as a positivity criterium.The virus detection results obtained by different methods were similar and quantitative determination depended on sensitivity of the method.The highest values were obtained by ELISA in both experimental sets (Tables 3 and 4).Rabies antibodies were detected in more than 50 % of animals on day 30 post-immunization in all three groups of animals of both experimental n -the number of animals in groups x -p < 0.05 sets.Only the values obtained by ELISA and RFFIT were expressed as percentage proportions as VNT was carried out on pool samples.The levels of rabies antibodies determined on day 90 were lower, except in the group of domestic swine, which were immunized orally by a dose15-times higher (p < 0.01).Detection and quantification of rabies antibodies in some animals that accepted vaccination bait Kamark provided negative results, because the animals probably swallowed the bait without puncturing the vaccine blister or accepted only a portion of the bait.The efficacy of live vaccine depends in general on both immunogenic and antigenic activity of the selected vaccination strain and the vaccination dose.The latter (at the same inoculum volume) depends on the concentration (titre of the live vaccination virus) in the cellular infectious medium (Wandeler 1991).It is very important to define the residual virulence for target and non-target animals (•vrãek et al. 1994, 1995a).Bioveta Ivanovice na Hané (the Czech Republic) has been using the vaccination strain Vnukovo-32/107 for production of live tissue vaccine for all species of domestic animals (Vrzal et al. 1988) and for production of inactivated vaccines for over 20 years.
For the development of oral rabies vaccine the strain Vnukovo-32 on the level of 107 th passage was used.This was preceded by extensive many years of experimental work.The experiments were carried out as model experiments on various species of laboratory animals, especially on white mice, but also on target animals -common foxes and related species -farm polar foxes (•vrãek et al. 1995b).
The first part of our study dealt with the apathogenicity of the vaccination strain Vnukovo-32/107 for non-target animals -swine, including wild boar, which compete with other animals for vaccination baits distributed in the field (Brochier et al. 1988) and can play an important role in their consumption.No less important is the conclusion that prevention and control of diseases in wild boars can be carried out by distribution of such baits (Aubert et al. 1994).The results of experiments performed in both species of animals demonstrate that the vaccination strain Vnukovo-32 is apathogenic at extremely high doses to non-target species -swine.Its high degree of attenuation and low residual virulence proves its suitability for the production of rabies vaccine.
The efficacy of rabies vaccines is evaluated by means of target and laboratory animals and detection of seroconversion.The relationship between the level of virus neutralizing antibodies and protection against rabies was described by Bunn (1987).The presence of specific rabies antibodies in the serum of vaccinated animals indicates the performed vaccination.However, one should realize, that also those animals that failed to produce sufficiently high titre of antibodies may be protected against rabies infection.Although the efficacy of correctly performed rabies vaccination is high, induction of immunity in all immunized animals should not be taken for granted (Bourhy et al. 1988).Some animals exhibit genetically inherited inadequate immune reaction (Blancou et al. 1989).

Table 1
Detection and quantification of rabies antibodies in experiments in domestic swine.Mean values are expressed in IU/cm 3 .

Table 3
Summarization of animals positive for rabies antibodies after vaccination.Comparison of ELISA and RFFIT tests.Titre of positivity ≥ 0.5 IU/cm 3 .Experiments in domestic swine.Summarization of animals positive for rabies antibodies after vaccination.Comparison of ELISA and RFFIT tests.Titre of positivity ≥ 0.5 IU/cm 3 .Experiments in wild boars.