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Many years of research and observations on bait uptake, efficacy, behavioural studies of foxes, reduction and elimination of rabies, population dynamics of foxes following oral vaccination, as well as annual exchanges of researchers' experiences, formed the scientific background for field trials extending over many years. These are the most extensive field studies for the testing of a rabies vaccine so far. The use of the techniques and strategies of oral immunisation of foxes against rabies using SAD B19 can eliminate wildlife rabies among foxes and raccoon dogs, as European experience has shown. The disease then also disappears completely in domestic animals and man.

Complementary DNA spanning the entire genome of the attenuated rabies virus strain SAD B19 which is used for oral immunization of foxes in Europe was cloned and sequenced. The viral genome comprises 11,928 nucleotides and encodes the five viral proteins N, NS, M, G, and L. Deduced protein sequences are highly similar to those of the pathogenic PV strain, homologies ranging from 90.6% for the M to 98.6% for the L protein. The five cistrons are separated by intergenic regions of 2, 5, 5, and 24 nucleotides, respectively. The G transcription stop/polyadenylation consensus signal in SAD B19 is destroyed by a deletion of three A residues. The strong conservation of both noncoding and coding nucleotide sequences indicates a high selective pressure on the primary structure of rabies virus genomic RNA.

Three attenuated rabies virus vaccines (SAD-B19, ERA/BHK-21, AZA 2) were compared for efficacy and safety in the striped skunk (Mephitis mephitis) by the oral and intranasal routes. The SAD-B19 and ERA/BHK-21 vaccines were given orally; all three vaccines were given intranasally. Oral administration of SAD-B19 and ERA/BHK-21 vaccines induced neither seroconversion nor significant protection against rabies challenge. One skunk which consumed a SAD-B19 vaccine-laden bait succumbed to vaccine-induced rabies. Intranasal instillation of the three vaccines resulted in the deaths of two of six (AZA 2), three of six (ERA/BHK-21) and six of six (SAD-B19) skunks.

The development of and the logistics used in campaigns for the oral immunization of foxes against rabies in the Federal Republic of Germany are described. The "Bavarian Model", which is outlined in detail, shows how local hunters were recruited for bait distribution and control measures at practically no cost. Over 10 million baits have been distributed in this way by hundreds of thousands of hunters in the Federal Republic of Germany as well as in the other European countries that participated in this large-scale field trial. As a result of the trial, rabies has been reduced or even eradicated in large parts of the Federal Republic of Germany.

In a field trial 181 high risk persons of both sexes between ages of 2--71 years were prophylactically vaccinated against rabies with the beta-propiolactone inactivated rabies-vaccine (hdc) mrc-5. 1 ml of vaccine was administered intramuscularly on days 0, 7 and 21. Serum samples were collected on days 0, 7, 21 and 35. Neutralizing antibody titers against the rabies virus were determined in the rapid fluorescent focus inhibition test, 7 days after the first injection measurable titers of antibodies were found in all sera with one exception. On day 21 all persons showed a 100% seroconversion with a mean titer of 1:1000 (8,3 IU/ml). On day 35 the final mean titer measured 1:2400 (20 IU/ml). Major side effects during the prophylactic vaccination were not observed.

The antirabies human diploid cell vaccine produced by 1'Institute Merieux, Lyon, France, was administered intradermally to 35 high-risk volunteers using 0.2-ml amounts and various immunization schedules. Three groups never before vaccinated against rabies developed virus-neutralizing antibodies, the titer of which was dose dependent. A single injection stimulated the formation of antibodies. Four inoculations induced the highest antibody levels and the longest persistence of antibody. The administration of a single intradermal booster inoculation was sufficient, even in the case of low-persisting antibody, to elicit a rapid increase of antibodies to high levels. A primary inoculation course of two injections induced a sufficient antibody level which, in case of exposure, could apparently be rapidly elevated by a 0.2-ml intradermal booster inoculation. Adverse side reactions were observed in 7 of 14 individuals after a 1- or 1.5-year intradermal booster inoculation. We therefore suggest that the intramuscular and subcutaneous routes continue to be used for primary vaccinations and that the highly effective intradermal route be restricted to booster inoculations. This is the first long term study of this vaccine and should be a guideline for the pre-exposure treatment of high-risk personnel.

The comparative studies undertaken by 7 laboratories in 6 countries show that the calculation I.U.s did not as anticipated minimize but actually enhanced the variability of results of Rabies antibody estaminations in the sera of HDCS vaccines. The high biological variance in the method(s) may not have been considered by individual laboratories and any neglect of fundamental biostatistical laws, unfortunately, diminishes the theoretical advantage of using the "International Standard (I.S.)" as a "tertium comparationis". Perhaps the intrinsic variability of the I.S. should be re-evaluated and it is conceivable that a pure IgG fraction of Rabies antiserum would show less variability. Intralaboratory variation might be reduced by agreeing that only a geometric mean of the I.S., and not a single value obtained in an individual test, should be used for calculation of I.U.s. Application of the principles of biochemical and pharmacological methods, such as test-to-test control of the I.S. and its analytical variances might well enhance the reproducibility of the results. MNT, RFFIT, PRT and CFT were unable to detect antibodies in HDCS vaccinees until 7 days after the first vaccination. The establishment of methods for detecting early antibody requires further investigation.

Since spring 1983 a field trial to immunize foxes by the oral route against rabies has been carried out in a heavily infected area of Hessen measuring about 1,500 km2. The vaccine strain SAD B 19, produced by the Federal Research Institute for Animal Virus Diseases at Tübingen and filled into 2 ml aliquots, was inserted into chicken heads and distributed by forest rangers in the trial area, 15 baits per km2. Up to the present day five baiting campaigns, two per year in spring and autumn, have been completed and in all 110,000 vaccine baits additionally marked with 150 mg tetracycline were layed out. Four baitings have been previously evaluated. With the exception of some rabies cases on the border to the unvaccinated region in the northwest, rabies was reduced impressively. A secondary outbreak in the region of Hausen/Kiedrich, caused by an extremely high density of the fox population, was eradicated by increasing the baits to 25 per km2. The number of rabies cases, which amounted to 238 within 15 months prior to the vaccination trial, went down from 38 in the first 6 months to 6 cases after the fourth baiting. As innocuity control 2,273 wild and domestic animals from the vaccination area were examined for the presence of the vaccine virus. The results were always negative indicating that rabies strain SAD B 19 had not established an infective cycle in wildlife. As could be shown by the marker studies, an average of 78 per cent of the foxes (variation of the baitings from 62 per cent to 82 per cent) had taken up one or more baits. Seroconversion had been achieved in 65 per cent of the foxes (variation from 43 per cent to 70 per cent) as demonstrated by the presence of neutralizing antibodies. From experiences in Switzerland it can be expected, that rabies will be extinguished, if 50 per cent of the population are successfully immunized. This value has been surpassed in the field trial in Hessen. These very promising results have led to the project of an enlargement of the vaccination area to 12,000 km2. The neighbouring states of Rheinland-Pfalz and Nordrhein-Westfalen have joined in this effort, which has started in autumn 1985 with a new artificial vaccine bait developed at the Tübingen research institute.

Unlike previous reports to the contrary, raccoons (Procyon lotor) were successfully vaccinated against rabies with a liquid SAD-B19 attenuated virus vaccine administered per os and given in vaccine-laden baits. There was neither evidence of vaccine-induced rabies in raccoons nor in a limited safety trial with opossums (Didelphis virginiana) given SAD-B19. Protection from lethal street rabies virus infection was not absolute: only three of nine raccoons given 1 x 10(6.0) TCID/ml were protected versus five of 10 raccoons given 1 x 10(7.0) TCID/ml of SAD-B19 and challenged 4 mo after consumption of vaccine-laden baits. Six of eight raccoons consuming 1 x 10(8.8) TCID/ml of SAD-B19 vaccine in baits survived street rabies virus challenge 2 mo postvaccination. Raccoon survivorship was not wholly dependent upon rabies virus-neutralizing antibody titer on the day of challenge. Vaccinated raccoons demonstrated a prominent anamnestic response within 1 wk following challenge. Surviving raccoons were observed for a minimum of 3 mo following street rabies virus challenge with neither clinical nor pathologic evidence of rabies. The SAD-B19 rabies vaccine administered within baits in captivity appears less effective for raccoons than for its demonstrated efficacy in the immunization of free-ranging foxes (Vulpes vulpes) in Europe.

Two candidate reference rabies vaccines were tested in a collaborative study involving seven laboratories. Using four different testing procedures it was determined that both vaccine preparations were highly potent and demonstrated satisfactory stability in the thermal degradation test.

The field trial for the oral immunization of foxes against rabies in the Federal Republic of Germany began in 1983 and since the end of 1985 has had the specific goal of the elimination of rabies in large interdependent areas of that country. To meet this goal it was necessary to develop methods for the large-scale production of the SAD (Street Alabama Dufferin) clone B19 vaccine as well as to develop a bait that could be mass-produced (the Tübingen fox bait). It has been shown that after a single vaccination campaign in the field approximately 75% of the fox population is immune to rabies and that after one or two repeated campaigns vaccination in many areas could be discontinued. Participation in the field trial in circumscribed border areas was extended to Italy in 1985 and to Austria, Luxembourg, Belgium, and France in 1986. For the first time, through the use of oral immunization, a method for the elimination of rabies is available that is effective, safe, inexpensive, and practical. Further, this method meets the requirements of animal-protection societies, does not interfere with the preservation of endangered species, and therefore represents a significant political factor.

The comparative studies undertaken by 7 laboratories in 6 countries show that the calculation of I.U.'s did not, as anticipated, minimize but actually enhanced the variability of results of rabies antibody estimations in the sera of HDCS vaccinees. The high biological variance in the method(s) may not have been considered by individual laboratories and any neglect of fundamental biostatistical laws unfortunately diminishes the theoretical advantage of using the "International Standard (I.S.)" as a "tertium comparationis". Perhaps the intrinsic variability of the I.S. should be re-evaluated and it is conceivable that a pure IgG fraction of rabies antiserum would show less variability. Intralaboratory variation might be reduced by agreeing that only a geometric mean of the I.S., and not a single value obtained in an individual test, should be used for calculation of I.U.'s. Application of the principles of biochemical and pharmacological methods, such as test-to-test control of the I.S. and its analytical variances might well enhance the reproducibility of the results. MNT, RFFIT, PRT and CFT were unable to detect antibodies in HDCS vaccinees until 7 days after the first vaccination. The establishment of methods for detecting early antibody requires further investigation.