EXPERIENCES WITH RABIES ERADICATION PROGRAMS

Oral vaccination as a method of rabies eradication in the field was first started in Switzerland in 1978 and after 1984 several other EU countries followed this practice. Due to oral vaccination some European countries are now rabies-free in terrestrial animals. In Slovenia, after the first experimental oral vaccination and study of vaccination models from 1988 –1992, the spring-autumn campaigns have been carried out since 1995. The model of oral vaccination of wildlife requires 16–20 baits per km2 in the vaccination area. The baits were distributed by plane. They were dispersed from a height of 300 – 500 m. The aeroplanes’ paths were 1000 metres apart. In the vaccination campaigns two vaccines were used. Lysvulpen®, produced by the Bioveta company at the Czech Republic, was laid down in the southwestern part of the country, and Fuchsoral®, produced by the German company Impfstoffwerk Dessau-Tornau, was placed in the eastern part of Slovenia. A rapid decline of rabies was evidenced from 1995 to 1999, when the oral vaccination program in the whole territory using the aircraft baits distributing system was practiced. In 1999, only 6 rabies cases were laboratory-confirmed, whereas in 1995, 1089 rabies cases were documented. Of the 14 rabies cases detected in 1998, 12 were found as an island in a circle with a radius of 30 km in the centre of the vaccinated area. In 2000 and 2001, rabies incidence increased again, so it was decided to change the baits distribution system in the year 2001. The vaccination by crossing flights in certain areas was introduced. In the next year (2002), after changing the vaccination strategy, positive cases rapidly dropped and only 15 cases in 2002, and 8 cases were found in 2003, near the non-vaccinated border with Croatia.

Rabies is a zoonotic disease of mammals, caused by infection with a negative-strained RNA virus belonging to the Lyssavirus genus of the Rhabdoviridae family, mainly transmitted via saliva following a bite from an infected animal Š1¹.In central and southeast Europe, the red fox (Vulpes vulpes) from the wild reservoir species is the main source of infection Š2, 3¹.In Slovenia, only the urbane rabies was present at the beginning of the 20th century.The sylvatic rabies was introduced in 1973, and the whole country had become infected by the end of 1980.The infection of domestic animals was always connected to the epizootics in wildlife.Immunization of dogs is obligatory and advisable for cats.In the period from 1997 to 2003, when the trend of epizootics was high, cattle, sheep and horses in pasture were also vaccinated against rabies.
Oral vaccination as a method of rabies eradication in the field was first started in Switzerland in 1978, and after 1984, several other EU countries followed this practice.Due to oral vaccination, some European countries are now rabiesfree in terrestrial animals; Finland (1991), Netherlands (1991), Italy (1997), Switzerland (1998), France (2000), Belgium (2001) and Luxembourg (2001) Š4¹.In other eastern and southern European countries, the number of reporting rabies cases has increased Š4¹.
A rabid animal was first registered in Slovenia in 1973.It was found in the Prekmurje region.The disease spread in the southwest direction.The nature of this breakout was that it only spread to the river Mura area and didn't go any further Š5¹.The disease reached its peak in Slovenia in 1979, when it crossed the borders in the upper Sava valley region and in the Carinthian region.In 1980, the first cases were reported on the outskirts of the capital Ljubljana and a year later it spread to the southern parts of the country.Ever since, we have had rabies occurrences among wild animals, some cases even in non-vaccinated domestic animals in the period before oral vaccination was carried out.The Institute for Microbiology and Parasitology at the Veterinary Faculty in Ljubljana annually examines 2,000 -4,000 samples for the rabies virus.The percentage of positive cases has varied from 20 to 45%.Cyclic oscillations in the numbers of positive cases have been a common feature for Slovenia as well as the rest of Europe.This cycle is repeated every 3 to 5 years and starts with a high occurrence followed with a fall in the number of positive cases and then again the number rises.This is closely associated to the size of the fox population at a given time.
The rabies surveillance has been in effect since the beginning of the 1970's.According to the Slovene legislation the veterinary organisations are obliged to send the animal carcases with neurological clinical symptoms for laboratory examination.In the territory of 21.000 km 2 from 1195 to 3787 samples were In Slovenia, after the first experimental oral vaccination and study of vaccination models from 1988-1992, the spring-autumn campaigns have been carried out since 1995.A Fuchsoral (Tubingen Institute, Germany) vaccine containing the attenuated SAD-B19 virus (titre 10 -7 TCID 50 /ml) raised on BHK21 cell culture was used.Each foil-wrapped ampoule contained 1 ml of vaccine and was covered with meat and fish flour mixed with fat.A biomarker in the form of tetracycline was also added to each bait.

Rabies surveillance in
The first oral vaccination in Slovenia was performed in 1988 (Gorenjska region).After carefully considering the geographical layout of Slovenia and examining the results acquired from Austria, West Germany, France and Italy, where this kind of vaccination started in the early eighties, we decided upon the Tubingen model of vaccination.This model requires laying 16 -20 baits per km 2 in the vaccination area.Some areas were laid using the "common sense" method where the baits were hand-laid in areas where foxes were registered.The other areas were laid systematically using a pattern of squares (each square being 250 m wide and 250 m long) in which 16 baits were to be laid (16 baits per km 2 ).The latter proved to be more efficient.The job of laying the baits by hand was given to the game wardens that also registered how many baits were actually taken from the spot where they had been laid (4, 8 and 12 days after the bait placement).The wardens also shot foxes, which were examined to the tetracycline biomarker.The laying of baits was completed in one day.The purpose of the control was to determine the vaccine intake and the vaccination level.Based on positive results from the Gorenjska region, we extended that method of bait laying to all Slovenia.Due to financial reasons, we baited the country gradually, starting at the west and ending in the east.The vaccination was performed in six-month intervals autumn/winter.The vaccination in the springs of 1990 and 1991 was cancelled due to financial difficulties.Each vaccination saw an enlargement of the area by 5 to 10 thousand km 2 taking into consideration the fox biology and ecological closures.Up to 1992, the entire country was covered in this way.

First period of oral vaccination in Slovenia / Prvi period oralne vakcinacije u Sloveniji
In the following section, we present brief data about oral antirabies vaccination of free living common foxes in Slovenia in the period from 1995 to 2004.The vaccination was performed first in autumn 1995, and then in spring and autumn 1996, in the surface of the whole country.In 1997, the vaccination was not done.Two campaigns per year have been carried out since 1998, the first two years in the whole country, later the vaccination was spread to 18.800 km 2 , but in the year 2000, it was reduced to 12.500 km 2 while the density of baits increased to up to 23-26 baits/km 2 .The Northwestern part of the country has not been vaccinated since 1999, because no rabies positive cases were found.In the vaccination campaigns two vaccines have been used.Lysvulpen® produced by the Bioveta company from the Czech Republic was laid down in the southwestern part of the country, and Fuchsoral® produced by the German company Impfstoffwerk Dessau-Tornau, was placed in the eastern part of Slovenia.The baits were distributed by plane.They were dispersed from a height of 300 -500 m.The aeroplanes' paths were 1000 metres apart in a parallel line.The pilots used the GPS navigation system for orientation and the discharge was monitored by a computer.A special computer program named FICO3J® (constructed by computer engineer Aleksander Modic) monitored the route and time of flying and calculated the density of distributed baits.Due to the increased incidence of rabies in 2000, the vaccination strategy in the year 2001 was changed.When a crossing flight instead of a single flight model was introduced, rabies cases dropped again in the year 2002, and positive cases were still found only in the non-vaccinated region near the border of Croatia.
In this field study the vaccine was used in Slovenia (Lysvulpen® and Fuchsoral®) both for oral vaccination, of foxes containing the live-modified rabies virus SAD -strain, and the thermo-stability of baits and the virus were observed.The vaccine baits were placed in three different locations: (a) in the open cutgrassing places, where direct sun was possible, (b) in the high grass, and (c) under trees and bushes where direct sunlight was not possible.This study was done during the summer period (from July 6, to August 14, 2003), when maximum daily temperatures were higher than 30 o C. Ten baits A and ten baits B in the open land (location a), thirty baits A and thirty baits B in the high grass (place b), and fift baits A and B in the shadow (location c) were placed.Baits were dropped directly on the ground and the distances between baits were at least 2 cm.

Thermostability of vaccine in the field / Termostabilnost vakcine na terenu
Oral antirabies vaccination in Slovenia / Oralna vakcinacija protiv besnila u Sloveniji The Rabies investigation showed that during the last 10 years, rabies prevailed among red foxes (2918 cases, 89,26%), rare rabies positive cases were found in other wildlife animals (194 cases, 8,01%; 68 martens, 58 badgers, 56 roe, 6 polecat, 3 deer, 2 wild boar, 1 lynx), and in domestic animals (158 cases, 4,85%; 81 cats, 59 dogs, 11 cattle, 4 sheep, 2 horses, 1 rabbit) (Table 1).In the period from 1992 to 1996, the territory of Slovenia was heavily affected by rabies.A rapid decline of rabies is evidenced from 1995 to 1999, when an oral vaccination program in the whole territory using the aircraft baits distributing system was practiced.In 1999, only 6 rabies cases were laboratory-confirmed, whereas in 1995, 1089 rabies cases were documented (181-fold decrease) (Table 1).Of the 14 rabies cases detected in 1998, 12 were found as an island in a circle with a radius of 30 km in the centre of the vaccinated area.In 2000 and 2001, rabies incidence increased again.In the next year ( 2002), after changing the vaccination strategy, positive cases rapidly dropped and only 15 cases were found near the nonvaccinated border with Croatia (Fig. 2).The number of positive cases then declined and 8 positive cases were found in 2003.
The stability of the baits matrix and virus titre was recorded over an 8week summer period (maximum daily temperatures were over    SAD B19.The loss of virus titre was highest when baits were placed in the sunlight, but when they placed in the shadow, the virus was detected still on the 53rd day of observation.After 24 hours, the bait matrix of Lysvulpen placed in the sunlight showed total destruction and the vaccine container was not covered with fat, paraffin-fish meal matrix.In the case of the vaccine Fuchsoral, the bait matrix was partially destructed and the vaccine container was visible, too.After 72 hours, the titre of the rabies virus in the vaccine Fuchsoral and after 48 hours the virus titter in the vaccine Lysvulpen were not detectible in the virus isolation test using BHK 21 cell culture, when baits were placed in the sunlight and the maximum daily temperature was 32 o C.
The evaluation of animal rabies cases in Slovenia during the last decade indicates that foxes represent the main source of infections among wild animals, and cats and dogs among domestic animals.Before the vaccination program of wildlife, rabies was detected all over Slovenia.After the vaccination of wildlife, the number of laboratory-confirmed rabies cases in animals in Slovenia decrease rapidly.A table 1

RABIES -2003
Tested: 993 Positive: 8 foxes During the years 1996 and 1997, rabies was dispersed practically in the whole territory of Slovenia, while in 1998, only 14 positive cases were found.Twelve of them were concentrated within a radius of 30 km in the centre of the vaccinated area.Only 2 cases occurred near the national border connected with the epidemic east.The persistence of rabies cases in the centre of the vaccinated area could be explained by the fact that during all three years of the vaccinating program, aircrafts were not allowed to fly in this area, and the baits were not distributed in this territory.All 6 rabies cases detected during the year 2000 have been found along the national border with Croatia.A significant increase of positive cases in 2000 and in 2001 in the southern part of the country was evidenced.The reason (lewer funds) for that increase could be the fact that in the year 1999, lewer baits were distributed (13 -15 baits/km 2 ) in this territory than in the years before (15 -17 baits/km 2 ).This territory is densely covered with forests and rich biotypes favourable to natural rabies vectors/reservoirs.After the increase of baits density per km 2 (23 -26 baits/km 2 ) the number of rabies positive animals decreased.In the year 2002, 15 rabies and in 2003, 8 rabies cases were confirmed.All were located near the Croatian border, except one badger, positive in the IF test, which was shot in the unvaccinated area.Later, this positive result in the IF test was not confirmed by the RT-PCR test.
We could notice that when 25 baits per km 2 were laid by single flight in parallel lines, the fox had less chances to find the bait.When the crossing flight distribution was used, the baits were more dispersed and more accessible for foxes.This strategy using the raster model for baits distribution was found suitable especially when a geographical configuration with high hills and deep valleys was present.After the introduction of this distribution model the number of rabies cases declined in 2002 and 2003.

AKNOWLEGEMENT / SRPSKI:
We acknowledge the constant support of Veterinary Administration of the Republic of Slovenia.Thank to Ales Brecelj and Emil Mlinaric for good collaboration and technical support.