Laboratory investigations and prevalence of rabies in Bulgaria Dr. Darinka Ilieva, PhD NRL of Rabies National Diagnostic Research Veterinary Institute (NDRVI), 1606 Sofia, Bulgaria Dr. Emil Iliev Laboratory activity Directorate Bulgarian Food Safety Agency ABSTRACT Laboratory surveillance is carried out to identify cases of rabies among animal populations. During 2009 – June 2011 in NRL of Rabies are studied 897 brain samples from different species (domestic and wild animals). In studies were used standard methods, recommended by the OIE: fluorescent antibody test (FAT) and mouse inoculation test (MIT). The last peak of morbidity of rabies were recorded 2009 with 59 cases and disease was diagnosed in 47 foxes, 2 jackals, 2 sheep, 1 goat, 1 cow, 3 dogs and 3 cats. Epizootic data from past years show, that the disease was found in 14 districts of Northern Bulgaria. During the period of cases of rabies were recorded in 5 districts of South Bulgaria. A significant improvement was recorded since 2009 after the introduction of oral vaccination of foxes, as a method to eradicate the disease. Declining of the epizootic process was observed Key words: rabies, foxes, prevalence, fluorescent antibody test INTRODUCTION Natural reservoirs of the rabies virus in Bulgaria are wild predators - mainly foxes and to a lesser extent, wolves, jackals, wild cats, badgers and others that infect primarily through bite domestic animals, dogs, cats and human. Prevails in our country "sylvatic form" of rabies, with the main distributor - the red fox (Vulpes vulpes). In , there were the first cases of "sylvatic rabies" recorded in northeastern Bulgaria, in district Silistra, it is considered that the infected animals have been passed from Constanta Romania. In 1977 it is reported a case of "sylvatic rabies" in northwestern Bulgaria, in district Montana, likely the infection is transmitted from Yugoslavia. During the period was registered rabies epizootic wave, which subsides with marked mortality in foxes to 50%, which clearly indicates that the rabies virus has been observed for the first time in the fox population. To interrupt the epidemiological chain in northeastern Bulgaria are annually over 3500 foxes shot. In 1982 occurs "second epizootia" of rabies in foxes, which continues until today. From , the epizootic process is characterized by high intensity and widespread rabies was registered in 14 districts (9 in northern Bulgaria and 5 in Southern Bulgaria). Rabies in Southern Bulgaria penetrates through the area of Kotel passage in district Sliven. From 1988 to the present moment rabies epizooties have peak of occurring at intervals of several years. Alarmingly in epidemiological situation in recent years (2007, 2008 and 2009) is not only a large number of identified positive cases of rabies for one year and spread of the disease south of the Balkan mountain in region of Sofia city (Novi Iskar town, Katina village, Mirovyane village and Vladaya quarter), Sofia district in area of Svoge town and Slivnitsa town, Pernik district (Rasnik village, Goljamo Buchino village and Baykalsko village); Kyustendil district (Dolna Koznitsa village, Lilyach village and Shatrovo village). The complicated epizootic situation was a prerequisite competent veterinary autorities ask the European Commission to approve and fund a program to vaccinate foxes, as the main vectors of rabies virus in Bulgaria. In 2009 and 2010 Decision 2008/897/EC and Decision 2009/883/EC of the EC in our country is conducted oral vaccination of foxes against rabies, which continues The purpose of this study is to analyze data 2009-June 2011, compared with the previous year related to territorial spread of rabies in our country and the epidemiological risk to humans. MATERIAL AND METHODS In the study used data from laboratory observations of the 897 samples submitted for diagnosis of rabies in the National Reference Laboratory (NRL) of Rabies - NDRVI, Sofia 2009-June Investigated samples from the brains of domestic and wild animals with suspicious clinical signs of rabies associated with the supervision of a disease in State prevention program (SPP) and samples from foxes shot for control of oral vaccination of foxes against rabies June Samples in relation to oral vaccination have been sent by the official veterinarian of the districts - Vidin, Montana, Vratsa, Pleven, Lovech, Veliko Tarnovo, Gabrovo, Ruse, Targovishte, Razgrad, Shumen, Silistra, Dobrich, Varna, Burgas, Sofia-city, Sofia-district, Pernik and Kyustendil. Laboratory investigations of rabies was made by direct Fluorescent antibody test (FAT) of brain impressions from animals and Mouse inoculation test (MIT) to confirm the diagnosis, acording the standard methods recommended by OIE (Manual of Diagnostic Tests and Vaccines for Terrestrial Animals, 2008). RESULTS Most registered positive cases of rabies were recorded in ; ; ; ; and (Table 1). The total number of infected from rabies wildlife and domestic animals increases to foxes 338, jackals 22, other species 22, domestic animals 210, dogs 68 and cats 33. The date show also that in 1988-June 2011 rabies infections in fox population comprised 48.7%, of all recorted cases, while the number of rabies cases in domestic animals was lower 30.3%. The percentage of rabies infections in jackals, other species, dogs and cats is shown in fig.1. Natural-focalis morbiditas in a given area allows us to identify trends and dynamics of spread of this deadly infection. On the Table 2 presents data from surveys conducted in the NRL of Rabies 2009-June 2011 in terms of prevalence of rabies on the territory of 19 districs in Bulgaria. There are investigated total of 897 brain samples from different animals. The results speak for a distinct tendency to increase infected with rabies virus domestic and wild animals to 59 cases in Detected cases of rabies have 2009 in 13 districs in 47 foxes, 2 jackals, 3 dogs, 3 cats, 2 sheeps, 1 goat and 1 cow. Data analysis in 2009 showed that most cases of rabies in animals are found in northeastern Bulgaria in Shumen -17. In northwestern Bulgaria in region of Montana have established the largest number of positive cases - 8. In 2009 an outbreak of rabies has been demonstrated in another district Burgas. In 2010 were recorded 6 cases of rabies in 4 districts of Vratsa, Rousse, Shumen and Veliko Tarnovo. At 2 foxes and 1 dog from district of Vratsa, 1 cat from Shumen, 1 cat from Veliko Tarnovo and 1 cat from Ruse. To June 2011 was found one case of rabies in foxes shot. In epidemiological relationship , diagnosed with rabies are 8 cats that have bitten 12 people. For the same period the rabies virus was demonstrated in 10 dogs, 2 of them were bitten 4 people. Persons exposed to exposition to the rabies virus was provided timely prevention with vaccine. In the special case of risk of infection (with single or multiple transdermal bites and scratches, or contamination of mucous membranes with saliva) persons were treated with combined rabies immunoglobulin and vaccine. Table1 Types and number of infected with rabies wildlife and domestic animals in Bulgaria 1988-June 2011 Fig.1 Percentage of rabies infection in wildlife and in domestic animals in Bulgaria ( June 2011) Table 2 Prevalence of rabies disease 2009–June 2011 in territory of Bulgaria Districs with rabies June Sofia town1-- 2 Sofia district Vidin Vraca Gabrovo Dobrich Lovech Montana8-- 9 Pleven Razgrad Ruse Silistra Turgovishte Shumen Veliko Turnovo Bourgas Kustendil Pernik Varna--- Total number of positive samples:5961 Spreading of the disease from North to South part of the country since 2007 DISCUSSION Our observations show that the largest number of infected animals are found in spring. From May to June 2009 are reported, respectively, 36 and 42 positive cases of rabies, fewer cases of rabies were autumn-winter season of the year-11, but at least the summer – 6 cases. Perhaps this is due to ekobiological features of fox populations in the country. The high incidence in spring is related to foxes mating season and the autumn-winter rise due to demand for living space of young foxes. On the other hand, high rates of disease in domestic animals due to breeding of pasture and hence the possible contact with wild animals. From studies conducted in , the rabies virus was found in areas where a shorter or longer period of disease is unknown. In 2009 in 2 districts - Veliko Tarnovo and Ruse were not detected cases of rabies. In Burgas previous case of rabies was registered in Laboratory studies show that there is no small number of rabies-infected cats that are actively involved in the chain of infectious disease transmission. Close contact between the cat and people and their way of farming is essential in the epidemiology of rabies. For containment and eradication of rabies is necessary to have strict control over population of cats and dogs which have to be vaccinated regularly. Also it is necessary realistic counting and measures to control the significant population of jackals and wolves with the help of hunters. Oral vaccination of foxes against rabies in Bulgaria is running 2009 and continued 2010–2011. There have been a total of 4 vaccination campaigns for foxes, two in (spring from march to may and autumn, september- october 2009), and a campaign in spring 2010 and 2011.Oral vaccination of foxes are performed with LISVULPEN vaccine type in dose of 20 pieces (vaccination bites with live vaccine) per 1 square kilometer covering up km2 total area. The vaccine baits are distributed by helicopters and planes over areas covered by the program. Program duration of 5-7 years (at least 2 years after the last positive case). Recorded cases of rabies in 2010-Juni 2011 are little. This can be considered as an indicator of successful implementation of the vaccination campaign. The results clearly show that the program of oral vaccination of foxes have to continue for a longer period of time to achieve eradication of rabies in Bulgaria. CONCLUSIONS: As a result of the implementation of oral vaccination of foxes in Bulgaria, cases of rabies in animals were decreased tenfold. The observed incidence of rabies in cats tells us that it is necessary to develop a program to control rabies in them. It is necessary to identify measures for inclusion in the population of jackals in the control program of rabies. REFERENCE 1. Community Summary Report on Trends and Sources of Zoonoses and Zoonotic Agents and Foot-borne Outbreaks in the European Union in The EFSA Journal, Program for control and eradication of rabies in R.Bulgaria 2009 and WAHID Interface, Rabies, Detailed country (ies) disease incidence, Jan 2006 – Dec 2010, YearnumberfoxesjackalsOther species domestic animals dogscats / Total:693338(48,7%)22(3,2%) 210(30,3%)68(9,8%)33(4,8%)