INVESTIGATION OF POSSIBILITIES FOR APPEARANCE OF AFRICAN HORSE SICKNESS VIRUS AND CHANGES IN SPECIES OF THE GENUS „ CULICOIDES ” IN BULGARIA

To date, Bulgaria has been free of African Horse Sickness (AHS). Contacts with and proximity to countries, in which it is assumed there are cases of the disease, necessitate surveillance of equine animals near the border, strict control of outgoing and incoming animals, especially those returning to the country after a long stay abroad of participation in races. Instructions for carrying out veterinary activities in instances when the disease occurs in Bulgaria have been worked out, since there were occurrences of Blue tongue disease (the disease has the same epizootiological vectors of spreading) in regions bordering with Turkey last year. The serological screening investigations of blood sera by ELISA of equine animals from the same villages and regions during one active period of flight of culicoides did not establish the presence of antibodies to this dangerous transmissive disease. The present study contributed to working out scientific-methodological diagnostic preparedness in Bulgaria as regards this exotic transmassive disease.

high lethality, up to 90 %.The disease is caused by a Reoviridae family virus of the Orbivirus genus.AHS has endemic and seasonal character.It can take a sporadic or epizootic course during the hot humid period of the year.Horses, donkeys and zebras are most susceptible to the virus.Dogs have been experimentally infected, while man is not susceptible to the disease Š1, 3, 4, 7, 14, 16¹.It is diagnosed in laboratory conditions by isolating the virus from hyparinized blood, spleen, lymph nodes or from collected culicoides.ELISA, Complement fixation test (CFT), Immunodiffusion (ID), PCR, etc. are also used as serological reactions Š2, 5, 6, 8, 9, 15, 16¹.Of over 1400 types of culicoides known to science, only a few dozen can transmit the AHS virus.To date, it has been found that the main vector for AHS transmission to equine animals in northern Africa and the Mediterranean basin is C. imicola, followed by C. obsoletus, C. punctatus, C. circumscriptus, C. pulicaris, C. fasciopennis, C. subfascipennis.Bulgaria has so far been free of AHS.Contacts with and proximity to countries, in which it is assumed that there are cases of the disease, necessitate surveillance of equine animals near the border, strict control of outgoing and incoming animals, especially of animals returning to the country after a long stay abroad or participation in races.Instructions for carrying out veterinary activities in instances when the disease occurs in Bulgaria have been worked out, since there were occurrences of Blue tongue disease (the disease has the same epizootiological vectors of spreading) in regions bordering with Turkey last year.Stringent control must be in place regarding equine animals in these regions -100 % within the 30 km zone bordering with Turkey and 50 % in the interior of the country.All import and export of animals must also be placed under strict control.
The objective of the study is to present etiological, entomological, faunistic and epizootiological investigations.The epizootiological data will be summarized and discussed in the light of known facts.
Serological investigations / Serolo{ka istra`ivanja ELISA and reagents produced in INIA-Valdeolmos, Spain, which is the reference laboratory and center for this disease, were used to find antibodies to the common group antigen of the AHS virus.We took as positive the samples showing average optical density (D OD) that was lower or equal to D OD of the positive control of the kit or calculated according to a formula giving as a result the percentage of ELISA reaction inhibition and the respective strength of antibodies in the serum tested against the AHS virus.Blood samples from 123 equine animals coming from the same regions and villages were investigated.Four-fold investigations were carried out in May, June, July and August 2000.

Materials and methods / Materijal i metode rada
Entomological investigations / Entomolo{ka istra`ivanja Entomological and faunistic investigations were carried out to determine the possibilities for the penetration of the AHS virus.During the period of observation 94 collections of culicoides were processed.The total number of identified culicoides was 10 623, which were collected from different spots by light traps with a light source capacity of 75 W from 22 p.m. until 6 a.m.The genus and sex of the caught specimens were determined.

Epizootiological observations / Epizootiolo{ka posmatranja
The method of epizootiological observation was used during the study.The data of the movement of animals in the border regions, as well as geographic and climatic factors that are favorable to the distribution of the main vector were taken into consideration.Climatic and geographic characteristics, relief, precipitation data (taken from the hydrometerological posts), direction and intensity of prevailing winds in the region were all considered.
On May 24, 2000, 123 blood sera samples from horses, donkeys and mules from regions bordering with Turkey were recived at the Laboratory of Viral Diseases of Equine Animals at the Central Research Veterinary Medical Institute in Sofia.
No antibodies to AHS were detected when investigating 21 blood sera samples from donkeys and 5 samples from mules (Table 1).

Results / Rezultati rada
After four-fold serological investigations on 123 equine animals, at the dates specified in Table 1, no antigens to the AHS virus were detected.
The results of the faunistic and entomological investigations are presented in Table 2.  7 -25.10.2000. 15.7 -20.11.2000 3.9 -25.10.2000 1.8. -1.11.2000The spreading of culicoides has so far included territories between latitudes 35 o South and 40 o North.The main carrier of the virus of AHS and Blue tongue disease, which transmits them to ruminants in South Africa is C. imicola Š10, 11, 12¹.The occurrences of Blue tongue disease in Bulgaria in 1999 and its spreading in the border regions indicates that disease that have characteristic biological carriers can occur in Bulgaria.The active zone of spreading of the transmissive Blue tongue disease has crossed the 42 o parallel (near the region of Bourgas and Yambol) and the appearance of species of culicoides not typical to the country reaffirms the danger of the penetration of the AHS virus, which has similar carriers.After concluding the epizootiological observation, it was found that sites in the proximity of the big rivers in southern Bulgaria are of a biotype which is extremely favorable to the reproduction of the culicoides.The climatic peculiarities, the constant trend for warming and the winds from Africa are contributory factors to the spreading of this transmissive disease.The detection of species of culicoides in Bulgaria, which until two years ago were not found at this geographic latitude -C.punctatas, C. subfascipennis, C. circumscriptus, C. nubeculosum, etc. confirm this.It is most disturbing that three of these species directly transmit the AHS virus.Usually, AHS appears two or three years after the appearance of Blue tongue disease at a given site Š10, 12, 13, 14, 15¹.
The lack of antibodies to the AHS virus, which was proven during the serological screening investigations of equine animals coming from the same regions and villages during the active period of flihgt of flight of culicoides is favorable as regards the epizootiological situation in Bulgaria.But the presence of possible vectors of transmission, the occurrence of a disease bearing similarities to the way of transmission os AHS, the favorable climatic and field conditions, necessitate strict measures and observation.Serological and entomological surveillance must be carried out in these regions and equine animals in the regions bordering with Turkey and Greece must be carefully monitored in the next two or three years.
1.An entomological investigation of the composition of culicoides in regions bordering with Turkey and Greece showed that C. imicola was not present there.But the detection of species directly transmitting the AHS virus to horses C. punctatas, C. subfascipennis, C. circumscriptus and C. nubeculosum for the first time in Bulgaria as a direct hazard to equine animals.
2. The serological screening investigations of blood sera of equine animals from the same villages and regions during one active period of flight of culicoides by ELISA did not establish the presence of antibodies to this dangerous transmissive disease.

Table 1 .
Results from the serological investigations carried out in the regions bordering with Turkey by ELISA for detection of antibodies to AHS virus in equine animalsTabela 1. Rezultati serolo{kih istra`ivanja u oblastima koje se grani~e sa Turskom putem ELISE radi otkrivanja antitela na AHS virus kod ekvida Chenchev et al.: Investigation of possibilities for appearance of african horse sickness virus and changes in species of the genus ...