Results of Seromonitoring in FMD Buffer Zone in the CIS Countries

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1 Appendix Results of Seromonitoring in FMD Buffer Zone in the CIS Countries T.A.Fomina, V.M.Zakharov, A.A.Gusev, N.E.Kamalova, S.R.Kremenchugskaya, O.A.Schekotova All-Russian Research Institute for Animal Health, Vladimir, Russia Summary In total 264 of blood sera and 617 of sheep blood were studied for the presence of FMD types A and O virus antibodies and for the presence of nonstructural polypeptides 3ABC during springautumn in Georgia, Armenia, Azerbaijan and in Volga region of Russian Federation. In these Republics the FMD epidemiological situation is controlled by emergency vaccination of contact using bivalent A-O vaccine supplied by ARRIAH. About 48% of the examined had FMD type A antibodies and 41% - FMD type O antibodies. During the expedition in had positive reaction with 3ABC antigene in three Transcaucasian Republics (6 in sheep) and 67 had positive reaction ( in and 12 in sheep) in 2. During the examination of 283 blood sera taken in three regions of Kalmykia and Krasnodar Territory (22 from and 81 from sheep) virus-specific antibodies to FMD types A and O virus were also found. Positive reacting with nonstructural polypeptides 3ABC were not found in the above mentioned regions of Russian Federation. Introduction Identification of infected in the herds and their differentiation from vaccinated livestock is one of the tasks of effective control of FMD. This identification is also important in examination of sera because the may have subclinical FMD and may infect the susceptible even if the agent present in their organisms in very small amount (1, 2,, 9). To solve this problem one can use the method of identification of nonstructural proteins antibodies (NP), which are not formed in the blood after vaccination. Not less than 6 NP which may cause the formation of antibodies found in reconvalescent sera (3A, 2C, 3AB, 3AC, 3D, 3ABC) are known (2, 4,, 7, 8, 9). One of them, 3D or VIA antigene, connected with RNA-polymerase activity of FMD virus, independent of agent s type, was used at the end of 8-ies in different laboratories of the world to differentiate convalescent in agar gel immunodiffusion test (, 9). However, this method had very low sensitivity could give false positive results, especially in the cases of revaccination or in the cases when concentrated vaccines were used. That is why many FMD laboratories of the world use ELISA with recombinant 2C or 3ABC antigene, controlling the antibodies formed against these antigenes in blood sera of different. This makes the control of epidemiological situation more adequate. The aim of this work was to carry out FMD seromonitoring in order to study the epidemiological situation in Transcaucasian Republics and in the regions of Russian Federation that have the border with FMD unfavourable regions and with great economic significance. To reach this aim we used ELISA, virusneutralization test (VNT) and 3ABC trapping-elisa. Materials and Methods Principles of blood sera sampling During our business trip in in order to take the blood we based on the following issues: - information about FMD outbreak in this region; - regions that were included in the National Programme on FMD vaccination; - regions having borders with FMD endemic zones where emergency vaccination is carried out; - geographical position of the region, i.e. situation to the borders with Iran and Turkey; - regions of Russian Federation were included into the study because they have borders with Transcaucasian Republics (Krasnodar Territory and Abkhazia) and because they are situated in the zone of military conflicts (Kalmykia, Dagestan, Chechnya). Besides, great amount of is has been sold from the regions of Kalmykia to the neigbouring Astrakhan, Volgograd and Rostov regions of Russian Federation. The blood was taken at random (1-1% from the tested ) before immunization in accordance with the plan of buffer zone functioning measures and also in 6-1 months after vaccination.

2 Sera testing All the sera were scrinning for the presence of FMD types A and O virus in liquid-phase blocking ELISA. Positive in ELISA were studied in VNT for the above mentioned strains. The sera were considered positive if the titre of antibodies was equal or above 1 : 16 in ELISA and in VNT. 746 (3%) were examined for the presence of antibodies against nonstructural 3ABC polypeptides of FMD virus using modified variant of ELISA developed in Istituto Zooprofilattico Sperimentale Della Lombardia e dell Emilia- Romagna, Brescia, Italy, and the kit of components kindly provided by E. Brocchi and F. De Simone for differentiation of postvaccinal and postinfectional antibodies. Index of optical density more than,2 was considered positive for this method. Results Antibodies against structural proteins In total 99 blood from and 236 from sheep were tested in Georgia, Armenia and Azerbaijan in Among them 249 (2,1%) FMDV type A were found in and 11 (4,6%) in sheep; FMDV type O 27 (2,9%) and 4 (22,8%), respectively. Moreover, higher percent of positive (41,4 and 4,) was found in in Armenia. In Georgia these figures were 11,7 and 16,3% for FMDV types A and O, in Azerbaijan 8, and 21,6%, respectively, in of both species. These data are shown in Table 1. During the expedition in blood of and sheep in Georgia, Armenia, Azerbaijan and also in Kalmykia and Krasnodar Territory of Russian Federation were taken after usage of bivalent FMD A- O vaccine produced in ARRIAH. FMDV type A antibodies in Georgia were found in 6,6% of and 34,8% - FMDV type O antibodies. In Armenia these figures were 72, and 7,%, respectively, in Azerbaijan 78,4 and,2%. Results of these studies are shown in Table 2. Results of the study of FMD immune status of in the regions of Russian Federation using ELISA showed that 8 and 66,6% of with positive reaction for FMD types A and O in Krasnodar Territory caused by prophylactic vaccination of with vaccine produced by ARRIAH. In VNT these figures were 9, and 83,3%, respectively. In Kalmykia these figures in were 6,8% and 1,9% for FMDV types A and O, respectively. Results of this study are shown in Table 3. Antibodies to nonstructural polypeptides of FMD virus In 1999 because of reduced number of components for 3ABC-ELISA 228 from and sheep were tested. Among them 88 positively reacting were found, moreover, 32,% formed sheep. Postinfectional FMDV antibodies were found in 4 localities (2 regions) of Georgia and in 3 localities (Nakhichevan Autonomous Republic) of Azerbaijan. In 2 18 were studied for the presence of antibodies against NP of FMD virus in three Transcaucasian Republics and in some regions of Russian Federation. None of positive were found in Krasnodar Territory and in three regions of Kalmykia. Among and sheep 39 positive were found in Georgia, 16 - in Armenia and 11 - in Azerbaijan. Half (21) of positive in Georgia are from Adigensk where in December 1999 an FMD outbreak caused by type A virus (it was typed in ARRIAH) was registered. OIE al Reference Laboratory for FMD in Vladimir didn t receive from Armenia and Azerbaijan for typing. Discussions Transcaucasian countries are very often the zone of FMD introduction into southern part of Russian Federation and then into Europe. Historical analysis showed that during USSR times a very effective method of control for FMD using FMD vaccines, veterinary specialists training and so on, existed. When USSR collapsed these methods became weaker, specially, very small percent of livestock was vaccinated. Taking into account all mentioned above it is obvious that buffer zone organization in this region under the control of ARRIAH is an extremely important moment. Nonstructural proteins presence during FMD virus reproduction and their absence after vaccination may become a reliable method for identification of with antibodies formed as a result of convalescence or carriage of viruses from postvaccinal antibodies (1, 2, 3, 4,, 6, 7, 8, 9). In order to eradicate the infection one of the most important conditions to solve the question about necessity of vaccination of against FMD in the herds where live agent is circulating, available sensitive method is essential. Such method should give an opportunity to identify the infected in vaccinated population. Modified variant of ELISA is such a method. With the help of this method one can identify the antibodies against nonstructural proteins of FMD virus 3AB, 3ABC, 3D and others. Distribution of sera positive for FMD virus in different regions of Transcaucasus and Russian Federation was different and depended on the fact whether the were vaccinated or infected with the virus.

3 From the given results one can see that in 1999 not very high percent of immune in all three Transcaucasian Republics was found among and sheep, i.e. 2 and 11% for A type and 2 and 24% for O type, respectively. The highest percent of positive was identified in Ararat of Armenia (7,2 74,7%). This fact can be explained by the following: annually Armenia bought in ARRIAH 8 thousand doses of FMD vaccine. In 2 after realization of buffer zone programme and usage of bivalent A-O vaccine this index significantly increased in all Republics up to 6,6 78,4% for type A and 34,8 7,% for type O, respectively. As for the data on nonstructural proteins, identified with the help of 3ABC-ELISA, 88 positively reacting were found. Among them 4 were found in Georgia, 41 in Armenia and 43 in Azerbaijan. That can be explained by the fact that in August 1998 FMD outbreak caused by A type virus which in antigenic relation is quite different from productional A 22 strain (r 1 =,22) was registered. In 2 the situation in Armenia and Azerbaijan became better, from 426 examined 16 and 11 positively reacting were found. In Georgia the number of positively reacting increased up to 39. Perhaps, that can be explained by the FMD outbreak in Adigensk and Goriysk s caused by O type. So, as a result of fulfilled work one can make a conclusion that seromonitoring carried out with the help of ELISA and VNT gives an objective analysis of epidemiological situation in the region under control. In this monitoring identification of antibodies against FMD virus nonstructural polypeptides, carried out with the help of 3ABC-ELISA which gives an opportunity to identify reconvalescent and carriages of virus in vaccinated population, plays very important role. Table 1: Results of FMD seromonitoring in Transcaucasian Countries, July 1999 Azerbaijan Astarinsk region Total number of Positive A% Positive O% 3ABC positive/total number 11 2 / / Dzhulphinsky region ,4,3 4/6 / Shakhbuzsky region ,2 1, 44,1 17/26 1/1 Babeksky region 71 14, 4, /14 17/23 Sharursk region 21 9, 7,7 /2 /1 Ordubadsky region 16 12, 6,3 /1 Georgia Adzharia 73 9,6 16,4 /19 Borzhom region* 4 31,4 22,3 1/7 Ahalkalak region 8 7,7 / - - Gardabansk region /8 Mtskhetsky region 4 4,4 7,6 /7 / Akhaltsikhsky region* 4 22,2 42,2 3/1 Ozurgetsky region 3 3/1 Armenia Artashatsky region* ,8 33,9 28,8 23,7 Ararat region* ,7 7, ,8 32,7 Note: * - regions where with positive reaction in 3ABC-ELISA were found 2/1 28/66 11/17

4 Table 2: Results of Seromonitoring in Transcaucasian countries, April 2 A, % O, % 3ABC Positive reacting 3ABC Georgia Goriysk region Cattle 46 82,6 17, Adzharia Cattle 17 64, 3, 34 Gardabansk region Cattle 6 6 1,8 6,7 28,6 3,3 1 2 Adigensk region Cattle 6 78,6 87, Armenia Aparansk region Cattle ,8 3, 71,3 1, Ararat region Cattle ,2 42,9 67,7 8,7 1 Echmiadzinsk region Cattle ,6 8 39, Abovyansk region Cattle ,6 6 9, Shiraksk region Cattle ,8 Azerbaijan Shakhbuzsk region Cattle 64 84,3 2 Sharursk region Cattle ,4 33,3 32 Babeksk region Cattle 88 63,6 4, Ordubadsk region Cattle 7 6,1 76,9 63,2 92, Dzhulphinsk region Cattle 4 7,6 44,4 1 Astarinsk region Cattle ,6 4,9 37 Table 3: Animal blood sera study for the presence of FMD antibodies in Krasnodar Territory of Russian Federation and in Kalmykia using ELISA positives A 22 O 1 % positives Kalmykia Gorodovikovsk region Cattle ,4 2 6,7 Chernozemelsk region Cattle , ,9 Priyutnensk region Cattle ,6 23, , 4,7 Krasnodarsk Territory Cattle ,3 %

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