Spatial and temporal distribution of bluetongue and its Culicoides vectors in Bulgaria.

Surveillance of Culicoides (Diptera: Ceratopogonidae) biting midges was carried out between 2001 and 2003, at 119 sites within a 50 x 50-km grid distributed across Bulgaria, using light trap collections around the time of peak adult midge abundance. Sentinel and ad hoc serum surveillance of hosts susceptible to bluetongue infection was carried out at around 300 sites between 1999 and 2003. Following the initial incursion of bluetongue virus 9 (BTV-9) into Bourgas province in 1999, affecting 85 villages along the southern border, a further 76 villages were affected along the western border in 2001, with outbreaks extending as far north as 43.6 degrees N. The BTV-9 strain in circulation was found to have a low pathogenicity for Bulgarian sheep populations, with less than 2% of susceptible individuals becoming sick and seroconversions detected up to 30 km from recorded outbreaks in the south. The major Old World vector Culicoides imicola Kieffer was not detected among over 70,000 Culicoides identified in summer collections, suggesting that BTV-9 transmission in Bulgaria was primarily carried out by indigenous European vectors. The most likely candidates, the Palaearctic species complexes - the Culicoides obsoletus Meigen and C. pulicaris L. complexes - were widespread and abundant across the whole country. The C. obsoletus complex represented 75% of all individuals trapped in summer and occurred in high catch sizes (up to 15,000 individuals per night) but was not found across all outbreak sites, indicating that both Palearctic complexes probably played a role in transmission. Within the C. pulicaris complex, only C. pulicaris s.s., C. punctatus Meigen and C. newsteadi Austen were sufficiently abundant and prevalent to have been widely involved in transmission, whilst within the C. obsoletus complex most trapped males were C. obsoletus s.s. Adult vectors were found to be largely absent from sites in west Bulgaria for a period of at least 3 months over winter, which, taken along with the spatiotemporal pattern of outbreaks in the region between years, indicates the virus may be overwintering here by an alternative mechanism - either by covert persistence in the vertebrate host or possibly by persistence in larval stages of the vector.

Epizootiological control of bluetongue disease in Bulgaria in 2002.

In accordance with the National Surveillance Programme for the control of bluetongue (BT) disease in 2002, serum surveillance was performed in 22 sentinel, seronegative animal herds located in western Bulgaria. These herds were at least 40 km outside the settlements affected by the 2001 epidemic. Another 42 sentinel villages (herds) were established in southern Bulgaria in a 10 km border strip zone in the Bourgas, Yambol, Haskovo, Kardjali, Smolyan and Blagoevgrad Districts. The implementation of the programme in 2002 commenced on 15 April and continued until 15 November. More than 7 200 serum samples were tested prior to 26 August and no evidence of active BT virus (BTV) infection detected. This was confirmed by further viral, serological, epidemiological and clinico-pathological observations. In addition, there was no evidence of transborder penetration of BTV into Bulgaria by infected livestock or by infected Culicoides. However, on 26 August 2002, BTV seropositive sentinel animals were detected close to the southern Bulgarian border. Subsequently, animals were detected in more than 20 villages, but clinical disease was not observed. Bulgaria was divided into 58 quadrants (50 km x 50 km) and a Culicoides surveillance programme established in 23 of these. A total of 92 Culicoides light-trap collections were made. During three years of Culicoides surveillance, not a single specimen of the principal BT vector C. imicola was captured. The dominant Culicoides species was C. obsoletus, followed by C. pulicaris and C. punctatus; in August 2001, C. puncticollis was recorded for the first time. Studies on the seasonal phenology of Culicoides were conducted in two villages (Vacsevo and Bersin in the District of Kiustendil) affected in the 2001 outbreak of BT. Here trapping of Culicoides commenced on 1 March 2002 and continued until 15 November 2002; midges became active during the third week of April to almost cease in the second half of November. There appeared to be three peaks of activity: one during the second half of May, another in August and a third at the beginning of October.

[Demonstration of specific VIA antibodies in foot-and-mouth disease]. / Dokazvane na spetsifichni VIA antitela pri shapa.

Experiments were carried out to obtain a specific VIA antigen of F.M.D. During the research, 7 series of VIA antigen were extracted with high purity and specificity. It was established that 90% of the infected animals build specific VIA antibodies against F.M.D.V. Up to 70% of the repeatedly vaccinated cattle against F.M.D. contain VIA antibodies. The percentage of the positive reagent depends on the age of the animals, on the number of the immunizations and on the sort of the used vaccine. The cause for the high percentage of the seropositive to VIA not vaccinated animals is not elucidated.

[Cultivation of cells in a medium with blood hydrolysate]. / Kultivirane na kletki v sreda s kruven khidrolizat.

Use was made of fresh blood of swine, diluted with an equal amount of distilled water to produce a protein hydrolysate. Enzyme hydrolysis was effected through extracellular alkaline protease produced by stain DI of Bacillus subtilis. The cell lines BHK-21, PK-15 and spzv were cultured in a nutrient medium containing 0.180-0.200 mg/cm3 alpha-amine nitrogen and relevant growth factors of nonprotein character. It was found that the cells cultured in this medium showed no differences with regard both to morphology and karyology as against the control cells which were treated with the classic medium of Eagle. It was also found that the medium could successfully be used instead of the imported nutrient media.

[Differentiation of foot-and-mouth disease viruses by counterimmunoelectrophoresis]. / Diferentsirane na shapnite virusi s nasreshtna imunoelektroforeza.

Studied were the opportunities for employing the method of counter immunoelectrophoresis (CIE) to differentiate F. M. D. viruses of different origin, comparing the results obtained with those reached through the immunodiffusion test (IDT). It was found that CIE could be used successfully for the serotyping of F. M. D. viruses, the method being as precise as IDT, however, the results are recorded at the 90th minute, while IDT requires at least 24 hours to read the results.

[Development of a method for the quantitative determination of the immunizing antigen (140S) of the foot-and-mouth disease virus]. / Razrabotvane na metod za kolichestveno opredeliane na imunizirashtiia (140S) antigen pri shapniia virus.

Attempts were made to work out a method for measuring the amount of the 140 S antigen in virus suspensions. Early postinfection sera were obtained from guinea pigs against the productional strains of the foot-and-mouth disease virus which were used in the radial immunodiffusion test. The investigated virus suspensions were concentrated 50 to 200 times and were placed in a CsCl gradient for gradient centrifugation. The 140 S antigen fractions obtained were titrated in a radial immunodiffusion test. The size of the resulting precipitation circles were defined for the individual dilutions of the antigen, and the method of least squares was employed to establish the standard straight lines for each of the investigated productional strains of the FMD virus. The new method makes it possible to fix the content of the 140 S antigen in productional series of the FMD virus intended for vaccine production.

[Binary ethyleneimine as an inactivator of the foot-and-mouth disease virus]. / Binerniiat etilenimin kato inaktivator na shapniia virus.

Experiments were carried out to inactivate F.M.D. viruses with the use of binary ethylene-imine. It was found that inactivation was optimal when the agent was used at the rate of 0.003 M for 18 hours at 26 degrees C. Its neutralization in the virus suspension was carried out with 3 mM sodium thiosulfate. The inactivated F.M.D. viruses retained their complement-fixing and immunogenic properties. Discussed are the advantages of using binary ethyleneimine as an inactivating agent as against other agents of the asiridine order.

[Experiments to produce an attenuated strain of the transmissible gastroenteritis virus and its use as a live vaccine]. / Opiti za polchavane na atenuiran shtam na virusa na transmisivniia gastroenterit i izpolzuvaneto mu kato zhiva vaksina.

Serial passages have been performed of a field virus of the transmissive gastroenteritis to obtain an attenuated strain adapted to a permanent cell line of pig kidney, "SPEV". The strain is innocuous for test animals, for pigs of all ages, and for swine during the entire period of pregnancy. It has proved genetically stable. The strain is immunogenic for swine and pigs both under laboratory and under field conditions (in new foci and on stationary farms) and can be used as a live vaccine against transmissive gastroenteritis, either untreated or in a freeze-dried state.

[Passive hemagglutination reaction in differentiating foot-and-mouth disease viruses]. / Reaktsiia pasivna khemaglutinatsiia pri diferentsirane na shapnite virusi.

Experiments were carried out with the use of the passive hamagglutination reaction in the differentiation of foot-and-mouth disease viruses. The investigations made use of purified sera and specific IgG antibodies obtained through column chromatography. Conjugates were prepared with the use of bis-diazotized benzidine and glutaraldehyde. In experiments with conjugates prepared with IgG and glutaraldehyde standard and reproducible results were obtained. The use of the passive hemagglutination test has led to the successful differentiation of F. M. D. viruses of various origins. Comparative investigations with the complements-fixation test and the passive hemagglutination test have shown that the latter is more readily applicable and rapid as well as more sensitive.

[Combined method (suspension-monolayer) of preparing an inactivated foot-and-mouth disease vaccine]. / Izgotviane na inaktivirana protivoshapna vaksina po kombiniran metod (suspenziia-monosloi).

Semiproductional and productional experiments were carried out to cultivate BHK21C13 cells in 30- and 250-litre fermentors, to produce F.M.D. virus in suspension and in roller, and to produce a F.M.D. vaccine in 500-litre automatic mixing containers, employing a combined method 'suspension-monolayer'. The F.M.D. virus that was replicated in suspension had a 10(-6,0-6,5) titer, and the virus that was obtained in monolayer - a 10(-7,0-8,0) titer. It was demonstrated that the F.M.D. vaccines that were produced after the combined method were sterile, innocuous, and immunogenic.

[Laboratory trials of culturing BHK 21 C 13 cells in a roller using an enzymatic egg hydrolysate-based nutrient medium]. / Laboratorni opiti po kultivirane na kletki BHK 21 C 13 v roler pri izpolzvane na khranitelna sreda na baza iaichen enzimatichen khidrolizat.

Experiments were carried out to find the optimal conditions for the replication of BHK 21 C 13 cells in one 1 roller glassware with the use of a nutrient medium containing products of the enzymatic breakdown of white of egg. Studied was the sensitivity of these cells to the type C strain 'Hungary' of the foot-and-mouth disease virus. Two series of F. M. D. vaccines were produced, which proved to be sterile and innocuous for guinea pigs. Immunogenicity trials with regard to these animals revealed that the VD50 (vaccinal dose) values ranged from 0.12 to 0.26 cm3.

[Isolation of anti-foot and mouth IgG by means of affinity chromatography]. / Izolirane na protivoshapen IgG s pomoshta na afinitetna khromatografiia.

The coupling of the F. M. D. antigen with unsoluble CNBr-activated sepharose (2B or 4B) induced the production of an active agent with which the specific F. M. D. antibody of the investigated solution may covalently couple. Coupling procedures prove most effective with the use of buffer solutions of high ion strength that is necessary for the reduction of a protein-protein adsorption initiated by the polyoelectrolite nature of proteins. The produced complexes of the type CNBr activated sepharose--12C antigen--antibody after elimination of the unspecific serum proteins are dissociated up to the release of a specific IgG antibody.

[Dynamics of multiplication of BHK21C13 cells in a suspension]. / Prouchvane dinamikata na razmnozhavane na kletki BNK21C13 v usloviiata na suspenziia.

In order to master a new method for the production of a F. M. D. vaccine the authors have studied the reproduction dynamics of BHK21C13 cells in the conditions of a suspension culture. The culturing of the cells has been carried out under laboratory conditions, investigating also the concurrent changes in the pH value along with the cell dynamics. The nutrient medium used and the conditions of cultivation has made it possible to study the properties and obtain a qualitative cell suspension. The cells obtained have shown equally good growth in a monolayer medium and in a suspension, and are appropriate for the reproduction of the F. M. D. virus.

[Production and use of a nutrient medium with egg hydrolysate for monolayer reproduction of BHK 21/13 cells]. / Poluchavane i izpolzvane na khranitelna sreda s iaichen khidrolizat za monosloino razmnozhavane na kletki BHK 21/13.

Experiments aiming to reproduce BHK 21/13 cells in Roux retorts by use of a semi-synthetic nutrient medium, based on protein hydrolyzate synthetized by the authors, were carried out. Results obtained show that the lower peptides and free amino acids found in ovarian hydrolizate meet the requirements of the cell line in respect to quality and quantity. The successfull cultivation of BHK 21/13 cells in hydrolyzate medium made possible a considerable reduction in the percentage involvement of serum, which is usually used in the classical nutrient media of the type Eagle BME intended for the propagation of diploid cell strains.

[Micromethod of determining the complement-binding properties of commercial series of the foot-and-mouth disease virus]. / Mikrometod za opredeliane komplement-svurzvashtite svoistva na proizvodstveni serii shapen virus.

Investigations were carried out to establish the possibility of using a micromethod of the complement-fixation test to determine the complement-fixing properties of productional series of the foot-and-mouth disease virus. It was found that the micromethod referred to is an economically profitable and practically simple one. It is readily applicable requiring no particular apparatuses and equipment, is specific, and can successfully be used instead of the routinely employed CFT method. The micromethod suggested is economical, requiring minimal amounts (0.025 cu.c7) of the components taking part.

[Relationship between the chemical structure and the biological activity of antibodies against the foot-and-mouth disease virus]. / Zavisimost mezhdu khimichna struktura i biologichna aktivnost na antitelata sresht virusa na shapa.

Investigated was the effect of various amino agents (stained and phthalic anhydride and sulfopicric acid) on the complement-and antigen-fixing activity of IgG and IgM antibodies isolated from guinea pig sera, the donor animals being hyperimmunzed with type O, strain Polyana, of the foot-and-mouth disease virus, At the moderate modification (75-86 per cent acetylation, and 36-40 per cent 'phthalation') of the primary amino groups the foot-and-mouth disease antibodies retained only half of the conjugation caused greater and complete loss of this activity. IgG and IgM showed almost equal specificity with regard to the antigen that had induced them. The lowered complement-fixing capacity was shown to have no specific effect on the primary relation of the foot-and-mouth disease virus to the antibody. Chemical modifying agents inactivated the specific small, strongly defined complement-fixing zones in the Fc-fragment of the foot-and-mouth disease antibody molecule.

[Effect of chemical modifiers on the antigen-binding capacity of antibodies]. / Vliianie na khimichnite modifikatori vurkhu antigen-svurzvashtata sposobnost na antitelata

The effect was followed up of the chemical modifications "carbamylation" and "benzylation" on the antigen-fixing activity of the foot-and-mouth disease immunoglobulins G and M. The chemical agents used - potassium cyanate and 2-hydroxi-5-nitrobenzylbromide--modified a considerable number of side lysine chains and tryptophane radicals, however, had no effect on the precipitation activity of the foot-and-mouth disease antibodies IgG and IgM manifested with the respective antigens. The lowered ability of the modified (to a higher degree) antibodies to form precipitations with the homologous virus was due to secondary reactions between the two components of the system, the functional groups of the active center remaining intact. The higher affinity of the IgM antibody to the homologous antigen was due to its polyvalent capacity (as compared to the bivalent immunoglobulin G).