Ferhat ABBAS, Faisal Ameer KHAN, Fayyaz AHMAD, Azhar HUSSAIN, Masroor AHMAD, Mohammed Arif AWAN, Mohammed Masood TARIQ, Mohammed Azam KAKAR, Abdul WADOOD, Mumtaz ALI

BHK-21 Hücre Hattı üzerinde Şap Hastalığı Virüs Aşısının (O tip) Üretilmesi

Şap Hastalığı “0” tipi aşısı, bebek Hamster Böbreğinin (BHK) 21 hücre hattında üretilmiştir. BHK-21 hücreleri doku kültürü şişelerinde dönüştürülmüştür. Otuzyedi 0C’ de 48 saatin sonunda mono katmanlar elde edilmiştir. Birleşen mono katmanlara, Şap hastalığı “O”tipi virüsü bulaştırılmıştır. Hücrelerin kümelenmesı ve ayrılması formunda, Sitopatik etki 48 saat sonra gözlenmiştir. Herhangi bir bakteri ve mikoplazma kontaminasyonu için aşı ücretsiz tedarik edilmiştir. Bu virüse (TCID 106/ml) maruz kalan sığır, fare ve kobaylarda sistematik reaksiyonlar gözlenmiştir. Yaklaşık 6-9 aylık yaşa sahip 9 buzağı, güvenlik testi için kullanılan aynı titrede aşı yapılmış ve öldürücü virüse (TCID 104.5/ml) meydan okunmuştur. Tüm hayvanlar bu zorluğa dayanmış ve herhangi bir reaksiyon göstermemiştir. Bu zorluktan sonra, iki aşılanmayan hayvanın her ikisi de tipik Şap hastalığı lezyonu göstermiştir. Şap hastalığı şüphesi olan 10 hayvandan alınan ağız lezyonları toplandı ve bu alan izolatlarının adaptasyonu, BHK 21. Hücre hattı üzerinden geçişlerle gerçekleştirilmiştir. Üç geçişten sonra bu alan izolatlarından herhangi bir viral sitopatik etki elde edilmemiştir. Şüpheli antijenler de Şap hastalığı için dikkate alınmıştır. ELISA testi ile O (60%), A (20%), ve Asia-1 (10%) tiplerinin varlık gösterdiği belirlenmiştir

Anahtar Kelimeler:

Adaptasyon, Antijenler, Aşı, BHK, direnç, virülent, adaptation, Sitopatik etki

Productıon of Foot and Mouth Dısease Vırus Vaccıne (O Type) on Bhk-21 Cell Line

Footh & Mouth Disease (FMD) “O” type vaccine was produced on Baby Hamster Kidney (BHK)-21 cell line. BHK-21 cells were grown in tissue culture flasks. Monolayers were obtained after 48 hours at 37 oC.Confluent monolayers were infected with FMD “O” type virus (TCID50 104.5/ml). Cytopathic effect (CPE) in theform of clumping and detachment of the cells was observed after 48 hours. Vaccine was found free of any bacterialand mycolplasma contamination. Systemic reaction was observed after injecting cattle, mice and guinea pigs withthe virus (TCID50 106/ml). Nine calves of about 6-8 month of age were vaccinated using the same titer used forsafety test and challenged with the virulent virus (TCID50 104.5/ml). All the animals withstood the challenge and didnot show any reaction. Both of the 2 unvaccinated animals after challenge showed typical FMD lesions. Mouthlesions from ten FMD suspected animals were also collected and adaptation of these field isolates was performedby passages on BHK-21 cell line. No viral CPE was obtained from these field isolates after 3 passages. Suspectedantigens were also got typed for FMD. The types O (60%), A (20%), and Asia-1 (10%) were found present usingELISA

Keywords:

BHK, vaccine, challenge, virulent, adaptation, cytopethic effect, antigens,

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Amarel-Doel, C.M.F., Owen, N. E., Feris, N. P.,. Kitching R. P and. Doel, T. R., 1993. Detection of foot-and-month disease viral sequences in clinical specimens and ethyleneimine-inac- tivated preparations by the polymerase chain reaction. Vac- cine, 11: 415-421.
Barteling, S. Z. and Vreeswijk, Z., 1991. Development in foot-and- mouth disease vaccine. Vaccine, 9: 75-88.
Bastos, A.D.S., 1998. Detection and characterization of foot-and –mouth disease virus in sub-Saharan Africa. J. Vet. Res., 65: 37-47.
Bergmann, I. E., Neitzert, B., Malirat, V., Ortiz, S., Colling, A., Sanchez C. and Correa Melo, E., 2003. Rapid serological profiling by enzyme-linked immunosorbent assay and its use in an epidemiological indicator of foot-and- mouth dis- ease viral activity. Arch. Virol., 148: 891-901.
Callahan, J. D., Brown, F.,. Csorio, J F. A., Sur, H., Kramer, E., Long, G. W., Lubroth, J., Ellis, S. J., Shoulers, K. S.,. Gaffney, K. L.,. Rock D. L.and Nelson, W. M., 2002. Use of a portable real-time transcriptase polymerase chain reaction assay for rapid detection for foot-and-mouth disease virus . J. Am. Vet. Med., 220 (11): 1636-1642.
Clarke, J.B. and Spier, R. E., 1980. Variation in the susceptibility of BHK populations and cloned cell lines to three strains of foot- and –mouth disease virus. Arch. Virol., 63:1-9.
Cunningham, C. H., 1973. A laboratory guide in virology. 7th ed. Burgess Publishing Co., Minneapolis, MN.
Doel, T. R. and Staple, R. F., 1982. The elution of FMD from vac- cines adjuvanted with aluminium hydroxide and with saponin. J. Biol. Stand., 10: 185-195.
Domingo, E., Baranowski, E., Escarmis C.,and Sobrino, F., 2002. Foot-and-mouth disease virus. Comp. Immunol. Microbiol. Infect. Dis., 25: 297-308.
European Pharmacopeia, 1986. Second Edition. Editions of the Council of Europe, Strasbourg, France.
European Pharmacopeia, 1993. Second Edition. Monograph No. 63. Editions of the Council of Europe, Strasbourg, France.
European Pharmacopeia, 2008. Version 6.1. Monograph No. 63. Editions of the Council of Europe, Strasbourg, France.
Ferris, N.P. and Dawsonm, M., 1988. Routine application of enzyme-linked immunosorbent assay in comparison with complement fixation for the diagnosis of foot-and-mouth and swine vesicular disease. Vet. Microbial., 16: 201-209.
Food and Agriculture Organization of the United Nations (FAO), 1984. Emerging Diseases of Livestock. Vol. 1. The Diseases and their Diagnosis, Geering W. A, ed. FAO, Rome, Italy. 1: 43-51.
Kitching, R.P. and Donaldson, A. I., 1987. Collection and transpor- tation of specimens for vesicular virus investigation. Rev. Sci. Tech. Off. Int. Epiz., 6:263-272
Kitching, R.P., 2005. Global epidemiology and prospects for con- trol of foot-and- mouth disease. Curr. Top. Microbiol. Immu- nol., 288: 133-148.
Mackay, D. K., Bulut, A. N., Rendle, T., Davidson F. and Ferris, N. P., 2001. A solid-phase competition ELISA for measuring antibody to foot-and-mouth disease virus. J. Virol. Methods., 97 (1-2): 33-48.
Ministry of Agriculture, Fisheries and Food, 1986. Foot-and-mouth disease. Ageing of lesions. Her majesty’s Stationery Office, London, UK.
Paton, D. J., Valarchar, J. F., Bergmann, I., Matlho, O. G., Zakharov, V. M., Palma E. L. and Thomson, G. R., 2005. Selection of foot-and- mouth disease vaccine strains-a review. Rev. Sci. Tech. Off . Int. Epiz., 24: 981-993.
Roeder, P.L. and Le Blanc Smith, P. M., 1987. The detection and typing of foot-and-mouth disease virus by enzyme-linked im- munosorbent assay: a sensitive, rapid and reliable technique for primary diagnosis. Res. Vet. Sci., 43: 225-232.
Skinner, H.H., 1960. Some techniques for producing and study- ing attenuated strains of the virus of foot-and-mouth disease. OIE., 53: 634-650.
Vianna Filho, Y.L., Astudillo, V.I., Gomes, G., Fernandez, C.E. Ro- zas, E., Ravison J. A. and Alanso, A., 1993. Potency control of foot-and-mouth disease vaccine in cattle. Comparison of the 50% protective dose and protection against generalization. Vaccine, 11(14): 1424-1428.
Villegas, P., 1989. Procedure for primary chicken embryo liver cells in tissue culture. In: Lab. Manual, avian virus diseases (AM 805). College of Vet. Medicine, University of Georgia, Ath- ens, Ga, 7p.
Woodbury, E.L., Ilott, M.C., Brown C.C. and Salt, J.S., 1995. Optimization of an In situ hybridization technique for the de- tection of foot-and-mouth disease virus in bovine tissues using the digoxigenin system. J. Virol. Methods., 51: 89-94.
Zulfiqar, M., 2003. Draft report for development of national disease control policy for foot and mouth disease in Pakistan under the FAO project “Support for Emergency Prevention and Control of Main Transboundry Animal Diseases in Pakistan Rinder- pest, FMD, PPR”.