Veli GÜLYAZ, Fahriye SARAÇ, Esra SATIR, Serdar UZAR, Eray ATIL

Contagious Ektima Aşı suşunun MDBK hücre kültürüne adaptasyonu ve kuzularda bağışıklık-stabilite çalışmaları

Contagious ecthyma (CE), kuzu ve oğlaklarda yaygın viral bir enfeksiyondur ve hala koyun ve keçi yetiştiriciliğinde önemini korumaktadır. Hücre kültürüne adapte canlı ve liyofilize CE aşıları mücadelede yaygın olarak kullanılmaktadır. Bu proje CE aşı suşunun Madin Darby Bovine Kidney (MDBK) hücre kültürüne adapte edilmesi ve MDBK hücre kültüründe üretimi yapılan aşısının raf ömrünü, stabilite ve kuzularda bağışıklık düzeyinin saptanması amacıyla gerçekleştirildi. MDBK hücre kültürüne adapte edilen aşı virüsünün titresinin DKID50 106.5/ml olduğu belirlenmiştir. Fareler ve kobaylarda yapılan zararsızlık çalışmalarında lokal veya genel reaksiyonlar gözlenmedi. Scarifikayon yoluyla aşılanan 10 kuzunun vücut ısılarının normal değerler (38.2-39.1°C) arasında olduğu bulundu. Aşılamayı izleyen 30. günde alınan kan serumlarında VNT ile CE virüsüne ait nötralize edici antikorlar tespit edilemedi. Patojen CE virüsü (E(P)CK5) ile yapılan challenge sonucunda aşılanmış kuzuların CE enfeksiyonuna karşı korunduğu gözlenmiştir. Stabilite çalışmasında, +4 /+8°C'de 24 ay süreyle muhafaza edilen liyofilize CE aşısı titrelerinin 18, 21 ve 24. aylarda DKID50 106.5/ ml ve 18, 21 ve 24 aylarda ise 106.0/ml olduğu tespit edildi. Aşı virüsünün 30, 33, 37 ve 40˚C'lerde DKID50 106.5/ml titrede 12 saat stabil kaldığı ve 12 saat sonra virüs titresinin 42 ve 45°C'lerde DKID50 105.75/ml'ye gerilediği tespit edildi. Bu çalışma sonucu, CE aşı virüsünün MDBK hücre kültürüne adapte edilmesi ile CE aşılarının MDBK hücre kültürlerinde üretilebileceği sonucuna varmıştır.

Anahtar Kelimeler:

Contagious ecthyma, virus, aşı, bağışıklık, immunity, innocuity

Adaptation of Contagious Ecthyma vaccine strain to MDBK cell culture and immunity-stability studies in lambs

Contagious ecthyma (CE) is a common viral infection in lambs and kids, and still maintains its importance in sheep and goat breeding. Attenuated, live and lyophilized CE vaccines adapted to cell culture are widely used in the fight against CE. The aim of this project was to adapt the CE vaccine strain to Madin Darby Bovine Kidney (MDBK) cell culture and to produce the vaccine, as well as determining the shelf life, stability, innocuity, and immune response in lambs. The titer of the vaccine virus adapted to the MDBK cell culture was determined to be TCID50 106.5/ml. In the innocuity study on mice and guinea pigs, no local or general reactions were observed. . Body temperature of 10 lambs vaccinated by scarification was found to be between normal values (38.2-39.1°C). Neutralizing antibodies belonging to CE virus with VNT could not be detected in blood sera taken on 30th day following the vaccination. As a result of challenge performed with pathogen (E(P)CK5) CE virus, the protection was observed against CE infection in vaccinated lambs. In the stability study, the titers of lyophilized CE vaccine were found to be TCID50 106.5/ml from 1 to 15 months and 106.0/ml at 18, 21 and 24 months during the 24-month storage period at + 4/+8°C. It was determined that the vaccine virus remained as stable at TCID50 106.5/ml titer for 12 hours at 30, 33, 37 and 40˚C and after 12 hours, the virus titer regressed to TCID50 105.75/ml. at 42°C and 45˚C. This study concluded that CE vaccines could be produced in MDBK cell cultures by adapting CE virus to MDBK cell culture.

Keywords:

Contagious ecthyma, virus, vaccine, immunity, inocuity, stability,

PDF

___

Buddle BM and Pulford HD. (1984). Effect of passively-acquired antibodies and vaccination on the immune response to contaqious ecthyma virus. Vet Microbiol. 9, 515-522.
Buddle BM, Dellers RW, Schuring GG. (1984). Heterogenecity of contagious ecthyma virus isolates. Am J Vet Res. 45, 75-79. Burleson FG, Chambers TM, Wiedbrauk DL. (1992). Virology Laboratory Manuel. First edition. Academic Press. California. p. 20-61.
Chan KW, Lin JW, Lee SH, Liao CJ, Tsai MC, Hsu WL, Wong ML, Shih HC. (2007). Identification and phylogenetic analysis of orf virus from goats in Taiwan. Virus Genes. 35 (3), 705-12.
Ergin H, Köklü A. (1973). Ektima virusunun doku kültürlerinde pasajı ve antijenik özelliklerinin incelenmesi. Pendik Vet Kont ve Araşt Enst. 6 (2), 12-20. Ergin H, Köklü A. (1975). Pendik orijinli ektima suşunun pasajı, aşı hazırlama imkanları ve bu aşının immunite süresinin tayini. TÜBİTAK (VHAG). Proje no: 207. Gameel AA, Abu Elzein EME, Housawi FMT, Agip A, Ibrahim A. (1995). Clinico-pathological observations on naturally occuring contagious ecthyma in lambs in Saudi Arabia. Revue Elev Med Vet Pays Trop. 48(3), 233-235
Gülyaz V, Hasöksüz M, Bulut M, Saraç F. (2012). Contagious ecthyma virus (CEV) oğlak izolatlarının moleküler karakterizasyonu ve ecthyma aşı suşu arasında çapraz koruma çalışmaları. TÜBİTAK. Proje no: 111O398.
Haig DM, Melnnes CJ. (2002). Immunity and counter-immunity during infection with the parapoxvirus orf virus. Virus Research. 88, 3-16.
Hosamani M, Bhanuprakash V, Scagliarini A, Sing RK. (2006). Comparative seguence analysis of major envelope protein gene (B2L) of indian orf viruses isolated from sheep and goats. Vet Microbiol.116, 317-324.
Housawi FMT, Abu Elzein EME, Gameel AA, Ala Faleg AI. (1993). A close comparative study on the response of sheep and goats to experimental orf infection. J Vet Med. 40, 272-282.
Hussain KA and Burger D. (1989). Invivo and invitro characteristics of contagious ecthyma virus isolates: host response mechanism. Vet Microbiol. 19, 23-36.
Inoshima Y, Morooka A, Sentsui H. (2000). Detection and diagnosis of parapoxvirus by the polymerase chain reaction. J Virol Methods. 84, 201-208.
Ivanov L, Hrıstov M, Peshev R. (2016). Studies on cultural characteristics of contagious ecthyma (orf) virus. Bulg. J Vet Med. 19, 308-316. Karki M, Venkatesan G, Kumar A, Kumar S, Bora PD.(2019). Contagious ecthyma of sheep and goats: A comprehensive review on epidemiology, immunity, diagnostics and control measures. Vet Arhiv. 89(3), 393-423.
Mazur C, Ferreira II, Rangel Filko FB, Galler R. (2000). Moleculer characterization of Brazilian isolates of orf virus. Vet Microbiol. 73, 253-259.
McKeever DJ, Reid HW, Ingus NF, Herring AJ. (1987). A qualitative and quantitative assessmen of the humoral antibody response of the sheep to orf virus infection. Vet. Microbiol. 15, 229-241.
Mercante MT, Lelli R, Gaetano Federico Ronchi GF, Pini A. (2008). Production and efficacy of an attenuated live vaccine against contagious ovine ecthyma. Vet Ital. 44 (3), 543‐547.
Mondal B, Bera AK, Hosamani M, Tembhurne PA, Bandyopadhyay SK. (2006). Detection of orf virus from an outbreak in goats and ıts genetic relation with other parapox viruses. Vet Res Commin. 30, 531-539.
Musser JMB, Waldron DF, Taylor CA. (2012). Evaluation of homologous and heterologous protection ınduced by a virulent field strain of orf virus and an orf vaccine in goats. Am J Vet Res 73(1), 86-90.
Musser JMB, Taylor CA, Gua J, Tizard IR, Walker JW. (2008). Development of a contagious ecthyma vaccine for goats. AJVR. 69(10),1366-1370.
Nashirudddullah N, Pathak DC, Barman NN, Ahmed JA, Rajbongshi G, Sharma RK, Borah P, Begum SS. (2016). Evaluation of orf virus (ORFV) isolation in continuous lamb testis cells (OA3.Ts) and development of a co-culture method with infected cells to increase infectivity. Indian J Anim Res. 50(6): 951-957.
Nettleton PF, Brebner J, Pow I, Gilray JA, Bell G.D, Reid HW. (1996). Tissue culture-propagated orf virus vaccine protects lambs from orf virus challenge. Vet Rec. 138, 184-186.
Nourani H and Maleki M. (2006). Contagious ecthyma: case report and review, Pak J Biol Sci. 9(13), 2543-2545. Pye D. (1990). Vaccination of sheep with cell culture grown orf virus, Aust Vet J. 67:182-183.
Rabinson AJ and Balassu TC. (1981). Contagious pustular dermatitis (orf). Vet Bull. 51(10), 771-782.
Rafii F and Burger D. (1984). Comparasion of contagious ecthyma virus genomes by restriction endonucleases. Arch Virol. 84, 283-289.
Scagliarini A, Pozzo FD, Gallina L, Guercio A, Clercq E.D, Snoeck R, Andrei G. (2005). Ovine skin organotypic cultures applied to the ex vivo study of orf virüs infection. Vet. Res. Commun. 29, 245-247.
Sullivan JT, Mercer AA, Fleming SB, Robinson AJ. (1994). Identification and characterization of an orf virus homologue of the vaccinia virus gene encoding the major envelope antigen p37K. Virology. 202, 968-973.
Wang G, Wang Y, Kong J, Li Y, Wu J, Chen Y, Liu X, Shang Y, Zhang Z. (2019). Comparison of the sensitivity of three cell cultures to ORFV. BMC Vet Res. 15:13.
Zamri SM, Rashidah I, AL-Ajeeli KSA. (1994). Experimental cross-infection of sheep and goats with different isolates of contagious ecthyma virus. Aust Vet J. 71(7) 218-220.