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CEVA’S APPROACH F O R CONTROLLING GUMBORO DISEASE

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  1. CEVA’S APPROACH FORCONTROLLING GUMBORO DISEASE By Dr. Vilmos PALYA Director of Viral Development, CEVA-PHYLAXIA BUDAPEST 19 October 2005

  2. KANATLI BAĞIŞIKLIK SİSTEMİ

  3. BAĞIŞIKLIK SİSTEMİNİN YAPISI VE İŞLEVİ • Lymphoid organlar • Primer: • Thymus (T lymphocytes) • Bursa fabricius (B lymphocytes) • Sekonder: • Dalak • Harderian bezi • Kemik iliği • Konjuctival lymphoid doku (CALT) • Bağırsak lymphoid dokusu (GALT) • Başta lymfoid dokular (HALT) • Bronşiyal lymphoid organlar (BALT)

  4. Immune cells • Lymphocytes: • T cells • B cells • Mononuclear phagocytes • Tissue macrophages • Follicular dendritic cells lymphocyte eosinophyl thrombocyte heterophyl monocyte

  5. Bursal B hücre morfogenesisi

  6. Bağışıklık hücrelerinin gelişimi • Civcivlerde inkübasyon periyodu: 21 days • Bursa’da B hücrelerinin gelişimi: • Follikule formasyonu (cortex, medulla) • B hücrelerinde gen değişimi • B hücrelerinin periferal bölgelere göçü • Thymus’ta T hücrelerinin gelişimi • Embryonasyonun 9.gününde T hücreleri gelişir • T hücreleri çıkımdan sonra perifere göç eder Tavuk embriyoları spesifik immun cevabı başlatma yeteneğine sahiptirler In ovoaşılama

  7. bağışıklık

  8. Innate and adaptive immunity

  9. Adaptive or acquired immunity • Active immunity • specific immune response against antigens • Primary response (first contact with antigen) • IgM IgY • Immune memory (B-ly) • Secondary response (second contact with antigen) • Rapid and longer immune response • IgY • Passive immunity • Maternal antibody (transmitted from hen to chick via egg yolk) • Transfusion of antibodies by sera

  10. Passive immunity • Maternally derived antibodies in offsprings • The level of the maternal antibodies can be increased by purposive immunisation of breeder flock • Protect the chicks against diseases at early stage of life • Antibodies decay 2-6 weeks after hatching • The maternally derived antibodies neutralise the live vaccine viruses → inhibition of active immune response

  11. Maternal immune transport in the chicken • FcRY transports IgY from yolk sac into embrionic blood (West et al., Immunity, 2004) • pH dependent IgY binding to the yolk sac membrane • the first IgY binding Fc receptor in birds

  12. Humoral immune response I. • Three Ig classes: IgM, IgY, IgA IgM: pentamer, expressed on the surface of B cells IgY: functionally homologous to mammalian IgG IgA: dimeric or tetrameric forms in mucosal secretion monomeric molecule in serum critical role in local immunity Specific immune response Antigen → IgM class switching mediated by T cells and cytokines IgY or IgA Strong immune memory Methods for determination of antibody response: ELISA, SN, IF, agglutination, hemagglutination

  13. Humoral immun cevap

  14. Cell mediated immunity T lympocytes are the principal cells in the cell mediated immunity Two subsets CD4+ cells: helper function – central role in humoral and cellular immunity CD8+ cells: cytotoxic function Important role in antiviral and antitumor immunity

  15. Activity of different factors of immune system after viral infection

  16. Role of the bursa in immune response Gene conversion in B cells → humoral immunity Intact Bursa Fabricius Chronic Bursitis after vvIBDV infection

  17. GUMBORO HASTALIĞI NEDİR

  18. Segment A Segment B VP3 VP2 VP4 VP 1 VP5 Infectious Bursal Disease Virus (IBDV) Taxonomy: Family: Birnaviridae Genus: Avibirnavirus Virus properties:Virion not enveloped, 60nm diameter, icosahedral symmetryTwo genome segments,Double stranded RNA genome Prof. Steward McNulty, Queen’s University of Belfast

  19. Infectious bursal disease (IBD, Gumboro disease) • Serotipeleri • Serotype 1 and 2 • Variant suşlar (serotip 1) • Virulans bazlı grublanırsa • Classical virulent IBDV • Very virulent (vvIBDV) • Moderately attenuated • „intermedier plus”, „hot” • Medium attenuated • „intermedier” • Heavily attenuated • „mild” ]vaccine strains

  20. Enfeksiyon ilk 3 hafta içerisinde immünosüpresyon nedenidir • 3-6 haftalar içerisinde hastalığın klınik formu oluşur: • Klınik bulgular enfeksiyondan 2-3 gün sonra • Mortalite enfeksiyondan 5-8 gün sonra

  21. IBDV infects cells of the cloacal bursa - primarily immature B-cells. • Immature B-cells mature into antibody producing cells. • If B-cells are destroyed early in life the bird may become immunosuppressed for life.

  22. Picture: Y. Gardin, 2003

  23. Klınik görünümler

  24. MORTALITE YAŞ(GÜN) Picture: Y. Gardin, 2003

  25. Sağlıklı Bursa histopatolojisi

  26. Bursa büyüklüğü Gün From U. Lohren, 2005

  27. IBD İMMÜNODEPRESSİF ETKİSİ Immunodepresyon (ND antikor cevaplarına göre) Enfeksiyon yaşı (Gün) 1 ÖNEMLİ 7 ORTA DERECEDE YOK 14 21 YOK (after Faragher et al, 1974)

  28. Characterisation of the vvIBDV strains • Pathotyping • Up to 100% mortality in SPF chickens • 50-60% mortality in layers, 25-30% in broilers • Break through higher levels of MDA  not possible anymore to protect passively broilers • Antigenicity • Panel of neutralizing Mabs  no (major) antigenic drift • High cross-neutralization indices measured in SPF embryonnated eggs • Classical serotype 1 vaccines still satisfactory on SPF birds • Molecular characterisation • Sequencing of the variable domain of the VP2 gene • Amino acid residues substitutions at positions 222Ala, 256Iso, 294Iso and 299Ser = Genetic fingerprints of vvIBDV • RT-PCR of vVP2 followed by sequencing and/or RE Analysis

  29. Mortality rates • Challenge of 5-week-old Lohmann SPF chickens (n=25-30) with 105 EID50 of IBDV strains of varying virulence in isolated conditions by the oculonasal route. • vvIBDV induce higher mortality rates than classical virulent IBDVs • Confusion in nomenclature • Need for standardized challenge model

  30. HİPERVIRULENT IBDV : MORTALİTENİN YAŞLA İLİŞKİSİ 120 Mortalite (%) 100 80 60 40 20 0 0 7 14 21 28 35 Bulaşma yaşı (gün) (after Meulemans et al, 1990)

  31. Dose effect in IBDV challenge Comparison of a classical virulent IBDV (Cu1wt) and a vvIBDV (HK46): Inoculation of increasing doses 103 107 EID50 Classical IBDV: maximum 60% mortality vvIBDV: up to 100% mortality Existence of a dose effect in IBDV challenge

  32. Multiplication rates • Titration of virus in BF of birds dying (or sacrificed) 3-4 days after challenge • Higher multiplication rates for more virulent IBDVs • 1 attenuated IBDV particle  10 particles • 1 classical virulent IBDV particle  102 to 103 particles • 1 vvIBDV particle  104 to 105 particles

  33. PATOGENEZ VIRUS ETKİMESİ GÜÇLÜ VIRUS LEZYON + MORTALITE LEZYON ZAYIF VIRUS ENFEKSİYON YOK 0 5 10 15 20 25 30 35 40 45 50 YAŞ (GÜN)

  34. WHAT FORM OF GUMBORO DISEASE AT FIELD LEVEL ? WHICH CONTROL STRATEGY ?

  35. Laboratory tools From Di Fabio et al, 2000

  36. OIE I.B.D.V. ClassificationN. Eterradossi - AFSSA Lab - Ploufragan France CAPSIDE ANTIGENS Characterisation by Monoclonal Antibodies (Mab) 8 Mab put in contact with the virus = bind or not. R.N.A. : Characterisation by sequencing: Sequence of Nucleotids and deduced a.a.

  37. OIE I.B.D.V. ClassificationN. Eterradossi - AFSSA Lab - Ploufragan France Specific Antigen-Capture ELISA  Revelation Mab specific Fixation or not Capture of the virus Polyclonal anti -IBDV

  38. OIE I.B.D.V. ClassificationN. Eterradossi - AFSSA Lab - Ploufragan France Classical IBD virus : All Mabs bindings positive Ag4 Ag5 Ag6 Ag3 Ag1 Ag9 Ag8 Ag7 VP2 Protein

  39. OIE I.B.D.V. ClassificationN. Eterradossi - AFSSA Lab - Ploufragan France vv IBD virus : Bindings to Mab 3 and 4 are negative - - Ag5 Ag6 Ag1 Ag9 Ag8 Ag7 VP2 Protein

  40. CEVA’S APPROACH OF GUMBORO CONTROL

  41. Results of the IBDV survey done by CEVA in 1999-2002 with AFSSA Origin Mab 1 Mab 3 Mab4 Mab5 Mab6 Mab7 Mab8 Mab9 F52/70 (Classic) 66% 91% 78% 75% 110% 114% 96% 77% 89 163 (vvIBD) 61% 1% 5% 68% 118% 113% 80% 53% Algeria 94% 4%24% 90% 143% 153% 131% 49% Bulgaria 89% 3%10% 77% 167% 172% 115% 78% Hungary 81% 1%14% 72% 166% 174% 135% 74% India 65% 1%10% 70% 121% 126% 81% 73% Iraq 74% 1%10% 70% 121% 126% 81% 73% Jordan 106% 1%11% 69% 206% 220% 167% 74% Kenya 104% 0%11% 63% 192% 177% 144% 86% Malaysia83%123%94% 61% 89% 106% 132% 38% Morocco 86% 1%5%83% 205% 189% 141% 62% Pakistan 94% 1%7% 60% 181% 175% 137% 76% Romania 60% 2%3% 37% 112% 105% 48% 78% Tanzania 79% 1% 20% 91% 131% 140% 118% 42% Tunisia 93% 0%12% 73% 155% 162% 120% 91% Turkey 69% 1%6% 54% 151% 152% 78% 102%

  42. Origin Mab 1 Mab 3 Mab4 Mab5 Mab6 Mab7 Mab8 Mab9 F52/70 (Classic) 66% 91% 78% 75% 110% 114% 96% 77% 89 163 (vvIBD) 61% 1% 5% 68% 118% 113% 80% 53% Cevac IBD L 64% 89% 74% 23% 94% 93% 91% 91% CEVAC IBD L derived from classical IBDV strain and NOT from vvIBDV AFSSA 2001 CEVA’S APPROACH OF GUMBORO CONTROL CEVAC IBD L Antigenic typing

  43. Segment A Segment B VP3 VP2 VP4 VP 1 VP5 Hypervariable region 1146 709 P2 1. PCRreaction 1229 587 P1 P5.3 2. PCRreaction 721 1176 P2.3 Molecular typing of IBDV strains PCR and sequencing Nucleotide numbering according to IBDV-STC strain (Kibenge et al, J. Gen. Virol. 1990. (71) 569-577.).

  44. OKYM VarE 52/70 D69 STC FS 500 400 300 200 100 50 CEVA’S APPROACH OF GUMBORO CONTROL Differentiation of IBDV strains (VP2 gene 414bp BseD I RFLP) CEVA 50 bp DNA ladder LIBDV Gp82 2512 P3 IBD B.WSV IBD B.WSV AEG1 D142 FS D69 450 400 Jakabszállás 2002.01.31. 350 300 250 200 150 250 100 150 50 2512 vv vaccine classical var very virulent subc

  45. CEVA’S APPROACH OF GUMBORO CONTROL MOH94/94H" MOH97/97H" Csengele/97H" PECS/97H" Phylogenetic study of IBDV strains OKYMT/95J DV86/86N vv OKYM/91J KABA/97H" UK661/86GB (based on the hypervariable region of the VP2 gene, 414bp) CS89/89GB FS/97H" AEG1/97H" Toszeg/97H" Diosjeno/97H" 849VB/87B vacc IBDL(2512)/80US" STC/67US classical P1II/75H" P1/75H" 52/70GB PBG98/76GB GP82/75H" D78" vacc TAD" Cu1/76D LIBDV/75H" P2/73D VarE/85US US var U28/88US VarA/85US 3212/88US serotype I GLS/87US P3/77H" P6/78H" subc P7/78H" P9/81H" P10/81H" serotype II P11/81H" 00273/73Aus OH/82US 19 18 16 14 12 10 8 6 4 2 0

  46. CEVA’S APPROACH OF GUMBORO CONTROL

  47. Aşı türleri • Canlı attenue • Mild • Intermedier • Intermedier plus („hot”) • İlk ve devamı aşılamalar için • Inaktive • Sadece canlı aşılama devamına • Yüksek titre ve uzun süreli korunma

  48. Conventional IBD Vaccines • Mild IBD Vaccine - Easily eliminated by low levels of maternal antibody. • Intermediate Vaccine - relatively safe to use in ovo or at hatch. Eliminated by low to moderate levels of maternal immunity. • Intermediate Plus Vaccine- not safe to give in ovo unless modified in some manner. Eliminated by high level maternal immunity.

  49. D78 group IBDV aşılarının filogenetikgrupları (Palya, 2003) P2 group 228E group 2512 group STC group MB group Lukert group V877 group