Response of the Embryo to in ovo

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1 Response of the Embryo to in ovo Vaccination with IBDV Jagdev Sharma a The Biodesign Institute Arizona State University Tempe, Arizona, U.S.A. BUDAPEST

2 37 Billion chickens produced yearly y

3 IBDV A highly stable virus Immunosuppressive Control by vaccination

4 IBDV-related Problems Mortality Gangrenous dermatitis Immunosuppression Tumors Ulcerative enteritis Stunting Poor vaccine efficacy

5 MǾ/DC Immune cells communicate with each other

6 24h PI 48h PI Replication of IBDV in the bursa

7 A B C Normal bursa Classical IBDV Variant IBDV D E Destruction of Bursa by IBDV

8 Days after IBDV IgM+ cells in the bursa

9 IBDV Control Virus (c) Spleen IgM+ 30 ining 25 ge positive sta cells percenta * IBDV-exposed control dpi Effect of IBDV on the spleen

10 Effect of IBDV on B Cells Virus replicates in B cells Cells destroyed Reduced antibody production Recovery slow

11 Effect of IBDV on the Thymus IBDV No IBDV Days PI Wt. Index Lesion Score Wt. Index Lesion Score 5 3.1* * 3.7* *Lower than control (P<0.05) Normal thymus IBDV thymus

13 CD3 in normal bursa CD3 in IBDV bursa CD4 in IBDV bursa CD8 in IBDV bursa T Cell Infiltration of Bursa After IBDV Exposure

14 Possible Role of T cells Infiltrating the Virus clearance Tissue injury Bursa

15 CPM Suppressor T cells induced by IBDV

16 Effect of IBDV on T Cells Resistant to infection and replication Function(s) impaired Suppressor cells

17 IBDV Infects and Activates Macrophages Bursal MØ Spleen MØ MØ Cell Line

18 Mechanisms of IBDV Immunosuppression Immune Cell Susceptibility to IBDV Replication Impaired Functions Functional Recovery Act as Immune Suppressor Cells B cell + + Slow? T cell - + Rapid + Macrophage + + (?) Rapid +

19 IBDV Control Passive Immunization ConventionalLive Vaccines Inactivated Vaccines Ag+Ab Mixtures Recombinant Live Vaccines

20 IBDV Vaccination In some flocks, early post hatch control of IBD is necessary Passively immunized chicks have variable levels of antibodies. Some have sub protective levels.

21 Effects of in ovo vaccination with IBDV Virus Virulence % Hatch % Mort % Protect TC IBDV Mild Intermed None From: Sharma, Av. Dis., 29, 1155, 1985

22 In ovo Vaccination with IBDV Mild IBDV rhvt IBDV Ag/Ab complex vaccines

23 In ovo Vaccination IBDV Resistant Chicken

24 % Hatchability following HVT exposure Incubation day Immune cells in spleen ility Poor hatchabi Interm mediate hatch ability Excellent hatchability T 5 O 6 L 7 E 8 R 9 A N 12 C B cells (IgM) (g 13 E T cells T cells IgG+ and IgA+ B cells Fig. 1. The relationship between the developmental stage of the embryo and resistance to prenatal exposure to HVT. The data on hatchability (left) are from preliminary studies (Zhang and Sharma, Dev. Comp. Immunol., 27:431, 2002); the data on immune cells (right) were summarized from published reports (Sharma, Section ed Avian Immunol (Data from In: Pastoret Sharma, et 1998; al Handbook Zhang and of Sharma, Vert Immunol 2002) 1998) The relationship between the developmental stage of the embryo and resistance it to prenatal tlexposure to HVT.

25 History of in ovo vaccination 1980 First successful experiment 1982 First paper published (Sharma and Burmester) 1984 Patent issued 1985 Patent licensed to Embrex, Inc. (now Pfizer) 1991 First commercial use 2000 Major hatcheries in the U.S.A; 27 countries 2002 Vaccine savers, layers, breeders 2009 >85% U.S. Broiler market; 36 countries in six continents t

26 Vaccine V i deposited d primarily il in the amniotic sac Amniotic fluid swallowed by the embryo Viruses infect embryonic tissues

27 In ovo Vaccination 50 70K eggs/hour

28 Objective To examine the effect of pathogenic and nonpathogenic IBDV on the embryonic lymphoid tissues and lymphoid cells

29 IBDV (HP and LP) HP IBDV: Highly ED18 Pathogenic IM IBDV ED21 LP IBDV: Low pathogenic vaccine strain (Bursine 2) Gross Lesions Weights Micro Lesions Histo Apoptosis T Cell Phenotype CD348 CD3,4,8 TCR1,2,3 T Cell Function Mitogenesis T Cell Activation Cytokine gene upregulation

30 Embryonation Day 21 (3dpi) Control Bursa HP IBDV Bursa LP IBDV Bursa

31 Virus in Control Bursa Virus in HP IBDV Bursa at Hatch Virus in LP IBDV Bursa at Hatch Virus in LP IBDV Bursa at 5 day old

32 Virus Load in Bursa C Ct LP-IBDV HP-IBDV h 48 h 72 h

33 Embryonation Day 21 (3dpi) Virus in Thymus of Control Virus in Thymus of HP IBDV Virus in Thymus of LP IBDV

34 Destruction ti of Embryonic Thymocytes by HP IBDV No. of cells per thymus (x10 6 ) Experiment Control LP-IBDV HP-IBDV ND ND 23 Mean 28±7 20±9 16±5* Control LP IBDV HP IBDV Apoptosis in Thymus at ED21

35 Proliferation Assay for Thymocytes Single cell suspensions of thymus Stimulate with anti CD28 mab, ionomycin and PMA Incubate for 48 h at 37 C Pulse cells with [3H] Thymidine during the last 5h of incubation

36 Mitogenic Inhibition by HP IBDV but not by LP IBDV CPM * Control Bursine IM IBDV Mitogens: PMA, inomycin, CD28 mab Virus inoculation at ED18, observations at ED21 *= P<0.05

Purification of T Cells from Embryonic Thymus PE-A 10 4 25.23 10 4 91.

37 Purification of T Cells from Embryonic Thymus PE-A FITC-A PE E-A FITC-A Before purification After purification

38 Mitogenesis of Purified CD3 Cells fromembryonicthymus 7000 Experiment Experiment CPM Control HP-IBDV 0 Control HP-IBDV

39 IBDV Induced Expression of Cytokines mrna in Thymocytes and Splenocytes Thymus Spleen 20 IFNγ IFNγ Embryonation Day 21 (3dpi) Vaccine IBDV Vaccine IBDV Fo old change Vaccine IL IBDV Fo old change Vaccine IL-8 12 IBDV IL IL LP-IBDV HP-IBDV LP-IBDV HP-IBDV

40 Summary: Response of Chicken Embryos to IBDV Highly Pathogenic IBDV Vaccine Strain of IBDV Bursal destruction + - Virus in bursa + (early) + (late) Thymic destruction + - Virus in thymus + - Thymocyte y lysis + - Alteration in relative - - numbers of T cell populations Impairment of T cell + - function T cell activation + + Protection against challenge + +

41 SUMMARY Highly immunosuppressive: B T M Flocks must be protected by vaccination Some vaccines may be administered in ovo Mild live vaccines cause minimal damage to embryo HVT (commonly used vector) caused no dt detectable tbl lesions in embryo

Infectious Bursal Disease, Immunosuppression and the role of VAXXITEK HVT+ IBD

Research note Infectious Bursal Disease, Immunosuppression and the role of VAXXITEK HVT+ IBD Grogan K. 1 1 Poultry Chicken Scratch, LLC, 30019 Dacula GA United States of America [from Hoerr F.J., 2010,