Non-Invasive Antibody Production in the Chicken
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Mayo, S.L., Tufvesson, M., Carlsson, H-E., Royo, F., Gizurar- son, S., Hau, J. 2005. RhinoVax® is an efficient adjuvant in oral immunization of young chickens and cholera toxin B is an effec- tive oral primer in traditional subcutaneous immunization with Freund’s incomplete adjuvant. In Vivo 19: 375-382. III. Mayo, S., Royo, F., Hau, J. 2005. Correlation between adjunc- tivity and immunogenicity of cholera toxin B subunit in orally immunized young chickens. APMIS 113: 284-287. IV. Mayo, S., Royo, F., Tufvesson, M., Carlsson, H-E. Hau, J. 2006. Immunospecific antibody concentration in egg yolk of chickens orally immunized with varying doses of bovine serum albumin and the mucosal adjuvant, RhinoVax®, using different immuni- zation regimes. Scand J. Lab Anim. Sci. 33(3): 163-169. V. Mayo, S., Carlsson, H-E., Zagon, A., Royo, F., Hau, J.2009. Enhancement of anamnestic immunospecific antibody response in orally immunized chickens. J. Immunol. Methods 342: 58-63. Reprints were made with permission from the publishers: © http://www.iiar-anticancer.org, 2003, 2005 © Wiley-Blackwell, 2005 © ScandLAS, 2006 ©Elsevier Limited, 2009 Contents INTRODUCTION.........................................................................................9 Importance of the Study .............................................................................9 Aim of the Study ......................................................................................11 The Avian Immune System......................................................................11 Function...............................................................................................11 Anatomy ..............................................................................................11 Types of Immune Response .....................................................................13 Innate (non-specific) immune response...............................................13 Adaptive (specific) immune response..................................................13 Antigen-Antibody Interaction ..................................................................14 Immunoglobulin Classes in Chickens (IgA, IgM, IgY) ...........................15 IgM ......................................................................................................15 IgA.......................................................................................................16 IgG and IgY.........................................................................................16 Transport of IgG from Maternal Serum to Egg Yolk...............................17 Antibody Production from Egg Yolk (IgY) .............................................17 Advantages of IgY Antibody Production.................................................18 Immunization Routes ...............................................................................19 Classical/Subcutaneous immunization ................................................19 Oral immunization...............................................................................19 Adjuvants .................................................................................................20 Freund’s Adjuvant (FCA and FIA)......................................................20 Cholera toxin B-subunit (CTB) ...........................................................21 RhinoVax® (RV)..................................................................................22 MATERIALS AND METHODS ...............................................................23 Animals and Husbandry ...........................................................................23 Age of Chickens.......................................................................................24 Treatment Groups and Immunization Schemes .......................................24 Paper I..................................................................................................24 Paper II. ...............................................................................................25 Paper III. ..............................................................................................26 Paper IV...............................................................................................27 Paper V. ...............................................................................................28 Collection of serum samples (Paper I, II, III)...........................................28 Collection of eggs (Paper IV and V)........................................................29 Antibody extraction from egg yolk (Paper IV and V)..............................29 ELISA quantification of immunospecific antibodies...............................29 Paper I and II .......................................................................................30 Paper III ...............................................................................................31 Paper IV and Paper V ..........................................................................32 Statistical analyses....................................................................................32 Ethics committee license..........................................................................33 RESULTS ....................................................................................................34 Paper I ......................................................................................................34 Paper II .....................................................................................................35 Paper III....................................................................................................36 Paper IV ...................................................................................................37 Paper V.....................................................................................................39 DISCUSSION ..............................................................................................41 SUMMARY .................................................................................................48 REFERENCES............................................................................................50 ACKNOWLEDGEMENTS .......................................................................61 ERRATA......................................................................................................63 Abbreviations AU arbitrary unit BSA bovine serum albumin CTB cholera toxin b subunit DPI days post immunization DPSI day post subcutaneous immunization ELISA enzyme linked immunosorbent assay FCA Freund’s complete adjuvant FIA Freund’s incomplete adjuvant IgA immunoglobulin A IgG immunoglobulin G IgM immunoglobulin M IgY immunoglobulin from egg yolk RV RhinoVax® SC subcutaneous immunization SF Softigen® Introduction Importance of the Study The majority of laboratory animals are used as model for man in the devel- opment of drugs, toxicity studies and studies of diseases. A large proportion, however, are used for the production of polyclonal antibodies which are used for laboratory and therapeutic purposes. Mice take up approximately 75% of the total animal species used for polyclonal antibody production followed by rabbits (10%), birds (5%) and other ungulates (2.5%) Domestic fowl or chickens accounted for 87% of all birds used (UK Statistics, 2008). In EU countries, Canada, the United States and Queensland Australia, birds, do- mestic foul and/or poultry make up 4% of the total animal species used in scientific research related to animal welfare or non-invasive procedures (EBRA 2002, USDA 2008; CACC, 2008, Queensland, 2004). This produc- tion is mainly carried out not only by commercial companies but also by academic and government research institutes. The reason why rabbits, birds and farm animals are classified as labora- tory animals is that the procedures used are associated with some discomfort for the animals. In antibody production, animals are injected on several oc- casions with antigens in combination with so-called adjuvant, i.e. the classic Freund’s Complete Adjuvant, which stimulate the immune system but often also result in unwanted side effects in the animals such as development of lesions including granulomas on the injection site (Alving, 2002; Gupta and Siber, 1995; Stills, 1994;