University of Alberta Anti-Bacterial Activities of Chicken Egg Yolk Antibody (IgY) against Enteric Pathogens Eun-Na Lee 62 A thesis subrnitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements for the degree of Master of Science in Food Science and Technology Department of Agricultural, Food and Nutritional Science Edmonton, Aiberta Fa11 2000 National Library Bibliothèque nationale 1+1 of Canada du Canada Acquisitions and Acquisitions et Bibliographic Services services bibliographiques 395 Wellington Street 395. nie Wellington Ottawa ON K1A ON4 Ottawa ON K1A ON4 Canada Canada Your lile Votre réUrenCe Our 6ie Norre refdrenu, The author has granted a non- L'auteur a accordé une licence non exclusive licence allowing the exclusive permettant à la National Libraty of Canada to Bibliothèque nationale du Canada de reproduce, han, distribute or sen reproduire, prêter, distribuer ou copies of this thesis in rnicrofom, vendre des copies de cette thèse sous paper or electronic formats. la forme de microfiche/film, de reproduction sur papier ou sur format électronique. The author retains ownership of the L'auteur conserve la propriété du copyright in this thesis. Neither the droit d'auteur qui protège cette thèse. thesis nor substantial extracts from it Ni Ia thèse ni des extraits substantiels may be printed or otherwise de celle-ci ne doivent être imprimés reproduced without the author's ou autrement reproduits sans son permission. autorisation. Abstract Hyperimmunized laying chickens against antizenic proteins, vimses or bacterial ongin produce antibodies (IgY) specific against the irnrnunized antigens both in the egs yoIk and serum. This system exploits a wide range of potential applications in food production. In this research, the anti-bacterial properties of IgY were investigated. A flock of Single Cornb White Leghorn (SCWL) chickens were hyperimmunized with four enteric pathogens, including Escherichia coii (E. coii) 0157:H7, E. coi[ 9S7P, Sczlmonellcr errteritidis or S. typhimrizrm, which are today's most threatening enteric rnicroorganisms associated with food poisoning pathogens in humans and animals. Polyclonal IgY loaded eggs were produced and then the water-soluble Fraction (WSF) containing IgY (average 28.8% of the total protein) was prepared. Specific binding activity and concentration (average 9.5% of total IgY content) of the specific antibody were determined by the ELISA method using bactenal whole cells. A high degree of cross-reactivity of anti-Sczimonelia IgY between S. enter-ititlis and S. typhzmw-itrm was demonstrated. S pecific IgY inhibited bacterial growth in virr-O to the maximum of 2.3 log CFU/ml reduction in bacterial counts. The specific binding actiçity of IgY was further demonstrated in the immunofluorescence and immunoelectron micrographs, which revealed the structural alterations of bacterial surface bound by specific IgY molecules. In conclusion, this research work shows potential applications of egg yolk antibodies as anti-bacterial prophylactic uses for infectious diseases and suggests an antibody farming concept as an alternative to conventional egg farrning. To God And 1Wy fmib Who are always with me 1 dedicate this thesis Acknowledgments 1 would like to thank my respectful supervisor, Dr. J. S. Sim, with al1 my heart for his guidance and inspiration throughout the course of my studies. 1 am also sincerely grateful to my committee members, Dr. M. R. Suresh and Dr. R. O. Bal1 for their academic direction. Special thanks are extended to Dr. J. S. Sim and Dr. M. R. Suresh, who have given me the valuable opportunity to fùrther my studies. Sincere thanks are expressed to Dr. H. H. Sunwoo for his invaluable advice and consideration, which aided me to complete my studies. I am indebted to Mr. K. Manninen for placing the facilities at my disposa1 and making revisions to my thesis. To my colleagues, Ms. L. Chui and Mr. Y. Wang, 1 am gratehl for their assistance and encouragement. The instruction and help of Dr. M. Chen for electron microscopy and other technical staff throughout various experiments are deeply acknowledged as well. 1 wish to thank my dear fiends for their companionship which provided me with cornfort and encouragement. Special thanks are also expressed to my uncle, aunt and cousin for their support and concern. To many other people, 1 am grateful for the enriching spare time that 1have had while studyinj. 1 wouId Like to express my deep appreciation to my family: Parents, sister, brother-in-law, brother, and niece. Without their prayer, love, concem, and inspiration, 1 would not have accomplished my studies. Most of all, gratitude and honor be to God with al1 my heart for his blessings upon me, and the opportunity to be thankfùl for al1 the above. Table of Contents Chapter 1. Generai Introduction ................................................................. 1.1, Chicken Egg Yolk Antibody (IgY) ............................................... 1.2. Characteristics of IgY ..................................................................... 1.2.1. Structure .......................................................................... ... 1-3-2- Stability ............................................................................... 1-2.3. Imrnunolo,oi cal Properties ................................................... Production of IgY .......................................................................... 1.3.1. Influencing Factors .............................................................. 1.3 .2. Cornparison to Marnmalian IgG production ................... .. .. Isolation of IgY ............................................................................. Advantages of IgY ........................................................................ Applications of &Y ....................................................................... 1.6.1 . Existing Application ............................................................ 1.6.1-1. Passive Immunization ............................................ 1-6.1.2. Irnrnunological Tool ............................................... 1.6.2. Future Application ............................................................... Plan of Studies ............................................................................... 1.7.1. Hypothesis ........................... ........ ..................................... 1.7.2. Objective of Studies ............................................................ 1-7.3. Outline of Studies ................................................................ Chapter 2 . Growth Inhibitory Effect of IgY on Escherichia coli 0157:87 2.1. Introduction ................................................................................... 2.2. Materials and Methods .................................................................. 2.2.1. Bacteria and Culture Conditions ........................................ 2 -2.2 . Immunization of C hickens .................................................. 2.2.3. Isolation of Water-Soluble Fraction (WSF) Containing IgY from Egg Yolk ....... ....*........................................................... 2.2.4. Preparation of IgY Powder .................................................. 2.2.5. Enzyme-Linked Immunosorbent Assay (ELISA) ............ .... 2.2.5.1. Specific Activity of IgY ......................................... 2.2.5.2. Cross-Reactivity of IgY ......................................... 2.2.5-3.Total IgY Concentration ......................................... 2.2.5 .4 . S pecific IgY Concentration .................................... 2.2.6. Protein Assay ....................................................................... 2.2.7. Growth Inhibition Assay ..................................................... 2-2.7.1. Preparation of Bacteria ........................................... 22.7.2. Bacterial Growth Curve ......................................... 2.2.7.3 . Preparation of IgY Solution ................................... 2.2.7.4. Growth of E . coli 0 157:H7 with IgY ..................... 2 -2. 8. Microscopie AnaIyses ......................................................... 2.2.8.1. Imrnunofluorescence Microscopy .......................... 2-28.2 . Immunoelectron Microscopy ................................. 2.3. Results and Discussion .................................................................. 2.3 .1 . Concentrations of Protein and Total IgY in the WSF ......... 2.3.2. Specific Activity of IgY against E. coli 0157:H7 .............. 2.3 .3 . Properties of IgY Powder ................................... .. ..... ... ....... 3.3 .4. Cross-Reactivity of IgY ...................................................... 2.3 .5 . Growth Inhibitory Effect of E. coli O 157:H7-Specific IgY 2.3.6. Microscopic Analyses of Specific IgY Binding to E. coii 0 157:H7 ....................................................................................... Chapter 3 . Anti-Bacterial Activity of IgY against Enterotoxigenic Escherichin coli 987P ................................................................... 3.1. Introduction ................................................................................... 3 .2 . Materials and Methods .................................................................. 3.2.2. Bacteria and Culture Conditions ......................................... 3 .2.2. Immunization of Chickens ................................................. 3 -2.3. Isolation of Water-Soluble Fraction (WSF) Containing IgY from Egg Yolk ......................................................................