VALIDATION OF AN IMPROVED ISOLATION AND DETECTION METHOD FOR CAMPYLOBACTER JEJUNI IN VARIOUS FOODS By ROSE ODONGO Bachelor of Science Wuxi Institute of Light Industry Wuxi, China 1992 Master of Science University of Western Sydney Sydney, Australi a 1998 Submitted to the Faculty of the Graduate College of the Oklahoma State University in partial fulfillment of the requirements for the Degree of DOCTOR OF PHILOSOPHY December , 2007 VALIDATION OF AN IMPROVED ISOLATION AND DETEC TION METHOD FOR CAMPYLOBACTER JEJUNI IN VARIOUS FOODS Thesis Approved: Dr . Stanley E. Gilliland Dr. K. D arrell Berlin Dr. Christina Dewitt Dr. William McGlynn Dr. A. Gordon Emslie Dean of the Graduate College ii ACKNOWLEDGEMENTS I am i ndebted to Dr. Stanley Gilliland who gave me an opportunity to work with him, I feel honored to have known and worked with him. I like to express my sincere thanks for his guidance and direction through out this study. My sincere appreciation goes to my supervisor, Dr. Siobhan Reilly, not only for her guidance and supervision during this study, but for her time and commitment towards this research. My sincere appreciation is also extended to my advisory committee members namely, Dr. K. D. Berlin, for his time and fatherly advice, Dr. Christina Dewitt, and Dr. William McGlynn for their guidance. I want to thank Miriam and Rien for their friendship and encouragement during this study. My sincere thanks and gratitude goes to my family and particularly to m y parents John and Janet Odon go , who have continuosly prayed, encouraged and supported me through the years. Last but not least I commend Paula, my daughter for the patience, love and encouragement. iii TABLE OF CONTENTS Chapter Page I. Introducti on ................................ ................................ ................................ .............. 1 II. Review of Literature History of Campylobacter ................................ ................................ ........................ 3 Characteristics ................................ ................................ ................................ .......... 5 Clinical manifestation ................................ ................................ ........................ 6 Pathogenesis and virulence determinants ................................ .......................... 7 Factors affecting growth of Campylobacter jejuni ................................ ................ 15 Influence of atmosphere ................................ ................................ ................... 15 Influence of temperat ure ................................ ................................ .................. 16 Influence of oxidative stress ................................ ................................ ............ 18 Influence of osmotic stress ................................ ................................ ............... 19 Influence of pH ................................ ................................ ................................ 20 Influence of of stationary phase and starvation ................................ ............... 20 The r ole of non -culturable cells ................................ ................................ ....... 21 Characterization of v iable -but -non culturable cells ................................ ... 21 Detection of viable -but -non culturable cells ................................ .............. 23 Recovery of viable -but -non culturable cells ................................ .............. 24 Recovery of viable -but -non culturable cells in vitro and in vivo models .. 24 Prevalence and sources of transmission of Campylobacter ................................ ... 26 Poultry as a source of transm ission ................................ ................................ .. 28 Drinking water as a source of transmission ................................ ..................... 30 Animals as a source of transmission ................................ ................................ 32 Fresh produce as a source of transmission ................................ ....................... 34 Isolation of Campylobacter from food products ................................ .................... 34 Currently used media for isolation of Campylobacter ................................ ..... 37 Media components ................................ ................................ ........................... 38 Development of Campylobacter plating media ................................ ............... 38 Selective agar media for isolation of Campylobacter ................................ ...... 45 Performance comparison of different media ................................ .................... 46 Standard methods for isolation of Campylobacter jejuni ................................ ...... 48 International Org anization for Standardization ................................ ............... 48 Food and Drug Administration /B acteriological Analytical Manual ................ 49 The Food and Agr icultural Products Center method for the detection of Campylobacter jejuni in foods ................................ ................................ ............... 50 References ................................ ................................ ................................ .............. 52 iv II. Validation of an improved isola tion and detection method for Campylobacter jejuni in various foods. ................................ ................................ ................................ 69 Abstract ................................ ................................ ................................ ........................ 70 Introduction ................................ ................................ ................................ .................. 72 Materials and Methods ................................ ................................ ................................ .74 Source, maintenance and preparation of cultures ................................ .................. 74 Preparation and inoculation of samples ................................ ................................ .75 Prepar ation of food samples ................................ ................................ ............. 77 Enrichment procedures ................................ ................................ .................... 78 Lysing of horse blood cells ................................ ................................ .............. 80 Enumeration procedure ................................ ................................ .................... 80 Confirmation of identity of detected organisms ................................ .................... 81 Gram Stain ................................ ................................ ................................ ....... 81 Oxidase test procedure ................................ ................................ ..................... 81 Identification of isolates using API Campy ®Identification system ................. 81 Dete ction of Campylobacter jejuni ATCC 29428 and 33560 in ground beef during storage ................................ ................................ ................................ ......... 82 Growth curves for Campylobacter jejuni ATCC 29428 and 33560 ...................... 83 Statistical Analysis ................................ ................................ ................................ .84 IV. Results and discussion ................................ ................................ ........................... 86 Confirmation of isolates of Campylobacter jejun i................................ ................. 86 Influence of supplementation of enrichment broth with horse blood on recovery of Campylobacter jejuni ................................ ................................ ........................ 88 Effect of enrichment time and storage period on the recovery of Campylobacter jejuni ................................ ................................ ................................ ..................... 89 Chicken and ground beef ................................ ................................ ................. 89 Effect of detection me thod and enrichment time on the recovery of Campylobacter jejuni ................................ ................................ ............................. 91 Chic ken skin and ground beef ................................ ................................ .......... 91 Effect of blood supplementation and storage period on the recovery of Campylobacter jejuni ................................ ................................ ............................. 92 Milk ................................ ................................ ................................ .................. 92 Effect of detection method, storage period a nd enrichment time on the recovery of Campylobacter jejuni ................................ ................................ ........................ 93 Beef and milk ................................ ................................ ................................ ... 93 Growth curve of Campylobacter jejuni cultured in Bolton broth at 37 oC and 42 oC................................ ................................ ................................ ....................... 94 Detection of Campylobacter jejuni ................................ ................................ ........ 96 Summary of findings ................................ ................................ .............................. 98 References ................................ ................................ ...............................