Ecology of Campylobacter Colonization in Poultry
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ECOLOGY OF CAMPYLOBACTER COLONIZATION IN POULTRY: ROLE OF MATERNAL ANTIBODIES IN PROTECTION AND SOURCES OF FLOCK INFECTION DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Orhan Sahin, D.V.M., M.S. ***** The Ohio State University 2003 Dissertation Committee: Professor Qijing Zhang, Advisor Approved by Professor Teresa Y. Morishita ________________________ Professor Linda J. Saif Advisor Professor Yehia M. Saif Department of Veterinary Preventive Medicine ABSTRACT Campylobacter jejuni, a gram-negative organism, is a leading bacterial cause of human foodborne enterocolitis worldwide. Although poultry are considered the major reservoir for this human pathogen, the ecology of Campylobacter in chicken flocks is poorly understood, hampering the design of effective intervention strategies at the preharvest stage. In the first part of this project, the prevalence, antigenic specificity, and bactericidal activity of poultry Campylobacter maternal antibodies were investigated. High levels of specific antibodies were detected in egg yolks, sera of broiler breeders, and young broiler chicks, as maternally-derived antibodies. These antibodies were against multiple outer membrane components of Campylobacter, and active in antibody- dependent complement-mediated killing of C. jejuni. These findings indicated the widespread presence of functional Campylobacter antibodies in the poultry production system, and prompted us to conduct the second part of the project to determine the effect of Campylobacter maternal antibodies on the colonization of young chickens by C. jejuni using challenge studies. Laboratory inoculation of commercial broilers of different ages showed that the onset of colonization occurred much sooner in birds challenged at the age of 21-days (which are naturally negative for Campylobacter antibody) than it did in the birds inoculated at 3-days of age (which are naturally positive for Campylobacter maternal antibody). Also, challenge experiments using laboratory-raised specific ii pathogen-free (SPF) chickens demonstrated that significant decreases in the percentage of colonized chickens were observed during the first week post inoculation in 3-day old SPF chicks with Campylobacter maternal antibody as compared with 3-day old chicks without Campylobacter-specific maternal antibody. These results indicated that Campylobacter maternal antibodies play a partial role in protecting young chickens against colonization by C. jejuni. In the last part of this study, we investigated the ability of Campylobacter to penetrate eggshells, its survival within eggs, and its prevalence in eggs from various sources to determine if vertical (egg-borne) transmission of Campylobacter occurred. Using a temperature differential method, it was shown that Campylobacter was either unable to penetrate the eggshell or did not survive up to 48 h inside the incubating eggs. C. jejuni survived for up to 14 days in eggs stored at 18°C following injection into the egg yolk; however, viability of the bacterium was dramatically reduced when injected into the albumen or the air sac. Although Campylobacter was detected in freshly-laid eggs obtained from an SPF flock colonized by C. jejuni, storage of eggs for 7 days at 18°C resulted in lack of detection of the organism. No Campylobacter was detected from 500 eggs laid by Campylobacter-colonized commercial breeders, and 1,000 eggs obtained from a commercial hatchery, suggesting that vertical transmission of Campylobacter through the egg is rare, and is unlikely to result in chicks infected with Campylobacter. iii Dedicated to my beloved family iv ACKNOWLEDGMENTS I would like to thank my advisor, Dr. Qijing Zhang, with sincere appreciation for his intellectual support, encouragement, enthusiasm, and great patience during the course of my degree program. His guidance and help made this dissertation possible, and will be cherished and remembered during my life. I also thank my previous sponsor, Ministry of Education of Republic of Turkey. Special thanks also go to my other committee members Dr. Yehia M. Saif, Dr. Linda J. Saif, and Dr. Teresa Y. Morishita for their efforts and valuable advice. I am grateful to Jerrel C. Meitzler for his technical support and his help in the animal studies. I thank Shouxiong Huang, Naidan Luo, and Sonia Pereira for their help in animal studies. I also wish to thank Dr. Linda O. Michel, Dr. Jun Lin, and Dr. Masato Akiba for their support and help. I thank Bob Dearth and Greg Myers for their support in the animal studies. Lastly, I would like express my deepest love and appreciation to my father, sister, and brother in heaven for their lifetime unconditional love and support. I miss you all! v VITA May 1, 1972 …………………… Born-Gaziantep, Turkey 1988-1993……………………… D.V.M. Ankara University, Ankara, Turkey 1996-1997……………………… M.S. The Ohio State University, Columbus, Ohio, USA 1998-present…………………… Graduate Research Associate, Food Animal Health Research Program, Department of Veterinary Preventive Medicine, The Ohio State University, OH, USA ACADEMIC HONORS AND AWARDS · Scholarship from the Ministry of Education, Turkey (1995 to 2001). · Second best poster Award in OARDC Annual Conference, Ohio, U.S.A. 2000 · Best Poster Award in Comparative Gastroenterology, the 82th Annual Conference of Research Workers in Animal Diseases, St. Louis, U.S.A. 2001 vi · Membership of Delta Chapter of the Phi Zeta Honor Society, U.S.A. 2000 to present · Membership of the American Society for Microbiology, U.S.A. 2000 to 2003 · Membership of Conference of Research Workers in Animal Diseases (CRWAD), U.S.A. 2001 to present PUBLICATIONS Research Publications 1. Sahin O, Zhang Q, Meitzler JC, Harr BS, Morishita TY, Mohan R. 2001. Prevalence, antigenic specificity, and bactericidal activity of poultry anti-Campylobacter maternal antibodies. Appl. Environ Microbiol.67:3951-7. 2. Naidan Luo, Orhan Sahin, Jun Lin, Linda Michel, and Qijing Zhang. 2003. In vivo selection of Campylobacter isolates with high levels of fluoroquinolone resistance associated with gyrA mutations and the function of the CmeABC efflux pump. Antimicrobial Agents and Chemothrapy.47:390-394. 3. Orhan Sahin, Teresa Y. Morishita, and Qijing Zhang. 2003.Campylobacter colonization in poultry: sources of infection and modes of transmission. Animal Health Research Reviews.3:95-105. 4. Orhan Sahin, Qijing Zhang, and Teresa Y. Morishita. 2002. Detection of Campylobacter. Book chapter. In press. 5. Orhan Sahin, Naidan Luo, Shouxiong Huang, and Qijing Zhang. 2003. Effect of Campylobacter-specific maternal antibodies on the colonization of young chickens by Campylobacter jejuni. Submitted to App. Environ Microbiol. 7. Orhan Sahin, Peter Kobalka, and Qijing Zhang. 2003. Detection and Survival of Campylobacter in chicken eggs. Submitted to J. Appl. Microbiol. 6. Jun Lin, Orhan Sahin, Linda O. Michel, and Qijing Zhang. 2003. Critical role of multidrug efflux pump CmeABC in bile resistance and in vivo colonization of Campylobacter jejuni. Submitted to Infect. Immun. vii Published Abstracts 1. O. Sahin, J.C. Meitzler, Q. Zhang, R. Mohan, B. Harr, and T.Y. Morishita. Prevalence of antibodies to Campylobacter jejuni in chicken flocks. Poster, 45P. In Proceedingsof the 80th Annunal Meeting of the CRWAD. 1999. Chicago, IL. 2. O. Sahin, J.C. Meitzler, Q. Zhang, , B. Harr, and T.Y. Morishita, R. Mohan. Prevalence of Campylobacter jejuni-specific antibodies in chicken flocks. Poster, 29. In OARDC Annual Conference. 2000. Wooster, OH. (Award winning). 3. O. Sahin, J.C. Meitzler, Q. Zhang, B. Harr, and T.Y. Morishita, R. Mohan. Prevalence of maternal antibodies to Campylobacter jejuni in poultry. Oral presentation (p. 37). In Proceedings of the 51st North Central Avian Disease Conference and Symposium on Emerging and Re-emerging Diseases. 2000. Columbus, OH. 4. O. Sahin, , Q. Zhang, and J.C. Meitzler. Effects of Anti-Campylobacter Maternal Antibody on the Colonization of Campylobacter jejuni in Poultry. Poster, Z-21. In Abstracts of American Society for Microbiology (ASM), 101st General Meeting. 2001. Orlando, FL. 5. O. Sahin, P. Kobalka, and Q. Zhang. Detection and Survivability of Campylobacter jejuni in chicken eggs. Poster, 47P. In Proceedings of the 82nd Annunal Meeting of the CRWAD. 2001. St. Louis, Missouri. (Award winning). 7. O. Sahin, and J. J. Kowalski. Development of a Selective Medium for Rhodococcus equi. Poster, 105P. In Proceedings of the 82nd Annunal Meeting of the CRWAD. 2001. St. Louis, Missouri. 8. O. Sahin, Q. Zhang, and T.Y. Morishita. Contributing factors for the lack of colonization of young chickens by Campylobacter jejuni. Poster, Z-13. In Abstracts of American Society for Microbiology, 102nd General Meeting. 2002. Salt Lake City, Utah. FIELDS OF STUDY Major Field: Veterinary Preventive Medicine Studies in Food Safety, Clinical Microbiology, Molecular Epidemiology viii TABLE OF CONTENTS Page Abstract …………………………………………………………………… ii Dedication ………………………………………………………………... iv Acknowledgments ………………………………………………………… v Vita ………………………………………………………………………... vi List of Tables ……………………………………………………………… xi List of Figures …………………………………………………………….. xii Chapters: 1. Literature review 1.1 The Genus Campylobacter: Taxonomy and general description ………………………………………………….. 1 1.2 Epidemiologic and clinical aspects of Campylobacter infections in humans ……………………………………….. 2 1.3 Pathogenesis of Campylobacter infections in humans …….. 4 1.4 Immunity to Campylobacter