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When You Get a Result That You Expect, You Have Another Result, but When You Get a Result You Don't Expect, You Have a Discove

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When You Get a Result That You Expect, You Have Another Result, but When You Get a Result You Don't Expect, You Have a Discove When you get a result that you expect, you have another result, but when you get a result you don’t expect, you have a discovery. -Frank Westheimer STUDIES ON MYCOBACTERIUM AVIUM SUBSP. PARATUBERCULOSIS: GENOTYPIC AND PHENOTYPIC VARIATIONS DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Alifiya H. Ghadiali, M.Sc. ***** The Ohio State University 2005 Dissertation Committee: Approved by Professor Srinand Sreevatsan, Adviser _______________________________ Professor Y. Mohamed Saif Adviser Professor William P. Shulaw Graduate Program in Professor Jeffrey T. LeJeune Veterinary Preventive Medicine Copyright by Alifiya Huzefa Ghadiali 2005 ABSTRACT The objective of the study was to understand the genotypic and phenotypic variations across Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis) isolates form diverse hosts and geographic locations. Multiplex PCR of IS900 loci (MPIL) fingerprint analysis of M. paratuberculosis isolates recovered from diverse hosts and geographic localities clustered 78% of bovine origin isolates into a major node, while isolates from human and ovine sources showed greater genetic diversity. Amplified fragment length polymorphism (AFLP) analysis clustered 75% of the isolates from bovine sources into 2 major nodes while those recovered from sheep or human clustered on distinct branches. This suggested that the M. paratuberculosis isolates were genotypically homogenous or were converging into a common fingerprint. To evaluate the alternate possibility that MPIL and AFLP analyses were inadequate for dissecting the M. paratuberculosis genotypes, short sequence repeat (SSR) analysis was performed on mycobacterial isolates obtained from 33 different host species and from environmental sources. Sequence based characterization of the G-repeat locus enabled differentiation of the M. paratuberculosis isolates from the major MPIL cluster into seven distinct alleles. Subsequent analysis of M. paratuberculosis isolates from human Crohn’s disease cases using two polymorphic SSR loci (G- and GGT- repeats) ii identified a limited number of genotypes amongst the human strains indicating an association of a few M. paratuberculosis types with the pathobiology of Crohn’s disease. SSR analysis using both polymorphic loci also enabled identification of varying levels of within-farm and between farm diversity and indicated that 7g-4ggt as the most common genotype in animals from Ohio dairy farms. To explore whether the differences in fingerprint profiles translated to variation in biological function and/or host adaptation, cDNA microarray analysis of a human macrophage cell line exposed to M. paratuberculosis isolated from cattle and human hosts was undertaken. Results indicated that the expression profiles induced by representative cattle and human M. paratuberculosis strains differed in several key inflammatory and apoptotic pathways suggesting that M. paratuberculosis strains with different genotypes induce variant transcriptional regulation. Taken together, the results of our genotypic and phenotypic analyses demonstrated that SSR analysis enabled the genetic characterization of M. paratuberculosis isolates from different host species, and provided support for a genotype-phenotype association in M. paratuberculosis infection. iii Dedicated to my father Saifuddin B. Motiwala iv ACKNOWLEDGMENTS …the words may be few but the gratitude is sincere and heart-felt… Grateful acknowledgment is due to my adviser, Dr. Srinand Sreevatsan, who encouraged and challenged me throughout my academic program. I have learned substantially from his uncompromising emphasis on quality and meaningful research. His refusal to accept anything less than my best efforts enabled me to achieve more than what I believed possible. I would also like to thank my committee members, Dr. Y. Mohamed Saif, Dr. William P. Shulaw and Dr. Jeffrey T. LeJeune, for providing me with constructive comments, advice and encouragement throughout the research program. Special thanks are due to Megan Strother for technical assistance at various stages in my research, for culturing and maintaining of samples and strains, for handling laboratory supplies at any time of the day, for locating precious samples saved ‘somewhere’ in the -70°C and -20°C freezers, and last but not the least for the laughs and tears and words of encouragement during these years and for sharing my frustrations in work with the fastidious M. paratuberculosis. v I also thank all my colleagues in the Sreevatsan Laboratory for sharing experiences and knowledge and for steady encouragement throughout the course of this work. Finally, I take this opportunity to express my profound gratitude to my mother, Zulekha S. Motiwala, and my sister, Mariyam M. Patni, for supporting me in my educational pursuits. I also want to thank my husband, Huzefa M. Ghadiali, for his unwavering patience and understanding in dealing with my crazy schedules and inexplicable emotional tantrums (which were inversely proportional to the success of my experiments)! vi VITA February 7, 1976 …….… Born – Mumbai (Bombay), India 1996 …………..……….. Bachelor of Science (Major-Microbiology and Biochemistry) Mithibai College, University of Mumbai, India 1998 …………..……….. Master of Science (Major-Biochemistry) Seth GS Medical College, University of Mumbai, India 1998 - 1999 …….……… Research Assistant Department of Clinical Pharmacology, Seth GS Medical College and KEM Hospital, Mumbai, India 1999 - 2001 …….……… Research Assistant and Co-ordinator Sneha-India and Medical Research Council - Environmental Epidemiology Unit, United Kingdom Mumbai, India 2001 – present …...…….. Graduate Research Associate Food Animal Health Research Program, Ohio Agricultural Research Development Center and Department of Veterinary Preventive Medicine, The Ohio State University, Columbus, Ohio PUBLICATIONS 1. Ghadiali A.H., M. Strother, N.E. Theus, R.W. Stich, B. Byrum, W.P. Shulaw, V. Kapur and S. Sreevatsan. Rapid detection and strain typing of Mycobacterium avium subsp. paratuberculosis from broth cultures. In press. J. Clin. Microbiol. 2. Rajeev S., Y. Zhang, S. Sreevatsan, A.S. Motiwala and B. Byrum. 2005. Evaluation of multiple genomic targets for identification and confirmation of Mycobacterium avium subsp. paratuberculosis isolates using real-time PCR. Vet. Microbiol. 105: 215-21. vii 3. Ghadiali A.H., M. Strother, S.A. Naser, E.J.B. Manning and S. Sreevatsan. 2004. Genetic analysis of polymorphic loci in Mycobacterium avium subspecies paratuberculosis isolated from Crohn’s disease patients and animal species exhibit similar polymorphic loci patterns. J. Clin. Microbiol. 42:5345-5348. 4. Motiwala, A.S., A. Amonsin, M. Strother, E.J.B. Manning, V. Kapur, and S. Sreevatsan. 2004. Molecular Epidemiology of Mycobacterium avium subsp. paratuberculosis Isolates Recovered from Wild Animal Species. J. Clin. Microbiol. 42:1703-1712. 5. Amonsin, A., L.L. Li, Q. Zhang, J.P. Bannantine, A.S. Motiwala, S. Sreevatsan, and V. Kapur. 2004. Multilocus Short Sequence Repeat Sequencing Approach for Differentiating among Mycobacterium avium subsp. paratuberculosis Strains. J. Clin. Microbiol. 42:1694-1702. 6. Ozbek, A., F.C. Michel, M. Strother, A.S. Motiwala, B.R. Byrum, W.P. Shulaw, C.G. Thornton, and S. Sreevatsan. 2003. Evaluation of two recovery methods for detection of Mycobacterium avium subsp. paratuberculosis by PCR: direct-dilution- centrifugation and C(18)-carboxypropylbetaine processing. FEMS Microbiol. Lett. 229:145-51. 7. Motiwala, A.S., M. Strother, A. Amonsin, B. Byrum, S.A. Naser, J.R. Stabel, W.P. Shulaw, J.P. Bannantine, V. Kapur, and S. Sreevatsan. 2003. Molecular epidemiology of Mycobacterium avium subsp. paratuberculosis: evidence for limited strain diversity, strain sharing, and identification of unique targets for diagnosis. J. Clin. Microbiol. 41:2015-26. PUBLISHED ABSTRACTS 1. Ghadiali A.H., M. Strother, N.E. Theus, B. Byrum, R.W. Stich, W.P. Shulaw, S. Sreevatsan. Molecular epidemiology of Mycobacterium avium subsp. paratuberculosis in Ohio dairy farms. Conference of Research Workers in Animal Diseases. 85th Annual Meeting, Chicago, Illinois, November 14-16, 2004. (Abstract # 99). 2. Ghadiali A.H., S.A. Naser, E.J.B. Manning and S. Sreevatsan. Short sequence repeat analysis of Mycobacterium avium subsp. paratuberculosis isolates from Crohn’s disease patients exhibit genetic similarity to extant clones of animal origin. 2004 OARDC Annual Research Conference, Wooster, Ohio, April 29, 2004 (Poster # 11). viii 3. Motiwala A.S., S.A. Naser and S. Sreevatsan. Short sequence repeat analysis of Mycobacterium avium subsp. paratuberculosis isolates from Crohn’s disease patients exhibit genetic similarity to extant clones of bovine and caprine origin. Conference of Research Workers in Animal Diseases. 84rd Annual Meeting, Chicago, Illinois, November 9-11, 2003. (Abstract # 18P). 4. Motiwala A.S., M. Strother, E.J.B. Manning and Sreevatsan S. Restricted Diversity within Mycobacterium avium subsp. paratuberculosis Isolated from Exotic Animal Species from Diverse Geographic Locations. American Society for Microbiology. 103rd General Meeting, Washington DC, May 18-22, 2003. (Abstract # U-39). 5. Motiwala A.S., M. Strother, E.J.B. Manning and Sreevatsan S. Restricted Diversity within Mycobacterium avium subsp. paratuberculosis Isolated from Exotic Animal Species from Diverse Geographic Locations. 2003 OARDC Annual Research Conference, Columbus,
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