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Evaluation of the Susceptibility and Humoral Immune Response of Foals to Rhodococcus Equi Infection

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Evaluation of the Susceptibility and Humoral Immune Response of Foals to Rhodococcus Equi Infection University of Kentucky UKnowledge Theses and Dissertations--Veterinary Science Veterinary Science 2014 EVALUATION OF THE SUSCEPTIBILITY AND HUMORAL IMMUNE RESPONSE OF FOALS TO RHODOCOCCUS EQUI INFECTION Macarena G. Sanz University of Kentucky, [email protected] Right click to open a feedback form in a new tab to let us know how this document benefits ou.y Recommended Citation Sanz, Macarena G., "EVALUATION OF THE SUSCEPTIBILITY AND HUMORAL IMMUNE RESPONSE OF FOALS TO RHODOCOCCUS EQUI INFECTION" (2014). Theses and Dissertations--Veterinary Science. 17. https://uknowledge.uky.edu/gluck_etds/17 This Doctoral Dissertation is brought to you for free and open access by the Veterinary Science at UKnowledge. It has been accepted for inclusion in Theses and Dissertations--Veterinary Science by an authorized administrator of UKnowledge. For more information, please contact [email protected]. STUDENT AGREEMENT: I represent that my thesis or dissertation and abstract are my original work. Proper attribution has been given to all outside sources. I understand that I am solely responsible for obtaining any needed copyright permissions. I have obtained needed written permission statement(s) from the owner(s) of each third-party copyrighted matter to be included in my work, allowing electronic distribution (if such use is not permitted by the fair use doctrine) which will be submitted to UKnowledge as Additional File. I hereby grant to The University of Kentucky and its agents the irrevocable, non-exclusive, and royalty-free license to archive and make accessible my work in whole or in part in all forms of media, now or hereafter known. I agree that the document mentioned above may be made available immediately for worldwide access unless an embargo applies. I retain all other ownership rights to the copyright of my work. I also retain the right to use in future works (such as articles or books) all or part of my work. I understand that I am free to register the copyright to my work. REVIEW, APPROVAL AND ACCEPTANCE The document mentioned above has been reviewed and accepted by the student’s advisor, on behalf of the advisory committee, and by the Director of Graduate Studies (DGS), on behalf of the program; we verify that this is the final, approved version of the student’s thesis including all changes required by the advisory committee. The undersigned agree to abide by the statements above. Macarena G. Sanz, Student Dr. David Horohov, Major Professor Dr. Daniel Howe, Director of Graduate Studies EVALUATION OF THE SUSCEPTIBILITY AND HUMORAL IMMUNE RESPONSE OF FOALS TO RHODOCOCCUS EQUI INFECTION ________________________ DISSERTATION ________________________ A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the College of Agriculture, Food and Environment at the University of Kentucky By Macarena G Sanz Lexington, Kentucky Director: Dr. David W. Horohov, Professor of Immunology Lexington, Kentucky Copyright © Macarena Sanz 2014 ABSTRACT OF DISSERTATION EVALUATION OF THE SUSCEPTIBILITY AND HUMORAL IMMUNE RESPONSE OF FOALS TO RHODOCOCCUS EQUI INFECTION While Rhodococcus equi (R. equi) remains the most common cause of subacute or chronic granulomatous bronchopneumonia in foals, development of a relevant model to study this bacterium has proven difficult. As a result, the reasons for the underlying foal’s susceptibility to this disease are not well understood. Furthermore, data regarding the immune response of foals to R. equi infection remains controversial. We hypothesized that foals are susceptible to R. equi early in life and that this susceptibility decreases with age. Also, we hypothesized that specific subclasses of IgG antibodies to the virulence- associated protein of R. equi, VapA, predict the outcome of exposure. The objectives of this study were: (1) to develop an R. equi challenge model that resulted in slow progressive disease in some foals as well as spontaneous regression of lesions in others, (2) using the developed model, to investigate the age-related susceptibility of young foals to R. equi, (3) to describe the humoral immune response of foals following experimental challenge and natural infection. The use of a low dose of R. equi to challenge neonatal foals resulted in slow, progressive disease characterized by pulmonary abscessation and spontaneous regression in approximately 50% of the foals. When this low dose was used in 1, 2 or 3-week-old foals, a marked decrease in disease susceptibility was observed as the foals aged. The immunological responses seen after experimental challenge reflect those observed after natural infection. While there was a significant increase of VapA-specific IgG and IgG subclasses over time in both pneumonic and healthy foals, use of VapA-specific IgG(T) showed good sensitivity and specificity when used as a diagnostic tool for R. equi pneumonia. In summary, this study shows that foal susceptibility to R. equi occurs early in life and decreases with age. Whereas all foals developed VapA-specific IgG antibodies post- exposure, IgG(T) appeared to be predictive of infection. KEY WORDS: Rhodococcus equi, foal, VapA, IgG Macarena G Sanz July 28, 2014 EVALUATION OF THE SUSCEPTIBILITY AND HUMORAL IMMUNE RESPONSE OF FOALS TO RHODOCOCCUS EQUI INFECTION By Macarena G Sanz Dr. David W. Horohov Director of Dissertation Dr. Daniel Howe Director of Graduate Studies June 2014 ACKNOWLEDGMENTS Completion of this doctorate was possible with the support of several people. I would like to express my sincere gratitude to all of them. I would like to begin by thanking my advisor, Dr. David Horohov, for giving me the tools to embark on a career as a research scientist as well as his unconditional support and advice. Special thanks to the members of my committee, Drs. John Timoney, Beth Garvy for their guidance and patience all along this learning experience and Dr. Alan Loynachan for the many hours spent on necropsy sampling and discussing protocols. I would like to express my gratitude to the UK farm personnel for their dedication looking after the mares and foals involved in these projects and to everybody in the immunology laboratory at the Gluck Equine Research Center for the help received with the many techniques involved in this work. Without the collaboration of Steeve Giguère from The University of Georgia, who provided the E. coli encoding recombinant VapA, much of the antibody work would not have been possible. I gratefully acknowledge the funding sources that made my Ph.D. work possible: Zoetis for providing my scholarship and Bioniche Life Science Inc. who partially funded many of these projects. Last, but not the least, I would like to thank my husband Martin for his unconditional love and support throughout this endeavor. iii TABLE OF CONTENTS ACKNOWLEDGMENTS ................................................................................................. iii LIST OF TABLES ............................................................................................................. vi LIST OF FIGURES .......................................................................................................... vii LIST OF ABBREVIATIONS ............................................................................................. x CHAPTER 1 LITERATURE REVIEW AND RESEARCH OBJECTIVES ..................... 1 CHAPTER 2 THE EFFECT OF BACTERIAL DOSE AND FOAL AGE AT CHALLENGE ON RHODOCOCUS EQUI INFECTION .............................................. 12 Summary ....................................................................................................................... 12 Introduction ................................................................................................................... 13 Materials and Methods .................................................................................................. 14 Results ........................................................................................................................... 22 Discussion .................................................................................................................... 36 CHAPTER 3 VALIDATION AND EVALUATION OF VapA-SPECIFIC IgG AND IgG SUBCLASSES ELISAs TO IDENTIFY FOALS WITH RHODOCOCCUS EQUI PNEUMONIA................................................................................................................... 40 Summary ....................................................................................................................... 40 Introduction ................................................................................................................... 42 Materials and methods .................................................................................................. 43 Results ........................................................................................................................... 48 CHAPTER 4 ADMINISTRATION OF COMMERCIAL RHODOCOCCUS EQUI SPECIFIC HYPERIMMUNE PLASMA RESULTS IN VARIABLE AMOUNTS OF IgG AGAINST PATHOGENIC BACTERIA IN FOAL’S SERUM ............................... 57 iv Summary ....................................................................................................................... 57 Introduction ................................................................................................................... 58 Materials and methods .................................................................................................. 59 Results ..........................................................................................................................
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