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Investigation of Immune Response to Sarcocystis Neurona Infection in Horses with Equine Protozoal Myeloencephalitis

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Investigation of Immune Response to Sarcocystis Neurona Infection in Horses with Equine Protozoal Myeloencephalitis Investigation of Immune Response to Sarcocystis neurona Infection in Horses with Equine Protozoal Myeloencephalitis Jibing Yang Thesis submitted to the faculty of the Virginia Polytechnic Institute and State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE In Biomedical and Veterinary Sciences Department of Large Animal Clinical Sciences Virginia-Maryland Regional College of Veterinary Medicine Approved: ___________________________________ Sharon G. Witonsky, Committee Chair ____________________________________ Robert M. Gogal, Jr., Committee Member ____________________________________ David S. Lindsay, Committee Member _________________________________________ Nammalwar Sriranganathan, Committee Member __________________________________ Yasuhiro Suzuki, Committee Member July 14, 2005 Blacksburg, Virginia Keywords: Horse, Equine Protozoal Myeloencephalitis, EPM, Sarcocystis neurona, immune response, lymphocyte proliferation assay, IFN-γ Copyright 2005, Jibing Yang Investigation of Immune Response to Sarcocystis neurona Infection in Horses with Equine Protozoal Myeloencephalitis Jibing Yang Abstract Equine Protozoal Myeloencephalitis (EPM) is a serious neurologic disease of horses in the United States. The primary etiologic agent is Sarcocystis neurona (S. neurona). Currently, there is limited knowledge regarding the protective or pathologic immune response to infection to the intracellular protozoa S. neurona. The objective of these studies was to determine the effects of S. neurona infection on the immune response of horses that had EPM due to natural infection (experiment 1) and experimental infection (experiment 2). In experiment 1, twenty- two horses with naturally occurring cases of EPM, which were confirmed positive based on detection of antibodies in the serum and/or CSF and clinical signs, and 20 clinically normal horses were included to determine whether S. neurona altered the immune responses, as measured by immune cell subsets (CD4, CD8, B-cell, monocytes, and neutrophils) and leukocyte proliferation (antigen specific and non- specific mitogens). Our results demonstrated that naturally infected horses had significantly higher percentages of CD4 and neutrophils (PMN) in peripheral blood mononuclear cells (PBMCs) than clinically normal horses. Leukocytes from naturally infected EPM horses had a significantly lower proliferation response, as measured by thymidine incorporation, to a non-antigen specific mitogen phorbol 12- myristate 13-acetate (PMA) / ionomycin (I) than did clinically normal horses (p=0.04). The implications of these findings will be discussed. In experiment 2, 13 horses were randomly divided into two groups. Baseline neurologic examinations were performed and all horses were confirmed negative for S. neurona antibodies in the CSF and serum. Then, one group with 8 clinically normal seronegative horses was inoculated intravenously with approximately 6000 S. neurona infected autologous leukocytes daily for 14 days. All the challenged horses showed neurologic signs consistent with EPM. PBMCs were isolated from the control and infected horses to determine how S. neurona alters the immune responses based on changes in immune cell subsets and immune function. There were no significant differences in the percentage of CD4 cells in peripheral blood lymphocytes or IFN-γ production by CD4 and/or CD8 cells. PMA/I stimulated proliferation responses in PBMCs appeared suppressed compared to that of uninfected controls. Additional studies are necessary to determine the role of CD4 and CD8 cells in disease and protection to S. neurona in horses, as well as to determine the mechanism associated with suppressed in vitro proliferation responses. This project was funded by Patricia Stuart Equine grants and paramutual racing funds from Virginia Tech. iii Acknowledgements There are lots of people I would like to thank for their help in finishing this work. First of all, I would like to take this opportunity to thank my advisor, Dr. Sharon Witonsky, for her unconditional support, outstanding scientific guidance, and cordial care for me. Without her help, I could not accomplish this work during the past three years. I also would like to thank my committee members: Dr. Robert M. Gogal, Dr. David S. Lindsay, Dr. Nammalwar Sriranganathan, and Dr. Yasuhiro Suzuki, for their valuable advice, kind encouragement and understanding. I would like to extend my thanks to Ms. Joan Kalnitsky for her kind help and excellent expertise in flow cytometry, and Dr. Dan Ward for his assistance in statistics. I sincerely thank Heather Norton, Chris Wakely, and Kevin Weaver for their technical support. I also thank everybody at the CMMID for their help and friendship. Finally, I thank my family members who always encouraged me to reach for the stars with their tremendous love and support. These studies were supported by Patricia Stuart Equine grants and paramutual racing funds from Virginia Tech and a graduate assistantship to Jibing Yang from the Office of Research and Graduate Studies at Virginia-Maryland Regional College of Veterinary Medicine. iv TABLE OF CONTENTS TITLE PAGE ……………………………………………………………………….і ABSTRACT ………………………………………………………………………...іі ACKNOWLEDGEMENTS ……………………………………………………….іv TABLE OF CONTENTS ………………………………………………………….v LIST OF FIGURES ………………………………………………………………vіі LIST OF TABLES ………………………………………………………………...ix LIST OF ABBREVIATIONS ……………………………………………………..x CHAPTER 1 Introduction ……………………………………………………..1 CHAPTER 2 Literature review of Equine Protozoal Myeloencephalitis …...3 2.1 History and etiologic agent ………………………………………….………3 2.2 Life cycle of Sarcocystis neurona ……………………………………….…..5 2.3 Epidemiology ……………………………………………………….……….8 2.4 Experimental Animal Models.……………………………...………………11 2.5 Clinical signs.……………………………………………………………….18 2.6 Diagnosis.………………………………………………………………...…19 2.6.1 Cytology and Cerebrospinal fluid indices (CSF/indices).……..…….20 2.6.2 Enzyme-linked immunosorbent assay (ELISA) to Sarcocystis neurona Surface Antigen 1 (SnSAG1).………………………………………..23 2.6.3 Immunoblot assay (western blot).……………………………………25 2.6.4 Immunofluorescence Assay (IFA)…………...……………………....26 2.6.5 Serum Agglutination Test (SAT)…………………………………….26 2.6.6 Polymerase Chain Reaction (PCR)….…...…………………………..27 2.7 Pathology.…………………………………………………………………..27 2.8 Treatment.…………………………………………………………………..28 2.9 Prevention.………………………………………………………………….30 2.10 Future goals.……………………………………………………………….31 CHAPTER 3 Immunophenotypes and lymphocyte proliferative responses in naturally infected EPM horses …………………………………………………..34 3.1 Introduction …………………………………………………………………….34 3.2 Materials and methods …………………………………………………………35 3.2.1 Animals ………….……………………………………………………….35 3.2.2 Collection of blood samples.………………………….……………….….36 v 3.2.3 Differential cell counts ………………………….………………………..36 3.2.4 Isolation of Peripheral Blood Mononuclear Cells (PBMCs)...………...…37 3.2.5 Cytospin preparation …………….……………………………………….37 3.2.6 Flow cytometry ………………………….……………………………….38 3.2.7 7-amino actinomycin D (7-AAD) staining……………………………….39 3.2.8 Live merozoite preparation …………………………………….……...…39 3.2.9 Lymphocyte Proliferation Assays………………………………………...40 3.2.10 SnSAG-1 (Sarcocystis neurona surface antigen-1) plate incubation …..41 3.3 Statistical Analysis ……………………………………………….…………….41 3.4 Results ……………………………………………………………………….....42 3.4.1 Effects of storage or shipping condition on immunological assays utilizing equine PBMCs…………………………………………………………...42 3.4.2 Analysis of immune cell subsets and changes in immune function between naturally infected EPM and clinically normal horses……………………44 3.5 Discussion ………………………………………………………………...……45 CHAPTER 4 Changes in immune subsets and function associated with experimental S. neurona infection of horses ……………………………………62 4.1 Introduction …………………………………………………………………….62 4.2 Materials and Methods …………………………………………………………64 4.2.1 Animals …………………………………………………………….…….64 4.2.2 Isolation of Peripheral Blood Mononuclear Cells (PBMCs)...…………...66 4.2.3 Cytospin preparation ………………………………………………….….66 4.2.4 Flow cytometry …………………………………………………….…….67 4.2.5 7-AAD staining …………………………………………………………..67 4.2.6 Live merozoite preparation …………………………………............……68 4.2.7 Lymphocyte proliferation assays ………………………………………...68 4.2.8 SnSAG-1 plate incubation ……………………………………….............69 4.2.9 γ-IFN intracellular cytokine staining ……………………………….……69 4.2.10 PMA/I induced lymphocyte apoptosis…………..……………............…70 4.3 Statistical Analysis..…………………………………………………….………71 4.4 Results..…………………………………………………………………………71 4.4.1 Effects of S. neurona infection on immunophenotypes of experimentally infected horses …………………………………………………………..71 4.4.2 Analysis of lymphocyte proliferation assays ………………………….....73 4.4.3 Analysis of γ-IFN intracellular cytokine staining ………………...……...73 4.4.4 Effects of PMA/I on lymphocyte apoptosis ………………………...…....74 4.5 Discussion.………………………………………………………………...…..74 CHAPTER 5 General Summary and Conclusions …………………………....101 FUTURE STUDIES …………………………………………………………..…105 LITERATURE CITED …………………………………………………………108 vi LIST OF FIGURES Figure 2.1 Phylogenetic classification of Sarcocystis spp ……………….………....4 Figure 2.2 Life cycle of Sarcocystis neurona …………………………….………...7 Figure 2.3 CSF-indices …………………………………………………….……...22 Figure 2.4 Calculation of AI SAG1 index ………………………………….…..…24 Figure 3.1 Percentage of CD8 T cells of total lymphocytes ……………….……..54 Figure 3.2 Pokeweed (PWM) stimulated proliferation assay comparing normal and EPM affected horses ……………………………………………….…56
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