Development of Molecular Diagnostic Tests for Inclusion Body Disease in Boid Snakes

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Development of Molecular Diagnostic Tests for Inclusion Body Disease in Boid Snakes
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english
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Chang, Li-Wen
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University of Florida
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Degree:
Doctorate ( Ph.D.)
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University of Florida
Degree Disciplines:
Veterinary Medical Sciences, Veterinary Medicine
Committee Chair:
Jacobson, Elliott R
Committee Members:
Abbott, Jeffrey
Mergia, Ayalew
Chen, Sixue
Farmerie, William G

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Subjects / Keywords:
antibody -- arenavirus -- body -- boid -- diagnostics -- disease -- ibd -- ibdp -- ihc -- inclusion -- monoclonal -- protein -- sequencing -- snake -- test -- validation
Veterinary Medicine -- Dissertations, Academic -- UF
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Veterinary Medical Sciences thesis, Ph.D.
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Abstract:
Inclusion Body Disease (IBD) is commonly seen in captive boa constrictors (Boa constrictor) and occasionally in other boid snakes. This disease is characterized by abnormal accumulation of an insoluble intracytoplasmic 68 KDa protein (IBDP). The exact causative agent(s) and pathogenesis remain unknown. Currently, diagnosis of IBD is based on the light microscopic identification of eosinophilic intracytoplasmic inclusion bodies in hematoxylin and eosin (H&E) stained tissues or blood smears. Better ante-mortem diagnostic tests are needed for screening IBD affected captive snakes, and preventing the introduction of IBD into wild populations. In this study, a mouse anti-IBDP monoclonal antibody (MAB) was produced against semi-purified IBD inclusion bodies. Using immunohistochemical (IHC) staining, the anti-IBDP MAB was validated by testing on a repository of IBD positive and negative paraffin embedded tissues collected from 1990 to 2011. In boa constrictors, the anti-IBDP MAB had a sensitivity of 80% and specificity of 100% in detecting IBD. The antibody also cross-reacted with IBD inclusion bodies in carpet pythons (Morelia spilota) and a ball python (Python regius). An improved H&E staining method was developed, and was used to stain the isolated peripheral white blood cells (PWBC) on microscopic slides. Using anti-IBDP MAB, antigen detection of the isolated PWBC samples were performed by IHC staining and western blots. The presences of antibody against IBDP in the plasma of boa constrictors were also evaluated by utilizing a previously developed anti-snake IgG antibody on western blots. A total of 78 blood samples from boa constrictors, rainbow boas (Epicrates cenchria) and ball pythons were evaluated for the presence of IBDP using both H&E staining and IHC staining.  The presence of IBDP antigen showed excellent agreement with the diagnosis of IBD. Plasma samples from IBD positive boa constrictors were negative for anti-IBDP antibody.  Finally, the IBD specific protein IBDP was sequenced using tandem mass spectrometry (MS/MS). The peptides of IBDP detected by MS/MS had a 90.5% amino acid coverage of the predicted nucleoprotein of a recently discovered arena-like virus (NCBI Gene ID: 13466438) that was identified in IBD positive boa constrictors.
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by Li-Wen Chang.
Thesis:
Thesis (Ph.D.)--University of Florida, 2012.
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Adviser: Jacobson, Elliott R.
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RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2013-12-31

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1 DEVELOPMENT OF MOLECULAR DIAGNOSTIC TESTS FOR INCLUSION BODY DISEASE IN BOID SNAKES By LI WEN CHANG A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2012

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2 2012 Li Wen Chang

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3 To researchers who are supportive and truly pas sionate in the study of Inclusion Body Disease and individuals who inspired me to pursue my graduate degree

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4 ACKNOWLEDGMENTS This project would not h ave been successful without support advice, and expertise provided by many collaborative institutes and indi viduals. Most importantly, I am grateful for the guidance and encouragement provided by my committee members: Elliott Jacobson, Ayalew Mergia, Jeffrey Abbott, Sixue Chen and William Farmerie. I am grateful for my sponsor, Ministry of Education, Republic o f China (Taiwan) that supported me for the first three years of graduate study at University of Florida. A major portion of my PhD research was funded by the Morris Animal Foundation, with out which this work would not have been possible. This funding was u sed to complete the following: 1) antibody validation; 2) d evelopment of the blood screening t est s; and 3) and s equencing the Inclusion Body Disease Protein (IBDP) This study was extra challenging since the causative agent of Inclusion Body Disease was un known. Additionally, initial attempts at sequencing IBDP were unsuccessful due to the absence of homologous sequences in the National Center for Biotechnology Information protein database. This study would not have been successfu l without the tremendous su pport I received from several core laboratories within the Interdisciplinary Center for Biotechnology Research, University of Florida. This included the individuals within the Proteomic Core ( Marjorie Chow, Cecilia Silva Sanchez, Ran Zheng, Carolyn Diaz an d Sixue Chen ), the Cellomic Core ( Linda Green and Diane Duke of the Hybridoma Laboratory ) and the Electron Microscopy Laboratory ( Karen Kelley ) Significant help was also provided by laboratories within College of Medicine, University of Florida This included the individuals within the Molecular Pathology Core (Ann Fu, Lynda Schneider and Martha Campbell Thompson) and McKnight Brain Institute (Joy Guingab and Firas Kobeissy ).

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5 I am thankful to several collaborated institutes outside of Universi ty of Florida. Joseph DeRisi and Mark Stenglein of t he DeRisi Lab at University of California San Francesco helped with virus identification u sing deep sequencing techniques. Also kind support from Freeland Dunker ( California Academy of Sciences, San Fran cisco, California, USA ), Drury Reavill ( Zoo/Exotic Pathology Services, West Sacramento, California, USA ), Gregory Rich ( The West Esplanade Veterinary Clinic, Metairie, LA USA), Thomas Boyer ( Pet Hospital of Penasquitos in San Diego San Diego, CA USA), A my Wells ( Avian and Exotic Clinic of Monterey Del Rey Oaks, CA ) Lauren P owers ( C arolina Veterinary Specialists, Huntersville, NC USA), Michael Garner (Northwest ZooPath Monroe, WA USA ) and many other veterinarians provided samples used in my project I am grateful to the Anatomic Pathology Service (Patrick Knisley, Micaela Barter and Jeffery Abbott) of the Veterinary Medical Hospitals, University of Florida. I am also grateful to the following individuals for their technical advice and encourageme nt: James Coleman, Francesco Origgi, Edward Wozniak, Jorge Hernandez, Rick Alleman, Nicole Stacy, David Allred, and Lynn Herrman. My graduate work would not have progressed and April Childress. I deeply appreciate their help. The following colleagues shared their friendship while in the graduate program, College of Veterinary Medicine: Astrid Grosche, Patricia Dingman, Claudio Verdugo, Takashi Uemura, Cruz Fan, Penni Chao, Ro nald Koh, Alice Chen, Galaxia Cortes, Katherine Saylor, Liliana Crosby, Alexa McDermott and Sylvia Tucker.

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6 I am extremely thankful for unlimited support by my family. Especially my parents, who deeply believed in my potential, provided financial and spiritual support throughout my academic studies.

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7 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ .......... 10 LIST OF FIGURES ................................ ................................ ................................ ........ 11 LIST OF ABBREVIATIONS ................................ ................................ ........................... 14 ABSTRACT ................................ ................................ ................................ ................... 18 CHAPTER 1 INTRODUCTION: INCLUSION BODY DISEASE IN BOID SNAKE ........................ 20 Review of the Literature ................................ ................................ .......................... 20 Background ................................ ................................ ................................ ...... 20 History, Hosts, and Geographic Range ................................ ............................ 20 Clinical Signs ................................ ................................ ................................ .... 21 Pathology and Disease ................................ ................................ .................... 22 Current Diagnostic Methods ................................ ................................ ............. 24 IBD Antigenic Protein ................................ ................................ ....................... 26 Possib le Causative Agents of IBD and Transmission ................................ ....... 27 Treatment and Prognosis ................................ ................................ ................. 29 Recent Findings ................................ ................................ ............................... 30 Research Goals ................................ ................................ ................................ ...... 30 The Needs of M olecular Diagnostic Tests ................................ ........................ 30 Better Understand the Relationship Between IBD and IBDP ............................ 31 Hypothesis and Objectives ................................ ................................ ..................... 32 Hypothesis ................................ ................................ ................................ ........ 32 Objectives ................................ ................................ ................................ ......... 32 2 THE PRODUCTION OF ANTI IBDP MONOCLONAL ANTIBODY ......................... 51 Introduction ................................ ................................ ................................ ............. 51 Materials and Methods ................................ ................................ ............................ 52 IBDP Purification ................................ ................................ .............................. 52 Polyclonal and Monoclonal Anti IBDP Antibody Production ............................. 55 Results ................................ ................................ ................................ .................... 59 IBDP Purification ................................ ................................ .............................. 59 Antibody Production and Monoclonal Antibody Selection ................................ 60 Discussion ................................ ................................ ................................ .............. 62 Insolubility of the IBD Inclusion Bodies ................................ ............................. 62 Inaccurate Estimation of Protein Concentration ................................ ............... 64 Challenges in Antibody Screening ................................ ................................ .... 64

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8 Conclusions ................................ ................................ ................................ ............ 65 3 VALIDATION OF AN ANTI IBD PROTEIN MONOCLONAL ANTIBODY FOR USE IN IMMUNOHISTOCHEMICAL STAINING ................................ ..................... 73 Introduction ................................ ................................ ................................ ............. 73 Material and Methods ................................ ................................ ............................. 74 Sample Collection and Management ................................ ................................ 74 Formalin Fixation and Embedding ................................ ................................ .... 75 H&E Staining for Paraffin Embedded Tissue ................................ .................... 76 IHC Staining for Paraffin Embedded Tissues ................................ ................... 77 IHC Evaluation ................................ ................................ ................................ 78 IHC Diagnostic Performance Evaluation ................................ .......................... 78 Results ................................ ................................ ................................ .................... 79 Standardization of IHC Staining Condition ................................ ....................... 79 Validation of anti IBDP MAB with IHC Staining ................................ ................ 81 a. Effects of prolonged formalin fixation ................................ ..................... 81 b. Effects of storage time in paraffin ................................ ........................... 82 c. Species cross reactivity ................................ ................................ .......... 83 d. Antigen cross reactivity ................................ ................................ .......... 84 e. IHC test diagnostic performance ................................ ............................ 84 Discussion ................................ ................................ ................................ .............. 85 Factors That may Affect IHC Staining ................................ .............................. 85 IHC Diagnostic Performance Evaluation ................................ .......................... 88 Cross Reactivity Among Non Boa Constrictors ................................ ................ 89 Conclusions ................................ ................................ ................................ ............ 90 4 IMMUNO BASED DIAGNOSTIC TESTS FOR SCREENING IBD .......................... 99 Introduction ................................ ................................ ................................ ............. 99 Materials and Me thods ................................ ................................ .......................... 100 Animal and Sample Collection ................................ ................................ ........ 100 Sample Preparation ................................ ................................ ........................ 100 Determine IBD Positive or Negative ................................ ............................... 101 Antigen Detection Using IHC Staining ................................ ............................ 101 Antigen Detection Using Western Blots ................................ .......................... 102 Antibody Detection Using Western Blots ................................ ........................ 104 Agreement and Association Analysis ................................ ............................. 106 Results ................................ ................................ ................................ .................. 107 Antigen Detection ................................ ................................ ........................... 107 Antibody Detection ................................ ................................ ......................... 108 Discussion ................................ ................................ ................................ ............ 109 Modified Bradford Protein Assay ................................ ................................ .... 109 Assessment of Using Blood Tests for Screening IBD ................................ ..... 110 Antigen detections ................................ ................................ ................... 110 Antibody detections ................................ ................................ .................. 112 Conclusions ................................ ................................ ................................ .......... 113

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9 5 SEQUENCING INCLUSION BODY DISEASE PROTEIN ................................ ..... 122 Introduction ................................ ................................ ................................ ........... 122 Material and Methods ................................ ................................ ........................... 123 Protein Preparation ................................ ................................ ........................ 123 Mass S pectrometry S ample P reparation ................................ ........................ 125 Tandem Mass Spectrometry ................................ ................................ .......... 126 Sequence Analysis ................................ ................................ ......................... 127 Results ................................ ................................ ................................ .................. 128 Protein Preparation ................................ ................................ ........................ 128 Sequence Analysis ................................ ................................ ......................... 128 Discussion ................................ ................................ ................................ ............ 129 Challenges in Sequencing IBDP and the Improvements in Methodology ....... 129 Arenaviruses and Their Protein Divergence ................................ ................... 131 Confirmation of the Linkage between IBDP and GGV NP .............................. 132 Future Diagnostic Tests Development for IBD ................................ ............... 133 Conclusions ................................ ................................ ................................ .......... 133 LIST OF REFERENC ES ................................ ................................ ............................. 138 BIOGRAPHICAL SKETCH ................................ ................................ .......................... 141

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10 LIST OF TABLES Table page 1 1 List of snake species IBD had been reported ................................ ..................... 34 3 1 The tissue processing procedure of two laboratories. ................................ ........ 91 3 2 IHC score of tissues processed by two laboratories using different AR treatments. ................................ ................................ ................................ .......... 91 3 3 Standardized IHC staining conditions for anti IBDP MAB ................................ ... 92 3 4 IHC scores of IBD positive tissues fixed in 10% NBF over different time period ................................ ................................ ................................ ................. 92 3 5 List of IBD positive non boa constrictors tested with anti IBDP MAB ................. 93 4 1 Summarized results of antigen detection in blood samples of three snake species ................................ ................................ ................................ ............. 113 4 2 Results of antigen detection in blood samples of three snake species ............. 114 4 3 Summarized results of H&E and Immuno tests ................................ ................ 115 4 4 Agreements between diagnosis by H&E stain and immuno based antigen detection tests in boa constrictors ................................ ................................ .... 115 4 5 Agreements between diagnosis by H&E stain and antibody detection test in boa constrictors ................................ ................................ ................................ 115

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11 LIST OF FIGURES Figure page 1 1 An IBD affected boa constrictor showing abnormal posture and sign of CNS disease. ................................ ................................ ................................ .............. 35 1 2 An IBD affected boa constrictor unable to right itself when placed in dorsal re cumbency. ................................ ................................ ................................ ....... 36 1 3 Photomicrograph of the H&E stained pancreas from an IBD affected boa constrictor with acinar cells containing eosinophilic in tracytoplasmic inclusion bodies ................................ ................................ ................................ ................. 37 1 4 Photomicrograph of the H&E stained liver from an IBD affected boa constrictor with hepatocytes containing eosinophilic in tracytoplasmic inclusion bodies ................................ ................................ ................................ .. 38 1 5 Photomicrograph of eosinophilic intracytopl asmic inclusion bodies in neurons and glial cells in the H&E stained brain of an IBD affected boa constrictor. ....... 39 1 6 Photomicrograph of amphophilic intracytoplasmic inclusion bodies in neurons of the H&E stained brain of an IBD affec ted boa constrictor. .............................. 40 1 7 Esophageal tonsils of a reticulated pyt hon. ................................ ....................... 41 1 8 Photomicrograph of H&E stained esophageal tonsil from an IBD affected boa constrictor, with numerous eosinophilic intracytop lasmic inclusion bodies withi n submucosal lympho id cells. ................................ ................................ ..... 42 1 9 Photomicrograph of a cytological impression of liver from an IBD affected boa constrictor. N umerous eosinophilic intracytoplasmic inclusion bodies can be seen in H &E stain. ................................ ................................ ........................ 43 1 10 Photomicrograph of cytological impressi on of liver from an IBD affected boa constrictor. ................................ ................................ ................................ .......... 44 1 11 A H&E stained peripheral blood film obtained from an IBD affected boa constri ctor with an erythrocyte and lymphocyte containing eosinophilic staining inclusion bodies. ................................ ................................ .................... 45 1 12 A H&E stained periphera l blood film obtained from an IBD affected boa constrictor with a lymphocyte containing an eosinophilic staining inclusion body ................................ ................................ ................................ ................... 46 1 13 Transmission electron photomicrograph of an enterocyte in the small intestine of an IBD affected boa constrictor. ................................ ....................... 47

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12 1 14 Transmission electron photomicrograph of an inclusion body within an enterocyte. ................................ ................................ ................................ .......... 48 1 15 Transmission electron photomicrograph of extracellular retroviral particles on the surface of primary kidney cells obtained from an IBD affected boa constrictor. ................................ ................................ ................................ .......... 49 1 16 S nake mite ( Ophionyssus natricis ). ................................ ................................ .... 50 2 1 The isolated inclusion bodies obtained from 3 g of IBD positive liver. ................ 66 2 2 The H&E stained IB prep on a microscopic slide. ................................ ............... 67 2 3 The resolved IB preps and liver homogenates on a NuPAGE. ........................... 68 2 4 Western blot showing the polyc lonal antibody reacted with the 68 KDa protein in the IB prep ................................ ................................ .......................... 69 2 5 Western blot showing presence of antibody that reacted with the 68 KDa protein in the cultured medium from hybr idoma mass culture 5B3 ................... 70 2 6 IHC staining of paraffin embedded pancre as from an IBD positive boa constrictor using polyclonal and monoclonal antibody. ................................ ....... 71 2 7 The collecting chamber of Model 422 Elec tro Eluter which attached inside of a tank that can be appli ed with electric current .. ................................ ................ 72 3 1 Pancreas of an IBD positive boa constrictor stained with standardized IHC condition. ................................ ................................ ................................ ............ 94 3 2 Mean IHC score of liver, kidney, and pancreas over fixation time in 10% NBF. ................................ ................................ ................................ ........................... 95 3 3 Differences in IHC staining between Trilogy and double AR treatment on prolong fixed tissues. ................................ ................................ .......................... 96 3 4 The performance characteristics and their 95% confident intervals of IHC test on paraffin embedded blocks of boa constrictors date from 1990 to 2011.. ....... 97 3 5 IHC staining of embedded tissue that strictly required double AR treatment. ..... 98 4 1 Cytospin preparation o f PWBC isolated from a boa constrictor. ....................... 116 4 2 Antigen detection by western blot using PWBC isolated from 8 boa constrictors. ................................ ................................ ................................ ...... 117 4 3 Western blots showing the reactivity of purified anti snake Ab to three plasma samples of boa constrictors. ................................ ................................ ............. 118

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13 4 4 Detection of antibody against IBDP within plasma of an IBD positive boa constrictor using western blots. ................................ ................................ ........ 119 4 5 The BSA standard curves with or without the background of LB measured by Bradford Protein Assay. ................................ ................................ .................... 120 4 6 Detection of antibody against IBDP within plasma of 4 IBD positive boa constrictors on Western blots. ................................ ................................ .......... 121 5 1 The total protein stain and western blot demonstrated a soluble form of IBDP that was im munoprecipitated ................................ ................................ ........... 134 5 2 The coverage of pepti des derived from IB preps over the predicted GGV NP sequence by MS/MS sequencing. ................................ ................................ .... 135 5 3 The coverage of peptides derived from immune precipitated IBDP over the predicted GGV NP sequence by MS/MS sequencing. ................................ ..... 136 5 4 The overall coverage of peptides derived from purified IBDP over the predicted GGV NP sequence by MS/MS sequencing. ................................ ..... 137

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14 LIST OF ABBREVIATION S 2 ME B eta mercaptoethanol 2 D Two dimensional a.a. A mino acids AB A mmonium bicarbonate ACN A cetonitrile AR A ntigen retrieval BF Blood film BSA B ovine serum albumin CASV California Academy of Sciences virus CI C onfident intervals CID C ollision induced dissociation CNS Central nervous system Co Collection CVV Collierville virus DAB D iaminobenzidine DDM D odecyl maltoside DMSO D imethyl sulfoxide DTT D ithiothreitol EDTA E thylenediaminetetraacetic acid ELISA E nyzyme linked immunosorbent assay EtOH E thanol FA F ormic acid g gram GGV Golden Gate virus

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15 Gu HCl G uanidine hydrochloride H&E H ematoxylin and eosin HB H omogenization buffer HEPES 4 (2 hydroxyethyl) 1 piperazineethanesulfonic acid HPLC High performance liquid chromatography HRP H orseradish peroxidase IACUC Institutional Animal Care and Use Committee IB prep I nclusion body preperation; semi purified inclusion bodies IBD Inclusion body disease IBD IBD negative IBD+ IBD positive IBDP Inclusion body disease protein ICBR Interdisciplinary Center for Biotechnology Research IDA I nformation dependent acquisition IgG Immunoglobulin G IHC I mmunohistochemical/immunohistochemistry IHC IHC stained negative IHC+ IHC stained positive Immuno Immuno tested negative Immuno+ Immuno tested positive IP Immunoprecipitation KDa K ilodalton L Li ter Lab1 L aboratory o f Anatomic Pathology Service, UF Veterinary Hospital Lab2 L aboratory of Molecul ar Pathology Core College of Medicine, UF

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16 LB L ysing buffer LC L iquid chromatography LDS L ithium dodecyl sulfate LSM L ymphocyte separtation media MAB M onoclonal antibody mL M ililiter mM M ilimolar mm M ilimeter MS/MS T andem mass spectrometry n S ample size N/A N ot available/ not applicable NBF N eutral buffered formalin NCBI National Center for Biotechnology Information Neg Negative nm nanometer NP N ucleoprotein nr N on redundant database, currently known as default OBG O ctyl beta glucoside OD O ptical densit y PAS P eriodic acid Schiff PBMC P eripheral blood mononuclear cells PBS P hosphate buffered saline PBST PBS containing 0.05% Tween 20 PCR P olymerase chain reaction PF P araformaldehyde

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17 PnPP para nitrophenyl phosphate substrate Pos Positive PTAH phosphotungsti c acid hematoxylin PWBC P eripheral white blood cells PyB1RV Python curtus endogenous retrovirus PyT2RV Python molurus endogenous retrovirus rpm R evolutions per minute RT R oom temperature RV Retrovirus SDS S odium dodecyl sulfate SDS PAGE S odium dodecyl sulfate polyacrylamide gel electrophoresis T Blue T oluidine blue TBS T ris buffered saline UF University of Florida g microgram L microliter m micrometer VH2 V iper ( Vipera russelli ) heart cell l ine WB Western blots

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18 Abstract of Dissertat ion Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy DEVELOPMENT OF MOLECULAR DIAGNOSTIC TESTS FOR INCLUSION BODY DISEASE IN BOID SNAKES By Li Wen Chang December 2012 Chair: Elliott R Jacobson Major: Veterinary Medical Sciences Inclusion Body Disease (IBD) is commonly seen in captive boa constrictors ( Boa constrictor ) and occasionally in other boid snakes. This disease is characterized by abnormal accumulation of an insoluble intracytoplasmic 68 KDa protein (IBDP). The exact causative agent (s) and pathogenesis remain unknown. Currently, diagnosis of IBD is based on the light microscopic identification of eosinophilic intracytoplasmic inclus ion bodies in hematoxylin and eosin (H&E) stained tissues or blood smears. Better ante mortem diagnostic tests are needed for screening IBD affected captive snakes, and preventing the introduction of IBD into wild populations In this study, a mouse anti IBDP monoclonal antibody (MAB) was produced against semi purified IBD inclusion bodies. Using immunohistochemical (IHC) staining, the anti IBDP MAB was validated by testing on a repository of IBD positive and negative paraffin e mbedded tissues collected from 1990 to 2011. In boa constrictors, the anti IBDP MAB had a sensitivity of 80% and specificity of 100% in detecting IBD. The antibody also cross reacted with IBD inclusion bodies in carpet pythons ( Morelia spilota ) and a ball python ( Python regius )

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19 An improved H&E staining method was developed and was used to stain the isolated peripheral white blood cells (PWBC) on microscopic slides. U sing anti IBDP MAB a ntigen detection of the isolated PWBC samples were performed by IHC staining and western blots. The presences of antibody against IBDP in the plasma of boa c onstrictors were also evaluated by utilizing a previously developed anti snake IgG antibody on western blot s A total of 78 blood samples from boa constrictors, rainbo w boas ( Epicrates cenchria ) and ball pythons were evaluated for the presence of IBDP using both H&E staining a nd IHC staining. The presence of IBDP antigen showed excellent agreement with the diagnosis of IBD. Plasma samples from IBD positive boa constric tors were negative for anti IBDP antibody. Finally, the IBD specific protein IBDP was sequenced using tandem mass spectrometry (MS/MS) The peptides of IBDP detected by MS/MS had a 90.5 % amino acid coverage of the predicted nucleoprotein of a recently di scovered arena like virus (NCBI Gene ID: 13466438) that was identified in IBD positive boa constrictors.

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20 CHAPTER 1 INTRODUCTION: INCLUS ION BODY DISEASE IN BOID SNAKE Review of the Literature Background Snakes make up approximately 19% of all reptiles maintained as pets. Of these, various boid snakes (members of the families Boidae and Pythonidae) are bred in large numbers for the pet trade. Although accurate numbers are not available, it is believed that several million of these snakes are classified as pets or maintained in breeding operations within the United States. Of illnesses affecting boid snakes, inclusion body disease (IBD) has surfaced as the most important worldwide disease, a condition charac terized by the formation of intracytoplasmic inclusion bodies. In Australia, where IBD has been identified in captive pythons and in other countries where boid snakes are being bred for release to the wild, there is concern that this disease will become es tablished in native wild populations. 1 History, Hosts, and Geographic Range In the 1970s, IBD was first identified in the United States, where it affected multiple species of boid snakes in private and zoological collections. When first recognized, Burm ese pythons ( Python bivittatus ) were the most common boid snake diagnosed with IBD. In 1998, IBD was reported in captive native carpet ( Morelia spilota variegata ) and diamond pythons ( M. spilota spilota ) in Australia, in captive boa constrictors in the Can ary Islands, Spain, and subsequently in Belgium. Beginning in the early 1990s, A p ortion of this chapter was reprinted from: Chang L, Jacobson ER. Inclusion body disease, a worldwide infectious disease of boid snakes: a review. Journal of Exotic Pet Medicine 2010; 19(3): 216 225, with permission from Elsevier.

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21 more cases of IBD were diagnosed in boa constrictors than pythons, but the cause of this epidemiologic shift is unknown. Additional species diagnosed with IBD include the green anaconda ( Eunectes murinus ), yellow anaconda ( Eunectes notaeus ), rainbow boa ( Epicrates cenchris ), Haitian boa ( Epicrates striatus ), Madagascan boa ( Acranthophis madagascariensis ), Indian python ( P. molurus molurus ), reticulated python ( P. reticulatus ), an d ball python ( P. regius ) (Table 1 1) In addition, a disease resembling IBD was diagnosed in an eastern king snake ( Lampropeltis getula ) that was housed with boa constrictors, and in a zoological collection of palm vipers ( Bothriechis marchi ). However, the correlation of the inclusion bodies in the king snake and viper cases to IBD has not been confirmed with molecular techniques or immunological reagents. 1 Clinical Signs Central nervous system (CNS) abnormalities. From the late 1970s and extending into the mid 1980s, Burmese pythons were the most common boid snake seen with IBD. Clinical signs of the disease in Burmese pythons primarily involved CNS abnormalities. 1 Signs of CNS disease observed in the IBD affected snakes were incoordination, disorientation, 2,3 head tremors, stargazing, 2 5 and opisthotonus (Figure1 1 and 1 2). 1, 2,4,5 The affected pythons with CNS disease often become anorectic, showing progressive loss of their motor function, posterior paresis and s ome had developed flaccid paralysis. 2,3 The neurologic disorders have been reported in Burmese pythons, green tree pythons ( Morelia viridis ), African rock pythons ( P. sebae ), 2 carpet python, diamond pythons, 3 boa constrictors, 2,4,5,6 and Haitian boas. 2 Th e sign of neurologic disorders were thought to be more pronounced in Burmese pythons than in boas. 2

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22 Non neurologic signs. Beginning in the early 1990s, more cases were diagnosed in boa constrictors in relation to Burmese and other pythons. Boa constrictors affected by IBD also regurgitated food items within several days of feeding, in addition to the CNS disease signs described for pythons. 1 In boas, the signs of CNS disease can be observed in some cases, but chronic intermittened regurgitation was the comm on first sign to be observed often before showing signs of the neurological disorder. 2,4,6 However, regurgitation was not a disease sign identified in Burmese pythons. 1 Some IBD affected boas progressively become more lethargic, and developed anorexia, chr onic weight loss, pneumonia, sever stomatitis, and lymphoproliferative disorders. 2,4,6 Subclinical infection. Although some snakes die within several weeks of first manifesting illness, others may survive for months. 1 Affected snakes may also appear healt hy, and not showing any clinical signs. 4,5 Four experimentally infected boa constrictors that were observed up to 52 weeks developed inclusion bodies within 10 weeks post infection, and none of infected boas showed clinical signs of IBD. 4 Those that showe d clinical signs may survive from several weeks to more than a year. 2 It is not known what percentage of IBD infected snakes will develop clinical signs, and how many of them will remain clinically normal. 1 Pathology and Disease Visceral tissues. The gross tissues of affected snakes may appear normal without observable lesion s or in some cases organs including spleen, pancreas, and thymus may atrophy 2 Using light microscopy eosinophilic to amphoph ilic intracytoplasmic inclusion bodies in hemato xylin and eosin (H&E) stain were identified in one or more visceral epithelial cells. 2 The inclusion bodies were commonly seen in pancreatic acinar cells (Figure 1 3), renal proximal and distal tubular epithelial cells, and

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23 hepatocytes (Figure 1 4). 1,2,3,5 Inclusion bodies were also observed in spleen, cardiomyocytes, 6 thyroid follicular cells, pars intermedia of adenohypophysis, thymic epithelial cells, thymocytes, gastric epithelial cells, 2 lining epithelial cells of the air passage, lymphoid cells in the esophageal tonsils and circulating lymphocytes. 1 3 The size of inclusion bodies can vary from 1 4 m 2 2 10 m 3 5 20 m 5 in diameter. S ome affected snakes ma y have numerous large inclusion bodies found in almost all organs, others may have fewer or smal ler inclusion bodies only in focal areas, 2 such as the CNS. 1 T he inclusion body bearing cells often have accompanying cytoplasmic vocuolation 2,4,6 Lymphoid depletion 2,3 and fibrosis were also commonly seen in IBD affected snakes. In some chronically infected boas, there were decreased number s of lymphoreticular cel ls and loss of splenic follicles 2 Nervous system. Eosinoph ilic to amphophilic intracytoplasmic inclusion bo dies were most commonly observed in neurons (Figure 1 5 and 1 6), especially in the hindbrain and the ependymal cells of infected snakes. 1 3 In clusion bodies were also found in the spinal cor d of infected snakes, with in the degenerating neurons of the gre y matter. 2 Segmental swelling of myelin sheaths and axons were observed in the proximal spinal cord of an affected carpet python. 3 Other lesions found in the CNS of affected snakes including, neuronal degeneration, gliosis, and demyelinization. 2,3 Of IBD i nfected sn akes with signs of CNS disease nonsu ppurative meningoencephalitis was commonly found with a corresponding infiltrate of lymphocyte s 2 In one IBD case of a carpet python, spongiform change of grey matter with occ asional mild multifocal gliosis an d perivescular lymphoplasmacytic cuffing were described. 3 Whereas a more pronounced inflammatory response is often seen in the CNS of Burmese pythons the response is

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24 only minimal to mild in boa constrictors. 2 However, the inclusion bodies were more numero us in the nervous tissue of boa constrictors than in pythons, although the inflammatory resp onse in pythons were greater. 2 Blood. Myeloid cells in the bone marrow of IBD infected snake often co ntain the cytoplasmic inclusion bodies 5 T he inclusion bodies have also been found in lymphocytes, 1,4 erythrocytes, and heterophils of IBD infected snakes. 1 In a survey of 13 IBD infected boa constrictors, the boas that showed signs of IBD for less than 2 months had lymphocytosis, whereas boas that showed signs over 2 months had lymphopenia. 2 Hematological and selected biochemical analyte values of acutely affected boa constrictors diagnosed with IBD included leukocytosis, relative lymphocytosis, lower total protein and globulin values, and significantly higher asparta te transaminase values compared with those of chronically affected snakes. 1 Current Diagnostic Methods Postmortem diagnosis. A postmortem diagnosis of IBD is based on the light microscopic identification of variably sized eosinophilic to amphophilic intra cytoplasmic inclusion bodies in H&E stained tissue sections. The tinctorial characteristics of the inclusion bodies may vary with the type of hematoxylin used and differences in staining methods. 1 B rain, liver, pancreas, and kidneys were the most commonly collected tissue s for making a diagnosis of IBD 1 6 Among snakes with IBD, the number of inclusion bodies seen in tissue may be quite variable. Whereas, some may have inclusion bodies in most epithelial cells, others m ay have very few inclusion bodies 6 In some snakes, in clusion bodies were only found in the brain. 1 A diagnosis of IBD is made when the characteristic inclusion bodies are detected. However, absence of inclusion bodies may not indicate that the snake is free of IBD. 5, 6

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25 Antemortem diagnosis. An antemortem diagnosis is made by demonstrating eosinophilic to amphophilic intracytoplasmic inclusion bodies in histologically processed and H& E stained biopsyspecimens. Boid snakes have well developed esophageal tonsils (Figure 1 7), and in snakes with IBD the tonsils may contain lymphoid cells or mucous epithelial cells with intracytoplasmic inclusion bodies (Figure 1 8). Using a flexible endos cope with a biopsy device, esophageal tonsils are easily biopsied, fixed, and routinely processed for light microscopy. Liver and kidney biopsy specimens can also be obtained for histological evaluation. For a more rapid diagnosis, cytological impression s mears of the liver and renal biopsy samples can be stained with H&E (Figure 1 9) and/or Wright Giemsa (Figure 1 10). 1 The inclusion bodies are easier to identify in H&E stained preparations. 1 Some snakes may have very few inclusion bodies that may be misse d in a biopsy specimen. 1 Thus, failure to identify inclusion bodies in the sampled tissue cannot completely rule out the snake as IBD positive. Inclusion bodies may be seen in erythrocytes (Figure 1 11), lymphocytes (Figure 1 11 and 1 12), and heterophils (Figure 1 13) in peripheral blood films of snakes with IBD. 1 Preparation of blood smears or buffy coats stained with H&E stain or Wright Geimsa stain had been used for inclusion body identification. 1 ,5 The inclusion bodies are more easily identified on bl ood films using H&E stain, compared to the Wright Geimsa stained inclusion bodies which is stain basophilic (light blue). 1 Using blood samples for examination is a relatively inexpensive method in making an antemortem diagnosis of IBD. 1 However, there is a lack of sufficient data to know whether all IBD snakes will develop inclusion bodies in peripheral blood cells, 1 or how long after infection will the inclusion bodies appear in the peripheral blood cells. Therefore, the

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26 absence of incl usion bodies in blood cells does not necessarily indicate that the snake is IBD negative. IBD Antigenic Protein Wozniak et al. demonstrated that IBD inclusion bodies were phosphotungstic acid hematoxylin (PTAH) positive, orthochromatic with toludine blue (T blue), periodic acid Schiff (PAS) negative and eosinophilic in H&E stain ed paraffin embedded tissue sections. 4 These profiles suggested that the materials within the inclusion bodies are proteinaceous. 4 The i nclusion bodies were found to consist of a n antigenically distinct 68KDa protein named IBD inclusion protein (IBDP). 4 The protein was semi purified b y protein electrophoresis of a liver homogenate, and electro eluted from the gel bands. 4 Monoclonal antibodies (MAB) against IBDP have been produce d that recognize an IBDP band in a western blot and IBDP antigen in frozen tissue sections using immunohistochemical (IHC) staining. 1 Using transmission electron microscopy, intracytoplasmic inclusion bodies identified within nerve cells in the CNS and vi sceral epithelial cells begin as polyribosome derived clusters of s mall round subunits (Figure 1 13 ). Inclusion bodies enlarge as additional subunits are deposited on the periphery of in dividual inclusion bodies (Figure 1 14 ). In some sections the inclusio n bodies have concentric profiles, with subunits observed on the surface. 1 Wozinak et al. demo nstrated the smaller inclusion bodies (< 2 m in diameter) were composed of none membrane limited intracytoplasmic aggregates of electron dense materials. The la rger inclusion bodies (3 6 m in diameter) appeared as membrane bound electron dense aggregates mixed with membrane like fragments. 4

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27 Possible Causative Agents of IBD and Transmission Retroviruses. I n the early 1990s, using transmission electron microscopy, Schumacher et al. identified 110 nm enveloped viral like particles in 2 of 17 boa constrictors having IBD affected s nakes by transmission e lectron microscopic examination. 2 The infected organs included brain kidney, and pancreas. 2 In this study, the virus was successfully isolated in cultured primary kidney cells obtained from the IBD affected snakes. 2 Further, two healthy Burmese pythons inoculated with th e cell free culture supernatant developed neurologic signs, nonsuppurative en cephalitis, and small inclusion bodies in the brain and pancreas that were visible under electron microscopy at 4 to 10 w ee k post inoculation 2 However, no viral particles were found in the inoculated snakes, and viral isolation a ttempt from the inoculated snakes were 2 In the late 1990s Wozniak et al. successfully infected healthy boas by inoculating them with 0.45 m filtered liver homogenates obtained from an I BD affected boa constrictor. V iral like particles that morphologically resembled t he IBD associated retrovirus were observed in all IBD positive livers from inoculated snakes, but not in the IBD negative snakes. 4 Subsequently, in another study, three viral isolates were partially characterized from two IBD affecte d boa constrictors and one clinically healthy Madagascan ground boa ( Acranthophis dumereli ) that was housed with IBD infected snakes. 6 The viruses were isolated by co culturing primary l ymphocytes with commercial viper heart (VH2) cells (named RV1), or by culturing primary kidney cells (named RV2 and RV3 ). 6 Based on size (80 110 nm) and morphology, the virus resembled C type retroviruses (Figure 1 15 ). 1 Reverse transcriptase activity was

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28 measured in infected cell cultures, and high activity levels were further evidence that the isolated viruses were retroviruses. 1 However, the VH 2 cells that were infected with isolated retrovirus did not form inclusion bodies. 1 The association between the identified retroviruses and IBD remained unclear, and the retroviruses were not sequenced. In the early 2000s, Huder et al. completely sequenced an actively expressing endogenous retrovirus (named PyT2RV) from Burmese pythons. 7 Using polymerase chain rea ction (PCR) techniques, a survey of the virus within peripheral blood mononuclear cells (PBMC) obtained from various snake species was conducted. 7 The viral sequence of PyT2RV was detected in all (18 out of 18) Burmese pythons whether or not they were show ing signs of IBD. 7 A closely related endogenous retrovirus (named PyB1RV) was discovered in blood pythons ( P. curtus ), but not in five other species that were tested. 7 Interestingly, the presences of PyT2RV sequences were not correlated with the presences of IBD. 7 Boa constrictors having IBD were negative for PyT2RV, and the isolated lymphocytes from boa constrictors were not susceptible to PyT2RV infection in vitro. 7 Using transmission electron microscopy, all attempts to visualize PyT2RV were unsuccessfu l. 7 Therefore, there was no evidence that the sequenced endogenous retroviruses was the causative agents of IBD 7 Relationship between retroviruses and IBD. Although retroviruses have been observed in inclusion bearing tissues by electron microscopy, many hours of searching are needed to locate mature retroviral particles. 1 For a number of IBD cases, neither viral particles nor reverse transcriptase activity have been demonstrated in tissues of affected snakes. 1 In one study young Burmese pythons that were inoculated with the supernatant of primary cultured kidney cells taken from an infected boa constrictor,

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29 developed clinical signs and microscopic lesions compatible with IBD. 2 In a second study, boa constrictor s that were administered filtered liver homogenate obtained from an IBD infected boa eventually developed intracytoplasmic inclusion bodies in hepatocytes. 4 Because purified virus was not used in these studies, it is impossible to implicate a retrovirus as the underlying etiology of IBD in the inoculated snakes. 1 Further, the presence of the inclusion bodies did not always co exist with the presence of the retrovirus within affected cells. 4 IBD may represent a protein storage disease induced by viral infect ion, or the protein itself may be behaving in a manner similar to that of a prion like disease. The protein and the isolated viruses must be sequenced to gain a better understanding of a possible causal relationship. 1 Transmission route and disease prevent ion. The route of transmission of IBD between snakes has not been determined, although it is believed that direct contact is involved. Because the snake mite ( Ophionyssus natricis ) (Figure 1 16 ) is present in many snake colonies experiencing an IBD outbrea k, mites may be involved in the transmission of the infectious agent. Thus, preventing mites from entering a collection and eliminating established infestations are essential components of a preventive medicine program. It is also possible the causative ag ent is passed through vertical transmission from mother to young in both egg laying and live bearing snakes. 1 Treatment and Prognosis There are no effective treatments for snakes infected with IBD. 2,5 The infected snakes that are showing clinical illness are always fatal, although the general condition can sometimes improve with force feeding and rehydration. 5 Euthanasia of the affected snake is recommended to reduce the risk of spreading the disease to other snakes in the collection. 2,5

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30 Recent Findings C oncurrent to this study, Stenglein et al. discovered three different arenavirus like viruses within IBD infected snakes by using deep sequencing method ology and the results were published in 2012 8 The virus found in annulated tree boas ( Corallus annulatus ) was named California Academy of Sciences virus (CASV), the other two viruses found in boa constrictors were named Golden Gate virus (GGV) and Collierville virus (CVV). 8 Using fluorescence IHC staining, a polyclonal antibody produced against the predicted peptide at the C terminal of GGV nucleoprotein detected IBD inclusion bodies within the paraffin embedded tissue sections. 8 The arenavirus like viruses were considered the candidate etiolo gical agents for IBD. 8 However, under electron microscopic examination of tissues from numerous IBD snakes, the mature arenavirus like virus were not seen in the inclusion body bearing tissues. The morphology of the arenavirus like viruses remained unknown It is unclear whether IBD is caused by the arenavirus like viruses alone? Or are other factors involved in the development of IBD? Research Goals The Needs of Molecular Diagnostic Tests Currently, a diagnosis of IBD is made by identifying H&E stained ch aracteristic eosinophilic to amphophilic intracytoplasmic inclusion bodies in the histologically processed tissues. 1 However, some IBD cases have only very few inclusion bodies or the inclusion bodies are very small and easily overlooked during microscopic examination. 1,2 Additionally it may be histologically difficult to differentiate IBD inclusion bodies from other intracytoplasmic protein accumulations. Therefore, a more sensitive and more specific molecular diagnostic test is need ed for a more specific diagnosis of IBD.

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31 Since infected snake may remain subcl inical for long periods of times, 4 a more specific and sensitive test is needed to screen snakes for IBD. A screening test is especially needed for the large collections in zoos, breeding collections, and those boid snakes kept as pets. Furthermore, a screening test is also needed for assessing the disease prevalence, or for determining pathogenesis in transmission studies. Therefore, a molecular diagnostic test using blood samples will be the ideal fo rmat for screening IBD affected snakes. A MAB was produced against IBDP and was capable of detecting IBD inclusion bodies in frozen tissue sections using IHC staining 4 Unfortunately, the original MAB producing clone was lost. 1 It is possible that a IBD P specific MAB can be reproduced and utilized in developing immuno based diagnostic tests. Better Understand the Relationship Between IBD and IBD P The key to understand the relationship between IBD and IBDP reside s in the sequence of IBDP. By obtaining the amino acid sequence of IBDP which can be back translated into mRNA sequences and DNA genomic sequences. Using the genomic sequence codes for IBDP, which will allowed veterinarians and researchers to unders tand the origin of this protein: whether it is a protein derived from foreig n organism? O r it is an overproduced self derivate protein being induced by an infection? O r the protein itself may be an infectious agent? In addition synthetic peptides of IBDP can be produced for developing immunoassays that requires large amount of consistent antigens, or for develop ing a better monoclo nal antibody Thus, sequencing IBDP is the key towa rd better understanding the disease mechanism and transmission of IBD. This will a lso provide better diagnostic tests and preventive programs for IBD.

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32 Hypothesis and Objectives Hypothesis The overall goal of this research was to develop better diagnostic tests for identifying IBD in boid snakes, and to further characterize the relations hip between IBDP and IBD. The central hypothesis for this research was : Based on the presence of antigenic novel protein (IBDP) that is present in IBD positive snakes, a monoclonal antibody can be produced against IBDP and be used for immunodiagnostic assa y developments. To better understand the nature of IBDP, this protein needs to be sequenced. In order to accomplish these goals, four specific research objectives were established: Objective 1. Produce MAB against semi purified IBDP. Objective 2. Validate the mouse MAB against IBDP for immunodiagnostic assay development. Objective 3. Assess whether MAB can detect circulating IBDP in the peripheral white blood cells (PWBC) and if measurable circulating antibody against IBDP can be detected in plasma of IBD i nfected snakes. Objective 4. Partially sequence the 68 KDa IBDP. Objectives 1. Produce MAB against semi purified IBDP. Isolation of the intracytoplasmic inclusion bodies from liver homogenates obtained from an IBD positive snake. Production of the anti IB DP antibodies by immunizing 2 mice with the semi purified preparation, and screen the monoclonal antibodies with the semi purified preparations. Eventually, develop a monoclonal antibody that recognizes the intracytoplasmic inclusion bodies under IHC stain ing, enyzyme linked immunosorbent assay (ELISA), and the 68 KDa IBDP band on western blots.

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33 2. Validate the mouse MAB against IBDP for immunodiagnostic assay development Paraffin embedded tissues from a repository of IBD positive or negative cases dated from 1990 to 2011 were tested using IHC staining. The performances of the MAB were evaluated in the following categories: a. Effects of prolonged formalin fixation; b. Effects of storage time in paraffin. c. Species cross reactivity; d. Antigen cross react ivity; e. IHC test diagnostic performance. 3. Assess whether MAB detect s circulating IBDP in the peripheral white blood cells (PWBC) and if measurable circulating antibody against IBDP can be detected in plasma of IBD infected snakes. Whole blood samples w ere obtained from three species of snakes including, boa constrictor, rainbow boa, and ball python. The snakes came from private collections with unkn own prevalence of IBD. The PWBC were sepa rated from the plasma, and were tested with H& E stain, IHC stain, and western blots respectively, to determine whether IBDP can be detected by an ti IBDP MAB in circulating PWBC The plasma of each samples were subjected for antibody detection using western blot to determine whether there are measurabl e antibodies produced against IBDP by the IBD infected snakes. 4. Sequencing IBDP. T he IBDP was purified by immunoprecipitation and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE). Digest t he collected 68 KDa protein band s with try psin, chymotrypsin, or Asp N. Analyze digested peptides by tendon mass spectrometry (MS/MS), and search t he MS/MS raw spectrums against known protein databases.

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34 T able 1 1. List of snake species IBD had been reported Family Scientific names Species c ommon names Boidae Acrantophis madagascariensis Madagascan ground boa 2 Boa constrictor Boa constrictor 2,4,5,6 ,8 Corallus annulatus Annulated tree boa 8 Epicrates cenchris Rainbow boa 2 Epicrates striatus Haitian boa 2 Eunectes murinus Green anaconda 2 Eunectes notaeus Yellow anaconda 2 Pythoninae Morelia spilota spilota Diamond pythons 3 Morelia spilota variegata Carpet pythons 3 Morelia viridis Green tree python 2 Python bivittatus Burmese python 2 Python curtus Blood python 2 Python molurus molurus Indian python 2 Python reticulatus Reticulated python 2 Python regius Ball python 2 Python sebae Afican rock python 2 Colubridae Lampropeltis getula Eastern king snake 1 Viperidae Bothriechis marchi Palm vipers 1

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35 Figure 1 1. An IBD affected b oa constrictor showing abnormal posture and sign of CNS disease. 1

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36 Figure 1 2. An IBD affected boa constrictor unable to right itself w h en placed in dorsal recumbency Courtesy of CRC Press. 1

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37 Figure 1 3. Photomicrograph of the H&E stained pancreas from an IBD affected boa constrictor with acinar cells containing eosinophil ic intracytoplasmic inclusion bodies. Courtesy of CRC Press. 1

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38 Figure 1 4. Photo micrograph of the H&E stained liver from an IBD affected boa constrictor with hepatocytes containing eosinophi lic intracytoplasmic inclusion bodies (arrows). Courtesy of CRC Press. 1

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39 Figure 1 5. Photomicrograph of eosinophilic intracytoplasmic inclusion bodies (arrows) in neurons and glial cells in the H&E stained brain of an IBD affected boa constrictor. 1

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40 Figure 1 6. Photomicrograph of amphophilic intracytoplasmic inclusion bodies in n eurons of the H&E stained brain of an IBD affected boa constrictor. Courtesy of Nikos Gurfield and CRC Press. 1

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41 Figure 1 7. Esophage al tonsils of a r eticulated python. Esophageal tonsils (arrows) are raised ovoid structures with a central cleft and covere d by a mucous epithelium. Courtesy of CRC Press. 1

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42 Figure 1 8. P hotomicrograph of H&E stained esophageal tonsil from an IBD affect ed boa constrictor, with numerous eosinophilic intracytoplasmic inclusion bodies (arr ows) within submucosal lymphoid cells. Courtesy of CRC Press. 1

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43 Figure 1 9. P hotomicrograph of a cytological impression of liver from an IBD affected boa constrictor N umerous eosinophi lic intracytoplasmic inclusion bodies can be seen in H&E stain. Courtesy of CRC Press. 1

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44 Figure 1 10. Photomicrograph of cytological impression of liver from an IBD affected boa constrictor Numerous basophi lic intracytoplasmic inclusion bodies (arrows) can be seen in Wright Giemsa stain. 1

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45 Figure 1 11. A H&E stained peripheral blood film obtained from an IBD affected boa constrictor with an erythrocyte (arrow) and lymphocyte (arro whead) containing eosinophilic staining inclus ion bodies 1

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46 Figure 1 12. A H&E stain ed p eripher al blood film obtained from an IBD affected boa constrictor with a lymphocyte containing an eosinophilic staining i nclusion body (arrow). 1

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47 Figure 1 13 Transm ission electron photomicrograph of an ente rocyte in the small intestine of an IBD affected boa constrictor During the initial stage of inclusion body formation, protein subunits from polyribosomes sta rt accumulating in the adjacent cytoplasm. Uranyl acetate and lead citrate stain. Courtesy of CRC Press. 1

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48 Figure 1 14 Transm ission electron photomicrograph of an inclusion body with in an enterocyte. De posited protein subunits have a virus like appearance. Tissue obtained from an IBD affected boa constrictor with u ranyl acetate and lead citrate st ain. Courtesy of CRC Press. 1

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49 Figure 1 15 Transm ission electron photomicrograph of extracellular retroviral particles on the surface of p rimary kidney cells obtained from an IBD affected boa constrictor. Uranyl acetate and lead citrate. Courtesy of CRC Press. 1

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50 Figure 1 16 Snake mite ( Ophionyssus natricis ). Photomicrograph of a mite removed from a snake. A single egg can be seen within the mite. Courtesy of CRC Press. 1

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51 CHAPTER 2 THE PRODUCTION OF AN TI IBDP MONOCLONAL ANTIBODY Introduction In boa constrictors with inclusion body disease (IBD) Wozniak et al. 1 identified a novel antigenically distinct protein within the characteri stic intracytoplasmic inclusion bodies The protein was approximately 68 KDa in molecular w eight, and named as inclusion body protein (IBDP). A monoclonal antibody (MAB) was produced against the electro eluted protein of the excised 68 KDa band obtained from sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE). 4 The MAB was used for immunohistochemical (IHC) staining of the IBD inclusion bodies in frozen liver sections. 4 An antibody recognizing IBDP can be used as a powerful tool for developing molecular or immunobased IBD diagnostic tests. Unfortunately, the hybridoma clone from this study was inactivated after transportation, and the MAB was lost (Wozniak, personal communication). In the study of Wozniazk et al. the immunogen (IBDP) was not considered to be a highly purified protein since it was obtained from resolved crude liver homogenates on SDS PAGE. 4 Further, the MAB would only react in sections of frozen material and n ot react with the I BD inclusion bodies in paraffin embedded tissues. 4 This limited its application for IBDP detection. In order to reproduce an anti IBDP antibody with broader application, a more purified inclusion body preparation was used a s the immunogen in developing a M AB. Immunohistochemical staining of IBD inclusion bodies in paraffin embedded tissues was one of the criteria for hybridoma clone selection. The inclusion A p ortion of this chapter was reprinted from: Chang L, Jacobson ER. Inclusion body disea se, a worldwide infectious disease of boid snakes: a review. Journal of Exotic Pet Medicine 2010; 19(3): 216 225, with permission from Elsevier.

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52 body purification procedure was modified from an unpublished project report of a research (Zachary Bissell 2004 ), which was adapted from a method for isolation of Mallory bodies. 9 The entire antibody production procedure was performed in the Hybridoma and Protein Core Laboratories, University of Florida Interdisciplinary Center for B iotechnology Research (ICBR), using the laboratory standard procedure s with necessary modifications due to the insolubility of IBDP. Materials and Methods IBDP Purification Tissue homogenization. Unfixed liver and kidney samples obtained from IBD positive or negative boa constrictors were stor ed in an ultrafreezer at 80C (IACUC approval # 201101156 ). Approximately, 5 g of liver was thawed, and cut into small portions (approximately 0.3 mm cubes) and homogenized in a Dounce tissue homogenizer with homogeniz ation buffer (HB) containing, 250 mM sucrose, 10 mM EDTA, 10 mM HEPES, pH 7.4. The tissue was processed 1 2 g at a time with 5 mL HB, and the liver homogenate was collected and filtered through two layers of gauze, to remove the larger tissue debris. The u nfiltered portion (retentate) was washed with additional 2 mL of HB. One milliliter of the filtered liver homogenate (diffusate) was collected as sample 1, and the remaining homogenate was centrifuged at 1000 x g for 10 minutes. The supernatant was separated from the soft pellet, and subjected to another round of ce ntrifugation using the same conditions as previously described. The supernatant was collected as sample 2, and soft pellets obtained from the first and second centrifugation was pooled together.

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53 Isolation of the inclusion bodies. The pooled soft pellet was resuspended with equal volume of HB, 1 mL of which was collected as sample 3. The remaining suspension was mixed with 1% sarkosyl ( Teknova 2S3380 ) at a 1:1 ratio, and incubated in 37 C for 30 minute s with frequent vortexing. The suspension was centrifuge d at 14,000 x g for 10 minutes in 4 C and the supernatant was collected as sample 4. The remaining pellet was resuspended with equal volume of 1% sarkosyl, and 500 L of which was collected as sample 5. The remaining suspension was subjected to one more r ound of the above procedure, with incubation followed by centrifugation. The supernatant was removed, and collected as sample 6. The pellet was carefully resuspended in 1 mL of HB, and will refer to hereafter as inclusion body preparation (IB prep). Ten microli ters of IB prep was collected as sample 7, the remaining preparation was stored at 4 C for future analysis. Cytospin preparation and hemotoxylin and eosin (H&E) stain. Samples 1 to 7 were diluted 50 fold with water, and 50 L of each diluted sample was placed in a cytocentrifuge chamber (Biomedical Polymers Inc., BMP CYTO S50), and centrifuged for 6 minutes at 800 rpm onto a glass microscopic slide using a cytospin centrifuge (Shandon, Cytospin 2). The slides were air dried, fixed in 10% neutral buf fered formalin (NBF; Fisher Scientific, SF100 20) for 10 minutes, washed with distilled water, and stained with H&E stain. The standard cytological H&E staining was performed using an automatic slide stainer (Thermo Shandon, Gemini Varistain), which staine d slides as follows: 1) hemotoxylin (Richard Allan Scientific, 7212) for 2 minutes; 2) incubation with clarifier 2 (Richard Allan Scientific, 7402) for 15 seconds; 3) followed by incubating with bluing reagent (Richard Allan Scientific, 7301) for 1 minute, and 4) staining with eosin

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54 (Richard Allan Scientific, 71311) for 1 minute. In between application of each reagent, the slides were washed with distilled water. Finally, the slides were dehydrated in graded ethanol, dipped i n xylene, and coverslipped by pe rmount. Electrophoresis and gel staining. Equal volume of 2X loading sample buffer (25 mM Tris, 4% SDS, 100mM DTT, and 30% glycerol) was added into aliquots of IB preps or crude liver homogenates, and heated at 95C for 10 minutes. Then the protein concent rations were estimated by standard protocol of Bradford P rotein A ssay ( Bio Rad Laboratories, Inc. ), using 1:100 dilution of the reduced IB prep. Five micrograms of IB preps and crude liver homogenates were mixed with loading dye, and resolved on a 4% 12% NuPAGE Bis Tris gel (Novex) with MES buffer (Novex) at 200V constant voltage. The resolved protein was visualized by SimplyBlue (Novex) stain, using the microwave protocol provided by the manufacturer (Novex). Electro e lution of IBDP. The resolv ed 68KDa IB DP bands were cut from the gels and packed inside of the glass tubes of the Model 422 Electro eluter (Bio Rad, 165 2976). The protein was electro eluted in Tris Glycine buffer without SDS, following the protocol provided by the manufacturer (Bio Rad). Prot ein s olubilization. Several solubilizing reagents were used alone or in combination in an attempt to solubilize the isolated insoluble inclusion bodies (IB preps). The tested solubilizing reagents included, 8 12 M urea, 6 M guanidine hydrochloride (Gu HCl) 1% Triton 100, 2% octyl beta glucoside (OBG), 1% dodecyl maltoside (DDM), 2 4% SDS, 20% lithium dodecyl sulfate (LDS), 1M DTT, dimethyl sulfoxide (DMSO), bicarbonate buffer and 1% acetic acid. Thirty microliters of IB prep was placed in a 1.5 mL tube, an d centrifuged at 12,000 rpm (15,294 x g) for 20 minutes in 4 C using an

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55 Eppendo rf 15 Amp Centrifuge Model 5810R ( Eppendorf North America Hauppauge New York ) After centrifugation, the supernatant was removed, and the pellet was resuspended by 30 L of a solubilizing reagent. After thorough vortexing the tube was maintained at room temperature (RT) for 30 minutes, followed by observation to see if any inclusion bodies precipitated at the bottom of the tube. Subsequently, the tube with or without precipita tion observed was being centrifuged again at 12,000 rpm for 20 minutes. After centrifugation, precipitation of inclusion bodies within the sample that form a pellet was visually confirmed. If the inclusion bodies were solubilized by the reagent, no visible pellet should be seen. If the inclusion bodies were not solubilized by the reagent, a pellet should be seen, and the size of the pellet should be equivalent to the untreated sample. If the portion of the inclusion bodies were solubilized by the reagent, t he pellet should appear smaller than the pellet of the untreated sample. Polyclonal and Monoclonal Anti IBDP Antibody Production Mouse immunization. Two female Balb/cByj mice were immunized with approximately 100 g of isolated inclusion bodies (IB p rep of #08 76) diluted in sterile physiologic phosphate buffered saline (PBS) and emulsified in Ribi MPL+TDM adjuvant. The immunogen was administered subcutaneously with a volume of 0.05 mL at both ventral groin sites and 0.1 mL on their dorsal midline on day 1, 21, 44, and 192. The test bleeds were collected 11 to 14 days after the second and third immunizations. Blood samples of 100 L or less were collected from the tail vein with the mice under a surgical plane of isoflurane anesthesia. The presence of anti IBDP antibodies in the post immunized serum were determined by western blots, eny zyme linked immunosorbent assay ( ELISA ) and IHC staining.

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56 Hybridoma cell production and screening. Six days after the fourth immunization mou se #1 was anesthetized with isoflurane and was exanguinated from the medial canthus of an eye. The mouse was subsequently euthanized using cervical dislocation. The spleen was removed, the ventral margin of the splenic capsule was incised and the B lymphoc yte rich, round cell constituents were flushed from the stroma with sterile serum free media. Splenic lymphocytes were fused with log phase Sp2/0 mouse myeloma cells with polyethylene glycol (PEG, 1500) at a ratio of 7:1. All resulting hybridomas were susp 11965118) with 1x HAT selective media (Sigma, H0262 10VL) supplemented with 25% sp2/0 conditioned media and 20% fusion tested horse serum (Sigma, H1270). Next they were dispensed into 96 well plates at a concentration of 2 x 10 5 cells per well and grown undisturbed for 5 to 7 days. Two feedings were done by replacing 50% of the cultured media with fresh media before sampling for antibody screening. The cultured media of the growing hybridoma mass cultur es were collected and screened for anti IBDP antibody production by ELISA. The wells that tested positive were transferred to 24 well plates. Western blot. Aliquots of IB preps were reduced by addition of 4X NuPAGE LDS sample buffer (Invitrogen, NP0008) an d 10X NuPAGE reducing agent (Invitrogen, NP0004) with 500 mM DTT, followed by heating over 95C for 10 minutes. The reduced IBDP was resolved on a 10% or 4%~12% NuPAGE Bis Tris gel (Invitrogen), with MOPS running buffer (Invitrogen) at 200V constant voltag e. The resolved protein was transblotted onto a nitrocellulose membrane using standard protocol of the iBlot dry blotting system (Invitrogen). The membrane was blocked overnight at 4C with 5% non

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57 fat dry milk dissolved in PBS, and subsequently washed thre e times the following day. Each wash was done by incubating the membrane with PBS containing 0.05% Tween 20 (PBST) on an automated rocker for 5 minutes. Each lane of the membrane was separated by a Fast Blot Developer manifold (Pierce, 88040) and incubated with serum of mouse #1 or mouse #2 in a dilution of 1:100 or 1:500 for 1 hour. For hybridoma mass culture and clone screening, each lane was incubated with undiluted cultured medium collected from each hybridoma clone. The blot was washed three times, and incubated with conjugated rabbit anti mouse antibody (Sigma, A1902) in a dilution of 1:1,000 for one hour. The blot was washed three times, followed by application of BCIP/NBT alkaline phosphatase substrate (Sigma, B5655), and developed for 3 to10 minutes until the color of the reacting band reached desired intensity. The blot was then rinsed with water and air dried. ELISA. Flat bottom 96 well assay plates were coated with IB prep that was diluted in optimal concentrations (10, 20, 30, 40 g/ mL ) with bica rbonate buffer. The IB preps isolated from liver and kidney of 2 different IBD positive snakes (#08 76, #08 122) were used as coating antigens on separate plates. The plates were loaded with 50 L diluted antigen per well, sealed, and incubated overnight a t 4C, followed by a wash procedure with PBST. The wash procedure was performed using a microplate washer (Biotek, ELx405 Select CW). Each well was filled and aspirated four times with 300 L wash buffer. The plate was blotted on a paper towel to remove t he r esidue buffer. Each coated well was blocked with 250 L of 1% bovine serum albumin (BSA) in PBS incubated overnight at 4C, and washed the following day. Fifty micr olit ers of diluted mouse serum or undiluted cultured medium of the hybridoma cells was a pplied to each

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58 well, and incubated for 1 hour at RT The plate was washed, incubated with 50 L per well of a 1:1000 dilution of alkaline phosphate conjugated rabbit anti mouse IgG antibody (Sigma A, 1902) for 1 hour at RT. After being washed again, 200 L of para nitrophenyl phosphate substrate (PnPP; Sigma, N2765) was added into each well and developed for 1 hour at RT. The reaction was stopped with 50 L of 3M NaOH. The direct optical density (OD) values of each well were recorded using a Spectramax Plus 384 plate reader (Molecular Devices) with the absorbance setting at 405 nm. The sample that had the highest OD reading on four different IB preps were further tested for reactivity to the 68 KDa IBDP by western blot, and by its reactivity to IBD inclusion bodies in tissues using IHC staining. IHC staining. Sections of paraffin embedded tissue on charged glass microscopic slides were deparaffinized in xylene, followed by rehydrating in graded ethanol, and finally rinsed with water. The slides were either tr eated or not treated with antigen retrieval (AR) procedure. The antigen in the embedded tissue was retrieved by incubating with trypsin (1:3; ZYMED Laboratory invitrogen immunodetection kit) for 5 minute s at 37C, quickly rinsed with water, and washed by t ris buffered saline (TBS) twice for 5 minutes. For frozen tissue s, sections were cut on a cr y ostat, mounted on glass slides, and air dried overnight. The frozen tissue sections were fixed in acetone for 5 minutes at 20C, air dried, and washed with TBS. T he slides were blocked with Sniper blocking reagent (Biocare Medical, BS966) for 15 minutes at RT, and washed with TBS. The blocked tissues were covered with diluted mouse serum in antibody dilutent (Invitrogen, 00 3218) or with medium collected from the g rowing hybridoma clones, and incubated overnight at 4 C. The following day, the slides were washed, the

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59 paraffin embedded tissues were incubated with 3% peroxide in methanol for 10 minutes, and the frozen tissue sections were incubated with Peroxo block so lution (Invitrogen) for 45 seconds. The slides were rinsed with water and washed by TBS before covering the tissue with HRP conjugated goat anti mouse antibody (Biocare Medical, MHRP520) for 30 minutes at RT. After being washed, the slides were developed w ith diaminobenzidine (DAB; Vector, SK 4100) for 1 to 5 minutes until the staining was visualized using a light microscope. The reaction was stopped by rinsing the slides with water. The tissues were counterstained with hematoxylin, dehydrated in graded eth anol, placed i n xylene, and cover slipped by Cytoseal XYL ( Thermo Scientific ) Monoclonal antibody purification. The final selected hybridoma clone was grown in a CL350 Celline Classic Bioreactor flask (Sigma, Z688037) in medium (BD Biosciences, 220511) co ntaining low IgG serum 10% low IgG fetal bovine serum (Invitrogen/Gib co, 16250 078) until the cell population reached a maximal density The culture medium was harvested, filtered, and circulated over a protein G column (GE Healthcare Protein G Sepharose 4 Fast Flow). The antibody was eluted, concentrated with Amicon Ultra 15 Centrifugal spin filters (Millipore), and buffer exchanged into PBS. The concentration of the purified monoclonal anti IBDP antibody was determined and stored at 4C for future validat ion. Results IBDP Purification Inclusion body preps and total liver homogenates from 3 IBD positive and 2 IBD negative boa constrictors were obtained. The IBD negative liver did not result in any solid pellet after the incubation with sar kosyl, whereas a tightly bound pellet was obtained from the IBD positive samples (Figure 2 1). The quality of each step in

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60 inclusion body isolation was evaluated by examine collected sample 1 to 7 under the light microscope. The isolated inclusion bodies were most concentr ated in sample 7 as anticipated. Among the IB preps, the best quality samples were those with the most inclusion bodies and containing the least amount of extraneous cellular materials. The quality of IB prep was determined by the presence or absence of pr otein bands other than the 68 KDa band, and the intensity of the resolved 68 KDa protein band on a NuPAGE gel. Under light microscopy, the H&E stained IB prep (sample 7) from snake #08 76 appeared as a suspension of primarily eosinophilic globules of vario us sizes with minimal extraneous material (Figure 2 2). When resolved on the gel, this IB prep consisted of a major intense band with a molecular weight of slightly less than 6 8 KDa (Figure 2 3, l ane 3). Despite the slight shift in molecular weight possibl y caused by the differences in the combination of gel and electrophoresis buffer and the weight marker references being used. This isolated protein was thought to be consistent with the previous ly described IBDP. 4 The 68 KDa protein isolated from snake #0 8 76 was highly concentrated from the liver tissue compared to the resolved crude liver homogenate (Figure 2 3, lane 4). All the attempts to completely solubilize the IB prep using the common reducing and solubilizing agents were un successful. Unfortunatel y further protein purifica tion by electro eluting the 68 KDa bands were not successful due to the insolubility of IBDP. Therefore, t he semi purified IBDP (IB prep) from snake #08 76 was decided to be the best immunogen available for antibody production. A ntibody Production and Monoclonal Antibody Selection Polyclonal antibody that was reactive to the 68 KDa protein band was detectable in the mouse serum by western blot on day 57 post immunization (F igure 2 4). Cultured media of hybridoma mass cultures deri ved from splenic lymphotyes of mouse #1 were

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61 screened for reactivity to the IB preps by ELISA. Four different IB preps were used as coating antigens, including liver and kidney isolates from two IBD positive snakes (#08 76 and #08 122). Each hybridoma mass culture was test ed for the reactivity against 2 to 3 different IB preps. Of 303 h ybridoma mass cultures screened, only 1 culture (5B3) showed a low positive reactivity (OD reading approximately 5 folds higher than the background or negative control) and c ross reacted with 3 different IB preps. Additional ly, 32 mass cultures that had significantly higher OD readings compared to the background were also selected. Collected cultured media of the 33 selected mass cultures were further tested for the reactivity to IB preps by western blots. Only antibodies produced by mass culture 5B3 showed reactivity to the 68 KDa protein band (Figure 2 5), and reacted to all four different IB preps. T he mass culture 5B3 was further cloned by limiting dilution and seeded at a single cell per well density. Of 72 single colony wells that were screened for reactivity to IB prep by ELISA, 10 wells that showed low positive reactivity (OD read approximately 5 fold of the negative control or background) were selected, and further test ed for their reactivity to all four IB preps. The monoclonal antibodies were isotyped and determined to be IgG subtype by ELISA. Seven out of the 10 selected clones were further tested for reactivity to the 68 KDa band of four IB preps by western blots, an d for their IHC reactivity to the inclusion bodies in liver and pancreas of snake #08 76 and #08 122. All the tested clones showed positive reactivity to the 68 KDa and inclusion bodies by western blots and IHC staining respectively. The clone 5B3 3D9 that showed less background in IHC staining was select ed (Figure 2 6) and grown to high density for subsequent purification. From a 120 mL of cultured medium t hat was harvested, and

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62 purified, a total yield of 16.9 mg anti IBDP monoclonal antibody at a concentration of 10.37 mg/ mL was obtained. This antibody w as isotyped as IgG1 with kappa light chain by IsoStrip. Discussion The most challenging issue encountered during protein purification was the insolubility of semi purifie d IBDP (IB prep). The inclusion bodies remained as solid insoluble particles that made it difficult for further purification, sequencing, and other downstream protein analysis. Insolubility of the IBD Inclusion Bodies Not solubilized by common solubilizing reagents. Multiple common protein solubilizing reagents were used in an attempt to solubilized the IB prep, including 8 12 M ur ea, 6 M Gu HCl, 1% Triton 100, 2% OBG, 1% DDM 2 4% SD S, 20% LDS 1 M DTT, DMSO bicarbonate buffer and 1% acetic acid. All reagents fail ed to solubilize the inclusion bodies completely. Even when combined with r educing agents in high concentration s, such as 4% SDS, 160 mM DTT, and 12 M Urea, the inclusion bodies still would not completely solubilize H eating the IB preps with combinations of reducin g agents over 95 C for at 5 to 20 minutes was strictly required to reduce the protein, and even with this only a portion of the sample solubilized. The extreme insolubility is a very unique feature of IBDP. Similar degree of protein insolubility had been described i n Huntingtin fragment a ggregates, a prion like protein. 11 Heating at 100C with combination of 1.25% SDS and 1.25% beta mercaptoethanol had been used for denaturing insoluble prion. 12 The insolubility of IBDP and the nature for aggregate formation may indicate that IBD share s a similar disease mechanism with other protein aggregate forming disease s

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63 Hydrophobic characteristics. Certain characteristics of the semi purified inclusion bodies observed during sample processing indicated that the inclusion bodies may have very hydroph obic characteristics When the IB preps were placed in aqueous solution s (such as water and normal saline), the inclusion bodies form a tight binding pellet a fter centrifugation, which was difficult to resuspend. The inclusion bodies also bound to the walls of plastic tubes and pipet tips that were subsequently lost during processing. When the IB preps were placed in solution containing urea, Gu HCl, OBG, sarkosyl and other detergents, the inclusion bodies pellet that was easier to resuspend. This also reduced the binding to plastics. In some cases, combinations of Gu HCl, urea, or OBG with DTT reduced the binding of IBDP. T he stickiness of IBDP in aqueous environme nt may explain the nature of forming cytoplasmic aggregates. And the high hydrophobicity of IBDP made protein analysis more difficult. Electro elution of IBDP was unsuccessful. band) obtained from liver homogenate was elect ro eluted from the excised gel, and used as immunogen to produce monoclonal anti body against IBDP. 4 When semi purified IBDP (IB prep) were used in this study, the electro eluted IBDP was not soluble. The protein coated onto the membrane inside collec ting chambers of the gel eluters (Figure 2 7), and was not possible to dissociate the protein from the membrane. The solution collected from the collecting chamber was tested for protein concentration using Bradford Protein Assay (Bio Rad) and was found too low to be effectively used for antibody production. Interestingly, this insolubility issue was not reported by Wozniak et al. 4 who first electro eluted the 68 KDa IBDP derive d from crude liver homogenates.

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64 Approximately 15 mg of IB prep was resolved into si x large SDS PAGE gels, all eluted protein was not retrievable. Inaccurate E stimation o f P rotein C oncentration Bradford Protein Assay is commonly used to estimate protein concentration in solutions by comparing the coloration of protein binding dyes in an unknown sample to a set of standard protein with known concentrations. 10 The insolubility made it difficult to estimate the concentration of IB preps. Heating the IB prep with reducing agent was crucial for the protein to be partially detectable in the Bradford Protein Assay. However, high concentration of detergents in the sa mple interfered with the color ation of the assay, and resulted in inaccurate estimation of the pr otein concentration For example, protein concentration of #08 76 liver IB prep was estimated at 7.6 mg/ mL whereas the HB (without any protein) was estimated to have protein concentration of 6.4 mg/ mL This inaccuracy may resulted in overestimating the amount of protein in the IB prep. For antibody production, protein quantity is very important in the immunization procedure, and for standardization of the assay s for antibody screening, such as, ELISA and western blot. Additionally, instead of solely relying on the estimated protein concentration, for each IB prep to be used as an antigen in assays, a standardization run was performed prior to the actual screenin g test to verify how much volume of IB prep was sufficient to react in assays. Challenge s in A ntibody S creening Ideally when screening for the monoclonal antibody, a purified protein should be used as the antigen for screening assays to avoid selecting an antibody that has non specific reaction to other undesired protein. Unfortunately, a purified IBDP was not available. Due to the insolubility, further purification methods such as 2 D

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65 electrophoresis, or liquid chromatography could not be performed on semi purified IBDP (IB prep). Alternatively, IB preps derived from different tissues (liver and kidney) and also from different boa constrictors were used as antigen for antibody screening. When screening the mass cultures, only those that reacted with all IB preps were selected to avoid choosing an antibody that is non specifically reacting to contaminants from liver or kidney. The specificity of the clones was confirmed by western blot and IHC staining, which ensured the antibody selected reacted to the 68 KD a protein and the inclusion bodies with minimal background staining. Conclusions During the ten month antibody production process, a monoclonal anti IBDP antibody producing clone was found in one out of 303 tested viable hybridoma mass cultures using ELISA and western blot. Despite the difficulties working with the insoluble protein, the final selected antibody produced by hybridoma clone 5B3 3C9 appeared to be very specific to the native and reduced IBDP. The MAB was purified, and was isotyped as IgG 1 wit h kappa light chain. This antibody can be used in various immune based assays, such as ELISA, western blot, IHC staining on frozen and paraffin embedded tissues. In contrast to the antibody produced by Wozniak et al 4 which did not react with IBDP in paraffin embedded tissues. The new anti IBDP antibody has greater advantages, which can be used in retrospective studies and diagnostics on paraffin embedded tissues.

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66 Figure 2 1. The isolated inclusion bodies obtained from 3 g of IBD positive liver. The arrows showing pelleted inclusion bodies in 1% sarkosyl after centrifugation. A. The pellet after first round of incubation with 1% sarkosyl. B. The same pellet after second round of incubation with 1% sarkosyl.

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67 Figure 2 2. The H& E stained IB prep on a microscopic slide. The semi purified inclusion bodies were used to immunize mice for antibody production. 1

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68 Figure 2 3. The resolved IB preps and liver homo genates on a NuPAGE. Ten microlit ers of protein were loaded on each lane. Lane 1 to 3 are three different IB preps obtained from three IBD positive boas. Lane 4 is the liver homogenate from the same boa as lane 3. Lane 5 and 6 are liver homogenates from two IBD negative boas. Lane 7 is the IB prep derived from an IBD negative bo a, which no pellet were left after incubating with 1% sarkosyl. Lane 8 is HB served as a blank control. The IB prep from #08 76 showed a major intense band approximately at 68 KDa (arrow), showing that the IBDP is concentrated in the IB prep compared to th e liver homogenate (Lane 4). M. Molecular weight marker (Invitrogen Mark 12 )

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69 Figure 2 4. Western blot showing the polyclonal antibody reacted with the 68 KDa protein in the IB prep (arrows). Each lane contained 16 g of IB prep and were detected by post immunized mouse serum collected on day 57 from mouse #1(lane 1 and 4) and mouse #2 (lane 2 and 5). Serum of an un immunized mouse was used as negative controls (lane 3 and 7). Lane 1 to 3 used mouse serum in 1:100 dilution. Lane 4 to 6 used mouse seru m in 1:500 dilution. Lane 7 was a blank control without primary antibody. M. Molecular weight marker (Invitrogen SeeBlue Plus 2 ).

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70 Figure 2 5. Western blot showing presence of antibody that reacted with the 68 KDa protein (arrow) in the cultured medium from hybridoma mass culture 5B3 (lane 8). A total of 140 g IB prep from liver was blotted onto the membrane, and each lane was detected with a different antibody separated by a Fast Blot Developer manifold. Lane 1 was detected with mouse #1 serum in 1:100 0 dilution as a positive control (arrow head). Lane 2 to 15 were detected with undiluted cultured supernatant collected from different hybridoma mass cultures, only mass culture 5B3 showed reaction to the 68 KDa protein band. M. Molecular weight marker (In vitrogen SeeBlue Plus 2 ).

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71 Figure 2 6. IHC staining of paraffin embedded pancreas from an IBD positive boa constrictor using polyclonal and monoclonal antibody. A. A negative control using a non specific antibody, the inclusion bodies were not stained ( arrows). B. A positive control using 1:800 diluted serum from mouse #1, the inclusion bodies were stained (arrows). C. The inclusion bodies are stained with undiluted cultured medium of clone 5B3 3D9, the antibody reacted specifically to the inclusion bodi es (arrows) with very little background staining.

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72 Figure 2 7. The collecting chamber of Model 422 Electro Eluter ( Bio Rad Instruction Manual M1652976B ) (Left), which attached inside of a tank that can be applied with electric current (Right). With electric current, the protein migrated from the gel slices into the collecting chamber, and a membrane cap in the bottom was used to trap the protein inside of the collecting chamber. After being eluted, the insoluble IBDP coated onto the membrane cap (red arrow) instead of staying in the solution within the collecting chamber P ermission to use the photograph and the illustration has been g ranted by Bio Rad Laboratories Inc.

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73 CHAPTER 3 VALIDATION OF AN A NTI IBD PROTEIN MONOCLON AL ANTIBODY FOR USE IN IMMUNOHISTOCHEMICAL STAINING Introduction Inclusion body disease (IBD) had been reported in captive boid snakes worldwide. The specific causative agent(s) and the transmission mechanism remain unclear. Currently, a diagnosis of IBD is made based on identifying characteristic eosinophilic intracytoplasmic inclusion bodies in hematoxylin and eosin (H&E) stained histological slides. In some case, inclusion bodies seen in IBD can be difficult to distinguish from other cellular proteinaceus inclusion bodies or cellular granules that may accumulate in the cytoplasm of affected cells. In some cases, the inclusion bodies may not be abundant in visceral tissue, or early developing smaller inclusion bodies may be o verlooked in an H&E stained section. An immunohistochemical (IHC) diagnostic test would be of great value in mak ing a more specific diagnosis 1,4 Immunohistochemical staining is a well recommended method with high sensitivity and specificity for diagnosing both infectious and non infectious diseases. With IHC staining, an antibody reacting to a specific antigen can be used to localize the antigen within affected tissue, which increases the accuracy of a diagnosis 1 3 In the study of Wozniak et al a 68 KDa protein (IBDP) that was found only in the tissue of IBD positive (IBD+) boa c onstrictors was used to produce a monoclonal antibody, and the antibody reacted to the inclusion bodies in the frozen tissue sections using IH C staining 4 Unfortunately, the antibody did not react to inclusion bodies in paraffin embedded tissues, and several years later the original hybridoma clone was lost. In my previous chapter, a n anti IBDP monoclonal antibody (MAB) was produced against a preparation of semi purified inclusion bodies of a bo a constrictor that was IBD+. On western blots

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74 this MAB reacted to the 68 KDa IBDP band s; i n IHC staining, the MAB reacted to the inclusion bodies in fresh frozen and paraffin embedded tissues. This antibody can be used as a valuabl e tool for developing immune based diagnostic tests for screening cases of IBD. A problem of IHC staining in veterinary medicine had been discussed by Ramos Vara et al that there is a lack of high quality antibody that is specifically made for an antigen but can be used among different species 1 3 This problem can be more significant for diagnosing diseases in zoological and exotic animal medicine and pathology where more diverse species are seen. Further, there appeared to be a lack of standardization in I HC staining methodology among different veterinary laboratories. The goal of standardization in IHC staining is to achieve reproducible and consistent results within and among different laboratories 1 3 Therefore, MAB needs to be properly validated before b eing used or offered as a diagnostic test. In this study, the MAB was validated using the format of IHC staining on paraffin embedded tissues following the guidelines s uggested by Ramos Vara et al 1 3 The conditions of IHC staining using an anti IBDP MAB we re standardized. The factors that may affect th e IHC staining, such as, the fixation time in formalin and the storage time in paraffin were evaluated. The cross reactivity of MAB in other snake species and other inclusion body li ke protein were also evaluated. Finally, the diagnostic p erformance of the IHC test using anti IBDP MAB was determined. Material and Methods Sample Collection and Management A repository of paraffin embedded tissue blocks were obtained for the period 1990 to 2007 from th e Anatomic Pathology Service and Zoological Medicine Infectious

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75 Disease Testing Laboratory, College of Veterinary Medicine, University of Florida (UF). Additional blocks were provided by a private exotic animal pathology service, Northwest ZooPath, Monroe, WA. A Pathology Report was available for each case, in which the diagnosis of IBD positive or negative (IBD ) was noted by certified pathologists based on histological examination of H&E stained sections. For each case, one block that best represented the IBD pathology was selected for IHC staining, otherwise a block that contained liver, kidney, or pancreas was selected. Twenty blocks were retrieved from the storage facility at a time, sectioned and submitted for one round of IHC staining. When completed, the blocks were returned, and another 20 blocks were retrieved for the next round of IHC staining. Fresh and formalin fixed tissue samples of year 2008 to 2011 were collected by veterinarians in private practices or UF Veterinary Hospitals, and transferre d to my laboratory for IBD diagnosis. Formalin Fixation and Embedding Fresh tissues including liver, kidney, and pancreas obtained from an euthanized boa constrictor were dissected into approximately 0.5 mm thick sections, placed in cassettes, fixed in 10% neutral buffered formalin (10% NBF) or 4% paraformaldehyde (4% PF) for 48 h ou rs, and finally embedded into paraffin (IACUC approval # 201101156 ) In the evaluation of effects of prolonged fixation, freshly obtained liver, kidney, and pancreas of an IBD+ b oa constrictor were each cut into 10 sections that were approximately 0.5 mm thick. One piece of each sectioned liver, kidney, and pancreas were placed in a cassette (ten sets of tissues), followed by fixation in ten identical containers filled with 10% NB F. On Day 2 (48 hours after initial fixation), Day 7, Day 8, Day 9, Day 15, Day 23, Day 32, Day 39, Day 50, and Day 58, one cassette was

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76 removed and the tissues were embedded in paraffin. For tissue set of Day 58, only kidney and pancreas were embedded. Ti ssues from the repository were embedded in the laboratory of Anatomic Pathology Service in the UF Veterinary Hospital (Lab 1). Using an automated processor (Thermo Electric Corporation, Shandon Excelsior), the fixed tissues were dehydrated in graded ethano l, followed by infiltration of xylene and paraffin. The processed tissues were manually mounted in paraffin blocks. In IHC staining standardization, the fixed tissues were embedded in two different laboratories, Lab 1 and the laboratory of Molecular Pathol ogy Core (Lab 2), College of Medicine, UF The embedding procedures of the two labs can be found in Table 3 1. H&E Staining for Paraffin Embedded Tissue The paraffin embedded tissues we re stained using an automatic slide stainer (Gemini Varistain, Thermo Shandon, Illinois, IL), which deparaffinized the slides with xylene, and rehydrated the tissue using graded ethanol. The rehydrated tissues were stained with hemotoxylin (Richard Allan Scientific, 7212) for two minutes, incubated with clarifier 2 (Richard Allan Scientific, 7402) for 30 seconds, followed by incubating with bluing reagent (Richard Allan Scientific, 7301) for one minute, then incubated one minute in 80% ethanol before staining with eosin (Richard Allan Scientific, 71311) for one minute. In be tween the application of each reagent, the slides were washed with running water. Finally, the slides were dehydrated in graded ethan ol, dipped in xylene, and cover slipped.

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77 IHC Staining for Paraffin Embedded Tissues Except for the steps described as follow the general IHC staining procedure remained the same as described in Chapter 2 ( IHC staining Polyclonal and Monoclona l Anti IBDP Antibody Production) Antigen Retrieval (AR). The deparaffined slides were either treated with AR reagents or were not treated with AR reagents. The following AR reagents were evaluated, trypsin (Invitrogen, Digest All2), Trilogy (Cell Marque), Citra (Biogenex), Dako Target Retrieval Solution, pH 6.0 (DAKO), Dako Target Retrieval Solution, pH 9.0 (DAKO). The trypsin AR was done by incubating th e slides for five minutes at 37 C. The AR treatments with other reagents were done by incubating the slides for 30 minutes at 95C. For double AR treatment, the slides were incubated with Trilogy for 30 minutes at 95C, followed by additional five minutes of incubation with trypsin at 37 C. For the standardized IHC staining protocol, Trilogy was used as the standard AR reagent. For some samples that the standard AR was not strong enough to retrieve the antigen, the double AR procedure was used. Incubation time for primary antibody. The slides were covered by anti IBDP MAB in a specific dilution (1:1,000, 1:2,000, 1:5,000, 1:10,000, 1:20,000), and incubated for 1 hour at room temp erature (RT), or overnight at 4 C. For the standardized IHC staining protocol, the slides were incubated with 1:10,000 diluted anti IBDP MAB for 1 hour. Automated staining machine. For the standardized IHC staining, after AR, blocking, th e washes and incubation of primary and secondary antibodies were performed in the automated staining machine (Autostainer Plus, DAKO). The slides

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78 were developed manually using procedures described in Chapter 2. Otherwise, the entire IHC staining procedure was done manually. Peroxidase chromogenic substrates. The HRP conjugated secondary antibody was visualized by development with diaminobenzidine (DAB; Vector Laboratory, SK 4100) or VECTOR NovaRED (Vector Laboratory, SK 4800) according to the protocol. For the standardized IHC staining, all slides were stained using NovaRED. IHC Evaluation Using light microscopy, the intensity of the IHC stain for each slide was given one of the four scores: 0 (no staining), 1 (faint, barely visible), 2 (moder ate), 3 (strong). For each IHC staining run, a positive control slide (IHC score 3) and negative control slides (IHC score 0) were run parallel to the slides to be scored. The negative control of each sample was a duplicated slide that stained with a comme rcial non specific mouse IgG instead of MAB (described in Chapter 2 : IHC staining, Polyclonal and Monoclona l Anti IBDP Antibody Production ) For evaluating the Effect of Storage Time in paraffin, Species Cross reactivity, and Antigen Cross reactivity, the IHC staining results were interpreted as either positive or negative A positive IHC stain (IHC+) was defined by a visible staining pattern (IHC score 1 to 3) compared to the negative control (IHC score 0). A negative IHC stain (IHC ) was defined by no vis ible staining pattern (IHC score 0) compared to the negative control (IHC score 0). IHC Diagnostic Performance E valuation Samples classified as IBD+ or IBD by H&E examination (current gold standard) were classified as true positive or true negative, respe ctively. The sensitivity, specificity,

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79 positive and negative predictive values of the IHC test, compared to H&E, were calculated f ollowing standard procedures 1 4 To assess the effect of storage time in paraffin, the samples collected during 1990 2000 and 2001 2011 that were classified as IHC+ were compared by using the Fisher Exact X 2 test following standard procedures 1 5 Results Standardization of IHC Staining Condition In order to establish a consistent staining performance, the IHC staining conditions were standardized according to the suggested gu idelines by Ramos Vara et al. 1 5 The IHC staining conditions for the use of anti IBDP MAB standardized by this study are summarized in Table 3 3. Species and tissue type. Tissue of the boa constrictors were as signed as the standard species, due to following reasons: 1. The anti IBDP MAB was produced using li ver tissue of a boa constrictor ; 2. Within the sample repository, the majority of the samples were from boa constrictors; 3. IBD in boa constrictors were mo re frequently diagnosed than other species. In IBD affected snakes, the inclusion bodies were most commonly observed in liver, kidney, and pancreas. Therefore, liver, kidney, and pancreas were selected as the standard material for IHC staining. In a clinic al perspective, the liver was preferable, since it is larger tissue compared to the pancreas and kidney, and is an easily accessible tissue for biopsy. Fixation. In this study, tissues that were fixed in 4% PF were stained with only mild AR treatment or no treatment at all. However, 10% NBF is the most commonly used fixative in routine histopathological evaluation of tissues, and was selected as the standard fixative in this study.

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80 Antigen Retrieval. A variety of AR reagents including, trypsin, Trilogy, Ci tra, Dako Target Retrieval solution pH 6.0, and Dako Target Retrieval solution pH 9.0 were tested to determine which reagent resulted in the best staining intensity in IHC staining on the selected tissues fixed with 10% NBF. When trypsin was used, the bloc ks that were made in Lab 2 stained strongly with MAB (IHC score 3), but the blocks that were made in the Lab1 stained faintly (IHC score 1) or no staining (IHC score 0). Using harsher AR treatment such as, Trilogy, Citra, Dako Target Retrieval solution (tw o different pH, pH 6.0 and pH 9.0), the staining intensity was improved to medium or high (IHC score 2 to 3) (Table 3 2). Trilogy was finally selected as the standard AR reagent, because it generated high staining intensity and less none specific backgroun d staining compared to the other reagents tested. Primary antibody. The standard primary antibody dilution was determined by testing different anti IBDP MAB dilutions until the staining intensity started to decrease. The staining intensity remained high with very minimal background when a dilution of 1:10,000 was used, but when a dilution of 1:20,000 was used the staining intensity decreased. Thus, the di luti on of 1: 10,000 was determined as the standard dilution for the anti IBDP MAB. The incubation of the primary antibody showed no significant difference s in the staining intensity when incubated 1 hour at RT or overnight at 4 C. Thus, the 1 hour incubati on protocol was selected as the standard condition, and was compatible with incubation time used in the automated staining machine. There were no significant differences in the staining intensity when IHC stains were performed manually or by machine. Thus, the automated staining procedure was selected as the

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81 Detection system. The chromogenic peroxidase detection system was used throughout all IHC staining, with HRP conjugated goat anti mouse antibody detected by DAB or NovaRED. The substrate NovaRED stained the reactive sites with a reddish purple color that contrasted better in the IHC stain than DAB. The substrate DAB stained reactive sites with a brown color that sometimes could be confused with the brown pigments of the hepatic macrophages in liver tissues. In this study, NovaRED was used as the standardized detection system, which showed better contrast with the blue background hematoxylin counter stain (Figure 3 1). Validati on of anti IBDP MAB with IHC Staining The anti IBDP MAB was validated using the standardized IHC staining conditions. The factors to be evaluated during the validation were determined based on the guidelines s uggested by Ramos Vara et al. 1 3 The factors in cluded the effects of prolonged formalin fixation and storage time in paraffin, and whether MAB cross reacted with IBD in other species or other inclusion body forming antigens. The overall IHC test sensitivity and specificity was compared against the resu lt of H&E stain, the current gold standard for IBD diagnosis. a. Effects of prolonged formalin fixation Ten blocks (Block 1 to 10) each containing liver, kidney, and pancreas that were fixed for a specific length of time (ranged from standard 48 hours to 5 8 days), were used in this study. Three slides were made from each block, two stained with anti IBDP MAB, and one stained with non specific mouse antibody as negative control. The inclusion bodies within the embedded tissue remained detectable by MAB up t o 58 days follo wing initial fixation (Table 3 4 ). The staining intensity of pancreas was constantly high (IHC score 3) throughout all tested fixation time period s The staining

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82 intensity of liver and kidney remained high (IHC score 2 3) up to 32 days of fi xation. Some uneven staining (IHC score 2 or 3) were observed in liver and kidney beyond 8 days of fixation. More severe unevenness in staining intensity were observed in the liver (IHC score 1 or 2) and kidney (IHC score 0 or 2 or 3) fixed for 39 days and 50 days. Some areas within the kidney fixed for 50 days were not stained. In all negative control slides, no staining of the inclusion bodies was observed. Overall, prolonged fixation up to 58 days did not affect the IHC staining in pancreas, but in kidne y uneven staining pattern were observed in fixation of 9 days, 39 days, and 50 days. In liver, the staining tended to be less intense compared to the staining in kidney and pancreas, and more inconsistence staining pattern was observed throughout the fixat ion time evaluated in this study (Figure 3 2). The double AR treatment restored the reactivity of anti IBDP MAB in liver up to 50 days of fixation in 10% NBF (Block 9), and the uneven staining pattern was not observed. When double AR treatment were used in staining liver tissues of Block 8 a nd 9 (Table 3 3), the inclusion bodies stained dark red and the intensity was stronger than IHC score 3 (IHC score 3+)(Figure 3 3A). Unfortunately, the double AR treatment was judged to be harsher on tissues, which resul ted in loss of cellular structural detail. In kidney and pancreas, double AR treatment reduced the staining of inclusion bodies (Figure 3 3B and C). b. Effects of storage time in paraffin The collection dates for paraffin embedded tissues of 94 (60 IBD+ and 34 IBD ) boa constrictors ranged from 1990 to 2011. Without having specific information, the assumption was that tissues were embedded after a standard fixation time of 48 hours in 10% N BF. One block of each case which included previously evaluated tissues (liver,

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83 kidney, or pancreas) was selected for IHC staining. If IBD inclusion bodies were not described in the standardized tissue, an additional block that contained inclusion body bear ing tissue was selected for IHC stain. If the standardized tissues were not available, the block conta ining tissue with IBD inclusion bodies that were noted in the Pathology Report was selected for IHC staining. The paraffin embedded tissues were divided into two groups: 1. tissue samples embedded within the time period 1990 2000 (Group 1: 29 IBD+ and 15 IBD ) and 2. Tissue samples embedded within the time period 2001 2011 (Group 2: 31 IBD+ and 19 IBD ). In Group 1, 50/60 IBD+ cases stained IHC+ and 15/15 IBD cases stained IHC which indicated a sensitivity of 82.8% and a specificity of 100%. In Group 2, 26/31 IBD+ cases stained IHC+ and 18/19 IBD cases stained IHC which indicated a sensitivity of 83.9% and a specificity of 94.7% (Figure 3 4). There w ere no significant differences (p value = 0.99) of sensitivity and specificity between two year groups analyzed by Fisher Exact test. This suggested that the storage time did not significantly affect the performance of MAB in IHC staining. c. Species c ross reactivity The reactivity of an anti IBDP MAB to IBD inclusion bodies in non boa constrictor species was evaluated by IHC staining using paraffin embedded tissues in the tissue repository. Inclusion body disease positive tissues of six different non b oa constrictor species were tested with IHC staining, which included annulated tree boas ( Corallus annulatus ) (n=3), ball pythons ( Python regius )(n=4), carpet pythons ( Morelia spilota )(n=2), emerald tree boa ( Corallus caninus )(n=1), palm viper ( Bothriechis marchi )(n=1), and rainbow boas ( Epicrates cenchria )(n=4) (Table 3 5 ). The sample s of annulated tree boas 8 and palm viper 1 6 were obtained from previously reported s tudies

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84 The anti IBDP MAB showed species cr oss reactivity to IBD inclusion bodies in ball pythons (1 out of 4) and carpet pythons (2 out of 2). d Antigen cross reactivity The reactivity of the anti IBDP MAB to IBD like inclusion bodies in previously reported corn snakes ( Elaphe guttata ) (n=2) 1 7 and boa constrictors with adenoviral in clusion bodies (n=1) and pox virus like inclusion bodies (n=1) were tested by IHC staining. No antigen cross reactivity of the anti IBDP MAB was observed in IHC staining. e. IHC test diagnostic performance Sixty samples from boa constrictors that were clas sified as IBD positive and 34 samples that were classified as negative by H&E stain were tested using IHC stain. The sensitivity of the IHC test was 50/60 or 83% (95% CI = 76%, 91%) (Figure 3 4). The specificity of the IHC test was 33/34 or 97% (94%, 100%) The negative predictive value of the IHC test was 10/43 or 77% (68%, 85%). The positive predictive value of the IHC test was 50/51 or 98% (95%, 100%). Out of 34 IBD negative cases, one case was suspected to be IBD+ based on clinical signs, but was determined to be IBD after examination of H&E stain ed sections. Using IHC stain, the anti IBDP MAB detected small inclusion bodies in liver and brain of this boa These small inclusion bodies were not recognized with H&E stain. Thus, the one IBD (H&E stained negative) case was actually IBD+. When included in with the positive group, the specificity of anti IBDP MAB in IHC staining was 100%. In testing boa c onstrictors, the IHC test is expected to perform with sensitivity above 75.8% and specificity above 93.6% on paraffin embedded tissues stored up to 22 years, and fixation in 10% NBF for up to 58 days.

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85 Discussion This study was designed to validate an anti IBDP MAB for use in IHC staining to diagnose IBD in snakes. This validation evaluated the following factors that could potentially affect the staining: 1. Tissue fixation time in 10% NBF; 2. Tissue storage time i n paraffin; 3. Antigen variation between species; 4. Reactivity to other antigens. The outcome of studies designed to evaluate these factors are discussed below. Factors That may Affect IHC Staining Fixation time in formalin. Prolonged fixation in form alin has been considered a limiting factor for IHC staining, because antigenic epitope(s) can be masked by cross link ing during formalin fixation 1 8 For immuno detection purposes, tissue fixation is typically treated very carefully, and fixation is done st rictly within the recommended time period (standard 48 hours), which is thought to be critical for obtaining a positive IHC staining reaction with some antigens. Prolonged formalin fixation is presumed to result in decreased antigen detection 1 8 However, t was not clearly defined. 18 In this study, multiple tissues from a snake with IBD were fixed in NBF for time periods up to 58 days following initial fixation. Interestingly, in this study the kidney and pancreas ha d moderate to strong reactivity to anti IBDP MAB up to 58 days of fixation, and the same in the liver up to 32 days of fixation. This finding corresponds to findings in a similar study, in which prolonged fixation affected the quality of the staining inten sity, however, under fixation affected the staining intensity more significantly 1 8 Tissue type. In the standardized staining condition using Trilogy AR treatment, the staining of liver was assessed to be less intense compared to the staining of pancreas and kidney regardless of the fixatio n time. In liver, the inclusion bodies located

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86 at the margin of the tissue stained more intensively then the inclusion bodies located near the center of the tissue section. In liver fixed beyond 7 days, more unevenness i n staining intensity was observed throughout the liver section. The uneven staining was also observed in kidney beyond 9 days of fixation. In fixation up to 50 days, the inclusion bodies were not stained in some areas within kidney. However, in pancreas th e staining was not affected at all for f ixation up to 58 days (Table 3 4 ). This suggested that the outcome of IHC staining following extended fixation time is tissue dependent. Possibly differences in pH among different tissues or other biochemical differe nces affected the efficiency of formalin infiltration. Webster et al. had discussed the possible differences in formalin penetrating rate that may caused the uneven IHC staining. 1 8 It is likely that the formalin penetration was slower in liver, compared to kidney and pancreas. Whether this is due to physical of biochemical properties of liver compared to the other tissues assessed remains unclear. Compared to the antigen at the margin of tissue, antigen located in the center of a tissue may be under fixed, resulting in less intensive staining compared to antigen at the margin of tissue. Webster et al. found that many antibodies had variable immune reactivity among differe nt tissue types 1 8 In this study, the validation of the anti IBDP MAB in IHC staining was evaluated using tissues that are most commonly collected for making a diagnosis in IBD. Selection of AR treatment. Antigen retrieval is known to be a critical step in IHC staining, which retrieves the cross linked antigenic epitopes especially when tissues are fixed or remain in fixatives for prolonged periods of time. In prolonged fixed tissue that showed mark decrease in staining intensity, opti mization of the AR treatment significantly restore d the immune reactivity (Figure 3 3). Prolong fixed liver of Block 9

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87 treated with double AR, showed much improved staining intensity without seeing the uneven staining between the margin and center of the tissue (Figure 3 3C). But when double AR tre atment was used, the structural details of the stained tissues were lost due to the harsh treatment. In pancreas and kidney, the staining of inclusion bodies faded when double AR was used (Figure 3 3A and B). Thus, the standard AR using Trilogy should be c onsidered as a routine procedure, but if the staining in liver is not satisfying, then double AR can be used. Variation of blocks made in different laboratories. During the process of standardization, the same tissue processed and embedded in two different laboratories (Lab1 and Lab2) was found to have different requirements for AR. The tissue processed in Lab2 stained strongly with mild AR (trypsi n) or even without AR treatment But the same tissue processed in Lab1 strictly required harsh AR treatments wi th incubation at high temperature (95C) in order for the MAB to stain the inclusion bodies. By comparing the tissue processing procedure between the two labs (Table 3 1) the procedures were not significantly different. It is possible that slight differen ces during tissue process, such as, temperature, incubation time, and reagents purchased from different suppliers may have affected the intensity of IHC staining. In a case collection of 9 IBD+ and 3 IBD boa constrictors, 3/9 IBD+ cases the inclusion bodi es within liver and kidney could only be stained using double AR treatment (Figure 3 5). This may also be caused by variation in sample processing procedures discussed above among different laboratories, however, the processing procedure of the laboratory was not documented. Fortunately, this study evaluated antigen retrieval methodologies and findings indicated the importance of selecting specific AR reagents. In this study the following AR reagents

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88 were evaluated, trypsin, Trilogy, Citra, Dako Target Retr ieval solution pH 6.0, and Dako Target Retrieval solution pH 9.0. Of these, Trilogy worked the best when evaluating tissues embedded in several different laboratories. Embedded tissue from most laboratories that submitted samples for testing stained with m oderate to high intensity. When staining was weak, double AR can be used to enhance the staining sensitivity. However, this may increase non specific background staining in the connective tissues of some samples. To ensure that insufficient AR was not t he cause of those cases that failed to stain with IHC, the blocks that did not stain positive (even though inclusion bodies were seen with H&E staining) in IHC stain (including the IBD cases) were retested with another run of IHC staining using the double AR treatment. Out of 60 IBD+ boa constrictor cases in the repository, 10 cases did not stain positive following standard AR. However, 3 of these 10 cases stained IHC positive when double AR was used. All IBD cases remained negative of IHC staining when r etested using double AR. IHC Diagnostic Performance E valuation False negative results. The sensitivity of the IHC test was not as high as a typical screening test due to the higher false negative value (10/60 or 16.7%) estimated by this study. When 10 prev iously diagnosed cases of IBD were re examined, the characteristic eosinoph ilic intracytoplasmic inclusion bodies were not found in five of these cases. All five stained negative with IHC. The diagnosis of these cases was classified as questionable. The nu mber of questionable cases resulted in the lower sensitivity of IHC test. Four of the questionable cases were within Group 1, and three of them were reported in 1995. This also suggested that a false positive diagnosis of IBD can likely be made by a pathol ogist, who may have limited experience diagnosing IBD.

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89 False positive results. The estimated specificity of IHC test was very high which is satisfactory for a screening test. Ho wever, the specificity remains under estimated, due to a high calculated false positive value (1/34 or 2.9%). There was one case that was diagnosed negative using H&E staining that clearly stained positive with IHC in liver and brain. The inclusion bodies were so small and sporadic that they were missed with H&E staining. This is an example demonstrated the value of IHC testing for IBD. With the exception of this case, the specificity of IHC test using anti IBDP MAB would be 100% and false positive would be 0. Application as a screening test for IBD. The high positive predicted value and low false positive of the IHC test indicated that a case having IHC + result can be confidently diagnosed as IBD+. However, the low negative predicted value and false negative of the IHC test indicated that for clinical applications when a sample tested by IHC is found to be negative, a diagnosis needs to be evaluated carefully in combination with the findings in H&E stain. In one IBD+ case, the inclusion bodies were only detected in the brain, not in any other tissues including spinal cord. This also ad dressed the importance of tissue selection for diagnosing IBD. If IBD is ranked high on the differential list of diseases in an ill snake, multiple tissues (including the brain) need to be sampled and tested. Based on the estimated sensitivity, specificity and positive and negative predictive values, the IHC test has more merit as a confirmatory test than as a screening test for diagnosis of IBD in boa constrictors. Cross Reactivity Among Non Boa Constrictors Of the total number of tissues available for this project, the number of IBD cases in non boa constrictors was limited. Only 15 cases were found in UF and collaborated laboratories between 1990 to 2011. Within six different non boa constrictor species

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90 test ed, only two carpet pythons and one ball python stained positive with IHC staining. Even when double AR was used, the other 12 cases remained unstained. This may suggest that different species may be infected with different str ains of the IBD causative age nt resulting in sufficiently different IBDP (epitopes) from the IBDP in boa constrictors. Therefore, the anti IBDP MAB produced in this study may not be able to recognize IBDP in all species of snakes that appear to be IBD+. Interestingly, the two species that anti IBDP MAB cross reacted were more distantly related to boa constrictors than other more close related species tested (Table 1 1) such as, rainbow boas, annulated tree boas and emerald tree boas. Nevertheless, the use of anti IBD P MAB in diagnosin g IBD in boa constrictors is well validated, but when testing in non boa constrictors the results need to be interpreted carefully. Conclusions The anti IBDP MAB was validated using IHC staining on paraffin embedded tissues of 60 IBD+ and 34 IBD boa const rictors. The IHC staining condition was standardized with the use of 10% NBF as fixative, Trilogy for AR treatment, 1 hr incubation with 1:1 0 ,000 diluted anti IBDP MAB, and NovaRED as detection substrate. Pancreas, liver, and kidney were used as the standa rd tissues for IHC testing. In kidney and pancreas the IBD inclusion bodies can be detected up to 58 days of fixation in 10% NBF, and in liver the inclusion bodies can be detected up to 50 days of fixation in 10% NBF. There were no significant differences in the IHC test performances between different storage times in paraffin of Group1 (1990 to 2000) and Group 2 (2001 to 2011). The anti IBDP MAB had species cross reactivity with IBDP in carpet pythons and ball pythons, and no antigen cross reactivity with inclusion bodies of adenovirus, pox virus, and IBD like inclusion bodies in corn snakes.

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91 In testing boa constrictors using anti IBDP MAB in IHC test, the sensitivity was 83% (95% CI = 76%, 91%), the specificity of was 97% (94%, 100%), the negative predict ive value was 77% (68%, 85%), and the positive predictive value was 98% (95%, 100%). In clinical applications, an IBD+ diagnosis can be made confidently when the sample tested IHC+. However, a diagnosis of IBD needed to be evaluated carefully in combinati on with the findings in H&E stain, when the sample tested IHC Table 3 1. Th e tissue processing procedure of two laboratories EtOH: ethanol. N/A: not applicable. In Lab1, the reagents used were Alcohol (Decon Laboratories), Histology Grade Xylene (Fisher), and Paraplast (Fisher). Formalin was made with 37% Formaldehyde (Fisher), Sodium Phosphate Monobasic anhy drous (MP laboratories), and Sodium Phosphate Dibasic, anhydrous (Fisher). In Lab2, the reagents used were NBF 10% buffered (Fisher), Ethanol (Fisher), Histology Grade Xylene (Fisher), and Paraffin Type9 (Fisher). Table 3 2. IHC score of tissues processed by two laboratories using different AR treatments. MAB: monoclonal anti IBDP antibody. Boa 08 76 and 08 122 were two IBD+ boa constrictors, the MAB was produced against the inclusion bodies isolated from boa 08 76. Lab1 Processor Lab2 Processor Reagent Inclubated Time (sec/run) Number of runs Temperature Inclubated Time (sec/run) Number of runs Temperature Formalin 30 2 Ambient N/A N/A N/A 70% EtOH 60 1 30C 1 1 Ambient 20 1 Ambient 80% EtOH 60 1 30C 20 1 Ambient 95% EtOH 60 1 30C 20 2 Ambient 100% EtOH 60 3 30C 20 2 Ambient Xylene 40 3 30C 20 2 Ambient Paraffin Wax 40 3 60C 20 3 60C Tissue embedded Primary antibody Sample origin AR reagents No AR Trypsin Trilogy Dako pH9.0 Dako pH6.0 Citrate Lab 1 MAB 08 122 Pancreas 2 3 3 2 2 2 Lab 2 MAB 08 122 Pancreas 0 0 3 2 2 2 Lab 2 MAB 08 76 Pancreas 1 1 3 2 3 3 Lab 1 None specific mouse IgG 08 122 Pancreas 0 0 0 0 0 0

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92 Table 3 3. Standardized IHC staining conditions for anti IBDP MAB Table 3 4 IHC scores of IBD positive tissues fixed in 10% NBF over different time period Block # 1 2 3 4 5 6 7 8 9 10 Fixation Time (day) 2 d 7 d 8 d 9 d 15 d 23 d 32 d 39 d 50 d 58 d IHC score Slide 1 Liver 2 2 2,3 3 2,3 3 2,3 1,2 1,2 N/A Kidney 3 3 3 3 3 3 3 2, 3 2,3* 3 Pancreas 3 3 3 3 3 3 3 3 3 3 Slide 2 Liver 2 3 2,3 2 2,3 3 3 1,2 2 N/A Kidney 3 3 3 2,3 3 3 3 2,3 3 3 Pancreas 3 3 3 3 3 3 3 3 3 3 Slide 3 Liver 0 0 0 0 0 0 0 0 0 N/A Kidney 0 0 0 0 0 0 0 0 0 0 Pancreas 0 0 0 0 0 0 0 0 0 0 Th e IHC score was not uniformed i n some sample s i n which two scores w er e given. *In some areas the inclusion bodies were not stained by anti IBDP MAB. N/A : s ample not available

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93 Table 3 5 List of IBD positive non boa constrictors tested with anti IBDP MAB a Sample of IBD cases reported by Stenglein et al. 8 b Sample of IBD cas e reported by Raymond et al. 1 6

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94 Figure 3 1. Pancreas of an IBD positive boa constrictor stained with standardized IHC condition. The tissue was fixed and stained under standardized IHC staining conditions using NovaRED as substrate. The negative control was stained with non specific mouse antibody. The cell nucleus stained dark blue with hematoxylin, and the inclusion bodies are indicated by arrows. A. The negative control in high magnification (400x). The inclusion bodies were not stained. B. The inclusion bodies stained dark red by MAB under high magnification (400x). C. The negative control under low magnification (100x). D. The positive stained pancreas by anti IBDP MAB under low magnification (100x).

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95 Figure 3 2. Mean IHC score of liver, kidney, and pancreas over fixation time in 10% NBF.

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96 F igure 3 3. Differences in IHC staining between Trilogy and double AR treatment on prolong fixed tissues. Tissue fixed up to 50 days (Block 9) in 10% NBF was stained with standardized Trilogy AR treatment (left) and double AR treatment (right). The inclusio n bodies are indicated by arrows. A. In pancreas, the staining of inclusion bodies decreased when double AR was used. B. In kidney, the staining of inclusion bodies decreased when double AR was used.

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97 C. In liver, the staining of inclusion bodies improved w hen double AR was used Figure 3 4. The performance characteristics and their 95% confident intervals (95% CI) of IHC test on paraffin embedded blocks of boa constrictors date from 1990 to 2011. Group1:1990 to 2000; Group2: 2001 to 2011; Overall: Group1 and Group2.

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98 Figure 3 5. IHC staining of embedded tissue that strictly required double AR treatment. The paraffin embedded kidney (left) and liver (right) of an IBD+ boa constrictor were stained with anti IBDP MAB using standard Trilogy treatm ent and double AR treatment. In this sample, the inclusion bodies (blac k arrows) were stained only using double AR treatment White arrow heads showed the pigmented granules in the kidney, and white arrows showed the pigmented macrophage in the liver. A. T he IHC staining of liver and kidney using standard Trilogy treatment. The inclusion bodies were not stained by anti IBDP MAB. B. The IHC staining of liver and kidney using double AR treatment. The inclusion bodies were stained with high intensity.

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99 CHAPTER 4 IMMUNO BASED DIAGNOSTIC TES TS FOR SCREENING IBD Introduction Inclusion body disease (IBD) is a commonly seen disease in captive boa constrictors, and occasiona lly in other non boa species. 1 However, without a screening test the prevalence of IBD is unk nown. The disease is characterized by accumulation of an insoluble antigenic unique 68 KDa protein, called IBD protein (IBDP). 4 Up until now, the diagnosis is based on identifying the characteristic intracytoplasmic eosinophilic inclusion bodies in hematox ylin and eosin (H&E) stained tissue obta ined from necropsy or biopsy. 1 However, the H&E will also stain inclusion bodies of other protein, and because of this a more specific diagnostic test is needed. A well validated specific and sensitive ante mortem sc reening test is still lacking. In IBD, inclusion bodies had been observed in lymphocytes of the infected snakes, and examination of blood film s or buffy coat cytological prepatations had been re commended for diagnosing IBD. 1 But until now there was a lac k of evidence that the inclusion bodies observed in the cytoplasm of peripheral blood cells were IBDP. A mouse monoclonal antibody (MAB) that is highly specific to IBDP in boa constrictors was produced and validated using immunohistochemistical (IHC) stai ning. This antibody can be used to develop an immuno based blood test, which can potentially become a screening test for diagnosi ng IBD. The goal of this study wa s to determine whether the circulating antigen (IBDP) within blood samples can be detected by the mouse anti IBDP MAB and whether there is detectable antibody against IBDP in infected boa constrictors. Being able to diagnose IBD using a blood sample will simplify

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100 and reduce the cost of screening collections of susceptible snakes for this insidious disease. Materials and Methods Animal and Sample Collection The following three different species of captive boid snakes were used in this study: boa constrictor ( boa constrictor ), rainbow boa ( Epicrates cenchria ), and ball python ( Python regius ). The sna kes were randomly selected from several snake collections with unknown prevalence of IBD. Approximately 1 mL of heparinzed whole blood samples were collected by veterinarians via cardiocentesis. The samples were transfer to the University of Florida on ice and stored in 4C until being processed. Sample Preparation Blood film. A blood film of each sample were made on a charged microscopic slide (Thermo Scientific, Shandon Colorfrost Plus Slides), air dried, and fixed in 100% methanol for 15 minutes. After air dried, the fixed blood film was stained with H&E stain, using the protocol described in Chapter 2 ( Cytospin prepara tion and H&E stain, IBDP Purification ) Plasma separation. The whol e blood samples were centrifuged for 10 minutes at 10,000 rpm on a Spectrafuge TM 16M Microcentrifuge (Labnet International). The plasma was separated from the blood cells, collected, and store in 20C for antibody detection experiments. Isolating peripher al white blood cells. The pelleted blood cells were resuspended with 500 L to 1 mL of phosphate buffered saline (PBS) and carefully layered over 300 L of lymphocyte separtation media (LSM) (Cellgro) in Microtainer TM tubes (BD, REF365971 ). The tubes were centrifuged for 30 minutes at 10,000 rpm.

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101 After centrifugation, the red blood cells were pelleted in the bottom of the tube, and the peripheral white blood cells (PWBC) were focused on the layer above LSM. All the supernatants in the tube were collected, a nd the isolated PWBC were pelleted by centrifugation. The PWBC were washed two times with 1 mL of PBS, and pelleted by centrifugation A p ortion of the isolated PWBC was resuspended in PBS, and cytospun onto three charged microscopic slides using a Shandon Cytospin2 Centrifuge. The remaining PWBC were stored in 20C for antigen detection experiments. One of the cytospu n slides with isolated PWBC was fixed in 10% neutral buffered formalin (10% NBF) for 15 minutes, rinsed with running water and air dried. Th e fixed PWBC slides were stained with H&E stain using a protocol described in Chapter 2 (Cytospin preparation and H&E stain, IBDP Purification) The two remaining PWBC slides were stored in a freezer at 20C until being stained by IHC staining using the m ouse anti IBDP MAB. Determine IBD Positive or Negative The H&E stained slides were examined under light microscopes (Figure 4 1 A). If the characteristic cytoplasmic eosinophilic inclusion bodies were identified, the sample was classified as IBD positive (IBD+), if not the sample was classified as IBD negative (IBD ). If the IBD inclusion bodies were identified in either H&E stained blood film or isolated PWBC, the snake was classified as IBD+. Antigen Detection U sing IHC Staining IHC staining on PWBC. The frozen slides with isolated PWBC were thawed, and air dried overnight in room temperature. The next day, the slides were fixed in 4% paraformaldeh yde for 5 minutes, and washed with Tris buffered saline (TBS). Each wash was done by incubating the slides tw o times in TBS, 5 minutes each time. The

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102 slides were incubated in 0.25% Triton X 100 for 5 minutes to permeabilized the cell wall. The slides were washed, and followed by incubating with Peroxo Block TM (Invitrogen) ready to use solution for 45 seconds. The slides were washed again, and the remaining IHC staining procedures were done manually as described in Chapter 3 ( IHC Staining for Paraffin Embedded Tissues ) A negative control slide was stained with non specific mouse IgG (Vector, I 2000) as primary ant ibody that was performed parallel to the testing slide. Result interpretation. The IHC positive was defined as positive staining specifically to the inclusion bodies in comparison to the negative control (Figure 4 1). The IHC negative was defined as absence of specific staining to inclusion bodies in comparison to the negative contr ol. Antigen Detection Using Western Blots Cell lysing and estimating protein concentration. The PWBC pellet was frozen and thawed three times, followed by addition of 1:1 volume of a 2X lysing buffer (LB) containing 2% sodium dodecyl sulfate (SDS) and 10% beta mercaptoethanol (2 ME), and solublized by incubation at 9 5C for 10 minutes. Two microli ters of the solublized cell pellet was removed, diluted 10 fold with water, and the protein concentration was estimated using the Bradford Protein Assay. The Quick Start TM (Bio Rad) Bradford Protein Assay used in this study was modified to increase the solubility of the IBDP. The bovine serum albumin (BSA) standards used in the Bradford P rotein A ssay contained the same background of SDS and 2 ME as the diluted PWBC samples. The protein concentration of the original PWBC lysate was estimated by multiplying the dilution factor to the concentration estimated by Bradford Protein Assay.

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103 Western blots. For each sample, 20 g of PWBC lysate was reduced by incubating with 4X sample buffer (Boston reagent, BP 110R) in 95 C for 10 minutes. The reduced PWBC were resolved on a 10% Tris Glycine sodium dodecyl sulfate polyacrylamide gel electropho resis (SDS PAGE). For each gel, the first well was loaded with molecular weight mark er (Bio Rad, 161 0374), the second well was loaded with semi purified i nclusion body protein preparation (IB prep) as the positive control, and the remaining eight wells each were loaded with a different PWBC isolate. The resolved proteins were transblotte d onto a supported nitrocellulose membrane (Bio Rad, 162 0254), and stained with MemCode TM (Pierce) reversible membrane stain to confirm the protein was properly transblotted. The membrane was destained, and blocked overnight with 5% non fat dried milk dis solved in wash buffer (0.1% Tween20 in PBS). After washing three times with wash buffer, the blot was cut into half, with the top part containing molecular weight >50 KDa, and the bottom part containing molecular weight < 50 KDa. The top blot was detected using the anti IBDP MAB in a dilution of 1:3,000 in dilution buffer (1% BSA in wash buffer), the bottom blot was detected by a commercial anti beta actin antibody (GenScript, A00702) in a dilution of 1:3,000 as loading controls. The blots are washed three times, and incubated with a HRP conjugated goat anti mouse antibody (Bio Rad, 170 6516) in a 1:3,000 dilution. Following three washes, the reactive bands were visualized by developing with Opti 4CN TM (Bio Rad) colorimetric substrate kit. The blots were rin sed with water and air dried. Result interpretation. The isolated PWBC sample that showed a 68 KDa reactive band detected with the mouse anti IBDP MAB was interpreted as western blot positive. The sample that did not show a 68 KDa reactive band, but was a ble to detect beta actin

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104 was interpreted as western blot negative. The positive band of 42 KDa detected by anti beta actin antibody demonstrated that equivalent amount of PWBC was loaded in each well (Figure 4 2). Antibody Detection Using Western Blots The collected plasmas from boa constrictors were tested for the presence of anti IBDP antibody. This test assay utilized the previously produced and validated mouse anti snake IgG monoclonal antibody, hereaf ter is refer as anti snake Ab 1 9 The application of anti snake Ab, followed by reaction to goat anti mouse antibody was used to determine if IBD+ produced anti IBDP antibody and could be visualized by Opti 4CN TM Standardizing anti snake Ab. The previously produced hybridoma clone HL1785 was thawed and cult ured, and the anti snake Ab was harvested and purified using the protocol described in Chapter 2 ( Monoclonal antibody purification, Polyclonal and Monoclonal Anti IBDP Antibody Production ) To confirm the reactivity of the anti snake Ab to the IgG of boa c onstrictors, the plasma samples were tested on western blots. Plasma of three boa constrictors were resolved on a SDS PAGE, transblotted onto a nitrocellulose membrane using the method described. The membrane was cut into portions, with each portion contai ning three resolved plasma samples. Each portion was detected with a specific dilution of anti snake Ab, and one portion was not treated with the anti snake Ab served as the negative control. The membranes were incubated by HRP conjugated antibody and deve loped as described in Antigen Detection using Western Blots. Test strip preparation. Three hundred micrograms of semi purified IB prep was reduced by incubating with 4X sample buffer in 95C for 10 minutes. The sample was

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105 resolved on a two well 10% Tris G lycing SDS PAGE, the reference well was loaded with molecular weight marker. The resolved protein was transblotted onto a nitrocellulose membrane, and the transblot was confirmed with reversible membrane stain. After being blo cked and washed, the membrane was cut into 5 mm wide strips. Antibody detection. For each plasma sample to be tested, two (1:500, 1:1,000) or three (1:100, 1:500, 1:1,000) different dilutions were prepared by diluting with 3 mL of diluting buffer. A positive control strip was performe d by using anti IBDP MAB alone in a dilution of 1:3,000. A negative control strip was performed using diluting buffer alone, using the same procedure as the strips receiving snake plasmas. Each strip was incubated with diluted plasma or anti IBDP MAB inside of a 15 mL nunc TM t ube (Thermo Scientific, 362695), and throughout the antibody detection procedures each strip was incubated and washed individually inside the tube to avoid cross contamination. The tubes were la id down in a container and rocke d ho rizontally on a rocker for 1 hour. The diluted plasma or anti IBDP MAB was ensured to cover the strip at all time during the incubation. The strips were washed three times with 5 mL of wash buffer on the rocker for 5 minutes. Next, each strip was incubated with 3 mL of anti snake Ab using a dilution of 1:5,000 or 1:30,000 for 1 hour. Then the strips were washed another three times, and incubated with HRP conjugated anti mouse IgG in a dilution of 1: 3,000 for 1 hour. After another three washes, the strips w ere developed by Opti 4CN TM Until the positive control strip was fully developed, the strips were rinsed with water, and air dried. Result interpretation. The tested plasma that reacted to the 68 KDa IBDP band aligned with the molecular weight indicated by the positive control was interpreted as

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106 positive. The plasma that did not react to the 68 KDa IBDP band was interpreted as negative. Standardizing the antibody detection system. Prior to testing the plasma samples of IBD positive and negative samp les, the optimal concentrations of each antibody were determined by western blots. Plasma samples of three index IBD positive boa constrictors were tested on transblotted IBDP, using combinations of plasma in three dilutions (1:100, 1:500, 1:1,000) and sna ke IgG Ab in two dilutions (1:1,000 and 1:500) to ensure the optimal conditions for antibody detection were covered. Agreement and Association Analysis Antigen detection. The sam ples that were tested positive f or IBD in H&E stained blood film or isolated P WBC were defined as IBD+ by H&E stain, otherwise were classified as IBD by H&E stain. The samples te sting positive f or IBD in IHC stain or western blots were defined as IBD+ by immuno based tests, otherwise were classified as IBD by immune based tests. T he agreement between H&E stain and the detection of IBD antigen by immune based test was estimated by using kappa statistic with standard proto col provided by Martin et al 20 Kappa values 0 was considered as no agreement; < 0.4 was considered as poor agreement; 0.4 to 0.75 as fair agreement; > 0.75 as excellent agreement; and 1 as perfect agreement. The 95% confident interval (95% CI) will be calculated by software Win Episcope 2.0. Antibody detection. The samples that tes ted positive of IBDP reactive antibody in the plasma were defined as positive in antibody detection. The samples that tested negative of IBDP reactive antibody in the plasma were defined as negative in antibody detection. The agreement between H&E stain an d antibody detection was estimated by kappa statistic as described for antigen detection.

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107 Results Antigen Detection A total of 78 blood samples were screened for the presence of IBDP, which included 39 boa constrictors, 20 rainbow boa and 19 ball pythons. The test results are listed in Table 4 1 and summarized in Table 4 2. Antigen detection in boa constrictors. In boa constrictors, inclusion bodies were detected in 13 (of 38) H&E stained blood films, and in 16 (of 39) H&E stained isolated PWBC. When detec ted with anti IBDP MAB, 16 (of 39) stained positive in IHC stain, and 15 (of 39) tested positive using western blots (Table 4 2). Using isolated PWBC preparation, inclusion bodies were found in three additional cases that tested negative in direct blood fi lms (Table 4 1). In one case (IB10 53), the inclusion bodies were very sporadic within the blood cells that were only detected in IHC staining (Table 4 1). Antigen detection in rainbow boas. In rainbow boas, inclusion bodies were detected in one (out of 2 0) H&E stained blood films, and three (out of 20) H&E stained isolated PWBC. No sample from rainbow boas tested positive in IHC staining and western blots. Antigen detection in ball pythons. In ball pythons, no inclusion bodies were detected in all 19 blo od samples tested. No samples from ball pythons test ed positive in IHC staining or W estern blots. Agreement between H&E stain and antigen detection. The diagnosis made by H&E stain and immune based tests were summarized in Table 4 3. The agreement in rain bow boas and ball pythons were not analyzed, since the reactivity of mouse anti IBDP MAB in rainbow boas were not validate, and in ball pythons no IBD positive cases

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108 were found. The agreement between H&E stain and immuno based tests determined by Kappa sta tistic was 0.894 (95% CI 0.58, 1.00), indicating an excellent agreement. Antibody Detection Standardizing the detection system. The reactivity of the purified anti snake Ab (10 mg/ mL ) to the IgG of boa constrictors were confirmed by western blots. The reac tive concentrations of anti snake Ab ranged from 3.33 g/ mL (dilution of 1: 3,000) to 0.33 g/ mL (dilution of 1: 30,000) (Figure 4 3). When being used for detecting antibody in the index samples, the anti snake Ab showed better sensitivity in 2 g/ mL (dilution of 1:5,000) than 0.33 g/ mL (dilution of 1: 30,000) without introducing non specific ba ckground (Figure 4 4). The plasma sample in the dilution of 1:100 showed darker non specific background, than the dilution of 1:500 and 1:1,000. The remaining plasma samples were tested with dilutions of 1:500 and 1:1,000 using anti snake Ab with a dilution of 1:5,000. Antibody detection in boa constrictors. The 39 boa constrictors (16 IBD+ and 23 IBD ) that had been tested f or the presence of IBDP, plasma coll ected from these snakes were test ed for the presence of anti IBDP antibody. By antigen detection in the isolated PWBC using anti IBDP MAB, 16 of the 39 snakes tested positive of IBDP, and 23 of the 39 snakes tested negative of IBDP. No antibody against IBD P antigen was detected in the plasmas of all 39 boa constrictors. Agreement between H&E stain and antibody detection. The diagnosis made by H&E stain and the results of antibody detection were summarized in Table 4 4. The agreement between H& E stain and immune based tests determined by Kappa statistic was 0, indicating no agreements.

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109 Discussion Modified Bradford Protein Assay Due to the insolubility of IBDP, IBDP needs to be treated by heating with reducing reagents at 95C prior to estimating protein concentration. The presence of some reducing reagents may interfere with coloration of the protein binding dye in Bradford Protein Assay, thus interfering in the estimation of protein concentrations. This issue had been discussed in Chapter 2 ( Ina ccurate Estimation of Protein Concentration ) In this study, using a 2X LB the cell pellets were solubilized by heating in 1% SDS and 5% 2 ME, followed by diluting the sample 10X before binding with the dye for Bradford Protein Assay. Thus, the diluted sam ple contained a background of 0.1% SDS and 0.5% 2 ME that was found not to cause severe interference in Bradford Protein Assay 10 21 The standard BSA protein of known concentrations contained the same background of LB ( 0.1% SDS and 0.5% 2 ME) as the 10 fo ld diluted testing samples, and the standard curve made by the BSA standards of known concentrations was used to estimate the pro tein concentration of the testing samples. The BSA standards with or without LB background were heated or unheated to demonstra te the differences in standard curve (Figure 4 5). Because the heated and unheated LB containing standards formed identical curves, the later standard curve (unheated standard) was used to estimate the protein concentration of isolated PWBC. Using this modified Bradford Protein Assay, protein concentration of the IBDP containing samples could be estimated much more precisely and this improved the accuracy of the assay. This modi fied assay may benefit other researchers that needed to analyze insoluble proteins.

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110 Assessment of U sing Blood Tests for Screening IBD Antigen detections H&E stain. Of the 39 boa constrictors examined using H&E stain, three additional cases with inclusion bodies were detected in the isolated PWBC preparation, that were not detected in the blood film. The isolated PWBC preparation increased the sensitivity (16/39 or 41%) of detecting inclusion body bearing cells compared to the sensitivity of blood films (1 3/38 or 34.2%). In the case of IB10 43 and IB10 44, inclusion bodies having very small and irregular shapes were sporadically seen in the blood films. They were difficult to identify and were missed in the H&E stained blood films. The isolated PWBC prepara tion concentrated the blood cells and improved the ability to identify cells having IBD inclusion bodies. Therefore, isolated PWBC preparations increase the ability of observing inclusion body bearing cells. In this study, red blood cells lysis occurred w hen films were fixed in 10% NBF. The morphology of the blood cells were better preserved when films were fixed in 100% methanol, and the color of cells stained with H&E appeared very similar to the cells fixed in 10% NBF. Fixing with 100% methanol is more versatile, because the fixed slides can also be stained with Wright Geimsa stain. Possible false positives in H&E stain. In H&E stain, sometimes it is difficult to differentiate IBD inclusion bodies from other intracellular protein. Especially in the case of aged sample s the phagocytosed hemoglobin may appear to be similar to IBD inclusion bodies. In one boa constrictor (IB10 75) and three rainbow boas (IB10 67, IB10 78 and IB10 81) inclusion bodies were observed in H&E stained PWBC, but did not stain with IHC. The inclusion bodies within these cases were found in monocytes, not in lymphocyte s where IBD inclusion bodies were more commonly found. Further,

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111 unlike most IBD cases, there were no signs of lymphocytosis in these cases. Thus, these samples are susp ected to be false positives caused by prolong storage or handling. Immuno based tests. In boa constrictors, except for 2 cases (IB10 53 and IB10 75), the test result of IHC staining and western blot all correlated with the diagnosis in H&E stained PWBC. In the case of IB10 53, the inclusion bodies were extremely sporadic (less than 5 in one preparation) that only IHC staining was able to detect IBDP. Using western blots, IBDP was readily detectable when using only 18 g of PWBC lysate. All the samples that were negative in western blots (20 g per load) were repeated with 30 g per loaded, and the results were consistently negative. In rainbow boas, the PWBC tested positive in H&E staining did not test positive in IHC staining or western blot. This may be du e to the following: 1) MAB may not cross react to IBDP in rainbow boas as previously discussed in Chapter 3 ( Cross Reactivity Among Non Boa Constrictors ) ; 2) the inclusion bodies observed in the samples of rainbow boas may represent some other accumulated protein that is unrelated to boa constrictor IBDP. Although the sensitivity of the immune based tests were relevant to the H&E stain in isolated PWBC, the immune based tests were much more specific in detecting IBDP, and provided various options for confir ming IBD diagnosis. For blood samp les with very small volume (0.2 to 0.5 mL ), the IHC staining using cytospin preparation of PWBC is a better choice of diagnosis. Western blots may be a more cost effective method when screening large numbers of samples wit h larger volumes. Or it can serve as a confirmatory test for samples that test IHC positive.

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112 Possible false positives in IHC stain. Although the inclusion bodies are very easy to identify in the IHC stain, the negative cases are sometimes difficult to def ine. The granules within heterophils sometimes stained positive in IHC stain, possibly caused by the reaction of the endogenous peroxidase to the substrate. The results needed to be evaluated carefully by comparing against the negative control, which also contained the non specific background staining. Possible false negatives in blood tests. Currently, without a proper transmission study, the disease course of IBD is unknown. Based on the inoculation studies done by Wozniak et al. and Schumacher et al. 2, 4 the inclusion bodies were visible in visceral organs approximately 10 week post infection. But the presence of inclusion bodies in blood cells were not determined. To date, there is no knowledge about whether all IBD infected snake will develop inclu sion bodies in the circulating blood cells. It is possible that a snake having IBD may tested negative by the blood tests, if inclusion bodies are not yet in the circulating blood cells. If a snake tested negative by the blood test, it is r ecommended to re test the snake 10 weeks later. Antibody detections Of 39 plasma samples of boa constrictors tested, which included 16 IBD+ and 23 IBD samples, none showed reactivity to the 68KDa IBDP. This finding indicated that no snakes had circulating antibody agains t IBDP, although the snakes are clearly infected with IBD. This agreed with the lack of immune responses at the histological level observed in most IBD snakes. However, when a recently made bulk IB prep was used, all of the tested plasma reacted to a band that was slightly above 50 KDa (Figure 4 6). It is possible the presence of this antibody in boa constrictor is not specific for IBD. It is also possible that additional proteins, other than the 68 KDa IBDP, may be present

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113 within the semi purified inclusio n bodies. While infected boas did not exhibit an immune response against IBDP, they did have antibody response to another protein(s) within the inclusion bodies. The reaction against the 50 KDa protein detected in the IBD boa constrictors may be due to ex posure to another structural protein in the causative agent of IBD. More work is needed to establish the significance of this finding. Conclusions The results of antigen detection in boa constrictor demonstrated that the observed inclusion bodies in H&E st ain agreed with the detection of IBDP in the immune based tests. Using antigen detection in whole blood samples can become the first line screening test for diagnosing IBD in boa constrictors. But the results of H&E stain need to be interpreted carefully. The confidence of the diagnosis will increase when the sample is confirmed using the immuno based test developed in this study. No antibody against IBDP was detected in the IBD positive boas, and there were no agreement between the H&E stain diagnosis and the detection of antibody against IBDP. The results of antibody detection in boa constrictors showed only moderate agreement. More investigation is needed to determine whether antibody detection can be used for screening IBD. Table 4 1 Summa rized resu lts of antigen detection in blood samples of three snake species Boa constrictors n=39 Rainbow boas n=20 Ball pythons n=19 Test Result Positive Negative Positive Negative Positive Negative Blood film H&E 13* 25 1 19 0 19 PWBC H&E 16 23 3 17 0 19 PWBC IHC 16 23 0 20 0 19 PWBC WB 15 24 0 20 0 19 *One sample the blood film was not available

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114 Table 4 2 R e sul ts of antigen detection in blood samples of three snake species Boa constrictors: n=39 Rainbow boas: n=20 Ball python: n=19 Test ID Co B F HE WBC HE WBC IHC WBC WB Test ID Co B F HE WBC HE WBC IHC WBC WB Test ID Co B F HE WBC HE WBC IHC WBC WB IB10 21 A Neg Neg Neg Neg IB10 26 A Neg Neg Neg Neg IB10 36 A Neg Neg Neg Neg IB10 22 G Neg Neg Neg Neg IB10 27 A Neg Neg Neg Neg IB10 37 A Neg Neg Neg Neg IB10 23 A Neg Neg Neg Neg IB10 28 A Neg Neg Neg Neg IB10 38 A Neg Neg Neg Neg IB10 25 B Pos Pos Pos Pos IB10 29 A Neg Neg Neg Neg IB10 39 A Neg Neg Neg Neg IB10 31 A Pos Pos Pos Pos IB10 30 A Neg Neg Neg Neg IB10 58 A Neg Neg Neg Neg IB10 32 A Neg Neg Neg Neg IB10 64 A Neg Neg Neg Neg IB10 59 A Neg Neg Neg Neg IB10 33 A Neg Neg Neg Neg IB10 65 A Neg Neg Neg Neg IB10 60 A Neg Neg Neg Neg IB10 34 A Neg Neg Neg Neg IB10 67 A Pos Pos Neg Neg IB10 62 A Neg Neg Neg Neg IB10 35 A Pos Pos Pos Pos IB10 77 A Neg Neg Neg Neg IB10 86 A Neg Neg Neg Neg IB10 41 A Neg Neg Neg Neg IB10 78 A Neg Pos Neg Neg IB10 92 A Neg Neg Neg Neg IB10 42 A Neg Neg Neg Neg IB10 79 A Neg Neg Neg Neg IB10 93 A Neg Neg Neg Neg IB10 43 A Neg Pos Pos Pos IB10 80 A Neg Neg Neg Neg IB10 94 A Neg Neg Neg Neg IB10 44 A Neg Pos Pos Pos IB10 81 A Neg Pos Neg Neg IB10 95 A Neg Neg Neg Neg IB10 45 A Neg Neg Neg Neg IB10 87 A Neg Neg Neg Neg IB10 96 A Neg Neg Neg Neg IB10 46 B Pos Pos Pos Pos IB10 88 A Neg Neg Neg Neg IB11 06 A Neg Neg Neg Neg IB10 47 B Pos Pos Pos Pos IB10 89 A Neg Neg Neg Neg IB11 07 A Neg Neg Neg Neg IB10 48 B Pos Pos Pos Pos IB10 90 A Neg Neg Neg Neg IB11 08 A Neg Neg Neg Neg IB10 49 B Neg Neg Neg Neg IB10 91 A Neg Neg Neg Neg IB11 09 A Neg Neg Neg Neg IB10 50 B Neg Neg Neg Neg IB11 12 G Neg Neg Neg Neg IB11 10 A Neg Neg Neg Neg IB10 51 B Pos Pos Pos Pos IB11 24 H Neg Neg Neg Neg IB10 52 B Pos Pos Pos Pos IB10 53 B N/A Neg Pos Neg IB10 54 B Pos Pos Pos Pos IB10 68 A Pos Pos Pos Pos IB10 75 A Neg Pos Neg Neg IB10 76 A Neg Neg Neg Neg IB10 84 A Neg Neg Neg Neg IB10 85 A Neg Neg Neg Neg IB11 11 F Neg Neg Neg Neg IB11 14 C Neg Neg Neg Neg IB11 15 C Pos Pos Pos Pos IB11 17 A Neg Neg Neg Neg IB11 18 A Neg Neg Neg Neg

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115 Table 4 2. Continued Boa constrictors: n=39 Rainbow boas: n=20 Ball python: n=19 Test ID Co B F HE WBC HE WBC IHC WBC WB Test ID Co B F HE WBC HE WBC IHC WBC WB Test ID Co B F HE WBC HE WBC IHC WBC WB IB11 22 E Pos Pos Pos Pos IB11 25 D Neg Neg Neg Neg IB11 26 D Pos Pos Pos Pos IB11 27 A Neg Neg Neg Neg IB11 28 A Neg Neg Neg Neg Co: snake collection of sample origin; BF: blood film; HE: H&E stain; WBC: peripheral white blood cell s ; IHC: immunohistochmistry stain; WB: Western blot; Pos: positive; Neg: negative. Table 4 3. Summarized results of H&E and Immuno tests Boa constrictors n=39 Rainbow boas n=20 Ball pythons n=19 Test Result Positive Negative Positive Negative Positive Negative H&E Stain 16 23 3 17 0 19 Immuno based test 16 23 0 20 0 19 Table 4 4. Agreement s between diagnosis by H& E stain and immun o based antigen detection tests in boa constrictors Immuno+ : immune based test positive ; Immuno : immune based test negative Table 4 5. Agreement s between diagnosis by H&E stain and antibody detection test in boa constrictors Antibody+: detected positive of antibody; Antibody : detected negative of antibody.

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116 Figure 4 1. Cytospin preparation of PWBC isolated from a boa constrictor. A. The IBD inclusion bodies (arrows) stained eosinophilic in H&E stain. B. Using IHC staining, the IBD inclusion bodies (arrows) stained dark brown with substrate DAB. C. The PWBC st ained with nonspecific mouse antibody as negative control for IHC staining. The inclusion bodies were not stained.

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117 Figure 4 2. Antigen detection by western blot using PWBC isolated from 8 boa constrictors. Lane 1 contained molecular weight marker ( Bio R ad #161 0347), lane 2 contained 20 g purified IBDP as positive control. Lane 3 to 10 each contained 20 g of PWBC lys ate from a boa constrictor. The top portion of membrane was detected by anti IBDP MAB, the thick arrow indicate the molecular weight of IBDP (68 KDa). The bottom portion of membrane was detected by anti beta actin antibody as loading control, showing that relevant amount of cell were loaded in each lane. The thin arrow indicated the molecular weight of beta actin (42 KDa). Lane 6 and 8 wer e interpreted as western blot positive, whereas others were interpreted as negative.

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118 Figure 4 3 Western blots showing t he react ivity of purified anti snake Ab (HL1785) to three plasma samples of boa constrictors. Lane 1 and 11 contained molecular weight marker. P lasma sample of three boa constrictors (a, b and c) were used l ane 2 to 10 and lane 12 to 17 each contained 10 g of resolved plasma from boa constrictor s. The purified anti snake Ab consistently react ed t o the light chain (arrow) of three boa constrictors The reactive concentrations ranged from 1 g / mL (dilution of 1:3 ,000) (lane 2 to 4) to 0.3 g / mL (dilution of 1:3 0, 000 ) (lane 12 to 14) Lane 15 to 17 were detected with PBS as negative controls.

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119 Figure 4 4 Detection of antibody against IBDP with in plasma of an IBD positive boa constrictor using western blots A. A ntibody detection using anti snake Ab with a dilution of 1:3 0, 000 dilution (0.3 g / mL ) and developed for 2 hours B. A ntibody detectio n using anti snake Ab with a dilution of 1:5 ,000 (2 g / mL ) and developed for 1 hour. The test strips were purposely over developed to ensure detection of all possible reactivity. The anti snake Ab with a dilution of 1:5 ,000 was more sensitive than the dilution of 1:3 0,000 that reached the maximal development in half of the incubation time. The anti IBDP MAB was used as positive controls in lane 2 and 7 to label the molecular weight of IBDP (68KDa) indicated by arrows Higher backgrounds can be seen in the strips detected with 1:100 diluted plasma, but no reactions specific to the 68 KDa IBDP band was detected in the condition listed. The test result of this plasma sample was interpreted as negative of anti IBDP antibody.

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120 Figure 4 5. The BSA standard curves with or without the background of LB measured by Bradford Protein Assay.

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121 Figure 4 6 Detection of antibody against IBDP within plasma of 4 IBD positive boa constrictors on Western blots. A membrane that was trans blotted with resolved IB prep, and cut into strips containing molecular weight marker on lane 1 and 20 g isolated inclusion bodies on each strip (lane 2 10). Lane 2 was detected with anti IBDP MAB as positive control reacted to the 68 KDa IBDP (arrow head). The remaining strips w ere d etected with plasma of 4 IBD positive boa constrictors, IB10 43 (lane 3 and 4), IB10 51 (lane 5 and 6), IB10 52 (lane 7 and 8), IB10 54 (lane 9 and 10), with dilutions of 1:500 and 1:1000. T h e plasma did not show reaction to the 68 KDa IBDP band, but react ed to a protein band approximately 50 KDa (arrow).

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122 CHAPTER 5 SEQUENCING INCLUSION BODY DISEASE PROTEIN Introduction Inclusion body disease (IBD) is a commonly seen disease in captive boid snakes. 1 This disease is characterized b y the accumulation of an antigenic 68 KDa protein, inclusion body disease protein (IBDP), which formed aggregates as insoluble inclusion bodies in the cytoplasm of affected tissues. 4 While several viruses have been identifi ed in snakes with IBD, 2, 4, 6, 7 a firm connection between these viruses and the formation of IBDP has not been made. Recently, three strains of arena like viruses within IBD positive tissues were identified using deep sequencing methods and bioinformatics analysis. 8 Although t he y have a genome organizat ion of a typical arenavirus, they belonged to a lineage that is very distinct from other known arenaviruses. 8 Additionally, they have a glycoprotein that is more closely related to filoviruses rather the known arenaviruses. 8 Until the mor phology of these viruses is verified, they are currently recognized as arena like viruses. The se arena like viruses were considered the candidate etiological agents for IBD. 8 The genomic sequences of two arena like viruses were annotated and available in t he NCBI GenBank database, which were California Academy of Science Virus (CASV) derived from IBD positive annulated tree boas and Golden Gate Virus (GGV) derived from IBD positive boa constrictors. 8 Stenglein et al. also produced a polyclonal antibody agai nst a synthetic peptide that was derived from the C terminus of the predicted nucleoprotein (NP) in GGV. 8 The polyclonal antibody reacted to the IBD inclusion bodies by immunohistochemistry and western blots. 8 However, direct amino acid sequencing of IBDP was not performed in this study.

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123 Up until the above recent findings revealing a link between arena like viruses and IBDP, it was unknown if IBDP represented a host protein that was induced by the causative agent or whether IBDP was a protein that was a com ponent of the causative agent. Sequencing IBDP will determine whether the inclusion b odies were derived from the GGV NP. Knowing the protein sequence of IBDP will help us better understand the nature of IBDP and its accumulation. A monoclonal anti IBDP ant ibody (anti IBDP MAB) was developed, and validated by immunohistochemical (IHC) staining. Due to the high specificity of this antibody to IBDP, it can be utilized to purify the soluble form of IBDP for protein sequencing. With knowledge of the protein sequ ence, better diagnostic tests can be developed and used as a tool for screening collections of snakes for IBD. Material and Methods Protein Preparation Sem i purified IBD inclusion bodies The semi purified IBD inclusion bodies (IB prep) using methods described in Chapter 2 ( IBDP Purification ) were reduced using methods described in Chapter 4 ( Antigen Detection Using Western Blots ) and resolved on a SDS PAGE or a NuPAGE. Each well was loaded with reduced IB prep with its maximum loading capacity. Immuno precipitation (IP) The frozen supernatants of the liver homogenates (boa 0906) made by the procedure described in Chapter 2 (IBDP Purification) were thawed. The protein concentration was determined by the method described in Chapter 4 (Antigen Dete ction Using Western Blots) For antibody binding, 30 L of protein A/G coated beads (Santa Cruz Biotechology, sc 2003) was added to each 1.5 mL tube, and incubated with 300 L of Tris buffered saline (TBS) containing 10 g of anti IBDP MAB.

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124 The tubes were placed on ice and agitat ed overnight. Next day, the beads were pelleted by centrifugation for 1 minute at 1,000 rpm in room temperature on a Spectrafuge TM 16M Microcentrifuge (Labnet International, Edison, NJ). The supernatant of each tube was removed, fol lowed by washing the beads three times. Each wash was done by resuspending the beads in 300 L RIPA buffer (Thermo Scientific) agitated for 15 minutes, and discarded the supernatant. Except for the negative controls, each tube containing anti IBDP MAB boun d beads were incubated with 500 L of IBD positive liver supernatant in RIPA buffer with a final protein concentration of 1 g/ mL For the IP negative control tube, the beads were incubated with RIPA buffer without liver supernatant. For the sham control t ube, the beads were incubated with 500 L of IBD negative supernatant in RIPA buffer with a final protein concentration of 1 g/ mL The tubes were placed on ice and agitated overnight. The following day, the supernatant of each tube was removed, and the be ads were washed four times with RIPA buffer. Finally, the beads in each tube were resuspended in 40 L of 1X LDS NuPAGE sample buffer (Novex) with addition of 1 L 1M d ithiothreitol (DTT). The tubes were heated at 95 C for 10 minutes, and the supernatants were resolved on a 4 12% NuPAGE gel (Novex). Each well was loaded with reduced IB prep with its maximum load ing capacity. Purified protein bands. The resolved IBDP derived from IB prep or IP was visualized by SimplyBlu e TM stain (Novex), using s protocol. The IBDP protein bands were cut out and two to three bands were pooled in a tube, and stored at 80 C for later use. Protein confirmation by western blots. To confirm the quality of protein purification, a portion of the gel containing resolved IBDP was transblotted and detected

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125 by anti IBDP MAB on western blots using the procedure described in Chapter 4 (Antigen Detection Using Western Blots) Mass S pectrometry S ample P reparation Reduction and a lk ylation. The gel bands containing purified IBDP protein were washed/distained with 50% sequence grade acetonitrile ( A C N ), then once with 100% A C N and twice with 50% ACN in 50 mM ammonium bicarbonate (AB) buffer pH 8.4 Each wash was done by agitating the gel with 200 L of above solutions for 30 minutes. The protein was reduced by incubating the gel bands in 100 L of 45 mM DTT in A B buffer at 95 C for 10 minutes, followe d by incubating the gel bands at 60 C for 50 minutes. The gel bands were chilled, and the DTT was removed. The alkylation was done by incubating with 100 L of 100 mM iodoacetamide in AB buffer for 1 hour at room temperature in dark. The iodoacetamide was removed, the gels were washed three t imes with 50% A C N in AB buffer, and dried completely by a CentriVap Vacuum Concentrator (Laconco, Kansas City, MO). Enzyme digestion. The dried gels were rehydrated either with 10 L of 12.5 ng/ L trypsin (Promega, V5111), chymotrypsin (Promega, V106A), o r Asp N (Roche, 11 054 589) in digestion buffer (100 mM Tris, 10mM CaCl 2 pH 8.0). The tubes were placed on ice for 45 minutes, and more enzyme solution was added as needed until the gels became completely rehydrated. The rehydrated gels were covered with 25 L digestion buffer. For complete digestion, the trypsin and Asp N digestion were incubated overnight at 37 C and the chymotrypsin digestions were incubated overnight at room temperature. For partial digestion, the gels were incubated for 4 hours only. The digestion was stopped by the addition of 5% acetic acid to a final concentration of 0.5%,

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126 and the tubes were store at 80 C until extraction. For each digestion condition, a gel band containing bovine serum albumin (BSA) was used as standard control, and was digested parallel with the IBDP samples using the same protocol. Peptide Extraction. The supernatants containing digested peptides were collected from the thawed samples in new clean tubes. The gels were extracted twice with 200 L of 50% A C N in 5% formic acid (FA), vortexed for 15 minutes, and the collected supernatant was combined with the previously collected supernatants. The extracted peptides were lyophilized in a CentriVap Vacuum Concentrator and stored at 80 C T a nd em Mass Spectrometry The dried peptide samples were resuspended in a loading buffer containing 3% ACN, 1% a cetic a cid, and 0.1 % trifluoranoacetic acid The extracted peptides were then injected onto a capillary trap (LC Packings PepMap) and desalted for 5 min with a flow rate 10 L /min ute of 0.1% v/v acetic acid. Samples were loaded onto an LC Packing C18 Pep Map HPLC column. The elution gradient of the HPLC column started at 3% solvent A, 97% solvent B and finished at 60% solvent A, 40% solvent B for 60 min for protein ident ification. Solvent A consisted of 0.1% v/v acetic acid, 3% v/v ACN, and 96.9% v/v H 2 O. Solvent B consisted of 0.1% v/v acetic acid, 96.9% v/v ACN, and 3% v/v H 2 O. Liquid chromatography tandem mass spectrometry (LC MS/MS) analysis was carried out on a hybri d quadrupole TOF mass spectrometer (QSTAR, Applied Biosystems, Framingham, MA). The focusing potential and ion spray voltage was set to 275 V and 2600 V, respectively. The information dependent acquisition (IDA) mode of operation was employed in which a su rvey scan from m/z 400 1200 was

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127 acquired followed by collision induced dissociation (CID) of the three most intense ions. Survey and MS/MS spectra for each IDA cycle were accumulated for 1 and 3 s econds respectively. Sequence Analysis Protein identificati on with Mascot and Scaffold Tandem mass spectra were extracted by ABI Analyst version 1.1. All MS/MS samples were analyzed using Mascot (Matrix Science, London, UK; version 2.0.01). Mascot was set up to search N CBInr database assuming the digestion enzym e trypsin. Mascot was searched with a fragment ion mass tolerance of 0.30 Da and a parent ion tolerance of 0.30 Da. Iodoacetamide derivative of Cys, deamidation of Asn and Gln, oxidation of Met, were specified in Mascot as variable modifications. Scaffold (version Scaffold 01 06 03, Proteome Software Inc., Portland, OR) w as used to validate MS/MS based peptide and protein identifications. Peptide identifications were accepted if they could be established at greater than 95.0% probability as specified by the P eptide Proph et al gorithm 22 Protein identifications were accepted if they were established at greater than 99.0% probability and contain at least 2 identified uni que peptides. Protein probabilities were assigned by the Protein Proph et al gorithm 2 3 Protein identification with Protein Pilot v4.2 Using Protein Pilot v4.2 software T he IPI bovine database (60814 total entries) was merge d with eight protein sequences t hat derived from genome of two Arenavirus s train s California Academy of Sciences virus ( CASV ) and Golden Gate virus ( GGV ) GenBank accession numbers JQ717261 to JQ717264 The eight protein sequences were formatted in the same way as the IPI database and the re were assigned IPI like numbers composed by the date of database creation followed by a later the name of the protein was input manually and all other

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128 parameters were defined as 0000 T he identification of proteins was performed using Software 4.2. The searching parameters were set as: protein identification cystein alkylation iodoacetamide, trypsin (or chymotrypsin, or Asp N) digestion and the identification focus for biological modifications. Results Protein Preparation The IB p reps made from liver of two IBD+ boa constrictors (0876 and 0906) were reduced, and the resolved 68 KDa IBDP gel bands were collected. Utilizing the anti IBDP MAB in IP procedure, a soluble form of IBDP within the supernatant of liver homogenate was demons trated (Figure 5 1) The quality of IP was monitored by the SimplyBlu e TM protein stain on the gels and western blots detected with anti IBDP MAB. The negative control showed adequate binding of anti IBDP MAB to the protein A/G beads. The IBDP was detected w ithin the IP product from the IBD+ liver, but not detected within the IP product from the IBD liver. The resolved 68KDa IBDP gel bands from IP preparations were collected. The protein bands were digested with either trypsin chymotrypsin, or Asp N, with digestion of a separated BSA control gel using the same protocol that was performed parallel with the IBDP samples. Sequence Analysis Th e t andem mass spectra d erived from digested peptides of each sample were searched against the NCBI nr database (upd ated July 2012). The peptides derived from BSA controls matched with the BSA sequence in the database with moderate to high coverage, which indicated a successful enzyme digestion, peptide extraction, and the adequate MS/MS analysis. The peptides derived from IBDP samples did not match with any known protein in the database.

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129 Searching the tandem mass spectra within the in house protein library built in Protein Pilot v4.2 software containing the complete Bovine proteins and 8 predicted protein s derived from t he genome s of CASV and GGV (each virus has 4 proteins: z protein, L protein, NP glycoprotein) The peptides derived from BSA controls matched with the BSA protein sequence within the library with high coverage. Each of the digested IBDP samples had produc ed peptide ( s ) which in various degrees the tandem mass spectra matched w ith in the predicted protein sequence of GGV NP (Figure 5 2, 5 3) Besides matching with GGV NP, the peptides derived from digested IBDP samples did not match with other predicted vira l protein s in the library which included NP of the related annulated tree boa strain ( CASV ) Compared to the IBDP samples derived from IB preps (Figure 5 2) the IBDP samples derived from IP generated more matching peptides that had coverage above 95% and more overall coverage of amino acids across the predicted GGV NP sequence (Figure 5 3) The entire length of the predicted GGV NP is 591 amino acids (a.a.) long (Figure 5 4). When combining all the matching peptides detected, a total of 535 a.a. within th e predicted GGV NP had been seen by MS/MS. Thus, t he combined coverage of the matching peptides derived from all sequenced IBDP samples indicated an overall coverage of 90.5% (535/591) to the predicted GGV NP sequence (Figure 5 4). Discussion Challenges in S equencing IBDP and the Improvements in Methodology Between the early 2000s and 2010, despite multiple attempts at sequencing IBDP, the protein identity and the origin of IBDP could not be determined. The most challenging aspects of sequencing and ident ifying a novel protein are the ability to obtain a high quality purified protein sample and the availability of a homolog protein or

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130 reference sequence in the database. Although Edman degradation (N terminal sequencing) is the ideal method for identifying protein sequences of a novel protein, in which a database or a reference sequence is not requ ired, 24, 25 m ultiple attempts to direct sequence IBDP by Edman degradation during the past 10 years all turned out unsucc essful. With the preparation of pooling m ultiple transblotted IBDP protein bands in one sample, no significant amino acid signal s was generated after Edman degradation This suggested that the N terminal of IBDP may be blocked, and the Edman degradation can not be used for sequencing IBDP. Thus, tandem mass s pectrometry became the next best sequencing method available for IBDP sequencing. Initial studies in the early 2000s used crude materials with less degree of purification for protein sequencing. Starting 2008, a semi purified inclusion body p reparation was used for sequencing. However, due to the insolubility of aggregated inclusion bodies, the efficiency of obtaining enough protein material for MS/MS sequencing was low, only a portion of the reduced IBDP within a sample could be resolved in a gel. Because of this, the amount of purified IBDP within a gel band was not sufficient for sequencing. Utilizing a newly developed anti IBDP MAB (Chapter 2) with IP methodology, the process of IBDP isolation was simplified, and the yield of purified IBDP was substantially increased. However, when sufficient amount of IBDP was initially subjected to sequencing (2008 to 2010), no match was found in the existing NCBI databases. At the time it was difficult to determine whether the unsuccessful sequencing was: 1) caused by insufficient sample preparation and processing; 2) the data analysis was insufficient; 3) or simply because of an absence of IBDP homolog proteins i n the

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131 database. Until the predicted arena like virus protein sequences were available in 2012 8 the previously abandoned MS/MS sequence data w ere found to match with the GGV NP. This demonstrated the difficulty in sequencing a protein de novo, and the imp ortance of having related proteins or genomic information in current databases. The IBDP samples derived from IP preparation generated more matching peptide s with high er coverage to the predicted GGV NP compared to the samples derived from IB preps (Figure 5 2, 5 3). It is possible that IP preparation improved the quantity and quality (purity) of purified IBDP. Thus, with less contaminants and more concentrated target protein (IBDP) in the sample, result ed in better peptide separation in liquid chromatography more intensive mass spectra signals, and lead to better protein identification s Arena viruses and T heir P rotein D ivergence Arenaviruses are enveloped viruses, with a bi segmented negative strand R NA genome. 2 6 According to Sebastien et al. in 2009, the Arenaviridae family comprises 22 viral species, each of them associated with a rodent species. 2 6 Rodents were thought to be the natural host of a renaviruses, and the infections in rodents were chronic and asymptomatic. 8 Prior to the discovery of the snake arena like viruses (CASV and GGV) the arenaviruses wer e though to infect only mammals. 8 S everal arenaviruses are known to cause severe disease in human, such as, th e lymphocytic choriomeningitis v irus (caused l ymphocytic choriomeningitis ) Lassa virus (caused Lassa fever) and Junin virus (caused Argentine hemorrhagic fever ) 8, 2 6 Protein sequences among arenaviruses are highly divergent as well as the snake arena like viruses 8, 2 6 The predicted NP sequence of the snake arena like viruses only shared approximately 25% amino acid identity between the predicted NP sequences of

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132 known a reaviruses. 8 Between CASV and GGV their predicted NP sequence only shared approximately 55% amino acid identity 8 Int e restingly, the peptides derived from IBDP of boa constrictors (GGV NP) did not share any amino acid sequence homology with the predicted NP sequence of CASV (the related annulated tree boa strain) This indicated possibly a high er than expected divergence between these two arena like viruses. Although currently there had been a lack of agreements in the criteria being used for species demarcation in Arenavirida e, 2 6 t hese IBD associated arena like viruses may eventually form a new clade. Additi onally t he diversity among different arena like viruses may explained the limited cross species reactivity discussed in Chapter 3 ( Cross Reactivity Among Non Boa Constrictors ) in which an antibody produced against NP of one arena like vi r us strain could not recognize the NP of other arena like virus strain s Confirmation of the Linkage between IBDP and GGV NP In addition to a high overall coverage (90.5%) of IBDP derived peptides to the predicted GGV NP, the repeated detection of peptides at the N te rminus, mid section, and C terminus of GGV NP indicated that the protein identification is highly confident. terminus of GGV NP (Figure 5 4) was repeatedly identified in the trypsin digested IBDP sample s derived from I P preparations (Figure 5 3 ). This peptide resembled a portion of the 14 amino acid peptides that were used to produced anti GGV NP antibody by Stenglein et al. that recognized IBD inclusion bodies using fluorescence IHC. 8 The sequence result obtained from pu rified IBDP had confirmed the accuracy of the GGV NP sequence prediction by genomic sequence analysis. F urther this protein sequence result s upported the linkage between GGV infection and the formation of IBD inclusion bodies

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133 Future Diagnostic Tests Dev elopment for IBD Knowing the protein sequen ce of IBDP, the marker protein for IBD, this made possible for the development of other higher quality molecular diagnostic tests. For immuno diagnostic approaches a better and more consistent immunogen (compar ed to the semi purified inclusion bodies) can be obtained through recombinant peptides. The recombinant peptides o f IBDP can be produced with consistency to serve as the antigen for serological tests/studies in enyzyme linked immunosorbent assay (ELISA) or western blots. Further, a better and more specific anti IBDP MAB can be produced using the recombinant peptides of IBDP for immune diagnostic tests, such as, IHC staining, ELISA and western blots. With the confirmation of the link between IBDP and GGV NP by this study, a polymerase chain reaction (PCR) screening test can be developed targeting the GGV NP. Further, a PCR test can be developed for IBD in other non boa constrictor species by designing primers targeting NP of the particular viral strains. Conc lusions Purified IBDP derived from 2 IBD+ boa constrictors using two preparation methods, reduced semi purified inclusion bodies (IB preps) and IP. Using MS/MS sequencing, all of the digested IBDP samples generated peptides that matched within the pr edict ed protein sequence of GGV NP. The combination of matching peptides derived from all sequenced IBDP samples generated a n overall coverage of 90.5% within the predicted GGV NP sequence. The protein sequence results indicated a highly confident identity of the IBDP to the GGV NP. This finding further suggested that the GGV is possibly the causative agent of IBD.

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134 Figure 5 1. The total protein stain and western blot demonstrated a soluble form of IBDP that was immunopreci pitated (white arrows). Lane 1 is the molecular weight marker, lane 2 is the purified IBDP with anti IBDP MAB to show the molecular weight of IBDP, heavy chain and light chain of anti IBDP MAB. Lane 3 is the IP product from IBD+ liver homogenate. Lane 4 is the IP product derived from IB D liver homogenate. Lane 5 is the IBD liver homogenate. A. The total protein stain of the gel with resolved IBDP derived from IP. The purified IBDP indicated by the white arrow is removed for sequencing. B. The western blot using anti IBDP MAB. The purif ied 68 KDa IBDP (large black arrow) purified from the IBD+ liver homogenate (lane 3), but not presence in purification from the IBD liver homogenate (lane 4). Anti IBDP MAB did not detect IBDP in the IBD liver homogenate (lane 5). The heavy and light cha ins (small black arrows) of the resolved anti IBDP MAB. were detected by the secondary antibody used for western blot.

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135 Figure 5 2. T he coverag e of peptides derived from IB preps (IBDP) over the predicted GGV NP sequence by MS/MS sequencing Green indica ted peptide with coverage equal or above 95%, yellow indicated peptide with coverage equal or above 50% but less than 95%, red indicated peptide with coverage less than 50%, and grey indicated peptide with no coverage.

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136 Figure 5 3. The coverage of peptides derived from immune precipitated IBDP over the predicted GGV NP sequence by MS/MS sequencing. Green indicated peptide with coverage equal or above 95%, yellow indicated peptide with coverage equal or above 50% but less than 95%, red indicated pept ide with coverage less than 50%, and grey indicated peptide with no coverage.

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137 Figure 5 4. The overall coverage of peptides derived f rom purified IBDP over the predicted GGV NP sequence by MS/MS sequencing. The peptides detected by mass spectrometry were underlined The C terminal of GGV NP was repeatedly detected in many IBDP preparations, which is a portion of t he peptide (highlight in yellow) that Stenglein et al. used to produce anti GGV NP polyclonal antibod y. 8

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138 LIST OF REFERENCES 1. Chang L, Jacobson ER. Inclusion body disease, a worldwide infectious disease of boid snakes: a review. J Exo Pet Med 2010;19:216 225. 2. Schumacher J, Jacobson ER, Homer BL, et al. Inclusion body disease in boid snakes. J Zoo and Wi ldlife Med 1994;25:511 524. 3. Carlisle Nowak MS, Sullivan N, Carrigan M, et al. Inclusion body disease in two captive Australian pythons (Morelia spilota variegata and Morelia spilota spilota). Aust Vet J 1998;76:98 100. 4. Wozniak E, McBride J, DeNardo D, et a l. Isolation and characterization of an antigenically distinct 68 kd protein from nonviral intracytoplasmic inclusions in Boa constrictors chronically infected with the inclusion body disease virus (IBDV: Retroviridae).Vet Pathol 2000;37:449 459. 5. Vancraeyn est D, Pasmans F, Martel A, et al Inclusion body disease in snakes: a review and description of three cases in boa constrictors in Belgium.Vet Rec 2006;158:757 760. 6. Jacobson ER, Oros J, Tucker SJ, et al. Partial characterization of retroviruses from boid snakes with inclusion body disease. Am J Vet Res 2001;62:217 224. 7. Huder JB, Boni J, Hatt JM, et al Identification and characterization of two closely related unclassifiable endogenous retroviruses in pyt hons (Python molurus and Python curtus).J Virol 2002;76:7607 7615. 8. Stenglein MD, Sanders C, Kistler AL, et al. Identification, characterization, and in vitro culture of highly divergent arenaviruses from boa constrictors and annulated tree boas: candidate etiological agents for snake inclusion body disease. MBio 2012;3(4):e00180 12. 9. French SD, Ihrig TJ, Norum BA. A method of isolation of Mallory bodies in a purified fraction. Lab Invest 1972;26(3):240 244. 10. Bradford M. A rapid and sensitive method for the qu antitation of microgram quantities of protein utilizing the principle of protein dye binding. Anal Biochem 1976;72:248 254. 11. Hazeki N, Tukamoto T, Goto J, et al. Formic acid dissolves aggregates of an N terminal huntingtin fragment containing an expanded po lyglutamine tract: applying to quantification of protein components of the aggregates. Biochem Biophys Res Commun 2000;277(2):386 393. 12. Bolton DC, McKinley MP, Prusiner SB. Identification of a protein that purifies with the scrapie prion. Science 1982;218(4 579):1309 1311.

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139 13. Ramos Vara JA, Kiupel M, Baszler T, et al. 2008. Suggested guidelines for immunohistochemical techniques in veterinary diagnostic laboratories. J Vet Diagn Invest 2008;20:393 413. 14. Dohoo I, Martin W, Stryhn H. Screening and diagnostic tests. In: McPike SM ed. Veterinary Epidemiologic Research,1st ed. Charlottetown,PEI,Canada:University of Prince Edward Island, AVC Inc.,2003;85 120. 15. Dohoo I, Martin W, Stryhn H. Measures of Association. In: McPike SM ed. Veterinary Epidemiologic Research,1st ed. Charlottetown,PEI,Canada:University of Prince Edward Island, AVC Inc.2003;121 138. 16. Raymond JT, Garner MM, Nordhausen RW, et al. A disease resembling inclusion body disease of boid snakes in captive palm vipers (Bothriechis marchi).J Vet Diagn Invest 2001; 13:82 86. 17. Fleming GJ, Heard DJ, Jacobson ER, et al. Cytoplasmic inclusions in corn snakes, Elaphe guttata, resembling inclusion body disease of boid snakes. J Herp Med Surg 2003;13:18 22. 18. Webster JD, Miller MA, DuSold D, et al. Effects of prolonged formali n fixation on diagnostic immunohistochemistry in domestic animals. J Histochem Cytochem 2009;57:753 761. 19. Lock BA, Green LG, Jacobson ER, et al. Use of an ELISA for detection of antibody responses in Argentine boa constrictors (Boa constrictor occidentalis).Am J Vet Res 2003;64(4):388 395. 20. Martin SW, Meek AH, Willeberg P. Measurement of disease frequency and production. In: Martin SW, Meek AH, Wil leberg P, eds. Veterinary epidemiology, principles and methods. 1st ed. Ames, Iowa: Iowa State University Press,1987;62 76. 21. Gotham SM, Fryer PJ, Paterson WR. The measurement of insoluble proteins using a modified bradford assay. Anal Biochem 1988;173:353 3 58. 22. Keller A, Nesvizhskii AI, Kolker E, et al. Empirical statistical model to estimate the accuracy of peptide identifications made by MS/MS and database search. Anal Chem 2002;74:5383 5392. 23. Nesvizhskii AI, Keller A, Kolker E, et al A statistical model fo r identifying proteins by tandem mass spectrometry. Anal Chem 2003;75:4646 4658. 24. Edman P. Method for determination of the amino acid sequence in peptides. Acta Chem Scand 1950;4:283 293. 25. Niall HD. Automated Edman degradation: the protein sequenator. Metho ds Enzymol 1973;27:942 1010.

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140 26. Emonet SF, de la Torre JC, Domingo E, et al. Arenavirus genetic diversity and its biological implications. Infect Genet Evol 2009;9(4):417 429.

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141 BIOGRAPHICAL SKETCH Li Wen Chang also known as Rita Chang was born in Taiwan in 1982. She had strong interests in exotic animals since her childhood, and sh e presented her first science project about Fresh Water Turtle s in the Middle Schools Scientific Exposition. During her high school years, she was actively involved in Taiwan Cetacean Society as a trained volunteer. She volunteered in events for bringing public awareness of the importance in Cetacean conservations. Following her interests, she perused her bachelor degree in Veterinary Medici ne at National Chung Hsing University (NCHU) Taiwan After graduation in 2005, she passed the national certification exam and became a board certified Veterinarian in Taiwan. Her interests had always been focused in the field of exotic and zoological medi cines. She completed externships at Taipei Zoo (mentored by Dr. An Hsing Lee), and at Ludwig Maximilian University of Munich under the Clinic of Animal Gynaecology, the Equine Clinic, and the Clinic of Small Animal Surgery. In 2006, she completed a one yea r residency at the Exotic Animal Clinic of Veterin ary Medical Teaching Hospital NCHU. She also spent one year in Europe America Biotechnology Co., Ltd as a specialized veterinarian to manage laboratory animals. In 2006, Dr. Chang received a governmental scholarship awarded by the Ministry of Education in Taiwan to pursu e advanced degree in USA In the spring of 2008, she was fortunate and honored to be admitted to the graduate program at Department of Small Animal Clinical Sciences, College of Veterinary Medicine, University of Florida Being mentored by Dr. Elliott Jacobson, she become de eply involved in the research of d iagnostic t ests development for Inclusion Body Disease. In 2012 fall, she received her Doctor of Philosophy in Veterinary Medical Scienc e from University of Florida. She will

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142 return to Taiwan and continue to devote herself in the clinical work of exotic animal medicine.