Relationships Between Severity of Histopathological Lesions of Aleutian Disease in Mink, Measured by Digital Image Analysis, and Antibody Titer and Serum Gamma-Globulin Level by Rojman Khomayezi Submitted in partial fulfilment of the requirements for the degree of Master of Science at Dalhousie University Halifax, Nova Scotia August 2018 © Copyright by Rojman Khomayezi, 2018 Table of Contents List of Tables ..................................................................................................................... v List of Figures ................................................................................................................... ix Abstract ............................................................................................................................. xi List of Abbreviations and Symbols Used ...................................................................... xii Acknowledgements ........................................................................................................ xiii Chapter 1. Introduction .................................................................................................. 1 Chapter 2. Quantitative measurement of the severity of histopathological lesions in AMDV-infected mink using digital image analysis. ...................................................... 4 2.1 Literature review ....................................................................................................... 4 2.1.1 The use of digital image analysis in histopathology ........................................... 4 2.1.2 Staining techniques ............................................................................................. 5 2.1.3 Validation of digital image analysis software .................................................... 7 2.1.4 Algorithms developed for Image Pro Plus ....................................................... 16 2.1.5 The use of DIA to measure the severity of Aleutian disease lesions ............... 19 2.2 Materials and methods ............................................................................................ 20 2.2.1 Image preparation and counting the infiltrated cells ........................................ 20 2.2.2 Software setup for counting total number of cells ............................................ 21 2.2.3 Using the Best-Fit tool to minimize variations among slides ........................... 24 2.2.4 Degree of similarity among different areas of a slide ...................................... 26 2.2.5 Statistical analysis............................................................................................. 26 2.3 Results ..................................................................................................................... 27 2.3.1 Finding the best color channel .......................................................................... 27 2.3.2 The effects of using the Best-Fit tool ............................................................... 29 2.3.3 Finding the best range score for different organs ............................................. 30 2.3.4 Degree of similarity among different areas of a slide ...................................... 34 2.4 Discussion ............................................................................................................... 42 Chapter 3. Relationships between the severity of histopathological and serological measurements in AMDV-infected mink. ................................................... 49 3.1 Literature review ..................................................................................................... 49 3.1.1 Aleutian disease in mink................................................................................... 49 3.1.2 Mink immune response to AMDV-infection .................................................... 50 3.1.3 Gross and histopathological lesions of AD ...................................................... 54 ii 3.1.4 Measuring severity of histopathological lesions of AD ................................... 63 3.1.5 Controlling Aleutian disease ............................................................................ 66 3.1.5.1 Vaccine ...................................................................................................... 66 3.1.5.2 Treatment ................................................................................................... 66 3.1.5.3 Virus eradication ........................................................................................ 67 3.1.5.4 Selection for tolerance ............................................................................... 67 3.2 Materials and methods ............................................................................................ 72 3.2.1 Source of animals and inoculation .................................................................... 72 3.2.2 Animal sampling............................................................................................... 72 3.2.3 Anti-AMDV antibody titers.............................................................................. 74 3.2.4 Serum gamma-globulin levels .......................................................................... 74 3.2.5 Measuring the severity of histopathological lesions......................................... 75 3.2.6 Data Analyzing ................................................................................................. 76 3.3 Results ..................................................................................................................... 77 3.3.1 Percentage of infiltrated cells in different organs ............................................. 77 3.3.2 Test of normality .............................................................................................. 80 3.3.3 Correlations between three images within each slide in organs ....................... 80 3.3.4 Factors affecting percent infiltrated cells and total number of cells ................. 82 3.3.5 Associations between anti-AMDV antibody titer and severity of histopathological lesion ............................................................................................. 85 3.3.5.1 The effects of antibody titer and duration of infection on percent infiltrated cells in the heart .................................................................................... 86 3.3.5.2 The effects of antibody titer and duration of infection on percent infiltrated cells in the kidneys ................................................................................ 91 3.3.5.3 The effects of antibody titer and duration of infection on percent infiltrated cells in the liver ..................................................................................... 93 3.3.5.4 The effects of antibody titer on percent infiltrated cells within sex and organs ..................................................................................................................... 94 3.3.6 Associations between serum gamma-globulin levels and severity of histopathological lesion ............................................................................................. 95 3.3.6.1 The effects of serum gamma-globulin level and duration of infection on percent infiltrated cells in the heart ................................................................... 95 3.3.6.2 The effects of serum gamma-globulin level and duration of infection on percent infiltrated cells in the kidneys ............................................................ 100 iii 3.3.6.3 The effects of serum gamma-globulin level and duration of infection on percent infiltrated cells in the liver ................................................................. 105 3.3.6.4 The effects of serum gamma-globulin level on percent infiltrated cells . 107 3.4 Discussion ............................................................................................................. 109 3.4.1 Differences among organs for response to AMDV-infection ........................ 109 3.4.1.1 The heart .................................................................................................. 110 3.4.1.2 The kidneys .............................................................................................. 111 3.4.1.3 The liver ................................................................................................... 113 3.4.2 Correlations between the three images of each slide within the organs ......... 114 3.4.3 Normality of the distributions of average measurements of the three images of each slide ................................................................................................. 115 3.4.4 Factors affecting the total number of cells ..................................................... 116 3.4.5 Factors affecting percent infiltrated cells ....................................................... 117 3.4.6 Associations between anti-AMDV antibody titer, gamma-globulin level and severity of histopathological lesion .................................................................. 120 3.4.6.1 The effects of antibody titer, gamma-globulin level and duration of infection on percent infiltrated cells in the heart ................................................. 122 3.4.6.2 The effects of antibody titer, gamma-globulin level and duration of infection on percent infiltrated cells in the kidneys ............................................. 126 3.4.6.3 The effects of antibody titer, gamma-globulin level and duration of infection on percent infiltrated