Studying the regulation of immune signalling molecules related to immunity during Avibacterium paragallinarum infection By Poojah Jawallapersand Dissertation submitted in fulfilment of the requirements for the Master’s degree Magister Scientiae in Biochemistry (Veterinary Biotechnology) Department of Microbial, Biochemical and Food Biotechnology Faculty of Natural and Agricultural Sciences University of the Free State Bloemfontein South Africa 20 June 2019 Supervisor: Dr. C.E. Boucher Co-supervisors: Dr. W.J. Janse Van Rensburg Dr. W.A. van der Westhuizen DECLARATION DECLARATION I, Poojah Jawallapersand, hereby certify that the dissertation submitted by me for the degree Magister Scientiae (M.Sc.) in Biochemistry (Veterinary Biotechnology), is my own independent work; and complies with the Code of Academic Integrity, as well as other relevant policies, procedures, rules and regulations of the University of the Free State. I hereby declare, that this research project has not been previously submitted before to any university or faculty for the attainment of any qualification. I further waive copyright of the dissertation in favour of the University of the Free State. Poojah Jawallapersand Date ACKNOWLEDGEMENTS ACKNOWLEDGEMENTS I would like to extend my sincere and heartfelt gratitude to the following people and organizations. The completion and success of my Master’s study would not have been possible without any of them. I am deeply grateful. • My supervisor and mentor, Dr. Charlotte Boucher, who took me under her wing, for the opportunity and privilege to be part of this exciting research, sharing her insight on molecular biology and veterinary immunology, her constant guidance, immense support, patience, mentorship, motivation, invaluable constructive criticism, for being my pillar of strength on this journey and for allowing me to grow and become an autonomous as well as humble scientist. • Dr. Wouter van der Westhuizen, for his guidance, immense support, unwavering mentorship, technical skills, expertise in Microbiology and indispensable assistance during the study. • Dr. Walter Janse Van Rensburg for his expertise, technical skills and knowledge on flow cytometry and haematology. • Prof. Robert Bragg, for his expertise on Av. paragallinarum, support and mentorship. • National Research Foundation (NRF) and the Post-Graduate School (University of the Free State) for generously funding my studies and enabling dreams to be realised. • National Health Laboratory Service (NHLS), Universitas, Bloemfontein, for enabling me to conduct my research. A tremendous thank you to Mrs. Gerda Le Roux and Mrs. Shireen Pretorius (Histopathology Department- Immunohistochemistry); as well as Mr. Mojalefa Setlai (Haematology Department- Blood smears and haematological tests) for their patience, support, immense assistance and expertise in their field of specialization in routine diagnostics. ACKNOWLEDGEMENTS • Dr. Errol Cason for his invaluable knowledge and expertise in bio-informatics. • Prof. Robert Schall for his immense contribution and assistance with the statistical analysis of the study. • Mr. Poifo Mokgatlhe (Qualified laboratory Animal Technician) for his indispensable phlebotomy skills and assistance during the study, Mr. Seb Lamprecht (Head of Animal Experimental Unit) and the rest of the team from the Animal Unit (UFS) • Dr. Hannes Swart (Avi-Farms Ltd. and Deltamune) for his expertise and assistance with chickens for the study. A special thank you to Mr. Julius (Eduan) Hellmuth and Mr. Sherwan van Jaarsfeld, for their immense contribution and help for the safe transportation of chickens all the way from Centurion to Bloemfontein. • My amazing colleagues and friends at the Department of Microbial, Biochemical and Food Biotechnology and my laboratory family at the Veterinary Biotechnology research cluster (University of the Free State), for sharing knowledge, memories, for their persistent support and uplifting my spirit when I needed it the most. The names are too many to mention, I thank you. • To my dearest friends and family, for being my ardent aficionados. Their continuous faith in me, prayers, moral support and motivation, gave me strength and inspiration during my studies. I am grateful for your love and support. • To my dearest aunts Kavita Ragoobar, Jankee Jawallapersand and Binta-Devi Jawallapersand for being my guardian angels and well-wisher, for your prayers, and for the words of encouragement and motivation during days of darkness and light. • To my late uncles bhai Ramesswar Jawallapersand, bhai Assoodeo Jawallapersand and grandmother Sarodah Jawallapersand, this dissertation is dedicated to you as a legacy to the family name, not a day goes by where you are all not thought of and dearly missed. ACKNOWLEDGEMENTS • To my father (Roheet Jawallapersand), mother (Dhanwantee Jawallapersand), sister (Shweta Jawallapersand) and Tutankhamun, for always believing in me, being my counsellors, being my pillars of support, loving me unconditionally and respecting my decisions. Thank you for nurturing me into who I am today. I am blessed to have all of you. Without all of you I would never have been where I am today, I can say with pride that I am immensely fortunate and blessed to have such an amazing family. • To my Creator, for His guidance, gracious blessings, loving protection, inner peace. and for providing whenever I asked. I glorify thee for giving me the chance to make a difference in this world and a true purpose in life. This study allowed me to share knowledge gained, travel, meet new people and most importantly discover who I really am. I believe that at the end you are competing against yourself and time, and that no one else is your contender. You are the master of your own destiny. You are the alchemist….…….. “Great spirits have always encountered violent opposition from mediocre minds.” - Albert Einstein “Arise, awake, sleep no more; within each of you there is the power to remove all wants and all miseries. Believe this, and that power will be manifested.” “All the powers in the universe are already ours. It is we who have put our hands before our eyes and cried out that it is dark.” - Swami Vivekananda CONTENTS CONTENTS Page LIST OF ABBREVIATIONS AND ACRONYMS VIII LIST OF FIGURES XXI LIST OF TABLES XXVIII LIST OF ANNEXURES XXX LIST OF APPENDICES XXXI ABSTRACT XXXII CHAPTER 1: LITERATURE REVIEW 1 1.1. Infectious coryza: An overview 1 1.2. History: Etiological agent 2 1.3. Epidemiology: Host, incidence and transmission 5 1.4. Clinical signs and symptoms of disease 7 1.5. Antigenic structure and virulence factors 9 1.6. Vaccines and treatment 10 1.7. Avibacterium paragallinarum 12 1.7.1. Serological classification 12 1.7.2. Cultivation and growth conditions 15 I CONTENTS 1.7.3. Morphology and staining 16 1.7.4. Colony morphology 17 1.7.5. Biochemical properties 17 1.7.6. Molecular methods of detection 20 1.8. Avian immunity 23 1.8.1. Host-pathogen interactions during Gram-negative bacterial invasion 23 1.8.2. Avian innate immune response 26 1.8.3. Avian adaptive immune response 33 1.8.4. Cytokines and chemokines of the avian immune system 40 1.9. Rationale and objectives of the study 42 References 45 CHAPTER 2: IMMUNOMICS: In silico MAPPING OF IMMUNE SIGNALLING PATHWAYS IN CHICKENS RELATED TO Avibacterium paragallinarum SEROVAR C-3 INFECTION 81 2.1. Introduction 81 2.2. Materials and methods 83 2.2.1. Animal ethics, experimental design and data analysis 83 2.2.2. Bio-statistical analysis 84 II CONTENTS 2.3. Results and discussion 86 2.3.1. Gene enrichment analysis 86 2.3.2. Functional annotation 91 2.3.3. Pathway analysis 104 2.3.4. Functional protein association network analysis 111 2.4. Conclusion 114 References 115 Annexure A 126 CHAPTER 3: A PILOT STUDY OF THE IMMUNE RESPONSE TO Avibacterium paragallinarum SEROVAR C-3 INFECTION IN Gallus gallus 131 3.1. Introduction 131 3.2. Materials and methods 133 3.2.1. Ethics statement, animal husbandry and study design 133 3.2.2. Bacterial isolate used for challenge 134 3.2.3. Microbial cultivation and identification 135 3.2.3.1. Cultivation of Av. paragallinarum serovar C-3 135 3.2.3.2. Genomic DNA extraction 136 3.2.3.3. Identification of bacterial strain 137 III CONTENTS 3.2.3.4. Agarose gel electrophoresis and visualisation of correct DNA fragment size 139 3.2.3.5. Sequencing of 16S rDNA PCR products 140 3.2.4. Challenge methods and clinical scoring 142 3.2.5. Blood collection and processing 144 3.2.6. Avian full blood counts, differential blood counts and microscopy 145 3.2.7. Staining and flow cytometry analysis 149 3.2.8. Direct Enzyme-linked immunosorbent assay (ELISA) 150 3.3. Results and discussion 152 3.3.1. Microbial cultivation and identification 152 3.3.1.1. Identification of bacterial strain 152 3.3.1.2. Sequencing of 16S rDNA PCR products 154 3.3.2. Challenge methods and clinical scoring 156 3.3.3. Avian full blood counts, differential blood counts and microscopy 162 3.3.4. Flow cytometry and antibodies 165 3.3.5. Direct enzyme-linked immunosorbent assay (ELISA) 167 3.4. Conclusion 169 References 172 Annexure B 179 IV CONTENTS Appendix A 199 Appendix B 203 CHAPTER 4: INFECTIOUS CORYZA AS AN INFECTION MODEL TO MONITOR IMMUNE CELLS AND MOLECULES DURING DISEASE PROGRESSION 205 4.1. Introduction 205 4.2. Materials and methods 207 4.2.1. Ethics approval, animal husbandry and study design 207 4.2.2. Bacterial isolate used for challenge 208 4.2.3. Microbial cultivation and identification 209 4.2.4. Challenge methods and clinical scoring 211 4.2.5. Blood collection and processing 212 4.2.6. Avian blood smears and microscopy 213 4.2.7. Flow cytometry and antibodies 213 4.2.8. Sandwich enzyme-linked immunosorbent assay (ELISA) 213 4.3. Results and discussion 215 4.3.1. Microbial cultivation and identification 215 4.3.2. Challenge methods and clinical scoring 222 V CONTENTS 4.3.3. Avian blood smears and microscopy 229 4.3.4. Flow cytometry and antibodies 235 4.3.5. Sandwich enzyme-linked immunosorbent assay (ELISA) 255 4.4. Conclusion 260 References 262 Annexure C 269 Annexure D 284 Appendix C 354 Appendix D 356 CHAPTER 5: POST-MORTEM EXAMINATION OF CHICKEN LYMPHOID TISSUES AFTER INFECTION WITH Avibacterium paragallinarum SEROVAR C-3 INFECTION 364 5.1. Introduction 364 5.2. Materials and methods 368 5.2.1. Study design and ethics approval 368 5.2.2. Necropsy, sample collection and formalin fixation 368 5.2.3. Microbial cultivation, isolation and identification 371 5.2.4. Immunohistochemistry (IHC) of samples collected 372 5.2.4.1. Anatomical grossing, tissue processing and paraffin wax impregnation 372 VI CONTENTS 5.2.4.2. Paraffin embedding and tissue sectioning 374 5.2.4.3. Antibody staining and microscopy 376 5.3. Results and discussion 377 5.3.1. Necropsy, sample collection and formalin fixation 377 5.3.2. Microbial cultivation and identification 379 5.3.3. Immunohistochemistry (IHC) of samples collected 384 5.4. Conclusions 401 References 403 CHAPTER 6: CONCLUDING REMARKS AND FUTURE STUDIES 410 Concluding remarks 410 Future research 413 VII LIST OF ABBREVIATIONS AND ACRONYMS LIST OF ABBREVIATIONS AND ACRONYMS $ Dollar unit of currency % Percentage < Less than = Equal to > Greater than ≤ Less than or equal to ® Registered trademark °C Degrees Celsius µg Microgram µg/µl Microgram per microlitre µl Microlitre µm Micrometre µM Micromolar ACD Acid citrate dextrose ACTH Adrenocorticotropic hormone AEC Animal Ethics Committee AIDS Acquired immune deficiency syndrome AIFM2 Apoptosis-inducing factor-2 AMP Antimicrobial peptide VIII LIST OF ABBREVIATIONS AND ACRONYMS APC Antigen-presenting cell APP Acute phase protein Arg Arginine Arp Actin related protein ATP Adenosine triphosphate BALT Bronchus associated lymphoid tissue BCR B cell receptor BLAST Basic Local Alignment Search Tool bp Base pair (nucleotide) BSA Bovine serum albumin BTA Blood tryptose agar CALT Conjunctiva-associated lymphoid tissue CASP6 Caspase-6 CAV Chicken anaemia virus CBC Complete blood count CC1 Cell conditioning solution CD Cluster of differentiation CFU Colony forming units CFU/ml Colony forming units per millilitre CH Cell haemoglobin IX LIST OF ABBREVIATIONS AND ACRONYMS CHCM Cell haemoglobin concentration mean CHr Reticulocyte haemoglobin content cIAP Cellular inhibitor of apoptosis protein CO2 Carbon dioxide COX Cytochrome c oxidase CRF Corticotropin releasing factor CRH Corticotropin-releasing hormone CRP C-reactive protein CSF Colony-stimulating factor CTL Cytotoxic T lymphocytes Cys Cysteine DAB Chromagen 3,3′-diaminobenzidine DAFF Department of Agriculture, Forestry and Fisheries DAVID Database for Annotation, Visualization and Integrated Discovery DC Dendritic cell DNA Deoxyribonucleic acid dNTP Deoxyribonucleotide triphosphate ds Double-stranded EASE Expression Analysis Systematic Explorer X LIST OF ABBREVIATIONS AND ACRONYMS EDTA Ethylenediaminetetraacetic acid EDTA-NA2.2H2O Ethylenediaminetetraacetic acid, disodium dihydrate ELISA Enzyme-linked immunosorbent assays ER Endoplasmic reticulum ERIC-PCR Enterobacterial repetitive intergenic consensus polymerase chain reaction ERK Extracellular signal-related kinase FADD Fas-associated protein with death domain FB Fibrinogen FBC Full blood count FDA Food and Drug Administration FFPE Formalin-fixed paraffin-embedded FITC Fluorescein isothiocyanate FSC Forward angle scatter g Relative centrifugal force GABRA-1 Gamma-aminobutyric acid type A receptor alpha1 subunit GALT Gut- associated lymphoid tissue GC Germinal centres GH Growth hormone XI LIST OF ABBREVIATIONS AND ACRONYMS Gln Glutamine GO Gene Ontology GPS Global positioning system h Hour H2SO4 Sulphuric acid HA Haemagglutinating antigen/hemagglutinin HCT Haematocrit HDW Haemoglobin distribution width HG Harderian glands HGB Haemoglobin HI Haemagglutination inhibition HPG Haemophilus paragallinarum HSP70 Heat shock protein 70 kilodalton IAV Influenza A viruses IBDV Infectious bursal disease virus IC Infectious coryza ICSH International Committee of Standardization in Haematology IEL Intraepithelial lymphocyte IFN Interferon XII LIST OF ABBREVIATIONS AND ACRONYMS IFNGR Interferon-gamma receptor Ig Immunoglobulin IGF-I Insulin-like growth factor-1 IHC Immunohistochemistry IL Interleukin IL-8L1 Interleukin 8-like 1 IM Inner membrane INF Interferon IPS1 Induced by phosphate starvation1 IRF3 Interferon regulatory factor-3 JAK Janus kinase kb Kilobase kDa Kilodalton KEGG Kyoto Encyclopedia of Genes and Genomes LP Lectin pathway LPS Lipopolysaccharide LTA Lipoteichoic acid M Molar Mab Monoclonal antibody XIII LIST OF ABBREVIATIONS AND ACRONYMS MALDI-TOF MS Matrix-assisted laser desorption ionization-time-of- flight mass spectrometry MAP2K3 Mitogen-activated protein kinase kinase 3 MAP3K4 Mitogen-activated protein kinase kinase kinase 4 MAPK Mitogen-activated protein kinase MBL Mannan-binding lectin MCH Mean corpuscular haemoglobin MCHC Mean corpuscular haemoglobin concentration MCV Mean corpuscular volume MCVr Reticulocyte mean corpuscular volume MD-2 Myeloid differentiation factor-2 MDV Marek’s disease virus MHC Major histocompatibility complex min Minute MIP-1β Macrophage inflammatory protein-1 beta MKK3/6 Mitogen-activated protein kinase kinase 3/6 ml Millilitres MLSA Multilocus sequence analysis MLST Multilocus sequence typing mM Millimolar XIV LIST OF ABBREVIATIONS AND ACRONYMS MN Mononuclear MW Molecular weight MyD88 Myeloid differentiation factor-88 N Normality NaCl Sodium chloride NAD+ Nicotinamide adenine dinucleotide oxidised form NADH Nicotinamide adenine dinucleotide reduced form NALT Nasal-associated lymphoid tissue NCBI National Centre for Biotechnology Information NF-κB Nuclear factor kappa B ng Nanogram NGS Next-generation sequencing NHLS National Health Laboratory Service NK Natural killer cell NKT Natural killer T cell nm nanometre NO Nitric oxide NOX2 Nicotinamide adenine dinucleotide reduced form oxidase 2 NS1BP Non-structural protein-1 binding protein XV LIST OF ABBREVIATIONS AND ACRONYMS NUDC Nuclear distribution gene C O −2 Superoxide anion OD Optical density OM Outer membrane p38 p38 mitogen-activated protein kinase PALS Peri-arteriolar lymphatic sheath PAMP Pathogen-associated molecular pattern PANTHER Protein Analysis Through Evolutionary Relationships PBMC Peripheral blood mononuclear cell PBS Phosphate-buffered saline PCR Polymerase chain reaction pg/ml Picogram per millilitre pH Power of hydrogen Phe Phenylalanine PI Post-infection PI3-K Phosphatidylinositol 3-kinase PLG Plasminogen PLT Platelet PMN Polymorphonuclear XVI LIST OF ABBREVIATIONS AND ACRONYMS PMN Polymorphonuclear leukocyte PRR Pattern recognition receptor qPCR Real-time/quantitative polymerase chain reaction R Rand unit of currency RANTES Regulated on activation, normal T Cell expressed and secreted RBC Red blood cell rDNA Ribosomal deoxyribonucleic acid RDW Red cell distribution width REA Ribotyping and restriction endonuclease analysis REVIGO Reduce + Visualize Gene Ontology RI Re-infected/re-infection RIP Receptor-interacting protein RNA Ribonucleic acid RNase Ribonuclease RNA-Seq Ribonucleic acid sequencing ROI Reactive oxygen intermediate RPE R-phycoerythrin RTX Repeats-in-toxin s Second XVII LIST OF ABBREVIATIONS AND ACRONYMS SANAS South African National Accreditation System SANS South African National Standards SDS Sodium dodecyl sulfate SE Salmonella enteritidis Sec61 Channel forming translocon SHISA5 Shisa Family Member 5 SOP Standard operating procedures SPF Specific pathogen free ss Single-stranded SSC Side angle scatter STAT Signal transducer and activator of transcription STRING Search Tool for the Retrieval of Interacting Genes TAE Tris(hydroxymethyl)aminomethane -acetate- ethylenediaminetetraacetic acid TAP Transporter associated with antigen-processing Taq Thermus aquaticus TCR T cell receptor TFTC Too few to count TGF-β Transforming growth factor beta 1 Th T helper XVIII LIST OF ABBREVIATIONS AND ACRONYMS TIR Translocated intimin receptor TIRAP Translocated intimin receptor domain containing adaptor protein TLR Toll-like receptors TLX3 T-Cell leukemia homeobox protein 3 TM/SN Test medium agar supplemented with chicken serum and NAD+ TMB Test medium broth TMB 3,3',5,5'-Tetramethylbenzidine TMTC Too many to count TNF Tumour necrosis factor TNFAIP3 Tumour necrosis factor α-induced protein 3 TNFR1 Tumour necrosis factor receptor 1 TNFSF Tumour necrosis factor superfamily TRADD Tumour necrosis factor receptor type 1-associated death domain TRAF2 Tumour necrosis factor receptor-associated factor- 2 Treg Regulatory T cells TRIF Translocated intimin receptor -domain-containing adapter-inducing interferon-β XIX LIST OF ABBREVIATIONS AND ACRONYMS Tris Tris(hydroxymethyl)aminomethane Tris-HCl Tris(hydroxymethyl)aminomethane-hydrochloric acid Trp Tryptophan TUBB Tubulin beta class 1 Tyr Tyrosine ™ Trademark U Enzyme activity (micromole per minute) URT Upper respiratory tract US United States UV Ultraviolet V/cm Volts per centimetre v/v Volume per volume VEGF Vascular endothelial growth factor w/v Weight per volume WASP Wiskott-Aldrich syndrome protein WAVE Wiskott-Aldrich syndrome protein family verproline- homologous protein WBC Diff White blood cell differential count WBC White blood cell XX LIST OF FIGURES LIST OF FIGURES Page Figure 1.1: Chickens infected with Av. paragallinarum. 8 Figure 1.2: Location of primers for amplification of regions within the HMTp210 open reading frame (ORF) region. 23 Figure 2.1: Flow diagram of bioinformatics pipeline indicating biological interpretation and outcome of in silico data of chickens infected with IC. 84 Figure 2.2: Biological process pie charts obtained from GO Enrichment Analysis linked to the PANTHER Classification System representing the percentage distribution of the biological process ontology in the comparison of the differentially expressed regulated gene cohorts of Group 2 (score 1) vs 1 (score 0), Group 3 (score 2) vs 1 (score 0) and Group 4 (score 3) vs 1 (score 0). 88 Figure 2.3: Biological process pie charts obtained from GO Enrichment Analysis representing the percentage distribution of the biological process ontology of Group 3 (score 2) vs 1 (score 0). 90 Figure 2.4: Scatterplot of biological process for Group 2 (score 1) vs 1 (score 0) with REVIGO providing a summarised and visual display of GO terms. 95 XXI LIST OF FIGURES Figure 2.5: Scatterplot of biological process for Group 3 (score 2) vs 1 (score 0) with REVIGO providing a summarised and visual display of GO terms. 98 Figure 2.6: Scatterplot of biological process for Group 4 (score 3) vs 1 (score 0) with REVIGO providing a summarised and visual display of GO terms. 101 Figure 2.7: Toll-like receptor pathway from KEGG pathways for Group 3 (score 2) vs 1 (score 0). 105 Figure 2.8: Phagosome pathway for the maturation of the phagocyte into a phagolysosome from KEGG pathways for Group 2 (score 1) vs 1 (score 0). 106 Figure 2.9: Influenza A virus pathway from KEGG pathways for Group 3 (score 2) vs 1 (score 0). 108 Figure 2.10: Oxidative phosphorylation pathway from KEGG pathways for Group 4 (score 3) vs 1 (score 0). 110 Figure 2.11: Functional association network of Group 2 (score 1) vs 1 (score 0), Group 3 (score 2) vs 1 (score 0) and Group 4 (score 3) vs 1 (score 0) correlated genes. 113 Figure 3.1: Handling of a bird by qualified personnel. 144 Figure 3.2: BTA plate with Av. paragallinarum. 153 Figure 3.3: HPG2-PCR for the SA-3 (C-3) reference isolate cultivated for experimental procedure, whereby amplification was observed for all samples, with an expected band size of 500 bp. 154 XXII LIST OF FIGURES Figure 3.4: 16S rDNA PCR for the SA-3 (C-3) reference isolate cultivated for experimental procedure, whereby amplification was observed for all samples, with an expected band size of 1500 bp. 155 Figure 3.5: The disease profile showing the daily mean disease score of the control (red) and experimental (green) cohorts. 158 Figure 3.6: Clinical signs and symptoms related to IC after the first injection. 159 Figure 3.7: Clinical signs and symptoms related to IC after the second injection. 161 Figure 3.8: Egg production indicated as the total number of eggs laid/chicken/day in the experimental and control groups. 161 Figure 3.9: Avian blood smear from a Day 0 chicken. 164 Figure 3.10: Flow cytometry profile of one of the control chickens showing the forward scatter (FSC-A) and side scatter (SSC-A) plot. 166 Figure 3.11: Whole blood separated by Ficoll-Paque PLUS (GE Healthcare) into different components after using a swing- bucket centrifuge at 400 x g. 167 Figure 3.12: Screening for antibodies using chicken plasma from both experimental and control chickens. 168 Figure 3.13: Graphical representation and statistical analysis of ELISA assay conducted on chicken plasma samples from the experiment (p<0.05). 169 Figure 4.1: Cattle blood tryptose agar plate with Av. paragallinarum. 216 XXIII LIST OF FIGURES Figure 4.2: Tryptic soy agar supplemented with 0.2% (v/v) NAD+ (TSA) plates showing growth of Av. paragallinarum serovar C-3 at different concentrations 100-10-8 when a serial dilution was performed in triplicate, however only one of the plates from each dilution was shown. 218 Figure 4.3: HPG2-PCR for the SA-3 (C-3) reference isolate cultivated for the experimental trial, whereby amplification was observed for all samples, with an expected band size of 500 bp. 219 Figure 4.4: 16S rDNA PCR for the SA-3 (C-3) reference isolate cultivated for the experimental trial, whereby amplification was observed for all samples, with an expected band size of 1500 bp. 220 Figure 4.5: Agarose gel visualisation of 16S rDNA PCR products for the SA-3 (C-3) reference isolate cultivated for the experimental trial when viewed using a UV transilluminator (Spectroline®). 222 Figure 4.6: The disease profile showing the daily mean disease score of the control (red) and experimental (green) cohorts. 224 Figure 4.7: Clinical signs and symptoms observed in chickens presented with IC. 226 Figure 4.8: Egg production indicated as the total number of eggs laid/chcicken/day in the experimental and control groups. 229 Figure 4.9: Haematological observations for blood slides showing different blood cells observed for infected chickens. 235 XXIV LIST OF FIGURES Figure 4.10: Flow cytometry profile of one of the experimental chickens (E3-score 0) showing the forward scatter (FSC-A) and side scatter (SSC-A) plot. 237 Figure 4.11: Flow cytometry profile of one of the experimental chickens (E3-score 0) showing the side scatter (SSC-A) vs CD45 PE-A plot. 238 Figure 4.12: Flow cytometry profile of one of the experimental chickens (E3-score 0) showing the CD45 PE-A vs CD4 FITC-A plot. 239 Figure 4.13: Flow cytometry profile of one of the experimental chickens (E3-score 0) showing the side scatter (SSC-A) vs CD8 PE-A plot. 240 Figure 4.14: Flow cytometry profile of one of the experimental chickens (E3-score 0) showing the CD45 PE-A vs CD4 FITC-A plot. 241 Figure 4.15: % Total leukocytes (CD45+ cells) plot gave all CD45+ cells detected via flow cytometry over 21 days in whole blood of both control and experimental chickens (p>0.05). 243 Figure 4.16: Blood smears used with the flow cytometry results obtained for % total leukocytes over 21 days showing cell morphology. 246 Figure 4.17: % CD4+ cells of total leukocytes plot gave all CD4+ cells detected via flow cytometry over 21 days in whole blood of both control and experimental chickens (p>0.05). 248 Figure 4.18: % CD8+ cells of total leukocytes plot gave all CD8+ cells detected via flow cytometry over 21 days in whole blood of both control and experimental chickens (p>0.05). 250 XXV LIST OF FIGURES Figure 4.19: Flow cytometry profile showing the CD4+/CD8+ ratio of cells detected via flow cytometry over 21 days in whole blood of both control and experimental chickens (p>0.05). 251 Figure 4.20: Plot with % B and NK (natural killer) cells of total leukocytes detected via flow cytometry over 21 days in whole blood of both control and experimental chickens (p>0.05). 252 Figure 4.21: Graphical representation and statistical analysis of the sandwich ELISA assay conducted on chicken plasma samples from the experimental trial for the cytokine IL-8 (p<0.05). 259 Figure 5.1: Necropsy and gross post-mortem examination was conducted on a chicken carcass. 370 Figure 5.2: Summarized flow diagram of steps followed for IHC staining. 372 Figure 5.3: Following the dissection, post-mortem examination of internal lymphoid organs. 378 Figure 5.4: Necropsy of trachea. 379 Figure 5.5: No growth observed on TSA supplemented with 0.2% NAD+ (v/v) plates for nasal swabs obtained from chickens E1-score 1, E23-score 1, E8-score 2, E20-score 2, E22-score 3 and E24-score 3. 380 Figure 5.6: Nasal swab samples cultured on BTA plates. 381 Figure 5.7: Liver swabs of chicken E20 showed that there was no growth observed on (A) BTA or (B) TSA supplemented with 0.2% NAD+ (v/v) plates. 382 Figure 5.8: Tracheal swabs of chickens Control 1, E15 RI and E19. 382 XXVI LIST OF FIGURES Figure 5.9: HPG2-PCR conducted from nasal and tracheal swab samples, whereby amplification was observed for all samples, with an expected band size of 500 bp, confirming the presence of Av. paragallinarum DNA. 383 Figure 5.10: Results for immunohistochemical staining with anti-CD3 marker for the liver, spleen and intestine of control and infected birds. 386 Figure 5.11: Results for immunohistochemical staining with anti-CD20 marker for the liver, spleen and intestine of control and infected birds. 387 Figure 5.12: Results for immunohistochemical staining of the trachea with anti-CD3, anti-CD20 and anti-CD68 marker for control, score 3 and score 3 RI birds. 399 Figure 5.13: Results for immunohistochemical staining of the uterus (shell gland) with anti-CD3 and anti-CD20 marker for control, score 3 and score 3 RI birds. 400 XXVII LIST OF TABLES LIST OF TABLES Page Table 1.1: Comparison of the classification of Av. paragallinarum using the original scheme by Kume et al. (1983) and the newly proposed scheme by Blackall et al. (1990b). 14 Table 1.2: Key characteristics and different biochemical tests on the genus Avibacterium. 19 Table 1.3: Three primers (two primer pairs) used for the species-specific PCR by Chen et al. (1996). 21 Table 3.1: Oligonucleotide primers for species-specific and 16S rDNA PCR amplification of target region. 139 Table 3.2: Nucleotide BLAST results for all 16S rDNA PCR products with species identification, GenBank® accession numbers, query length, query coverage, E-value and high sequence identities. 156 Table 4.1: Cell count and CFU/ml results for triplicate plating of bacterial culture for the first injection. 216 Table 4.2: Cell count and CFU/ml results for plating of bacterial culture for the second injection. 217 Table 4.3: Nucleotide BLAST results for all 16S rDNA PCR products for the experimental trial with species identification, GenBank® accession numbers, query length, query coverage, E-value and high sequence identities. 221 XXVIII LIST OF TABLES Table 4.4: Summary of disease progression over the course of 21 days (3 weeks) and the total number of chickens with clinical scores from each cohort. 225 Table 5.1: Reagents and conditions used for processing of tissue samples with Tissue‐Tek® VIP5 automated processor (Sakura Finetek) as per the SOP of the NHLS. 373 Table 5.2: IHC results for the detection of cells expressing CD3, which is the T lymphocyte population in the liver, spleen and intestine for control, score 1, score 2 and score 3 chickens. 388 Table 5.3: IHC results for the detection of cells expressing CD20, which is the B lymphocyte population in the liver, spleen and intestine for control, score 1, score 2 and score 3 chickens. 392 Table 5.4: IHC results for the detection of cells expressing CD3, CD20 and CD68; which represent the T lymphocyte, B lymphocyte and macrophage/monocyte population within the trachea of control, score 3 and score 3 RI birds. 396 Table 5.5: IHC results for the detection of cells expressing CD3 and CD20; which represent the T lymphocyte and B lymphocyte population within the uterus (shell gland) of control, score 3 and score 3 RI birds. 398 XXIX LIST OF ANNEXURES LIST OF ANNEXURES Page Annexure A 126 Annexure B 179 Annexure C 269 Annexure D 284 XXX LIST OF APPENDICES LIST OF APPENDICES Page Appendix A 199 Appendix B 203 Appendix C 354 Appendix D 356 XXXI ABSTRACT ABSTRACT Infectious coryza is a contagious and acute upper respiratory poultry disease caused by Avibacterium paragallinarum, that plagues predominantly layers but also affects broiler breeds. The chicken’s immune response to Av. paragallinarum serovar C-3 (SA-3) infection (reported to be most virulent in South Africa), and the underlying genetic mechanisms involved, are poorly understood and not well documented. The aim of the study is to understand the complexity of the regulation of immune functions by identifying the molecules that are expressed during Av. paragallinarum serovar C-3 (SA-3 strain) infection. In this study, chickens (control versus experimental groups) were directly challenged via infraorbital injection with Av. paragallinarum serovar C-3 (SA-3 strain) and the immune response was monitored. The mean disease score and mean daily egg production score were recorded and calculated. Blood and sera were obtained for blood microscopy, leukocyte population profiling by flow cytometry analysis, and antibody/cytokine screening with ELISA assays. Finally, control and experimental chickens were sacrificed based on the clinical scores obtained (0, 1, 2 or 3). Post-mortem examination was conducted, and organs were harvested for immunohistochemistry staining for identification of distinct immune cell populations. The in vivo results obtained from the experimental studies in combination with the in silico results obtained from bioinformatics tools for the generation of immune signalling pathway maps may provide insight and a birds-eye view into the immune mechanisms between host-pathogen interactions for this disease. Results from our study could potentially assist with diagnostic tests for serovar C-3 and provide insight towards more efficient vaccine development. Hence, if vaccine practices are improved this will limit importations of birds from huge global markets, thus preventing carry-over poultry diseases and zoonosis as well as maintaining a safe and sustainable economy. Key words: Avibacterium paragallinarum; infectious coryza; poultry, immunity; microscopy; flow cytometry, ELISA; post-mortem examination; immunohistochemistry. XXXII CHAPTER 1: LITERATURE REVIEW CHAPTER 1 LITERATURE REVIEW Introduction 1.1. Infectious coryza: An overview In South Africa, the poultry industry comprises of the largest agricultural sector and is a major contributor to the country’s economy having a gross income of R37.8 billion per annum (South African Poultry Association, 2012). One of the main aims of the poultry industry is to provide food security and quality. Economic growth and development are correlated with the consumer demand for protein, which implies that as income increases, consumers will enhance their nutritional requirements with protein rich foods such as eggs and chicken meat (Pattison et al. 2008). Currently, there is a high consumer demand for poultry meat and egg production, due to dietary preferences, cost of living, processing technology, change in lifestyle and cultural or religious constraints (Taha and Hahn, 2015). Thus, it can be difficult to increase egg and poultry supplies, whereby birds are raised under commercial conditions making them vulnerable to environmental exposure and susceptible to infection by pathogenic microorganisms, eventually leading to disease (Sharma, 1999). One notorious and frequently encountered poultry disease is infectious coryza (Blackall, 1999). The disease has worldwide distribution and economic significance, pertaining to poor growth performance in growing broods and a marked 10-40% decrease in egg production (Yamamoto, 1984; Blackall, 1999; Vargas and Terzolo, 2004; Blackall and Soriano, 2008). An example of an IC outbreak was over a three-year period from 1986 to 1988 in China, whereby IC caused economic losses of about 100 million yuan (approximately US$ 15 million) at the 2018 exchange rate (Chen et al. 1993). 1 CHAPTER 1: LITERATURE REVIEW Infectious coryza (IC) is characterized as a fast-spreading and acute upper respiratory disease caused by Avibacterium paragallinarum (previously called Haemophillus paragallinarum) (Blackall et al. 2005). During the early phases of infection, IC may be acute to sub-acute, progressing to a chronic respiratory disease state with complications from the presence of other pathogens such as Mycoplasma synoviae, Mycoplasma gallisepticum, Salmonella spp., Pasteurella spp., chronic Escherichia coli and infectious bronchitis virus (Rimler et al. 1978; Reid and Blackall, 1984; Droual et al. 1992a; Droual et al. 1992b; Sandoval et al. 1994; Badouei et al. 2014). In 1968, the first IC outbreak was reported on a multi-age farm with approximately 100 000 layers in South Africa (Buys, 1982; Bragg, 2005). Consequently, several vaccinations have been developed since 1975 until today, based on the limited knowledge surrounding virulence factors and antigenicity of affecting serovars (Bragg et al. 1996; Mena-Rojas et al. 2004). Irrespective, infectious coryza still plagues layer and broiler breeds, especially during the winter season (Bragg et al. 1996). Hence, the characteristics and functions of the avian immune response to antigenic and immunogenic proteins from Av. paragallinarum, is yet to be studied and elucidated (Mena-Rojas et al. 2004; Boucher et al. 2015). 1.2. History: Etiological agent In the 1920s, IC was recognized as a unique and separate clinical entity (Beach, 1920). However, for several years, IC evaded identification and detection, as the disease was masked by other sources of infections, such as fowl pox (Blackall and Soriano, 2008). Early literature clinically describes IC as roup, contagious or infectious catarrh, cold and uncomplicated coryza (Yamamoto, 1972; Blackall and Soriano, 2008; Akter et al. 2013). Furthermore, during that era the disease was called Coryza infectiosa gallinarum (De Blieck, 1932; Nelson, 1933; Elliot and Lewis, 1934). De Blieck (1932) was the first to isolate and name the etiological agent Bacillus hemoglobinophilus coryzae gallinarum (De Blieck, 1932; Hinz 2 CHAPTER 1: LITERATURE REVIEW and Kunjara, 1977). Due to the infectious nature of the disease, and that solely the nasal passages were affected, the term “Infectious Coryza” was coined (Beach and Schalm, 1936). However, similar closely resembling microorganisms were also reported (McGaughey, 1932; Nelson, 1933). The genus Haemophilus (derived from Greek nouns haima, meaning “blood”, and philia, meaning “loving or fondness”), meaning “blood-loving”, was created to classify bacteria growing optimally and specifically in the presence of blood, haemoglobin, serum or protein- containing fluids (Winslow et al. 1920). Therefore, the proposed name Haemophilus gallinarum as the causative agent of infectious coryza was eagerly accepted (Elliot and Lewis, 1934; Delaplane et al. 1934). Knowledge pertaining to the growth essentials of haemophili, and the introduction of the terms X (hemin) and V (nicotinamide adenine dinucleotide (NAD+) factors was advancing at a fast pace (Thjötta and Avery, 1921; Lwoff, 1937; Lwoff and Lwoff, 1937; Hinz and Kunjara, 1977). McGaughey (1932) reported that the strains isolated only required factor V and not X, whereas Beach and Schalm (1936) and Delaplane et al. (1938) instead reported on strains that were both X and V dependant organisms (Beach and Schalm, 1936; Delaplane et al. 1938). The work conducted by McGaughey (1932) was largely disregarded and H. gallinarum was proposed as an X and V factor dependant strain (Blackall, 1989). Following reports and evidence as of the 1960s onwards, based on avian haemophili requiring solely V factor, Biberstein and White (1969) suggested a new species for those bacteria that were V factor dependent and X factor independent that came to be known as Haemophilus paragallinarum. Recently, using methods described by Beach and Schalm (1936), it was shown that X factor independent and V factor dependent strains of H. paragallinarum were found to be X and V factor dependent (Blackall and Yamamoto, 1989). Indicating that the authenticity and validity of the work by Beach and Schalm (1936) was under speculation and that there were limitations in their techniques (Blackall, 1989). Moreover, evidence that was once considered as the most credible on X and V factor dependency of H. 3 CHAPTER 1: LITERATURE REVIEW gallinarum is now an unsettled matter and has been questioned whether it ever existed, as the strains used by Beach and Schalm (1936) were lost (Biberstein and White, 1969). Additionally, McGaughey (1932) recognized that more than one haemophili can exist in chickens, where he found two bacterial groups based on growth characteristics, this work was also largely overlooked, until Page (1962) indicated that the strains he worked with comprised of two avian haemophili groups (Blackall, 1989). These two groups comprised of catalase positive and catalase negative haemophili. The catalase positive group was aerophilic and non-pathogenic to experimental birds, whereas the catalase negative group would not grow in oxygen (capnophilic or microaerophilic) and caused typical infectious coryza symptoms on inoculation of susceptible birds (Blackall, 1989). H. paragallinarum belongs to a unique group within the bacterial family, Pasteurellaceae (Bisgaard, 1993). Blackall et al. (2005) investigated the genotypic and phenotypic taxonomy of avian 16S rDNA cluster 18 of the Pasteurellaceae family, using DNA sequencing as a tool. The study showed that the avian-associated species H. paragallinarum, Pasteurella gallinarum, Pasteurella avium and Pasteurella volantium, forms a monophyletic group, with a sequence similarity of 96.8% (Blackall et al. 2005). Based on these results H. paragallinarum, P. gallinarum, P. avium and P. volantium were reclassified into a new genus termed Avibacterium (Blackall et al. 2005). Due to the occurrence of Avibacterium paragallinarum (H. paragallinarum) and Avibacterium gallinarum (P. gallinarum) within a single genus, this raised the opportunity for confusion (Blackall et al. 2005). Per traditional nomenclature, within the genus Haemophilus the prefix ‘para’ was used to highlight a species similar to an existing species, however these two species Av. paragallinarum and Av. gallinarum differ in growth factor requirements (Blackall and Yamamoto, 1989). 4 CHAPTER 1: LITERATURE REVIEW The Pasteurellaceae family consists of the genus Avibacterium, which further consists of five genuinely named species: Av. gallinarum, Av. paragallinarum, Av. avium, Av. volantium and Av. endocarditidis and one unnamed taxon, Avibacterium species A (Blackall et al. 2005; Bisgaard et al. 2007). Classification of Avibacterium isolates can be challenging when using phenotypic identification due to variable species characteristics (Blackall, 1988a; Blackall and Nørskov-Lauritsen, 2008). A study by Bisgaard et al. (2012), using multilocus sequence analysis (MLSA) and multilocus sequence typing (MLST) of Avibacterium, successfully identified and confirmed the existence of the species Av. paragallinarum. However, the identification of species of other members of Avibacterium could not be resolved, even by DNA sequencing (Bisgaard et al. 2012). Hence, the validity of the current members of Avibacterium, except Av. paragallinarum, has been questioned, whereby the discrepancies could have been due to original misclassification of the isolates or the use of a diverse bacterial strain collection during investigations (Bisgaard et al. 2012; Alispahic et al. 2014). Furthermore, the authors hypothesised that members of Avibacterium might be incipient species, due to the close phylogenetic relationship and high similarity observed between some of the Avibacterium isolates, with the exception of Av. paragallinarum (Bisgaard et al. 2012). A study by Alispahic et al. (2014) confirmed the findings suggested by Bisgaard et al. (2012) and also showed that matrix-assisted laser desorption ionization–time-of flight mass spectrometry (MALDI-TOF MS) can be used as a fast and reliable method for the correct identification and separation of Av. paragallinarum. Thus, further taxonomic investigation and re-organization is required for the genus Avibacterium, whereby whole genomes will need to be compared among different isolates (Bisgaard et al. 2012; Alispahic et al. 2014). 1.3. Epidemiology: Host, incidence and transmission The natural host for Av. paragallinarum is the chicken (Gallus gallus) (Blackall et al. 1997; Blackall and Soriano, 2008). There have been numerous reports of IC in other avian species 5 CHAPTER 1: LITERATURE REVIEW such as pheasants, Japanese quails and guinea fowls, however these reports have not been validated by phenotypic or genotypic studies and should be regarded and interpreted with caution (Yamamoto, 1991; Thenmozhi and Malmarugan, 2013). Species like turkey, pigeon, sparrow, duck, crow, rabbit, guinea pig, and mouse are refractory to experimental infection, although, there have been reports on experimentally infected Japanese quail with Av. paragallinarum in Australia (Yamamoto, 1972, Yamamoto, 1978; Reece et al. 1980; Thenmozhi and Malmarugan, 2013). Upon referral, a flock of peafowl were diagnosed with IC at the University of Ilorin Veterinary Teaching Hospital (Ilorin, Nigeria), this was the first reported case of infectious coryza present in peafowl (Adenkola et al. 2016). Av. paragallinarum is non-pathogenic to humans, and hence does not have any serious implications on public health (Blackall and Soriano, 2008). A study by Byarugaba et al. (2007), showed that turkeys and guinea fowls from Uganda were susceptible to IC and only chickens were infected, however transmission of IC could not be demonstrated from infected chickens or from infected turkeys to turkeys or chickens in close proximity, respectively, even though there was sharing of water and feed in the same containment. Similarly, Yamamoto and Clark (1966) conducted studies on sparrows, and were unsuccessful in showing transmission from infected sparrows to in-contact chickens or transfer of infection from inoculated chickens to the in-contact sparrows under experimental conditions. Thus, it is undoubtedly apparent that Av. paragallinarum is the etiological agent of infectious coryza in chickens, and that the role of other avian species in its epidemiology is yet to be revealed. IC is a cosmopolitan disease and is prevalent wherever chickens are raised and bred (Vargas and Terzolo, 2004). IC predominantly affects layers, however there have been reports in broiler breeds in North and South America, usually found with other bacteria or pathogens (Droual et al. 1990a; Droual et al. 1990b; Sandoval et al. 1994; Conde et al. 2011). IC has a global distribution and has been reported in several countries such as Argentina (Linzitto et al. 1988), Australia (Arzey, 1987), Bulgaria (Giurov, 1984), Canada (Kerr and Hammarlund, 6 CHAPTER 1: LITERATURE REVIEW 1982), Egypt (Aly, 2000), United Kingdom (Roberts et al. 1964), Guatemala (Matzer, 1974), Holland (De Blieck, 1932), India (Sobti et al. 2001), Indonesia (Takagi et al. 1991), Iraq (Rashid and Poeiecha, 1984), Switzerland (Baumann, 1982), United States of America (Rooney, 1979; Cutler, 1980; Droual et al. 1990a; Droual et al. 1990b; Hoerr et al. 1994; Matsumoto, 1999), Mexico (Guzman et al. 1980; Soriano et al. 2001), South Africa (Buys, 1982; Bragg, 2005) and Peru (Mendoza-Espinoza et al. 2009). Although, it is a disease mostly associated with the ever growing, expansive and intensive poultry industry, it can also occur in less industrialised situations such as in the kampong (village) chickens in Indonesia and other Asian countries that are just as susceptible to infection as commercial breeds (Zaini and Kanameda, 1991; Zaini et al. 1992, Poernomo et al. 2000). New Zealand is the only country reported globally that seems to be free of IC (Vargas and Terzolo, 2004). The main reservoirs of IC are both healthy and chronically infected birds (Blackall and Soriano, 2008). IC has been proposed to be air-borne and can spread through contact with infected chickens, and ingestion of contaminated water and feed (Yamamoto, 1991). However, it has been demonstrated that Av. paragallinarum cannot survive too long outside of its host thereby becoming inactivated (Bragg et al. 2004). No other vectors- mechanical or biological, have been reported other than healthy and chronic birds (Blackall and Soriano, 2008). Interestingly, IC is non-transmissible through eggs (Blackall and Soriano, 2008). 1.4. Clinical signs and symptoms of disease Infectious coryza occurs commonly during autumn and winter months in subtropical climatic areas or during the rainy season in tropical regions (Blackall and Soriano, 2008). All age groups are susceptible to IC, which usually occurs within 1-6 weeks after exposure, whereby birds are segregated, moved and quarantined from the brooder house to another cage with 7 CHAPTER 1: LITERATURE REVIEW more mature infected birds (Clark and Godfrey, 1961). However, in juvenile birds the disease is less severe (Blackall and Soriano, 2008). A short incubation period, fast and highly contagious spread; high morbidity (20-50%) and low mortality (5-20%) are typical characteristics of IC (Chen et al. 1993; Blackall and Soriano, 2008, Pattison et al. 2008). The disease develops within 24-48 hours after infection with Av. paragallinarum culture or tissue fluid (exudate), followed by a longer progression of the disease in adult birds, especially with active egg-laying hens (Yamamoto, 1984; Blackall and Soriano, 2008, Pattison et al. 2008). Susceptible and infected birds will show signs within 1-3 days of infection, whilst flocks will show signs at 7-10 days, depending if the infection is mild or severe, with severe infection taking 3 weeks to display (Pattison et al. 2008). A B Figure 1.1: Chickens infected with Av. paragallinarum. (A). Conjunctivitis with closed eyes and facial swelling. (B). Excessive frothy facial and nasal discharge. (Taken from Akter et al. 2013). Mostly, IC affects the upper respiratory tract, whereas the lower respiratory tract (lungs and air sacs) is affected by chronic complications from other infectious microorganisms (Alder and Page, 1962; Reid and Blackall, 1984; Akter et al. 2013). The most typical clinical signs and symptoms include conjunctivitis (Figure 1.1A), nasal and ocular discharge (Figure 1.1B), facial oedema, excessive secretion of tears, swelling of the sinuses, swollen-head syndrome 8 CHAPTER 1: LITERATURE REVIEW (especially in males), anorexia, diarrhoea, poor appetite and water consumption, dyspnoea, fetid odour of exudates and poor growth in younger chickens (Blackall, 1999; Pattison et al. 2008). Rales may be heard due to infection in the lower air tract, and lesions can be present leading to acute catarrh (Blackall and Soriano, 2008). 1.5. Antigenic structure and virulence factors There is limited knowledge pertaining to the virulence factors of Av. paragallinarum. Gyles and Thoen (1993) showed that the bacterial capsule is linked with virulence, where bacteria with capsules are more virulent than those that are non-encapsulated. Av. paragallinarum has a capsule that mediates adhesion to the surface of mucous membranes of its host, leading to colonization and is also considered to be involved in resistance to bactericidal activity of normal chicken serum (Ueda et al. 1982; Sawata and Kume, 1983; Sawata et al. 1984; Sawata et al. 1985a; Sawata et al, 1985b, Nakamura et al. 1993). Sawata and Kume (1983) demonstrated that a capsular antigen and a haemagglutinating antigen (HA) are responsible for pathogenicity. Kume et al. (1984) showed that the hyaluronic-like component has an important function in the capsule since treatment with hyaluronidase leads to degradation of the capsule. A gene encoding for hemagglutinin has been recognized and completely sequenced known as hagA, which functions as an adhesion-binding constituent to respiratory mucin (Hobb et al. 2002). Moreover, putative compounds have been identified such as repeats-in-toxin (RTX) proteins (other virulence factors), metalloproteases and haemocin (a toxin secreted by the bacteria that inhibits growth of closely related strains) that may have implications on virulence, moreover it has been reported that these substances may be secreted via membrane vesicles by Av. paragallinarum (Terry et al. 2003; Rivero-Garcia et al. 2005; Ramón Rocha et al. 2006; Blackall and Soriano, 2008). In addition, it had been demonstrated that Av. paragallinarum can form biofilms due to the presence of HMTp210, a haemagglutinin / adhesion protein (Tokunaga et al. 2005; Noro et al. 2007; Noro et al. 2008; 9 CHAPTER 1: LITERATURE REVIEW Wang et al. 2014). Biofilms are accretions of bacteria forming a sedimentary layer on biotic or abiotic surfaces which aids the bacteria to develop resistance towards the host immune system (Wang et al. 2014). It is still unclear whether the biofilm formation contributes towards virulence in Av. paragallinarum (Wang et al. 2014). Thus, future studies can be conducted to affirm whether biofilm formation contributes to virulence. 1.6. Vaccines and treatment Early intervention strategies may be of value in the control and management of IC, which involves the use of biosecurity, medication and vaccination (El-Ghany, 2011). Effective biosecurity measures include minimising exposure of chickens to IC infection. Recovered birds are potential carriers of the disease. Therefore, to eradicate the possibility of IC contamination on farms it is imperative to depopulate infected or recovered birds, especially in cases of an outbreak (Blackall and Soriano, 2008). Moreover, parental stock and starting chicks should not be purchased from unknown sources. On multi-age farms chickens, should be reared and housed in segregation away from older stock (Blackall and Soriano, 2008). Furthermore, to reduce the severity and duration of IC; proper and regular cleaning, fogging and disinfection of equipment, water and premises should be conducted, whereby premises should be left vacant for a few weeks before replenishing with clean or specific-pathogen-free (SPF) chickens (Bragg and Plumestead, 2003; Bragg, 2004). The treatment of IC involves the use of sulfonamides and antibiotics such as erythromycin, oxytetracycline, streptomycin, sulfodimethoxine, tylosin tartrate, spectinomycin and norfloxacin to reduce the severity of the disease (Blackall et al. 1997, Lublin et al. 1993). Currently, antibiotic resistance is a major concern due to intensive and widespread utilisation of antibiotics leading to negative consequences that has an impact on both human or animal 10 CHAPTER 1: LITERATURE REVIEW health and food safety (Moyane et al. 2013). Antibiotic drug resistance to Av. paragallinarum is common as such strains have been revealed to carry plasmids, thus conducting tests on antimicrobial sensitivity is strongly advised (Blackall, 1988b). As per, the Food and Drug Administration (FDA) withdrawal periods should be strictly adhered to by individuals in poultry farming and husbandry (Donoghue, 2003). The withdrawal period refers to the amount of time that must pass after the last antibiotic treatment has cleared the animal’s system, before the animal is slaughtered (chicken meat) or its product used (eggs) for human consumption (Food and Drug Administration, 1958; Donoghue, 2003). Withdrawal times can vary from a few days to weeks depending on the antibiotic used, whereby the withdrawal times are printed on the antibiotic’s label when sold to a farmer or veterinarian (Food and Drug Administration, 1958; Donoghue, 2003). Moreover, farmers are expected to adhere to the specific antibiotic withdrawal times and keep a track record for future reference that verifies compliance. However, compliance to the withdrawal period does not give complete assurance that antibiotic residues are no longer present in the animal tissue (chicken meat) and as such implies that the minimum amount of residue remaining in the organism’s system is not considered harmful or a public health concern by governing authorities (Food and Drug Administration, 1958; Donoghue, 2003). In addition, the use of some antibiotics in layers is prohibited in some countries. Some of the drugs could also lead to adverse side and toxic effects in birds; hence dosages need to be administered correctly at appropriate time intervals. However, if treatment is discontinued a relapse can occur, as some birds may still be carriers of the bacteria and harbour the disease (Yamamoto, 1978). The safest and most effective prevention method against IC is vaccination (Blackall and Reid, 1987; Reid and Blackall, 1987). Infectious coryza vaccination programs do not exempt chickens from getting infected, however the severity of clinical manifestations and progression of the disease is lessened by the reduction of shedding and spreading of the bacteria (El- Ghany, 2011). IC vaccines are manufactured from inactivated and internationally recognised 11 CHAPTER 1: LITERATURE REVIEW reference strains of Av. paragallinarum; however such vaccines do not provide protection against the local variants of the bacteria (Rimler et al. 1977a; Blackall, 1999). Commercialized IC bacterins (a suspension of killed or attenuated bacteria) are commonly available on the market, usually derived from broth-grown cultures (Blackall and Soriano, 2008). The mode of delivery of these bacterins is by injection via subcutaneous or intramuscular routes directly into the leg or breast muscle (Matsumoto and Yamamoto, 1975; Davis et al. 1976; Iritani et al. 1984; Blackall and Reid, 1987). Whole cell vaccines should consist of 108 colony-forming units/ml to be considered effective (Matsumoto and Yamamoto, 1975; Rimler et al. 1975; Davis et al. 1976; Coetzee et al. 1982; Iritani et al. 1984; Blackall and Reid, 1987). Some vaccines also contain adjuvants such as aluminium hydroxide gel (Alhydrogel®), mineral oil (Whiterex 307), purified saponin (Quil A®), oil-based emulsion (oil-in-water or water-in-oil) and a combination of aluminium hydroxide and mineral oil (Stone et al. 1981; Reid and Blackall, 1987). Adjuvants play a vital role in the enrichment or modulation of immunogenicity of weak antigens by enhancing the speed and duration of immunity, controlling antibody avidity, specificity, isotype or subclass distribution by stimulation of cell-mediated response, as well as improving immune response in immature or senescent individuals (Blackall and Matsumoto, 2003; Lindblad, 2004, Rajput et al. 2007). In ovo vaccination against IC may become a possible option for farmers, which could allow the birds to develop early protection and immunity. Moreover, in ovo vaccination decreases the chances of handling chicks, it reduces stress and anxiety of chicks from manual injection, there is a lesser risk of needle contamination, it is less labour intensive, and the volume dosage delivery is more accurate. 1.7. Avibacterium paragallinarum 1.7.1. Serological classification 12 CHAPTER 1: LITERATURE REVIEW Mainly two interconnected classification schemes have been developed to serotype Av. paragallinarum, namely the Page and the Kume schemes (Page, 1962; Kume et al. 1983). Page (1962) was the first to perform serological classification of Av. paragallinarum in the United States, whereby a slide agglutination test was used to identify three serotypes: A, B, and C. Although the plate agglutination test was developed by both Page (1962) and Kato and Tsubahara (1962) there were major disadvantages. Firstly, there was a problem of spontaneous agglutination (Barnard, 2001). Secondly, several field isolates of Av. paragallinarum were found to be “untypable” (Blackall et al. 1990a). The Haemagglutination (HA) and Haemagglutination inhibition (HI) tests are currently the only tests available for classification of Av. paragallinarum, whereby Kato et al. (1965) was the first to demonstrate the haemagglutinating ability of Av. paragallinarum. Kume et al. (1983) developed a typing system based on the haemagglutinating antigens. The first step is to obtain the haemagglutinating antigens by treating bacterial cells with potassium thiocyanate (KSCN) and sonication of the cells, this is followed by a HA test (Kume et al. 1983). The HA test comprises of agglutinating fresh haemagglutinating antigens with glutaraldehyde-fixed chicken erythrocytes (GA-fixed RBC) (Kume et al. 1983). Finally, the different isolates were treated using the haemagglutination inhibition (HI) test with fresh rabbit raised antisera against each isolate (Kume et al. 1983). Based on the HA/HI tests, the Kume scheme identified three different serogroups, termed I, II and III, and seven different serovars, HA-1 to HA-7 (serovars HA-1 to HA-3 (serogroup I), serovars HA-4 to HA-6 (serogroup II) and serovar HA-7 (serogroup III). Later on, two additional serovars were found; HA-8 belonging to serogroup I by Eaves et al. (1989) and HA- 9 allocated to serogroup II by Blackall et al. (1990b). Moreover, Blackall et al. (1990b) altered the nomenclature on the Kume scheme, since it was demonstrated that the Kume serogroups were linked to the Page serovars A, B and C. Hence, the Kume scheme was modified to nine serovars, which we still use today (Blackall et al. 1990b) (Table 1.1). 13 CHAPTER 1: LITERATURE REVIEW Table 1.1. Comparison of the classification of Av. paragallinarum using the original scheme by Kume et al. (1983) and the newly proposed scheme by Blackall et al. (1990b). (Taken from Blackall et al. 1990b). Original scheme (Kume et al. 1983) New scheme (Blackall et al. 1990b) Reference isolates Serogroup Serovar Serogroup Serovar 0083/221 I HA-1 A A-1 2403 I HA-2 A A-2 E-3C I HA-3 A A-3 HP14 I HA-8 A A-4 H18 II HA-4 C C-1 Modesto II HA-5 C C-2 SA-3 II HA-6 C C-3 HP60 II HA-9 C C-4 0222 III HA-7 B B-1 Currently, several laboratories around the world can perform Page serotyping and this is the most common serotyping scheme in use. The Kume scheme is not used on a routine basis since this scheme is too technically demanding. Serotyping is vital for the production of vaccines, and incorrect typing of a serovar could result in failures in the administration of vaccines for IC (Soriano et al. 2004a). However, serological typing is quite time-consuming, cumbersome, expensive and laborious. Moreover, monoclonal antibodies (Mab) are not easily available and difficult to produce. Various haemophilic species exist in chickens as part of the normal microbiota, as a result it is difficult to obtain pure cultures of Av. paragallinarum (Mutters et al. 1985; Chen et al. 1996). Hence, there is a risk of contamination or outgrowth 14 CHAPTER 1: LITERATURE REVIEW by other haemophili when isolating and culturing Av. paragallinarum (Mutters et al. 1985; Chen et al. 1996). Therefore, the use of molecular techniques would be most ideal, as it would be possible to type several serovars simultaneously, it would require less time and it would be cost-effective. 1.7.2. Cultivation and growth conditions Av. paragallinarum is considered to be a fastidious organism, however it is not difficult to isolate (Blackall and Soriano, 2008). Two or three chickens should serve as specimens, for isolation of the bacterium (Blackall and Soriano, 2008). Swabbing can occur in two ways: a small and precise incision is made into the sinus cavity of infected birds using sterile scissors, proceeded by the insertion of a sterile cotton swab into the sinus cavity (invasive technique performed when the bird is dead) or a sterile cotton swab can be used around the nasal areas where nasal discharge is prominent (non-invasive technique performed when bird is still alive) (Blackall and Soriano, 2008). The sinus cavity is rich in Av. paragallinarum in its unadulterated form (Blackall and Soriano, 2008). The swab is streaked onto a suitable agar plate or inoculated into media. Av. paragallinarum grows optimally on blood tryptose agar (BTA) plates or test medium agar supplemented with chicken serum and NAD+ (TM/SN) or the broth version of TM/SN (test medium broth, TMB) (Yamamoto, 1984; Reid and Blackall, 1987; Eaves et al. 1989; Chen et al. 1993). BTA plates contain cattle, horse, rabbit, chicken or sheep inactivated serum (Yamamoto, 1984). TM/SN media is a mixture of oleic-albumin complex at 5% (v/v), chicken serum at 1% (v/v) and NADH at 0.0025% (w/v) (Eaves et al. 1989). NAD+-dependent Av. paragallinarum strains need NADH (reduced form) or NAD+ (oxidized form) to grow. Hence, the Av. paragallinarum is cross-streaked with a Staphylococcus culture and incubated. 15 CHAPTER 1: LITERATURE REVIEW Staphylococcus cultures such as Staphylococcus epidermidis or Staphylococcus aureus, on the agar plate acts as a “feeder” culture, as this bacterial strain has the ability to haemolyse blood resulting in NAD+ release (Page, 1962; Vargas, 2004). Media should also contain sodium chloride (NaCl) at 1.0–1.5%, which is necessary for growth of the bacterium (Rimler et al. 1977b). The pH of the media should be between 6.9 to 7.6 (Blackall and Soriano, 2008). Av. paragallinarum grows ideally in microaerobic or microaerophilic conditions under an atmosphere of 5-10% carbon dioxide (CO2) (Rimler et al. 1976; Vargas, 2004). In the laboratory, a candle is placed and allowed to burn out in a jar with a tightly fitted lid together with growing cultures, to mimic oxygen-deprived conditions (Bragg et al. 1997). The minimal and maximal temperatures of growth for Av. paragallinarum are 25 and 45°C, with the optimal range being 34-42°C for 16-24 hours after incubation, provided that the correct CO2 levels, supplements and media is used (Yamamoto, 1984; Vargas, 2004; Blackall and Soriano, 2008). 1.7.3. Morphology and staining Av. paragallinarum is a Gram-negative and non-motile bacterium. Properly grown bacterial cultures appear as short rods or coccobacilli in shape (Blackall and Soriano, 2008). The dimensions for Av. paragallinarum include the length at 1–3 µm and the width at 0.4–0.8 µm, where filament development is possible (Blackall and Soriano, 2008). An outer membrane capsule may be present in virulent strains of Av. paragallinarum (Hinz, 1973; Sawata et al. 1980). After, 48-60 hours Av. paragallinarum degenerates and becomes fragmented, hence it is advisable to work with fresh cultures on a daily basis for laboratory work. 16 CHAPTER 1: LITERATURE REVIEW 1.7.4. Colony morphology Depending on optimal conditions and selective media used, colonies belonging to Av. paragallinarum appear as tiny dewdrops that are non-haemolytic in nature at 0.3 mm in diameter (Blackall and Soriano, 2008). Some colonies are mucoid (smooth) and iridescent, whereas some colonies are irregular (rough) and do not display iridescence (Hinz, 1973; Rimler, 1979; Sawata and Kume, 1983). Moreover, colonies (NAD+-dependent strains) show satellitic behaviour in the presence of feeder cultures such as S. epidermidis (Blackall et al. 1997; Chen et al. 1998). 1.7.5. Biochemical properties Av. paragallinarum though a Gram-negative bacterium, has completely different biochemical properties from its other avian Avibacterium counterparts (Table 1.2.) (Vargas, 2004; Kuhnert and Christensen, 2008). Species from the genus Avibacterium have the potential to reduce nitrate to nitrite, ferment glucose via specific metabolic pathways without the production of gas and possess oxidase activity, however they are unable to produce indole, hydrolyse urea or gelatine (Blackall et al. 1998). Av. paragallinarum can produce acid from maltose, D-mannitol and D-sorbitol and lacks catalase activity, unlike other avian haemophili (Blackall and Soriano, 2008). Moreover, Av. paragallinarum lacks the ability to ferment galactose or trehalose, as well as produce formazan from 3, 3, 5-triphenyl-tetrazoil chlohydrate (Terzolo et al. 1993). There is considerable misinterpretation surrounding carbohydrate fermentation patterns of V- factor dependent species due to variability in results recorded in literature (Blackall and Soriano, 2008). As such, false-negative results obtained are mainly attributed to poor growth (Blackall and Soriano, 2008). For routine identification for the determination of carbohydrate fermentation patterns, a medium consisting of phenol red broth with 1% (w/v) NaCl, 25 g/ml 17 CHAPTER 1: LITERATURE REVIEW NADH, 1% (v/v) chicken serum and 1% (w/v) carbohydrate is used (Blackall, 1983; Terzolo et al. 1993). 18 CHAPTER 1: LITERATURE REVIEW Table 1.2. Key characteristics and different biochemical tests on the genus Avibacterium. (Taken from Kuhnert and Christensen, 2008). Characteristics Av. Av. Av. Av. Av. gallinarum endocarditidis paragallinarum volantium avium Haemolysis (ovine blood) − − − − − CO2 improves growth + − + − − Symbiotic growth (-NAD − − V + + requirement) Catalase + + − + + Urease − − − − − Indole − − − − − ODC (Ornithine decarboxylase) − − − V − Acid from: (+)-L-Arabinose − − − − − (+)-D-Arabinose V − − + − (+)-D-Galactose + + − + + Lactose V − − V − Maltose + + V + − (-)-D-Mannitol − + + + − (+)-D-Mannose + + + + + (-)-D-Sorbitol − + V V − Trehalose + + − + + ONPG (ortho-Nitrophenyl-β- V + − + − galactoside) - Fucosidase − + − − − -Galactosidase − + − − − -Glucosidase + + − + + -Glucosidase − + − − − -Xylosidase − + − − − Host Birds Chickens Chickens Birds Birds Data based on: Avibacterium gallinarum (Christensen et al. 2002; Bisgaard et al. 2005); Avibacterium endocarditidis (Bisgaard et al. 2007); Avibacterium paragallinarum (Hinz, 1980; Blackall and Reid, 1982; Blackall et al. 2005); Avibacterium volantium (Mutters et al. 1985); Avibacterium volantium (Mutters et al. 1985). Characters are scored as: +,  90% positive; −,  10% positive; V, 11–89% positive. 19 CHAPTER 1: LITERATURE REVIEW 1.7.6. Molecular methods of detection Technology has advanced at a highly fast pace during the past decades that DNA fingerprinting and molecular techniques have become widely available for the identification of Av. paragallinarum. Ribotyping and restriction endonuclease analysis (REA) have been beneficial in identifying and linking NAD-independent strains from South Africa, using an rDNA 16S probe (a 16S rDNA operon inserted into a plasmid vector PUC19) (Miflin et al. 1995). However, there are only a limited number of molecular typing techniques that have been reported for the differentiation of different serovars of Av. paragallinarum, which include Enterobacterial Repetitive Intergenic Consensus (ERIC)-polymerase chain reaction (PCR) (Soriano et al. 2004b), multiplex PCR (mPCR) and PCR-Restriction Fragment Length Polymorphism (RFLP) (Sakamoto et al. 2012). ERIC-PCR is a molecular serotyping technique that has been suggested for molecular differentiation of various bacterial species, whereby ERIC sequences are intergenic consensus sequences that are highly conserved, and are located at different loci within a genome for each species or strain (Sharples and Lloyd, 1990; Hulton et al. 1991; Chatelut et al. 1995; Khan et al. 1998; de Souza et al. 2015). During amplification using PCR with ERIC sequences as the target sequence, oligonucleotide primers ERIC1R (reverse primer) and ERIC2 (forward primer) are used, which produces different band sizes (Versalovic et al. 1991). The different band sizes result in unique banding profiles which can be compared to group isolates, thus subtyping of strains based on the banding patterns obtained is possible (Versalovic et al. 1991). Although, Soriano et al. (2004b) reported that different Av. paragallinarum serovars could be distinguished from one another using ERIC-PCR, the results were difficult to interpret as ERIC-PCR showed several patterns even within each serovar (Sakamoto et al. 2012). Additionally, the results obtained from the findings of Soriano et al. (2004b) and Khan et al. (1998) differ from each other in terms of dissimilar banding patterns 20 CHAPTER 1: LITERATURE REVIEW reported. A study by Hellmuth et al. (2017) confirmed that ERIC-PCR is unsuitable for the differentiation or for molecular typing of Av. paragallinarum serovars, as the banding patterns of field isolates and reference strains cannot be correlated to one another, although isolates of similar origin have unique banding patterns that are shared. Species-specific PCR or HPG2-PCR is a PCR-based technique that was developed by Chen et al. (1996). HPG stands for H. paragallinarum. The technique used 4 DNA probes and 2 PCR tests (HPG1-PCR and HPG2-PCR) designed specifically for Av. paragallinarum. HPG1- PCR (combination of F1 and R1 primers) yielded a PCR product of 1.6 kb and HPG2-PCR (combination of N1 and R1 primers) yielded a 0.5 kb amplicon (Table 1.3.) (Chen et al. 1996). However, the HPG1-PCR gave several false negatives and was unreliable due to the lengthy PCR fragment, whereas HPG2-PCR gave better results even in the presence of normal microbiota (Chen et al. 1996). Although, species-specific PCR can detect whether or not Av. paragallinarum is present, it cannot be used to distinguish between different serovars A, B and C (Sakamoto et al. 2012). Table 1.3. Three primers (two primer pairs) used for the species-specific PCR by Chen et al. (1996). Primer Amplicon size Primer Name Primer sequence combinations (bp) F1 5’-CAA TGT CGAT CCT GGT ACA ATG AG-3’ F1 and R1 1600 R1 5’-CAA GGT ATC GAT CGT CTC TCT ACT-3’ N1 5’-TGA GGG TAG TCT TGC ACG CGA AT-3’ N1 and R1 500 R1 5’-CAA GGT ATC GAT CGT CTC TCT ACT-3’ 21 CHAPTER 1: LITERATURE REVIEW The gene hmtp210 of Av. paragallinarum encodes for a 210-kDa outer-membrane protein that functions as a haemagglutinin (HA), and is a major protective antigen playing a role in pathogenicity (Tokunaga et al. 2005; Noro et al. 2007, Wang et al. 2014). HA is a trimeric autotransporter adhesin functioning not only in hemagglutination but also in cell adherence and biofilm formation (Wang et al. 2014). The hmtp210 gene consists of three regions based on DNA sequence homology (Figure 1.2) (Sakamoto et al. 2012). Regions 1 and 3 are highly conserved regions for serovars A and C, whereas the homology of region 2 between serovars A and C was found to be approximately 50% (Figure 1.2) (Wu et al. 2011; Sakamoto et al. 2012). Region 2 is also known as the hypervariable region of the hmtp210 gene (Figure 1.2). Therefore, region 2 can be seen as a serovar-specific region, and as such is the best region in the htmp210 gene to study for protection, against serovar A and C (Figure 1.2) (Sakamoto et al. 2012). Sakamoto et al. (2012) developed and used multiplex PCR and PCR-RFLP based on region 2 of the HMTp210 gene. It was found that the DNA sequence homology of this region corresponded to more than 99.8% within each serovar, the strains used in the study were: 221, 083, W, Georgia and Germany (serovar A); Spross and 0222 (serovar B); and 53– 47, Modesto and HK-1 (serovar C), respectively (Sakamoto et al. 2012). A study by Morales- Erasto et al. (2014) showed that mPCR yields poor performance in terms of low sensitivity for recognition of serogroup C isolates and has a relatively high level of inaccuracy pertaining to the results of serogroups A and B. Wang et al. (2016) made comparisons between their study and the research conducted by Morales-Erasto et al. (2014), whereby both studies found that for certain strains/isolates the mPCR gave correct results across all Page serogroups A, B and C. Furthermore, both studies found that complications arose when the mPCR gave a serogroup B result (Wang et al. 2016). Additionally, the previous findings by Morales-Erasto and co-workers indicated that most false serogroup B mPCR results were associated with serogroup C, whereas Wang and co-workers implicated false mPCR serogroup B results to serogroup A (Wang et al. 2016). Moreover, it was suggested that different geographic sources of the bacterial isolates/strains of the two studies, was a contributing factor for the difference in results (Wang et al. 2016). Both studies suggested that the mPCR and PCR-RFLP 22 CHAPTER 1: LITERATURE REVIEW molecular assays are challenging for routine diagnostic use and not suitable for identifying the serogroups of Av. paragallinarum isolates (Wang et al. 2016). Figure 1.2: Location of primers for amplification of regions within the HMTp210 open reading frame (ORF) region. (Taken from Sakamoto et al. 2012). 1.8. Avian immunity 1.8.1. Host-pathogen interactions during Gram-negative bacterial invasion Chickens like many avian species or other vertebrates are endlessly surrounded by micro- organisms and pathogens (Akira et al. 2006; Genovese et al. 2013). Prior to the publication of the chicken genome that was first sequenced in 2004, the avian immune system and its unique structural features, such as the absence of lymph nodes and the presence of the bursa of Fabricius for B-lymphocyte development, were still a mystery (International Chicken Genome Sequencing Consortium, 2004; Kaiser, 2010). In order to understand host-pathogen interactions in avian species, we first need to have a comprehensive understanding of the 23 CHAPTER 1: LITERATURE REVIEW immunological aspects unique to birds as well as those shared among other species (Kaiser, 2010). However, with the publication of chicken genomic data on genes involved in immune- regulation, comparisons between the chicken, human and murine immune systems have made it possible to understand the evolution, and some of the mechanisms of the avian immune system with its counterparts (Kaiser, 2010). The avian immune system is composed of the innate (non-specific) and adaptive (specific) immune responses (Erf, 1997). Evolution has highly impacted the development of complex systems used by organisms to sense and respond to an array of stimuli from their surrounding environment (Medzhitov and Janeway, 1997; Keestra et al. 2013). The systems primarily consist of sensory receptors that detect the occurrence of specific environmental cues and transduce these signals to intracellular effectors that elicit the applicable cellular response (Keestra et al. 2013). A critical feature of the immune system is its capability to recognize pathogens (non-self) while remaining unresponsive to self-antigens (Janeway, 1992). These receptor systems can be found in three distinct forms as endocytic receptors, cell-associated receptors expressed on the cell surface, cytoplasm or intracellularly in immune cells, and are found circulating as humoral proteins in plasma collectively called pattern recognition receptors (PRRs) (Janeway, 1989; Fearon and Locksley, 1996; Janeway and Medzhitov, 2002). These germ line encoded PRRs detect and bind to invading pathogens of microbial but not vertebrate origin based on a series of conserved molecular structural motifs such as lipopolysaccharide (LPS), lipoteichoic acid (LTA), peptidoglycans, flagellin and molecules of viral origin including double and single- stranded RNA and oligonucleotides, known as pathogen associated molecular patterns (PAMPs) (Janeway, 1989; Medzhitov and Janeway, 1997; Ginsburg, 2002; Iqbal et al. 2005; Kannaki et al. 2010). Thus, the fundamental mechanism pertaining to innate immune recognition is highly conserved from species to species (Akira et al. 2006). 24 CHAPTER 1: LITERATURE REVIEW Pathogens face an enormous challenge when invading the host’s immune system, they need to attach to the host tissue and replicate as fast as they can before the host’s system responds and recruits immune cells and molecules against foreign invaders (Gioannini and Weiss, 2007). Our focus will be primarily on PAMPs related to Gram-negative bacteria, as Av. paragallinarum belongs to this group of bacteria. Lipopolysaccharide (LPS) is a well-studied PAMP, which is found in Gram-negative bacteria (Miyake, 2004). During bacterial invasion there are primarily two outcomes; damage or no damage to the host (Casadevall and Pirofski, 1999). Gram-negative bacteria are composed of two lipid bilayers and have the following subcellular components; the outer membrane (OM), the peptidoglycan cell wall, the inner membrane (IM) and the periplasm (Silhavy et al. 2010). Secreted products in the form of vesicles from the OM, known as OM proteins, that contain virulence factors and other immunomodulatory compounds, contribute to the survival of the pathogen and lead to damage of the host (Kuehn and Kesty, 2005; Galdiero et al. 2012) LPS has long been known for its potent immune stimulatory activity but how this response is induced has remained enigmatic for decades until the discovery of a member of a large family of pathogen sensors or PRRs called Toll-like receptors (TLRs) (Janeway and Medzhitov, 2002; Keestra et al. 2013). TLRs function by detecting conserved microbial particles and in turn command eukaryotic cells to respond effectively by producing antimicrobial peptides, cytokines and chemokines (Kawai and Akira, 2007). Therefore, the host is protected from invading pathogens and their corresponding virulence factors are neutralised (Medzhitov and Janeway, 1997). To date at least 10 chicken TLRs have been identified that are orthologous and share gene repertoires with vertebrates (TLR3, 4, 5 and 7), mammals (TLR2A and 2B), fish and amphibians (TLR21) and that are unique to chickens (TLR1LA, 1LB, 15) (Kannaki et al. 2010). These TLRs can bind PAMPs, where binding of PAMPS to PRRs triggers the activation of immune cells, which in the case of macrophages leads to the production of inflammatory cytokines but also to activate signals for the adaptive immune system. In order 25 CHAPTER 1: LITERATURE REVIEW to identify the broad range of microorganisms additional PRR-families have evolved during evolution and are found in birds as in mammals (Kaiser, 2010). Due to their portend immune system activating activity, PAMPs are now intensively investigated as potential adjuvants by the vaccine industry and have shown promising results under experimental conditions (Powell et al. 2015). In a study by Boucher et al. 2014, it was indicated that initial pathogen recognition for Av. paragallinarum serovar C-3 occurs via TLR 2 and 4 respectively, there was also up- regulation of TLR 7, which the research group indicated and proposed could be as a result of prophages and their remnants, which could contribute to the severe inflammatory immune response observed with IC. In a study by Leveque et al. 2003, it was shown that the chicken TLR4 protein shows 46% identity (64% similarity) to human TLR4 and 41% similarity to other TLR family members. Moreover, using linkage studies they showed that TLR4 is associated with resistance to Salmonella enterica serovar Typhimurium (Leveque et al. 2003). Additionally, they showed using Northern blot techniques that TLR4 is expressed in the same concentrations in the brain, thymus, kidney, intestine, muscle, liver, lung, bursa of Fabricius, heart and spleen (Leveque et al. 2003). 1.8.2. Avian innate immune response The innate immune response in chickens is similar to that in human and the murine system, however there are a few differences in terms of the unique structural features present in the avian immune system. In the field of avian immunology, the chicken is the most researched species however, very little is known about the innate system with regards to other avian species. In order to understand host-pathogen interactions in birds, it is vital to have an in- depth understanding of features that are unique to birds, as well as those that are shared with other species (Kaiser, 2010). Compared to mammals, chickens have different repertoires of Toll-like receptors, cytokines, defensins, antibodies and other immune molecules (Kaiser et al. 2005; Boyd et al. 2007; Hughes et al. 2007; Lynn et al. 2007; Kaiser, 2007; Temperley et 26 CHAPTER 1: LITERATURE REVIEW al. 2008; Cormican et al. 2009; Kaiser, 2010). The innate immune system was previously mistaken as a mere scavenger or non-specific system that mainly involved phagocytosis and cell lysis (Playfair and Bancroft, 2013). However, today it is more apparent that there exists a complex interplay between both the innate and the adaptive immune responses involving immune cells, co-stimulatory molecules and cytokines (Schat et al. 2014). Mechanisms pertaining to innate immunity is found in a myriad of guises, ranging from the initial non-specific antimicrobial response of bactericidal enzymes, phagocytes and interferon to physical and chemical attack by pathogens to the co-ordinated recruitment of innate immune cells such as macrophages, dendritic cells (DCs), heterophils and natural killer cells (NKs) that are induced via PRRs eventually leading to the release of effector molecules namely cytokines and chemokines that impact inflammatory and acute phase responses, as well as influencing the aftermath effects of the adaptive immune response via the major histocompatibility complex (MHC) through antigen-presenting cells (APCs) (Kogut et al. 2005; Kaiser, 2010). The first-line of defence of the innate system following pathogen invasion consists of a highly effective constitutive physical barrier which includes the epithelial surfaces, mucus, ciliary movement in the airways, fatty acids on skin, peristaltic movement of the intestine, the gastric acidic pH, secretion of mucus and antimicrobial peptides (AMP) (Kaiser, 2010). Following, penetration of these barriers by pathogens this may result in lesions in the skin, the airways and other mucosal surfaces with increased risk of further infection (Schat et al. 2011). The normal microbiota, though not of host origin, present on body surfaces also help to prevent colonization by pathogens, and as such prophylaxis with undefined mixtures of normal gastrointestinal microbiota, designated as “competitive exclusion” is fed to day-old chickens to prevent Salmonella infections (Van Immerseel et al. 2005). Antimicrobial peptides (AMPs) are natural components and have been isolated from most living organisms. AMPs react by forming pores in the membrane of bacteria and fungi leading to cell death (Kagan et al. 1990). These peptides have antibacterial, anti-fungal, antiviral or anticancer properties and can 27 CHAPTER 1: LITERATURE REVIEW influence inflammation, proliferation, wound healing, release of cytokines, redox homeostasis and chemotaxis (Bals, 2000). Avian AMPs have been shown to be active against a number of microorganisms. In chickens, two classes of AMPs have been identified, cathelicidin-like proteins (Fowlicidin-1 and -2) and defensins (gallinacins (Gal)-1 to -13), showing cytotoxic effects and binding capacity to LPS, resulting in complete blockage of LPS-mediated pro- inflammation (Davison et al. 2011). Gal-1 and Gal-1α is reported to be effective in killing Staphyloccocus aureas, Escherichia coli, Candida albicans, Salmonella enterica, and Campylobacter jejuni but not Pasteurella multocida or infectious bronchitis virus (Harwig et al. 1994; Evans et al. 1995). Gal-11 was found to be primarily active against Salmonella typhimurium and Listeria monocytogenes (Higgs et al. 2005). Granulocytes and lymphocytes are immune cells mostly involved in the immune system that are derived from lymphoid stem cells (Playfair and Bancroft, 2013). The most important immune cells in the avian innate immune response are the phagocytes: macrophages and heterophils. NK cells are very poorly phagocytic, however they employ cytotoxic tactics to target and kill pathogens (Playfair and Bancroft, 2013). Macrophages belong to the mononuclear phagocytic system lineage derived from the bone marrow and are the first-line of defence against pathogenic invasion (Skamene and Gros, 1983; Qureshi, 2003). During cell development and differentiation, it takes about 6 days for a monoblast to develop into a pro-monocyte and then into a monocyte under the influence of colony stimulating factors (CSF) (Qureshi, 2003). After entering the bloodstream, it takes a further 3 days for blood monocytes, to seed various tissues and organs, whereby they differentiate into macrophages (Qureshi, 2003). Hence, macrophages are the tissue forms of blood monocytes and are known by various names such as alveolar macrophages (lungs), Kupffer cells (in liver), microglia cells (brain), osteoblasts (bones) and histiocytes (connective tissue) (Qureshi, 2003). Moreover, macrophages participate in a variety of functions, such as phagocytosis (engulfment) of foreign particles, opsonisation, chemotactic targeting of pathogens, annihilation of bacterial 28 CHAPTER 1: LITERATURE REVIEW and tumour cells, secretion of prostaglandins and cytokines that regulate activity of lymphocytes and other macrophages, as well as interact with the adaptive aspect of the avian immune response (Bonney and Davies, 1984; Qureshi et al. 1986; Kimball, 1990; Qureshi and Miller, 1991;). Phagocytosis is a complex and multi-step process, which starts off with the phagocyte migrating towards the site of infection via chemotactic gradients or chemokines secreted from other cells (Playfair and Bancroft, 2013). Once in the presence of the microbe, the phagocyte attaches to the bacterial cell wall via complement or antibody and engulfs the bacterium into a phagosome (Playfair and Bancroft, 2013). Once the bacterium is in the phagosome, lysosomes are released and come into contact with the phagosome, whereby oxidative and non-oxidative killing mechanisms via reactive oxygen intermediates (ROI) and nitric oxide (NO) pathways, are employed to kill the bacterium (Playfair and Bancroft, 2013). Finally, the act of phagocytosis ends by the internal clearance of the remains by digestion involving enzymes (Playfair and Bancroft, 2013). Additionally, macrophages process antigens and present antigenic fragments via antigen-presenting cells (APCs) to T lymphocytes within the framework of both MHC class I and II cell surface antigens (Unanue and Allen, 1987; Qureshi, 1998). Polymorphonuclear leukocytes (PMNs) are very important cellular components of innate immunity, and function by killing pathogenic microbes following phagocytosis (Kogut et al. 2005). The heterophil is the primary PMN in poultry, the avian equivalent to the mammalian neutrophil (Kogut et al. 2005). Like the neutrophil, avian heterophils are involved in the phagocytosis of invading microbes and foreign particles (Genovese et al. 2013). Heterophils lack myeloperoxidase, their bactericidal activity has low oxidative burst and their granular constituents seem to differ from those in mammalian neutrophils (Penniall and Spitznagel, 1975; Montali, 1988). Heterophils amass and infiltrate inflamed tissue, whereby tissue damage is caused leading to the formation of heterophil granulomas that are morphologically similar to inflammatory lesions found in reptiles (Montali, 1988; Harmon, 1998). It was found that during 29 CHAPTER 1: LITERATURE REVIEW receptor-mediated phagocytosis of opsonized and non-opsonized Salmonella enteritidis (SE), avian heterophils differentially expressed transcripts encoding proinflammatory and Th1 cytokines (Kogut et al. 2003). Heterophils and monocytes of the innate immune system develop earlier than T and B lymphocytes at hatching and after 2 weeks after hatching the population of PMNs increases in the gut- associated lymphoid tissue (GALT) (Burton and Harrison, 1969; Wells et al. 1998; Bar-Shira and Friedman, 2005). It was found that inflammatory stimuli such as LPS, turpentine or various infectious conditions like E. coli airsacculitis, staphylococcal tenosyvitis cause a notable influx of heterophils (Harmon, 1998). Moreover, the activation of heterophils by pathogens or by cytokines seem to induce the expression of various pro-inflammatory cytokines such as interleukins (IL) (IL-1, IL-6 and IL- 8) (Kogut et al. 2005, Kogut et al. 2006). Dendritic cells (DCs) in innate immunity play a unique participating role by activating naïve T cell subsets depending on the availability of high levels of MHCs and co-stimulatory molecules whether of lymphoid or myeloid progenitors (Playfair and Bancroft, 2013). Moreover, DCs determine the type of T cell response, which is dependent on stimulatory cytokines such as interleukin (IL)-10, IL-12 and IL-18 (Davison et al. 2011). Immature DCs are kept dormant in tissues and upon pathogen exposure this in turn activates DCs, and mobilise to naïve T-cell rich areas such the lymphoid organs in birds (Davison et al. 2011). During this migration phase, the DCs reach maturation differently and have the ability to influence downstream T cell responses, consequently manifested as lowered phagocytic capacity, increase in MHC molecules and accumulation of surface co-stimulatory molecules. However, it should be noted that activation of DCs rely solely on the type of PAMP-PRR interaction (Davison et al. 2011). Natural killer cells (NKs) are morphologically characterised as large lymphocytes with electron dense granula that share many features with cytotoxic T lymphocytes (Göbel et al. 1994). In 30 CHAPTER 1: LITERATURE REVIEW contrast, to B and T cell development, NKs have thymus-independent development (Bucy et al. 1989). Some infections can cause a severe, yet a temporary increase in the NK cell frequency, such a case was reported for Mycoplasma gallisepticum infection leading to an accumulation of NK (also CD8+) cells in the tracheal mucosa during the first week of infection (Gaunson et al. 2006). The serum complement system is the primary component of the innate response, and as such was observed as a heat-sensitive factor in serum that was complementary to heat-stable antibody during lysis of bacterial and red blood cells (Carroll, 2004).These complement proteins are produced by hepatocytes (liver cells) and macrophages with early synthesis of components such as C1, C2, C4 and C3 (Carroll, 2004). Today, the complement system is known to comprise of approximately 25 serum proteins, 10 or more cell surface complement receptors and regulatory proteins found on numerous host cells (Davison et al. 2011). Similar to the mechanism pertaining to PRRs, complement proteins circulate in an inactive form, however upon recognition of molecular motifs from pathogens, they become activated prompting a cascade-like effect, in which the binding of one protein promotes the binding of the next in a sequential cascade (Carroll, 2004). There are three main pathways in the complement system, with the pathways differing in manner of activation, however eventually through proteolytic cleavage reactions they lead to the production of C3 convertase, a key enzyme and factor in the complement system (Carroll, 2004). The classical pathway (CP) is activated by antibody-antigen complexes. The lectin pathway (LP) is activated by microbial carbohydrates in serum and tissue fluids, and the alternative pathway is activated by binding of C3b to microbial surfaces and antibodies (Carroll, 2004). Proteins that cause a change in plasma concentration by 25%, fever, drowsiness, an augmentation in cytokine production and haematological and metabolic alterations, upon inflammatory stimuli are called acute phase proteins (APP) (Schat et al. 2014). In addition, APP causing an increase during the response are positive APP while those that cause a decrease in the response are negative APP 31 CHAPTER 1: LITERATURE REVIEW (Davison et al. 2011). APP play a role in host adaptation or defence, inhibition of serine proteinases, and transport of proteins with antioxidant activity (Davison et al. 2011). Other factors such as MBL (mannan-binding lectin), FB (fibrinogen) and CRP (C-reactive protein), including C3 are upregulated via pro-inflammatory cytokines and thus also act as APP (Carroll, 2004; Playfair and Bancroft, 2013). Factor B is the only alternative pathway factor that has been characterised thus far and findings indicate that a factor B dependent alternative pathway is active in chickens (Kjalke et al. 1993). The complement system is important in the innate response and adaptive response since it is involved in the induction of inflammatory responses, boosts effects of opsonin activity (phagocytosis) and enhances the direct killing of target cells, as well as B and T cell responses (Playfair and Bancroft, 2013). The end result of complement activation is to trigger inflammation, chemotactically attract phagocytes to the site of infection, promote opsonization, cause lysis of Gram-negative bacteria and cells, expressing of foreign epitopes, participate in B-cell activation and remove harmful immune complexes from the body (Schat et al. 2014). There is a plethora of chemical acute responses during the innate immune response in chickens that serve to protect the host. Serine proteinases regulate extracellular matrix turnover, fibrinolysis and complement activation (Davison et al. 2011). However, to down- regulate the activity and effects of these enzymes, serpin inhibitors such as α1-proteinase inhibitor which inhibits neutrophil elastase, α1-antichymotrypsin which inhibits chrymotrypsin- like serine proteinases, and C1 inhibitor which inactivates the blood coagulation factors XIIa and XIIf, protects the integrity of host tissues (Davison et al. 2011). APP protects host tissues from toxic oxygen metabolites released from phagocytic activity during inflammation (Davison et al. 2011). CRP has both pro- and anti-inflammatory effects in vitro and in vivo involved in the clearance of damaged tissue, prevention of autoimmunity and regulation of the inflammatory response (Davison et al. 2011). In chickens, natural infection with the protozoan parasites such as Eimeria spp. and Histomonas induce high levels of CRP (Chamanza et al. 32 CHAPTER 1: LITERATURE REVIEW 1999). α1-Acid glycoprotein is a natural anti-inflammatory agent that inhibits neutrophil activation, increases the secretion of an IL-1 receptor antagonist by macrophages and might be involved in the clearance of lipopolysaccharide (LPS) by neutralizing its toxicity (Murata et al. 2004). In chickens, elevated levels of α1-acid glycoprotein have been recorded during infections with infectious bronchitis, infectious laryngotracheitis, infectious bursal disease viruses, E. coli, and Salmonella enterica serovar Enteritidis (Chamanza et al. 1999). Fibrinogen (FB) has a primary role in homeostasis, during fibrin formation and in tissue repair, by providing a substrate or matrix for the migration of inflammatory-related cells (Murata et al. 2004). Mannan-binding lectin (MBL) is synthesised in the liver and released into the blood stream, however, low expression of MBL has been demonstrated in organs such as the lung, thymus, kidney, small intestine and testis (Wagner et al. 2003). At the onset of pathogen invasion, the innate response is considered the most critical, preventing spread of the pathogen until the adaptive response becomes active with recruitment and mobilization of B and T cells (Juul-Madsen et al. 2003). In birds, the activation of complement by the alternate pathway, the action of C reactive protein, and of properdin which contributes to innate resistance in mammals, have yet to be described (Davison et al. 2011). The interaction of the lymphoid tissues with the infectious organism is known as specific immunity and is mediated by immune cells and the production of antibodies (Juul-Madsen et al. 2003). Thus, innate defence is critical to provide protection during the first weeks of life when the adaptive immune system is still under development. 1.8.3. Avian adaptive immune response The adaptive immune system is highly specific, it involves the interaction of the lymphoid organs, the lymphocytes, antigen recognition and immunoglobulin production (Playfair and 33 CHAPTER 1: LITERATURE REVIEW Bancroft, 2013). Specific immunity encompasses two main immune responses. This entails humoral immunity carried out by B lymphocytes producing antibodies, and the cell-mediated response which refers to T lymphocyte activity in conjunction with the MHCs (Erf, 1997). The B and T lymphocytes develop in the bone marrow of the chicken via haematopoiesis, thereafter they migrate and undergo maturation in unique lymphoid organs, whereby B lymphocytes mature in the bursa of Fabricius whereas T lymphocytes mature in the thymus (Davison et al. 1996). The immune system of a newly hatched chick is underdeveloped and therefore is unable to provide complete protection against pathogenic infection upon first encounter with the external environment, and as such, very few lymphocytes are found in the secondary lymphoid organs such as the spleen, caecal tonsil, Harderian gland or the bronchus associated lymphoid tissue (BALT) (Davison et al. 2011). Atrophy or prior surgical removal of these organs leads to severe immunosuppression and the birds become more susceptible to disease, as seen in IBDV (infectious bursal disease virus), MDV (Marek’s disease virus) or CAV (chicken anaemia virus) infection or in response to (experimental) glucocorticoid treatment (Dohms and Metz, 1991; Davison et al. 2011). T cells mature in the thymus and start to seed the periphery around hatching (Schat et al. 2014). Antigen recognition by T cells is a highly complex and remarkable process mediated by the T cell receptors (TCRs) (Chen et al. 1989). Unlike other surface receptors that are pre- committed to a specific ligand, T cell antigen recognition via the TCRs are randomly generated, whereby there is recognition of a set of diverse peptides complexed to MHC molecules (Chen et al. 1989). The TCR is a disulphide-linked membrane bound heterodimeric protein normally consisting of α and β chains expressed as part of a complex with the invariant CD3 chain molecules (Chen et al. 1989). T cells expressing these two chains are referred to as αβ T cells, although a small population of T cells do express an alternate receptor, formed by variable γ and σ chains, referred as γσ T cells (Chen et al. 1989; Siu et al. 1990). Additionally, to the TCR, the T cell has other accessory molecules or co-receptors in antigen 34 CHAPTER 1: LITERATURE REVIEW recognition that were defined using monoclonal antibodies and are helpful as T lymphocyte specific markers (Siu et al. 1990). CD4 a molecule, originally referred to as L3T4, is expressed on all T cells that are restricted to MHC class II molecules and is a T helper cell that serves to escalate B and T cell responses through the release of cytokines such as interferon-gamma (INF-), IL-4, IL-5, IL-13, IL-17 (Littman, 1987; Davison et al. 2011; Playfair and Bancroft, 2013). Cells expressing the CD8 molecule function as cytotoxic T cell, originally known as Lyt 2,3 in mice, which control viral infections and tumour formation via cytokines such as INF- (Littman, 1987; Spellberg and Edwards, 2001). CD8 is expressed on all MHC class I molecules (Littman, 1987). T cells must recognize antigen bound to MHC gene products and antigen- presenting cell (APC), before they can partake in any immune-related response (Erf, 2004). Like lymphocytes APCs, develop and mature in the bone marrow and migrate to peripheral lymph organs (Davison et al. 2011). Initiation of TCR signalling requires these co-receptors CD4 and CD8 that act as cellular adhesion molecules that bind to their respective MHC molecules and stabilize the interaction of T cells and APCs (Siu et al. 1990). T helper cells can be discriminated from each other on basis of function, in particular by cytokine secretion profiles (Spellberg and Edwards, 2001). T helper 1 (Th1) cells produce cytokine interferon-γ (INF-), which potently activates macrophages to combat a range of intercellular pathogens such as Salmonella, Mycobacteria and Listeria (Spellberg and Edwards, 2001). Th2 cells secrete cytokines IL-4 and IL-13, helping B-cells to develop into antibody producing cells (Spellberg and Edwards, 2001). These T cells are the classical T helper cells. More recently, additional subsets of CD4 T cells were identified (Chen et al. 1994; Fukaura et al. 1996; Powrie et al. 1996; Hafler et al. 1997). Th17 cells release IL-17, which can attract granulocytes into tissues infected by bacteria (Chen et al. 1994; Fukaura et al. 1996; Powrie et al. 1996; Hafler et al. 1997). Thus, Th1 and Th17 cells closely interact with innate immune system cells through cytokine secretion to control intra- and extracellular bacteria, respectively (Chen et al. 1994; Fukaura et al. 1996; Powrie et al. 1996; Hafler et al. 1997). While these cells of the CD4 lineage induce an immune response and inflammation, CD4 positive regulatory T cells (Tregs) 35 CHAPTER 1: LITERATURE REVIEW provide signals (IL-10, transforming growth factor beta 1 (TGF-β)), which control other T cell subsets to prevent immunopathology, auto reactivity, and down-regulation of an immune response after pathogen clearance (Davison et al. 2011). However, convincing evidence for the existence of Tregs is still lacking in avian species, due to the difficulties associated with the cultivation and isolation of T lymphocytes (Davison et al. 2011). T cells are tasked to monitor intracellular compartments of the host, whereas B cells are responsible for patrolling extracellular spaces such as blood and tissue fluid (Playfair and Bancroft, 2013). In contrast, B cell maturation shows striking differences to T cells and takes place in a unique organ, the bursa of Fabricius (Schat et al. 2014). The molecular mechanisms taking place during avian B cell development are well understood (Davison et al. 2011). However, their regulation is still largely unclear. As a net result of B cell maturation millions of B lymphocytes develop, each of them producing a unique antibody that differs from those antibodies generated by other B lymphocytes (Davison et al. 2011; Schat et al. 2014). Through this mechanism, the entire B cell pool of chicken can generate millions of different antibodies which theoretically should bind any antigen encountered by the bird (Davison et al. 2011). However, B cells with a specific antibody are rare and without further activation and enhancement of the immune system, insufficient amounts of antibodies will be generated (Davison et al. 2011). During a bacterial invasion, the immunoglobulin (Ig) surface molecule binds antigen, whereby the antigen-Ig complex is endocytosed and digested by enzymes (Playfair and Bancroft, 2013). Following digestion, the constituting peptides are collected and bind to MHC class II molecules (Playfair and Bancroft, 2013). The MHC-peptide complex either binds to the surface of a B cell or T helper cells recognise the MHC-peptide complex presented to them by DCs (Playfair and Bancroft, 2013). Once T helper cells recognise the MHC-peptide complex they activate into T effector cells and finally proliferate into clones (Playfair and Bancroft, 2013). Consequently, the effector T cell secretes cytokines such as IL- 2, IL-4, IL-5 and IL-6 to attract a B cell carrying the same MHC-peptide complex, which 36 CHAPTER 1: LITERATURE REVIEW activates the B cell (Playfair and Bancroft, 2013). The B cells proliferate into clones. Finally, some B cells differentiate into plasma cells and secrete antibody, while others remain as memory B cells (Playfair and Bancroft, 2013). A hallmark of antigen-specific B cell responses is the formation of germinal centres (GC) (Davison et al. 2011). In birds GCs are found in all secondary lymphoid tissues (Davison et al. 2011). Antigen transported to the spleen is presented to T and B cells thereby inducing a complex immune response resulting in the generation of high affinity antibodies (Davison et al. 2011). Specialized DCs, T helper cells and B cells physically interact with each other and exchange signals through cell surface receptors and secreted cytokines (Davison et al. 2011). During this response GCs form and provide an environment for the selection and promotion of B cells which produce the best antibodies to the presented antigen (Davison et al. 2011). Several cytokines involved in B cell maturation and function (antibody production) have recently been identified with the help of genomic data. Some of them have been cloned and shown to have potent B cell activating activities in cell culture systems. These cytokines have enabled extended B cell cultures and provided in vitro systems to investigate the interaction of host cells and pathogens with B cell tropism such as MDV or IBDV (Schermuly et al. 2015). Immunoglobulins (Ig) are synthesized by B cells and are globular glycoproteins that have antibody (Ab) activity and are found in the blood, lymph and vascularized tissues of vertebrates (Marchalonis, 1977; Litman et al. 1993). Antibodies have the basic unit structure that consists of four polypeptide chains: two heavy (H) and two light (L) that form the monomeric unit (H2L2) (Davison et al. 2011). Immunoglobulins are found in two forms, either as membrane-bound antigen receptors or soluble secreted molecules (Davison et al. 2011). Some antibodies such as IgG consists of a basic unit, however others like IgM and IgA are more complex and are made up from multiples of the basic unit (Schat et al. 2014). Only three main classes of antibodies that have been described for birds: IgM, IgA and IgY (Carlander et al. 1999). Both 37 CHAPTER 1: LITERATURE REVIEW IgG and IgY antibodies are more systemic antibodies, with IgA being a secretory antibody (Hopkins et al. 1987; Carlander, 2002). The structure and function of chicken IgM is homologous to its mammalian counterpart (Davison et al. 2011). IgM is the most prevalent antibody and is the first isotype to be expressed during embryonic development in developing chicks and after initial exposure to a novel antigen (Davison et al. 2011). The molecular weight (MW) of chicken IgM is in the range 823–954 kDa (Davison et al. 2011). Free M chains have been found in sera of bursectomized chickens and in survivors of infectious bursal disease virus (Choi and Good, 1971; Gauldie et al. 1973; Ivanyi, 1975; Higgins, 1976). Like in mammals, the response with IgM is usually transient, although for some chronic bacterial diseases, such as Bordetella avium in turkeys, the effect was reported to be active for several weeks (Suresh et al. 1994). However, due to the evolutionary conservation of IgM and its transient effect in the immune response, the area of avian IgM research has been rather limited. Chicken IgY has been found in duodenal contents, tracheal washings and seminal plasma (Carlander, 2002). Chicken IgY in sera is monomeric with MW ranging between 165–206 kDa (Davison et al. 2011). IgY is a unique avian maternal antibody transported from the hen to the offspring, involving a two-step process (Carlander, 2002). Firstly, IgY is transferred from the hen’s serum into the egg yolk, which is then followed by transmission of IgY from the yolk sac to the developing chick (Carlander, 2002). Phylogenetic studies have shown that the avian IgY is a homologue of mammalian IgG, sharing similarities with both mammalian IgG and IgE (Warr et al. 1995). IgY is the predominant and main isotype produced in sera in the secondary antibody response, after IgM is secreted in the primary antibody response (Davison et al. 2011). Both IgY and IgG are used interchangeably in literature. Avian immunologists prefer to use the term IgY because this Ig appears to be the evolutionary predecessor of both IgG and 38 CHAPTER 1: LITERATURE REVIEW IgE, sharing homology with each of these mammalian isotypes (Warr et al. 1995). The major difference between the chicken IgY and the mammalian homologue is the longer H chain in the chicken molecule (Davison et al. 2011). Avian IgY consists of five domains (V, C1–C4) and does not have a genetically encoded hinge, in contrast to the four domains that are found in mammalian IgG (Davison et al. 2011). Thus, avian IgY has limited flexibility, which may account for some of the unique biochemical properties, such as the inability to precipitate antigens at physiological salt concentrations, seen in chickens and ducks (Davison et al. 2011). Although IgY is the major avian systemic antibody active in infections, complete characterization has only been carried out in the chicken and in the duck (Davison et al. 2011; Schat et al. 2014). IgA is found in birds, predominantly in bodily secretions such as in the respiratory system, urogenital system and intestine, and play a major role in mucosal immunity (Playfair and Bancroft, 2013). IgA is a dimer that possesses a J chain that binds to a receptor on the surface of epithelial cells (Underdown and Schiff, 1986; Kerr, 1990). This receptor integrates with IgA as a secretory component (SC), whereby the IgA complex gets transported through epithelial cells and is secreted into the lumen of the designated organ (Solari and Kraehenbuhl, 1985). SC provides adhesion of IgA to the epithelial surface and protection from proteolytic degradation within the cells (Solari and Kraehenbuhl, 1985). The phylogenetic origins of IgA are still unknown. A secretory molecule IgX from the African clawed frog (Xenopus laevis) has been reported, that has antigenically similar properties to IgM and is secreted into the intestinal tract (Hsu et al. 1985). However, IgX is considered an analogue of IgA because of sequence differences and that it does not possess a J chain nor SC molecule (Mussman et al. 1996). Birds such as pigeons, penguins and flamingos produce a specific secretion in the crop sac called crop milk, which is regurgitated to feed to the young (Davison et al. 2011). Crop milk is rich in nutrients such as fats and proteins, however unlike mammalian milk it does not contain lactose (Davison et al. 2011). Moreover, the production of crop milk is under the control of 39 CHAPTER 1: LITERATURE REVIEW prolactin (Anderson et al. 1984). Crop milk is also rich in IgA with concentrations in the range of 1.5 mg/ml, though it contains little IgY (Davison et al. 2011). An increased uptake of IgA from crop milk leads to IgA accumulation in the intestinal tract which cannot pass through the epithelial gut lining into circulation, thus providing local immunity against pathogenic microorganisms within the gut (Davison et al. 2011). 1.8.4 Cytokines and chemokines of the avian immune system Cytokines are peptides having a molecular weight less than 30 kDa that controls the regulation of extracellular signals between cells during the onset and course of immunological responses (Davison et al. 2011). Cytokines have various functions and effects on cells as they elicit, modulate, and regulate immune as well as inflammatory responses (Davison et al. 2011). Cytokines are generally secreted; however, they also act as cell surface molecules influencing the cells of the immune system and consist of interleukins (IL), interferons (IFN), transforming growth factor-β (TGF-β) family, tumour necrosis factor (TNF) superfamily (TNFSF), colony- stimulating factors (CSF) and chemokines (Davison et al. 2011). The IL series have functional roles involving lymphocytes and the IFN series have antiviral effects (Davison et al. 2011). TGF-β, TNFSF, CSF and the TGF-β family has a crucial role in regulating inflammatory reactions, whereas TNFSF does not have any anti-tumour activity (Davison et al. 2011). Various cytokines and chemokines have been identified in mammals also present in chickens, however there are some exceptions with regards to multigene families, whereby the chicken seems to have fewer members than in mammals, which also explain the unique and fundamental differences in the organs and cells of the avian immune system (Davison et al. 2011). 40 CHAPTER 1: LITERATURE REVIEW Both IL-1β and IL-18 having functionality in inflammatory responses and have been identified in the chicken genome (Weining et al. 1998; Schneider et al. 2000). However, no other IL-1 family has been identified in the chicken genome and it is also likely that there are fewer IL-1 family members in chickens compared to mammals (Kaiser et al. 2005). Genes encoding IL- 2, IL-15 and IL-21, which play a role in T cell proliferation, all lie on chromosome 4 of the chicken, however none of the genes are in synteny (Sundick and Gill-Dixon, 1997; Lillehoj et al. 2001; Kaiser et al. 2005). IL-12α and IL-12β of IL-12 family, have a biological role in driving inflammatory Th1 responses (Balu and Kaiser, 2003). The chicken has only four members of IL-10 family: IL-10 and IL-19 on chromosome 26, and IL-22 and IL-26 on chromosome 1 (Rothwell et al. 2004; Davison et al. 2011). IL-10 seems to be conserved in the chicken and acts as an anti-inflammatory cytokine, downregulating the effects of IFN-γ (Rothwell et al. 2004). In mammals, IL-19 is a Th2 cytokine, whereas IL-22 and IL-26 are involved in inflammatory responses (Rothwell et al. 2004). In the IL-17 family only four genes have been identified in the chicken genome: IL-17A, IL-17B, IL-17D and IL-17F, that seem to play a role in Th17 cell responses, are known to be pro-inflammatory cytokines in both chickens and mammals and are produced early after infection as part of the induced innate immune response (Min and Lillehoj, 2002; Kaiser et al. 2005; Chen et al. 2006; Harrington et al. 2006). Type I IFN is divided into three subcomponents in chickens: IFN-α, IFN-β and IFN-λ (Sekellick et al. 1994; Sick et al. 1996). IFN-α and IFN-β both have antiviral activity (Sekellick et al. 1994; Sick et al. 1996). The chicken IFN-γ gene a Type II IFN was identified and in vitro was found to protect chicken fibroblasts from undergoing virus-mediated lysis, was capable of inducing nitrite secretion from macrophages, and showed enhanced MHC class II expression on macrophages (Digby and Lowenthal, 1995; Lowenthal et al. 1997). Chicken IFN-γ gene has a crucial role in mediating Th1-controlled responses (Digby and Lowenthal, 1995; Lowenthal et al. 1997). The TGF-β family in chickens, consisting of TGF-β1, TGF-β2, TGF-β3 and TGF-β4 are vital players in immune-regulation (Burt and Jakowlew, 1992; Jakowlew et al. 1988; 41 CHAPTER 1: LITERATURE REVIEW Jakowlew et al. 1990; Jakowlew et al. 1997; Pan and Halper, 2003). Members of the TNFSF and TNF receptor (TNFR) superfamily (TNFRSF) have central roles in both innate and adaptive immunity, including inflammatory responses, apoptosis, cell proliferation, and stimulation of the immune system (Davison et al. 2011). Some TNFSF members should be considered as co-stimulatory molecules, rather than cytokines, however members which can be considered as cytokines are TNF-α, lymphotoxin (LT)-α, LT-β and B cell activating factor of the TNF family (BAFF) (Davison et al. 2011). Chicken BAFF has been cloned and was found to mediate B cell survival (Koskela et al. 2004; Schneider et al. 2004). Chemokines have a role in the migration of leukocytes (homeostasis) and have a role in the recruitment of cells to sites of inflammation (inflammatory responses) (Davison et al. 2011). Chemokines can be divided into four groups – XC, CC, CXC and CX3C –, and are categorised on the basis of the spacing of the first two conserved cysteine residues at the amino termini of these chemotactic proteins, with the exception being XC, which lacks a first cysteine residue (Davison et al. 2011). For chemokines, the suffix “L” represents ligands, whereas their receptors are given the suffix “R” (Davison et al. 2011). Lymphotactin, the chicken orthologue of mammalian XCL, has been cloned and acts as a chemoattractant for splenic B cells (Rossi et al. 1999). The chicken lacks orthologues of CCL11, CCL24 and CCL26 (eotaxin 1–3), which are chemoattractants in mammals for eosinophils and basophils through the receptor, CCR3, which is also absent in the chicken (Davison et al. 2011). This clearly indicates that a lack of functional eosinophils in the chicken fits with the lack of eosinophil attracting chemokines (Davison et al. 2011). 1.9. Rationale and objectives of the study 42 CHAPTER 1: LITERATURE REVIEW There are a plethora of published works on various notable avian diseases related to disease management, vaccine development and gene expression profiling such as Newcastle disease, Marek’s disease, chicken anaemia virus, infectious bursal disease (Dohms and Metz, 1991; Davison et al. 2011). However, very few challenge studies specifically with regards to the monitoring of the avian immune response have been conducted encompassing disease progression at a genetic, cellular, tissue, and clinical level as a whole. Notable studies conducted on the avian model with regards to immune mechanisms were conducted on Salmonella serovars and avian influenza virus (Withanage et al. 2004, Withanage et al. 2005; Xing et al. 2008; Nerren et al. 2010). The aim of this study is to understand the complexity of the regulation of immune functions by identifying immune cells and molecules that are expressed during the chickens’ response to Av. paragallinarum serovar C-3 (SA-3 strain) infection. This knowledge is important since the exact immune defence mechanisms are poorly understood for this infection. It should be noted that by simply looking at cytokines, immune cells and antibodies, does not give a full understanding of the complexity of the immune mechanisms of Av. paragallinarum serovar C- 3 (SA-3 strain) infection, however it can provide some insight on how these cells and molecules fit or link as a puzzle into a greater picture involving immunological modulating pathways and immune cell interactions and how the organism is affected by the pathogen causing infection, specifically to serovar Av. paragallinarum C-3 (SA-3 strain). As such, this research is a stepping-stone to understanding a disease having dire consequences in the poultry industry and only has a small part to play in a bigger project that has a never-ending scope, which implies that there will always be a drive for future research work in this field, since there is much to discover. Moreover, this project will also further improve diagnostic tests for Av. paragallinarum serovar C-3 (SA-3 strain) (reported most virulent serovar in South Africa) in the veterinary field and also provide more perception with regards to vaccine development, since failed vaccination attempts is still a major problem with regards to IC. 43 CHAPTER 1: LITERATURE REVIEW Thesis layout with study objectives: Chapter 2: To perform in sillico data analysis, to identify potential immune signalling pathways using existing bioinformatics pathway databases and tools; for understanding future comprehensive experimental data and the systematic workings of the chicken immune system. Chapter 3: To conduct a pilot study of the immune response of control versus experimental chickens to Av. paragallinarum serovar C-3 (SA-3 strain); perform bacteriological cultivation, isolation and identification of the infecting serovar C-3 (SA-3 strain); monitor the chickens’ immune response for 21 days; conduct full and differential blood counts; to prepare blood smears; conduct haematological analysis of avian blood performing CD4+ (T helper cell population) and CD8+ (cytotoxic T cell population) cell population profiling using flow cytometry; conduct antibody profiling using direct ELISA; and establish research techniques to be used in further chapters. Chapter 4: To use infectious coryza as an infection model for poultry, monitor disease progression by clinical signs and symptoms, immune cells and molecules; perform bacteriological cultivation, isolation and identification of the infecting serovar Av. paragallinarum C-3 (SA-3 strain); conduct cytokine profiling using commercially available ELISA kits; perform microscopy on avian blood smears and conduct morphological classification of immune cells; and conduct haematological analysis of avian blood performing CD45+(pan-leukocyte), CD4+ (T helper cell population) and CD8+ (cytotoxic T cell population) cell population profiling using flow cytometry. Chapter 5: To conduct necropsy of lymphoid and non-lymphoid organs, as well as perform immunohistochemical staining of tissues obtained. Chapter 6: Concluding remarks and future studies. 44 CHAPTER 1: LITERATURE REVIEW REFERENCES Adenkola, A.Y., Jegede, H.O., Adeyemi, A.B., Raji, L.O., Kolapo, T.U., Oyedipe, E.O. 2016. 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W., Reid, W. M., Yoder, H. W., Jr. (eds). Iowa State University Press: Ames, Iowa, USA. pp. 186-195. Yamamoto, R., Clark, G.T. 1966. Intra- and interflock transmission of Haemophilus gallinarum. American Journal of Veterinary Research, 27(120): 1419-1425. Zaini, M.Z., Iritani, Y. 1992. Serotyping of Haemophilus paragallinarum isolated in Malaysia. Journal of Veterinary Medical Science, 54(2): 363–365. Zaini, M.Z., Kanameda, M. 1991. Susceptibility of the indigenous domestic fowl (Gallus gallus domesticus) to experimental infection with Haemophilus paragallinarum. Jurnal Veterinar Malaysia, 3(1/2): 21-24. 80 CHAPTER 2 IMMUNOMICS: In silico MAPPING OF IMMUNE SIGNALLING PATHWAYS IN CHICKENS RELATED TO Avibacterium paragallinarum SEROVAR C-3 INFECTION 2.1. Introduction A major problem faced by the poultry industry is that chickens are prone to opportunistic pathogens when reared under intensive conditions (Sharma, 1999). This factor is critical especially for chickens during the first week of life, as their immune system has not yet fully matured and the levels of maternal antibodies on which they are dependent are diminishing (Lowenthal et al. 1994). The aftermath, is that there is loss of productivity due to disease and substantial resources are required to treat and maintain the health status of these birds (Lowenthal et al. 2000). Infectious coryza is an important avian disease that has the ability to cause major economic losses in both layer and broiler breeds (Blackall et al. 1999). Av. paragallinarum forms part of the Pasteurellaceae family, however to date factors leading to pathogenicity, immunogenicity and serotyping are not well understood, even among the different serogroups (A, B and C). Over a thirty year period, it was observed that the incidence of serovar C-3 had been notably increasing (Bragg et al. 1996). IC, like many other avian infectious diseases, is the 81 CHAPTER 2: IMMUNOMICS: IN SILICO MAPPING OF IMMUNE SIGNALLING PATHWAYS IN CHICKENS RELATED TO AV PARAGALLINARUM SEROVAR C-3 INFECTION consequence of a complex set of interactions between the pathogen and host responses (discussed in Chapter 1). For more than 100 years, the chicken has been used as a model organism in developmental biology (Stern, 2005). The publication and availability of chicken genomic sequence information has resulted in detailed analysis and characterization of various and numerous genes related to immune signalling and regulation, which can provide insight into the mechanisms that drive the immune system leading towards either elimination of the invading pathogen or reduction in damage to the host caused by bacterial burden (International Chicken Genome Sequencing Consortium, 2004; Medzhitov, 2009; Boucher et al. 2014). There have been studies conducted on immune-related gene expression and the genetic mechanisms that control immunity for the avian model on Salmonella serovars, Eimeria parasite, Marek's disease virus and avian influenza virus (Xing and Schat, 2000; Withanage et al. 2004, Withanage et al. 2005; Hong et al. 2006; Xing et al. 2008; Adams et al. 2009; Nerren et al. 2010). Initially, the genetic mechanisms that elicit the immune responses of chickens infected with Av. paragallinarum were unknown, until a study by Boucher et al. (2015), using high through-put microarray technology screened for genes and regulated biological pathways that correlated to the immunity of birds during disease progression of IC. A study by Boucher and co-workers (2015), highlighted that the regulation of chicken genes to serovar C-3 infection, occurs via Toll-like receptor 4 (TLR4) through the myeloid differentiation factor-88 (MyD88)-dependent pathway, leading to activation of NF-κβ (nuclear factor kappa B), thus resulting in the production of inflammatory cytokines. This finding is similar to studies conducted, whereby C3H/HeJ mice expressing deficient levels of TLR4 showed higher mortality after challenge with live Salmonella enterica serovar Typhimurium or Escherichia coli, which are Gram-negative bacteria, suggesting that LPS/TLR4 downstream signalling is crucial for host protective immune responses (O’Brien et al. 1980; Cross et al. 1995; Vazquez-Torres et al. 2004). 82 CHAPTER 2: IMMUNOMICS: IN SILICO MAPPING OF IMMUNE SIGNALLING PATHWAYS IN CHICKENS RELATED TO AV PARAGALLINARUM SEROVAR C-3 INFECTION The chicken’s immune response to Av. paragallinarum SA-3 serovar C-3 (most virulent in South Africa) infection causing infectious coryza, is still poorly understood and not well documented. A gap in the exact immune defence mechanism involving the regulation of immune signalling molecules against infectious coryza (SA-3 strain) has prompted the need to identify the major antibodies, cytokines, chemokines and cells involved in immunity against this disease, and to study the regulation of immune signalling pathways. The aim of the study is to conduct gene enrichment, functional annotation of differentially regulated genes and identify potential immune signalling pathways using existing bioinformatics pathway databases and tools, for understanding future comprehensive experimental data and the systematic workings of the chicken immune system. The findings obtained in silico will enable us to predict potential immune responses in vivo during disease progression of chickens affected with IC. 2.2. Materials and methods 2.2.1. Animal ethics, experimental design and data analysis There was no animal experimental work involved or conducted in this study, rather existing biological data and resources collected from a previous trial that had already been published was used, as described (Boucher et al. 2014; Boucher et al. 2015). For this study, differentially expressed microarray data that had been processed and validated from Boucher et al. (2014, 2015), was obtained, whereby stringent filtering was performed (p- value≤0.02). Stringent filtering targets differentially expressed regulated genes having a higher confidence level. A flow diagram summarizing the bioinformatics pipeline used in the study for the interpretation of up- and down- regulated genes in avian immunity of birds infected with IC is shown (Figure 2.1). 83 CHAPTER 2: IMMUNOMICS: IN SILICO MAPPING OF IMMUNE SIGNALLING PATHWAYS IN CHICKENS RELATED TO AV PARAGALLINARUM SEROVAR C-3 INFECTION Figure 2.1: Flow diagram of bioinformatics pipeline indicating biological interpretation and outcome of in silico data of chickens infected with IC. Chicken images were published with permission from Dr C.E. Boucher. 2.2.2. Bio-statistical analysis Enrichment analysis of differentially expressed regulated genes was conducted using GO Enrichment Analysis connected to the PANTHER (Protein Analysis Through Evolutionary Relationships) Classification System PANTHER™ GO slim v14.0 (http://geneontology.org/). The enrichment analysis scouts for GO terms that are both over- or under- expressed using annotations for the gene list provided (Ashburner et al. 2000; Mi et al. 2017; Gene Ontology Consortium et al. 2017). Functional annotation of differentially regulated genes in networks 84 CHAPTER 2: IMMUNOMICS: IN SILICO MAPPING OF IMMUNE SIGNALLING PATHWAYS IN CHICKENS RELATED TO AV PARAGALLINARUM SEROVAR C-3 INFECTION was performed using the web-based tool DAVID v6.8 (Database for Annotation, Visualization and Integrated Discovery) (https://david.ncifcrf.gov/) whereby enriched functional-related gene groups, biological themes and meaning can be obtained from the gene list provided using a set of annotation tools (Huang et al. 2008a; Huang et al. 2008b). Moreover, DAVID has a larger database for enriched GO terms, which can assist with GO terms not provided using GO Enrichment Analysis. Results from high-throughput experiments such as microarrays interpreted by statistical testing and generating enriched GO terms may be extensive in size and highly redundant, making it difficult to comprehend (Supek et al. 2011). Hence, REVIGO (Reduce + Visualize Gene Ontology) (http://revigo.irb.hr/), a web server was used to reduce redundancy and allow GO terms to be summarised and visualised by searching for a representative subset of the terms via a clustering algorithm based on a measure of semantic similarity (Supek et al. 2011). To understand GO term terminology, QuickGO (https://www.ebi.ac.uk/QuickGO/) was used (Binns et al. 2009). A KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway identified as significantly differentially expressed, was visualised in a KEGG pathway plot using Pathview (https://pathview.uncc.edu/), whereby fold change was used that involves information on up- or downregulation (fold change >1 for upregulation and <1 for downregulation), an up-regulated gene was shown in red and a down-regulated gene in green (Luo and Brouwer, 2013; Luo et al. 2017). Functional association studies were performed using the manually curated STRING (Search Tool for the Retrieval of Interacting Genes) v11.0 database (https://string-db.org/) (Szklarczyk et al. 2014). For the primary study conducted by Boucher et al. (2014), birds were sacrificed according to the severity of IC symptoms and at different time periods post challenge. The reason for the 12-24 h intervals between time periods, was to initially allow for adequate time for the 85 CHAPTER 2: IMMUNOMICS: IN SILICO MAPPING OF IMMUNE SIGNALLING PATHWAYS IN CHICKENS RELATED TO AV PARAGALLINARUM SEROVAR C-3 INFECTION disease to run its course during the infection of chickens with the purpose of properly scoring and assessing IC symptoms, as each chicken would react differently when injected with the Av. paragallinarum culture. Moreover, the different time periods are the approximate times when the different clinical symptoms ranging from mild to severe were observed. As per literature, the disease develops in chickens within 24-48 h following infection with Av. paragallinarum culture, whereby infected birds will show IC symptoms within 24-72 h of infection (Pattison et al. 2008). Group 1 was represented by score 0 chickens, which were also control birds. Group 2 represented score 1 chickens with mild IC symptoms, whereby chickens with mild clinical symptoms were sacrificed at 36-48 h post-infection (PI). Group 3 consisted of chickens with score 2 showing moderate IC symptoms, whereby chickens with moderate clinical symptoms were sacrificed at 72-96 h PI. Group 4 had score 3 chickens with severe symptoms for IC and these chickens were sacrificed at 108-132 h PI. For comparative purposes the differentially expressed and regulated genes from the microarray data of experimental birds (Group 2, 3 and 4) were compared to control birds (Group 1). 2.3. Results and discussion 2.3.1. Gene enrichment analysis From the GO Enrichment Analysis for the enrichment of differentially expressed regulated genes from the microarray data, pie charts were obtained representing the percentage distribution of the biological process ontology for the comparison of the differentially expressed regulated gene cohorts of Group 2 (score 1) vs 1 (score 0), Group 3 (score 2) vs 1 (score 0) and Group 4 (score 3) vs 1 (score 0). Figure 2.2 shows that as the clinical score progressed during IC infection, from score 1 (mild) to score 3 (severe), there was an increase in the regulation of genes related to the immune response (0.5% → 2.2% → 2.6%). 86 CHAPTER 2: IMMUNOMICS: IN SILICO MAPPING OF IMMUNE SIGNALLING PATHWAYS IN CHICKENS RELATED TO AV PARAGALLINARUM SEROVAR C-3 INFECTION A gradual transition from innate to adaptive immunity was also observed from score 1 to score 3. From the gene enrichment for Group 2 (score 1) vs 1 (score 0), it was found that for 810 differentially expressed genes that was queried, only 422 had been mapped and annotated. The main immune response for Group 2 (score 1) vs 1 (score 0) was found to be the innate immune response. This was in line with the host’s response to Av. paragallinarum, whereby the innate immune response is the first line of defence to pathogenic infection consisting of physical barriers, the serum complement system, immune cells (natural killer (NK) cells, heterophils, macrophages and dendritic cells) and molecules (cytokines and chemokines) (Kaiser, 2010; Carroll, 2004; Davison et al. 2011). For Group 3 (score 2) vs 1 (score 0), out of 1095 genes that were differentially expressed, only 571 genes were upregulated, mapped and annotated. GO identifiers with percentage distribution associated with the immune response (Figure 2.2) were represented as pie charts (Figure 2.3). Genes representing both innate and adaptive immune responses, as well as the immune effector process were seen to be up-regulated for Group 3 (score 2) vs 1 (score 0), whereas with Group 2 (score 1) vs 1 (score 0) only the innate immune response was found to be up-regulated, showing that there was a transition from innate to adaptive immune responses from score 1 to score 2 (Figure 2.2). Moreover, T cell proliferation and differentiation indicated that the main lymphocytes involved in the immune response were T lymphocytes (Figure 2.3). T lymphocytes are mediators of both the innate and adaptive responses and are expressed as either CD4+ (helper T cells) or CD8+ (cytotoxic T cells) cells, whereby antigen-presentation occurs via MHC class II for CD4 molecules and MHC class I for CD8 molecules on the surfaces of T cells leading to the activation of these cells (Davison et al. 2011). During the innate response, CD4+ cells such as Th1 cells produce 87 CHAPTER 2: IMMUNOMICS: IN SILICO MAPPING OF IMMUNE SIGNALLING PATHWAYS IN CHICKENS RELATED TO AV PARAGALLINARUM SEROVAR C-3 INFECTION INF- activating macrophages, Th2 secretes IL-4 and IL-3 which assist in the differentiation of naïve B cells into plasma cells that secrete antibody (Spellberg and Edwards, 2001). CD8+ cells display cytotoxic activity against pathogens during the adaptive immune response (Zhang and Bevan, 2011). Figure 2.2: Biological process pie charts obtained from GO Enrichment Analysis linked to the PANTHER Classification System representing the percentage distribution of the biological process ontology in the comparison of the differentially expressed regulated gene cohorts of Group 2 (score 1) vs 1 (score 0), Group 3 (score 2) vs 1 (score 0) and Group 4 (score 3) vs 1 (score 0). As the clinical score progressed during IC infection, from score 1 (mild) to score 3 (severe), there was an increase in the regulation of genes related to the immune response (section shown with arrows). This suggests that following the innate and adaptive immune responses there are various mechanisms from different systems (metabolic, endocrine, gastrointestinal, pulmonary, neurological, blood, etc.) involved that prevent the after effects of the immune system from damaging the tissues of the host organism while also deterring infection. For Group 4 (score 3) vs 1 (score 0), out of 194 genes that were differentially expressed, only 114 genes were upregulated, mapped and annotated. However, for the GO term for immune response (Figure 2.2), further hits for lower level categories could not be obtained. 88 CHAPTER 2: IMMUNOMICS: IN SILICO MAPPING OF IMMUNE SIGNALLING PATHWAYS IN CHICKENS RELATED TO AV PARAGALLINARUM SEROVAR C-3 INFECTION Thus, further annotations between the immune response of score 3 versus control chickens could not be established. 89 CHAPTER 2: IMMUNOMICS: IN SILICO MAPPING OF IMMUNE SIGNALLING PATHWAYS IN CHICKENS RELATED TO AV PARAGALLINARUM SEROVAR C-3 INFECTION Lymphocyte Leukocyte activation Immune effector process activation (100%) (12.5%) (12.5%) (GO:0002252) (GO:0046649) (GO:0045321) Lymphocyte Lymphocyte proliferation (50%) differentiation (50%) Immune response process(75%) (GO:0006955) (GO:0046651) (GO:0030098) Innate immune Adaptive immune response (75%) response (25%) (GO:0045087) (GO:0002250) T cell proliferation (100%) T cell differentiation (100%) (GO:0042098) (GO:0030217) Figure 2.3: Biological process pie charts obtained from GO Enrichment Analysis representing the percentage distribution of the biological process ontology of Group 3 (score 2) vs 1 (score 0). The different GO identifiers for the immune response section shown in Figure 2.2 for Group 3 (score 2) vs 1 (score 0), which was divided into three main activities: immune response process, leukocyte activation and immune effector process (SEE TOP RIGHT PIE CHART). Ultimately, the lymphocyte that had a major role in the immune response was T lymphocytes, seen with T cell proliferation and d ifferentiation. 90 CHAPTER 2: IMMUNOMICS: IN SILICO MAPPING OF IMMUNE SIGNALLING PATHWAYS IN CHICKENS RELATED TO AV PARAGALLINARUM SEROVAR C-3 INFECTION 2.3.2. Functional annotation Differentially expressed genes between cohorts of Group 2 (score 1) vs 1 (score 0), Group 3 (score 2) vs 1 (score 0) and Group 4 (score 3) vs 1 (score 0) showed highly significant enrichment of genes involved in the regulation of defense and immune responses. A full list of enriched and functionally annotated gene sets of Group 2 (score 1) vs 1 (score 0), Group 3 (score 2) vs 1 (score 0) and Group 4 (score 3) vs 1 (score 0), based on gene ontology (GO) terms using the DAVID bioinformatics tool is provided (Annexure A), whereby the p- values observed in the tables represent the EASE (Expression Analysis Systematic Explorer) score or a modification of the Fisher Exact p-value, used as a measure of gene enrichment in terms of functional annotation (Huang et al. 2008a; Huang et al. 2008b). The lesser the p-value, the more enriched the genes that have been queried (Huang et al. 2008a; Huang et al. 2008b). For Group 2 (score 1) vs 1 (score 0) and Group 3 (score 2) vs 1 (score 0), there were potential functional and enriched genes for the immune response, however for Group 4 (score 3) vs 1 (score 0) there were more molecular function and cellular component hits obtained as GO terms. For visualization of the GO terms obtained from DAVID, REVIGO was used for better interpretation. The results for the gene list of Group 2 (score 1) vs 1 (score 0), Group 3 (score 2) vs 1 (score 0) and Group 4 (score 3) vs 1 (score 0) were shown as illustrations from REVIGO (Figure 2.4 - Figure 2.6). Each of the GO terms in the scatterplots (Figure 2.4 - Figure 2.6) is represented as a node or bubble (Supek et al. 2011). In the scatterplot view of Figure 2.4 - Figure 2.6, the X- and Y- axes represent the two-dimensional semantic space of the graph, whereby the x and y coordinates of the bubbles were derived by implementing multidimensional scaling to a 91 CHAPTER 2: IMMUNOMICS: IN SILICO MAPPING OF IMMUNE SIGNALLING PATHWAYS IN CHICKENS RELATED TO AV PARAGALLINARUM SEROVAR C-3 INFECTION matrix of the GO terms' semantic similarities (Supek et al. 2011). The colour of the bubble represents the user-provided p-value, where blue and green bubbles are GO terms with more significant p-values than orange and red bubbles (Supek et al. 2011). Moreover, the size of the bubble indicates the frequency of the GO term, where bubbles of more general terms are larger in size and smaller bubbles have more specific terms (Supek et al. 2011). Additionally, the closeness between the bubbles in the X- and Y- semantic spaces reflects their closeness in semantic similarity, hence similar nodes are found closer together and highly similar GO terms are designated by edges in the graph (Supek et al. 2011). Figure 2.4 shows the REVIGO scatterplot for Group 2 (score 1) vs 1 (score 0), whereby a summarised and visual display of GO terms relating to biological process for mild symptoms to IC infection was observed. Interestingly, some of the immune responses included: response to lipopolysaccharide, xenophagy, cellular response to interferon-gamma, lymphocyte chemotaxis, chemokine-mediated signalling pathway, cellular response to tumour necrosis factor, cellular response to IL-1, reactive oxygen species metabolism and intracellular transport of viral protein in host cell. Most of the immune responses obtained were associated with innate immune responses against IC infection for Group 2 (score 1) vs 1 (score 0) (Figure 2.4). Despite the fact, that chickens with mild symptoms from Group 2 (score 1) vs 1 (score 0) were sacrificed at 36-48 h post challenge, the REVIGO results of Group 2 (score 1) vs 1 (score 0) (Figure 2.4) suggested that during that time period the innate immune responses of chickens was still active, even though innate responses are known to be immediate. The response to lipopolysaccharide (LPS) refers to the binding of LPS to the TLR4-myeloid differentiation factor-2 (MD2) complex, the impetus required for the activation of pro- inflammatory signalling pathways and leading to a change in the state or activity of the host 92 CHAPTER 2: IMMUNOMICS: IN SILICO MAPPING OF IMMUNE SIGNALLING PATHWAYS IN CHICKENS RELATED TO AV PARAGALLINARUM SEROVAR C-3 INFECTION such as movement, secretion, enzyme production and gene expression, as well as a cascade of signalling pathways leading to the initial immune responses against Gram- negative bacteria (Figure 2.4) (Bryant et al. 2010). Xenophagy is a type of autophagy (self- eating) against antibacterial and antiviral defenses (Figure 2.4) (Mao and Klionsky, 2017). This process is mediated by the autophagosome, whereby organelles in specific regions of the cytosol are sequestered for targeted infected cells and the contents are delivered to lysosomes for degradation (Mizushima, 2007; Xu and Eissa, 2010). Additionally, it was found that autophagy is part of the innate and adaptive immune responses respectively (Xu and Eissa, 2010). It was shown that TLR4 acts as the primary environmental receptor for the process of autophagy during pathogen invasion (Xu et al. 2007; Delgado et al. 2008; Xu et al. 2008). Moreover, studies have shown that LPS induces regulation of TIR-domain- containing adapter-inducing interferon-β (TRIF)-dependent, myeloid differentiation factor-88 (MyD88)-independent TLR4 of human and murine macrophages resulting in autophagy (Xu and Eissa, 2010). The cellular response to interferon-gamma (INF-), suggests the mediation of immunity and inflammation by INF- a type II interferon that uses the Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathway to activate STAT1, promote inflammation, limit tissue damage, modulate T helper (Th) and regulatory T (Treg) cells, activate Th1 responses, activate macrophages, facilitate host defense and tumour surveillance (Figure 2.4) (Hu and Ivashkiv, 2009; Green et al. 2017). Lymphocyte chemotaxis refers to the directed migration of a lymphocyte towards a specific stimulus and is important for physiological conditions during adaptive immune responses (Figure 2.4) (Cabrero et al. 2006). Lymphocyte chemotaxis requires the synchronized activity of adhesion and chemotactic receptors, cytoskeleton and signalling molecules (Vicente-Manzanares and Sánchez-Madrid, 2004). The cellular response to tumour necrosis factor (TNF) relates to the proinflammatory activities of this cytokine through the activation of macrophages or 93 CHAPTER 2: IMMUNOMICS: IN SILICO MAPPING OF IMMUNE SIGNALLING PATHWAYS IN CHICKENS RELATED TO AV PARAGALLINARUM SEROVAR C-3 INFECTION monocytes (Figure 2.4) (Liu and Han, 2001). TNF also has important cellular functions such as the release/induction of other cytokines, cell proliferation, cell differentiation, modification of the anticoagulant properties of endothelial cells and apoptosis (Liu and Han, 2001). Most TNF-induced responses are via tumour necrosis factor receptor 1 (TNF-R1) mediated pathways (Liu and Han, 2001). Tumour necrosis factor receptor type 1-associated DEATH domain (TRADD) is enlisted to the TNF-R1 complex and consequently effectors such as TNF receptor-associated factor-2 (TRAF2), receptor-interacting protein (RIP), Fas- associated protein with death domain (FADD), cellular inhibitor of apoptosis protein-1 (cIAP1), cellular inhibitor of apoptosis protein-2 (cIAP2) and tumour necrosis factor α- induced protein 3 (TNFAIP3)/(A20), are recruited to the complex, that act as mediators in the activation of proteases, phospholipases, protein kinases and transcription factors via the pathways described (Liu and Han, 2001). Interleukin (IL)-1 is a highly inflammatory cytokine found in two forms IL-1α and IL-Iβ, whereby they co-function with TNF (Figure 2.4) (Dinarello, 1997). IL-1 has systemic effects and during innate immunity has an effect on all innate immune cells through several functions such as leukocyte recruitment, leukocyte migration, cortisol regulation, humoral innate immunity through the activation of acute phase proteins, lymphoid cell-mediation, as well as survival and effector functions (Garlanda et al. 2013). For the regulation and amplification of innate immunity and uncontrolled inflammation, there are four signalling receptors, two decoy receptors (IL-1R2, IL-18BP), two negative regulators (toll interleukin-1 receptor (IL-1R) 8 (TIR8)/ single immunoglobulin interleukin-1 receptor related molecule (SIGIRR), interleukin-1 receptor accessory protein (IL-1RAcPb), seven ligands with agonist activity (IL-1α and IL-1β, IL-18, IL-33, IL-36α, IL-36β, IL-36γ), three receptor antagonists (IL- 1Ra, IL-36Ra, IL-38) and an anti-inflammatory cytokine (IL-37) in the IL-1 family (Garlanda et al. 2013). 94 CHAPTER 2: IMMUNOMICS: IN SILICO MAPPING OF IMMUNE SIGNALLING PATHWAYS IN CHICKENS RELATED TO AV PARAGALLINARUM SEROVAR C-3 INFECTION Figure 2.4: Scatterplot of biological process for Group 2 (score 1) vs 1 (score 0) with REVIGO providing a summarised and visual display of GO terms. The scatterplot shows the initial immune responses of chickens with mild IC symptoms. Some of the notable biological processes were as follows: response to lipopolysaccharide, xenophagy, cellular response to interferon-gamma, lymphocyte chemotaxis, chemokine- mediated signalling pathway, cellular response to tumour necrosis factor, cellular response to IL-1, reactive oxygen species metabolism and intracellular transport of viral protein in host cell. In the scatterplot view, each bubble represents a GO term, the X- and Y-axes represent the two-dimensional semantic space of the graph, the colour of the bubble represents the user-provided p-value, the size of the bubble indicates the frequency of the GO term and the closeness between the bubbles in the X- and Y- semantic spaces reflects their closeness in semantic similarity (Supek et al. 2011). Reactive oxygen species (ROS) metabolism pertains to the chemical reactions and pathways involving a ROS, whereby ROS exist as highly reactive free radicals or minute short-lived oxygen-containing molecules (Figure 2.4) (Chen et al. 2016). Moreover, they have a key role in microbicidal activity of phagocytes such as macrophages and serve as second messengers in cell signalling (Bae et al. 2009). Upon the transformation of macrophages into phagolysosomes during pathogenic engulfment, NADPH-dependent phagocytic oxidase (NADPH oxidase 2 (NOX2)) is assembled to reduce oxygen to 95 CHAPTER 2: IMMUNOMICS: IN SILICO MAPPING OF IMMUNE SIGNALLING PATHWAYS IN CHICKENS RELATED TO AV PARAGALLINARUM SEROVAR C-3 INFECTION superoxide anion (O −2 ), involved in the immediate killing and degradation of bacteria inside the phagolysosomes (Slauch, 2011). Phagocytes are the main immune cells involved in innate immunity during pathogenic infection, which would explain ROS metabolism during mild symptoms of chickens during IC infection. The GO term that was interesting to find was that of intracellular transport of viral protein in host cell (Figure 2.4), as similar results were found by Boucher et al. (2014). During a study by Boucher et al. (2014) there was up- regulation of TLR7 activated by ss-RNA viruses, it was proposed that there was a possibility that the bacterial genome of Av. paragallinarum consisted of prophages and prophage remnants that was previously discovered in a study by Roodt et al. (2012). Boucher and co- workers suggested that the prophage remnant in Av. paragallinarum might contribute to the severe symptoms observed with serovar C-3 (SA-3 strain). All the processes described above might potentially contribute to innate immunity against IC invasion in chickens. Figure 2.5 shows the REVIGO scatterplot for Group 3 (score 2) vs 1 (score 0), with GO terms relating to biological process. Some of the GO terms obtained from the scatterplot for Group 3 (score 2) vs 1 (score 0) were similar to Group 2 (score 1) vs 1 (score 0), such as response to lipopolysaccharide and cellular response to tumour necrosis factor for the biological process category, which implies that even during moderate symptoms some of the initial innate responses continue to occur (Figure 2.4 and Figure 2.5). In comparison, to Group 2 (score 1) vs 1 (score 0), Group 3 (score 2) vs 1 (score 0) generated more GO terms, due to a combination of both innate and adaptive immune responses (Figure 2.4 and Figure 2.5). A new series of biological process GO terms was obtained: chemotaxis, inflammatory response, apoptotic signalling pathway, haemopoiesis, angiogenesis, regulation of catalytic activity, regulation of ERK1 and ERK2 cascade, negative regulation of protein tyrosine kinase activity, positive regulation of peptide hormone secretion, regulation of cell size, positive regulation of cell proliferation, regulation of cell differentiation, negative regulation of phosphatidylinositol 3-kinase signalling and skeletal system development 96 CHAPTER 2: IMMUNOMICS: IN SILICO MAPPING OF IMMUNE SIGNALLING PATHWAYS IN CHICKENS RELATED TO AV PARAGALLINARUM SEROVAR C-3 INFECTION (Figure 2.5). The chickens with moderate symptoms from Group 3 (score 2) vs 1 (score 0), were sacrificed at 72-96 h, at this time period both late innate and initial adaptive immune responses seemed to occur, as shown with REVIGO results for Group 3 (score 2) vs 1 (score 0) (Figure 2.5). The findings obtained are suggested to be in line with the mechanisms of the avian immune response, since both the innate and adaptive immune responses are interconnected. Following the activation of the avian innate immune response, downstream signalling pathways and molecules trigger adaptive immunity at a later stage during IC infection, which was why moderate symptoms from Group 3 (score 2) vs 1 (score 0) were observed at 72-96 h. Among the immune responses for Group 3 (score 2) vs 1 (score 0) was inflammatory response, which is a biological response of the immune system that can be activated via nuclear factor kappa B (NF-κB), mitogen-activated protein kinase (MAPK), and JAK-STAT pathways, whereby different organs in the host may be affected (Figure 2.5) (Chen et al. 2018). The apoptotic signalling pathway refers to molecular cell signalling pathways controlling apoptosis or programmed cell death, in leukocytes and other cells of the immune system (Figure 2.5) (Siegel and Lenardo, 2002). There are two apoptotic pathways, whereby active apoptosis involves TNF-related receptors known as death receptors which leads to antigen-induced cell death, whereas passive apoptosis occurs when stimulated lymphocytes are denied access to essential growth cytokines and thus death receptors are not needed (Siegel and Lenardo, 2002). Both pathways are carried out by intracellular cysteine proteases referred to as caspases, whereby any defects in either of these apoptotic pathways produce unique pathologies within the host organism (Siegel and Lenardo, 2002). 97 CHAPTER 2: IMMUNOMICS: IN SILICO MAPPING OF IMMUNE SIGNALLING PATHWAYS IN CHICKENS RELATED TO AV PARAGALLINARUM SEROVAR C-3 INFECTION Figure 2.5: Scatterplot of biological process for Group 3 (score 2) vs 1 (score 0) with REVIGO providing a summarised and visual display of GO terms. The scatterplot shows both innate and adaptive responses of chickens with moderate IC symptoms. Some of the notable biological processes were shared with Group 2 (score 1) vs 1 (score 0) such as response to lipopolysaccharide and cellular response to tumour necrosis factor. Distinguished biological process GO terms included: chemotaxis, inflammatory response, apoptotic signalling pathway, haemopoiesis, angiogenesis, regulation of catalytic activity, regulation of ERK1 and ERK2 cascade, negative regulation of protein tyrosine kinase activity, positive regulation of peptide hormone secretion, regulation of cell size, positive regulation of cell proliferation, regulation of cell differentiation, negative regulation of phosphatidylinositol 3-kinase signalling and skeletal system development. In the scatterplot view, each bubble represents a GO term, the X- and Y-axes represent the two-dimensional semantic space of the graph, the colour of the bubble represents the user-provided p-value, the size of the bubble indicates the frequency of the GO term and the closeness between the bubbles in the X- and Y- semantic spaces reflects their closeness in semantic similarity (Supek et al. 2011). In both innate and adaptive immune responses cells undergo apoptosis primarily with phagocytes, neutrophils and effector T cells for clearance and for elimination of pathogens. Furthermore, the presence of GO terms chemotaxis and angiogenesis suggest the release of neuropeptides during moderate symptoms (Figure 2.5). Neuropeptides from the brain may have similar potent functions to the angiogenic cytokine, vascular endothelial growth factor 98 CHAPTER 2: IMMUNOMICS: IN SILICO MAPPING OF IMMUNE SIGNALLING PATHWAYS IN CHICKENS RELATED TO AV PARAGALLINARUM SEROVAR C-3 INFECTION (VEGF) and cause a concentration gradient that attracts immune cells (macrophages, monocytes, heterophils) within the avian host (Fischer-Colbrie et al. 2005). Neurotransmission implies that the nervous system is involved and one of the symptoms of IC involves lethargy and disorientation in infected chickens. Although, regulation of catalytic activity usually pertains to enzymatic activity, it might also refer to antibody-mediated catalysis by natural antibodies (abzymes) that have the capacity to degrade nucleic acids, protein, and polysaccharide substrates in infection and immunity (Nevinsky et al. 2000; Bowen et al. 2017). Haemopoiesis, regulation of cell size, positive regulation of cell proliferation and regulation of cell differentiation showed that there was an influx of cells since the avian immune response consisted of various immune cells to target and eliminate pathogens; and cellular differentiation occurred whereby cells transformed from naïve cells to effector immune cells such as monocytes to macrophages, naïve B cells to plasma cells, naïve T cells to Th or cytotoxic T cells during the innate and adaptive immune phases of chickens with score 2 (Figure 2.5) (Playfair and Bancroft, 2013.). Efferent signals from the brain to the nervous system and finally to the immune system are transmitted by the neuroendocrine and nervous systems, which is possible via shared ligands and receptors, neurotransmitters, neuropeptides, growth factors, neuroendocrine hormones and cytokines (Kelley et al. 2007). Therefore, hormones secreted by the neuroendocrine system such as growth hormone (GH) and insulin-like growth factor-1 (IGF-I) play an vital role in the communication and regulation of the cells of the immune system, hence the GO term, positive regulation of peptide hormone secretion was generated from the gene cohort of Group 3 (score 2) vs 1 (score 0) (Kelley et al. 2007). The cluster of GO terms: regulation of ERK1 and ERK2 cascade, negative regulation of protein tyrosine kinase activity and negative regulation of phosphatidylinositol 3-kinase signalling, relates to the mechanisms of the TLR2 or TLR4 signalling pathway (Dahle et al. 2004). TLR2 signalling commences with the assembly of 99 CHAPTER 2: IMMUNOMICS: IN SILICO MAPPING OF IMMUNE SIGNALLING PATHWAYS IN CHICKENS RELATED TO AV PARAGALLINARUM SEROVAR C-3 INFECTION large membrane complexes and results in the stimulation and nuclear translocation of NF- κB, whereby pro- and anti-inflammatory cytokines are activated in the immune response (Dahle et al. 2004). Moreover, this nonspecific mechanism of cytokine gene induction has been extended on through the recognition of multiple signalling kinases such as p38 mitogen-activated protein kinase (MAPK), extracellular signal-related kinase (ERK1 and ERK2), phosphatidylinositol 3-kinase (PI3-K) and protein tyrosine kinases (Dahle et al. 2004). Although, it is known that LPS- mediated signalling is conducted via TLR4, it can also occur via TLR2, which is dependable as both peptidoglycan and LPS are constituents of the Gram-negative cell walls (Arbibe et al. 2000; Akira, 2001; Takeuchi and Akira, 2001). This finding, is consistent to that of Boucher et al. (2014), whereby initially for mild symptoms, both TLR2 and TLR4 were expressed in expression profiles. Nevertheless, TLR 4 was found to be up-regulated during moderate symptoms. However, there is still a discrepancy related to species-specific differences in the role of the TLRs, since LPS signalling is mediated by TLR4 in mice and by TLR2 in humans, hence it is possible that in avian models LPS signalling is mediated by both TLR2 and TLR4 (Akira, 2001). Figure 2.6 shows the REVIGO scatterplot for Group 4 (score 3) vs 1 (score 0), with GO terms relating to biological process. In comparison to the scatterplots for Group 2 (score 1) vs 1 (score 0) and Group 3 (score 2) vs 1 (score 0), there are less GO terms for biological process (Figure 2.4 - Figure 2.6). Moreover, since fewer GO terms were obtained for Group 4 (score 3) vs 1 (score 0), this indicated that there were less differentially expressed genes. The results obtained for Group 4 (score 3) vs 1 (score 0) (Figure 2.6) suggested that mechanisms leading to disease tolerance and protection to the host from systemic hyperinflammation of the innate and adaptive immune responses were involved, rather than defence mechanisms against pathogenic infection, as seen with mild and moderate IC symptoms (Figure 2.4 - Figure 2.5). The biological process GO terms for Group 4 (score 3) vs 1 (score 0) were as follows: cellular response to amino acid stimulus, positive regulation 100 CHAPTER 2: IMMUNOMICS: IN SILICO MAPPING OF IMMUNE SIGNALLING PATHWAYS IN CHICKENS RELATED TO AV PARAGALLINARUM SEROVAR C-3 INFECTION of cortisol secretion, protein heterotrimerization, female sex differentiation, gamete generation, regulation of protein catabolism (Figure 2.6). Figure 2.6: Scatterplot of biological process for Group 4 (score 3) vs 1 (score 0) with REVIGO providing a summarised and visual display of GO terms. The scatterplot shows the different biological responses of chickens with severe IC symptoms. The GO terms obtained for Group 4 (score 3) vs 1 (score 0), did not correlate to the immune responses as observed with Group 2 (score 1) vs 1 (score 0) and Group 3 (score 2) vs 1 (score 0). There were fewer GO terms compared to those of mild and moderate IC symptoms (Figure 2.4 - Figure 2.5). Moreover, the GO terms obtained for Group 4 (score 3) vs 1 (score 0) related more to mechanisms leading to disease tolerance and protection to the host from systemic hyperinflammation of the innate and adaptive immune responses, rather than defence against pathogenic infection, as seen with mild and moderate IC symptoms (Figure 2.4 - Figure 2.6). Biological process GO terms included: cellular response to amino acid stimulus, positive regulation of cortisol secretion, protein heterotrimerization, female sex differentiation, gamete generation, regulation of protein catabolism . In the scatterplot view, each bubble represents a GO term, the X- and Y-axes represent the two-dimensional semantic space of the graph, the colour of the bubble represents the user-provided p-value, the size of the bubble indicates the frequency of the GO term and the closeness between the bubbles in the X- and Y- semantic spaces reflects their closeness in semantic similarity (Supek et al. 2011). 101 CHAPTER 2: IMMUNOMICS: IN SILICO MAPPING OF IMMUNE SIGNALLING PATHWAYS IN CHICKENS RELATED TO AV PARAGALLINARUM SEROVAR C-3 INFECTION The GO term cellular response to amino acid stimulus refers to any alteration in cell morphology or cellular activity due to an amino acid stimulus, however not much could be deduced from this general GO term (shown with a light blue medium sized bubble in Figure 2.6). However, from the scatterplot (Figure 2.6), it was observed that the bubbles for GO terms cellular response to amino acid stimulus, positive regulation of cortisol secretion, female sex differentiation and gamete generation, were closely grouped to the right side of the graph and consisted of a small green bubble (having a GO term that was specific and that had a significant p-value), blue and green bubbles (having a GO term that had significant p-values) as well as an orange and a dark blue bubble that were in close proximity (indicating high semantic similarity). Therefore, one probable hypothesis how these four GO terms were similarly connected would have been via the neuroendocrine system of the host organism. Additionally, the GO term cellular response to amino acid stimulus could refer to neuropeptides that have a role to play in the adaptive immune response during IC infection. Neuropeptides are neurotransmitters that possess short amino acid chains, involved in neurological transmission and communication, and may affect cell activity due to having potent effects on immunoglobulin synthesis both in vivo and in vitro, as well as in the proliferation of mast cells and granulocytes (Stanisz et al. 1987). Other neurotransmitters such as vasoactive intestinal peptide, substance P, and somatostatin, that are also peptides, have regulatory functions on immune effector cells in gut-associated lymphoid tissue (GALT) and thus are crucial in gastrointestinal physiology (O’Dorisio, 1986). Vasoactive intestinal peptide regulates lymphocyte migration and natural killer (NK) cell activity via a cyclic adenosine monophosphate (cAMP)-dependent mechanism, somatostatin inhibits the effects of both vasoactive intestinal peptide and substance P using a process that appears to include inhibitory guanine nucleotide binding proteins (O’Dorisio, 1986). Another inference to cellular response to amino acid stimulus (Figure 2.6), would be the interplay of catabolic mechanisms of essential amino acids and modulation of the host’s robust immune system to prevent hyperinflammation in innate, adaptive, and regulatory responses to infections, as well as prevent infection-driven immunopathology (Grohmann et al. 2017). The amino acids 102 CHAPTER 2: IMMUNOMICS: IN SILICO MAPPING OF IMMUNE SIGNALLING PATHWAYS IN CHICKENS RELATED TO AV PARAGALLINARUM SEROVAR C-3 INFECTION tryptophan (Trp) and arginine (Arg) modulate immune reactivity, whereas phenylalanine (Phe), glutamine (Gln) and cysteine (Cys) balance immune reactivity (McGaha et al. 2012). Ultimately, mammals through evolution have acquired a mechanism to regulate pathogen infection by increasing amino acid catabolism thereby limiting the availability of intracellular nutrients to colonizing pathogens, whereby amino-acid sensing and degradation in immunometabolism occurs via the Trp- (kynurenine (Kyn) pathway) and Arg-catabolic pathways (arginine decarboxylase (ADC), intestinal-renal axis, citrulline-nitric oxide (NO) pathways) (McGaha et al. 2012; Grohmann et al. 2017). Amino acid catabolism described is also in line with the GO term regulation of protein catabolism obtained with REVIGO (Figure 2.6). The GO term positive regulation of cortisol secretion may refer to the activity of corticotropin releasing factor (CRF) or corticotropin-releasing hormone (CRH) from the extrahypothalamic areas in the brain, that stimulates the anterior of the pituitary gland to produce adrenocorticotropic hormone (ACTH) which travels in the bloodstream to the adrenal glands inducing secretion of cortisol, a stress hormone (Figure 2.6) (Ohmura and Yoshioka, 2009). During an immune and inflammatory response, there is stimulation of the neuroendocrine stress system, whereby a Th2 shift is induced which causes stress hormones to electively suppress Th1 responses instead of immunosuppression (Elenkov, 2002). In doing so, the Th2 shift provides protection to the host from systemic hyperinflammatory responses by countering the tissue-damaging effects of macrophages and Th1 cells, as well as Th1/ proinflammatory cytokines (Elenkov, 2002). Nevertheless, the activation of CRH/substance P(SP)‐histamine axis, may assist inflammatory responses through initiation of IL‐1, IL‐6, IL‐8, IL‐18, TNF‐α, and c-reactive protein (CRP) synthesis (Calcagni and Elenkov, 2006). Female sex differentiation and gamete generation, is an invalid GO term, since 10-week old White Leghorn male birds (cocks) with no prior clinical history of IC were obtained for the study and not hens, therefore these biological processes probably occur during embryonic development (Figure 2.6). 103 CHAPTER 2: IMMUNOMICS: IN SILICO MAPPING OF IMMUNE SIGNALLING PATHWAYS IN CHICKENS RELATED TO AV PARAGALLINARUM SEROVAR C-3 INFECTION 2.3.3. Pathway analysis The Pathview results for differentially regulated genes using fold change for Group 2 (score 1) vs 1 (score 0), Group 3 (score 2) vs 1 (score 0) and Group 4 (score 3) vs 1 (score 0) generated KEGG pathways. The KEGG pathways were selected based on the relevance to some of the GO terms obtained in Section 2.3.2. Genes that were upregulated were shown as red and genes that were downregulated were shown as green. Due to stringent filtering, not many genes were found to be up- or downregulated. Response to lipopolysaccharide was the shared GO term between Group 2 (score 1) vs 1 (score 0) and Group 3 (score 2) vs 1 (score 0) (Figure 2.4 and Figure 2.5). Hence, the result for toll-like receptor signalling pathway was obtained for both Group 2 (score 1) vs 1 (score 0) and Group 3 (score 2) vs 1 (score 0) (Figure 2.7). It was found that IL-8, regulated on activation, normal T Cell expressed and secreted (RANTES/CCL5) and macrophage inflammatory protein-1 beta (MIP-1β/CCL4) were upregulated for Group 2 (score 1) vs 1 (score 0), which are inflammatory cytokines that function as chemoattractants for mild symptoms of IC. It seems that these molecules mediate the initial responses of innate immunity that eventually culminate into the adaptive immune responses. MIP-1β is a chemoattractant for CD4+ and CD8+ cells which also induces chemotaxis and cell adhesion of T cells, IL-8 attracts T cells and neutrophils, and RANTES has attractant activities for T cells and monocytes of memory phenotype (Schall et al. 1990; Schall et al. 1993; Tanaka et al. 1993). For Group 3 (score 2) vs 1 (score 0) which was for moderate symptoms for IC, TLR2, myeloid differentiation factor 2 (MD-2) complexed to TLR4 and TIR domain containing adaptor protein (TIRAP) were upregulated, showing that TIRAP acts as a bridge between MyD88 to the receptor complex for TLR-2 and TLR4 signalling that mediates NF-κB proinflammatory responses (Figure 2.7) (Verstak et al. 2009). Therefore, both TLR2 and TLR4 seem to mediate pathogen recognition in chickens during IC infection for moderate 104 CHAPTER 2: IMMUNOMICS: IN SILICO MAPPING OF IMMUNE SIGNALLING PATHWAYS IN CHICKENS RELATED TO AV PARAGALLINARUM SEROVAR C-3 INFECTION symptoms (Figure 2.7). TLR4 recognizes LPS and utilises both MyD88 and TRIF upstream, and TIRAP downstream as signalling adaptors to stimulate the activation of NF-κB and MAPK (Figure 2.7) (Takeuchi and Akira, 2010). The TRIF pathway also activates interferon regulatory factor-3 (IRF3), whereby IFN-β and -α are produced that stimulate CD8 T cells as a response to viral infection, antigen-presenting cells (APCs) and other T cells (Figure 2.7) (Welsh et al. 2012). Figure 2.7: Toll-like receptor pathway from KEGG pathways for Group 3 (score 2) vs 1 (score 0). The genes that were upregulated (red) included: TLR2, MD-2, Ras-related C3 botulinum toxin substrate 1 (Rac1), phosphoinositide 3-kinase (PI3K), TIRAP, mitogen-activated protein kinase kinase (MKK) 3/6, p38 mitogen- activated protein kinase (p38) and TRIF. The pathway shown was for moderate symptoms, hence both innate and adaptive responses were involved. Figure 2.8 shows the signalling pathway of the phagosome for Group 2 (score 1) vs 1 (score 0), an important defence mechanism during innate immunity for the elimination of invasive pathogens. Macrophages are highly motile cells that carry out phagocytosis and chemotaxis 105 CHAPTER 2: IMMUNOMICS: IN SILICO MAPPING OF IMMUNE SIGNALLING PATHWAYS IN CHICKENS RELATED TO AV PARAGALLINARUM SEROVAR C-3 INFECTION (Rougerie et al. 2013.). Genes that were upregulated included F-actin (linear polymer microfilament), TAP (transporter associated with antigen-processing), TUBB (tubulin beta class 1) and Sec61(channel forming translocon), whereas CD36 was downregulated (Figure 2.8). Figure 2.8: Phagosome pathway for the maturation of the phagocyte into a phagolysosome from KEGG pathways for Group 2 (score 1) vs 1 (score 0). The genes that were upregulated (red) included: F-actin, TAP, TUBB and Sec61, whereas CD36 was downregulated (green). The pathway shown was for mild symptoms, during innate immunity. The differentially regulated genes have a role in the different stages of phagosome maturation (Figure 2.8). During phagocytosis, actin cytoskeletal remodeling is required for the formation of lamellipodia and phagocytic cups that are F-actin rich, requiring regulation of 106 CHAPTER 2: IMMUNOMICS: IN SILICO MAPPING OF IMMUNE SIGNALLING PATHWAYS IN CHICKENS RELATED TO AV PARAGALLINARUM SEROVAR C-3 INFECTION actin polymerization via actin related protein (Arp) 2/3 activation and nucleation promoting factors like the Wiskott-Aldrich syndrome protein (WASP)/ WASP family Verproline- homologous protein (WAVE) family, as well as remodeling of these actin networks (Rougerie et al. 2013). Therefore, the F-actin gene was up-regulated to facilitate the engulfment of the pathogen (Figure 2.8). In addition, TAP transports peptides of foreign origin (from the pathogen), from the cytosol into the endoplasmic reticulum (ER) to MHC class I molecules, whereby the peptide is presented to CD8+ cytotoxic T cells (Ritz and Seliger, 2001). Interestingly, the pathway for influenza A virus was one of the pathways obtained for Group 3 (score 2) vs 1 (score 0) for moderate symptoms with IC infection, that affects the respiratory system within alveolar and bronchial epithelial cells, alveolar macrophages and type II pneumocytes of both humans and birds alike (Figure 2.9). Genes that were upregulated included 70 kilodalton heat shock proteins (HSP70), MKK3/6, p38, IL-8, TRIF, PI3K, influenza virus non-structural protein-1 binding protein (NS1BP), plasminogen (PLG), interferon-gamma receptor (IFNGR) and tumour necrosis factor receptor 1 (TNFR1), whereas induced by phosphate starvation1 (IPS1) was downregulated (Figure 2.9). Fibrinolysis is the process of dissolving fibrin into a soluble form conducted by the serine protease plasmin (Berri et al. 2013). Plasmin is produced through cleavage of plasminogen generated in the liver and found in the bloodstream, whereby PLG/plasmin has a key role in fibrinolysis-mediated inflammation (Berri et al. 2013). Influenza A viruses (IAV) may influence the specific binding and conversion from PLG into plasmin by allowing the virus a substitute protease for cleavage in the form of its hemagglutinin molecule, which is a crucial stage in viral replication leading to disease (Goto and Kawaoka, 1998; Goto et al. 2001; LeBouder et al. 2008; LeBouder et al. 2010). Additionally, PLG may lead to pathogenesis of IAV infections, by facilitating virus replication or via induction of a fibrinolysis-dependent 107 CHAPTER 2: IMMUNOMICS: IN SILICO MAPPING OF IMMUNE SIGNALLING PATHWAYS IN CHICKENS RELATED TO AV PARAGALLINARUM SEROVAR C-3 INFECTION damaging inflammatory response in the respiratory tract (Berri et al. 2013). PLG was found to be upregulated during IC infection of Group 3 (score 2) vs 1 (score 0) (Figure 2.9). Figure 2.9: Influenza A virus pathway from KEGG pathways for Group 3 (score 2) vs 1 (score 0). The genes that were upregulated (red) included: HSP70, MKK3/6, p38, IL-8, TRIF, PI3K, NS1BP, PLG, IFNGR and TNFR1, whereas IPS1 was downregulated (green). The pathway shown was for moderate symptoms, during innate and adaptive immunity. Ironically, although Av. paragallinarum serovar C-3 is a Gram-negative bacteria, like IAV it also has virulence factors in the form of hemagglutinin (HA) antigens associated with pathogenicity and immunogenicity (Figure 2.9) (Yamaguchi et al. 1993; Gamblin and Skehel, 108 CHAPTER 2: IMMUNOMICS: IN SILICO MAPPING OF IMMUNE SIGNALLING PATHWAYS IN CHICKENS RELATED TO AV PARAGALLINARUM SEROVAR C-3 INFECTION 2010). Therefore, it is possible that both Av. paragallinarum serovar C-3 and IAV share common mechanisms and genes leading to pathogenesis of the host, in this case the chicken. Since, the mapped pathways of IAV share numerous up-regulated genes (shown in red Figure 2.9) and one down-regulated gene (shown in green Figure 2.9) with IC, this may also explain the severe symptoms observed with IC infection, especially to Av. paragallinarum serovar C-3 (SA-3), which is the most virulent strain in South Africa (Figure 2.9). The reason for this shared trait might also be due to the prophage and prophage remnants in the genome of Av. paragallinarum discussed in Section 2.3.2. The presence of prophages in Av. paragallinarum serogroups has been investigated (Coetsee, 2014). However, since only one of the genes was found to be down-regulated and far more numerous genes were found to be unaffected (Figure 2.9), shared pathogenesis between IC and IAV could only be speculated, whereby this area of research would need further investigation. Surprisingly, NS1BP was also an up-regulated gene observed with moderate symptoms during IC infection (Figure 2.9). NS1BP that is encoded by the virus genome, blocks IFN signalling (Jia et al. 2010). This is achieved by inhibiting the intracellular sensor retinoic acid- inducible gene 1 protein (RIG-I), whereby association with the downstream regulatory proteins IPS-1 (shown as downregulated in Figure 2.9) and IRF3 are disrupted leading to impaired transcriptional activation of IFN-β (Jia et al. 2010). Thus, INF-β and other pro- inflammatory cytokines are inhibited and their functions, thereby suggesting a similar mechanism for IC infection (Jia et al. 2010). There was a significant downregulation of numerous genes in the oxidative phosphorylation pathway of differentially regulated genes of Group 4 (score 3) vs 1 (score 0) (Figure 2.10), which might indicate a septic state in the host, which indicates selective inhibition of 109 CHAPTER 2: IMMUNOMICS: IN SILICO MAPPING OF IMMUNE SIGNALLING PATHWAYS IN CHICKENS RELATED TO AV PARAGALLINARUM SEROVAR C-3 INFECTION downstream kinases and phosphatases that regulate the activity of the oxidative phosphorylation complexes leading to a reduction in adenosine triphosphate (ATP) production, which might cause symptoms observed with IC infection, such as neurological disorientation, lethargy, laboured breathing, weight loss, poor growth and an unthrifty appearance. Figure 2.10: Oxidative phosphorylation pathway from KEGG pathways for Group 4 (score 3) vs 1 (score 0). The genes that were downregulated were shown in green). The pathway shown was for severe symptoms, following innate and adaptive immunity. Inhibition of the oxidative phosphorylation process causes a reduction in metabolism leading to energy deficiency causing dire consequences to the host. Score 3 chickens face severe symptoms to IC infection such as hemorrhage and conjunctivitis, which might be indications of upper respiratory sepsis to Av. paragallinarum C-3 serovar. Sepsis is predominantly caused by bacterial infections that can originate from any region of the host’s body and may even result in unspecific responses such as tachycardia 110 CHAPTER 2: IMMUNOMICS: IN SILICO MAPPING OF IMMUNE SIGNALLING PATHWAYS IN CHICKENS RELATED TO AV PARAGALLINARUM SEROVAR C-3 INFECTION (abnormally rapid heart rate) and tachypnoea (abnormally rapid breathing) leading to more drastic consequences such as organ dysfunction and failure (Lee and Hüttemann, 2014). During sepsis and acute inflammation, it was proposed that administration of LPS in animal models causing inflammatory signalling results in alterations in the oxidative phosphorylation state of mitochondrial proteins such as tyrosine (Tyr) 304 phosphorylation of cytochrome c oxidase (COX) catalytic subunit I (Lee and Hüttemann, 2014). Subsequently, there is inhibition of the oxidative phosphorylation process, causing reduced metabolism, a decline in the mitochondrial membrane potential, and an energy deficiency, which can cause organ dysfunction and fatality (Lee and Hüttemann, 2014). The down-regulation of numerous genes (shown in green in Figure 2.10) for the oxidative phosphorylation pathways might lead to the inhibition of various oxidative phosphorylation processes, leading to a dysfunction in the host’s metabolism during IC infection. 2.3.4. Functional protein association network analysis Correlations of differentially expressed gene cohorts of Group 2 (score 1) vs 1 (score 0), Group 3 (score 2) vs 1 (score 0) and Group 4 (score 3) vs 1 (score 0), may reflect differences in the immune response composition between each group. Functional association analysis was conducted to elucidate the functional relevance of this biological variation, and to visualise the networking and interactions between the differentially expressed gene cohorts, using the STRING database (Szklarczyk et al. 2014). The STRING functional association network for Group 2 (score 1) vs 1 (score 0), Group 3 (score 2) vs 1 (score 0) and Group 4 (score 3) vs 1 (score 0) are shown (Figure 2.11 A – Figure 2.11 C). The coloured nodes represent the query proteins and the first shell interactors, whereas the white nodes represent secondary shell interactors. 111 CHAPTER 2: IMMUNOMICS: IN SILICO MAPPING OF IMMUNE SIGNALLING PATHWAYS IN CHICKENS RELATED TO AV PARAGALLINARUM SEROVAR C-3 INFECTION For Group 2 (score 1) vs 1 (score 0), 589 gene products were obtained (Figure 2.11 A). For Group 2 (score 1) vs 1 (score 0) associated with innate immune responses during mild symptoms, the correlating genes included: inflammatory cytokines (IL-4, interleukin 8-like 1 (IL-8L1) and IL-18), acute phase response protein (CRP) and mediators of apoptosis (caspase-6 (CASP6), apoptosis-inducing factor-2 (AIFM2), shisa Family Member 5 (SHISA5), and PLG) (Figure 2.11 A). For Group 3 (score 2) vs 1 (score 0), 796 gene products were obtained (Figure 2.11 B). Some of the correlating genes for Group 3 (score 2) vs 1 included: IL-8L1, TIRAP and MAPK family (mitogen-activated protein kinase kinase kinase 4 (MAP3K4), mitogen-activated protein kinase kinase 3 (MAP2K3) and mitogen- activated protein kinase 11 (MAPK11)) (Figure 2.11 B). Group 3 (score 2) vs 1 correlating genes from the STRING database consists of numerous unknown chicken genes. For Group 4 (score 3) vs 1 (score 0), 151 gene products were obtained (Figure 2.11 C). T-Cell leukemia homeobox protein 3 (TLX3), nuclear distribution gene C (NUDC) and gamma-aminobutyric acid type A receptor alpha1 subunit (GABRA-1) are genes mostly associated with neuronal signalling responses found as correlating genes for Group 4 (score 3) vs 1 (score 0) (Figure 2.11 C). From the analyses conducted, it was evident that the most genes that were differentially expressed were found in Group 3 (score 2) vs 1 (score 0), which occurred during moderate symptoms of IC (Figure 2.11 B). This clearly showed that both innate and adaptive responses were at play. However, Group 4 (score 3) vs 1 (score 0) had the least number of differentially expressed genes (Figure 2.11 C). Similar results were observed, whereby fewer GO terms correlated to less differentially expressed genes (Figure 2.6). These findings suggest that a lack of up-regulated genes, resulted in more severe disease due to relative suppression of the immune response. Thus, it is possible that in severely ill-birds there may be failure of the immune system as a result of fewer up-regulated genes. 112 CHAPTER 2: IMMUNOMICS: IN SILICO MAPPING OF IMMUNE SIGNALLING PATHWAYS IN CHICKENS RELATED TO AV PARAGALLINARUM SEROVAR C-3 INFECTION A B C Figure 2.11 (A-C): Functional association network of Group 2 (score 1) vs 1 (score 0), Group 3 (score 2) vs 1 (score 0) and Group 4 (score 3) vs 1 (score 0) correlated genes. The STRING functional association network was generated for differentially expressed genes in the cohorts. Shown here is the confidence view. From the analyses conducted, it was evident that the most genes that were differentially expressed were found in Group 3 (score 2) vs 1 (score 0), which occurred during moderate symptoms of IC, clearly showing that both innate and adaptive responses were at play, whereas Group 4 (score 3) vs 1 (score 0 ) had the least number of differentially expressed genes. 113 CHAPTER 2: IMMUNOMICS: IN SILICO MAPPING OF IMMUNE SIGNALLING PATHWAYS IN CHICKENS RELATED TO AV PARAGALLINARUM SEROVAR C-3 INFECTION 2.4. Conclusion In this study, gene enrichment, functional annotation and identification of potential immune signalling pathways of differentially regulated genes was successfully conducted for mild, moderate and severe symptoms obtained from chickens challenged with IC. Focus on functional pathways and interaction networks was conducted to gain comprehensive insights into biological processes relevant to protection and pathogenesis in IC infection which can be harnessed for relevant biomarker signatures. Moreover, pathogenesis of IC infection with serovar C-3, was found to be similar to that of influenza A virus, suggesting that the presence of prophages and prophage remnants may have a crucial role to play in the virulence of the bacterial pathogen, as proposed by Boucher et al. (2014). The role of prophages in the virulence of Av. paragallinarum still needs further study. IL-8 a pro- inflammatory cytokine and chemoattractant for immune cells was up-regulated during IC infection throughout all symptoms, hence its potential use as a biomarker in IC infection can be considered. It was also found that the metabolism of the host altered during IC infection, whether this was caused by sepsis due to bacterial infection or as a mechanism for protecting the host from hyperinflammatory responses as well as depriving nutrients from the invasive pathogen, remains to be discovered. The in silico results obtained from bioinformatics tools for the generation of immune signalling pathway maps may assist in predicting in vivo immune responses during IC infection. These findings provide a better understanding of the immune pathology in IC infection, while combined biosignatures may be utilized in future into the development of a pathogenesis-based point-of-care test to monitor and maintain disease in the poultry industry. 114 CHAPTER 2: IMMUNOMICS: IN SILICO MAPPING OF IMMUNE SIGNALLING PATHWAYS IN CHICKENS RELATED TO AV PARAGALLINARUM SEROVAR C-3 INFECTION REFERENCES Adams, S.C., Xing, Z., Li, J. Cardona, C.J. 2009. 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Immunity, 35(2): 161-168. 125 ANNEXURE A ANNEXURE A GO terms for biological process, molecular function and cellular component for Group 2 (score 1) vs 1 (score 0) GO term Description p-Value GO:0006955 immune response 4.32E-03 GO:0006954 inflammatory response 4.63E-03 GO:0072593 reactive oxygen species metabolic process 6.59E-03 GO:0045595 regulation of cell differentiation 6.59E-03 GO:0070098 chemokine-mediated signaling pathway 8.16E-03 GO:0048247 lymphocyte chemotaxis 9.18E-03 GO:0006526 arginine biosynthetic process 1.20E-02 GO:0008637 apoptotic mitochondrial changes 1.57E-02 GO:0055114 oxidation-reduction process 1.67E-02 GO:0071356 cellular response to tumor necrosis factor 2.12E-02 GO:0030097 hemopoiesis 2.62E-02 GO:0043161 proteasome-mediated ubiquitin-dependent protein catabolic process 3.06E-02 GO:0002548 monocyte chemotaxis 3.49E-02 GO:0071347 cellular response to interleukin-1 3.81E-02 GO:0009060 aerobic respiration 4.10E-02 GO:0051289 protein homotetramerization 4.26E-02 GO:0071346 cellular response to interferon-gamma 5.46E-02 GO:0030308 negative regulation of cell growth 5.73E-02 GO:0042787 protein ubiquitination involved in ubiquitin-dependent protein catabolic 6.15E-02 process GO:0046627 negative regulation of insulin receptor signaling pathway 6.21E-02 GO:0006099 tricarboxylic acid cycle 6.21E-02 GO:0006633 fatty acid biosynthetic process 7.00E-02 GO:0045454 cell redox homeostasis 7.40E-02 GO:0098792 xenophagy 7.93E-02 GO:0008285 negative regulation of cell proliferation 8.63E-02 126 ANNEXURE A GO:0006744 ubiquinone biosynthetic process 8.87E-02 GO:0014054 positive regulation of gamma-aminobutyric acid secretion 9.02E-02 GO:0061732 mitochondrial acetyl-CoA biosynthetic process from pyruvate 9.02E-02 GO:0071421 manganese ion transmembrane transport 9.02E-02 GO:0006121 mitochondrial electron transport, succinate to ubiquinone 9.02E-02 GO:0019060 intracellular transport of viral protein in host cell 9.02E-02 GO:0045901 positive regulation of translational elongation 9.02E-02 GO:1902361 mitochondrial pyruvate transmembrane transport 9.02E-02 GO:0032496 response to lipopolysaccharide 9.63E-02 GO:0016874 ligase activity 2.59E-02 GO:0008137 NADH dehydrogenase (ubiquinone) activity 2.94E-02 GO:0008009 chemokine activity 3.22E-02 GO:0048020 CCR chemokine receptor binding 6.57E-02 GO:0016279 protein-lysine N-methyltransferase activity 6.57E-02 GO:0030955 potassium ion binding 6.57E-02 GO:0046872 metal ion binding 7.06E-02 GO:0016491 oxidoreductase activity 7.85E-02 GO:0009055 electron carrier activity 8.41E-02 GO:0000287 magnesium ion binding 9.28E-02 GO:0015086 cadmium ion transmembrane transporter activity 9.34E-02 GO:0005384 manganese ion transmembrane transporter activity 9.34E-02 GO:0004056 argininosuccinate lyase activity 9.34E-02 GO:0005743 mitochondrial inner membrane 2.38E-07 GO:0043209 myelin sheath 3.43E-06 GO:0005747 mitochondrial respiratory chain complex I 1.11E-05 GO:0005739 mitochondrion 4.39E-03 GO:0031463 Cul3-RING ubiquitin ligase complex 1.07E-02 GO:0005759 mitochondrial matrix 4.07E-02 GO:0045177 apical part of cell 5.64E-02 GO:0005764 lysosome 6.71E-02 GO:0043657 host cell 8.88E-02 127 ANNEXURE A GO:0033018 sarcoplasmic reticulum lumen 8.88E-02 GO:0070062 extracellular exosome 9.44E-02 Go terms for biological process, molecular function and cellular component for Group 3 (score 2) vs 1 (score 0) GO term Description p-Value GO:0006954 inflammatory response 1.09E-06 GO:0032496 response to lipopolysaccharide 4.91E-04 GO:0015986 ATP synthesis coupled proton transport 1.97E-03 GO:0008284 positive regulation of cell proliferation 2.41E-03 GO:0090277 positive regulation of peptide hormone secretion 1.19E-02 GO:0015991 ATP hydrolysis coupled proton transport 1.72E-02 GO:0042127 regulation of cell proliferation 1.81E-02 GO:0006094 gluconeogenesis 2.04E-02 GO:0045595 regulation of cell differentiation 2.12E-02 GO:0005980 glycogen catabolic process 2.31E-02 GO:0014067 negative regulation of phosphatidylinositol 3-kinase signaling 2.31E-02 GO:0050790 regulation of catalytic activity 2.32E-02 GO:0070372 regulation of ERK1 and ERK2 cascade 3.04E-02 GO:0045087 innate immune response 3.22E-02 GO:0051683 establishment of Golgi localization 3.69E-02 GO:0061099 negative regulation of protein tyrosine kinase activity 3.69E-02 GO:0006096 glycolytic process 3.71E-02 GO:0010628 positive regulation of gene expression 3.77E-02 GO:0001501 skeletal system development 3.82E-02 GO:0007155 cell adhesion 3.99E-02 GO:0050727 regulation of inflammatory response 4.19E-02 GO:0008361 regulation of cell size 4.78E-02 GO:0043401 steroid hormone mediated signaling pathway 4.78E-02 GO:0046328 regulation of JNK cascade 7.11E-02 128 ANNEXURE A GO:0030836 positive regulation of actin filament depolymerization 7.11E-02 GO:0071356 cellular response to tumor necrosis factor 7.35E-02 GO:0018108 peptidyl-tyrosine phosphorylation 7.35E-02 GO:0005975 carbohydrate metabolic process 7.38E-02 GO:0001525 angiogenesis 7.42E-02 GO:0030097 hemopoiesis 7.52E-02 GO:0006935 chemotaxis 7.52E-02 GO:0030593 neutrophil chemotaxis 7.52E-02 GO:0002548 monocyte chemotaxis 8.11E-02 GO:0035987 endodermal cell differentiation 8.11E-02 GO:0003382 epithelial cell morphogenesis 9.08E-02 GO:0050873 brown fat cell differentiation 9.39E-02 GO:0034446 substrate adhesion-dependent cell spreading 9.44E-02 GO:0097190 apoptotic signaling pathway 9.44E-02 GO:0031532 actin cytoskeleton reorganization 9.72E-02 GO:0070062 extracellular exosome 2.70E-05 GO:0043209 myelin sheath 3.44E-05 GO:0045261 proton-transporting ATP synthase complex, catalytic core F(1) 2.40E-03 GO:0005615 extracellular space 4.45E-03 GO:0005623 cell 5.00E-03 GO:0005829 cytosol 9.81E-03 GO:0009986 cell surface 1.72E-02 GO:0005753 mitochondrial proton-transporting ATP synthase complex 2.27E-02 GO:0005759 mitochondrial matrix 2.59E-02 GO:0005743 mitochondrial inner membrane 3.17E-02 GO:0000139 Golgi membrane 4.66E-02 GO:0005925 focal adhesion 5.40E-02 GO:0005887 integral component of plasma membrane 6.04E-02 GO:0016324 apical plasma membrane 7.41E-02 GO:0032982 myosin filament 8.94E-02 GO:0031012 extracellular matrix 9.50E-02 129 ANNEXURE A GO:0046961 proton-transporting ATPase activity, rotational mechanism 1.43E-03 GO:0046933 proton-transporting ATP synthase activity, rotational mechanism 4.79E-03 GO:0005351 sugar:proton symporter activity 1.22E-02 GO:0005003 ephrin receptor activity 1.22E-02 GO:0042802 identical protein binding 1.87E-02 GO:0003779 actin binding 2.08E-02 GO:0000287 magnesium ion binding 2.23E-02 GO:0005031 tumor necrosis factor-activated receptor activity 4.86E-02 GO:0005385 zinc ion transmembrane transporter activity 5.90E-02 GO:0008083 growth factor activity 5.92E-02 GO:0008009 chemokine activity 7.03E-02 GO:0015078 hydrogen ion transmembrane transporter activity 7.03E-02 GO:0009055 electron carrier activity 7.53E-02 Go terms for biological process, molecular function and cellular component for Group 4 (score 3) vs 1 (score 0) GO term Description p-Value GO:0046660 female sex differentiation 3.53E-02 GO:0071230 cellular response to amino acid stimulus 4.34E-02 GO:0051464 positive regulation of cortisol secretion 4.68E-02 GO:0070208 protein heterotrimerization 5.81E-02 GO:0007276 gamete generation 8.04E-02 GO:0042176 regulation of protein catabolic process 9.13E-02 GO:0005793 endoplasmic reticulum-Golgi intermediate compartment 4.46E-02 GO:0005581 collagen trimer 6.74E-02 GO:0005634 nucleus 6.78E-02 GO:0030686 90S preribosome 7.29E-02 GO:0005782 peroxisomal matrix 7.29E-02 GO:0035256 G-protein coupled glutamate receptor binding 2.40E-02 130 CHAPTER 3 A PILOT STUDY OF THE IMMUNE RESPONSE TO Avibacterium paragallinarum SEROVAR C-3 INFECTION IN Gallus gallus Sections of Chapter 3 have been used for manuscript for submission in a peer-reviewed journal, with the title “Omens and Remnants of Infectious Coryza: A Macabre Tale of Necropsy and Immunohistopathology of Chicken Lymphatic Tissues after Infection with Av. paragallinarum serovar C-3” and “The Infectious Coryza Diaries: Disease Monitoring of Immune Cells and Molecules during Av. paragallinarum serovar C-3 Infection”. 3.1. Introduction Infectious coryza caused by the Gram-negative bacterium Av. paragallinarum, is a well- recognized and commonly encountered upper respiratory tract disease in chickens (Blackall, 1999). The occurrence of outbreaks has emphasized the significance of the disease in both broiler and layer chickens (Blackall, 1999). In South Africa, IC is regarded as one of the most serious diseases of layers, with C-3 being the most predominant serovar (Bragg et al. 1996). The economic impact of IC is primarily observed in the layer industry, with a marked drop in egg production and a significant number of culls due to morbidity in growing broods (Blackall, 1999). 131 CHAPTER 3: A PILOT STUDY OF THE IMMUNE RESPONSE TO Avibacterium paragallinarum SEROVAR C-3 INFECTION IN Gallus gallus The genetic mechanisms that govern the immune response of birds infected with Av. paragallinarum are still unknown and poorly studied. Thus far, the only studies conducted on the avian model with regards to immune mechanisms were conducted on Salmonella serovars and avian influenza virus (Withanage et al. 2004, Withanage et al. 2005; Xing et al. 2008; Nerren et al. 2010; Matulova et al. 2013). Notable studies investigated the role played by candidate genes and genomic regions in disease resistance, through challenge methods with pathogenic Salmonella that mimicked the natural routes of exposure to the pathogen, thus allowing the opportunity to study mucosal immunity and the immune response (Lamont, 1994; Lamont, 1998; Kaiser and Lamont, 2002; Lamont et al. 2002; Kramer et al. 2003). Studies by Boucher et al. (2014, 2015), investigated the up- and down- regulation of genes using microarray technology and RNA isolated from chicken sinuses presented with clinical symptoms when infected with serovar C-3, thus opening the way to the immune mechanisms pertaining to Av. paragallinarum host-pathogen interactions. Moreover, with regards to innate and adaptive immunity in birds challenged with Av. paragallinarum, it is still uncertain which immune cells and molecules are involved that serve as immune mechanisms against pathogenic infection. This prompted us to investigate and study the regulation of immune signalling molecules related to immunity during Av. paragallinarum infection. However, before we could commence, a pilot study was conducted to validate whether the study would be feasible. The pilot study would also provide a robust and logical workflow and experimental design, that will enable us to troubleshoot any discrepancies or errors encountered and take further necessary measures for the actual experiment to be conducted. For this research project, a pilot study was conducted in accordance with clear study aims and objectives, as an exploratory tool to enable us to determine a suitable experimental design, master and understand techniques, implement correct laboratory procedures and practices, involving chickens challenged with a highly virulent Av. paragallinarum serovar C-3 (SA-3 strain). This was done to elicit an immune response and monitor disease progression. A pilot 132 CHAPTER 3: A PILOT STUDY OF THE IMMUNE RESPONSE TO Avibacterium paragallinarum SEROVAR C-3 INFECTION IN Gallus gallus study is essential since it would encourage the necessity of the study, establish a framework within which to work and therefore guarantee that it is scientifically valid (Lancaster et al. 2004). 3.2. Materials and methods 3.2.1. Ethics statement, animal husbandry and study design The research conducted and handling of animals in the study was performed in accordance with current South African legislation, The South African National Standard for the Care and Use of Animals for Scientific Purpose (SANS 10386:2008), and the Animals Protection Act, 1962 (Act No. 71 of 1962). Prior to experiments, permission to perform animal research was applied for and clearance was obtained under Section 20 of the Animal Diseases Act, 1984 (Act No. 35 of 1984) from the Department of Agriculture, Forestry and Fisheries (DAFF), South Africa. The study and specific experiments were conducted and monitored according to an approved ethics protocol by the Interfaculty Animal Ethics Committee (AEC) of the University of the Free State, Bloemfontein, South Africa (Project number: UFS-AED2016/0105). IC affects predominantly layer breeds. Hence, layer chickens were chosen for the study (Droual et al. 1990). A total of 40 specific pathogen free (SPF)/unvaccinated White Leghorn chickens at 20 weeks of age, were obtained from Deltamune (Lyttleton, Centurion, Pretoria, South Africa). The chickens were transported in carton crates, with each carton containing 10 chickens. Special care was taken during transportation of the chickens such as proper air ventilation throughout the vehicle, limited exposure to the environment and disinfection of surface areas with a pressure sprayer (2% dilution Virukill®, ICA Laboratories, Stellenbosch, South Africa) to prevent any contamination or carry-over diseases. 133 CHAPTER 3: A PILOT STUDY OF THE IMMUNE RESPONSE TO Avibacterium paragallinarum SEROVAR C-3 INFECTION IN Gallus gallus The chickens were separated into two cohorts namely; the experimental group consisting of 30 birds and the control group consisting of 10 birds. The chickens were housed and maintained by the University of the Free State Animal Unit (University of the Free State, Bloemfontein) (GPS Coordination: 2906’49.143”S, 2611’2.169”E). Cages and isolators were properly cleaned and thoroughly disinfected using a pressure sprayer (2% dilution Virukill®, ICA Laboratories, Stellenbosch, South Africa) prior to the arrival of chickens. Care was taken to prevent over-crowding in cages; hence, the experimental chickens were placed into individual cages, whereas the control chickens were placed into isolators of 5 chickens each. Isolators had filtered air that supplied ventilation and air to the chickens, thus implying that the chickens were not in direct contact with the experimental chickens nor the outside environment. The experimental set-up was similar to that described by Bragg (2002). Cages were in rows of five cages in the facility. The drinking water system consisted of an adjoining water supply that travelled through each cage sourced from a header tank; within each cage a nipple drinker was provided. There was also a communal feed trough that passed each row of the cage. The birds were given animal feed (Layer mash, Senwes) and water daily ad libitum. Cages were cleaned on a regular basis and kept clean through disinfection of cages with a pressure sprayer (2% dilution Virukill®, ICA Laboratories, Stellenbosch, South Africa). Regular check-ups were conducted to ensure the well-being of the chickens. Additionally, a qualified laboratory animal technician was on standby for any emergencies or assistance needed. The chickens were kept under observation and were monitored over a week before the experimental phase commenced. 3.2.2. Bacterial isolate used for challenge Av. paragallinarum SA-3 strain is indigenous to South Africa, however to ensure that a mixed culture or the wrong strain was not used which could interfere with the disease progression of the study, a reference isolate and not a field isolate was needed. Import permits and all 134 CHAPTER 3: A PILOT STUDY OF THE IMMUNE RESPONSE TO Avibacterium paragallinarum SEROVAR C-3 INFECTION IN Gallus gallus necessary documentation required for the shipment of the strain was conducted in accordance with the Department of Agriculture, Forestry and Fisheries (DAFF). The SA-3 (or C-3 under Blackall classification) reference isolate was obtained from Prof. Patrick Joseph Blackall at the University of Queensland, Australia. The reason for choosing or working with SA-3, was primarily because it was reported as the most virulent strain of Av. paragallinarum in South Africa (Bragg et al. 1996). 3.2.3. Microbial cultivation and identification 3.2.3.1. Cultivation of Av. paragallinarum serovar C-3 Av. paragallinarum is a fastidious organism and is typically grown in test media with supplements such as chicken serum and nicotinamide adenine dinucleotide (NAD+) to simulate the environment of the host (Miflin et al. 1995). Av. paragallinarum serovar C-3 (SA- 3 strain) was cultured on blood tryptose agar (BTA) plates containing cattle blood (Onderstepoort Biological Products, Pretoria) and cross-streaked with S. epidermidis obtained from the culture collection of Prof. Celia Hugo at the University of the Free State, South Africa. This serves as a feeder culture and nourishes the organism with a supply of NAD+ (Page, 1962). The culture was passaged every 2 days on BTA plates to keep the bacterial culture viable. Tryptic soy broth (TSB) (Merck) supplemented with 0.2% (v/v) NAD+ (Merck) was the growth media used to culture Av. paragallinarum serovar C-3 (SA-3 strain). TSB media was autoclaved at 121C for 15-20 min and the supplements were added, only after the media was cooled down, by filter sterilisation using a syringe filter with a pore-size of 0.20 m (GVS ABLUO). A pre-inoculum was prepared that contained a bacterial culture of less than 24 h of age. This was then inoculated into a flask containing 250 ml of TSB supplemented with 0.2% NAD+ (v/v) 135 CHAPTER 3: A PILOT STUDY OF THE IMMUNE RESPONSE TO Avibacterium paragallinarum SEROVAR C-3 INFECTION IN Gallus gallus for a further 10-14 h with continuous shaking of the flask at 120 rpm at 37C (Labwit Scientific), grown to an optical density (OD600) of 1.0. The bacterial culture (<24 h) obtained was then centrifuged at 3000 x g for 10 min, to obtain a pellet which was re-suspended in 10 ml of 1X phosphate-buffered saline (PBS) (pH 7.4, Merck) which was kept at 4C overnight. A volume of 1-3 ml of the bacterial suspension was kept for bacterial identification and the rest was kept for the infection of chickens. Thus, a 48 h old bacterial culture previously re-suspended in 1X PBS, was used to infect the chickens for the first and second injections respectively, whereby for the second injection, fresh bacterial culture was prepared again and re-injected on Day 9. 3.2.3.2. Genomic DNA extraction To identify the bacterial species that would be used for infecting the chickens, genomic DNA needed to be extracted from the 1-3 ml of the bacterial culture that was reserved, using a modified version of the phenol-chloroform DNA extraction technique by Labuschagne and Albertyn (2007). Bacterial cells were harvested by centrifugation at 3000 x g at 4C for 15 min. Once a pellet was obtained, the cells were re-suspended in 500 l of cell lysis buffer [100 mM Tris-HCl, Roche Diagnostics, Merck; 50 mM ethylenediaminetetraacetic acid (EDTA), Merck; 1% SDS, BDH Laboratory Supplies, pH 8.0]. The cell suspension was then incubated at 37C for 20 min. After incubation, the mixture was vortexed for 30 s, followed by 30 s on ice and repeated for a period of 4 min. A volume of 275 l of ammonium acetate (7 M; pH 7.0, Merck) was added to precipitate any proteins bound, followed by a quick vortex or slow inversion of the Eppendorf tube with contents. The mixture was incubated for a further 5 min at 65C on a heating block (FMH instruments, Labotec), followed by 5 min on ice. A volume of 500 l of chloroform (Sigma-Aldrich) was added to dissolve proteins in solution, followed by a quick vortex and centrifugation at 20 000 x g for 2 min at 4C. The mixture forms three distinct layers: the aqueous phase (supernatant) containing DNA, the interphase containing proteins and the organic phase containing RNA and lipids (Köchl et al. 2005). The upper aqueous phase was 136 CHAPTER 3: A PILOT STUDY OF THE IMMUNE RESPONSE TO Avibacterium paragallinarum SEROVAR C-3 INFECTION IN Gallus gallus carefully transferred to a clean 1.5 ml tube without touching other phases of the mixture. An equal volume of isopropanol (Merck) was added to the supernatant to precipitate the DNA out of solution, and the mixture was centrifuged at 20 000 x g for 2 min at 4C. The supernatant was discarded, and the pellet obtained was washed with 70% ethanol (ice-cold) and centrifuged at 20 000 x g for 2 min at 4C, this procedure was repeated. Once, the supernatant was discarded, the pellet was either air dried or placed in the miVac centrifugal vacuum concentrator (SP Scientific, Genevac Ltd., UK). The air-dried pellet was then re-dissolved in 50 l of TE buffer [10 mM Tris-HCl, Roche Diagnostics, Merck; 1 mM EDTA, Merck, pH 8.0], to which 2 l of RNase (10 mg/ml, Qiagen®) was added. The solution was incubated for 1 hour at 37C in a water bath (Labotec®), to digest any residual RNA that might be present. The NanoDrop 1000 (Thermo Scientific) was used to measure the concentration and purity of DNA extracted from bacterial samples. The extracted DNA was then stored at -20C for long- term storage or until further use. 3.2.3.3. Identification of bacterial strain The isolate was first phenotypically identified based on the shape and appearance of the colonies formed on BTA plates, as well as the satellitic behaviour observed when crossed- streaked with “feeder” culture Staphylococcus species (De Blieck, 1932; Page, 1962; Vargas and Terzolo, 2004). Molecular identification involved carrying-out a species-specific PCR also known as the HPG2-PCR described by Chen et al. (1996). The species-specific PCR consisted of 50 l reaction volumes. The reaction mixture consisted of: 1l of each of the primer set as described by Chen et al. (1996) (0.2 M, Table 3.1) (Integrated DNA Technologies Inc.), 5 l of 1x ThermoPol® reaction buffer (New England BioLabs® Inc.), 1 l of dNTPs (100 M, Thermo Scientific), 0.4 l of Taq DNA polymerase (2U/ 50 l PCR, New England BioLabs® Inc.), 36.6 l of nuclease-free water and 5 l of the appropriate amount of template DNA (4-1000 ng/µl) were added. The PCR reaction was performed in the 2720 137 CHAPTER 3: A PILOT STUDY OF THE IMMUNE RESPONSE TO Avibacterium paragallinarum SEROVAR C-3 INFECTION IN Gallus gallus Thermal Cycler (Applied Biosystems, Life Technologies) with an initial 1 min denaturation step at 95C. The PCR amplification and cycling conditions had 35 cycles that consisted of the following steps: denaturation at 95C for 25 s, annealing at 55C for 30 s, and an elongation step at 68C for 45 s, followed by a final elongation/holding step at the end of the cycle at 68C for 7 min. The samples were then stored at -20C until further use. To confirm that the bacterial species that were used during experimentation was indeed Av. paragallinarum serovar C-3, a 16S rDNA PCR was conducted. The 16S rDNA PCR consisted of 50 l reaction volumes. The reaction mixture consisted of: 1l of each of the primer sets as described by Edwards et al. (1989) and Mendoza-Espinoza et al. 2008 (0.2 M, Table 3.1) (Integrated DNA Technologies Inc.), 5 l of 1x ThermoPol® reaction buffer (New England BioLabs® Inc.), 1 l of dNTPs (100 M, Thermo Scientific), 0.4 l of Taq DNA polymerase (2U/ 50 l PCR, New England BioLabs® Inc.), 36.6 l of nuclease-free water and 5 l of the appropriate amount of template DNA (4-1000 ng/µl) were added. The PCR reaction was performed in the G-Storm GS482 (Gene Technologies Ltd.) with an initial 5 min denaturation step at 94C. The PCR amplification and cycling conditions had 30 cycles that consisted of the following steps: denaturation at 94C for 30 s, annealing at 50C for 30 s, and an elongation step at 72C for 1 min 45 s, followed by a final elongation/holding step at the end of the cycle at 72C for 5 min. The samples were then stored at -20C until further use. Both the species-specific and 16S rDNA PCR reactions consisted of a negative control (no template control) and two positive controls from Av. paragallinarum (SA-3 strain serovar C-3 (C3+) and Modesto strain serovar C-2 (C2+)). The negative control is used primarily to check for contaminants, as it contains all PCR reagents except the template. A positive control is a 138 CHAPTER 3: A PILOT STUDY OF THE IMMUNE RESPONSE TO Avibacterium paragallinarum SEROVAR C-3 INFECTION IN Gallus gallus sample that had previously been amplified using the same PCR reagents and conditions but is used to test for inhibitors. Table 3.1: Oligonucleotide primers for species-specific and 16S rDNA PCR amplification of target region. Expected Molecular Primers Sequence amplicon Authors technique size (bp) Species- HPG2 forward 5’-TGA GGG TAG TCT TGC ACG CGA AT-3’ Chen et al. specific 500 1996 PCR HPG2 reverse 5’-CAA GGT ATC GAT CGT CTC TCT ACT-3’ 8F 5’-AGA GTT TGA TCA TGG CTC AG-3’ Edwards et al. 1989; 16S rDNA 1500 Mendoza- PCR 1525R 5’-AAG GAG GTG ATC CAG CCG CA-3’ Espinoza et al. 2008 3.2.3.4. Agarose gel electrophoresis and visualisation of correct DNA fragment size The agarose gel was prepared with SeaKem® LE Agarose (Lonza), 1X TAE buffer [50X TAE stock: 0.1 M Tris, 0.05 M Na2EDTA.2H2O, pH 8.0, 0.1 mM glacial acetic acid; Merck] and stained with ethidium bromide (0.3 μg/μl). PCR products from the species-specific PCR and 16S rDNA PCR were resolved on a 1% (w/v) agarose gel, with each well containing 1 l of 6X Orange Loading Dye (Thermo Scientific) and 4 l of PCR product. The O'GeneRuler DNA Ladder Mix (100-10,000 bp, Thermo Scientific) served as a molecular marker and was run in parallel with samples on agarose gels respectively, this was done to determine the relative 139 CHAPTER 3: A PILOT STUDY OF THE IMMUNE RESPONSE TO Avibacterium paragallinarum SEROVAR C-3 INFECTION IN Gallus gallus sizes of the DNA fragments by comparing their electrophoresis site of mobility with that of the molecular marker. Gel electrophoresis was performed in 1X TAE buffer for 30 min at 9 V/cm. The gel and visualisation of correct DNA fragment size was conducted under ultraviolet (UV) light using the Gel Doc™ EZ Imager and Image Lab™ Software (Bio-Rad). 3.2.3.5. Sequencing of 16S rDNA PCR products To confirm the identity of the bacterial strain and that the nucleotide sequence indeed corresponded to Av. paragallinarum, sequencing needed to be performed. Following the visual confirmation of the correct fragment size of the 16S rDNA PCR product, the gel band was cut out using the UV transilluminator (Spectroline®). The agarose gel slice containing the 16S rDNA PCR product was purified with the Wizard® SV Gel and PCR Clean-Up System (Promega) according to the manufacturer’s instructions. The gel slice was weighed-off and a volume of 1 l of Membrane Binding Solution was added per 1 mg of gel slice. The mixture was vortexed thoroughly and incubated at 65C until the excised agarose gel had been completely dissolved. An SV column was placed into a collection tube per purification to be performed. The dissolved gel mixture was transferred to the column assembly and incubated at room temperature for 1 min. The mixture was then centrifuged at 16 000 x g for 1 min, the flow-through was discarded and the column was placed back into the collection tube. A volume of 700 l of Membrane Wash Solution was added to the column, to remove excess dNTPs, enzymes and primers not used during amplification and the column was centrifuged at 16 000 x g for 1 min. The flow-through was discarded and the column was re-inserted back to the collection tube. The wash step was repeated with a volume of 500 l of Membrane Wash Solution and the contents centrifuged at 16 000 x g for 5 min. The collection tube was emptied, and the column assembly was re-centrifuged for 1 min with 140 CHAPTER 3: A PILOT STUDY OF THE IMMUNE RESPONSE TO Avibacterium paragallinarum SEROVAR C-3 INFECTION IN Gallus gallus the lid open to allow evaporation of any residual ethanol. The column was re-inserted to a clean 1.5 ml tube. A volume of 50 l of nuclease-free water was then added to the column. This was followed by incubation of the column assembly at room temperature for 1 min and centrifugation at 16 000 x g for 1 min, to elute and recover the purified PCR product. Purified DNA samples were stored at -20°C until needed. PCR sequencing reactions were performed using the BigDye® Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems™). Since the size of our PCR product is approximately 1500 bp, a final reaction quantity of 10-40 ng of the PCR product was needed. The sequencing PCR consisted of 10 l reaction volumes. The reaction mixture consisted of: 1l of the 8F and 1525R primers in separate reactions as described by Edwards et al. (1989) and Mendoza- Espinoza et al. 2008 (0.32 M, Table 3.1) (Integrated DNA Technologies Inc.), 1 l of BigDye® Terminator v3.1 Ready Reaction Mix (premix), 2 l of 5X Sequencing Buffer, 4 l of nuclease- free water and 2 l of the appropriate amount of template DNA (10-40 ng) were added. The sequencing PCR was performed in the G-Storm GS482 (Gene Technologies Ltd.) with an initial 1 min denaturation step at 94C. The sequencing PCR amplification and cycling conditions had 30 cycles that consisted of the following steps: 96C for 10 s, 50C for 5 s and 60C for 4 min, followed by a final holding step at the end of the cycle at 4C for 5 s. The BigDye® EDTA/ethanol precipitation post-sequencing reaction clean-up protocol was carried-out according to manufacturer’s instructions for a clean and consistent signal during sequencing analysis, as well as to remove unused primers and unincorporated dye-labelled terminators. The sequencing reaction volume was adjusted to 20 l by adding a volume of 10 l of sterile nuclease-free water to the 10 l PCR products obtained from the sequencing reaction. The mixture was transferred to a clean Eppendorf tube. A volume of 60 μl of 100% 141 CHAPTER 3: A PILOT STUDY OF THE IMMUNE RESPONSE TO Avibacterium paragallinarum SEROVAR C-3 INFECTION IN Gallus gallus (v/v) ethanol (final concentration: 70.6% (v/v)) and 5 μl of 125 mM EDTA at pH 8.0 (final concentration 7.35 mM) was added to precipitate DNA, remove unincorporated BigDye® terminators and to chelate divalent ions for inactivation of DNases. The mixture was vortexed for 5 sec and left to precipitate at room temperature for 15 min. DNA was pelleted by centrifugation at 4C at 20 000 x g for 15 min and the supernatant was completely aspirated. The pellet was washed with a volume of 60 μl of ice-cold 70% (v/v) ethanol. The suspension was centrifuged at 20 000 x g for 5 min. The supernatant was discarded, and the samples were dried using the miVac centrifugal vacuum concentrator (SP Scientific, Genevac Ltd., UK). Once dried, the samples were stored in the dark at 4C. Sequencing was performed with the Applied Biosystems 3130xl Genetic Analyzer at the Department of Microbial, Biochemical and Food Biotechnology, University of the Free State. Sequence analysis was performed using Geneious® 9.8.1 (Biomatters Ltd.) (Kearse et al. 2012). The forward and reverse sequences were aligned, and the consensus sequence was compared with known sequences in GenBank, using a nucleotide BLAST analysis (Altschul et al. 1990). 3.2.4. Challenge methods and clinical scoring Handling of the chickens, by scientists in training and a qualified animal technician, was done to prevent any injury to the chickens, by holding the chicken’s wings and feet securely under the arm, thereby restricting movement (Figure 3.1). Infection of the chickens was conducted via infra-orbital injection directly into the sinus cavity on the left side of the facial area as described by Boucher et al. (2014), as IC is an upper respiratory disease and the sinus cavity is an ideal location for infection. For the first trial, the control group was injected using 100 l of 1X PBS (pH 7.4, Merck) only and the experimental group was injected with 100 l of 1X 142 CHAPTER 3: A PILOT STUDY OF THE IMMUNE RESPONSE TO Avibacterium paragallinarum SEROVAR C-3 INFECTION IN Gallus gallus PBS (pH 7.4, Merck) solution containing a suspension of Av. paragallinarum serovar C-3 (SA- 3 strain) bacterial cells at an optical density (OD) of 1.0. The bacterial culture used for the first injection was 48 h old (Section 3.2.3.1). After infection, the signs and symptoms were monitored, to ensure that the chickens had been exposed and infected. On Day 9, all experimental birds except control birds were re-infected with a 48 h old bacterial culture as described above (Section 3.2.3.1), the disease was allowed to progress, and signs and symptoms were monitored. Following infection of the chickens, we allowed the signs and symptoms of IC to progress to score 1, 2 and 3, the data was recorded on a daily basis on a monitoring sheet for both groups (Appendix A). The adapted criteria and clinical manifestations of IC by Bragg (2002) was used, based on a scoring system (Matsumoto and Yamamoto, 1971). A score of 0 is used to indicate “No clinical signs”. A score of 1 indicates “Mild clinical signs with nasal discharge with or without mild facial oedema”. A score of 2 indicates “Moderate signs with nasal discharge on both left and right sides and slight facial oedema” and a score of 3 describes “Severe signs with severe bilateral oedema with or without haemorrhage and conjunctivitis”. Blood was collected from 5-7 chickens after every 2-4 days (48-96 h), based on the disease score and symptoms observed in chickens. For each scoring system, chickens were unbiasedly/randomly selected from the total population of experimental subjects, where 2-4 chickens showing the same score were sacrificed for histopathology, for observation of lymphoid organs and the rest of the chickens were left for the disease to progress further. This process was repeated until a score of 3 was observed. The total number of eggs produced, and the total number of eggs laid per bird per day from each group were carefully recorded. 143 CHAPTER 3: A PILOT STUDY OF THE IMMUNE RESPONSE TO Avibacterium paragallinarum SEROVAR C-3 INFECTION IN Gallus gallus Figure 3.1: Handling of a bird by qualified personnel. Utmost care was taken to ensure that the well-being of the birds was considered and that the chickens were treated as humanely as possible during injection procedures, as well as during bleeding times without causing stress, pain, suffering, anxiety or physical injury. 3.2.5. Blood collection and processing Blood samples (2-3 ml) were collected via the branchial vein (wing vein) into commercial 4 ml EDTA coated SGVac PET Blood Collection Tubes (The Scientific Group). An additional volume of EDTA (0.5 M, Merck) of 800 l- 1 ml was supplemented to the EDTA tubes, to prevent quick coagulation of the blood collected, as chicken blood coagulates quickly due to high levels of calcium present (Lewis and Stoddart, 1971; Mikaelsson, 1991; Preda et al. 2014). Following blood collection, the EDTA tubes were quickly inverted 3-4 times to thoroughly mix the blood and EDTA together to prevent coagulation and to minimise clots. The collected blood was then separated into three parts into separate 1 ml EDTA coated SGVac PET Blood Collection Tubes (The Scientific Group) with a maximum volume of approximately 1 ml per tube for flow cytometry, blood microscopy and to obtain plasma to perform direct enzyme-linked immunosorbent assays (ELISAs). The blood samples were 144 CHAPTER 3: A PILOT STUDY OF THE IMMUNE RESPONSE TO Avibacterium paragallinarum SEROVAR C-3 INFECTION IN Gallus gallus processed within 6-8 h. To obtain plasma, one of the tubes containing whole blood, was centrifuged at 3000 x g for 15 min, to separate the blood components from plasma. The plasma obtained was then stored at -80C for long-term storage and until further use. 3.2.6. Avian full blood counts, differential blood counts and microscopy EDTA tubes containing a volume of 1 ml of the blood sample collected from each experimental subject, were sent to the National Health Laboratory Service (NHLS, Universitas, Bloemfontein, South Africa) and to Pathcare Vetlab (Westdene, Bloemfontein, South Africa), respectively for preliminary complete/full blood counts (CBC/FBC) and white blood cell differential counts (WBC Diff). The NHLS mainly conducts diagnostic testing for human patients and the Pathcare Vetlab caters for animal diagnostics. These laboratories were chosen as we did not have the equipment, expertise or facilities in our laboratory to carry out these tests. We also needed to conduct blood tests and the smears as soon as blood was drawn according to the experimental design, whereby a laboratory in close proximity was ideal to prevent a delay in the blood processing which could lead to erroneous and inaccurate results. Moreover, these laboratories are South African National Accreditation System (SANAS) accredited laboratories, whereby calibration, quality assurance and controls and standard operating procedures (SOPs) are followed and used on a routine basis. Blood smears of the avian specimens at the NHLS (Universitas, Bloemfontein, South Africa) were performed according to the SOP described by the NHLS below. The EDTA tube containing whole blood was gently inverted 8 times. An applicator stick was used to check for blood clots. Using a glass capillary tube or applicator stick, a small drop of blood was placed on the glass slide at 1 cm from one end in the midline. A spreader was placed on a flat surface 145 CHAPTER 3: A PILOT STUDY OF THE IMMUNE RESPONSE TO Avibacterium paragallinarum SEROVAR C-3 INFECTION IN Gallus gallus and held down firmly with the thumb and forefinger at opposite ends. The spreader was then held at an angle of 45, and was pulled backwards until it touched the drop of blood, which allowed the blood to spread across the breadth of the slide on both ends. The spreader was dragged forward smoothly and rapidly, maintaining contact between the two slides. The blood film formed is 3-4 cm long, evenly spread with no ragged tails. Moreover, the thickness should be such as to allow the erythrocytes to be separated from each other in the last quarter of the film. The film was left to air-dry before staining the slide. Staining was conducted using the slide stainer Hematek® 3000 System (Siemens Healthineers) with Hematek Modified Wright’s stain and Hematek Wright-Giemsa stain to stain peripheral blood and identify different blood cells. Visualisation of the slides was conducted with the Eclipse 50i microscope, DS-Fi1 digital microscope camera and NIS-Elements F 4.00.06 Build 786 microscope imaging software (Nikon), whereby images were taken at a 100X magnification. Complete/Full blood counts (CBC/FBC) and white blood cell differential counts (WBC Diff) were conducted as described by the SOPs followed by the NHLS (Universitas, Bloemfontein, South Africa) below. The ADVIA® 2120i (Siemans Healthineers) was used according to manufacturer’s instructions and operator’s manual, which is an automated flow-cytometry based analyser that provides complete blood counts, complete differential count, reticulocyte absolute and reticulocyte percentage in a single run with a single blood sample provided (Harris et al. 2005a; Harris et al. 2005b). There are several processes involved in the analyser such as cytochemical reactions, cytometric measurements of specific cell properties and algorithms converting data into familiar results for cell classification, cell count, cell size and haemoglobin. Firstly, a volume of approximately 175 l of the EDTA anti-coagulated whole blood was aspirated by an automatic/manual sampler, this is followed by the anti-coagulated whole blood sample being passed and separated into different reaction chambers comprising of different reagents and reaction settings (HGB, BASO, RBC/PLT, PEROX and RETIC). For the haemoglobin concentration (HGB), the haemoglobin method was used which is a 146 CHAPTER 3: A PILOT STUDY OF THE IMMUNE RESPONSE TO Avibacterium paragallinarum SEROVAR C-3 INFECTION IN Gallus gallus modification of the manual cyanmethaemoglobin method developed by the International Committee of Standardization in Haematology (ICSH) (ICSH Standard EP6/2, 1977; ICSH Standard EP6/3, 1977; Dacie and Lewis, 2006). For a white blood cell (WBC) count, ADVIA® 2120i BASO reagent containing acid and surfactant was mixed with the whole blood (Cremins and Orlik, 1996). ADVIA® 2120i BASO reagent causes haemolysis of RBCs, whereby the white blood cells were able to be analysed using 2 angle laser light scatter signals (Cremins and Orlik, 1996). For red blood cell (RBC) and platelet (PLT) counts, whole blood was mixed with ADVIA® 2120i RBC/PLT reagent. The red blood cells (RBCs) were lightly fixed with glutaraldehyde present in the reagent to preserve their isovolumetrically spherical shape (Kim and Ornstein, 1983). Both RBCs and PLTs were detected from a single optical cytometer/detector with 2 gain settings, whereby platelet signals are amplified considerably more than the RBCs. A coincidence correction was performed to each of the counts so that the accurate counts are conducted over a wide range of each cell type. For sizing RBCs and platelets, simultaneous measurement of laser light scattered at 2 different angular intervals was performed, which eliminates the effect of variation in the cellular haemoglobin concentration when determining cell volume (Kerker, 1969; Groner and Tycko, 1980). Moreover, red cell indices like the mean corpuscular haemoglobin (MCH) and mean corpuscular haemoglobin concentration (MCHC), were derived from mathematical calculations based on the RBC count, the total haemoglobin and the mean corpuscular volume (MCV) determination (Wintrobe, 1932). The haematocrit (HCT) values were calculated from the RBC count and the MCV (Wintrobe, 1932). The red cell distribution width (RDW) and haemoglobin distribution width (HDW) values were calculated from the cell-by-cell measurement of the cell volume and haemoglobin concentration (Bessman, 1981). The CH represents the cell haemoglobin content histogram from internal complexity of intact RBC by side scattered of a low angle laser light and cell haemoglobin concentration mean (CHCM) is calculated as the mean of the RBC haemoglobin concentration histogram (Mohandas et al. 1986). 147 CHAPTER 3: A PILOT STUDY OF THE IMMUNE RESPONSE TO Avibacterium paragallinarum SEROVAR C-3 INFECTION IN Gallus gallus The WBC Diff was performed based on the peroxidase method (Cremins et al. 1990). Leukocytes possess the enzyme peroxidase that is active in cells. In the presence of hydrogen peroxide and an appropriate electron acceptor chromogen, peroxidase develops a darkly coloured precipitate in the cells. The peroxidase cytochemical reaction involves two steps and is conducted simultaneously with the CBC. In the first step, ADVIA® 2120i PEROX 1 reagent is mixed with whole blood, whereby surfactants and thermal stress cause lysis of the red blood cells. Moreover, ADVIA® 2120i PEROX 1 reagent contains formaldehyde that fixes the WBCs. In the second step, ADVIA® 2120i PEROX 2 reagent and ADVIA 120 PEROX 3 reagent were added to the peroxidase reaction chamber. ADVIA 120 PEROX 2 reagent contains 4-chloro- 1-naphthol and ADVIA 120 PEROX 3 reagent contains hydrogen peroxide, which stain the sites of peroxidase activity in the granules of neutrophils, eosinophils, and monocytes. There are no granules with peroxidase enzyme activity in lymphocytes, basophils, and large unstained cells. The cell suspension from the Perox reaction chamber passes through the flowcell, whereby the two fluids flow independently and without mixing, with the ADVIA 120 PEROX SHEATH stream encasing the sample stream. The absorbance and the forward light- scattering measurements of each blood cell are captured, and the optical signals are converted to electrical pulses by photodiodes. The raw data is processed, and the information is presented in two histograms namely, Perox Y histogram containing the forward-scattering data (cell size) and Perox X histogram containing the absorption data (peroxidase staining). These two histograms are merged to form the Perox cytogram, from which cells are identified and counted. The basophil/lobularity method was performed and was based on Cremins and Orlik (1996), whereby basophils are unaffected by lysis using a combination of acid and surfactant. This method provides rapid recognition of basophils, accurate basophil counts and a measure of cellular lobularity. During this reaction, EDTA anticoagulated whole blood was mixed with ADVIA 120 BASO reagent in the BASO reaction chamber, where RBCs and leukocytes were lysed or have their cytoplasm stripped from them, except basophils. Thereafter, a two-angle laser light scattering detection method was performed to analyse the cell suspension using a laser diode. Finally, the leukocytes could be classified into three 148 CHAPTER 3: A PILOT STUDY OF THE IMMUNE RESPONSE TO Avibacterium paragallinarum SEROVAR C-3 INFECTION IN Gallus gallus categories: basophils, mononuclear (MN) cells, and polymorphonuclear (PMN) cells respectively. The reticulocyte cell count was conducted using a nucleic acid dye (oxazine 750). EDTA anticoagulated whole-blood sample was mixed with the ADVIA 120 autoRETIC reagent. The ADVIA 120 autoRETIC reagent preserves the isovolumetrically sphere shape of erythroid cells and stains cellular RNA. This was followed by a low-angle laser light scatter, and high-angle laser light scatter, whereby the absorption characteristics of all cells were counted and measured. The absorption data was used to categorize each cell as a reticulocyte or mature red blood cell depending on its RNA content. The sizing of reticulocytes was conducted via the simultaneous measurement of laser light scattered at two (2) different angular intervals, whereby variation in cellular haemoglobin concentration on the determination of the MCVr parameter is eliminated. The last parameter analyzed was the CHr, which was the mean of cellular haemoglobin content (CH) histogram for the reticulocyte population (Brugnara et al. 1994; Fishbane et al. 1997). The CHr is an indicator of functional iron deficiency in human patients (Brugnara et al. 1994; Fishbane et al. 1997). Blood smears, CBC and WBC Diff were also requested at Pathcare Vetlab (Westdene, Bloemfontein, South Africa). Unfortunately, protocols and standard operating procedures could not be obtained from Pathcare Vetlab (Westdene, Bloemfontein, South Africa). 3.2.7. Staining and flow cytometry analysis Fresh chicken blood was obtained that were anticoagulated from venesection as described above (Section 3.2.6) and processed no later than 8 h. Flow cytometric analysis was performed using the following mouse anti-chicken antibodies: CD4-FITC (2-35 clone, MCA2164F) and CD8-RPE (11-39 clone, MCA2166PE) (Bio-Rad). A modified version of the 149 CHAPTER 3: A PILOT STUDY OF THE IMMUNE RESPONSE TO Avibacterium paragallinarum SEROVAR C-3 INFECTION IN Gallus gallus method described by Macey et al. (1999) was used. A volume of 5 l of each antibody was added to 25 l of whole blood in a round bottom, snap cap tube (Greiner Bio-One International). The labelled cells were incubated for 10 min in the dark at room temperature. A volume of 2 ml of working strength 1X FACS Lysing Solution (Becton Dickinson, Immunocytometry Systems, San Jose, CA) was added and the cells were lysed for 10 min. The cells were centrifuged for 3 min at 2000 x g and the supernatant discarded. The cells were then washed once with 2 ml of 1X PBS (pH 7.4), vortexed and centrifuged for 3 min at 2000 x g and the supernatant discarded. Finally, the cells were resuspended in 1 ml of 1X PBS (pH 7.4) and vortexed thoroughly. Sample analysis were performed on the BD FACSCanto II and BD FACSDiva 8.0.1 software (Becton Dickinson), whereby calibration and set-up were performed on a routine basis using BD FACS 7-color setup beads (Becton Dickinson). To separate RBCs from lymphocytes in whole blood for flow cytometry: either lysis of RBCs was conducted, whereby 2 ml of 1X FACS Lysing Solution (Becton Dickinson, Immunocytometry Systems, San Jose, CA) was added to 25 µl of whole blood and the cells were lysed for 10 min, or Ficoll-Paque PLUS (GE Healthcare) according to the manufacturer’s instructions was used. A volume of 4 ml of Ficoll-Paque PLUS (GE Healthcare) was layered to a diluted blood sample consisting of 2 ml of whole blood and 2 ml of balanced salt solution. The blood sample was centrifuged at 400 x g for 30-40 min at room temperature. Following centrifugation, the formation of different blood cell types was obtained. The upper layer consisting of plasma was drawn off, followed by the extraction of the lymphocyte/ peripheral blood mononuclear cell (PBMC) layer using aseptic techniques. 3.2.8. Direct Enzyme-linked immunosorbent assay (ELISA) 150 CHAPTER 3: A PILOT STUDY OF THE IMMUNE RESPONSE TO Avibacterium paragallinarum SEROVAR C-3 INFECTION IN Gallus gallus A volume of 500 ml of broth, containing strain SA3 (C-3) was cultivated as described above (Section 3.2.3.1) and centrifuged at 3000 x g for 10 min to obtain a pellet. The pellet was re- suspended in 10 ml of PBS. The bacterial culture was standardised to an OD of 1.0 at a wavelength of 600 nm and kept in the fridge at 4C until further use, for a period of not more than 1 week due to gradual degradation of the antigens with time. Enzyme-linked immunosorbent assays (ELISAs) were performed in 96-well Costar® high-binding polystyrene plates (Corning Inc.). The method described had been previously optimised. The Costar® plates were coated with 100 l of antigen with the bacterial cells prepared. The tests were conducted in quadruplicate and the controls were performed in duplicate. Two wells per batch tested had only PBS added (no antigen controls) and 2 wells per batch tested were coated with antigen with no plasma added (no plasma controls). The coated plates were incubated overnight at 37C or for 1 h, before the removal of excess liquid by shaking-off excess liquid by decanting in one motion or through complete aspiration of the well contents. The wells were washed 6 times with 200 l of PBS-Tween® 20 0.1% (v/v) (Sigma-Aldrich), to remove any unbound antigen and to wash the cells. Blocking was performed using 200 l of 3% (w/v) Bovine Serum Albumin Fraction V (BSA) (Roche) in PBS-Tween® 20 0.1% (v/v) (Sigma- Aldrich) to prevent non-specific binding of antibodies and antigens to the microtiter well (Xiao, 2012), followed by incubation for 1 h at 37C. Following blocking, another washing step was performed, whereby wells were washed 6 times with 200 l of PBS-Tween® 20 0.1% (v/v) (Sigma-Aldrich) and shaking-off excess liquid by decanting in one motion. Chicken plasma was diluted at 1:100 with PBS-Tween® 20 0.1% (v/v) (Sigma-Aldrich) and a volume of 100 l was added to appropriate wells except the control wells, this was followed by incubation at room temperature for 1 h with gentle mixing on the Mini BioMixer (Benchmark Scientific). The wells were washed 8 times with 200 l of PBS-Tween® 20 0.1% (v/v), followed by shaking-off excess liquid by decanting in one motion. Anti-chicken IgY (IgG) (whole molecule) peroxidase antibody produced in rabbit (Sigma-Aldrich) was diluted at 1:10 000 with PBS-Tween® 20 0.1% (v/v) and a volume of 50 l was added to appropriate wells and incubated for 1 h at room 151 CHAPTER 3: A PILOT STUDY OF THE IMMUNE RESPONSE TO Avibacterium paragallinarum SEROVAR C-3 INFECTION IN Gallus gallus temperature with gentle mixing on the Mini BioMixer. A final washing step was conducted, whereby the wells were washed thoroughly 8 times with 200 l of PBS-Tween® 20 0.1% (v/v), followed by 5 min intervals on the Mini BioMixer and shaking-off excess liquid by decanting in one motion after every washing step. A volume of 50 l of 3,3’,5,5’- Tetramethylbenzidine (TMB) (Roche) was added as a substrate to appropriate wells and incubated at room temperature for 15 min, until a gradient colour change was observed, this final incubation time should not exceed 30 min. A volume of 50 l of 2N (1M) sulphuric acid (H2SO4) (Merck) was added to all the wells to stop the reaction. The absorbance values of the wells were measured at a wavelength of 450 nm using the ELx800 plate reader with Gen5™ software (BioTek). Statistical analysis was performed to determine the statistical significance of the data using Microsoft Excel. The mean and standard deviation of the data were calculated, and a p<0.05 was found to be statistically significant, from which a graph was plotted. This was a preliminary screening for antibodies present in plasma of control and IC infected chickens, hence only 16 samples were used for the ELISA from Day 0, 3, 7 and 14. There were 4 samples for Day 0, 4 samples for Day 3, 6 samples for Day 7 (consisting of 4 post- infected (PI) and 2 control chickens) and 2 samples (consisting of 1 control and 1 re-infected (RI) chicken) for Day 14. There were no known positive or negative control serums available for the ELISA. However, no antigen and no plasma controls were included for quality control. 3.3. Results and Discussion 3.3.1. Microbial cultivation and identification 3.3.1.1. Identification of bacterial strain 152 CHAPTER 3: A PILOT STUDY OF THE IMMUNE RESPONSE TO Avibacterium paragallinarum SEROVAR C-3 INFECTION IN Gallus gallus Tiny dewdrop colonies and satellitic behaviour adjacent the S. epidermidis “feeder” cultures on the BTA plates were observed, typical of Av. paragallinarum (Figure 3.2) as described in literature (Blackall et al. 1997). As bacterial cultures were grown in 2-5 flasks, only flasks with an OD600 closest to 1.0 was selected for the first (3IC: OD600 (flask)= 0.938; OD600 (1X PBS)= 0.834) and second injection (5IC: OD600 (flask)= 0.804; OD600 (1X PBS)= 0.972). Turbidity in cultures was checked for any bacterial contamination. Av. paragallinarum S. epidermidis Figure 3.2: BTA plate with Av. paragallinarum. Tiny dewdrop colonies are observed near the cross-streaked “feeder” cultures thus displaying satellitic behaviour. A species-specific PCR was performed, using the DNA extracted from bacterial cultures from the flasks containing broth (TSB) (Merck) supplemented with 0.2% (v/v) NAD+ (Merck) and the bacterial suspension of C-3 in 1X PBS (pH 7.4, Merck) which was kept at 4C, for the first and second injection respectively. This was done as a precautionary measure from the flask stage to the injection stage, to ensure that the bacterial cultures used were sterile and contamination- free, as in the past we had experienced an outbreak of spore-forming Bacillus species. The species-specific PCR/ HPG2-PCR was performed with the DNA extracted from the reference 153 CHAPTER 3: A PILOT STUDY OF THE IMMUNE RESPONSE TO Avibacterium paragallinarum SEROVAR C-3 INFECTION IN Gallus gallus isolate SA-3 (C-3) cultivated and an expected amplicon size of 500 bp was obtained (Figure 3.3). M 1 2 3 4 C3 + C2+ NC 600 bp 500 bp 400 bp Figure 3.3: HPG2-PCR for the SA-3 (C-3) reference isolate cultivated for experimental procedure, whereby amplification was observed for all samples, with an expected band size of 500 bp. Lane M- molecular marker O'GeneRuler DNA Ladder; lane 1: SA-3 strain in 1X PBS used for first injection; lane 2: SA-3 strain in 1X PBS used for second injection; lane 3: SA-3 strain in supplemented TSB for first injection; SA-3 strain in supplemented TSB for second injection; lane C3+: SA-3 strain serovar C-3 positive control; lane C2+: Modesto strain serovar C- 2 positive control; lane NC: negative control. 3.3.1.2. Sequencing of 16S rDNA PCR products The identity of the Av. paragallinarum reference isolate was confirmed using 16S rDNA amplification based on the 8F and 1525R region, which is a highly conserved evolutionary region amongst bacterial species used to study bacterial phylogeny and taxonomy. PCR products of an amplicon size of 1500 bp were obtained for all samples (Figure 3.4). Following amplification of samples with 16S rDNA PCR, Sanger sequencing was conducted. The sequences obtained, were analysed using Geneious® 9.8.1 (Biomatters Ltd.) (Kearse et 154 CHAPTER 3: A PILOT STUDY OF THE IMMUNE RESPONSE TO Avibacterium paragallinarum SEROVAR C-3 INFECTION IN Gallus gallus al. 2012). Both the forward and reverse sequences of each sample were aligned, and the consensus sequence was compared with known sequences in GenBank, using a nucleotide BLAST (Basic Local Alignment Search Tool) analysis program (Altschul et al. 1990). A sequence identity of >97%, indicated DNA-DNA relatedness/homology and as such we were able to identify the bacterial strain to a species level, which matched the identity of our bacterial strain of interest. The sequencing results for the samples were recorded in Table 3.2 Using the results from both the species-specific PCR and 16S rDNA sequencing results from the National Centre for Biotechnology Information (NCBI) website (https://www.ncbi.nlm.nih.gov/), we were successful in identifying the bacterial strain used for the study, which coincides with the Av. paragallinarum SA-3 strain, which was a crucial step for the entire experimental study. M 1 2 3 4 C3+ C2+ NC 1500 bp Figure 3.4: 16S rDNA PCR for the SA-3 (C-3) reference isolate cultivated for experimental procedure, whereby amplification was observed for all samples, with an expected band size of 1500 bp. Lane M- molecular marker O'GeneRuler DNA Ladder; lane 1: SA-3 strain in 1X PBS used for first injection; lane 2: SA-3 strain in 1X PBS used for second injection; lane 3: SA-3 strain in supplemented TSB for first injection; SA-3 strain in supplemented TSB for second injection; lane C3+: SA-3 strain serovar C-3 positive control; lane C2+: Modesto strain serovar C-2 positive control; lane NC: negative control. 155 CHAPTER 3: A PILOT STUDY OF THE IMMUNE RESPONSE TO Avibacterium paragallinarum SEROVAR C-3 INFECTION IN Gallus gallus Table 3.2: Nucleotide BLAST results for all 16S rDNA PCR products with species identification, GenBank® accession numbers, query length, query coverage, E-value and high sequence identities. Sample Isolate/Species Accession Query Query E-value Identity number length (bp) coverage 3IC Avibacterium paragallinarum KC951277.1 653 100% 0.0 100% strain SA-3 16S ribosomal RNA gene, partial sequence 5IC Avibacterium paragallinarum KC951277.1 529 100% 0.0 100% strain SA-3 16S ribosomal RNA gene, partial sequence 1SD Avibacterium paragallinarum KC951277.1 697 100% 0.0 100% strain SA-3 16S ribosomal RNA gene, partial sequence 2SD Avibacterium paragallinarum KC951277.1 664 100% 0.0 100% strain SA-3 16S ribosomal RNA gene, partial sequence C3+ Avibacterium paragallinarum KC951277.1 644 100% 0.0 100% strain SA-3 16S ribosomal RNA gene, partial sequence C2+ Haemophilus paragallinarum AY498870.1 546 100% 0.0 100% strain Modesto 16S ribosomal RNA gene, partial sequence 3.3.2. Challenge methods and clinical scoring 156 CHAPTER 3: A PILOT STUDY OF THE IMMUNE RESPONSE TO Avibacterium paragallinarum SEROVAR C-3 INFECTION IN Gallus gallus Once all chickens were injected accordingly, they were closely monitored for a period of 21 days for any visible IC related symptoms, the daily mean score was recorded during disease progression for both the experimental and control groups (Figure 3.5). After 24 h, very slight swelling at the site of injection was observed, which is a typical inflammatory response due to the protective barrier of the skin being damaged by a foreign object (needle) or introduction of the pathogen. However, following 24-48 h, we still could not observe any IC related symptoms and the slight swelling subsided around the site of infra-orbital injection near the sinus cavity. Only on 72 h, did we see the mean disease score change to 0.1 (Figure 3.5), which indicated that 3 in 30 chickens showed symptoms which was still a very low number. Furthermore, on 72 h the chickens were on a score 1 with facial swelling and slight nasal discharge on the left side of the nasal area, diarrhoea and lethargy (Figure 3.6). On Day 4-7, the mean disease score increased until a mean disease score of 0.5 was reached on Day 7, which could be because of the innate immune response being at its peak, with supplementary symptoms in addition to the symptoms mentioned on Day 3 such as sneezing as well as swollen wattles and combs (Figure 3.6). In addition, on Day 7 only 1 chicken reached a score 2, with nasal discharge on both sides of the nasal region. However, after Day 7, there was a gradual drop in the mean disease score, indicating that the chickens were recovering. Unfortunately, for a duration of 1 week a mean disease score of 1 could not be reached in the experimental group, the chickens started to recover, and the disease did not progress any further. Hence, we had to reinject the experimental chickens on Day 9, to “boost” the disease progression and to see whether this strategy had any effect. The scoring for the control group during the first week (Day 0-7), was stable at a constant mean disease score 0, which indicated that no clinical signs and symptoms of IC were observed nor did any cross-contamination occur between the two cohorts. For the second injection, after 24 h (Day 10), most chickens in the experimental group gave facial/inter-mandibular swelling, but no nasal discharge and only experimental chickens 157 CHAPTER 3: A PILOT STUDY OF THE IMMUNE RESPONSE TO Avibacterium paragallinarum SEROVAR C-3 INFECTION IN Gallus gallus labelled E10 and E17 (E10 and E17: E stands for experimental chicken and the number 10 and 17 indicate the cage number) showed prominent nasal discharge. E10 was at a score 1 and had very slight nasal discharge on the left side only with mild facial swelling (Figure 3.7). E17 was on a score 2 and had nasal discharge on both sides with mild facial swelling (Figure 3.7). The disease did gradually progress after the second injection and on 72 h (Day 12), the mean disease score was 0.9 (Figure 3.5), showing that the second injection did stimulate the adaptive phase of the immune response. However, after 72 h the chickens started to recover from the infection and inter-mandibular swelling was diminished. The scoring for the control group during the duration of the study after the second injection (Day 9-21), was once again stable at a mean disease score 0, no clinical signs and symptoms of IC were observed, and this was a good indication that no cross-contamination between the two cohorts occurred. The disease did not progress for most of the experimental group and a score 3 was not reached once again with a second injection, which was quite puzzling given that the SA-3 strain (serovar C-3) is a virulent, indigenous strain to South Africa. Figure 3.5: The disease profile showing the daily mean disease score of the control (red) and experimental (green) cohorts. The trend of the control group was consistent at a score 0 throughout the duration of 21 days, as they were not challenged or exposed to C-3 isolate. The experimental group had a different trend whereby an initial innate immune response was observed on Day 7 with the first injection and an adaptive immune response due to a secondary exposure occurred on Day 12. In between Day 7 and Day 12, there is a gradual drop in the daily mean disease score, which was mainly due to recovering chickens given that IC does not cause mortality. 158 CHAPTER 3: A PILOT STUDY OF THE IMMUNE RESPONSE TO Avibacterium paragallinarum SEROVAR C-3 INFECTION IN Gallus gallus According to literature, IC symptoms usually develop in chickens between 24-72 h after exposure to the bacteria and symptoms last over a period of days (Blackall and Soriano, 2008). However, the lack of clinical signs and symptoms indicated either there was a good initial immune response or there was an issue with the bacterial inoculum with the first and second injections respectively. Our results were contrary to the study conducted by Bragg et al. (2004), whereby in a chicken population of untreated and unvaccinated birds challenged with C-3 isolate yielded a rapid disease progression whereby on Day 5 based on a slightly different scoring reached the status of “moderately affected” similar to a score 2 in this study. As the disease progressed by Day 9 the chickens had “severely affected” clinical signs and symptoms equivalent to score 3. This indicated that there were issues in the current study and that immediate troubleshooting needed to be performed to resolve the problem. A B C D Figure 3.6: Clinical signs and symptoms related to IC after the first injection. (A) Male presented with swollen wattles and combs. (B) Diarrhoea frequently occurring in sick birds. (C) Nasal discharge observed on left side of facial region and slight facial swelling can be seen. (D) IC leads to lethargy in birds. 159 CHAPTER 3: A PILOT STUDY OF THE IMMUNE RESPONSE TO Avibacterium paragallinarum SEROVAR C-3 INFECTION IN Gallus gallus We suspected that various reasons could have led to the experiment not being successful. Firstly, the culturing conditions needed improvement. Although, the SA-3 strain bacterial culture was grown to an OD600 of 1.0, we did not establish whether the culture had enough viable bacterial cells to cause infection. In other words we did not ensure that in the 100 l that was injected into the experimental chickens, there were 108 colony forming units (CFU) (Byarugaba et al. 2007), implying that there might not have been enough viable bacterial cells to breech the infection threshold that would cause and prolong disease progression. Moreover, after we re-suspended the bacterial pellet in 1X PBS (pH 7.4, Merck), we kept the suspension at 4C, which could have shocked the bacterial cells and caused the bacterial cells to either die or become dormant. Av. paragallinarum, being a poultry pathogen, thrives best at a temperature similar or close to the body temperature of a chicken, which is 41.8C (Bolzani, 1979). Secondly, the sinuses of the chickens at 20 weeks of age, were not yet well developed, being the site of infection, which is why we could not observe immediate signs and symptoms of IC and the disease could not spread or progress. Lastly, it could be that the chickens were not SPF and had been exposed to Av paragallinarum or other pathogens prior to using them in the experiment, although they were obtained from a reliable source. Thus, implying previous exposure to other organisms indicates that the immune system was already established to pathogenic infection and any other exposure would cause a fast-immune response to occur, which is why the birds recovered faster, as opposed to if they were SPF whereby an infection would take longer to progress and heal. If the chickens, were indeed not SPF or were previously exposed to other micro-organisms, chicken plasma before infection would contain antibodies. As such, we decided to conduct a direct ELISA screening as described (Section 3.2.8) and the results were recorded (Section 3.3.5). 160 CHAPTER 3: A PILOT STUDY OF THE IMMUNE RESPONSE TO Avibacterium paragallinarum SEROVAR C-3 INFECTION IN Gallus gallus A B C D Figure 3.7: Clinical signs and symptoms related to IC after the second injection. (A) and (B) Birds with inter- mandibular swelling, with no nasal discharge. (C) E10 with a score 1 showing very slight nasal discharge on the left side only with mild facial swelling and swollen wattles and comb. (D) E17 with a score 2 presented with nasal discharge on both sides of the nasal cavity with mild facial swelling. Figure 3.8: Egg production indicated as the total number of eggs laid/chicken/day in the experimental and control groups. Both cohorts showed highly variable egg-laying trends, making it difficult to deduce any significant weekly or daily egg-laying patterns. 161 CHAPTER 3: A PILOT STUDY OF THE IMMUNE RESPONSE TO Avibacterium paragallinarum SEROVAR C-3 INFECTION IN Gallus gallus The total number of eggs laid/ chicken/ day was recorded for both experimental and control groups. Both experimental and control groups had highly variable egg-laying trends (Figure 3.8). As such, we could not observe any stable or consistent trend with regards to the control group or experimental group infected with IC. Moreover, we did not observe any significant decline in egg production. 3.3.3. Avian full blood counts, differential blood counts and microscopy We had a major setback during blood collection with chicken blood, it coagulated very rapidly once we collected the blood, which was ideal for serum, but not whole blood collection. During collection of blood samples of 2-3 ml collected via the branchial vein into commercial 4 ml EDTA coated SGVac PET Blood Collection Tubes (The Scientific Group). Supplemented was 800 l- 1 ml of additional EDTA (0.5 M, Merck) into the EDTA coated SGVac PET Blood Collection Tubes followed by quick inversions of the tubes 3-4 times. This was conducted to prevent quick coagulation of the blood collected, as chicken blood coagulates quickly due to high levels of calcium present in chicken blood. EDTA chelates free calcium ions present in the blood, hindering coagulation. Previously, we had also tried acid citrate dextrose (ACD), heparin and Alsever's solution as anticoagulants for supplementing into the EDTA tubes however, none of these solutions worked as effectively as EDTA. The high levels of calcium were due to the rich layer feed provided for egg-laying chickens intended for egg development. Calcium plays a role in the blood coagulation cascade, however high levels of calcium could lead to conformational changes in proteins such as factor V and factor VIII, which has an increased effect on pro-coagulation, leading to clotting occurring at a faster rate (Michaelsson, 1991). Chickens could not be bled daily, and blood was drawn every 2-4 days. This was due to hematoma development, which is a collection of clotted blood outside of a blood vessel as a result of a collapsed vein at the sight of bleeding. This takes from a few days up to a week 162 CHAPTER 3: A PILOT STUDY OF THE IMMUNE RESPONSE TO Avibacterium paragallinarum SEROVAR C-3 INFECTION IN Gallus gallus to heal. Thus, bleeding was done on one side of the wing and the other wing was left available for bleeding upon healing. If both wings were bled from no blood was drawn until one of the wings had healed. We had obtained the CBC and WBC Diff results from NHLS (Universitas, Bloemfontein, South Africa) and Pathcare Vetlab (Westdene, Bloemfontein, South Africa) respectively (Annexure B). However, we learned that the analysers from both NHLS and Vetlab were not ideal for avian blood but only human blood. The reason was that avian blood have nucleated RBCs and human RBCs do not. Thus, when human whole blood is used, the analysers can distinguish RBCs from WBCs as well as other immune cells. Regrettably, with the avian whole blood that was provided, the analysers mistook the nucleated RBCs with WBCs leading to erroneous CBC and WBC Diff counts. Hence, on the result statements there was an escalated number of WBCs in comparison to RBCs, which was not the case. As such, the results were inaccurate. Pathcare Vetlab (Westdene, Bloemfontein, South Africa), sent blood smears to be evaluated manually by a veterinary pathologist, since the analyser counts were incorrect. However, this could still not provide any insight as to what the true counts were. Moreover, after querying the results for clarity, the veterinary pathologist at Vetlab indicated that they do not offer avian CBC and WBC Diff, even though these options were available on the request form (Appendix B) with prior enquiry and consultation about whether these haematology tests could be performed before commencing the project. Furthermore, blood smears prepared at Vetlab (Westdene, Bloemfontein, South Africa) for our study, were discarded without any notification and we could not obtain results. The NHLS (Universitas, Bloemfontein, South Africa) told us that they could only use human samples and they also could not help us further, however we did obtain blood smears (Figure 3.9) which showed us the cell morphology in whole blood, which could provide insight during IC infection. 163 CHAPTER 3: A PILOT STUDY OF THE IMMUNE RESPONSE TO Avibacterium paragallinarum SEROVAR C-3 INFECTION IN Gallus gallus After a week, we had to cease and abandon the CBC and WBC Diff from the NHLS and Vetlab, for the pilot study due to high cost with inaccurate results being obtained. Figure 3.9: Avian blood smear from a Day 0 chicken. Peripheral blood film showing nucleated RBCs and some leukocytes (100X Magnification). According to literature, the correct way for counting erythrocytes of chickens is by using the erythrocyte Unopette 5850 system (Becton Dickinson) with Neubauer-ruled haemocytometer or using Natt and Herrick’s method. To count leukocytes of chickens the eosinophil Unopette brand 5877 system (Becton Dickinson) with Neubauer-ruled haemocytometer or the direct leukocyte count using Natt and Herrick’s method can be performed (Natt and Herrick, 1952; Campbell, 1995). These manual techniques are the best alternatives to the automated analysers. However, the disadvantage is that the process is laborious, time-consuming and has a high cost, especially with a large sample size. For the study, the RBC count was not important for us, however the WBC count was. Hence, the combination of blood smears with flow cytometry results were looked at (Chapter 4), as an alternative to obtain different cells counts (excluding RBCs), as an indication of disease progression. 164 CHAPTER 3: A PILOT STUDY OF THE IMMUNE RESPONSE TO Avibacterium paragallinarum SEROVAR C-3 INFECTION IN Gallus gallus 3.3.4. Flow cytometry and antibodies Cell population profiles of the T-cell population mainly the CD4 T cells (T helper cells) and CD8 T cells (cytotoxic T cell) using flow cytometry were studied and generated. Flow cytometry profiles were generated when fluorescently labelled cells passing through the interrogation point interact with a laser, whereby light scattering is produced that could be measured and correlated with relative cell size and structures inside the cell. The measurements were termed forward angle scatter (FSC) which is based on the size of the cell and side angle scatter (SSC) which is based on the granularity/complexity of the cell. During the runs on the BD FACSCanto II (Becton Dickinson) while simultaneously performing the analysis on the BD FACSDiva 8.0.1 software (Becton Dickinson), it was difficult to locate and gate the precise location of the entire leukocyte population and lymphocyte population. This was due to the nucleated RBCs that overlapped with the leukocyte and lymphocyte population, thus making it difficult to gate the leukocyte population, which was also where the lymphocyte population was, containing both CD4 and CD8 cells (Figure 3.10). Hence, the cell counts of the cell populations of interest could not obtain, as gating could not be performed. To resolve this problem, the RBCs were separated from the lymphocytes either by lysing the RBCs or using the Ficoll-Paque PLUS (GE Healthcare) according to the manufacturer’s instructions (Figure 3.10), however this was to no avail the same plots as in Figure 3.10 were obtained. Ficoll-Paque PLUS (GE Healthcare) separates whole blood into different components present in blood and plasma where platelets are also found and the lymphocyte/ peripheral blood mononuclear cell (PBMC) layer, Ficoll-Paque PLUS layer, granulocyte layer and erythrocyte layer (Figure 3.11). Finally, an anti-CD45 pan-leukocyte marker was used upon repeat of experiments (Chapter 4), which would be the best solution to tackle the 165 CHAPTER 3: A PILOT STUDY OF THE IMMUNE RESPONSE TO Avibacterium paragallinarum SEROVAR C-3 INFECTION IN Gallus gallus avian nucleated RBC dilemma, CD45 would only be present on leukocytes (granulocytes and lymphocytes) but not erythrocytes (RBCs). Moreover, avian nucleated RBCs are much smaller in size in comparison to leukocytes, hence when the anti-CD45 pan-leukocyte marker would be used they would be easily separated and distinguished from the leukocytes, making it easy for gating purposes. Figure 3.10: Flow cytometry profile of one of the control chickens showing the forward scatter (FSC-A) and side scatter (SSC-A) plot. The sequestered region outlined in green shows the RBC population, this area overlaps with the gated region P1 (shown in red) which is the approximate location of the lymphocyte population. It was very difficult to gate and perform further analysis, since we could not differentiate between the different cells and gate the precise location of the lymphocytes where the overlapping occurred. 166 CHAPTER 3: A PILOT STUDY OF THE IMMUNE RESPONSE TO Avibacterium paragallinarum SEROVAR C-3 INFECTION IN Gallus gallus Plasma/platelets Lymphocytes/PBMCs Ficoll-Paque PLUS Granulocytes/heterophils Erythrocytes Figure 3.11: Whole blood separated by Ficoll-Paque PLUS (GE Healthcare) into different components after using a swing-bucket centrifuge at 400 x g. Despite using this technique, we still could not gate the lymphocyte population and there was still overlapping occurring. 3.3.5. Direct enzyme-linked immunosorbent assay (ELISA) The ELISA assay was carried out (p<0.05) and the results are shown in (Figure 3.12 and Figure 3.13). The results showed that although the chickens were supposedly SPF, they had a well-established immune system, since they were able to produce antibodies even before exposure to SA-3. Day 0 chickens were not yet infected, yet we could still see high absorbance values in chickens 1, 2, 3 and 5. Although, the antibody production was different for each chicken that was randomly bled, we could see that post-infection (PI) with the first injection the antibody titres against the anti-chicken IgY (IgG) (whole molecule) peroxidase antibody produced in rabbit (Sigma-Aldrich), did increase for some of the chickens PI such as E12 and E19 on Day 3 and E28 on Day 7. 167 CHAPTER 3: A PILOT STUDY OF THE IMMUNE RESPONSE TO Avibacterium paragallinarum SEROVAR C-3 INFECTION IN Gallus gallus Chicken plasma testing positive Negative controls Figure 3.12: Screening for antibodies using chicken plasma from both experimental and control chickens. A coloured yellow product indicates presence of antibodies in the chicken plasma sample used, whereas the clear wells are the negative controls showing that no antibodies are present. It was surprising that despite there being no cross-contamination or IC related symptoms in the control group we still obtained antibody production on Day 7 for the chickens control 1 and 2, and a very high antibody titre for bird control 3 on Day 14. One of the chickens (E7) that was re-infected (RI) with the second injection, also showed a high antibody titre on Day 14. From the results, it was apparent that the chickens might have been previously exposed to microorganisms. The only improvements in our ELISA assay would be to include plasma positive and negative controls to validate results, and to perform the experiment for each sample for each score obtained in duplicate, triplicate or quadruplicate. 168 CHAPTER 3: A PILOT STUDY OF THE IMMUNE RESPONSE TO Avibacterium paragallinarum SEROVAR C-3 INFECTION IN Gallus gallus Day 0 Day 3 Day 7 Day 7 Control Day 14 (RI) (PI) (PI) Figure 3.13: Graphical representation and statistical analysis of ELISA assay conducted on chicken plasma samples from the experiment (p<0.05). Day 0 (beige), chickens were not infected, yet antibody titres were still observed. On Day 3 (light green), high antibody titres were observed for chickens E12 and E19, as symptoms for these chickens aggravated with SA-3. On Day 7 (blue), chicken E28 at a score 1 showed high antibody titres. Antibody production could be seen in Day 7 (light blue) and 14 (yellow) control chickens as well. Overall, we saw an increase in antibody production in experimental chickens as they were infected with SA-3. On Day 14 (yellow), with the re-infected chicken E7 we saw a high antibody titre due to a second injection with SA-3. 3.4. Conclusion The pilot study was not as successful as planned. This was because disease progression with regards to IC infection was not observed in chickens infected with SA-3 (serovar C-3). As such, no IC related signs and symptoms of injected chickens were seen. On the contrary, the injection of serovar C-3 in the chickens, yielded a fast and effective (good) immune response leading to quick recovery, which we suspect was due to prior immunity established in the chickens before the study, from previous exposure, whereby memory cells had been formed. Therefore, the clinical signs and symptoms that was anticipated and expected of an IC 169 CHAPTER 3: A PILOT STUDY OF THE IMMUNE RESPONSE TO Avibacterium paragallinarum SEROVAR C-3 INFECTION IN Gallus gallus infection was not fully observed during the course of the disease progression, as the chickens recovered swiftly following infection with serovar C-3, which is the reason why a score 3 was not attained. These hypotheses, were proven, based on existing antibodies present in chicken blood of Day 0 chickens (which were supposedly SPF), of Day 7 chickens control 1 and 2, as well as a very high antibody titre for the chicken control 3 on Day 14 (Section 3.3.5). It should be emphasized that the control chickens were not infected or exposed to serovar C-3. However, despite the negative outcome of the study, we were still able to find valuable insight that could further improve the outcome of the project for future studies (Chapter 4 and 5). We tried to troubleshoot all the shortcomings of the methodology and have made a few core findings. Firstly, there was a problem with the culturing of the bacterial cells, whereby we must ensure that the final bacterial culture has 108 CFU to ensure that there are enough bacteria to cross the disease threshold to cause disease. Moreover, the temperature should be kept at 37C before injecting the chickens in the next study, to simulate the environment of the chicken host to ensure that Av. paragallinarum SA-3 remains viable. Moreover, we were not certain if the SA-3 serovar C-3 strain might have lost its virulence during multiple passaging in our laboratory, hence we should cultivate a fresh bacterial culture from a new vial of the freeze- dried bacterium that had not been passaged. Furthermore, in younger birds, the sinuses are not well developed, which could have contributed to the poor immune response obtained in the study, hence in the next experiment (Chapter 4 and 5) older SPF birds would be used, as their sinuses would be more developed. Lastly, from our ELISA results it was apparent that the alleged SPF chickens were previously exposed to microorganisms or even vaccinated, as their immune system was already well established observed from the antibody titres on Day 0 and for control chickens on Day 7 and 14 respectively, leading to no IC related symptoms. However, due to the lack of positive and negative control serums, it is difficult to have any confidence in the serology and the interpretation of the detected absorbances, hence the ELISA’s should be repeated in future studies with known positive and negative control serums. 170 CHAPTER 3: A PILOT STUDY OF THE IMMUNE RESPONSE TO Avibacterium paragallinarum SEROVAR C-3 INFECTION IN Gallus gallus From these core findings, we hoped that the next trial would be successful. The main reason for conducting a pilot study is chiefly to determine initial data for the primary outcome measure, as well as to perform a sample size calculation and to further improve the techniques for the next round of experimentation. 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Journal of General Virology, 89: 1288-1299. 178 ANNEXURE B ANNEXURE B 179 - NHL$'Universitas ADVIA 212OiB : stD HU00398543 Patient Name CHICKEN t , ttO runVt Aspiration Date/Time 03-May-1 7 12 21:42 P Sample Type PATIENT Rack&Position 00 lnstiument Number lR35'1 114'12 Sample Selectivity CBC\DIFF ".. 1\se__$- s._9-x. 1.-utsx a,&moRAtoRV u$ri"or,![Y i Ptatelei Vot t'6 6CTwroIc=;H:5'10.72 I RBg" L',, 2 io i .. I /T/t I HGB | 11,6 ;ji I IlCe llular HG /l ,oBi : !,,7 6 o )s 1., ." rrr9Y: H:114:7 ,%Micro ,3,7 . . i MCH: Hi 53. 0 RBC fpgmgnts , j o oo xr oi, ietiiilI MCHC: /j1 t .l-1".. 49 2 RBC Ghosts 0.01 xi 0r2 cells /L /,,/lI CHCM Li 30. 5 o Neut X i J"t! cH: i 32,I oq NeutYi I ! i 'lI RDW, H, ?7 i /o MNx i 8.5 Ii gow, a ldl I iI ,-. *-,; l, B, ,i *- - PLT' L, 5 xl0s cells/L I %MN , "09.0! ".i -i t | -MPV" :9. i I fL [ -z;eur-ru, 's-s.,s: itq"t PDW L:'16. 5' -.-- L &!!gErHe9-l.o lI PCT .ir:s00 ToBasg WB,.C WBCQ WBCy %BASOu #BASOu CrrcMcelCrrdiiilrcf HoW nCB Mi-H McHC MCV RBc nowcn cnoW WBC-CE WBC WBCu PX I.IO *I-UC Y" TUC #LYMPH O/"LYMPH WBCP #NEUT %NEUT #MONC i%MONO #EOS %EOS %NEUTu #NEUTu %LYMPHu #LYMPHu #EOSu %LUCu #LUCu #EOS %EOS %NEUTu #NEUTu %LYMPHu #LYMPHu #MONOu %EOSu #EOSu o/oLUCu #LUCu #EOS %EOS %NEUTu #NEUTu o/oLYMPHu #LYMPHu NRCELL #BASO %Baso #LUC %LUC #LYMPH %LYMPH WBC #NEUT .%NEUT #MONO %MONO #EOS %EOS %NRBC #NRBC Baso : slD HU00398547 : Patient Name .CHICKEN 2 , NO NAM lAspiration Date/Time : 03-May-17 12.22.16 P , Sample Type PATIENT RBC HC : Rack & Position 0 0 lnstrument Number 1R351 1 1 412 Sample Selectivity isCtotrr Age & Sex U - . . . r"*$.!j$mqry{T_$sY RBCbH [ " "lJ$$ *Nr,Y.. . :l,j G" '*; RBCI l/ll{C i. ;:,. :, .":r WBC :H 257.41 ).ix10e cells/L 'Additional Routine Parameters RBC L 1.81 vl O12 rells /l : %Blast Suspect 0.0 HGB L 77 .: o /dL %Hyper 12.6 lellular HGB 5.8 o /dL HCT L 0191.L/L -fL I YoMaCroi285.t : - MCV H105.4 .ps ; ,ZqMlgro,1,3;1 ... :i Mv,CCH AHn 4n2z7:t .'p,qe/dl iRBC Fragments l : : 0.00 tx10r, cells/11i MvcCuHCc H, 40,,55 L 5 ,.,9/dL RBC Ghosts 026 i it0 , cellslt CHCM , 30 :- ,g ldL L1,__ _,___.:-_-::i.i_Nt,_._e,...-u tx:, -, :,, ;i __ .__ __-, _._,,--l i, CHH . 30.66 *',p oqo l i Neu1Y, i 4: ..-... .--R. DW:H 250 '*:%n : HDW H 8,06 ..q/dl ...l, MNx 3.7 iMNy 6O i If P!.f LT Lli , :2- 4 . I, *- :xflL0 , Lc,Eetllrs)//L %MN 0.0^!Y i1 UeV H: 35.2 i %PMN 99 6 _ Cellular HGB .:i " " 5r8- ^ g/d! l*--"*-r^9l-!,-..q,g-e- .:ft* "*- - MICRO ,+ MACRO +++ HVPO +++ HVPER - ,J++ RBCIFR :CHCM HCT HDW MCH MCHC MCV MPV PCT PDW PLT RBC jANISO i+++ RDW CH CHDW i : iATYPS + PX-NV :#LUC %LUC #LYMPH %LYMPH WBCP #NEUT %NEUT #MONO ||ARGE ?LT +++ %MONO #EOS %EOS %NEUTu #NEUTu %LYMPHu #LYMPHu i IPLT CLUMPS.+ :%MONOu #MONOu %EOSu #EOSu o/oLUCu #LUCu i PLT.CL MPV PCT PDW PLT WBCP 2.56 iBP.9'9lg tr. ,".-,- -i+.2 t) PLJ No .:MPi4"?-9r.P-DW ?!r_ ; lisd NRaELL ,*anSo z"aaso #LUC %[uc #LVMPH %LYMPH wBc #NEUr 7a : -l,Xl.Ql--cSlLslL NR-LPD #BASO %Baso #LUC %LUC #LYMPH o/oLYMPH WBC #NEUT j .,.-.-:y':,!!-E-u] jUQNg.f !!-ol!9}.E9F-"7:*F"o*91/tIBq-c-f liuq--.-. ' NHLS Univeisitas Abvin 2t2oi a RBC Volume I srD HU00398549 --T.-r--: Patient Name ,CHICKEN 3 t:l, NO NAM Aspiration Date/Time 03-May-1 7 12.22.52 I Bl,_ ,P -.I-.Al-l-- Sample Type PATIENT RBC Hc Rack&Position 00 --*:I t lnstrument Number 1R351 1 1412 T.s 1" ;El" :- Sample Selectivity CBC\DIFF 9 { d: i_U, -- F tg"-f:.Agq Q ; -s,ex I Cq LAtsT RA ] IJR\' ,J$E CIfu1 1 . nec eu I ,*i - r f€lr ili&Sr{Lr*l*@ -.-,-,1*i:u... -*",,,i BC WBC .19 xl0s cells/L Routine Parameter$ liFiffliei,':,Vpj g RBC L 1.73 xl0r2 cells/L %Blast SusDect . HGB L 7.9 ridL %Hyper : ,16.4 . lellular HGB 5.9 ridL ToHypo . 19,! r;i*;.-- " --..i HCT L 0.1 89 ltt %Macro : 29.6 MCV 'I 09.1 Y"tvtlCro 2 iffi$:.iiFiq l=r$C MCH 45.4 no RBC Fragments q,90 r1 Otz rplls / I Ghosts : ::r . :ll !: :::.!MCHC 41.6 RBC 0.24 x10r2 cells/[ CHCM 31.4 Neut X lfHi9l f.,:;ii l ;ir,l i*lrir,lCH 32.8 oo trteut YRDW l- 22.6 )/ MNx 40HDW 8.48 olrll MNy , 6Q i PLT 1 xl Oe rells/l r %MN ' 0.0 iMPV 19.2 Y"PMN rsg.a L+;:6fir;El*,*-., ; -^r^ Il , i PDW 34.7 % t* 60 r /dL L PCT % - ^-- _t .---"-"-j Routine WBC Diferential j ltle*Foltuv- rBul :,i im Flaos %# iMACRO '+++ l B.NO #BASO %Baso WBC WBCB WBCu %BASOu #BASOu I wBC' ' -'..H 222.rs ;,*f[;;;,11;;r; iuvPo +++ l B-NV _t ; Neut :1, 0 0 :- L 0 09 -:x]0, cells/L] jHvern ,+++; ::CHCM HCT HDW HGB MCH MCHC MCV RBC RDW CH CHDW Lymph H 84.3 * H 187.34 - xl 0o cells/.l lRtttso +++ l RBCIFR JHL;M HU I HUW MUH MUHU MUV MPV PU I I'UW I-L I HBU! I Mono Hl 9.5 :-rH 21.18 .lxt O, cells/L :HC VAR +++ i RDW CH CHDW! I -ros | 0 0 ,:: 0 p0.-.11 0, cells/L /VBC-CE IWBC WBCu ] Baso PLT rX-NOI t.00 l, Io1 cells/Li 1LARGE ,+ +LUC %LUC #LYMPH %LYMPH WBOP #NEU I %NtU I #MONCl,XtQ? LUC H 6.'1 'H 13.54*xt0ecellsrL ill]9-lu\4ts t+ + l ,t ToMONO #EOS %EOS %NEUTu #NEUTu %LYMPHu #LYMPHu NRQ9 I I _:. * ., cellsl - /oMONOu #MONOu %EOSu #EOSu %LUCu #LUCuI L1,0-1 L ; |BP-Q -Ql:-g:t:" -:: "r trXIFR : t-t I 'i 246- +LUC %LUC #LYMPH %LYMPH WBCP #NEUT %NEUT #MONC] MPXI L .47,1 ToMONO #EOS %EOS %NEUTu #NEUTu %LYMPHu #LYMPHu j ToMONOu #MONOu %EOSu #EOSu %LUCu #LUCui"t1vep,r .-. _. .... _.*,. J.l_?s. 1.. x"l.-0", ee.[ u r rX-NV +LUU %LUU #LYMI-H %LYMPH WUUP #NEU I voNtrU I *MUNL /oMONO #EOS %EOS %NEUTu #NEUTu %LYMPHu #LYMPHu %lt4ONOr r #l\ilONOr r o/^trOSr r ttrOSr r o/^l I lCrr #l I lCr r WBCP l-- \RCELL +tsASO %tsaso #LUC %LUC #LYMPH %LYMPH WBU #NtU I % NEUT #MONO %MONO #EOS %EOS %NRBC #NRBC \R-LPD rBASU %tsASO #LUU %LUC #LYMPH %LYMPH WUU #NEU I ,/ONEUT #MONO %MONO #EOS %EOS %NRBC #NRBC , _ - _ _ - _s_!"q- i tl9"*q_0""9_e9"_5"9 1 Patient Name TCHICKEN 4, NO NAM Aspiration Date/Time 03-May-17 12.23.24 P Sample Type PATIENT Rack&Position 00 lnstrument Number 1R35111412 Sampte Selectivity CaCtotrF nge & Sex U ,, rQs r&qq$$l*ry\: Li$sl,ryL,], .": . #,4, 1 ;.,..,"ffii),tt**.-i {ffi FH HffiffifiHfl MfiHfl.f lEEHflffitrflI} :fiflt tV ifittHil iiiiLriiiirIiiti] illl*WHffiiiliiI WBC H'242.23 xt0q cells/L Additional Routine irs . lril t 'itiRBC,L 184 .xl0t2cells/.L, %Blast Suspect il / I r' HGB L 8.1 . q/dl %Hvoer i / / /:l Cellular HGB 6.7 q r dL i /DO 4 *i-". :l***- l '' , / !11 HCT L 0.213-LlL tcro .t J! /E MCV H 115.9 .,fL --j : lro-pq : ll l/ /iH 441 "aomenls ,y16r) l) ll)/i J) i J J ;MCH -q/dl 00 1s11571 | H 380 lJ J / / .!,MCHC ) Ghosts 0.01 ixl0r2 cells/L ,)(/1,/,/t CHCI\,I L 31 5 tqrdf Neut X CH 34.8 ^ p9 Neut Y RDW H 2'l .7 r o/o MNx 67 MNY 6.5 PLT L 3 .xl0ocellsL %MN 0.0 MPV H 13,3 'fL %PMN E9.7 t_ PDW j Cellular HGB 6.7 olrllL-: !: J3:9, :,ri PCT L: 000 -% : fiffiffi # WBC 242.23 I 0e cells/L Neut 0.1 0.12 rl 0e cells/L Lvmoh :F OE ' 206 61 (l 0e ce Mono 'F 9,5 23.13 e cells/l ]PX-NO ]#LUC %LUC #LYMPH %LYMPH WBCP #NEUT %NEUT #MON( Eos 00 0.00 s cells i lxrvoNo / #Eos %Eos %NEUTu #NEUTu %LyMpHu #LyMpHuI I %o/^Ml\OIONOrr#l\Baso 0.0 009 xl0s cplls/l NOu #M ilOONNOOrru o%/^EtrOOSSrur ##EtrOoSrur o%/^Ll UllCrur #{lL UllCuil LUC 5.1 12.27 'l Os re . /l NRBC Os cpllq /l #EOS %EOS %NEUTu #NEUTu %LYMPHu #LYMPHu1 LI 2.47 MPXI -30.5 WBCP 279.59 xl0e cells/L #MONO %MONO #EOS %EOS O/ONRBC #NRBC #MONO %MONO #EOS O/OEOS %NRBC hac c+{ t i,a:lni ).--" .iit#W .a ,-. .,-*, RBC V/HC . WBC H42O82 "xl0qcells/L Routine Psrsmelers ]lreffi B-BC l: _2 14 ,st Suspect 0.0 ., , li:., i , --xl0rrcellsrlrcB L: eg lgldL r'per 13 9 "*iz 1t;ia:., : _.. /n r'po :47.2 , ${r: ;;....r. tl HCT L 0.237.LlL %Macro :36.1 6:.:,,-, lJ& Mev uirog "fL /licro 'l2.9 )3 MCH H 458 .p9 nents 0.0c cells/11 /3.,x10r2 MCHC H 4'l .3 -o'dL /.;-t RBC GhoStS 0.01 .x10lr- cells/L] './, c!{cM_! 9Qa "s/dr .1 Neut X Neut Y Rqw.H 2q8 .% :10.7 ::t.rl MNx ii, HDW,H 8.34 L 1 '.Oxl0l'Ogcfe- MNv 6.0 PLT lls/L] 1 %MN 0.0 %PMN 99.5 Baso Rate Peioi Raie PDW H 69.0 *o/o olrll I ICellular HGB 7_2 i'li !:,, ,i.:.j. ..11,:i:lii:: iri i Routile W€C Differential , 'ok# i *uc. t, .n-+zoa2 ;,ifo;..liiIL: i Neut lL: 0.0 t-. L: 0 05].rxl0, - cells/L].lyrpn H 9'l B H 3S6.44 xl0scetls/L ] Mono , , 41 : H: 17 14 * xt0e cells/Li I fqp , . Q,0 ,-- , , 0.00 - xl0, cells/L] i aas,o I q.g ; o oz - , ir o' cettill i : LUC ,H, a 1 ,- H, 17,12,,* x10, cels/l1 llnaC - ] , ,lt* ,. xt0, ceils/Li ;MPX| t, -a.2,1 , rryedn i 338 28 - x I 0, cellsl L Drs Voigt & Vennote Medi-Clinic Hospitaal Derde Laan. Bloemfontein Tel : 05 I 401 4616 Prac.No. :5200539 FINALE VERSLAG Lab Verw :750527228 - Rekening No : F6019652 Dokter Pasient AFSKRIF VERSLAG BFN (OTHER) AVIAN CHICKEN BOUCHER AVIAN BLOEMFONTEIN LABORATORIUM DEPT OF MICROBIAL.BIOCHEMICAL P O BOX 339 93OO BLOEMFONTEIN 93()() BLOEMF-ONTEIN Tel (H) 0514013253 Iel (S) 078363453 I Tel (W) 0514012214 Monster 0503:HAO5267U ID Nommer NIE BESKII(BAAR NIE Versameldatum 2011-05-03 Nie verskaf nie Oud:Gslg:DvG 1zl4Y:M Ontvangsdatum 2017-05-04 20:10 LeeT NT NIE BESKIKBAAR NIE Verslagdatum 2011-05-13 1l26 Hooflitl DR C BOUCHER llediese Fonds VET ACC'S DIRECT TO OWNER Med.Fnds Nr NOT AVAILABLE Kliniese Data: URGENT I!I FAX RESULTS TO: CI"IARLOTTE / POOJAH E-MAIL RESULTS TO: jawallapersand(@gmail.com CONTROL(NOT CHALLENGED) CFIICKEN BLOOD. TOTAL OF 5 SAMPLES IN TOTAL - HEALTHY CHICKENS BREED: LEGHORN CHICKENS. AGE: 2lu'eeks 'f oetse Arngevra: PLAATJIETELLING, HAEM SIGN P,i, Toets Resultaat Wyser Verwysing BLOEDTELLING-GEBN PLAATJIES Rooisettelling l .81 I .8 - 2.3 xllEl2lL Hemoglobien 8.1 L 9.0 - 13.0 g/dl- Hematokrit 0.21 L 0.21 - 0.31 LIL Gkv I l8 fl Gkh 45 ckhk 38 g/dL Rdw 24.0 *H 10-15% Witseltelling 235.8 H 5.0 - 10.0 x10E9/L Let asseblief daarop dat as gevolg van rooibloedselle met kerne, is hierdie witseltelling nie korrek nie. Stuur asseblief'n bloedsmeer aan die Vetlab om te evalueer. Neutrofiele 5.7 % t3.44 xl0E9/L Limfbsiete 92.8 % 218.82 x I 0E9/L Monosiete 1.3 0 3.0'7 xl0E9/L Eosinofiele o.1 % 0.24 x10E9/L Vbt Kommentaar PLAATJIETELLING Plaatjietelling l5 x 10E9,4- Goedgekeur deur: DR LUCIA LANGE op 2017-05-05 13:03:00 Veearts Patoloog: 082 8088773 ^, H=Hoog, L=Laag, *H:Kritiek Hoog, +L:Kritiek Laag - Liasseer [ ] Bel Pasient [] Maak Afspraak [] Voorskrif [ ] Trek Leer [] Drs Voigt & Vennote rr4ecli-Clinic Hospitaal Derde Laan, Bloemfontein Ww* Tel : 051 .+01 4616 Prac.No. :5200539 FINALE VERSLAG - Lab Verw :750527227 - Rekening No : F6019643 Dokter Pasient AFSKRIF VERSLAG BFN (OTHER) AVIAN CHICKENS BOUCHER AVIAN BLOEMF'ONTEIN LAB ORA.TORIUM DEPT OF MICROBIAL.BIOL]HEMICAL P O ROX 339 93OO BLOEMFONTEIN. 93OO BLOEMFONTEIN Tel (H) 05r4013253 Tel (S) 078363453 I rel (w) 0514012211 Monster 0503:HA05266U ID Nommer NIE BESKIKBAAR NIE Yersameldatum 2011-05-03 Nie verskaf nie Oud:Gslg:DvG 144y:M Ontvangsdatum 2017-05-04 20:12 Leer Nr NIE BESKIKBAAR NIE Yerslagdatum 2017 J)5-13 11 .27 Hoollid DR C IIOUCHER Medicse Fonds VET ACC'S DIRECT TO OWNER ]Ied.Fnds Nr NOT AVAILABLE Kliniese Data: URGENT !I! FAX RESULTS TO:CHARLOTTE/POOJAH E-MAIL RESULTS TO:JAWALLAPERSAND(@GMA IL.COM BREED: LEGHORN CHICKENS AGE: 21 WEEKS CONTROL(NOT CHALLENGED) CHICKEN BLOOD .fOTAL OF 5 SAMPLES IN TOTAL, HEALTHY CHIC]KENS Toetse Aangevra: PLAATJIETELLING, HAEM SIGN QUEUt,'IRICGER I. BLOEDTELLING-GEEN PLAA TJIES Prirncre lcDl0 Ko(lcls):z;() q ---------HE\,rATggg51g--------------_ Toets Resultaat WYser VerwYsing BLOEDTELLING-GEEN PLAATJIES Rooiseltelling 2.16 1.8-23xl0F1l'L Hemoglobien 9.8 9.0- 13.0g/dl- Hematokrit 0.r3 L O.2t - 0.31 LiL Gkv r08 fl Gkh pg Gkhh 42 gr'dL 27.8 xH Rdrv l0 - 15 % Witseltelling 3 99.3 H 5.U - lU.0 x lUFei L Please note - due to nucleated red cells the white cell count is incorrect Please send blood smear to the Vetlab fbr DilT count Neutroflele 0.1 % 0.40 x I 089/L Limfbsiete 89.5 % 357.37 x l0E9iL Monosiete 10.4 %, 41.53 x 10E9/L Eosinofiele 0.0l x I 0E9/L Basofiele 0.01 x I 0E9/L Morfblogie Kommentaar On the blood smear there is a relative decrease in rvhite cells. Those present are mostly (granolocytes) heterophils ancl ll,mPhocYtes. Vbt Kommentaar PLAATJIETELLING Plaadietelling x 10E9iL Vir konsultasie: Dr Lucia Lange Veearts Patoloog: 082 8088773 .' H:Hoog, L:Laag, *H-Kritiek Hor"lg, *L=Kritiek Laag .-Liasseer [ ] Bel Pasient [ ] Maak Aftpraak [ ] Voorskrif I I Trek Leer [ ] Drs Voigt & Vennote Medi-Ctinic Hospitaal W Derde Laan, Bloerrfontein Tel:051 401 4616 Prac.No. :5200539 FINALE VERSLAG - Lab Verrv z 750527226 - Reliening No : F6020518 Dokter Pasient AFSKRIF YERSLAG BFN (OTHER) AVIAN CHICKENS BOUCHER AVIAN BLOEMFONTEIN LABORATORIUM DEPT OF MICROBIAL. UF-S [, o Box 339 93Ot) BLOEMFONTEIN 93()O BLOEMFONTEIN Tet (H) 05 140 I 3253 Tel (S) 078363453 I Tel (w) 05140t2274 Monster 0503:HA05268U ID Nommer NIE BESKIKBAARNIE Yersameldaturn 2017-05-03 Nie verskaf r.rie Oud:Gslg:DvG l44y:M Ontvangsdatum 2017-05-0420:13 Leer Nr NIE BESKIKBAAR NIE Verslagdatum 2017-05-13 1l:28 Hooflid DR C BOUCHER Mediese Fonds VET ACC'S DIRECT TO OWNER NOT T\VAILABLE URGENT !!! FAX RESULTS TO:CIIARLOTTE/POOJAH E-MAIL RESULTS TO:JAWALLAPERSAND@IGMAIL.COM BREED: LEGHORN CHICKENS AGE: 21 WEEKS CONTROL(NOT CHALLENGED) CHICKEN BLOOD TOTAL OF 5 SAMPLES IN TOTAL, HEALTHY CHICKENS Toetse Aangevra: PERIPHER,\L BLOOD FILM, PLAATJIEI'ELLINC, IIAEM SIGN QUEUE TzuCGER 1, BLOEDTELLING-GEEN PLAATJIES ntt'nt" oloc tg-----.-------_ Toets Resultaat Wyser Verwysing BLOEDTELLING-GEEN PLAATJIES Rooiseltelling 1.50 L 1.8-2.3x10E121L Hemoglobien 6.1 L 9.0 - 13.0 gidl- Hematokrit 0. l6 L 0.21 - 0.31 LtL Gkv 107 f1 Gkh 41 vs Ckhh 38 g/dL Rdw 21 .0 *H t0 - 15 % wirseltelling 1 66.1 H 5.0 - 10.0 x10E9/L Please note: Due to the nucleated red cells the $,hite csll count is incorrect. Please send a blood smear to the Vetlab for evaluatiotr Neutrofiele 6.5 y" 10.1J(-) x 10E9/L Limfbsiete 9o.t o 150.65 x I 0E9/L Monosiete 2.1% 3.99 x I 0E9/L Eosinofiele 0.3 %, 0.50 x10E9/L Basofieie 0.1 % 0.r7 x I 0E9/L Vbt Kommentaar PLAATJIETELLING Plaatjietelling 24 x 10E9/L Vir konsultasie: Dr Lucia Lange Vecarts Patoloog: 082 8088773 -' H=Hoog, L-Laag, *H-Kritiek Hoog. *L-Kritick Laag - Liassei:r [] Bel Pasient [] Maak Afspraak [] Voorskril'[ ] Trek Leer [] Drs Voigt & Vennote Medi-Clinic Hospitaal Derde Laan, Bloemfontein W Tel:051 401 4616 Prac.No. :5200539 FINALE VERSLAG - Lab Verw :750527265 - Rekening No : F6020530 Dokter Pasient AFSKRIF VERSLAG BFN (OTHER) AVIAN CHICKENS BOUCHER AVIAN BLOEMFONTEIN LABORATORIUM DEPT OF MICROBIAL,UFS P O BOX 339 93OO BLOEMFONTEIN 93OO BLOEMFONTEIN Tel (H) 0s 140 I 3253 Tel (S) 078363453 1 Tel (w) 05t40t2274 Monster 0503:HA05269U ID Nommer NIE BESKIKBAAR NIE Versameldatum 2011-05-03 Nie verskaf nie Oud:Gslg:DvG 144y:M Ontvangsdatum 2017-05-0420:14 Leer Nr NIE BESKIKBAAR NIE Verslagdatum 2011-05-13 ll:28 Hooflid DR C BOUCHER Mediese Fontls VET ACC'S DIRECT TO OWNER Med.Fnds Nr NOT AVAILABLE Kliniese Data: URGENT !!! FAX RESULTS TO:CHARLOTTEi?OOJAH E-MAIL RESULTS TO:JAWALLAPERSAND@GMAIL.COM BREED: LEGHORN CHICK-ENS AGE:21 WEEKS CONTROL(NOT CHALLENGED) CHICKEN BLOOD TOTAL OF 5 SAMPLES IN TOTAL, HEALTHY CHICKENS Toetse Aangevra: PEzuPHERAL BLOOD FILM, PLAATJIETELLING, HAEM SIGN QUEUE TRIGGER 1, BLOEDTELLING.GEEN PLAATJIES @ Toets Resultaat Wyser Verlvysing BLOEDTELLING-GEEN PLAATJIES Rooiseltelling 1.17 L 1.8-2.3x10E12/L -1.2 Hemoglobien L 9.0 - 13.0 grdl- Hematokrit 0.20 L 0.21 -0.31 LtL Gkv ri1 fl Gkh 4t Ghhk 31 g/dL Rdw l ).+ *H l0 - t5 % Witseltelling 209.1 H 5.0 - 10.0 xlOE9/L Plsase note: Due to the nucleated red blood cells the machine count ibr the white cells is incorect. Please send blood smear to the Vetlab lor evaluation. Neutrofiele tt.5 % 24.05 x10E9/L Limfbsiete 85.9 % 179.62 x 10E91L It4onosiete /.+ lo J.vJ r l0E9/L Eosinofiele 0.1 %, 0.21 x I 0E91L Vbt Kommentaar PLAATJIETELLTNG Plaatjietelling 20 x l0E9/I- Vir konsultasie: Dr Lucia Lange Veearts Patoloog: 082 8088773 -' H:Hoog, L:Laag, *H:Kritiek Hoog. EL:Kritiek Laag -- Liasseer [ ] Bel Pasient [ ] Maak Afspraak [ ] Voorskrif [ ] Trek Leer [ ] P,.'Y"rg'f-Y"nno,", Medi-Clinic Hospitaal Derde Laan. Bloemtbntein ffiW** Tel:051 1ol 4616 PracNo':5200539 FINALE VERSLAG - Lab Verrv :750527264 - Rehening Nrro^ -: nF6z0n2.0^5r4/5r Dokter Pasient AFSKRIF VERSLAG BFN (OTHER) AVIAN CHICI(ENS BOUCHERAVIAN BLOEMFONTEIN LABORATORIUM DEPT OF MICROBIAL. UFS P O BOX 339 93OO BLOEMFONTEIN 93OO BLOEMFONTE]N Tel (H) 05 14013253 Tel (S) 0783634531 Tel (W) 0514012274 MONSIET O5O3:HAO527OU ID NOINMCT NIE BESKIKBAAR NIE Yersameldatuln 2011-05-03 Nie verskaf'nie Oud:Gslg:DvG lzl4y:M ontvangsdatum 2017-05-04 20:16 Leer Nr NIE BESKIKBAAR NIE Yerslagdatum 2017-05-13 11:30 Hooflid DR C BOUCHER Mediese Fonds VET ACC'S DIRECT TO OWNER Med'Fnds Nr NOT AVAILABLE r$rrt"r" Drt* URGENT !!! FAX RESULTS TO:CHARLOTTE/POOJAH E-MAIL RESULTS TO :JAWALLAPERSAND(4GMAIL.C]OM RREED: LEGHORN CI{ICKENS AGE: 21 WEEKS CONTROL(NOT CHALLENGED) CHICKEN BLOOD TOTAL OF 5 SAMPLES IN TOTAL, HEALTHY CHICKENS Toetse Aangevra: PEzuPHER,\L BLOOD FILM, PLAATJIETELLING, HAEM SIGN QUEUE TR1CCER I, BLOEDTELLING-GEEN PLAATJIES Primere ICDI0 Kode(s) : 276.9 Toets Resultaat Wvser Verlvysing BLOEDTELLING-GBEN PLAATJIES Rooiseltelling 2.6i H 1.8-2.3x10E12/1. Hemoglobien I 3.5 H 90-il0sLlL Hematokrit 0.32 0.21 - 0.3i LtL Gkv lt8 fl Gkh 50 pg Gkhk 9-) grdL +H Rdu, 25.2 10-15% Witseltelling 57 0.2 H 5.0 - 10.0 xlOE9i'L Please note: Due ro the nucleated red cells the rr.rachine white cell count is incorrect. Please scnd blood smear to the Vetlab 1or cornment and evaluation Neutrot'iele 4.2 % 23.95 x I 0E9/L Limfbsiete 95.3 % s43.40 x l0E9/L Monosiete 01% 228 x10E9/L Vbt Kommentaar PLAATJIETELLING Plaatjietelling x 10E9rL Vir konsultasie: Dr Lucia Lange Vecarts Patoloog: 082 8088773 - H=Hoog, L:Laag, +H:Kritiek Hoog, *L-Kritiek Laag .' Liasseer [ ] Bel Pasient [ ] Maak Afspraal< | I Voorsl300 TMTC - 10-1 >300 TMTC - 10-2 >300 TMTC - 10-3 >300 TMTC - 10-4 >300 TMTC - 10-5 >300 TMTC - 10-6 109 30<109<300 1.09 x 109 10-7 12 TFTC - 10-8 2 TFTC - 216 CHAPTER 4: INFECTIOUS CORYZA AS AN INFECTION MODEL TO MONITOR IMMUNE CELLS AND MOLECULES DURING DISEASE PROGRESSION Table 4.2: Cell count and CFU/ml results for plating of bacterial culture for the second injection. Serial dilution Average plate count/cell count Within Range of 30 and 300 cells CFU/ml 100 >300 TMTC - 10-1 >300 TMTC - 10-2 >300 TMTC - 10-3 >300 TMTC - 10-4 >300 TMTC - 10-5 >300 TMTC - 10-6 69 30<69<300 6.90 x 108 10-7 2 TFTC - 10-8 0 TFTC - *TMTC: Too many to count; TFTC: Too few to count The cell count from 100 l from each of the cell cultures for the first and second injection was determined by plating out (Figure 4.2) and calculated, whereby the results were recorded (Table 4.1 and Table 4.2). Hence, using the formula for cell count per ml= (cell count (30CFU300) x dilution factor x factor of 10 (to convert a 100 µl sample to a 1000 µl sample)), the cell count per ml was determined (Table 4.1 and Table 4.2). Since, 500 ml of the bacterial culture was grown and was then concentrated to a volume of 5 ml, the cell concentration increased 100x. From the cell count per ml results obtained, it was evident that there was greater than 108 CFU/ml of viable cells to invade the upper respiratory system of the host to cause disease following infra-orbital injection, especially since the microorganism is also a highly virulent serovar (Table 4.1 and Table 4.2). The cell count also enabled us to know approximately how many viable cells were injected into the chickens, 217 CHAPTER 4: INFECTIOUS CORYZA AS AN INFECTION MODEL TO MONITOR IMMUNE CELLS AND MOLECULES DURING DISEASE PROGRESSION instead of relying solely on the OD600 based on the turbidity of bacterial growth (Table 4.1 and Table 4.2). TMTC TMTC TMTC TMTC TMTC WR WR TFTC TFTC Figure 4.2: Tryptic soy agar supplemented with 0.2% (v/v) NAD+ (TSA) plates showing growth of Av. paragallinarum serovar C-3 at different concentrations 100-10-8 when a serial dilution was performed in triplicate, however only one of the plates from each dilution was shown. Tiny colonies visible to the naked eye can be seen and the cell count can be determined. *TMTC: Too many too count; WR: Within the range of 30 and 300 colonies; TFTC: Too few to count. A species-specific PCR was performed, using the DNA extracted from 800 l of the bacterial culture obtained from the flasks with broth (TSB) (Merck) supplemented with 0.2% (v/v) NAD+ (Merck) containing a bacterial suspension of C-3, for the first and second injection 218 CHAPTER 4: INFECTIOUS CORYZA AS AN INFECTION MODEL TO MONITOR IMMUNE CELLS AND MOLECULES DURING DISEASE PROGRESSION respectively. The species-specific PCR/ HPG2-PCR was performed with the DNA extracted from the reference isolate Av. paragallinarum serovar C-3 (SA-3 strain) cultivated, whereby an expected amplicon size of 500 bp was obtained indicative that the desired microorganism was present (Figure 4.3). M 1 2 C3+ NC 500 bp Figure 4.3: HPG2-PCR for the SA-3 (C-3) reference isolate cultivated for the experimental trial, whereby amplification was observed for all samples, with an expected band size of 500 bp. Lane M- molecular marker O'GeneRuler DNA Ladder; lane 1: SA-3 strain used for first injection (1IC); lane 2: SA-3 strain used for second injection (2IC); C3+: SA-3 strain serovar C-3 positive control; lane NC: negative control. The identity of the Av. paragallinarum serovar C-3 (SA-3 strain) reference isolate was confirmed using 16S rDNA amplification (Section 3.2.3.5, Chapter 3; Section 4.2.3, Chapter 4). PCR products of an amplicon size of 1500 bp were obtained for all samples (Figure 4.4), indicating positive amplification of the DNA template. Following amplification of samples with 16S rDNA PCR, the gel slices (Figure 4.5) which were excised were then purified (Section 3.2.3.5, Chapter 3; Section 4.2.3, Chapter 4) and Sanger sequencing was conducted on the purified DNA samples. Sequences obtained, were analysed using Geneious® 9.8.1 219 CHAPTER 4: INFECTIOUS CORYZA AS AN INFECTION MODEL TO MONITOR IMMUNE CELLS AND MOLECULES DURING DISEASE PROGRESSION (Biomatters Ltd.) (Kearse et al. 2012). Sequences of each sample were aligned and the consensus sequence was compared with known sequences in GenBank, using a nucleotide BLAST analysis program (Appendix C) (Altschul et al. 1990). A homology of 100% between reference sequences and the queried sequences, indicated DNA-DNA relatedness and as such the identity of the bacterial strain of interest could be known to a species level. M 1 2 C3+ NC 1500 bp Figure 4.4: 16S rDNA PCR for the SA-3 (C-3) reference isolate cultivated for the experimental trial, whereby amplification was observed for all samples, with an expected band size of 1500 bp. Lane M- molecular marker O'GeneRuler DNA Ladder; lane 1: SA-3 strain used for the first injection (1IC); lane 2: SA-3 strain used for the second injection (2IC); lane C3+: SA-3 strain positive control; lane NC: negative control. The sequencing results for the samples were recorded in Table 4.3. Finally, using the results from both the species-specific PCR and the BLASTn analysis of the 16S rDNA sequencing results, it was found that the data corresponds to that of Av. paragallinarum strain SA-3 in the database. BLASTn results show that the strain of interest shares 100% correlation to the 220 CHAPTER 4: INFECTIOUS CORYZA AS AN INFECTION MODEL TO MONITOR IMMUNE CELLS AND MOLECULES DURING DISEASE PROGRESSION Av. paragallinarum strain SA-3 16s rDNA gene from the database, hence the results coincide and support that the strain used for the study was that of Av. paragallinarum serovar C-3 (SA-3 strain) (Table 4.3). The identification of the bacterial species of interest was very important, since during the infection of the chickens it was crucial that the Av. paragallinarum SA-3 strain be the main avian pathogenic organism, as having other microorganisms in the bacterial culture injected might have led to secondary infections or no infection, and could have jeopardised the validity of the study, since clinical symptoms of IC might have been masked or not seen at all. Table 4.3: Nucleotide BLAST results for all 16S rDNA PCR products for the experimental trial with species identification, GenBank® accession numbers, query length, query coverage, E-value and high sequence identities. Sample Isolate/Species Accession Query Query E-value Identity number length (bp) coverage 1IC Avibacterium KC951277.1 640 100% 0.0 100% paragallinarum strain SA-3 16S ribosomal RNA gene, partial sequence 2IC Avibacterium KC951277.1 649 100% 0.0 100% paragallinarum strain SA-3 16S ribosomal RNA gene, partial sequence C3+ Avibacterium KC951277.1 644 100% 0.0 100% paragallinarum strain SA-3 16S ribosomal RNA gene, partial sequence 221 CHAPTER 4: INFECTIOUS CORYZA AS AN INFECTION MODEL TO MONITOR IMMUNE CELLS AND MOLECULES DURING DISEASE PROGRESSION 1500 bp Figure 4.5: Agarose gel visualisation of 16S rDNA PCR products for the SA-3 (C-3) reference isolate cultivated for the experimental trial when viewed using a UV transilluminator (Spectroline®). The correct gel fragments were cut out using a sterile surgical scalpel blade, the excised gel slices were then purified, whereby the purified DNA samples were used for Sanger sequencing. 4.3.2. Challenge methods and clinical scoring Once all chickens were injected accordingly, they were closely monitored for a period of 21 days for any visible IC related symptoms, the daily mean disease score was recorded during disease progression for both the experimental and control groups and a disease profile was plotted (Figure 4.6) and a summary of the total number of chickens and scores can be viewed (Table 4.4). After 24 h (Day 1), facial oedema as a result of an initial inflammatory response, was observed around the site of injection with all experimental chickens. Thus, all experimental chickens reached a mean disease score of 1. Symptoms associated with a score of 1 in the experimental chickens also included lethargy and diarrhoea, however the most spot-on indication of a score of 1 was the serous to mucoid nasal discharge observed on the left side of the facial area, which corresponds to the side which was injected (Figure 4.7). 222 CHAPTER 4: INFECTIOUS CORYZA AS AN INFECTION MODEL TO MONITOR IMMUNE CELLS AND MOLECULES DURING DISEASE PROGRESSION Contrary to the study in Chapter 3, the signs and symptoms of the experimental chickens infected with IC did progress from mild (score 1) to moderate (score 2) to severe (score 3) within a period of 18 days similar to the disease progressions as described in literature (Bragg, 2004; Blackall and Soriano, 2008). After Day 4, some chickens recovered, while the rest progressed to a score of 2. Affected chickens with score 2 had a cheese-like mucoid nasal discharge on both sides of the nasal cavity with slight facial oedema, lethargy, diarrhoea and swollen combs and wattles (swollen head-like syndrome). Chicken E14 at a score of 2, had difficulty in breathing, rales and disorientation in addition with symptoms such as bilateral nasal discharge, poor appetite and facial oedema. Chicken E29 was presented with rales and sneezing in addition to facial oedema and bilateral nasal discharge On Day 8 and 9 respectively, the mean disease score was 1.9, which implies that the majority of the experimental cohort reached a score 2 and score 3 respectively (Figure 4.6). After Day 14, some chickens remained on a score 1 and 2, while very few chickens progressed to a score 3. Only a minority of chickens reached a score of 3, which can be observed on Day 18, where the daily mean disease score was 2.5, whereby 12 out of the remaining 23 chickens had a score 3, 10 out of 23 had a score 2 and 1 out of 23 had a score 1. Chickens presented with score 3 had bilateral facial oedema with or without haemorrhage, poor appetite and conjunctivitis (Figure 4.7). There were no mortalities from IC in this study. On two occurrences, on the disease profile there was a sharp decline or drop observed in the disease progression trend, resulting in recovering chickens (Figure 4.6). The first decline on the disease profile was seen on Day 4 where the mean disease score changed from a score 1 on Day 3 to a score 0.6 on Day 4, which is then followed by an increase in the mean disease score on Day 5 to a score of 1.7 followed by a relatively stable disease progression trend over the next 8 days (Figure 4.6). The reason for this sharp decline on Day 4, may be attributed to the innate immune response that is active during those first few days following infection, whereby the first-line of defence includes release of immune cells (phagocytes, 223 CHAPTER 4: INFECTIOUS CORYZA AS AN INFECTION MODEL TO MONITOR IMMUNE CELLS AND MOLECULES DURING DISEASE PROGRESSION dendritic cells, heterophils and natural killer cells) and immune molecules (complement, collectins, acute phase proteins, cytokines) that have antibacterial properties, that are involved in inflammatory responses and that interact with the adaptive phase of the avian immune system (Playfair and Bancroft, 2013). The second significant drop on the disease profile was observed on Day 14, whereby from Day 11 the mean disease score gradually declined from a score of 1.9 to a score of 1.1, over the course of 3 days (Figure 4.7). After Day 14, the mean disease score then increases from a score of 1.1 to a score of 2 on Day 15 (Figure 4.7). Figure 4.6: The disease profile showing the daily mean disease score of the control (red) and experimental (green) cohorts. The trend of the control group was consistent at a score 0 throughout the duration of 21 days, as they were not challenged or exposed to C-3 isolate. The experimental group had a different trend whereby an initial innate immune response was observed on Day 4 and an active adaptive immune response occurred on Day 14, resulting in recovering chickens given that IC does not cause mortality. 224 CHAPTER 4: INFECTIOUS CORYZA AS AN INFECTION MODEL TO MONITOR IMMUNE CELLS AND MOLECULES DURING DISEASE PROGRESSION Table 4.4: Summary of disease progression over the course of 21 days (3 weeks) and the total number of chickens with clinical scores from each cohort. Chickens randomly selected were sacrificed after a clinical score was obtained. At the end of the study the control chickens, with no IC related symptoms at a score 0 were sacrificed. Experimental chickens (NE= 30) Control chickens (NC=8) Day Score 0 Score 1 Score 2 Score 3 Score 0 Score 1 Score 2 Score 3 0 30/30 8/8 1 30/30 8/8 2 30/30 8/8 3 29/29 8/8 4 11/29 18/29 8/8 5 9/26 17/26 8/8 6 13/26 13/26 8/8 7 11/26 15/26 8/8 8 8/26 12/26 6/26 8/8 9 11/26 8/26 7/26 8/8 10 10/26 13/26 3/26 8/8 11 8/24 11/24 5/24 8/8 12 1/23 11/23 8/23 3/23 8/8 13 2/23 15/23 5/23 1/23 8/8 14 6/23 9/23 8/23 8/8 15 7/23 9/23 7/23 8/8 16 4/23 6/23 6/23 7/23 8/8 17 2/23 5/23 9/23 7/23 8/8 18 1/23 10/23 12/23 8/8 19 1/23 1/23 17/23 4/23 8/8 20 1/23 1/23 18/23 3/23 8/8 21 1/23 1/23 18/23 3/23 8/8 *NE= Initial total number of chickens in the experimental group; NC= Initial total number of chickens in the control group 225 CHAPTER 4: INFECTIOUS CORYZA AS AN INFECTION MODEL TO MONITOR IMMUNE CELLS AND MOLECULES DURING DISEASE PROGRESSION Clinical Signs and Symptoms of IC Score 1 Control A B C J Score 2 D E K Score 3 F G H I Figure 4.7: Clinical signs and symptoms observed in chickens presented with IC. Score 1 chickens displayed mild clinical signs and symptoms such as (A) facial oedema, (B) nasal discharge on one side of the nasal cavity and (C) lethargy. Score 2 chickens showed moderate symptoms such as (D) bilateral nasal discharge with swollen comb and (E) diarrhoea. Score 3 chickens were afflicted with (F) severe bilateral facial oedema, (G and H) conjunctivitis and bloody nasal exudates due to haemorrhage. Control chickens were kept separate in isolators and did not have any IC related symptoms, hence they were given a score 0 (J and K). 226 CHAPTER 4: INFECTIOUS CORYZA AS AN INFECTION MODEL TO MONITOR IMMUNE CELLS AND MOLECULES DURING DISEASE PROGRESSION This second decline in the disease trend is due to the adaptive immune system that is highly active during the last stages of IC infection following the innate immune response, whereby the humoral response associated with B-cells and antibody production, and the cell- mediated response involving T-cells associated with cell proliferation, cell differentiation, cytotoxic activity and cytokine secretion; have a vital and active role to play (Playfair and Bancroft, 2013). The adaptive immunity also leads to memory B and T cells, important since a second exposure to IC would lead to a more rapid immune response or even resistance to IC infection, as a result of an already well-established immune response. The scoring for the control group over the course of 21 days, was stable at a constant mean disease score of 0. No clinical signs and symptoms of IC were observed, with all the control chickens being healthy, as they were not challenged with or exposed to Av. paragallinarum serovar C-3 (SA-3 strain). This was a good indication that no cross-contamination occurred between the two cohorts and that all necessary measures taken while handling the two cohorts was effective. The control chickens also had a good appetite with feed and water provided on a daily basis, compared to the infected chickens that had a poor appetite for feed with increased water uptake. Following the first injection, 5 chickens E15 (score 2), E16 (score 2), E24 (score 3), E26 (score 2) and E28 (score 1) respectively, were injected a second time on Day 8. The chickens were injected a second time to observe whether the second dose of bacterial culture would lead to disease escalation. However, after 24 h (Day 9), the chickens which received the second injection of Av. paragallinarum serovar C-3 (SA-3 strain) only suffered from inflammation at the site of injection and diarrhoea. Re-injection of the 5 chickens did not cause the disease to progress any further, and as such the disease progression of the re- infected chickens was the same as the rest of the experimental group that were at score 2. 227 CHAPTER 4: INFECTIOUS CORYZA AS AN INFECTION MODEL TO MONITOR IMMUNE CELLS AND MOLECULES DURING DISEASE PROGRESSION Chicken E24 stayed at a score 3, 2 days after being injected. Chickens E15 and E16 did reach a score of 3, 2 days after being injected, with the rest of the experimental group. Chicken E26 remained on score 2 and E28 on score 1, were there was no disease progression. A probable explanation for disease progression not becoming severe could be due to an actively running innate immune response that has already activated the adaptive immune response within the first week of infection, since these two systems are integrated. The innate immune system makes an important contribution to the activation of adaptive immunity via the inflammatory response caused by macrophages that secrete cytokines that increase vascular permeability when they encounter bacteria (Janeway et al. 2005). Vascular permeability allows antigen to flow into lymphoid tissues, thereby activating lymphocytes (Janeway et al. 2005). Moreover, the induction of the adaptive immune system starts when the bacteria is phagocytosed by an immature dendritic cell (Janeway et al. 2005). Upon phagocytosis, the dendritic cell becomes activated and matures into an antigen presenting cell (APC), that presents antigen to T lymphocytes (Janeway et al. 2005). The total number of eggs laid per chicken per day was recorded for both experimental and control groups. On Day 9 and 18, there was a slight drop in the egg-laying trend for the experimental group (Figure 4.8). However, no statistically significant decline in egg production with infected chickens was observed, as mentioned in literature, as the chicken sample size in this study was too small to deduce the effect of IC on egg production, whereas in the extensive chicken industry farmers have larger flocks to work with (Blackall and Soriano, 2008). Moreover, as the study was conducted for only 21 days, the long-term effects on egg production caused by IC was not investigated. For the control group, over the course of 3 days (Days 1 to 3), it was observed that the chicken egg production was higher than expected, resulting in no eggs being laid on Day 4. However, after Day 4, a normal egg laying trend was observed resulting in 1 egg laid per day per chicken. The reason for this phenomenon could be attributed to human error, as it was possible that eggs laid the night 228 CHAPTER 4: INFECTIOUS CORYZA AS AN INFECTION MODEL TO MONITOR IMMUNE CELLS AND MOLECULES DURING DISEASE PROGRESSION before could have remained hidden from view and counted with the rest of the eggs laid on the next day, resulting in more eggs being counted, as four chickens were kept together in one isolator. Compared to the control group, the experimental group had a more stable egg- laying trend, whereas the control group had a highly variable egg-laying trend (Figure 4.8). Figure 4.8: Egg production indicated as the total number of eggs laid/chicken/day in the experimental and control groups. The experimental group had a more consistent egg-laying trend in comparison to the control group that had a highly variable egg-laying trend, thus making it difficult to deduce any significant egg- laying patterns. 4.3.3. Avian blood smears and microscopy Following the principles of the “Three Rs” in animal research (Reduction, Refinement and Replacement), to minimise stress, harm and pain to the chickens during blood collection, blood was drawn from the chickens every 1-4 days and the blood was collected in EDTA coated SGVac PET Blood Collection Tubes (The Scientific Group). Unfortunately, the chickens could not be bled every day, this was due to a haematoma being formed each time a needle was inserted into a vein and the vein collapsed, during blood collection. The haematoma formed takes approximately a week to recover. Hence, to overcome this 229 CHAPTER 4: INFECTIOUS CORYZA AS AN INFECTION MODEL TO MONITOR IMMUNE CELLS AND MOLECULES DURING DISEASE PROGRESSION problem chickens were randomly selected for bleeding based on the clinical scoring usually from one wing. In case the same chicken had to be bled again the other wing would be available for phlebotomy. The experimental chickens were bled on Days 0, 1, 3, 4, 7, 10, 11, 15, 18 and 21. For the control group, as there were only a few chickens and we did not want to risk Av. paragallinarum serovar C-3 (SA-3 strain ) exposure by opening and closing the doors sealing the incubators frequently as well as causing hematoma formation in a few chickens, hence blood was drawn on Days 0, 11, 15, 18 and 21. To collect the blood, an additional volume of EDTA (0.5 M, Merck) of 1 ml was supplemented to the EDTA tubes to prevent quick coagulation of the blood collected, as chicken blood coagulates quickly due to high levels of calcium present. Following blood collection, the EDTA tubes were quickly inverted 3-4 times to thoroughly mix the blood and EDTA together to prevent coagulation and to minimise clots. Whole blood samples were collected and processed within 6-8 h, so that the samples were not compromised and thus making them unsuitable for flow cytometry analysis (Section 4.3.4) and blood smears. Blood smears were prepared by and obtained from the NHLS. The blood smears were visualised using the Eclipse 50i microscope, DS-Fi1 digital microscope camera and NIS- Elements F 4.00.06 Build 786 microscope imaging software (Nikon), whereby images were taken at a 100X magnification. The blood smears prepared and obtained from the blood of chickens at different clinical scores on Day 0, 1, 3, 4, 7, 10, 11, 15, 18 and 21, were compiled (Annexure C). From the blood smears, cell morphology of different blood cells based on the staining technique used could be evaluated (Hematek Modified Wright’s Stain and Hematek Wright-Giemsa was used) for the classification and differentiation of those blood cells. Figure 4.9 (A-F) shows some of the blood cells observed under the microscope 230 CHAPTER 4: INFECTIOUS CORYZA AS AN INFECTION MODEL TO MONITOR IMMUNE CELLS AND MOLECULES DURING DISEASE PROGRESSION that have interesting features and functions in the avian immune response following infection with A. paragallinarum serovar C-3 (SA-3 strain). A toxic heterophil (h) was observed with a lymphocyte (ly) indicated by an arrow and a teardrop-shaped red blood (tds) cell in chicken E8 with score 1 on Day 1 (Figure 4.9 A), after 24 h following infection. Heterophils are characterised by brick-red granules found in the cytoplasm and possessing a bilobed nucleus (Fudge, 1998; Harrison and Lightfoot, 2006). However, with the toxic heterophil shown there was a loss of nuclear lobulation and the cytoplasm shows very few granules (Figure 4.9 A). During pathogenic invasion, induction of the innate immune response occurs, which triggers heterophils as the main effector cells to respond to the site of infection via chemokines released and that are actively involved in the phagocytosis of these invading pathogens (Kaiser, 2010), which is also the reason why the toxic heterophil on Day 1 was observed. Moreover, heterophils are the counterparts of the mammalian neutrophil and are polymorphonuclear cells (along with basophils and eosinophils) (Harrison and Lightfoot, 2006). In addition to phagocytosis, heterophils are involved in bactericidal activity in processes such as respiratory burst and degranulation (Kaiser, 2010). Teardrop-shaped red blood cells observed are usually an indication of toxicosis, septicaemia or anaemia due to bacterial endotoxins released from gram-negative bacteria (lipopolysaccharide-LPS) (Fudge and Joseph, 2000; Raetz and Whitfield, 2002). On Day 7, the blood sample from chicken E22 with score 2, there was an infiltration of lymphocytes (ly) indicated by several arrows and monocytes (mo), and a heterophil (h) as seen in (Figure 4.9 B). The infiltration of lymphocytes and monocytes which were observed, was an indication of an infection, but it could also explain the adaptive immune response that was actively at play on Day 7, as lymphocytes consist of B and T cells (Figure 4.9 B). Lymphocytes are round in shape with a centrally positioned nucleus with a pale blue 231 CHAPTER 4: INFECTIOUS CORYZA AS AN INFECTION MODEL TO MONITOR IMMUNE CELLS AND MOLECULES DURING DISEASE PROGRESSION cytoplasm when stained with Wright-Giemsa stain. Monocytes are large sized and typically have a round shape with a ratio of 3:1 cytoplasm to nucleus. A normal monocyte with a kidney shaped nucleus eccentrically positioned was observed on the blood smear of chicken E17 with score 3 on Day 10 (Figure 4.9 C). On Day 11, for chicken E15 with score 3, a bilobed heterophil (h) with lymphocytes (ly) in the periphery blood smear along with teardrop-shaped red blood cells (tds), was observed (Figure 4.9 D). During the activation of heterophils by pathogens or cytokines, there is expression of pro-inflammatory cytokines such as interleukins (IL) (IL-1, IL-6 and IL-8) (Kogut et al. 2005; Kogut et al. 2006). IL-1 and IL-6 target T and B cells, thus causing acute phase responses, recruitment of other immune cells, phagocyte activation and proliferation of antibody secreting B cells (Playfair and Bancroft, 2008). In addition to this, IL-8 is a potent pro-inflammatory cytokine and stimulator of neutrophil activation and chemotaxis leading to inflammatory reactions in humans, not much is known about IL-8 in avian immunology except that it is involved in mucosal immunity (Borrmann et al. 2007). Perhaps this explains why there are lymphocytes in the vicinity of heterophils for Day 1, 7 and 11, which could be due to cytokines acting as chemoattractants for B and T lymphocytes. Thus, this shows that there is an interaction of both the innate immune system via heterophils and cytokines; and the adaptive immune system through the recruitment of B and T lymphocytes leading to the antibody and cell-mediated responses. A basophil (ba) was shown with an unlobed nucleus for chicken E22 with score 3 on Day 11 (Figure 4.9 E). The basophil observed was characterised according to the presence of variable large and small, round and dark purple granules widespread across the cytoplasm, with an unlobed nucleus. Basophils have a role in early inflammatory and immediate hypersensitivity responses (Maxwell and Robertson, 1995). Moreover, there seems to be a 232 CHAPTER 4: INFECTIOUS CORYZA AS AN INFECTION MODEL TO MONITOR IMMUNE CELLS AND MOLECULES DURING DISEASE PROGRESSION correlation between severe stress, an increased heterophil/lymphocyte (H/L) ratio, heteropenia and basophilia, which may be a physiological response unique to birds (Maxwell and Robertson, 1995). If that was the case, the chickens during the experimental procedure were also stressed (other than being infected), especially when bled, it is possible that basophilia or leucocytosis could be an indicator of stress. The blood sample from chicken E22 at score 3 on Day 11 (Annexure C), basophilia can be observed by numerous basophils in the blood periphery, which is not normally seen. In the blood smear prepared from blood of chicken E13 with score 3 on Day 15, there were several blood cells such as a monocyte (mo), an eosinophil (eo), thrombocytes (th), hypochromic (hc) and teardrop-shaped (tds) red blood cells (Figure 4.9 F). The hypochromic and teardrop-shaped red blood cells are indicative of anaemia and gram-negative septicaemia respectfully. Thrombocytes are smaller than lymphocytes and monocytes and are oval to rectangular in appearance (Figure 4.9 F). Thrombocytes are the haemostatic counterparts of mammalian platelets (Ferdous et al. 2016). Thrombocytes have an important function in the immune response such as phagocytic ability, inflammation mediation, antimicrobial activity and other immune modulating activities, as well as haemostatic function and blood coagulation (Ferdous et al. 2016). Monocytes are naïve precursors having limited effector and regulatory capabilities, however upon stimulation they develop into macrophages (Klasing, 1998). Macrophages are more capable of phagocytic activity and mediating the host defence mechanism that is crucial in defining the type and intensity of specific innate immune responses (Klasing, 1998). The disrupted eosinophil observed were of a medium size and there was loss of nuclear lobulation, as the cytoplasm stained pale blue with red-orange granules (Figure 4.9 F). The role of avian eosinophils in literature is still unclear, however avian eosinophils may have a role in delayed hypersensitivity responses and as such could have similar function as their mammalian counterparts such as immediate 233 CHAPTER 4: INFECTIOUS CORYZA AS AN INFECTION MODEL TO MONITOR IMMUNE CELLS AND MOLECULES DURING DISEASE PROGRESSION hypersensitivity responses and modulation against parasitic infestation (Montali, 1988; Grasman, 2002). Unfortunately, the automatic slide stainer Hematek® 3000 System (Siemens Healthineers) with Hematek Modified Wright’s stain and Hematek Wright-Giemsa stain used for staining of human blood smears, did not produce adequate quality with the staining of avian blood smears, for the differentiation of subtle blood structures. Although, the avian blood smears were of poor quality, the different morphologies of red blood cells could still be distinguished. However, the morphological characteristics of heterophils, monocytes, leukocytes and granulocytes were very difficult to classify and thus could not be properly distinguished. This indicates that the Hematek Modified Wright’s stain and Hematek Wright-Giemsa stain, which are rapid stains, are not suited for staining of avian blood samples for morphological characterisation and classification, as they yield poor quality staining. Therefore, stains such as Wright stain, Giemsa stain, Wright-Giemsa stain, Leishman stain, Wright-Leishman stain, May-Grünwald stain and May-Grünwald-Giemsa stain, are highly recommended for future studies. Moreover, the avian blood slides analysed, cannot be used to interpret disease progression solely on their own, as they do not provide adequate information on the immune response in the affected organism except the morphological characteristics of blood cells and possible causes. Thus, in future studies, the blood slides need to be supported by complete/full blood counts (CBC/FBC) and white blood cell differential counts (WBC Diff) to obtain an overall view in making a diagnosis and prognosis. In our study, CBC/FBC and WBC Diff could not be performed, however the data from the flow cytometry results with the blood smears obtained was used to show the change in blood morphology and the evolution of the immune response during IC disease progression in the chickens. 234 CHAPTER 4: INFECTIOUS CORYZA AS AN INFECTION MODEL TO MONITOR IMMUNE CELLS AND MOLECULES DURING DISEASE PROGRESSION ly mo mo mo h h tds A B tds ly mo h ly tds C D hc tds mo ba eo th th tds E F Figure 4.9: Haematological observations for blood slides showing different blood cells observed for infected chickens. (A) A heterophil (h) was observed with a lymphocyte (ly) indicated by an arrow and a teardrop-shaped red blood (tds) cell in chicken E8 with score 1 on Day 1. (B) For the blood sample from chicken E22 with score 2 on Day 7, there was an infiltration of lymphocytes (ly) indicated by arrows and monocytes (mo), and a heterophil (h) was seen. (C) The blood smear of chicken E17 with score 3 on Day 10, showed a normal monocyte (mo) with a kidney-shaped nucleus and a lymphocyte (ly) indicated by an arrow. (D) A bilobed heterophil (h) was seen, with lymphocytes (ly) in the periphery blood smear along with teardrop-shaped red blood cells (tds) for chicken E15 with score 3 on Day 11. (E) A basophil was shown with an unlobed nucleus for chicken E22 with score 3 on Day 11. (F) For this particular blood smear belonging to chicken E13 with score 3 on Day 15, there were several blood cells such as a monocyte (mo), an eosinophil (eo), thrombocytes (th), hypochromic (hc) and teardrop-shaped (tds) red blood cells. Magnification X100. 4.3.4. Flow cytometry and antibodies 235 CHAPTER 4: INFECTIOUS CORYZA AS AN INFECTION MODEL TO MONITOR IMMUNE CELLS AND MOLECULES DURING DISEASE PROGRESSION The cell population profiles of the leukocytes (composed of lymphocytes and granulocytes) using a CD45 pan-leukocyte marker were studied and generated; to evaluate a specific lymphocyte population, the T-cell population mainly the CD4 T cells (T helper cells) and CD8 T cells (cytotoxic T cell) using the CD4 and CD8 markers respectively. Flow cytometry was conducted as described (Section 3.2.7, Section 4.2.7) using the BD FACSCanto II (Becton Dickinson) with analysis on the BD FACSDiva 8.0.1 software (Becton Dickinson), however two separate flow cytometric tubes were run on the BD FACSCanto II (Becton Dickinson) simultaneously for each chicken blood sample, whereby the first tube consisted of CD45 (total leukocyte) and CD4 (T helper cell) markers and the second tube consisted of CD4 (T helper cell) and CD8 (cytotoxic T cell) markers respectively. All three mouse anti-chicken antibodies: CD4-FITC (2-35 clone, MCA2164F), CD8-RPE (11-39 clone, MCA2166PE), CD45-RPE (UM16-6 clone, MCA2413PE) (Bio-Rad) could not be added, into one tube as both CD8 and CD45 antibodies had the same fluorophore RPE (R-phycoerythrin), whereas the CD4 antibody had a different fluorophore FITC (Fluorescein isothiocyanate). Having the same fluorophore would imply that the fluorescent labels would have the same peak excitation (496 nm) and emission wavelength (578 nm, green in colour) with a 488 nm blue argon laser from the BD FACSCanto II (Becton Dickinson), which can be problematic as CD8+ cells are also CD45+ cells, thus the differentiation between CD8+/CD45+ (cytotoxic T cells) cells from CD45+ cells would not be possible. Moreover, CD8+ cells would be masked and be solely shown as CD45+ cells due to the same fluorescent labels being used, therefore analyses of each chicken blood sample in two separate tubes (Annexure D) had to be conducted, which was very laborious. The flow cytometry profiles were generated from the excitation of labelled cells with fluorescent antibodies and the interrogation point with the laser, whereby light scatter is produced that could be measured and correlated with relative cell size and structures inside the cell. The measurements were termed forward angle scatter (FSC) which was based on 236 CHAPTER 4: INFECTIOUS CORYZA AS AN INFECTION MODEL TO MONITOR IMMUNE CELLS AND MOLECULES DURING DISEASE PROGRESSION the size of the cell and side angle scatter (SSC) which was based on the granular complexity of the cell. All the flow cytometry profiles of both control and experimental chickens were compiled (Annexure D) that were bled on the days mentioned in Section 4.3.3. In the previous study (Chapter 3, Section 3.2.7 and Section 3.3.4), during our runs, it was difficult to locate and gate the precise location of the entire leukocyte population and lymphocyte population. This was due to the nucleated RBCs that overlapped with the leukocyte and lymphocyte population, thus making it difficult to gate the leukocyte population, which was also where the lymphocyte population was containing both CD4 and CD8 cells. However, the problem of overlapping RBCs with leukocytes, was overcome with the use of the CD45 pan-leukocyte marker as observed in Figure 4.10 compared to Figure 3.10 (Chapter 3), there is better separation of the RBCs from the leukocytes. Figure 4.10: Flow cytometry profile of one of the experimental chickens (E3-score 0) showing the forward scatter (FSC-A) and side scatter (SSC-A) plot. Two distinct populations can be seen within the sequestered region gated as region P1 (shown in red) which is the approximate location of the lymphocyte population. The RBC population can be seen outlined in green and the leukocyte population can be seen outlined in orange. 237 CHAPTER 4: INFECTIOUS CORYZA AS AN INFECTION MODEL TO MONITOR IMMUNE CELLS AND MOLECULES DURING DISEASE PROGRESSION Firstly, a CD45/CD4 run (whole blood stained with CD45 and CD4) was performed, to enable us to locate and gate the correct region where the total leukocyte cells were situated, whereby a forward scatter (FSC-A) and a side-scatter (SSC-A) plot (Figure 4.10) was generated. From there onwards, a SSC-A versus a CD45 PE-A plot was generated, whereby proper gating of the total leukocyte population (total CD45 count) could be performed, which consists of both lymphocytes and granulocytes (Figure 4.11). Once this was done, it was easier to locate and gate the region where the lymphocytes were situated within the total CD45 population, based on the size and complexity of the cells (Figure 4.11). Finally, after the lymphocyte population were gated, a CD45 PE-A vs CD4 FITC-A analysis was performed, to obtain the cell counts of the total lymphocytes, CD45+ cells (CD8 T cells/B lymphocytes/ Natural killer (NK) cells), cells that are CD4+/CD45+ (T helper cells) and the total CD45 cells (total leukocytes) (Figure 4.12). Figure 4.11: Flow cytometry profile of one of the experimental chickens (E3-score 0) showing the side scatter (SSC-A) vs CD45 PE-A plot. Two distinct populations can be seen the RBC population (shown in red) and the total CD45 (total leukocyte) population outlined (shown in blue). The lymphocyte population is outlined within the total CD45 population. 238 CHAPTER 4: INFECTIOUS CORYZA AS AN INFECTION MODEL TO MONITOR IMMUNE CELLS AND MOLECULES DURING DISEASE PROGRESSION Figure 4.12: Flow cytometry profile of one of the experimental chickens (E3-score 0) showing the CD45 PE-A vs CD4 FITC-A plot. In the first quadrant (Q1) above quadrant three (Q3), CD45+ cells are shown. In the second quadrant (Q2) above quadrant four (Q4), CD4+/CD45+ cells are shown. The cell counts (#Events) can be seen in the table for each of the cell components such as total lymphocytes, CD45+ cells (CD8 T cells/B lymphocytes/ Natural killer (NK) cells), cells that are CD4+/CD45+ (T helper cells) and the total CD45 cells (total leukocytes). Following the first run, a CD4/CD8 run (whole blood stained with CD4 and CD8), was conducted using the same chicken blood sample. The same steps as in the first run were performed where a FSC-A and a SSC-A plot (similar to Figure 4.10) was generated, to locate and gate the exact region where the lymphocytes were situated, known from the CD45/CD4 run. A SSC-A versus a CD8 PE-A plot was then generated, whereby proper gating of the CD8 cells could be performed, which was also the same region, where the CD4 cells could be located (Figure 4.13). Once this was done, the CD8 and CD4 populations based on the emission wavelength produced by the two different fluorophores: 525 nm (FITC, green in colour) and 578 nm (R-PE, yellow in colour) with a blue argon laser from the BD FACSCanto II (Becton Dickinson) were gated and analysed. A CD8 PE-A vs CD4 FITC- A plot was generated, to obtain the cell counts of CD8+ cells (cytotoxic T cells) and CD4+ (T helper cells) (Figure 4.14), whereby for chicken E3 with score 0 on Day 0, there were no CD8+ cells but there were 10 CD4+ cells. The whole process was then conducted and 239 CHAPTER 4: INFECTIOUS CORYZA AS AN INFECTION MODEL TO MONITOR IMMUNE CELLS AND MOLECULES DURING DISEASE PROGRESSION repeated for all chicken blood samples for the CD45/CD4 and CD4/CD8 runs, as described above. However, the data obtained from the first and second analyses (CD45/CD4 and CD8/CD4 runs) were not reliable as is, as findings need to be reported as the percentage of total leukocytes. Hence, using the data from both the first and second analyses conducted, ratio calculations (Appendix D) were used to obtain the following: % total leukocytes, % CD8 of total leukocytes, % CD4 of total leukocytes, the CD4/CD8 ratio and % B and NK cells of total leukocytes. The data was graphically represented to understand the trend of the avian immune response elicited by Av. paragallinarum serovar C-3 via immune cells, which could be inferred or detected from the flow cytometry analysis (Figure 4.15 - Figure 4.20). Statistical analysis was performed to determine the statistical significance of the data. The mean and standard deviation of the data were calculated, and a Student’s t-test was conducted to validate the results obtained, where a p<0.05 was found to be statistically significant. Figure 4.13: Flow cytometry profile of one of the experimental chickens (E3-score 0) showing the side scatter (SSC-A) vs CD8 PE-A plot. From the plot there were very few lymphocytes. However, once the same region as in Figure 4.11 was gated, lymphocytes of interest could be obtained. 240 CHAPTER 4: INFECTIOUS CORYZA AS AN INFECTION MODEL TO MONITOR IMMUNE CELLS AND MOLECULES DURING DISEASE PROGRESSION Figure 4.14: Flow cytometry profile of one of the experimental chickens (E3-score 0) showing the CD45 PE-A vs CD4 FITC-A plot. In the first quadrant (Q1) above quadrant three (Q3), CD8+ cells would have been shown. In the fourth quadrant (Q4), CD4+ cells are shown. In the third quadrant (Q3), RBCs are shown. The cell counts (#Events) can be seen in the table for the CD8 and CD4 count. However, as observed there are no CD8+ cells. The % total leukocytes (CD45+ cells) plot gives all CD45+ cells detected via flow cytometry over 21 days in whole blood of both control and experimental chickens (p>0.05) (Figure 4.15). For better visualisation of how the leukocyte population looks like in whole blood, blood smears (Section 4.3.3) were compiled from each day from the control group (Days 11 and 21) and experimental group (Days 0-21), that correlates to the results obtained for the flow cytometry results as represented by the % total leukocyte plot (Figure 4.16). On Day 0, it seemed that the chickens had a high leukocyte percentage, which could be due to stress or previous exposure to microorganisms before the study. In the experimental group: On Day 1, following infection of experimental chickens, it was observed that there was a decrease in the percentage of total leukocytes, followed by an increase on Day 3 and a decrease on Day 4. The chickens were injected on Day 0, and a score 1 was observed in the majority of chickens, the innate immune system was triggered as an immediate response to infection and inflammation caused, that had a key role through innate immune cells such 241 CHAPTER 4: INFECTIOUS CORYZA AS AN INFECTION MODEL TO MONITOR IMMUNE CELLS AND MOLECULES DURING DISEASE PROGRESSION as epithelial cells, dendritic cells, monocytes, macrophages, heterophils and natural killer (NK) cells. Monocytes matured into inflammatory macrophages, whereby both macrophages and heterophils were involved in phagocytosis of the Gram-negative bacteria, Av. paragallinarum serovar C-3 (Bellingan and Laurent, 2008). However, the fate of the innate immune cells was short-lived, hence the decline observed on Day 1 and 4, which could be due to necrosis, apoptosis and subsequent phagocytosis of these innate immune cells (Bellingan and Laurent, 2008). Moreover, macrophages might be involved in phagocytosis of apoptotic heterophils, thus causing their numbers to decline with the clearance of phagocytosed cells, allowing the tissue to return to their normal structure and function (Bellingan and Laurent, 2008). There could also be possible degranulation of the heterophils, as observed previously with Gram-negative bacteria such as Salmonella enterica serovar Typhimurium (Lam and Munn, 2002). Furthermore, the heterophil numbers might have peaked earlier than macrophages during the inflammatory response, with simultaneous activation of the adaptive immune response having a slow effect (requiring several days to even a few weeks to respond to inflammation via the B and T cells) (Davison et al. 2011). Hence, on Day 3, an increase in the total leukocyte population was observed, as infected macrophages increase MHC class II expression to activate additional antigen- specific Th cells and produce co-stimulatory cytokines (IL-12, IL-18) that help in the differentiation of Th1 effector cells, leading to an efflux of leukocytes as seen on Day 3 (Erf, 2004). On Days 7 and 10, the percentage total leukocyte stayed stable. On Days 11 and 18, a high percentage of total leukocytes was observed, due to the adaptive immune system actively playing a role in the humoral and cell-mediated responses with a release of antibodies. This was followed by low percentages on Days 15 and 21, respectively, due to clearance of the immune cells following the cell-mediated response with cytotoxic T cells, natural killer (NK) cells and phagocytosis of infected cells via macrophages. In the control group: A stable trend 242 CHAPTER 4: INFECTIOUS CORYZA AS AN INFECTION MODEL TO MONITOR IMMUNE CELLS AND MOLECULES DURING DISEASE PROGRESSION of percentage total leukocytes over the course of 11 days was observed, followed by a slight decrease in percentage leukocytes on Days 15, 18 and 21 respectively. On Day 11, there was a high leukocyte percentage, which could imply that there was possible exposure, however the bacterial load was not sufficient to cause disease, hence the chickens did not develop IC related signs or symptoms. The high leukocyte percentage on Day 11, could also be stress induced. Figure 4.15: % Total leukocytes (CD45+ cells) plot gave all CD45+ cells detected via flow cytometry over 21 days in whole blood of both control and experimental chickens (p>0.05). In the experimental group: On Day 1, following infection of experimental chickens, it was observed that there was a decrease in the percentage of total leukocytes, followed by an increase on Day 3 and a decrease on Day 4. On Days 7 and 10, the percentage leukocyte stayed stable. On Days 11 and 18, a high percentage of leukocytes was observed. This was followed by low percentages on Days 15 and 21, respectively. In the control group: A stable trend of percentage leukocytes over the course of 11 days was observed, followed by a slight decrease in percentage leukocytes on Days 15, 18 and 21 respectively. Antibodies can prevent disease and provide protection through mucosal surfaces (secretory IgA) and in the elimination of pathogens that are found in the extracellular environment of the host (Erf, 2004). However, when antigens have infiltrated cells (endocytic mechanisms; exogenous antigens) or are produced within the cell (viral or neoplastic proteins; 243 CHAPTER 4: INFECTIOUS CORYZA AS AN INFECTION MODEL TO MONITOR IMMUNE CELLS AND MOLECULES DURING DISEASE PROGRESSION endogenous antigens) the humoral immune response which involves direct antibody-antigen contact is no longer effective in eliminating antigen (Abbas et al. 2014). In such a situation, the cell-mediated immune mechanisms that lead to intracellular elimination of the antigen or elimination of the host cell are the most promising strategies in antigen elimination (Erf, 2004). In poultry, as in humans and other mammals, T cells are the antigen-specific components of the cell-mediated immunity (CMI) and express T-cell receptors (TCR) that are collectively able to recognise various antigens (Chen et al.1991). All T cells express CD3 complexes together with the TCR molecules, which makes CD3 a pan-T cell marker; whereby its presence on a cell indicates that the cell is a T cell (Erf, 2004). T cells, having a primary and regulatory role in the adaptive immune response, whether cell-mediated or humoral, are referred to as T helper (Th) cells and express CD4 molecules on their surface (Chen et al. 1991; Arstila et al. 1994). Thus, upon specific recognition of the antigen-peptide expressed on the cell surface of an antigen-presenting cell (APC) in association with a self-MHC class II complex to the TCR, Th cells are activated that secrete cytokines and express cell surface molecules providing crucial activation signals to cells of innate and adaptive immunity, thereby propelling the mechanisms of the immune response towards pathogen elimination (Arstila et al. 1994). There are 2 types of Th cells that are highly specialised and specific to the type of infection (Arstila et al. 1994). Type-1 Th (Th1) cells which are effective in directing the innate immune response towards a cell-mediated response for intracellular pathogens whereby macrophage activation is required (Playfair and Bancroft, 2013). Th1 cells secrete macrophage-activating cytokines such as interferon- (IFN-), tumour-necrosis factor- (TNF-) and interleukin- 2 (IL-2) (Erf, 2004; Playfair and Bancroft, 2013). Contrary to Th1, type-2 Th (Th2) cells favour the development of a humoral response for antibody production to deal with extracellular pathogens with further specialisation towards particular antibody subclasses such as IgA for mucosal infections (Arstila et al. 1994; Playfair and 244 CHAPTER 4: INFECTIOUS CORYZA AS AN INFECTION MODEL TO MONITOR IMMUNE CELLS AND MOLECULES DURING DISEASE PROGRESSION Bancroft, 2013; Abbas et al. 2014). Th2 cells secrete B cell-activating cytokines such as transforming growth factor- (TGF-), IL-4, IL-5 and IL-10 (Erf, 2004; Playfair and Bancroft, 2013). The % CD4+ cells of total leukocytes plot gave all CD4+ cells detected via flow cytometry over 21 days in whole blood of both control and experimental chickens (p>0.05) (Figure 4.17). For the experimental group: On Day 3 and from Day 7 onwards, high CD4+ percentages (Day 3- 11.41% and Day 7- 15.73%), which was slightly higher than the CD8+ percentage (Day 3- 8.04% and Day 7- 5.94%), were observed (Figure 4.17 and Figure 4.18). This was mainly because CD4+ cells upon being activated during IC infection, they proliferate and transform into effector Th1 and Th2 cells or memory cells, mediating both the innate and adaptive immune responses. Hence, Th cells are found in more numbers as they recruit macrophages, granulocytes, B-cells and CD8+ cells in different tissue locations, compared to CD8+ cells involved in cytotoxic activity and apoptosis. On Days 0, 1 and 4 low CD4+ percentages (Day 0- 6.12%, Day 1- 6.87%, Day 4- 5.01%) were observed, with the same trend seen with CD8+ percentages (Day 0- 2.06%, Day 1- 2.90%, Day 4- 1.73%) (Figure 4.17 and Figure 4.18), this was mainly due to the fact that T cells are mainly involved in the adaptive immune response requiring a few days to be fully functional following the innate immune response. Moreover, unlike B cells that secrete antibodies, T cells need to physically travel to the site of an infection to perform their respective functions, implying they will travel to peripheral tissues, hence their numbers in whole blood could decline as they disperse. In the control group: high CD4+ percentages were observed on Days 11, 15 and 18, which could be due to previous exposure to microorganisms, thus the adaptive immune system was observed to already have been activated (Figure 4.17). 245 CHAPTER 4: INFECTIOUS CORYZA AS AN INFECTION MODEL TO MONITOR IMMUNE CELLS AND MOLECULES DURING DISEASE PROGRESSION Day 0: E3-Score 0 Day 1: E7-Score 1 Day 3: E21-Score 1 Day 4: E4-Score 1 Day 7: E15-Score 2 Day 10: E6-Score 1 Figure 4.16: Blood smears used with the flow cytometry results obtained for % total leukocytes over 21 days showing cell morphology. (Continued on next page) 246 CHAPTER 4: INFECTIOUS CORYZA AS AN INFECTION MODEL TO MONITOR IMMUNE CELLS AND MOLECULES DURING DISEASE PROGRESSION Day 11: E15-Score 3 Day 11: Control 2-Score 0 Day 15: E2-Score 3 Day 18: E4-Score 2 Day 21: E9-Score 2 Day 21: Control 12-Score 0 Figure 4.16: Blood smears used with the flow cytometry results obtained for % total leukocytes over 21 days showing cell morphology. 247 CHAPTER 4: INFECTIOUS CORYZA AS AN INFECTION MODEL TO MONITOR IMMUNE CELLS AND MOLECULES DURING DISEASE PROGRESSION Figure 4.17: % CD4+ cells of total leukocytes plot gave all CD4+ cells detected via flow cytometry over 21 days in whole blood of both control and experimental chickens (p>0.05). For the experimental group: On Day 3 and from Day 7 onwards high CD4+ percentages were observed, which was slightly higher than the CD8+ percentage. On Days 0, 1 and 4 low CD4+ percentages were observed, with the same trend seen with CD8+ percentages. For the control group: high CD4+ percentages on Days 11, 15 and 18 were observed. Cytotoxic T lymphocytes (CTLs) of this lineage, are specialised effector cells that will eliminate target cells such as virus-infected cells and neoplastic cells that contain endogenous antigen (Erf, 2004). CTLs typically express CD8 molecules on their cell-surface in the form of either αβ heterodimers or αα homodimers (Erf, 2004). CD8+ T cells are MHC class I restricted, unlike CD4+ Th cells that are MHC class II restricted and act by recognising antigen peptides bound to MHC class I molecules and APC and interacting with the complex, this in turn triggers the CD8+ cell causing the release or activation of molecules that gives a “suicide signal” (Playfair and Bancroft, 2013; Abbas et al. 2014). CD4+ Th cells may also activate CTLs via IL-2 that promotes cell proliferation and differentiation of functional CTLs (Playfair and Bancroft, 2013). CTLs kill in two ways. The first tactic is the most rapid and important mechanism, whereby granzymes (granule-derived enzymes) are transferred to the target cell through the actions of perforin whereby holes are punctured into the target cell cytoplasm, this in turn activates caspase enzymes that induce apoptosis or 248 CHAPTER 4: INFECTIOUS CORYZA AS AN INFECTION MODEL TO MONITOR IMMUNE CELLS AND MOLECULES DURING DISEASE PROGRESSION “cell suicide” (Playfair and Bancroft, 2013). Secondly, a Fas ligand is present on the surface of CTLs, whereby they use this molecule to trigger apoptosis in the target cell via the Fas receptor (“death receptor”) (Playfair and Bancroft, 2013). In these ways CTLs, are the “serial killers” of the adaptive immune response, as they bind and release cytotoxic mediators with the delivery of a lethal dose, after which they seek other infected target cells as their next victims (Playfair and Bancroft, 2013). The % CD8+ cells of total leukocytes plot shows all CD8+ cells detected via flow cytometry over 21 days in whole blood of both control and experimental chickens (p>0.05) (Figure 4.18). For the experimental group: On Days 0, 1 and 4 very low CD8+ percentages were observed (Figure 4.18), which shows that CD8+ cells are part of the cell-mediated response and adaptive immunity, employed for cytotoxic killing of target cells and the clearance of infected cells, which is why on Days 7 to 21, high CD8+ percentages were observed (Figure 4.18). However, on Day 3, high CD8+ percentages were also observed which might be due to CD8+ cells being recruited by CD4+ Th cells and dendritic cells to become activated and fully functional, during the innate response. It is also possible that natural killer (NK) cells were present on Day 3 leading to an increase in CD8+ percentage, as they share the same cell surface molecules as T cells such as CD8 marker, a putative interleukin‐2 receptor, CD45 and a receptor for IgG, but do not express CD4, major histocompatibility complex class II or immunoglobulin (Göbel et al. 1994). On Day 11 and 15, the control group had high CD8+ percentages, and it was evident from the results that there was previous exposure since cytotoxic T cells are primarily recruited to kill host cells that are infected. However, severe stress induction could also be a contributing factor. It was found that 7% of the bacterial genome of Av. paragallinarum consisted of prophages and/or prophage remnants (Roodt et al. 2012). Moreover, in a study by Boucher et al. 249 CHAPTER 4: INFECTIOUS CORYZA AS AN INFECTION MODEL TO MONITOR IMMUNE CELLS AND MOLECULES DURING DISEASE PROGRESSION (2014), TLR7 was found to be up-regulated, whereby they hypothesized that the prophages and/or prophage remnants could be stimulating TLR7 expression, thus a more infallible and potent immune response was triggered. CD8+ cells are activated specifically for viral invasion, as viruses invade host cells and replicate, hence it is possible that due to the prophages and/or prophage remnants found in the Av. paragallinarum genetic make-up, that CD8+ cells recognise Av. paragallinarum infections as viral, instead of bacterial. There might also be a correlation between TLR7 expression and CD8+ T cells, as TLR7 has been highly expressed in CD8+ cells of individuals with HIV (human immunodeficiency virus) type-1 infection compared with healthy control individuals (Song et al. 2009). Figure 4.18: % CD8+ cells of total leukocytes plot gave all CD8+ cells detected via flow cytometry over 21 days in whole blood of both control and experimental chickens (p>0.05). On Day 3 and from Day 7 onwards high CD8+ percentages were observed, in the experimental group. On Day 11 and 15, the control group had high CD8+ percentages. Cells expressing the CD8 marker are cytotoxic T cells. 250 CHAPTER 4: INFECTIOUS CORYZA AS AN INFECTION MODEL TO MONITOR IMMUNE CELLS AND MOLECULES DURING DISEASE PROGRESSION The CD4+/CD8+ ratio measures the ratio of CD4 helper/inducer cells and CD8 cytotoxic/suppressor cells which are 2 phenotypes of T lymphocytes. In humans, the CD4/CD8 ratio is routinely evaluated and used as a prognostic factor for disease progression in patients with AIDS (acquired immune deficiency syndrome) as well as in viral acute diseases such as cytomegalovirus, Epstein-Barr virus and influenza virus infections (Amadoni et al. 1995). Furthermore, it was found that MHC genes are known to determine the CD4/CD8 ratio in rats (Damoiseaux et al.1999). Similarly, the CD4/CD8 ratio of the peripheral T cells in various chicken lines had been detected which seemed to be dependent on the MHC haplotype (Hala et al. 1991). In chickens, as in humans, the normal CD4/CD8 ratio is still poorly established (Amadoni et al. 1995). Figure 4.19: Flow cytometry profile showing the CD4+/CD8+ ratio of cells detected via flow cytometry over 21 days in whole blood of both control and experimental chickens (p>0.05). For the experimental group: On Days 1, 4 and 7 had high CD4+/CD8+ ratios compared to the rest of the days that had decreased to stable CD4+/CD8+ ratios. For the control group: The CD4+/CD8+ ratios throughout the days were stable with a consistent trend, with a slightly higher ratio on Day 18. The CD4/CD8 ratios in Figure 4.19, were based on the estimated cell counts obtained from the flow cytometry calculations (Appendix D). Figure 4.19 shows the flow cytometry profile 251 CHAPTER 4: INFECTIOUS CORYZA AS AN INFECTION MODEL TO MONITOR IMMUNE CELLS AND MOLECULES DURING DISEASE PROGRESSION with the CD4+/CD8+ ratio of cells detected via flow cytometry over 21 days in whole blood of both control and experimental chickens (p>0.05). For the experimental group: On Days 1, 4 and 7 had high CD4+/CD8+ ratios compared to the rest of the days that had decreased to stable CD4+/CD8+ ratios. The high ratio indicates that the CD4+ percentage was higher than CD8+ cell percentage and that there was an inflammatory response (Figure 4.17 and Figure 4.18). Therefore, the immune system was active and resilient towards IC infection due to Th cell mediation with cytotoxic T cells. For the control group: The CD4+/CD8+ ratios throughout the days were stable with a consistent trend, with a slightly higher ratio on Day 18. The slightly higher ratio could be attributed to an inflammatory response due to suspected exposure or stress. Figure 4.20: Plot with % B and NK (natural killer) cells of total leukocytes detected via flow cytometry over 21 days in whole blood of both control and experimental chickens (p>0.05). For the experimental group: Days 3, 18 and 21 had high percentages of B and NK cells compared to the rest of the days that had low to moderate percentages. For the control group: Days 11 and 18 had high percentages of B and NK cells, compared to the rest of the days that had a stable trend. 252 CHAPTER 4: INFECTIOUS CORYZA AS AN INFECTION MODEL TO MONITOR IMMUNE CELLS AND MOLECULES DURING DISEASE PROGRESSION NK cells are large granular lymphocytes, also involved in the elimination of viral and neoplastic infected cells (Erf, 2004). However, unlike CTLs they do not have the classical TCR for specific antigen recognition and are not MHC restricted, hence being non-specific, NK cells are part of the innate immunity (Playfair and Bancroft, 2013). NK cells are activated via Th1-mediated activity with type-1 cytokines such as IFN-γ, IL-2 and IL-12, which enhances cell proliferation and cytotoxic activity of the NK cells (Erf, 2004; Abbas et al. 2014). Additionally, NK cells share similarities including phenotypic expression with T cells such as the cell surface molecules other than TCR-CD3 complexes (CD8 and CD25) and cytotoxic killing mechanisms (Abbas et al. 2014). NK cells target cells via activating receptors that recognise target-cell surface motifs such as viral products or stress-related proteins, thus initiating a kill signal (Playfair and Bancroft, 2013). However, upon recognition of MHC class I molecules, inhibitory receptors of the NK cells are put on hold or prevent, thus only when MHC molecules are absent or altered can NK cells carry out their functions (Playfair and Bancroft, 2013). Lysis of cells by NK cells is carried out by the granules of NK cells such as perforin and granzymes, whereas induction of apoptosis and intracellular killing are carried out by granulysin (Playfair and Bancroft, 2013). B cells are produced in the bone marrow and mature in the bursa of Fabricius (Davison et al. 2011). Remarkably, only 5% of B cells from the bursa of Fabricius, survive to emigrate to the periphery and participate in active humoral immunity (Scott, 2004). B cells are activated in the lymphoid organs, depending on the route by which the antigen arrives. IC is associated with the upper respiratory tract of chickens, hence mucosal lymphoid tissue such as the paraocular Harderian glands (HG), conjunctiva-associated lymphoid tissue (CALT) and nasal-associated lymphoid tissue (NALT) are regions where activated B cells would be present (Davison et al. 2011). B cells possessing B cell receptors (BCRs) are activated in different ways: via costimulatory molecules (CD21, CD19 and CD81), through direct activation with mitogenic antigens (bacterial endotoxins), repeating antigens (bacterial 253 CHAPTER 4: INFECTIOUS CORYZA AS AN INFECTION MODEL TO MONITOR IMMUNE CELLS AND MOLECULES DURING DISEASE PROGRESSION capsular polysaccharides) that cross-link the immunoglobulin (Ig) surface molecules on B cells eliciting recognition and lastly responses to typical proteins that need to cross-link the Ig surface with further stimuli or assistance from CD4+ bearing T cells (T helper cells-Th2) for B cell proliferation and antibody production/secretion which may include IgM, IgA and IgY (Davison et al. 2011; Playfair and Bancroft, 2013). Thus, antibodies bind to antigen that have a principle biological functions such as agglutination, mucosal surface mediators via FcαR receptor, B cell triggering, opsonization for phagocytosis and can activate the classical complement pathway (Woof, 2004; Playfair and Bancroft, 2013). Figure 4.20 represents a plot with % B and NK (natural killer) cells of total leukocytes detected via flow cytometry over 21 days in whole blood of both control and experimental chickens (p>0.05). For the experimental group: Days 3, 18 and 21 had high percentages of B and NK cells compared to the rest of the days that had low to moderate percentages. On Day 3, there were high percentages due to: NK cells that were recruited and proliferated due to cytokines released by Th1 cells thereby performing cytotoxic activity; and B cells being recruited by Th2 cells via secretion of B cell- activating cytokines such as IL-4, IL-5 and IL-10 that would be actively involved in the adaptive immunity mainly the humoral response. On Day 18 and 21, B cells proliferated, bound to antigen via the B-cell receptor (BCR) and secreted immunoglobulins whereby some B cells became plasma cells and others remained as memory cells. In chickens, there are only three classes of antibody IgA, IgM and IgY, whereby each of the antibodies have specialised functions. For the control group: Days 11 and 18 had high percentages of B and NK cells, compared to the rest of the days that had a stable trend. The control group showed both innate and adaptive immune responses since the plot shows the percentages of B and NK cells, hence the chickens were indeed exposed to pathogen before or during the study, however from our daily monitoring scores the control chickens did not develop IC related symptoms, hence exposure could be due to other microorganisms or the bacterial load of Av. paragallinarum serovar C-3 was not sufficient to 254 CHAPTER 4: INFECTIOUS CORYZA AS AN INFECTION MODEL TO MONITOR IMMUNE CELLS AND MOLECULES DURING DISEASE PROGRESSION cause disease. Fortunately, the incident from the infection model was detected, showing that the infection model was correctly set-up and designed, and discrepancies could be accounted for despite the drawbacks. However, severe stress could also be a possible contributing factor in the overall immune responses observed, as the chickens were in isolation and when bled, their physical and mental wellness could have been compromised during the study. Moreover, birds may develop a leukopenia and lymphopenia in the initial stress response, however after 12h later may show leucocytosis and heterophila (Davison and Flack, 1981). Unfortunately, for statistical analysis using the Student’s t-test we failed to reject the null hypothesis (p>0.05), that there is no difference between the means of the experimental and control groups (Appendix D). Furthermore, the t-statistics were not statistically significant, hence it was concluded that there was not sufficient evidence available to suggest the null hypothesis false at a 95% confidence level. From our data analysis, a considerable variation between birds of the control and experimental groups was observed. This could be due to a small sample size used for the study (n=38), thus the sample size was not large enough to allow the null hypothesis to be rejected at the p<0.05 level. Hence a larger sample size should be chosen for future studies. Moreover, there were bird-to-bird differences as it seemed that some birds were previously exposed prior to the study despite being SPF, implying that their immune system was already well established. 4.3.5. Sandwich enzyme-linked immunosorbent assay (ELISA) The ELISA assay was carried out (p<0.05) and the results are shown in (Figure 4.21). Day 0 chickens that were SPF, were not yet infected when they were bled at a score 0, however moderate levels of IL-8 titres for chickens E5, Control 1 and 2 were observed; with a very 255 CHAPTER 4: INFECTIOUS CORYZA AS AN INFECTION MODEL TO MONITOR IMMUNE CELLS AND MOLECULES DURING DISEASE PROGRESSION high IL-8 titre for chicken E2. A possible explanation for the Day 0 chickens with moderate to high IL-8 expressions could be attributed to previous exposure to microorganisms before the study started, which could have been transmitted before collection from suppliers, during transportation or while they were being housed at the experimental facility in spite of necessary precautions and disinfection routines which were implemented during transportation and while housing at the facility at the University of the Free State. The control groups had a stable trend over the duration of the study, showing low expression of IL-8 on Days 3, 7 and 15. However there were moderate levels of IL-8 titre at the beginning (Day 0) with Control 1 and 2 chickens at score 0 and at the end of the trial (Day 21) with Control 10 and 11. Control 1 and 2 chickens could have been previously exposed as mentioned. Control 10 and 11 (named after the nth time they were bled, where n is a number), could have also been exposed to IC during the study, however the threshold host density was not met and was not sufficient to cause disease, leading to a lowered infection rate. Although the Day 0 and control chickens had moderate to high cytokine titres of IL-8, they did not show any IC related signs or symptoms (asymptomatic). For the experimental group it was observed that IL-8 expression from moderate to high titres was found across all IC clinical scores 1, 2 and 3. Chicken E13 with score 1 on Day 3 had a moderate IL-8 titre. High cytokine titres were observed for experimental chickens presented with score 3: E15 (Day 7), E16 (Day 7) and E2 (Day 15). Moderate levels of IL-8 production were observed, for chicken E19 with score 3 on Day 15. Only at a later stage were moderate levels of IL-8 production for chickens presented with score 2 observed: E11 (Day 15), E6, E9, E11 and E26 (Day 21); but not during the initial stages of score 2, as seen with chickens E8 and E26, having low cytokine titres. 256 CHAPTER 4: INFECTIOUS CORYZA AS AN INFECTION MODEL TO MONITOR IMMUNE CELLS AND MOLECULES DURING DISEASE PROGRESSION In mammals, IL-8 is a CXC chemokine and has therefore been renamed CXCL8 and is involved in the recruitment, activation and movement of human neutrophils (Cacalano et al. 1994; Davison et al. 2011). The chicken orthologue of IL-8/CXCL8 was suggested to be 9E3/CEF4, known as cCAF (chicken chemotactic and angiogenic factor) (Martins-Green and Feugate, 1998; Kaiser et al, 1999). However, it was proposed that 9E3/CEF4 would be called chicken CXCLi2 (chCXCLi2, “i” denoting inflammatory function) (Kaiser et al. 2005). To date, IL-8 has been mostly associated with mucosal and gut-associated immunity, linked to diseases, such as rheumatoid arthritis, inflammatory bowel disease and highly pathogenic avian influenza (H5N3 and H7N9) in human hosts, as well as Salmonella enteritidis and Campylobacter jejuni infections in avian hosts (Luster, 1998; Kogut, 2002; de Jong et al. 2006; Smith et al. 2008; Zhou et al. 2013). chCXCLi2/IL-8 is a potent chemoattractant and during an inflammatory response, there is a dramatic increase in the secretion of this cytokine resulting in the selective recruitment and influx of specific leukocytes into inflamed tissue or site of infection such as monocytes, heterophils and macrophages (Kaiser et al., 2008). IC is an upper respiratory disease, whereby for score 1 there is mild facial swelling, score 2 there is bilateral facial oedema and score 3 there is severe bilateral oedema and conjunctivitis with or without haemorrhage, whereby the chickens also develop diarrhoea. The results with regards to the expression of IL-8 with sandwich ELISA, coincide with the observed clinical scores of IC in infected chickens, whereby as the disease becomes more severe the expression and cytokine titres of IL-8 increases. Moreover, the findings also correlate to a study by Boucher et al. (2015), where there was a significant up-regulation for IL-8 during disease score 2 with Av. paragallinarum serovar C-3 infection. Furthermore, the flow cytometry (Figure 4.15 - Figure 4.20) and IL-8 assay (Figure 4.20) results showed that although the chickens were supposedly SPF before the start of the study, the chickens 257 CHAPTER 4: INFECTIOUS CORYZA AS AN INFECTION MODEL TO MONITOR IMMUNE CELLS AND MOLECULES DURING DISEASE PROGRESSION already had a well-established immune system possibly due to prior exposure, since they were able to generate innate and adaptive immune cells as well as produce cytokines even before being exposed to the experimental Av. paragallinarum serovar C-3 (SA-3 strain), fortunately this was detected. Hence, it was possible that due to prior exposure, severe symptoms as described in literature were not observed in this study, which might also be the reason why some chickens recovered quickly after a score was reached with only a minority of chickens reaching a score of 3. In future studies, it is recommended to screen for antibodies even if suppliers guarantee that the chickens provided are SPF, as the immunity of chickens for the study could be compromised, especially if the effect of a drug or challenge study is being conducted which could lead to misleading or even erroneous results. 258 CHAPTER 4: INFECTIOUS CORYZA AS AN INFECTION MODEL TO MONITOR IMMUNE CELLS AND MOLECULES DURING DISEASE PROGRESSION Figure 4.21: Graphical representation and statistical analysis of the sandwich ELISA assay conducted on chicken plasma samples from the experimental trial for the cytokine IL-8 (p<0.05). Day 0 chickens were not yet infected when they were bled at a score 0, however, moderate levels of cytokine titre for chickens E5, Control 1 and 2 were observed; with a very high cytokine titre for chicken E2. Chicken E13 with score 1 on Day 3 had a moderate cytokine titre. High cytokine titres were observed for experimental chickens presented with score 3: E15 (Day 7), E16 (Day 7) and E2 (Day 15). Moderate levels of IL-8 production, for chicken E19 with score 3 on Day 15 were observed. Only at a later stage were moderate levels of IL-8 production for chickens presented with score 2 observed: E11 (Day 15), E6, E9, E11 and E26 (Day 21); but not during the initial stages of score 2, as seen with chickens E8 and E26 having low cytokine titres. The control group had a stable and low expression of IL-8 on Days 3, 7 and 15. However there were moderate levels of cytokine titre at the beginning (Day 0) with Control 1 and 2 chickens at score 0 and at the end of the trial (Day 21) with Control 10 and 11. Although, the Day 0 and control chickens had moderate to high cytokine titres of IL-8 expressed, they did not show any IC related signs or symptoms. 259 CHAPTER 4: INFECTIOUS CORYZA AS AN INFECTION MODEL TO MONITOR IMMUNE CELLS AND MOLECULES DURING DISEASE PROGRESSION 4.4. Conclusion Older birds were used in this study, as the sinuses of the birds at 25 weeks would be more developed than those at 20 weeks. This preliminary and novel study on IC infection, was successful, as disease progression was able to be monitored and each clinical score pertaining to IC related signs and symptoms observed (score 1-3), correlating the progression of the disease at different scores during different time intervals (days), thus deducing the approximate days of innate and/or adaptive mechanisms at play. Peripheral whole blood smears of chickens at different scores were also obtained, which provided us with the different cell morphologies and their respective functions in controlling disease. Furthermore, it was observed that the innate immunity was fast acting requiring less than a few hours or days to be fully functional, occurring within the first week of infection (7 days), compared to the adaptive response which took longer requiring several days (at Day 11 onwards). The leukocyte, CD4 and CD8 flow cytometry profiles of chicken blood samples at different scores were evaluated, giving us insight into the innate, humoral and cell-mediated immune responses involved in pathogen elimination. Moreover, the CD4+/CD8+ ratio was higher during the innate immune phase, indicating that Th cells have a major role to play in IC related infections, which could also be observed with the flow cytometry profile of CD4+ cells percentages, however this area needs further investigation to determine whether the response was Th1 or Th2 mediated. With regards to the data obtained from egg production, highly variable egg production trends in both the control and experimental groups were observed. No statistically significant decline in egg production as stated in literature was noted. However, this anomaly could be as a result of a very small sample size in our study compared to the intensive chicken industry. Results with the expression of IL-8 with the sandwich ELISA coincide with all observed clinical scores (score 1-3) of IC in infected chickens, whereby as the disease becomes more severe the expression and cytokine titres of IL-8 increases, due to facial oedema severity as the diseases progresses, eventually leading to haemorrhage with severe facial oedema and conjunctivitis. 260 CHAPTER 4: INFECTIOUS CORYZA AS AN INFECTION MODEL TO MONITOR IMMUNE CELLS AND MOLECULES DURING DISEASE PROGRESSION Strangely, the SPF chickens had been injected with Av. paragallinarum serovar C-3 and had developed clinical symptoms pertaining to IC whereby all scores were reached, however the disease was not as severe as observed in literature with field chickens, which is mainly because Av. paragallinarum infections in chickens from the field or extensive chicken industry often occur as mixed infections, hence the presence and possibility of other bacteria or viruses as secondary infections lead to complicated IC. However, from our findings only a minority of chickens reached a score of 3, whereas the rest of the chickens recovered. The control group, from the flow cytometry profiles and IL-8 assay results obtained, showed that they had an already well-established immune system suspected due to exposure to microorganisms before or during the study, however this did not lead to pathology as the chickens were asymptomatic for IC. If the entire cohort had been exposed to microorganisms before the study was conducted or during transportation of the chickens, memory cells had been formed. Therefore, the clinical signs and symptoms that was anticipated and expected of an IC infection was not fully observed during disease progression, as the chickens recovered swiftly following infection with Av. paragallinarum serovar C-3. In this study, after each clinical score was obtained, 2-4 chickens were sacrificed, whereby post-mortem examination and immunohistochemistry were performed (Chapter 5). The IC infection model in this study could be used as a prognostic tool to monitor disease progression or the effect of therapeutic products (vaccines), for future studies. Failed vaccination attempts are still a major problem with IC, hence the knowledge gained from the study could help improve diagnostic testing for Av. paragallinarum serovar C-3 and/or other poultry diseases due to a better understanding of the avian immune system, which can also aid in the development of novel products such as an infectious coryza (IC) specific ELISA kit, avian haematology kits or CD4 and CD8 biomarkers, as well as in vaccine development. 261 CHAPTER 4: INFECTIOUS CORYZA AS AN INFECTION MODEL TO MONITOR IMMUNE CELLS AND MOLECULES DURING DISEASE PROGRESSION REFERENCES Abbas, A.K., Lichtman, A.H., Pillai, S. 2014. Cellular and Molecular Immunology E-book. Elsevier Health Sciences. pp. 544. Altschul, S. F., Gish, W., Miller, W., Myers, E. W., Lipman, D. J. 1990. Basic local alignment search tool. 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Comparative pathology of inflammation in the higher vertebrates (reptiles, birds and mammals). Journal of Comparative Pathology, 99 (1): 1-26. Playfair, J. H. L., Bancroft, G. J. 2008. Infection and immunity, 3rd ed. edn. Oxford University Press: USA. pp. 360. Playfair, J.H.L., Bancroft, G.J. 2013. Infection and Immunity, 4th edn. Oxford University Press, Oxford. pp. 400. Raetz, C.R., Whitfield, C. 2002. Lipopolysaccharide endotoxins. Annual Review of Biochemistry, 71(1): 635-700. Roodt, Y., Bragg, R.R., Albertyn, J. 2012. Identification of prophages and prophage remnants within the genome of Avibacterium paragallinarum bacterium. Sequencing. Scott, T.R. 2004. Our current understanding of humoral immunity of poultry. Poultry Science, 83(4): 574-579. Smith, C.K., AbuOun, M., Cawthraw, S.A., Humphrey, T.J., Rothwell, L., Kaiser, P., Barrow, P.A., Jones, M.A. 2008. Campylobacter colonization of the chicken induces a proinflammatory response in mucosal tissues. FEMS Immunology and Medical Microbiology, 54(1): 114-121. 267 CHAPTER 4: INFECTIOUS CORYZA AS AN INFECTION MODEL TO MONITOR IMMUNE CELLS AND MOLECULES DURING DISEASE PROGRESSION Song, Y., Zhuang, Y., Zhai, S., Huang, D., Zhang, Y., Kang, W., Li, X., Liu, Q., Yu, Q., Sun, Y. 2009. Increased Expression of TLR7 in CD8+ T Cells Leads to TLR7-Mediated Activation and Accessory Cell-Dependent IFN-γ Production in HIV Type 1 Infection. AIDS Research and Human Retroviruses, 25(12): 1287-1295. Srinivasa, C.S., Reddy, P.K., Aruna, D. 1989. Incidence of poultry diseases in Kurnool District (AP). Poultry Adviser, 22: 45-48. Thitisak, W., Janviriyasopak, O., Morris, R. S., Srihakim, S., Kruedener, R. V. 1988. Causes of death found in an epidemiological study of native chickens in Thai villages. In: Proceedings of the 5th International Symposium on Veterinary Epidemiology and Economics. Acta Veterinaria Scandinavica (Denmark). pp. 200-202. Woof, J.M., Kerr, M.A. 2004. IgA function–variations on a theme. Immunology, 113(2): 175- 177. Zhou, J., Wang, D., Gao, R., Zhao, B., Song, J., Qi, X., Zhang, Y., Shi, Y., Yang, L., Zhu, W., Bai, T. 2013. Biological features of novel avian influenza A (H7N9) virus. Nature, 499(7459): 500. 268 ANNEXURE C ANNEXURE C 269 Day 0 Control 1-Score 0 Control 2-Score 0 E3-Score 0 E4-Score 0 E5-Score 0 Day 1 E6-Score 1 E7-Score 1 E8-Score 1 E9-Score 1 E11-Score 1 Day 3 E12-Score 1 E13-Score 1 E16-Score 1 E20-Score 1 E21-Score 1 Day 4 E4-Score 1 E11-Score 1 E17-Score 1 E24-Score 1 E25-Score 1 Day 7 E15-Score 2 E16-Score 2 E22-Score 2 E24-Score 2 E26-Score 2 E28-Score 1 E30-Score 1 Day 10 E6-Score 1 E9-Score 2 E11-Score 2 E17-Score 3 E22-Score 3 Day 11 Control 1-Score 0 Control 2-Score 0 Control 3- Score 0 E15-Score 3 E22-Score 3 E26-Score 2 E28-Score 2 Day 15 Control 4-Score 0 Control 5-Score 0 Control 6-Score 0 E2-Score 3 E11-Score 2 E13-Score 3 E19-Score 3 E21-Score 2 Day 18 Control 7-Score 0 Control 8-Score 0 Control 9-Score 0 E4-Score 2 E11-Score 3 E15-Score 3 E19-Score 3 E27-Score 3 Day 21 Control 10-Score 0 Control 11-Score 0 Control 12-Score 0 Control 13-Score 0 E6-Score 2 E9-Score 2 E11-Score 3 E15-Score 2 E17-Score 2 E21-Score 2 E26-Score 2 E27-Score 0 E28-Score 2 ANNEXURE D ANNEXURE D 284 Printed by: Admtnistrator Printed by: Admintstrator BD FACSDiva 8.0.1 BD FACSD|Va 8.0.1 E3 Day 0,CD4/CD8 9 5 o aa 20 30 FSC-A 2A 30 40 50FSC.A (x 1 000) Experiment Name: Experiment Name: Chicken new batchChicken new batch Specimen Name: Name: E3 Day Specrmen 0E3 Day 0 Namei Tube Namer Tube CD4/CD8CD45/CD4 Record Date Date: Aug2 2011 Record 11 32:00..Aug2,2011 it:31r12 AM $oP: $oP: AdministratorAdminrstrator #Events o/oParent Bgpu.lgLo-t, PEvelrsX coa YIVPHOCYTES 90 0.0 X co+ cD45 1064 26 28.9 297 Chicken new batch Page 1 of 2 Printed on: lvlon Nov 6, 2017 11:59t20 CAT Chicken new batch Page 2 of 2 Printed on: Mon Nov 6,2017 11159:20 CAT Printed by: Admintstrator Printed by: Administrator BD FACSDiva 8.0.1 BD FACSD|Va 8.0.1 20 30 40 50 FSC-A (x 1,000) Experiment Name: Chicken new batch Experiment Name: Chicken new batch Specimen Namer E4 Day 0 Specimen Name: E4 Day 0 Tube Namei CD45/CD4 Tube Name: CD4/CD8 Record Date: Aua 2, 201 t 1134:A8 RecordDate: Auo2,201/11:35:'13..... $oP: Admrnistrator $OP: Administrator Populdlplr" - - 4Lvpnts LYMPHOCYTES 27 KcD8" 18 15 5s6 X co+ 12 44.4 232 0.1 Chicken new batch Page 1 of 2 Printed onr Mon Nov 6, 2017 12102:29 CAT Chicken new batch Page 2 of 2 Printed on: N,lon Nov 6,2017 12.O2t29 CAT Printed by: Administrator Prlnted by: Adminlstrator BD FACSD|Va 8.0.1 BD FACSDiva 8.0.1 20 3a 40 50 FSC-A 20 30 40 50 (x 1,000) FSC-A (x 1,000) Experiment Name: Chicken new batch Specimen Name: E5 Day 0 Tube Name: CD45/CD4 Experiment Namet Chicken Record new Date: batchAua2 2017 11.4A34 Specimen Namer E5 Day 0 $oP: Administrator Tube Namer CD4/CD8 Record Date: Aug2,2017 11:41:3. . $oP: Admrnistrator LYIVIPHOCYTES 14 0.0 X coas 7 500 Popuralron qEvenrs X co+rco+s * 1 50.0 Xcd8__ -*-? 1s8 0.'1 X coa Chicken new batch Page 1 of 2 Printed ont Mon Nov 6,2017 12t06:58 CAT Chicken new batch Page 2 of 2 Printed on: Mon Nov 6,2017 12t06;58 CAT Printed by: Administrator Printed by: Administrator BD FACSDiVa 8.0.1 tqqntrol 1 Dly 0-CDrt5/C!4 5 aoa 70 80 (x 1 000) 70 80 (x 1,000) Experimeni Name: Chicken new batch Specimen Name: Control 1 Day O Tube Name: Experiment Name: Chicken new CD45/CD4 batch Record Datei Specimen Name: Aug 2 Control 1 Day 20,17 010:56:3. $OP: Tube Name: Administrator CD4/CD8Record Date Auo2 2O1l 10:58:23. . $oP: Administrator Populdhon A[ vents X coa lss Chicken new batch Page 1 of 2 Printed onr Mon Nov 6,2017 12:10;10 CAT Chicken new batch Page 2 of 2 Printed on: l4on Nov 6. 2017 12:10r10 CAT Printed by: Administrator Printed byi Administrator BD FACSDiva 8.0.1 BD FACSD|Va 8.0.1 2A 20 3030 FSC-A FSC-A Experiment Namer Chicken new batch Specimen Name Control 2 Day 0 Experiment Name Chicken new batch Tube Namei CD4/CD8 Specimen Name: Control 2 Day 0 Record Date: Aug2 2017 11:27:4 .. Tube Name: CD45/CD4 $OP: Administrator Record Date: Auo 2,2017 11:26:00 $oPi Admrnistralor PoouldLo'n* " tEventsD{"coa rr X cD4 139 LYI\,/IPHOCYTES 261 X coas 132 50.6 X co+lco+s 129 49.4 1,006 0.2 Chicken new batch Page 1 of 2 Printed on: Mon Nov 6, 2017 10:33r05 CAT Chicken new batch Page 2 ol 2 Printed on: l4on Nov 6,2017 10:33:06 CAT Printed by: Administrator Printed by: Adminrstrator BD FACSDiva 8.0.1 BD FACSDiva 8.0.1 30 40 50 30 40 50 FSC-A (x 1,000) FSC-A (x 1 000) Experiment Name: Chicken new batch Specimen Name: E6 Day 1 Expeflment Name: Chicken new batch Tube Name: CD4/CD8 Specrmen Name: E6 Day 1 Record Date: Aug2,2011 . Tube Name 11:44 CD45/CD4 Record Dater Aua2 2017 11:425.. $OP: Administrator Populdtion ftvenrs trTD8 Xco+ 149 LYI\,lPHOCYTES 246 1 CD45 112 45.5 x CD4/CD45 134 54.5 I rotnl co+s 1,315 0.1 -sl Chicken new batch Page 1 of 2 Printed on: Mon Nov 6, 2017 12:07:53 CAT Chicken new batch Page 2 of 2 Printed on: Mon Nov 6, 2017 12107:53 CAI Printed by: Adminrstrator Printed by: Administrator BD FACSDiva 8.0.1 BD FACSDiva B.0.1 : O a a 20 30 20 30 40 50 FSC-A FSC-A (x 1,000) Experiment Name: Chicken new batch Experiment Namei Chicken new batch Specimen Name: E7 Day 1 Specimen Name: E7 Day 1 Tube Name: CD45/CD4 Tube Namer CD4/CD8 Record Datei Auo 2, 2O17 1 1 i45r26 A[,4 Record Date: Aul2,2017 11:46:2 $OP: Administrator Iffi LYI\,4PHOC X co+s 't05 X co+lco+s 70.8 I rorAl cD45 2,840 1.3 Chicken new batch Page 1 of 2 Printed on: Mon Nov 6,2017 12:11:55 CAT Chicken new batch Page 2 ol 2 Printed on: Mon Nov 6,2017 12:11t55 CAT Printed by: Administrator Printed by: Adm nistrator BD FACSDiVa 8.0.1 BD FACSD|Va 8.0.1 E8 Day 1-CD45/CD4 5 5 O Oa a a 10 20 10 2A 30 40 5030 FSC.A FSC.A (x 1 000) Experiment Name: Chlcken new batch Expenment Name: Chicken new batch Specimen Name: E8 Day 1 Specimen Namer E8 Day 1 Tube Name: CD45/CD4 Tube Name: CD4/CD8 Record Date: Auq2,2011 11:47:4... Record Date: AuA2,2017 11.49:... $OP: Administrator Populdtron *"* LYMPHOCYTES '' FEv"p*n5 s 3 X"cD8 cD45 48.6 Xcoa 5 cD4/CD45 51.4 TOTAL CD45 1, 1.0 Chicken new batch Page 1 of 2 Printed on: Mon Nov 6, 2017 12:1,2t46 CAT Chicken new batch Page 2 ot 2 Printed on: Mon Nov 6, 2017 12:12;46 CAT Printed by: Admlnistrator Printed by: Admrnrstrator BD FACSD|VA 8.0.1 20 30 40 50 FSC-A (x 1,000) Experiment Name: Chicken new batch Specimen Name: Eg Day '1 Experiment Namer Chicken new batch Tube Namel CD45/CD4 Specimen Namer E9 Day'l Record Date Au92,2011 11r50:'18.. Tube Name: CD4/CD8 $oP, Administrator Record Date: Aug 2,2017 11:51:... $OPi Administrator #Events LYMPHOCYTES 151 pooulat-io n #EvPnts""28X co+s 84 55.6 Mcoa X co+ico+s 6t 44.4 X co+ 2,339 1.2 Chicken new batch Page 2 af 2 Prlnted on: N4on Nov 6, 2017 !2114:44 CAT Chicken new batch Page 1 of 2 Printed on: l4on Nov 6, 2017 l2i!4143 CAf nistrator Printed byr AdministratorPrinted by: Adm BD FACSDiva 8.0.1 BD FACSDiva 8.0.1 aoa 30 40 50 1O 20 30 40 50 FSC-A (x 1 000) FSC-A (r I ooo) Experiment Name: Chicken new batch Specrmen Name: E1 1 DaY 1 Tubc Name CD45/CD4 Experiment Name Chlcken new batch Record Date: Au92,2011 11 5248 Specimen Name E11 DaY 1 $OPr Administrator Tube Name: CD4/CD8 Record Date: Aua2 2O1'l 11 54:0... $OP. Adm nistraror %Parent t Lvupuocvrcs 0.1 X coas Pooulalon EcDs- "-*'si dfvenls X co+lco+s L torar co+s Printed on: Mon Nov 6 12i14i53 CAT Chicken new batch Page 1 of 12 Printed on: N4on Nov 6,2017 12:14:53 CAT Chicken new batch Page 2 of 12 ,2017 Pnnted by: Administrator Printed byr Administrator BD FACSDiva 8.0.1 BD FACSDiva 8.0.1 20 20 3030 FSC-A FSC-A Experiment Name: Chicken new batch Experiment Name: Chicken new batch Specimen Namer E12 Day 3 Specimen Name E12 Day 3 Tube Name: CD45/CD4 Tube Name: CD4/CD8 Record Date: Auo 4,2017 10:48:5 Record Dater AuA4,2A17 10:4949... $OP Administrator $OP: Admrnistrator oopulation -- *'--_-tE-v enls"r1 LYI\,lPHOCYTES 0.2 X"CD8* cD45 54.8 Xl coa CD45/CD4 45.2 TOTAL CD45 05 Chicken new batch Page 1 of 2 Printed on: Mon Nov 6, 2017 121!6:47 CAf Chicken new batch Page 2 of 2 Printed on: Mon Nov 6, 20L7 L2tL6i47 CAf Pnnted by: Administrator Pnnted by: Admin strator BD FACSDiva 8.0,1 BD FACSDiva 8.0.1 20 40 20 30 40 5030 50 FSC-A FSC-A (r j ooo)(x 1 000) Experiment Name: Chicken new batch Specimen Namer E13 Day 3 Tube Name: CD45/CD4 Date: Experiment Namer Chicken new batchRecord Aua 4,201'1 10:50:37 AM $OP: Specimen Name: E13 Day 3Admrnistrator Tube Name: CD4/CD8 Record Date: Aug 4,20'17 10:51:... $OP Adm nistrator LYIi]PHOCYTES 212 0.3 CD45 1s3 Populat X o-n- --- dEventsco+srco+ 59 21.8 L rorel co+s Xa-D8 502 0.7 X coa Chicken new batch Page 1 of 2 Printed on: l4on Nov 6, 2017 12r 17:58 CAT Chicken new batch Page 2 of 2 Printed on: Mon Nov 6, 2017 12:17:59 CAT Printed by: Administrator Printed by: Administrator BD FACSD|VA 8.0.1 BD FACSDiVa 8.0.1 oo o 20 30 40m FSC-A (r 1,@0) Experiment Name: Chicken new batch Experiment Name: Chicken new batch Specimen Name: E16 Day 3 Specimen Name: El6Day3 Tube Name: CD4/CD8 Tube Name: cD45/CD4 Record Date: Aug 4,2017 10:53:... Record Date: Aug4,2017 10:52:19 AM $OPr Administrator $oP: Administrator Population fEvents Population #Events ffi I t-vtrlpnocrrcs 90 ,.#1 Xcoa 133, EI coas 59 X coa 3'1 Chicken new batch Page 1 of 2 Printed on: lYon Nov 6, 2017 12r18:45 CAT Chicken new batch Pa9e2 of 2 Printed on: l4on Nov 6,2017 12r18:45 CAT Printed by: Administrator Printed byr Admin strator BD FACSD|Va 8.0.1 BD FACSDiva B.0.1 O 40 50 (x 1,000) Experiment Name Chicken new batch Exoenment Name: Chicken new batch Specimen Name: E20 Day 3 Specimen Name: E20 Day 3 Tube Name: CD45/CD4 Tube Name: CD4/CD8 Record Date: AUA 4,2017 10153:55 AN.4 Record Date: Auq 4, 2017 10:54:45 $OP: Administrator #Events o/oParent LYMPHOCYTES 212 CD45 161 61.4 cD45/CD4 '10s 38.6 TOIAL CD45 625 0.'l til Chicken new batch Page 1 of 2 Printed on: Mon Nov 6,2017 12:18:55 CAT Chicken new batch Page 2 of 2 Printed on: Mon Nov 6, 2017 12:18:56 CAT Printed by: Administrator Printed by: Adminrstrator BD FACSDiva 8.0.1 BD FACSD|Va B.0.1 o Oa @ a a Experiment Name: Ch cken new balch Specimen Name E21 Day 3 Tube Name: CD45/CD4 Experiment Name: Chicken new batch Record Date: Auq 4,2017 10:55:3 Specimen Name: E21 Day 3 $OP Administrator Tube Name: CD41CD8 Record Date: Aug 4,2017 10:56:... $OP: Administrator Populat,oa 4fvents Xcrt;* - ""*'* -"id' Xco+ 61 Chicken new batch Page 1 of 12 Printed on: Mon Nov 6, 2017 l2t!9i15 CAf Chicken new batch Page 2 of 12 Printed on: I4on Nov 6, 2017 12:19:15 CAT Printed by: Administrator Printed by: Admrnistrator BD FACSDiva 8.0.1 BD FACSDiva 8.0.1 20 30 40 50 A a0 90FSC (x 1,ooo) (x 1,000) Experiment Name: Chicken new batch Experiment Namer Chicken new batch Specimen Name: E4 Day 04/08 Specrmen Name: E4 Day 04/08 Tube Namer CD45/CD4 Tube Name: CD4/CD8 Record Date: Aug 7, 2017 9:28:16 AM Record Date: Au97,2017 9:29:19.. $OP: Administrator $OP Admrnr\traLor #Events o/o Parent Pop-latron tl vpnls LYMPHOCYTES 200 0.1 X'Cii8--- -**^ -16 X coas 121 X coa I co+slco+ 19 Chicken new batch Page 1 of 3 Printed onr Mon Nov 6,2017 12:19i31 CAT Chicken new batch Page 2 of 3 Printed on: Mon Nov 6. 2017 12:19:32 CAT Printed byr Administrator Printed by: Administrator BD FACSD|VA 8.0.1 BD FACSDiva 8.0.1 E1 1 Dav 04/08-CD4/CD8 I .:. ... .',., i' 20 30 40 50 20 30 FSC-A (x 1 ooo) FSC A Experiment Namer Chicken new batch Specimen Namer E1 1 Day 04/08 Tube Namer CD4/CD8 Experiment Name: Chicken new batch Record Dater Auq 7, 2017 9 3312.. Specimen Name: El1 Day 04/08 $OP: Administrator Tube Name: CD45/CD4 Record Daiel Augl .2017 9:31.2.. $OP: poouldtton 4fven,sAdministrator FX9 cl'rD---8--' ' 43 L 1 cD45 61.8 CD45/CD4 38.2 TOTAL CD45 0.4 Chicken new batch Page 1 of 2 Printed on: Nlon Nov 6, 2017 12:19:50 CAT Chicken new batch Page 2 of 2 Printed on: Mon Nov 6,2017 12:19:51 CAT Pnnted by: Administrator Printed byj Administrator BD FACSDiVa 8.0.1 BD FACSDiva 8,,i.1 20 30 10 20 30 40 FSC-A FSC-A Experiment Name: Chlcken new batch Experimenl Name: Chicken new batch Specimen Name: E17 Day 04/08 Specimen Name: E17 Day 04/08 Tube Name: CD4/CD8 Tube Name: CD45/CD4 Record Date: Auq 7, 2017 9136:1 .. Record Date: Aug 7.2017 9:34 5.. $OPi Administralor $OP: Administrator Pooula[on #E{ents Fx4 cD8 93 L 0.1 X co+ 134 cD45 56.6 cD45tCD4 43.4 TOTAL CD45 0.7 Chicken new batch Page 1 of 2 Printed on: Mon Nov 6,2017 12:20:52 CAT Chicken new batch Page 2 of 2 Printed on: Mon Nov 6, 2017 12:20:53 CAT Printed by: Administrator Printed by: Administrator BD FACSDiva 8,0.1 BD FACSDiva 8.0.1 20 30 FSC-A Experiment Name: Chicken new batch Specimen Name E24 Day 04lOB Experiment Name: Chrcken new batch Tube Name: CD45/CD4 Specimen Name E24 Day 04108 Reco.d Date: AuT7,2017 9:37:2 Tube Namer CD4/CD8 $OP: Administrator Record Date: Aug 7.2017 9:38:30 LYI\,4PHOCYTES cD45 42.2 cD45/CD4 57.8 TOTAL CD45 0.6 Chicken new batch Page 1 of 2 Printed on: Mon Nov 6, 2017 12:21:04 CAT Chicken new batch Page 2 of 2 Printed on: Mon Nov 6, 2017 12:21i05 CAf Printed by: Admrnistrator Printed by: Administrator BD FACSDiva 8.0.1 BD FACSDiva 8.0.1 : o o @ @ a a 20 30 FSC.A Expe.iment Name: Chicken new batch Specimen Name: E25 Day 04108 Tube Namer CD45/CD4 Experiment Name: Ch cken new batch Record Date: Auo 7,2017 9r40r5. Specimen Namer E25 Day 04/08 $OP: Administrator Tube Name: CD4/CD8 Record Date: Auo 7,2017 9:4313... $OP: Adminrstrator #Events ahPa.enl E Lvrr,rpHocvrrs 346 | 0.1 X co+s 't17 I 33.8 Populalion tLvenls X co+slco+ 2291 66.2 x cD8 22 X co+ Chicken new batch Page 1 of 2 Printed on: Mon Nov 6, 2017 12i21i15 CAI Chicken new batch Page 2 of 2 Printed on: l4on Nov 6,2017 12:21:15 CAT Prlnted by: Admlnistrator Printed by: Administrator BD FACSD|Va 8.0.1 BD FACSDiva 8.0.1 a o @ @ 20 30 40 50 FSC,A (x 1 oo0) Experiment Namer Chicken new batch Specimen Name: El5 Day 07/08 Tube Name: CD4/CD8 Experiment Chicken new Record Date: Augi,2011 4:03:42. .Namel batch Administrator Specimen Namel E15 Day 07/08 $OPr Tube Name: cD45/CD4 Record Daiei Auq7,2017 4:02:5. Bestcl-ao-tl9st" -..-.., .. .....--^ ,........{B-s$t$oPr Adminrstrator X 16 X co+ #Events o/oParent LYI\,4PHOCYTES 199 0.s X co+s 105 i s2.8 X co+srco+ 94 47.2 Chicken new batch Page 1 of 2 Printed on: Mon Nov 6, 2017 12:21125 CAI Chicken new batch Page 2 of 2 Printed on: N4on Nov 6, 2017 12:21:25 CAT Printed by: Administrator Printed by: Administrator BD FACSD|Va 8.0.1 BD FACSD|Va 8.0.1 30 40 50 10 20 30 40 50 FSC.A FSC-A(x 1,000) (x 1,000) E16 Day 07i08-CD4lC!! Experiment Name: Chicken new batch Spec men Name: E16 Day 07/08 Expenmenl Name: Chicken new batch Tube Name: CD45/CD4 Specrmen Namei E'16 Day 07/08 Record Date: Aua 1,2017 4:04130.. Tube Namer CD4/CD8 $OP: Administrator Record Date: Auq 7, 2017 4:05:1.. $OP: Administrator LYMPHOCYTES Popu'atro. 512 EL vents cD45 361 63.1 Elcob ?a CD45/CD4 211 36.9 E co+ 116 TOTAL CD45 1,645 2.O Chicken new batch Page 1 of 2 Printed on: l'4on Nov 6, 2017 L2:21:36 CAf Chicken new batch Page 2 of 2 Printed onr lvlon Nov 6,2017 12i21:36 CAT Printed byi Adminrstrator Printed by: Administrator BD FACSDiva 8.0.1 BD FACSDiva 8.0.1 20 40 5030 FSC-A (x 1,000) Experiment Name: Chicken new batch Experiment Name: Chlcken new batch Specimen Name: E22 Day 07108 Specimen Name: E22Day 01108 Tube Name: CD4/CD8 Tuhe Name: CD45/CD4 Record Date: Aug 7,2017 4:06:56... Record Date: Aug 7.2017 4:06 10.. $OP: Administrator $OP: Adm nistrator Populaiion fEvents ffi LYMPHOCYT 445 X co+ cD45/CD4 251 56.4 cD4 194 43.6 TOIAL CD45 1,541 21 Chicken new batch Page 1 of 2 Printed on: Mon Nov 6,2017 12:21:45 CAT Chicken new batch Page 2 of 2 Printed on: N4on Nov 6, 2017 12t21146 CAT Printed by: Admrnistrator Printed byr Administrator BD FACSDiva 8.0.1 BD FACSDiva 8.0.1 10 20 30 40 50 FSC-A (x 1,000) E24Day 01|08-CD4ICDL l.'tr;::+ei::ri ffi Experiment Name: Chicken new batch Experiment Name: Chicken new Specimen Name batchE24 Day OTIOB Specimen Namer E24 Day O7lO8 Tube Name: CD45/CD4 Tube Name: Record Dater CD4/CD8Auo1,2011 4.07.46. Record Date Auq 7, 20'17 4:08:3. . $OP Administrator $OPr Administrator Populatron uEvents LYMPHOCYTES '- 1,10 0.4 cD4s Xcoa 273 66.4 co+ CD45/CD4 X 31 33.6 TOTAL CD45 360 1.2 .H Chicken new batch Page 1 of 2 Printed on: Mon Nov 6, 2017 12:21:54 CAT Chicken new batch Page 2 of 2 Printed on: Mon Nov 6, 2017 12r21:55 CAT Printed by: Administrator Printed by: Admlnrstrator BD FACSDiva 8.0.1 BD FACSDiva 8.0.1 40 50 40 50 (x 1 000) (x 1,000) Experiment Name: Chicken new batch Specimen Name E26 Day A7lA8 Experiment Namer Chicken new batch Tube Name: CD45/CD4 Specimen Name: E26 Day 07/08 Record Date: Aus7,2O1/ 4.49:4. Tube Name: CD4/CD8 $oP: Administrator Record Date: Aul7,2017 4.10.4. #Events %Parent LYIVPHOCYTES 633 0.5 Populat-ro-"n' " '- - H-E vents X coas ztt | 43.8 XcD irs X co+ 356 562 X co 8 + 366 3.781 28 Chicken new batch Page 1 of 2 Printed on; Mon Nov 6,2017 12i22i07 CAf Chicken new batch Page 2 of 2 Printed on: Mon Nov 6, 2017 12:22:OB CAf Printed by: Administrator Printed byr Adminrstrator BD FACSDiva 8.0.1 BD FACSD|Va 8.0.1 O a a 20 3A FSC-A Experiment Name: Chicken new batch Experiment Name Chicken new batch Specimen Namer 828 Namer Day 07/08Specimen E28 Day AllA8 Tube Name: CD4/CD8 Tube Namei CD45/CD4 Record Date: Auq7,201-1 4:14.0. Record Datei Auq1.2011 4.123.. $OP: Admlnistrator Populdlron FEvpns Elc'oa - * --i;e LYI\,4PHOCYIES 524 02 cD45 X co+ 359212 40.5 cD4 312 59.5 TOTAL CD45 4,481 1.7 Chicken new batch Page 1 of 2 Printed on: l.,lon Nov 6 , 2017 12:22:17 CAT Chicken new batch Page 2 of 2 Printed on: Mon Nov 6,20L7 121221L7 CA't Printed by: Administrator Printed by: Administrator BD FACSDiva 8.0.1 BD FACSDiva 8.0.1 O aa 20 30 10 20 30 FSC-A FSC-A Experiment Name: Chicken new batch Specimen Name E30 Day 07108 Tube Name CD45/CD4 Record Date: Aue7,2017 4:15:2... Experiment Name: Chicken new batch $oP Administrator Specimen Name: E30 Day 07/08 Tube Namer CD4/CD8 Record Date: AuAl,2017 4.16.37... $op: Adm nrstrator LYI\,4PHOCYTES I coas a: i e.6 X Populalron qEvenlsco+srco+ B coa 186 I co+ Chicken new batch Page 1 of 6 Printed on: Mon Nov 6, 2017 !2i22:34CAf Chicken new batch Page 2 of 6 Printed on: Mon Nov 6, 2017 12:22t34 CAf Printed by: Administrator Printed by: Administrator BD FACSDiva 8.0.1 BD FACSD|Va 8.0,1 10 20 30 40 20 30 FSC-A FSC A Experiment Name: Chicken new batch Experiment Name: Specrmen Namer Chicken new E6 Day 10/08batch Specimen Name: Tube Name: CD4/CD8E6 Day 10/08 Tube Name: Record Date: CD45/CD4 Aug 11,2011 3:27:... Record Date $OP: AdministratorAug 11,2017 3:26:. $OPr Administrator Populal on tEvents fficoa " "-= 7i coa Lymphocytes X CD45 150 CD45/CD4 '132 46.8 TOTAL CD45 874 0.4 Chicken new batch Page 1 of 2 Printed on: lYon Nov 6, 2017 12:22:44 CAf Chicken new batch Page 2 of 2 Printed on: Mon Nov 6, 2017 12r22:.t4 CAT Printed by: Administrator Printed by: Administrator BD FACSDiva 8.0.1 BD FACSD|Va 8.0.1 5 oaa 1A 20 30 40 FSC-A Experiment Namei Chlcken new batch Experimcnt Name Chicken new batch Specrmen Name: E9 Day 10/08 Specimen Name: E9 Day 10/08 Tube Name: CD4/CD8 Tube Name: CD45/CD4 Record Date: Aug 1 1,2017 3:3015... Record Date: Aug 11 2017 3:29:1. $OP: Administrator $OP: Administrator Populalio-n glvents tr"cD8 "js LYi\,IPHOCYTES X co+ 109 cD45 59.1 CD45/CD4 40.9 TOIAL CD45 0.2 Chicken new batch Page 1 of 2 Pdnted on: Mon Nov 6,2017 12:22:57 CAT Chicken new batch Page 2 ol 2 Printed on: Mon Nov 6,20L7 !2:22157 CA\ Printed by: Administrator Printed by: Administrator BD FACSDiva 8.0.1 BD FACSDiva 8.0.1 20 30 40 20 3050 FSC A FSC-A(x 1 000) Experiment Name: Chicken new batch Specimen Name: E11 Day'10/08 Experiment Name: Chicken new batch Tube Name: CD45/CD4 Specimen Name E1'1 Day 10/08 Record Dale: Aug '1 Tube Namer 1 , 20'17 3:31:5. CD4/CD8 $OP: Administrator Record Dater Auq 11,2017 3:33:1... $op: Administrator LYMPHOCYTES Population *-*-*-r?#Events CD45 186 57.9 ECix CD45/CD4 13s 42.1 X coa - fi4 TOTAT CD45 1 ,'t59 0.6 Chicken new batch Page 1 of 2 Printed onr Mon Nov 6,2017 12t23:13 CAT Chicken new batch Page 2 of 2 Printed on: Mon Nov 6, 2017 12:23:13 CAT Printed by: Administrator Printed by: Adminiskator BD FACSD|Va 8.0,1 BD FACSD|Va 8.0.1 20 10 20 30 40 5030 FSC-A FSC-A (x 1,000) Experiment Name Chicken new batch Experiment Name: Chicken new batch Specimen Name: E17 Day 10/08 Specimen Name: E17 Day 10/08 Tube Name: CD45/CD4 Tube Name: CD4/CD8 Record Date: Auq 1 1, 201 7 3r34r 1. . Record Date: Aug 1 1 2017 3 $OP: 35:'1Adminlstrator Populanon FLvents LYIVPHOCYTES 1 ffioa--- 43 cD45 55.4 X co+ io 44.6 TOTAL CD45 0.5 Chicken new batch Page 1 of 2 Printed on: Mon Nov 6, 2017 12123122 CAf Chicken new batch Page 2 ol 2 Printed on: Mon Nov 6, 2017 12t23:23 CA'l Printed by: Administrator Printed by: Administrator BD FACSD|Va 8.0,1 BD FACSDiVa 8.0.1 50 (x 1,000) Experiment Name: Chicken new batch Specrmen Namei E22 Day 10108 Tube Name: CD45/CD4 Date: Expenmeni Name: Chicken new batchRecord Aug '1 1 2017 3:36:1 $OP: Specimen Name: E22 Day 10108Administrator Tube Name: CD4/CDB Record Date: Aug 11,2017 3:3713... $OP: Administrator L 0.1 cD45 48.4 cD45/CD4 51.6 PXgsqcl.solr-cla]..". ..... *- 20 TOTAL CD45 0.3 X coa Chicken new batch Page 1 of 2 Printed on: l.4on Nov 6,2017 12:23;31 CAT Chicken new batch Page 2 of 2 Printed on: Mon Nov 6, 2017 12i23:31 C f Printed by: Administrator Printed by: Administrator BD FACSD|Va 8.0.1 BD FACSD|Va 8.0.1 40 50 (x 1 000) Experiment Name: Chicken new batch Specimen Name: Conrol 1 Day 1 1/08 Tube Name CD4/CD8 Experiment Name: Chicken new batch Record Dater Aug 11,2011 3:40:4.. Specimen Name: Control 1 Day '1'1108 $OP: Administrator Tube Name: cD45/CD4 Record Date: Aug 1'1,2017 3:39:1 egpcuoatrso- 1r....-.--.-.-......---.--.-.-fEy9$i.$oP: Administrator X 106 El co+ LYMPHOCYTES X coas X coasrco+ Chicken new batch Page 1 of 2 Printed on: Mon Nov 6,2017 12:23:56 CAT Chicken new batch Pagezol 2 Printed on: Mon Nov 6 , 2017 12123i56 CA]. Printed by: Administrator Printed by: Administrator BD FACSDiVa 8.0.1 BD FACSDiva 8.0.1 5 oaa 2C 30 50 FSC.A (x I 000) Namei Experiment Name: Chicken new batchExperiment Chicken new batch Specimen Name: Control 2 Day 1'1108 Specimen Name Control 2 Day 1 1/08 Tube Name: Tube Name: CD4/CD8CD45/CD4 Record Date: Record Date: Aug 11,2017 3:42:4Aug 11.20173141:. $OPr Administrator %Parent LYI\,'IPHOCYTES 353 cD45 161 4s6 CD45/CD4 192 54.4 TOTAL CD45 1.179 0.7 Chicken new batch Page 1 of 2 Printed on: Mon Nov 6,2017 12:24:05 CAT Chicken new batch Page 2 of 2 Printed on; Mon Nov 6,2017 12:24:05 CAT Printed by: Administrator Printed by: Administrator BD FACSDiva 8.0.1 BD FACSD|Va 8,0.1 a o aa 2A 30 (x I 000) FSC-A Expenment Name: Chicken new batch Name: Experiment fJarne: Chicken new batchSpecimen Control 3 Day 1 1/08 Namei Specimen Name: Control 3 Day 1 1/08Tube CD45/CD4 Date: Tube Name: CD4/CD8Record Auq 11,2017 3143:4 $OP: RecordDate: Aug'11,20173:4415...Administrator $OP: Adh,nrstrator LYI\,lPHOCYTES 0.5 # P-o--o'ulanon CEvents 143 cD45 XCD8 566 58.2 41.8 X co+CD45/CD4 407 IOIAL CD45 1,792 0.8 Chicken new batch Page 1 of 2 Printed on: Mon Nov 6,20t7 L2i24ifs CAf Chicken new batch Page 2 of 2 Printed on: Mon Nov 6, 20!7 !2124if5 CA'l Printed byi Administrator Printed by: Administrator BD FACSDiVa 8.0,1 O a a 20 30 40 20 30 40 50 FSC.A FSC.A (x 1 000) Expenment Name: Chicken new batch Specimen Namel El5 Day 11/08 Tube Name: CD45/CD4 Experiment Name: Chicken new batch Record Datel Aug'11,2017 3:46:0.. Specimen Name: E15 Day 11/08 $oP, Admrnistrator Tube Namei CD4/CD8 RecordDate: Aug'11,20173:47:0... SOP] Administrator LYMPHOCYTES CD4FIT,, CD8 PE,, El coas I Pooulanon ,Events Mean l\,4eancoavco+ X cos 84 26 l.001 X coa 2s1 2.141 182 Chicken new batch Paqe 1 of 2 Printed on: Mon Nov 6, 2017 f2i24:24 CAf Chicken new batch Page 2 of 2 Printed onr Mon Nov 6, 2017 12124t24 CAf Printed by: Administrator Pfl nted by: Administrator BD FACSDiva 8.0.1 BD FACSD|Va 8.0.1 20 30 10 20 30 40 50 FSC A FSC-A (x 1,000) Experiment Nsme Chicken new batch Experiment Namel Chicken new batch Specimen Name: E22 Day 11108 Specimen Namel E22 Day 11,08 Tube Name: CD4/CD8 Tube Name: CD45/CD4 Record Date: Auo 11,2017 3:49:1.. Record Date: Aug 1 1, 2017 3 48rl $OP: Admrlistrato- $oP: Administrator Pooulat-o n #Ev-e n's Population #Events %Parent Hcbri aJ E rvupnocvres 465 0.6 E coa X co+s 172 37.0 X co+slco+ 293 63.0 Chicken new batch Page 1 of 2 Printed on : Mon Nov 6 , 2Ol7 12124135 CN Chicken new batch Page 2 ol 2 Printed on: Mon Nov 6, 2017 f2124:35 CAf Printed by: Administrator Printed by: Administrator BD FACSDiva 8.0.1 BD FACSDiva 8.0.1 20 30 FSC-A rd3 CD8 PE.A Experiment Name: Chicken new batch Experiment Namer Chicken new batch Specimen Namer E26 Day 11/08 Specimen Namer E26 Day 11/08 Tube Namei CD45/CD4 Tube Name: CD4/CD8 Record Date: Auq 1 1, 2017 3:50:... Record Datei Aug 11,2017 3:52:4. $OP: Administrator $OPr Administrator Pooulalron #Evenl5 LYMPHOCYTES 4 mCD8- --*'- iso cD45 33.0 X co+ 446 CD45/CD4 67.0 TOTAL CD45 1.0 Chicken new batch Page 1 of 2 Printed on: l4on Nov 6, 20t7 12:24144 CA-|. Chicken new batch Page 2 of 2 Printed on: lYon Nov 6,2017 l2t24t44CAf Printed byr Administrator Printed byr Administrator BD FACSDiVA 8.0.1 BD FACSDiva 8.0.1 O a a 20 30 40 50 FSC-A (x 1,000) Expenment Name: Chicken new batch Specimen Name: E28 Day '11108 Experiment Name Chicken new batch Tube Name: CD45/CD4 Specimen Name E28 Day '1'1108 Record Date Aug 1 1,20'17 3154:1. Tube Name CD4/CD8 $oP: Adminlstrator Record Dater Auq 11,2017 3:55:5.. $OP: Administrator Pooulation dEvents Mcoa " - rs6 X co+ Chicken new batch Page 1 of 2 Printed on: Mon Nov 6, 2077 72:24154 CAf Chicken new batch Page 2 of 2 Printed on: l4on Nov 6, 2017 12124155 CAI Printed by: Administrator Prnted by: Administrator BD FACSDiva 8.0.1 20 30 40 50 2A 30 FSC-A (x FSC.A 1,000) Experiment Name: Chicken new batch Specimen Namei Control 4 Day 15/08 Tube Name: cD45/CD4 Experiment Name: Chicken new batch Record Date: Aug16,2017 2423.. Specimen Name: Control 4 Day'15/08 $oP: Administrator Tube Name: CD4/CD8 Record Date: Aug 16,2017 2:44:2 .. Population $OPr Administrator Lymphocytes 191 X co+s 105 55.0 Pooulaton #Fvenls X coavco+ 86 45.0 X'cos 169 0.7 I co+ Chicken new batch Page 1 of 2 Printed on: Mon Nov 6, 20L7 12i25i28 CAT Chicken new batch Page 2 of 2 Printed on: Mon Nov 6,2017 12:25:29 CAT Printed by: Administrator Printed by: Administrator BD FACSDiVa 8.0.1 BD FACSDiva 8.0.1 oq @ 20 30 40 50 FSC-A (x 1,000) Experiment Name: Chicken new batch Specimen Name: Control 5 Day 15/08 Experiment Name Chicken new batch Tube Name: CD4/CD8 Speclmen Namer Control 5 Day 15/08 Record Date: Aul16,2017 2:48:3... Tube Namei CD45/CD4 $OP Administrator Record Date: Aug 16,20'17 2 46:3 $oP: Administrator PooLIanon fFvents fficoa 113 EI coa LYMPHOCYTES 510 cD45 240 47.1 CD45/CD4 210 TOTAL CD45 1,331 0.3 Chicken new batch Page 1 of 2 Printed on; Mon Nov 6,2017 12:25:38 CAT Chicken new batch Page 2 of 2 Printed on: Mon Nov 6, 2017 12'.25139 CAf Printed by: Administrator Printed by: Adminrstrator BD FACSD|Va 8.0.1 BD FACSD|Va 8.0.1 O a a 10 20 30 40 50FSC-A (x 1,ooo) Experiment Name: Chicken new batch Specimen Name: Control 6 Day 15/08 Experiment Name: Chicken new batch Tube Name: CD4/CD8 Specimen Name Control 6 Day 15/08 Record Date: Aug 16,2017 2:50:4... Tube Name: CD45/CD4 $OPr Administrator Record Date Auo 16,2017 2:49:4 $OPi Administrator Pooulailon ,Events McD8" - ios X coa LYMPHOCYTES CD45 40.2 cD45/CD4 59.8 TOTAL CD45 0.5 Chicken new batch Page 1 ol 2 Printed on; Mon Nov 6, 2017 f2125i47 CA'l Chicken new batch Page 2 ot 2 Printed on: Mon Nov 6,2017 12'.25t47 C r Printed by: Administrator Printed byr Administrator BD FACSD|Va 8.0.1 BD FACSDiva 8.0.1 40 50 (x 1 000) Experiment Name: Chicken new batch Specimen Namer E2 Day'15/08 Experiment Name: Chicken new batch Tube Name: CD45/CD4 Specimen Name: E2 Day 15/08 Record Date: Aua 16,2017 2152:2.. Tube Name: CD4/CD8 $OP: Administrator Record Date: Auq 16,2017 2:54:0... $OP: Administrator L Pcp.uLe!g1--**-- cD45 6.1 X cos 3't CD45/CD4 93.3 X co+ 149 TOTAL CD45 0.2 Page2 2 Printed on: Mon Nov 6, 2017 12:26:00 CAT Chicken new batch Page 1 of 2 PrinGd on: Mon Nov 6,2017 12:25:59 Uf Chicken new batch of Printed by: Administrator Printed by: Administrator BD FACSDiva 8.0.1 BD FACSDiva 8.0.1 5 o O a a a a 20 30 FSC.A Experimenl Name: Chicken new batch Specimen Name: E1 1 Day 15/08 Experiment Name: Chicken new batch Tube Name: CD45/CD4 Specimen Namei El'1 Day 15/08 Record Dater Aug 16,2017 2:55:3... Tube Namer CD4/CD8 + $OP: Administrator Record Date: Aua16,2017 2:51:1... cU $OP: Administrator oo Iffi LYMPHOCYT . Populatron ...M' :i{:rps - -" "" - --d*f"viernetscoas 47.9 X'cD8 cD/l5f@4 X X co+srco+ X co+ I rornr co+s 0.s , Q3 Q4 o 10' lou ii rd' +s r,r"-o 'docJoi rrrc-l "Jdi Chicken new batch Page 1 of 2 Printed onr Mon Nov 6, 2017 12126107 CAf Chicken new batch Page 2 of 2 Printed on: Mon Nov 6, 2017 12:26108 CAT Printed by: Administrator Printed by: Admlnistrator BD FACSDiva 8.0.1 BD FACSD|Va 8.0,1 oa @ 40 50 (x 1,000) Experiment Name: Chicken new batch Experiment Name: Chicken new batch Specimen Name: E13 Day'15/08 Specimen Name: E13 Day 15/08 Tube Name CD4/CD8 Tube Name: CD45/CD4 Record Date: Aug 16,20'17 3100:5... Record Dater Aug'16,2017 2:59:0. $OPr Administrator $OP: Administrator .: ^'j ..CDg puoulanon fEvenls X CD8 31 LYMPHOCYTES 0.0 X co+ CD45 43.1 co45tcDA 56.9 TOTAL CD45 0.3 Printed on: Mon Nov 6,2017 12:26:16 CAT Chicken new batch Page 2 ol 2 Printed on: I4on Nov 6, 2017 12:26:16 CAT Chicken new batch Page 1 of 2 Printed by: Administrator Printed by: Administrator BD FACSDiva B.0.1 BD FACSD|Va 8.0.1 oaa fi2A304050 to 20 30 40 50 FSC-A FSC-A (x I ooo)(x i ooo) Experiment Name: Chicken new batch Experiment Name: Chicken new batch Specimen Name: E19 Day 15/08 Specimen Namer E19 Day 15/08 Tube Name: CD4/CD8 Tube Name: CD45/CD4 Record Date: Au}16,2017 3:04:2... Record Date: l\ug 16 2017 3t02 4. . $OP: Administrator $OP: Administrator PooJlauon dEvenls #Events %Parent X CD8 389 E LvnrpHocvtes 1,228 0.4 X co+s 903 X coavco+ Chicken new batch Page 1 of 2 Printed on: Mon Nov 6,2017 12:26:25 CAT Chicken new batch Page 2 of 2 Printed on: Mon Nov 6, 2017 t2i26i25 CAf Printed by: Administrator Printed by: Admrnistrator BD FACSDiva 8.0.1 BD FACSDiva 8.0.1 20 30 10 2A 30 40 50 FSC-A FSC A (x 1,000) Expenment Name: Chicken new batch Experiment Name: Chicken new batch Specimen Name: E21 Day 15/08 Specimen Namei E21 Day 15/08 Tube Name: CD4/CD8 Tube Namer CD45/CD4 Record Date: Aug 16,2017 3107:1... Record Datel Auq 16,20'17 3:0515... $OP: Administrator $OPr Administrator Pooulatron 4Fvelis Mco6 -" eo L X co+ cD45 66.3 cD45/CD4 33.7 TOTAL CD45 0.8 Chicken new batch Page 1 of 18 Printed on: Mon Nov 6,20L7 12t26t43 CAf Chicken new batch Page 2 of 18 Printed on: lYon Nov 6, 2017 f2t26i43 CA-l Printed by: Administrator Printed by: Administrator BD FACSD|VA 8.0.1 BD FACSD|Va 8.0.1 20 30 40 50 10 20 30 40 50 FSC-A (r FSC-A 1 ooo)tx i ooo) Experiment Name: Ch cken new batch new Specimen Namer Experiment Namer Chicken batch Conrol 7 Day 18/08 7 Day 18/08 Tube Name: Specimen Name: Conrol CD45/CD4 Tube Name: CD4/CD8 Record Date: Aug 18,2017 2:48:. Record Date: Auq 18,2017 2.49:. $OP: Admrnistrator $OPr Adminrstrator PoDJlat on 4fvPnls LYMPHOCYTES 688 *--.i; cD45 440 X co+ cD45/CD4 248 TOTAL CD45 1,325 Chicken new batch Page I of 2 Printed on: Mon Nov 6,2017 11:00:44 CAT Chicken new batch Page 2 of 2 Printed on: Mon Nov 6, 2017 11r00:44 CAT Printed by: Administrator Printed by: Administrator BD FACSD|Va 8.0.1 BD FACSD|Va 8.0,1 1o 20 30 40 50 FSC-A (x 1 000) Conrol 8 Day 18/08-CD4/CD8 Experiment Name: Chicken new batch Name: Experiment Name: Chicken new batchSpecimen Contro 8 Day 18/08 Name: Specimen Name: Control 8 Day 18/08Tube CD45/CD4 Daier 2:49: Tube Name: CD4/CD8Record Auo 18,2017 . Record Date: Aug 18 2017 2:51:... $OP: Admlnistrator n9.pc!.1o.?!ser --.---..- ^,"--.-" ---.--."-" --. tEY.e$;.X 109 Chicken new batch Page 1 of 2 Printed on: Mon Nov 6, 2017 11:06:36 CAT Chicken new batch Page 2 of 2 Printed on: Mon Nov 6, 2017 11:06:36 CAT by: Administrator Printed by: AdrninrstratorPnnted BD FACSDiVA 8,0.1 BD FACSDiva 8.0.1 20 30 40 50 30 40 50 FSC A . .ooo, FSC,A (x 1,000) Experlment Namei Chicken new batch Namer Chicken new Specimen Name: Control I Day 18/08Experment batch Specrmen Name: Control 9 Day 18/08 Tube Name: CD4/CD8 Tube Name CD45/CD4 Record Dater Aug 18,2017 2:53r. . Record Date Aug 18 2011 2 52 $OP: Administrator.. $OP: Administrator pooJlauon 4Events x cD8 152 LYMPHOCYTES X co+ cD45 CD45/CD4 TOTAL CD45 Chicken new batch Page 1 of 2 Printed on: Mon Nov 6, 2017 11:09:42 CAT Chicken new batch Page 2 of 2 Printed on: l4on Nov 6, 2017 11:09:42 CAT Printed by: Administrator Printed by: Admlnlstrator BD FACSDiva 8.0.1 20 30 40 FSC.A 18/08-Cp45/eD4 Experiment Name: Chicken new batch Name: Day 18/08Experiment Chicken new batch Specimen Namer E4 Name: E4 Day 18/08 Tube Namer CD4/CD8Specimen Name: CD45/CD4 Record Date: Aua 18,2017 2:55 4 .u Tube L Record Date: Aug 18,2017 2:54.4 $OP, Administrator $OPr Administratora O Pffoiocdr;a d stio n ' Evants iio 40s X coa 116 2,315 Chicken new batch Page 1 of 2 Printed on: Mon Nov 6,20t7 lli!2124 CAf Chicken new batch Page 2 af 2 Printed on: Mon Nov 6, 2017 11i12:24 CAf Printed by: Admin strator Printed by: Adminlstrator BD FACSDiva 8.0.1 BD FACSDiva 8.0.1 20 30 1A 20 30 40 50 FSC-A FSCA (xiooo) Experiment Name Chicken new batch Experiment Namei Chicken new batch Specimen Namer El 1 Day 18/08 Specimen Name: El 1 Day 18/08 Tube Name: CD45/CD4 Tube Name: CD4/CD8 Record Dare Aua 18.201 / 2:57:1 Record Dater Aug 18.2017 2:58:2.. $OP: Administrator PoDJlar on #EvF4ls LYI\,4PHOCYTES 1,561 X'coa 254 cD4s 1,028 X co+ 446 cD4s/cD4 533 TOTAL CD45 3,139 Chicken new batch Page 1 of 2 Printed on: lYon Nov 6, 2017 t1:15:00 CAT Chicken new batch Page 2 ol 2 Printed on: [4on Nov 6,2017 11:15:00 C-AT Printed by: Administrator Printed by: Admlnistrator oaa 30 40 50 30 40 50 FSC-A (x 1,000) FSC-A G 1.ooo) Experiment Name Chicken new batch Name: Experimenl Name Chrcken new batch Specimen E15 Day 18/08 Name: Specimen Name: E15 Day 18/08 Tube CD45/CD4 Tube Namer CD4/CD8 Record Date: Auq 18,2017 2:59:... Record Date: Aug 18,20'17 3 00:. $OP: Admin strator $OP: Administrator Pooulatton qFvenls L,lLYMPHocYItS 744 epJc-uo-Lae !.19-n...-,--,,----..*,..- #f-Yeili- x coas X 81436 X co+slcoa X co+ 281308 @ roreL co+s 1,489 Chicken new batch Page 1 of 2 Printed on: Mon Nov ,2017 11:17:28 CAI Chicken new batch Page 2 ol 2 Printed on: Mon Nov 6, 2017 11:17r28 CAT6 Printed by: Administrator Printed by: Administrator BD FACSDiva 8.0.1 BD FACSD|VA 8.0,1 o @ @ 10 20 30 2a 30 FSC-A FSC-A Expenmeni Name Ch cken new batch Experiment Name: Chicken new batch Specimen Name: E19 Day 18/08 Specimen Name: E19 Day 18/08 Tlbe Namer CD4/CD8 Tube Name CD45/CD4 Record Date: Aug 18,2017 3:0313... Record Date: l\ua 18 2017 3:02:.. $OPr Administrator $OP Administrato. Poouidt,on CFvPnts PoDJlatron ,Evenls x cD8 234 Ll L /N,,IPHoCYTES 984 8 co+ 251 X cD45 73't X coaslco+ 241 BB rorlr coas 1,761 Nov 6,2017 11:20:36 CAT Chicken new batch Page 1 of 2 Printed on: Mon Nov 6, 2017 11:20:35 CAT Chicken new batch Page 2 of 2 Printed on: Mon Prln[ed by: Administrator Pr nted by: Admin strator BD FACSDiVa B.0.1 5 O a 20 30 40 50 FSC.A (x 1,000) Experiment Name: Chicken new batch Spec men Name E27 Day 18148 Experiment Name: Chicken new batch Tube Namer CD45/CD4 Specrmen Name: E21 Day 18108 Record Date: Auq 18.2017 3:04:. Tube Name CD4/CD8 $OP: Admin strator Record Date: Auo 18,2017 3:06: #Events L 114 CD45 403 cD45/CD4 311 TOTAL CD45 1,549 Chicken new batch Page 1 of 2 Printed on: Mon Nov 6, 2017 71,:23114 CAf Chicken new batch Page 2 ol 2 Printed on: Mon Nov 6,2017 11:23114 CAI by: Admrnistrator Printed by: AdmlnlstratorPrinted BD FACSD|Va 8.0.1 BD FACSDiva 8.0.1 5 a O O @ a a a 20 3A 40 50 20 30 40 50 FSC-A (x 1,000) FSC-A (x 1,000) Experiment Name: Chicken new batch Expenment Name Chicken new batch Specimen Name: Control '10 DaY 2'1108 Specimen Namer Control 10 Day 21108 Tube Name: CD4/CD8 Tube Name: CD45/CD4 Record Date: Au922,2017 3:38:0. . Record Date: Auo22 2017 3:35.5. . $OP Adminlstrator $OP: Admlnistrator pooJldton {fven.S Pooulatron dEvenl5 fficos 13t L,l lvtVlo{OCV tS 17 X co+s 1't X co+srcoa 6 I rotnr co+s 103 lvlon Nov 6, 2017 11:25:36 CAT Chicken new batch Page 1 of 2 Printed onr l4on Nov 6, 2017 11:25:35 CAT Chicken new batch Page 2 of 2 Printed on: Printed by: AdmLnistrator Printed by: Administrator BD FACSDiva 8.0.1 BD FACSDiva 8,0.1 aoa 30 40 50 FSC.A (x 1.000) Experiment Name: Chicken new batch Specimen Name: Control 1 1 Day 21108 Tube Name CD45/CD4 Date Experiment Name: Chicken new batchRecord Au\22.2011 3:4O2.. $OP Specimen Name Conrol 1 1 Day 21108Adminlstrator Tube Name: CD4/CD8 Record Date: Au922,2A11 3:42:.. $OP Administrator LYMPHOCYTES 845 cD45 499 rP+o-o ulaton dEvelrs cD45/CD4 346 -X cD8 '159 TOTAL CD45 3,412 2017 11:28:18 CAT Chicken new batch Page 1 ol 2 Printed on: Mon Nov 6,2017 11:28:18 CAT Chicken new batch Page 2 ol 2 Printed on: Mon Nov 6, Printed by: Admlnrstrator Printed byi Administrator BD FACSD|Va 8.0.1 BD FACSDiVa B.0.1 Experimenl Name Chicken new batch Experiment Name: Chicken new batch Specimen Name: ControL 12 Day 21108 Specrmen Name: Control 12 Day 21lOB Tube Name: CD45/CD4 Tube Name: CD4/CD8 Record Date: Aug22 2017 3:44:1 Record Date: Aug22 201'1 3:46:... $OP Administrator $OP: Administrator PfofoiJc douosn CEvenls LYI\,IPHOCYTES 216 1) cD45 103 X co+ CD45/CD4 1 13 TOTAL CD45 1,511 Chicken new batch Page 1 of 2 Printed on: Mon Nov 6, 2017 11:30:38 CAT Chicken new batch Page 2 ol 2 Printed on: Mon Nov 6, 2017 11r30:38 CAT Printed byr Administrator Pnnted by: Administrator BD FACSDiva 8.0.1 BD FACSDiva 8.0.1 9 l O a a 1a 20 30 40 10 20 3050 FSC_A FSC A(x I u00) CollellEDayz l/0E-cp,Uc!8 Exper ment Name: Ch cken new batch Experiment Name Ch cken new batch Specimen Name: Control 13 Day 21108 Specimen Name: Control 13 Day 21l08 Tube Name CD45/CD4 Tube Name: CD4/CD8 Record Dater AuT22,2011 3:48:5.. Record Date: Au022,2017 3:5A:0.. $OPr Administrator $OP: Administrator Poprlanon dEvents Populal-ion CFvenls L_lLY[/ProcYils 2] XcD8 ***- X co+s 16 X co+ X co+slco+ s M rornr co+s 143 #., Chicken new batch Page 1 of 2 Printed on: Mon Nov 6, 2017 11t33:00 CAT Chicken new batch Page 2 ol 2 Prlnted on: Mon Nov 6,2017 11:33i00 CAT Printed by: Administrator Printed by: Administrator BD FACSDiva 8.0.1 BD FACSDiva 8.0.1 5 aoa 20 30 30 40 50 FSC-A FSC-A (x 1,000) 1/08-CD45/CD4 TOTAL CD45 Experiment Name: Chicken new batch Specimen Name: E3 Day 21l08 Tube Name: CD45/CD4 Expeflment Name: Chicken new batch Record Date: Auq22 2017 3.51.0. Specimen Name: E3 Day 21l08 $OP: Administrator Tube Name: CD4/CD8 Record Date: Au922,2017 3:52 LYMPHOCYTES 63 CD45 39 cD45/CD4 24 TOTAL CD45 s36 Chicken new batch Page 1 of 2 Printed on: Mon Nov 6, 2017 11:36:54 CAT Chicken new batch Page 2 ol 2 Printed on: Mon Nov 6, 2017 11:36:54 CAT Prlnted by: Administrator Pr nted by: Administrator BD FACSDiva 8.0.1 BD FACSD|VA 8,0.1 5 O 2A 30 20 30 4Q 50 FSC.A FSC-A (x 1 000) EO Day 2 1i08-CD4ICD8 Experiment Name: Chicken new batch Experiment Name Chicken new batch Specimen Name: E6 Day 2'1108 Spec.mPn \ane: f6 Day 21l08 Tube Namei CD45/CD4 Tube Name: CD4/CD8 Record Date: Au022,2011 3:53 1 Record Date: AuT22.2011 3:54 1... $OP: Admln st.ator $oP: Administrator PoDulaton ,Evenls Pooulanon fEvenls L] I YVPHOC/TES 104 X cos 23 I co+s 58 X co+srcoa 46 B rorlr co+s 340 Chicken new batch Page 1 of 2 Printed on: Mon Nov 6, 2017 11:39:24 CAI Chicken new batch Page2 ol 2 Printed on: Mon Nov 6, 2017 11:39:25 CAT Printed by: Administrator Printed by: Adminrstrator BD FACSDiva 8.0.1 BD FACSD|Va 8.0.1 O O a @ 20 30 FSC-A Experiment Name: Chicken new batch Experiment Name: Chicken new batch Specimen Name: E9 Day 21l08 Specimen Name Eg Day 21l08 Tube Name: CD4/CD8 Tube Name: CD45/CD4 Record Date: Au922,201-7 3:55:4. . Record Date: Aug22 2011 3:55:.. $OP Administrator $OP: Administrator Pooulaton {Eveals pooJtaton CEven.: fficoa -" " -' 28 L,lt\MPHocYrrs 99 X co+ X cD45 67 X co+srco+ 32 ffi rornr coas 344 Chicken new batch Page 1 of 2 Printed on: 1"10n Nov 6,2017 11:41:52 CAT Chicken new batch Page 2 of 2 Printed on: Nlon Nov 6, 2017 11:41:52 CAT Printed by: Administrator Printed by: Administrator BD FACSDiva 8.0.1 BD FACSD|Va 8.0,1 E11 Day 21l08-cD4lQPE O aa 20 30 40 50 FSC-A (x 1,000) Experiment Namer Chicken new batch Specimen Name: E1'l Day 21108 Experiment Name: Chicken new batch Tube Name: CD45/CD4 Specimen Namer E11 Day 21108 Record Date: Au\22.2011 3:56:4 Tube Name: CD4/CD8 $OP: Administrator Record Date: Auo22,2017 3:5/:4... $OP: Administrator LYMPHOCYTES pooulaton #Evpnts102 cD45 61 X cD8 40 CD45/CD4 41 8 coa 10 TOTAL CD45 458 Chicken new batch Page 1 of 2 Printed on: l40n Nov 6. 2017 11:44:06 CAT Chicken new batch Page 2 of 2 Printed on: Mon Nov 6, 2017 11t44106 CAT Printed by: Adm nlstrator Printed by: Administrator BD FACSDiva 8.0.1 BD FACSDiva 8.0.1 o aa 20 30 FSC.A Experiment Name Chicken new batch Specimen Name E15 Day 21l08 Experiment Namer Chicken new batch Tube Namer CD45/CD4 Specimen Name: E15 Day 21l08 Record Dater Au922,2017 3:58: Tube Name: CD4/CD8. Record Date: Aua22 2011 3159:2. . $OP: Administrator e,o!!!g!tgn ..,_-_.,.,,,.--,..-..-,..-......_"..".". *."..._{E_JglI!" 24 Xcoa 12 20 X coa 318 Chicken new batch Page 1 of 2 Printed on: Mon Nov 6, 2017 11:46:26 CAT Chicken new batch Page 2 ol 2 Printed on: l4on Nov 6, 2017 1t146:27 CAI Printed by: Administrator Printed by: Administrator BD FACSDiva 8.0.1 El1 Oay 21t48 CD45/CD4 20 30 40 20 30 40 50 FSC-A FSC-A (x 1,000) Name: Experiment Name: Chicken new batchExperiment Chicken new batch Specimen Name: E17 Day 21108 Specimen Name: El1 Day 21loa Name: Tube Name: CD4/CD8Tube CD45/CD4 Date: Record Date: Aug22,2017 4 01 ...Record AuT22,2417 4:40:2... $OP: Administrator$OPr Administrator Bgr,q ls!rol,-,-----*-",--* coe " ., "fEv-gtLi: I 19 LYMPHOCYTES 127 cD45 78 cD45/CD4 49 TOTAL CD45 Chicken new batch Page 1 of 2 Printed on: lYon Nov 6,2017 11:49105 CAT Chicken new batch Page 2 ol 2 Printed onr [4on Nov 6,2017 11:49:06 CAT Printed by: Administrator Printed by: Administrator BD FACSDiva 8.0.1 BD FACSDiva 8.0.1 Oa ao @a 1A 20 30 40 50 20 3a 40 FSC-A (x 1,000) FSC A . . ,, ,t'1'DqY-21l'08-cD4/cD8 :i#'#'r ,,' ffi,','' Experiment Name: Chicken new batch Experiment Namer Chicken new batch Specimen Name: E21 Day 21108 Specimen Name E21 Oay 21108 Tube Name: CD45/CD4 Tube Name: CD4/CD8 Record Date: Auq22,2017 4:021 . Record Dater Au922,2017 4:43:0.. $OP Admin strator $OP Administrator Pooulalon ,fvenls L-1. /MPHocYrt s 89!qleli9," .. ..:.{E_u".snl"9=164 coa 37 X co+s X 1oo X co+srcol X coa64 M rornl coas 585 Chicken new batch Page 1 of 2 Printed on: l4on Nov 6, 2017 11r51:16 CAT Chicken new batch Page 2 of 2 Printed on: l40n Nov 6,2017 11:51:16 CAT Printed by: Adminrstrator Printed by: Adminrstrator BD FACSDiVa 8.0,1 BD FACSDiVA 8.0.1 aoa 20 30 20 30 40 50 (x 1,000) (x I 000) E26 Dav ?1q8-CD41CD8 21l08 CD4/CD8 Experiment Name: Chicken new batch Experiment Namer Chicken new batch Specimen Name: 826 Day 21108 Specimen Name: E26 Day 21108 Tube Name: CD4/CD8 Tube Name: CD45/CD4 Record Date: Au922.2017 4:04:3... Record Date Auo22,2017 4 03 5 . $OP: Administrator $OP: Administrator Pooula[on Ffvenls Pooulanon #fve'ls tXs "-Ct:D -8 32 Ll L\t4Phoc/rES 168coas :ffi X co+ 102X 12 X co+slcoa 96 M rornr co+s 442 Chicken new batch Page 1 of 2 Printed onr Mon Nov 6, 2017 11:53:28 CAT Chicken new batch Page 2 of 2 Printed on: l4on Nov 6,2017 11r53:28 CAT by: Administrator Printed by: AdministratorPrinted BD FACSDiva 8.0.1 BD FACSD|Va 8.0.1 2A 30 40 50 10 20 30 40 50 FSC-A (x 1,000) FSC A (x i,ooo) Name: Experiment Name Chicken new batch Experiment Chicken new batch Name: Specimen Namer E2l Day 21lOB Specimen E27 Oay 21108 Name: Tube Name: CD4/CD8 Tube CD45/CD4 Reco.d Date: Aua22,2017 4:06: .. Record Datei Au922,2011 4.05:3.. Adminisl.ator $OPr $OP: Administrator PooJldtion dEvFrls Poourdr.oa lEvents 1e Ll LYt\,lPFoc,TES ffib;a 1?8 X co+s 85 X coasrco+ 43 @ roral co+s 498 new batch Page 1 of 2 Printed on: Mon Nov 6,2017 11r55:18 CAT Chicken new batch Page 2 of 2 Printed on: l.4on Nov 6, 2017 11:55:18 CAT Chicken Printed by: Admin strator Printed by: Admin strator BD FACSDiva 8.0.1 BD FACSDiva 8.0.1 E28 Qql 21l08-CD4/Qp8 : Oq a 2A 30 30 40 50 FSC-A FSC-A (x 1 000) Expeflment Name: Chicken new batch Experiment Name: Chicken new batch Specimen Name: E28 Day 21108 Specimen Name: E28 Day 21108 Tube Name: CD45/CD4 Tube Name: CD4|CD8 u+ Record Date: Auq 22 2A11 4!7 .2. Record Date: Aug22 2017 40a1... L $OP: Administrator oo Populdilon lEvplls Populdhol - ^sEveS X"cD8 ^^2 n2rs LI LYIVPFOCYTI 10] Xcoas 54 X coa X co+srco+ 4i M rorar coas 323 Chicken new batch Page 1 of 2 Printed on: Mon Nov 6,2017 1L:57:27 CAf Chicken new batch Page 2 of 2 Printed on: l4on Nov 6,2017 11.57127 CAf APPENDIX C APPENDIX C 1STC3PJR_2018-03-16.ab1 (1st Injection with Av. paragallinarum SA-3 Strain- 1IC) GCCCGAGAACGTATTCACCGCAACATTCTGATTTGCGATTACTAGCGATTCCGACTTCA TGGAGTCGAGTTGCAGACTCCAATCCGGACTTAGATGCACTTTCTGAGATTCGCTCCC CCTCGCAGGCTCGCTTCCCTCTGTATGCACCATTGTAGCACGTGTGTAGCCCTACTCG TAAGGGCCATGATGACTTGACGTCATCCCCACCTTCCTCCAGTTTATCACTGGCAGTCT CCTTTGAGTTCCCACCCGAAGTGCTGGCAACAAAGGATAAGGGTTGCGCTCGTTGCGG GACTTAACCCAACATTTCACAACACGAGCTGACGACAGCCATGCAGCACCTGTCTCTAA GCTCCCGAAGGCACAAACTCATCTCTGAGTTCTTCTTAGGATGTCAAGAGTAGGTAAGG TTCTTCGCGTTGCATCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCA ATTCATTTGAGTTTTAACCTTGCGGCCGTACTCCCCAGGCGGTCGATTTATCACGTTAG CTACGGGCACCAAGCCTAAAGCCCAATCCCCAAATCGACAGCGTTTACAGCGTGGACT ACCAGGGTATCTAATCCTGTTTGCTCCCCACGCTTTCGCACATGAGCGTCAGTAG 2NDC3PJR_2018-03-16.ab1 (2nd Injection with Av. paragallinarum SA-3 Strain- 2IC) TGTACAAGGCCCGAGAACGTATTCACCGCAACATTCTGATTTGCGATTACTAGCGATTC CGACTTCATGGAGTCGAGTTGCAGACTCCAATCCGGACTTAGATGCACTTTCTGAGATT CGCTCCCCCTCGCAGGCTCGCTTCCCTCTGTATGCACCATTGTAGCACGTGTGTAGCC CTACTCGTAAGGGCCATGATGACTTGACGTCATCCCCACCTTCCTCCAGTTTATCACTG GCAGTCTCCTTTGAGTTCCCACCCGAAGTGCTGGCAACAAAGGATAAGGGTTGCGCTC GTTGCGGGACTTAACCCAACATTTCACAACACGAGCTGACGACAGCCATGCAGCACCT GTCTCTAAGCTCCCGAAGGCACAAACTCATCTCTGAGTTCTTCTTAGGATGTCAAGAGT AGGTAAGGTTCTTCGCGTTGCATCGAATTAAACCACATGCTCCACCGCTTGTGCGGGC CCCCGTCAATTCATTTGAGTTTTAACCTTGCGGCCGTACTCCCCAGGCGGTCGATTTAT CACGTTAGCTACGGGCACCAAGCCTAAAGCCCAATCCCCAAATCGACAGCGTTTACAG 354 APPENDIX C CGTGGACTACCAGGGTATCTAATCCTGTTTGCTCCCCACGCTTTCGCACATGAGCGTC AGTAGC PCC3PJR_2018-03-16.ab1 (Av. paragallinarum SA-3 strain positive control- C3+) CAAGGCCCGAGAACGTATTCACCGCAACATTCTGATTTGCGATTACTAGCGATTCCGAC TTCATGGAGTCGAGTTGCAGACTCCAATCCGGACTTAGATGCACTTTCTGAGATTCGCT CCCCCTCGCAGGCTCGCTTCCCTCTGTATGCACCATTGTAGCACGTGTGTAGCCCTAC TCGTAAGGGCCATGATGACTTGACGTCATCCCCACCTTCCTCCAGTTTATCACTGGCAG TCTCCTTTGAGTTCCCACCCGAAGTGCTGGCAACAAAGGATAAGGGTTGCGCTCGTTG CGGGACTTAACCCAACATTTCACAACACGAGCTGACGACAGCCATGCAGCACCTGTCT CTAAGCTCCCGAAGGCACAAACTCATCTCTGAGTTCTTCTTAGGATGTCAAGAGTAGGT AAGGTTCTTCGCGTTGCATCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCC GTCAATTCATTTGAGTTTTAACCTTGCGGCCGTACTCCCCAGGCGGTCGATTTATCACG TTAGCTACGGGCACCAAGCCTAAAGCCCAATCCCCAAATCGACAGCGTTTACAGCGTG GACTACCAGGGTATCTAATCCTGTTTGCTCCCCACGCTTTCGCACATGAGCGTCAGTAG 355 APPENDIX D APPENDIX D 356 Flow cytometry ratio calculations for one of the chickens, Control 2 with score 0 on Day 0 1st Page Cell Populations Cell Counts % of Total % of Total Leukocytes Lymphocytes Total Lymphocyte count 261 25.94 - CD8 (Cytotoxic T cells)/ B lymphocytes/NK cells 132 13.12 50.57 Estimated CD8/CD45 (Cytotoxic T cells) 29 2.86 11.02 CD4/CD45 (T helper cells) 129 12.82 49.43 Total Leukocyte Count (CD45) 1006 - - 2nd Page Cell Populations Cell Counts % of Total Lymphocytes CD8 (Cytotoxic T lymphocytes) 31 11.02 CD4 (T helper lymphocytes) 139 49.43 Calculated Total Lymphoctye Count 281 - Ratio of CD4:CD8 4.5 % of Total Summary: Leukocytes CD4 (T helper lymphocytes) 12.82 CD8(Cytotoxic T cells) 2.86 T cells (CD4 and CD8) 15.68 B cells and Natural killer cells 10.26 Ratio of CD4:CD8 4.5:1 Page 1 Page 2 Values might be subjected to slight changes due to using Excel spreadsheet and formulas for calculations: Fomula: T= C/%TL x 100 T= Calculated Total Lymphocyte count or Leucocyte count C= Cell Count from table of populations/results from first or second page provided %TL= % of Total Lymphocytes or Leucocytes Calculations: CD4 (T helper lymphocytes) cell count= 139 will have the same % of Total Lymphocytes of 49.34% from page 1 Estimated Total Lymphocyte count using data from page 2= 139/49.34 x 100 = 281.4 ~ 281 cells We then use the Calculated Total Lymphocyte count to Calculate the % of Total Lymphocytes of CD8 (Cytotoxic T lymphocytes)= CD8 cell count/ estimated total lymphocyte count X 100= 31/281 x 100= 11.02% We then calculate the Estimated CD8/ CD45 (Cytotoxic T cells) cell count for page 1= % of Total Lymphocytes of CD8 (Cytotoxic T lymphocytes)/100 x Total Lymphocyte count from page 1= 11.02/100 x 261= 28.71 ~ 29 cells Finally, we calculate the CD8/ CD45 (Cytotoxic T cells) % of Total Leukocytes= Estimated CD8/ CD45 (Cytotoxic T cells) cell count for page 1/ Total Leukocyte count from page 1= 28.71/1006 x 100 = 2.85% Summary (As explained) CD4 (T helper lymphocytes) % of Total Leukocytes= CD4 cell count/ total leukocyte count X 100= 129/1006 x 100 = 12.82% CD8 (Cytotoxic T lymphocytes) % of Total Leukocytes= CD8 cell count/ total leukocyte count X 100= 28.71/1006 x 100 = 2.85% T cell % of Total Leukocytes= (CD4 + CD8) % of Total Leukocytes= (12.82 + 2.85)% = 15. 67% B cells and Natural killer cells % of Total Leukocytes= Lymphocyte % of Total Leukocytes - T cell % of Total Leukocytes= (25.94 - 15.65)%= 10.29% Ratio of CD4: CD8 count= 139 (139/31):31 (31/31) = 4.48:1 ~ 4.5:1 % of Total Leukocytes- Student t-Test Day Cell % of Total % of Total Chicken ID Chicken ID Cell count Count Leukocytes Leukocytes % of Total Leukocytes 0 Chicken( Control 2) 261 25.94 Chicken( Control 2) 261 25.94 Day 0 0 Chicken( Control 1) 102 100.00 Chicken( Control 1) 102 100.00 t-Test: Two-Sample Assuming Unequal Variances 0 E3 90 30.30 E3 90 30.30 Variable 1 Variable 2 0 E4 27 11.64 E4 27 11.64 Mean 35.348 35.348 0 E5 14 8.86 E5 14 8.86 Variance 1389.38932 1389.38932 1 Chicken( Control 2) 261 25.94 E6 246 18.71 Observations 5 5 1 Chicken( Control 1) 102 100.00 E7 359 12.64 Hypothesized Mean Difference 0 1 E3 90 30.30 E8* 37 2.34 df 8 1 E4 27 11.64 E9 151 6.46 t Stat 0 1 E5 14 8.86 E11 281 23.42 P(T<=t) one-tail 0.5 3 Chicken( Control 2) 261 25.94 E12 42 32.06 t Critical one-tail 1.859548038 3 Chicken( Control 1) 102 100.00 E13 212 42.23 P(T<=t) two-tail 1 3 E3 90 30.30 E16 90 25.00 t Critical two-tail 2.306004135 3 E4 27 11.64 E20 272 43.52 However: p=1.000 3 E5 14 8.86 E21 387 16.77 Null Hypothesis is rejected (p>0.05) Data is not statistically significant 4 Chicken( Control 2) 261 25.94 E4 200 7.92 4 Chicken( Control 1) 102 100.00 E11 241 12.13 Day 1 4 E3 90 30.30 E17 221 7.66 t-Test: Two-Sample Assuming Unequal Variances 4 E4 27 11.64 E24 147 8.98 Variable 1 Variable 2 4 E5 14 8.86 E17 221 7.66 Mean 35.348 12.714 4 E24 147 8.98 Variance 1389.38932 74.32708 4 E25 346 15.56 Observations 5 5 7 Chicken( Control 2) 261 25.94 E15 199 42.34 Hypothesized Mean Difference 0 7 Chicken( Control 1) 102 100.00 E16 572 34.77 df 4 7 E3 90 30.30 E22 445 28.88 t Stat 1.322872061 7 E4 27 11.64 E24 110 30.56 P(T<=t) one-tail 0.128218026 7 E5 14 8.86 E26 633 16.74 t Critical one-tail 2.131846786 7 E28 524 11.64 P(T<=t) two-tail 0.256436053 7 E30 447 42.09 t Critical two-tail 2.776445105 10 Chicken( Control 2) 261 25.94 E6 282 32.27 However: p=0.256 10 Chicken( Control 1) 102 100.00 E9 208 25.43 Null Hypothesis is rejected (p>0.05) Data is not statistically significant 10 E3 90 30.30 E11 321 27.7 10 E4 27 11.64 E17 202 23.63 Day 3 10 E5 14 8.86 E22 155 26.14 t-Test: Two-Sample Assuming Unequal Variances 11 Chicken (Control 1) 507 34.05 E15 453 32.97 Variable 1 Variable 2 11 Chicken (Control 2) 353 29.94 E22 465 49.21 Mean 35.348 31.916 11 Chicken (Control 3) 973 54.3 E26 636 40.54 Variance 1389.38932 129.57113 11 E28 782 38.39 Observations 5 5 15 Chicken (Control 4) 191 34.54 E2 104 14.59 Hypothesized Mean Difference 0 15 Chicken (Control 5) 510 38.32 E11 647 38.04 df 5 15 Chicken (Control 6) 346 29.35 E13 167 17.08 t Stat 0.196906045 15 E19 1228 43.19 P(T<=t) one-tail 0.425827421 15 E21 578 29.69 t Critical one-tail 2.015048373 18 Chicken (Control 7) 688 51.92 E4 405 17.05 P(T<=t) two-tail 0.851654842 18 Chicken (Control 8) 20 33.33 E11 1561 49.73 t Critical two-tail 2.570581836 18 Chicken (Control 9) 758 28.9 E15 744 49.97 However: p=0.852 18 E19 984 55.69 Null Hypothesis is rejected (p>0.05) Data is not statistically significant 18 E27 714 46.09 21 Chicken (Control 10) 17 16.5 E3 63 11.75 Day 4 21 Chicken (Control 11) 845 24.34 E6 104 30.59 t-Test: Two-Sample Assuming Unequal Variances 21 Chicken (Control 12) 216 13.7 E9 99 28.78 Variable 1 Variable 2 21 Chicken (Control 12) 21 14.69 E11 102 22.27 Mean 35.348 9.841428571 21 E15 44 13.84 Variance 1389.38932 8.772147619 21 E17 127 20.22 Observations 5 7 21 E21 164 28.03 Hypothesized Mean Difference 0 21 E26 168 38.01 df 4 21 E27 128 25.7 t Stat 1.52668004 21 E28 101 31.27 P(T<=t) one-tail 0.100773566 t Critical one-tail 2.131846786 P(T<=t) two-tail 0.201547132 t Critical two-tail 2.776445105 However: p=0.202 Null Hypothesis is rejected (p>0.05) Data is not statistically significant Day 7 t-Test: Two-Sample Assuming Unequal Variances Variable 1 Variable 2 Mean 35.348 29.57428571 Variance 1389.38932 139.0688619 Observations 5 7 Hypothesized Mean Difference 0 Null hypothesis: df 5 H0: There is no significant difference between the means of the experimental and control chicken groups (µd=0) t Stat 0.334605568 HA: There is a significant difference between the means of the experimental and control chicken groups (µd≠0) P(T<=t) one-tail 0.375751268 t Critical one-tail 2.015048373 P(T<=t) two-tail 0.751502537 t Critical two-tail 2.570581836 However: p=0.752 Null Hypothesis is rejected (p>0.05) Data is not statistically significant Day 10 t-Test: Two-Sample Assuming Unequal Variances Variable 1 Variable 2 Mean 35.348 27.034 Variance 1389.38932 10.70463 Observations 5 5 Hypothesized Mean Difference 0 df 4 t Stat 0.496839862 P(T<=t) one-tail 0.322684319 t Critical one-tail 2.131846786 P(T<=t) two-tail 0.645368638 t Critical two-tail 2.776445105 However: p=0.645 Null Hypothesis is rejected (p>0.05) Data is not statistically significant Day 11 t-Test: Two-Sample Assuming Unequal Variances Variable 1 Variable 2 Mean 39.43 40.2775 Variance 170.0607 45.60689167 Observations 3 4 Hypothesized Mean Difference 0 df 3 t Stat -0.102707565 P(T<=t) one-tail 0.462337783 t Critical one-tail 2.353363435 P(T<=t) two-tail 0.924675566 t Critical two-tail 3.182446305 However: p=0.925 Null Hypothesis is rejected (p>0.05) Data is not statistically significant Day 15 t-Test: Two-Sample Assuming Unequal Variances Variable 1 Variable 2 Mean 34.07 28.518 Variance 20.2809 158.03167 Observations 3 5 Hypothesized Mean Difference 0 df 5 t Stat 0.89633965 P(T<=t) one-tail 0.205572708 t Critical one-tail 2.015048373 P(T<=t) two-tail 0.411145416 t Critical two-tail 2.570581836 However: p=0.411 Null Hypothesis is rejected (p>0.05) Data is not statistically significant Day 18 t-Test: Two-Sample Assuming Unequal Variances Variable 1 Variable 2 Mean 38.05 43.706 Variance 149.1889 233.84208 Observations 3 5 Hypothesized Mean Difference 0 df 5 t Stat -0.57577146 P(T<=t) one-tail 0.294856307 t Critical one-tail 2.015048373 P(T<=t) two-tail 0.589712614 t Critical two-tail 2.570581836 However: p=0.590 Null Hypothesis is rejected (p>0.05) Data is not statistically significant Day 21 t-Test: Two-Sample Assuming Unequal Variances Variable 1 Variable 2 Mean 17.3075 25.046 Variance 23.32449167 66.01873778 Observations 4 10 Hypothesized Mean Difference 0 df 10 t Stat -2.194668235 P(T<=t) one-tail 0.026458188 t Critical one-tail 1.812461123 P(T<=t) two-tail 0.052916375 t Critical two-tail 2.228138852 However: p=0.053 Null Hypothesis is rejected (p>0.05) Data is not statistically significant CD8 Cytotoxic T cell Results- Student t-Test Day Cell % of Total % of Total Chicken ID Chicken ID Cell count Count Leukocytes Leukocytes % CD8 Cells of Total Leukocyte 0 Chicken( Control 2) 29 2.86 Chicken( Control 2) 29 2.86 t-Test: Two-Sample Assuming Unequal Variances Day 0 0 Chicken( Control 1) 23 22.13 Chicken( Control 1) 23 22.13 0 E3 0 0.00 E3 0 0.00 Variable 1 Variable 2 0 E4 11 4.90 E4 11 4.90 Mean 6.230212193 6.230212193 0 E5 2 1.27 E5 2 1.27 Variance 82.31026361 82.31026361 1 Chicken( Control 2) 29 2.86 E6 48 3.62 Observations 5 5 1 Chicken( Control 1) 23 22.13 E7 49 1.74 Hypothesized Mean Difference 0 1 E3 0 0.00 E8* 19 1.20 df 8 1 E4 11 4.90 E9 23 1.00 t Stat 0 1 E5 2 1.27 E11 83 6.95 P(T<=t) one-tail 0.5 3 Chicken( Control 2) 29 2.86 E12 9 7.05 t Critical one-tail 1.859548038 3 Chicken( Control 1) 23 22.13 E13 84 16.79 P(T<=t) two-tail 1 3 E3 0 0.00 E16 26 7.12 t Critical two-tail 2.306004135 3 E4 11 4.90 E20 48 7.66 However: p=1.000 3 E5 2 1.27 E21 37 1.59 Null Hypothesis is rejected (p>0.05) Data is not statistically significant 4 Chicken( Control 2) 29 2.86 E4 36 1.43 4 Chicken( Control 1) 23 22.13 E11 29 1.48 t-Test: Two-Sample Assuming Unequal Variances Day 1 4 E3 0 0.00 E17 67 2.31 4 E4 11 4.90 E24 31 1.91 Variable 1 Variable 2 4 E5 2 1.27 E17 67 2.31 Mean 6.230212193 2.903843087 4 E24 31 1.91 Variance 82.31026361 6.188317129 4 E25 17 0.78 Observations 5 5 7 Chicken( Control 2) 29 2.86 E15 26 5.52 Hypothesized Mean Difference 0 7 Chicken( Control 1) 23 22.13 E16 82 4.98 df 5 7 E3 0 0.00 E22 72 4.69 t Stat 0.790655484 7 E4 11 4.90 E24 1 0.40 P(T<=t) one-tail 0.232488373 7 E5 2 1.27 E26 213 5.63 t Critical one-tail 2.015048373 7 E28 129 2.89 P(T<=t) two-tail 0.464976746 7 E30 186 17.47 t Critical two-tail 2.570581836 10 Chicken( Control 2) 29 2.86 E6 70 8.06 However: p=0.465 10 Chicken( Control 1) 23 22.13 E9 62 7.53 Null Hypothesis is rejected (p>0.05) Data is not statistically significant 10 E3 0 0.00 E11 91 7.83 10 E4 11 4.90 E17 55 6.47 t-Test: Two-Sample Assuming Unequal Variances Day 3 10 E5 2 1.27 E22 16 2.62 11 Chicken (Control 1) 104 6.97 E15 87 6.31 Variable 1 Variable 2 11 Chicken (Control 2) 68 5.76 E22 79 8.36 Mean 6.230212193 8.042268577 11 Chicken (Control 3) 151 8.41 E26 181 11.57 Variance 82.31026361 30.04620348 11 E28 178 8.73 Observations 5 5 15 Chicken (Control 4) 33 5.95 E2 20 2.83 Hypothesized Mean Difference 0 15 Chicken (Control 5) 131 9.84 E11 192 11.29 df 7 15 Chicken (Control 6) 94 7.98 E13 42 4.28 t Stat -0.382258958 15 E19 373 13.12 P(T<=t) one-tail 0.356805079 15 E21 85 4.35 t Critical one-tail 1.894578605 18 Chicken (Control 7) 59 4.44 E4 107 4.51 P(T<=t) two-tail 0.713610158 18 Chicken (Control 8) 2 4.12 E11 304 9.67 t Critical two-tail 2.364624252 18 Chicken (Control 9) 116 4.41 E15 89 5.96 However: p=0.714 18 E19 230 13.03 Null Hypothesis is rejected (p>0.05) Data is not statistically significant 18 E27 102 6.57 21 Chicken (Control 10) 4 3.52 E3 7 1.22 t-Test: Two-Sample Assuming Unequal Variances Day 4 21 Chicken (Control 11) 154 4.44 E6 16 4.71 21 Chicken (Control 12) 34 2.17 E9 19 5.66 Variable 1 Variable 2 21 Chicken (Control 12) 2 1.21 E11 23 5.12 Mean 6.230212193 1.730639787 21 E15 8 2.36 Variance 82.31026361 0.298119753 21 E17 22 3.45 Observations 5 7 21 E21 27 4.65 Hypothesized Mean Difference 0 21 E26 30 6.81 df 4 21 E27 23 4.56 t Stat 1.107562391 21 E28 21 6.53 P(T<=t) one-tail 0.165082972 t Critical one-tail 2.131846786 P(T<=t) two-tail 0.330165943 t Critical two-tail 2.776445105 However: p=0.330 Null Hypothesis is rejected (p>0.05) Data is not statistically significant Null hypothesis: H0: There is no significant difference between the means of the experimental and control chicken groups (µd=0) t-Test: Two-Sample Assuming Unequal Variances Day 7 HA: There is a significant difference between the means of the experimental and control chicken groups (µd≠0) Variable 1 Variable 2 Mean 6.230212193 5.939015205 Variance 82.31026361 29.29543978 Observations 5 7 Hypothesized Mean Difference 0 df 6 t Stat 0.064085117 P(T<=t) one-tail 0.475492094 t Critical one-tail 1.943180281 P(T<=t) two-tail 0.950984188 t Critical two-tail 2.446911851 However: p=0.951 Null Hypothesis is rejected (p>0.05) Data is not statistically significant t-Test: Two-Sample Assuming Unequal Variances Day 10 Variable 1 Variable 2 Mean 6.230212193 6.502341318 Variance 82.31026361 5.084669262 Observations 5 5 Hypothesized Mean Difference 0 df 4 t Stat -0.065090394 P(T<=t) one-tail 0.475612623 t Critical one-tail 2.131846786 P(T<=t) two-tail 0.951225246 t Critical two-tail 2.776445105 However: p=0.951 Null Hypothesis is rejected (p>0.05) Data is not statistically significant t-Test: Two-Sample Assuming Unequal Variances Day 11 Variable 1 Variable 2 Mean 7.05007715 8.741107781 Variance 1.760500027 4.683449724 Observations 3 4 Hypothesized Mean Difference 0 df 5 t Stat -1.275497525 P(T<=t) one-tail 0.129089209 t Critical one-tail 2.015048373 P(T<=t) two-tail 0.258178418 t Critical two-tail 2.570581836 However: p=0.258 Null Hypothesis is rejected (p>0.05) Data is not statistically significant t-Test: Two-Sample Assuming Unequal Variances Day 15 Variable 1 Variable 2 Mean 7.921592332 7.175067084 Variance 3.789244603 21.87513125 Observations 3 5 Hypothesized Mean Difference 0 df 6 t Stat 0.31439658 P(T<=t) one-tail 0.381932505 t Critical one-tail 1.943180281 P(T<=t) two-tail 0.76386501 t Critical two-tail 2.446911851 However: p=0.764 Null Hypothesis is rejected (p>0.05) Data is not statistically significant t-Test: Two-Sample Assuming Unequal Variances Day 18 Variable 1 Variable 2 Mean 4.322230807 7.948570958 Variance 0.032322139 11.61546819 Observations 3 5 Hypothesized Mean Difference 0 df 4 t Stat -2.373725106 P(T<=t) one-tail 0.038254966 t Critical one-tail 2.131846786 P(T<=t) two-tail 0.076509933 t Critical two-tail 2.776445105 However: p=0.077 Null Hypothesis is rejected (p>0.05) Data is not statistically significant t-Test: Two-Sample Assuming Unequal Variances Day 21 Variable 1 Variable 2 Mean 2.833224733 4.507670421 Variance 2.04228688 3.081561401 Observations 4 10 Hypothesized Mean Difference 0 df 7 t Stat -1.850551947 P(T<=t) one-tail 0.053340327 t Critical one-tail 1.894578605 P(T<=t) two-tail 0.106680654 t Critical two-tail 2.364624252 However: p=0.107 Null Hypothesis is rejected (p>0.05) Data is not statistically significant CD4 T-Helper cell Results- Student t-Test Day Cell % of Total Cell % of Total Chicken ID Chicken ID Count Leukocytes count Leukocytes % CD4 Cells of Total Leukocytes 0 Chicken( Control 2) 129 12.82 Chicken( Control 2) 129 12.82 Day 0 t-Test: Two-Sample Assuming Unequal Variances 0 Chicken( Control 1) 53 51.96 Chicken( Control 1) 53 51.96 0 E3 26 8.75 E3 26 8.75 Variable 1 Variable 2 0 E4 12 5.17 E4 12 5.17 Mean 16.62816965 16.62816965 0 E5 7 4.43 E5 7 4.43 Variance 401.2230467 401.2230467 1 Chicken( Control 2) 129 12.82 E6 134 10.19 Observations 5 5 1 Chicken( Control 1) 53 51.96 E7 254 8.94 Hypothesized Mean Difference 0 1 E3 26 8.75 E8* 19 1.20 df 8 1 E4 12 5.17 E9 67 2.86 t Stat 0 1 E5 7 4.43 E11 134 11.17 P(T<=t) one-tail 0.5 3 Chicken( Control 2) 129 12.82 E12 19 14.50 t Critical one-tail 1.859548038 3 Chicken( Control 1) 53 51.96 E13 59 11.75 P(T<=t) two-tail 1 3 E3 26 8.75 E16 31 8.61 t Critical two-tail 2.306004135 3 E4 12 5.17 E20 105 16.80 However: p=1.000 3 E5 7 4.43 E21 124 5.37 Null Hypothesis is rejected (p>0.05) Data is not statistically significant 4 Chicken( Control 2) 129 12.82 E4 79 3.13 4 Chicken( Control 1) 53 51.96 E11 92 4.63 Day 1 t-Test: Two-Sample Assuming Unequal Variances 4 E3 26 8.75 E17 96 3.33 4 E4 12 5.17 E24 85 5.19 Variable 1 Variable 2 4 E5 7 4.43 E17 96 3.33 Mean 16.62816965 6.873794094 4 E24 85 5.19 Variance 401.2230467 20.48292063 4 E25 229 10.30 Observations 5 5 7 Chicken( Control 2) 129 12.82 E15 94 20.00 Hypothesized Mean Difference 0 7 Chicken( Control 1) 53 51.96 E16 211 12.83 df 4 7 E3 26 8.75 E22 194 12.59 t Stat 1.062134709 7 E4 12 5.17 E24 37 10.28 P(T<=t) one-tail 0.174023218 7 E5 7 4.43 E26 356 9.42 t Critical one-tail 2.131846786 7 E28 312 6.96 P(T<=t) two-tail 0.348046435 7 E30 404 38.04 t Critical two-tail 2.776445105 10 Chicken( Control 2) 129 12.82 E6 132 15.10 However: p=0.348 10 Chicken( Control 1) 53 51.96 E9 85 10.39 Null Hypothesis is rejected (p>0.05) Data is not statistically significant 10 E3 26 8.75 E11 135 11.65 10 E4 12 5.17 E17 90 10.53 Day 3 t-Test: Two-Sample Assuming Unequal Variances 10 E5 7 4.43 E22 80 13.49 11 Chicken (Control 1) 238 15.98 E15 259 18.85 Variable 1 Variable 2 11 Chicken (Control 2) 192 16.28 E22 293 31.01 Mean 16.62816965 11.40810659 11 Chicken (Control 3) 407 22.71 E26 426 27.15 Variance 401.2230467 20.75629948 11 E28 423 20.77 Observations 5 5 15 Chicken (Control 4) 86 15.55 E2 97 13.60 Hypothesized Mean Difference 0 15 Chicken (Control 5) 270 20.29 E11 337 19.81 df 4 15 Chicken (Control 6) 207 17.56 E13 95 9.71 t Stat 0.568218218 15 E19 325 11.43 P(T<=t) one-tail 0.300133048 15 E21 195 10.02 t Critical one-tail 2.131846786 18 Chicken (Control 7) 248 18.72 E4 229 9.64 P(T<=t) two-tail 0.600266097 18 Chicken (Control 8) 7 11.67 E11 533 16.98 t Critical two-tail 2.776445105 18 Chicken (Control 9) 242 9.23 E15 308 20.69 However: p=0.600 18 E19 247 13.98 Null Hypothesis is rejected (p>0.05) Data is not statistically significant 18 E27 311 20.08 21 Chicken (Control 10) 6 5.83 E3 24 4.48 Day 4 t-Test: Two-Sample Assuming Unequal Variances 21 Chicken (Control 11) 346 9.97 E6 46 13.53 21 Chicken (Control 12) 113 7.17 E9 32 9.30 Variable 1 Variable 2 21 Chicken (Control 12) 5 3.50 E11 41 8.95 Mean 16.62816965 5.013806908 21 E15 20 6.29 Variance 401.2230467 6.238089061 21 E17 49 7.80 Observations 5 7 21 E21 64 10.94 Hypothesized Mean Difference 0 21 E26 96 21.72 df 4 21 E27 43 8.63 t Stat 1.289404609 21 E28 47 14.55 P(T<=t) one-tail 0.133382123 t Critical one-tail 2.131846786 P(T<=t) two-tail 0.266764247 t Critical two-tail 2.776445105 However: p=0.267 Null Hypothesis is rejected (p>0.05) Data is not statistically significant Null hypothesis: Day 7 t-Test: Two-Sample Assuming Unequal Variances H0: There is no significant difference between the means of the experimental and control chicken groups (µd=0) HA: There is a significant difference between the means of the experimental and control chicken groups (µd≠0) Variable 1 Variable 2 Mean 16.62816965 15.7304878 Variance 401.2230467 113.4651399 Observations 5 7 Hypothesized Mean Difference 0 df 6 t Stat 0.091403434 P(T<=t) one-tail 0.465073634 t Critical one-tail 1.943180281 P(T<=t) two-tail 0.930147268 t Critical two-tail 2.446911851 However: p=0.930 Null Hypothesis is rejected (p>0.05) Data is not statistically significant Day 10 t-Test: Two-Sample Assuming Unequal Variances Variable 1 Variable 2 Mean 16.62816965 12.23183724 Variance 401.2230467 4.116470998 Observations 5 5 Hypothesized Mean Difference 0 df 4 t Stat 0.488276757 P(T<=t) one-tail 0.325455902 t Critical one-tail 2.131846786 P(T<=t) two-tail 0.650911803 t Critical two-tail 2.776445105 However: p=0.651 Null Hypothesis is rejected (p>0.05) Data is not statistically significant Day 11 t-Test: Two-Sample Assuming Unequal Variances Variable 1 Variable 2 Mean 18.32697422 24.44306188 Variance 14.44435684 31.73320181 Observations 3 4 Hypothesized Mean Difference 0 df 5 t Stat -1.712975724 P(T<=t) one-tail 0.073695298 t Critical one-tail 2.015048373 P(T<=t) two-tail 0.147390596 t Critical two-tail 2.570581836 However: p=0.147 Null Hypothesis is rejected (p>0.05) Data is not statistically significant Day 15 t-Test: Two-Sample Assuming Unequal Variances Variable 1 Variable 2 Mean 17.7980962 12.91541191 Variance 5.646104845 17.22468582 Observations 3 5 Hypothesized Mean Difference 0 df 6 t Stat 2.115526318 P(T<=t) one-tail 0.039384442 t Critical one-tail 1.943180281 P(T<=t) two-tail 0.078768885 t Critical two-tail 2.446911851 However: p=0.079 Null Hypothesis is rejected (p>0.05) Data is not statistically significant Day 18 t-Test: Two-Sample Assuming Unequal Variances Variable 1 Variable 2 Mean 13.2032416 16.27260453 Variance 24.29011216 20.91830185 Observations 3 5 Hypothesized Mean Difference 0 df 4 t Stat -0.875875978 P(T<=t) one-tail 0.215272402 t Critical one-tail 2.131846786 P(T<=t) two-tail 0.430544804 t Critical two-tail 2.776445105 However: p=0.431 Null Hypothesis is rejected (p>0.05) Data is not statistically significant Day 21 t-Test: Two-Sample Assuming Unequal Variances Variable 1 Variable 2 Mean 6.613172031 10.619842 Variance 7.292403933 24.45567502 Observations 4 10 Hypothesized Mean Difference 0 df 10 t Stat -1.939265882 P(T<=t) one-tail 0.040589798 t Critical one-tail 1.812461123 P(T<=t) two-tail 0.081179597 t Critical two-tail 2.228138852 However: p=0.081 Null Hypothesis is rejected (p>0.05) Data is not statistically significant Ratio of CD4:CD8 Results- Student t-Test Day Chicken ID Cell Count Chicken ID Cell count CD4/CD8 ratio 0 Chicken( Control 2) 4.48 Chicken( Control 2) 4.48 Day 0 t-Test: Two-Sample Assuming Unequal Variances 0 Chicken( Control 1) 2.35 Chicken( Control 1) 2.35 0 E3 0.00 E3 0.00 Control Variable 2 0 E4 1.06 E4 1.06 Mean 2.277582274 2.277582274 0 E5 3.50 E5 3.50 Variance 3.261943097 3.261943097 1 Chicken( Control 2) 4.48 E6 2.81 Observations 5 5 1 Chicken( Control 1) 2.35 E7 5.15 Hypothesized Mean Difference 0 1 E3 0.00 E8* 1.00 df 8 1 E4 1.06 E9 2.86 t Stat 0 1 E5 3.50 E11 1.61 P(T<=t) one-tail 0.5 3 Chicken( Control 2) 4.48 E12 2.06 t Critical one-tail 1.859548038 3 Chicken( Control 1) 2.35 E13 0.70 P(T<=t) two-tail 1 3 E3 0.00 E16 1.21 t Critical two-tail 2.306004135 3 E4 1.06 E20 2.19 However: p=1.000 3 E5 3.50 E21 3.39 Null Hypothesis is rejected (p>0.05) Data is not statistically significant 4 Chicken( Control 2) 4.48 E4 2.19 4 Chicken( Control 1) 2.35 E11 3.14 4 E3 0.00 E17 1.44 4 E4 1.06 E24 2.72 Day 1 t-Test: Two-Sample Assuming Unequal Variances 4 E5 3.50 E17 1.44 4 E24 2.72 Control Variable 2 4 E25 13.18 Mean 2.277582274 2.684207704 7 Chicken( Control 2) 4.48 E15 3.63 Variance 3.261943097 2.525788472 7 Chicken( Control 1) 2.35 E16 2.58 Observations 5 5 7 E3 0.00 E22 2.68 Hypothesized Mean Difference 0 7 E4 1.06 E24 26.00 df 8 7 E5 3.50 E26 1.67 t Stat -0.377942165 7 E28 2.41 P(T<=t) one-tail 0.357652189 7 E30 2.18 t Critical one-tail 1.859548038 10 Chicken( Control 2) 4.48 E6 1.87 P(T<=t) two-tail 0.715304379 10 Chicken( Control 1) 2.35 E9 1.38 t Critical two-tail 2.306004135 10 E3 0.00 E11 1.49 However: p=0.715 10 E4 1.06 E17 1.63 Null Hypothesis is rejected (p>0.05) Data is not statistically significant 10 E5 3.50 E22 5.15 11 Chicken (Control 1) 2.29 E15 2.99 Day 3 t-Test: Two-Sample Assuming Unequal Variances 11 Chicken (Control 2) 2.83 E22 3.71 11 Chicken (Control 3) 2.70 E26 2.35 Control Variable 2 11 E28 2.38 Mean 2.277582274 1.909460056 15 Chicken (Control 4) 2.62 E2 4.81 Variance 3.261943097 1.060994583 15 Chicken (Control 5) 2.06 E11 1.75 Observations 5 5 15 Chicken (Control 6) 2.20 E13 2.27 Hypothesized Mean Difference 0 15 E19 0.87 df 6 15 E21 2.30 t Stat 0.395901862 18 Chicken (Control 7) 4.21 E4 2.14 P(T<=t) one-tail 0.352935916 18 Chicken (Control 8) 2.83 E11 1.76 t Critical one-tail 1.943180281 18 Chicken (Control 9) 2.09 E15 3.47 P(T<=t) two-tail 0.705871831 18 E19 1.07 t Critical two-tail 2.446911851 18 E27 3.06 However: p=0.706 21 Chicken (Control 10) 1.66 E3 3.67 Null Hypothesis is rejected (p>0.05) Data is not statistically significant 21 Chicken (Control 11) 2.25 E6 2.87 21 Chicken (Control 12) 3.30 E9 1.64 Day 4 t-Test: Two-Sample Assuming Unequal Variances 21 Chicken (Control 12) 2.90 E11 1.75 21 E15 2.67 Control Variable 2 21 E17 2.26 Mean 2.277582274 3.834196915 21 E21 2.35 Variance 3.261943097 17.41403556 21 E26 3.19 Observations 5 7 21 E27 1.89 Hypothesized Mean Difference 0 21 E28 2.23 df 9 t Stat -0.878433357 P(T<=t) one-tail 0.20127548 t Critical one-tail 1.833112933 P(T<=t) two-tail 0.40255096 t Critical two-tail 2.262157163 However: p=0.403 Null Hypothesis is rejected (p>0.05) Data is not statistically significant Null hypothesis: H0: There is no significant difference between the means of the experimental and control chicken groups (µd=0) Day 7 t-Test: Two-Sample Assuming Unequal Variances HA: There is a significant difference between the means of the experimental and control chicken groups (µd≠0) Control Variable 2 Mean 2.277582274 5.877862358 Variance 3.261943097 79.0808419 Observations 5 7 Hypothesized Mean Difference 0 df 7 t Stat -1.041498536 P(T<=t) one-tail 0.16613675 t Critical one-tail 1.894578605 P(T<=t) two-tail 0.3322735 t Critical two-tail 2.364624252 However: p=0.332 Null Hypothesis is rejected (p>0.05) Data is not statistically significant Day 10 t-Test: Two-Sample Assuming Unequal Variances Control Variable 2 Mean 2.277582274 2.303614547 Variance 3.261943097 2.56595284 Observations 5 5 Hypothesized Mean Difference 0 df 8 t Stat -0.024112442 P(T<=t) one-tail 0.490676759 t Critical one-tail 1.859548038 P(T<=t) two-tail 0.981353517 t Critical two-tail 2.306004135 However: p=0.981 Null Hypothesis is rejected (p>0.05) Data is not statistically significant Day 11 t-Test: Two-Sample Assuming Unequal Variances Control Variable 2 Mean 2.605716785 2.855964513 Variance 0.077575787 0.41194195 Observations 3 4 Hypothesized Mean Difference 0 df 4 t Stat -0.697168737 P(T<=t) one-tail 0.262046287 t Critical one-tail 2.131846786 P(T<=t) two-tail 0.524092574 t Critical two-tail 2.776445105 However: p=0.524 Null Hypothesis is rejected (p>0.05) Data is not statistically significant Day 15 t-Test: Two-Sample Assuming Unequal Variances Control Variable 2 Mean 2.292443839 2.400475425 Variance 0.082982723 2.142831752 Observations 3 5 Hypothesized Mean Difference 0 df 4 t Stat -0.15994112 P(T<=t) one-tail 0.440339595 t Critical one-tail 2.131846786 P(T<=t) two-tail 0.88067919 t Critical two-tail 2.776445105 However: p=0.881 Null Hypothesis is rejected (p>0.05) Data is not statistically significant Day 18 t-Test: Two-Sample Assuming Unequal Variances Control Variable 2 Mean 3.047084454 2.29846139 Variance 1.159691122 0.942754544 Observations 3 5 Hypothesized Mean Difference 0 df 4 t Stat 0.98715648 P(T<=t) one-tail 0.18972524 t Critical one-tail 2.131846786 P(T<=t) two-tail 0.379450481 t Critical two-tail 2.776445105 However: p=0.379 Null Hypothesis is rejected (p>0.05) Data is not statistically significant Day 21 t-Test: Two-Sample Assuming Unequal Variances Control Variable 2 Mean 2.524878656 2.451977451 Variance 0.522949231 0.421241119 Observations 4 10 Hypothesized Mean Difference 0 df 5 t Stat 0.175341884 P(T<=t) one-tail 0.43384533 t Critical one-tail 2.015048373 P(T<=t) two-tail 0.86769066 t Critical two-tail 2.570581836 However: p=0.868 Null Hypothesis is rejected (p>0.05) Data is not statistically significant % B and NK cells of Total Leukocytes Results- Student t-Test Day % of Total % of Total Chicken ID Chicken ID Leukocytes Leukocytes % B and NK cells of Total Leukocytes 0 Chicken( Control 2) 10.26 Chicken( Control 2) 10.26 Day 0 t-Test: Two-Sample Assuming Unequal Variances 0 Chicken( Control 1) 25.91 Chicken( Control 1) 25.91 0 E3 21.55 E3 21.55 Variable 1 Variable 2 0 E4 1.57 E4 1.57 Mean 12.49082912 12.49082912 0 E5 3.16 E5 3.16 Variance 118.3860105 118.3860105 1 Chicken( Control 2) 10.26 E6 4.89 Observations 5 5 1 Chicken( Control 1) 25.91 E7 1.96 Hypothesized Mean Difference 0 1 E3 21.55 E8* -0.06 df 8 1 E4 1.57 E9 2.59 t Stat 0 1 E5 3.16 E11 5.30 P(T<=t) one-tail 0.5 3 Chicken( Control 2) 10.26 E12 10.50 t Critical one-tail 1.859548038 3 Chicken( Control 1) 25.91 E13 13.69 P(T<=t) two-tail 1 3 E3 21.55 E16 9.27 t Critical two-tail 2.306004135 3 E4 1.57 E20 19.06 However: p=1.000 3 E5 3.16 E21 9.81 Null Hypothesis is rejected (p>0.05) Data is not statistically significant 4 Chicken( Control 2) 10.26 E4 3.37 4 Chicken( Control 1) 25.91 E11 6.03 Day 1 t-Test: Two-Sample Assuming Unequal Variances 4 E3 21.55 E17 2.02 4 E4 1.57 E24 1.88 Variable 1 Variable 2 4 E5 3.16 E17 2.02 Mean 12.49082912 2.935408133 4 E24 1.88 Variance 118.3860105 4.87194586 4 E25 4.48 Observations 5 5 7 Chicken( Control 2) 10.26 E15 16.82 Hypothesized Mean Difference 0 7 Chicken( Control 1) 25.91 E16 16.97 df 4 7 E3 21.55 E22 11.60 t Stat 1.924541793 7 E4 1.57 E24 19.88 P(T<=t) one-tail 0.063303958 7 E5 3.16 E26 1.69 t Critical one-tail 2.131846786 7 E28 1.84 P(T<=t) two-tail 0.126607915 7 E30 -13.42 t Critical two-tail 2.776445105 10 Chicken( Control 2) 10.26 E6 9.10 However: p=0.127 10 Chicken( Control 1) 25.91 E9 7.51 Null Hypothesis is rejected (p>0.05) Data is not statistically significant 10 E3 21.55 E11 8.22 10 E4 1.57 E17 6.63 Day 3 t-Test: Two-Sample Assuming Unequal Variances 10 E5 3.16 E22 10.03 11 Chicken (Control 1) 11.09 E15 7.81 Variable 1 Variable 2 11 Chicken (Control 2) 7.89 E22 9.85 Mean 12.49082912 12.46560658 11 Chicken (Control 3) 23.17 E26 1.82 Variance 118.3860105 16.52569409 11 E28 8.89 Observations 5 5 15 Chicken (Control 4) 13.04 E2 -1.85 Hypothesized Mean Difference 0 15 Chicken (Control 5) 8.19 E11 6.93 df 5 15 Chicken (Control 6) 3.81 E13 3.08 t Stat 0.00485567 15 E19 18.64 P(T<=t) one-tail 0.498156764 15 E21 15.32 t Critical one-tail 2.015048373 18 Chicken (Control 7) 28.77 E4 2.90 P(T<=t) two-tail 0.996313528 18 Chicken (Control 8) 17.55 E11 23.08 t Critical two-tail 2.570581836 18 Chicken (Control 9) 15.26 E15 23.32 However: p=0.996 18 E19 28.68 Null Hypothesis is rejected (p>0.05) Data is not statistically significant 18 E27 19.45 21 Chicken (Control 10) 7.16 E3 6.05 Day 4 t-Test: Two-Sample Assuming Unequal Variances 21 Chicken (Control 11) 9.93 E6 12.34 21 Chicken (Control 12) 4.36 E9 13.81 Variable 1 Variable 2 21 Chicken (Control 12) 9.98 E11 8.20 Mean 12.49082912 3.096531323 21 E15 5.19 Variance 118.3860105 2.642038323 21 E17 8.97 Observations 5 7 21 E21 12.44 Hypothesized Mean Difference 0 21 E26 9.48 df 4 21 E27 12.51 t Stat 1.915424011 21 E28 10.19 P(T<=t) one-tail 0.063971955 t Critical one-tail 2.131846786 P(T<=t) two-tail 0.12794391 t Critical two-tail 2.776445105 However: p=0.128 Null Hypothesis is rejected (p>0.05) Data is not statistically significant Day 7 t-Test: Two-Sample Assuming Unequal Variances Variable 1 Variable 2 Mean 12.49082912 7.912095065 Null hypothesis: Variance 118.3860105 141.4999874 H0: There is no significant difference between the means of the experimental and control chicken groups (µd=0) Observations 5 7 HA: There is a significant difference between the means of the experimental and control chicken groups (µd≠0) Hypothesized Mean Difference 0 df 9 t Stat 0.69112289 P(T<=t) one-tail 0.253460711 t Critical one-tail 1.833112933 P(T<=t) two-tail 0.506921422 t Critical two-tail 2.262157163 However: p=0.507 Null Hypothesis is rejected (p>0.05) Data is not statistically significant Day 10 t-Test: Two-Sample Assuming Unequal Variances Variable 1 Variable 2 Mean 12.49082912 8.29654543 Variance 118.3860105 1.760577632 Observations 5 5 Hypothesized Mean Difference 0 df 4 t Stat 0.855632127 P(T<=t) one-tail 0.220213786 t Critical one-tail 2.131846786 P(T<=t) two-tail 0.440427572 t Critical two-tail 2.776445105 However: p=0.440 Null Hypothesis is rejected (p>0.05) Data is not statistically significant Day 11 t-Test: Two-Sample Assuming Unequal Variances Variable 1 Variable 2 Mean 14.05201545 7.091066158 Variance 64.92628342 13.05003184 Observations 3 4 Hypothesized Mean Difference 0 df 3 t Stat 1.394853714 P(T<=t) one-tail 0.128697657 t Critical one-tail 2.353363435 P(T<=t) two-tail 0.257395313 t Critical two-tail 3.182446305 However: p=0.257 Null Hypothesis is rejected (p>0.05) Data is not statistically significant Day 15 t-Test: Two-Sample Assuming Unequal Variances Variable 1 Variable 2 Mean 8.347924082 8.42529618 Variance 21.32571717 72.06294129 Observations 3 5 Hypothesized Mean Difference 0 df 6 t Stat -0.01667829 P(T<=t) one-tail 0.493617017 t Critical one-tail 1.943180281 P(T<=t) two-tail 0.987234034 t Critical two-tail 2.446911851 However: p=0.987 Null Hypothesis is rejected (p>0.05) Data is not statistically significant Day 18 t-Test: Two-Sample Assuming Unequal Variances Variable 1 Variable 2 Mean 20.52655085 19.48484963 Variance 52.22917637 96.8178662 Observations 3 5 Hypothesized Mean Difference 0 df 6 t Stat 0.171781693 P(T<=t) one-tail 0.434628271 t Critical one-tail 1.943180281 P(T<=t) two-tail 0.869256542 t Critical two-tail 2.446911851 However: p=0.869 Null Hypothesis is rejected (p>0.05) Data is not statistically significant Day 21 t-Test: Two-Sample Assuming Unequal Variances Variable 1 Variable 2 Mean 7.859758109 9.919146138 Variance 7.184933854 8.39440116 Observations 4 10 Hypothesized Mean Difference 0 df 6 t Stat -1.268505552 P(T<=t) one-tail 0.125804844 t Critical one-tail 1.943180281 P(T<=t) two-tail 0.251609688 t Critical two-tail 2.446911851 However: p=0.252 Null Hypothesis is rejected (p>0.05) Data is not statistically significant CHAPTER 5 POST-MORTEM EXAMINATION OF CHICKEN LYMPHOID TISSUES AFTER INFECTION WITH Avibacterium paragallinarum SEROVAR C-3 INFECTION Sections of Chapter 5, have been used for manuscript for submission in a peer-reviewed journal, with the title “Omens and Remnants of Infectious Coryza: A macabre tale of necropsy and immunohistopathology of chicken lymphatic tissues after infection with Av. paragallinarum serovar C-3 infection”. 5.1. Introduction Infectious coryza (IC) is a contagious poultry disease with the causative agent being Avibacterium paragallinarum (Yamamoto, 1984). Infectious coryza affects the upper respiratory tract (URT) of chickens, however in rare cases where the disease is severe or complicated, the infection spreads to the trachea, air sacs and lungs, leading to pneumonia and air sacculitis, even in the absence of other avian pathogens (Droual et al. 1990; Hoerr et al. 1994; Deshmukh et al. 2015). Although, infectious coryza is a disease pertaining to the URT, there are a multitude of clinical signs and symptoms (Blackall and Soriano, 2008). The initial stage of the disease, following an infection starts off with nasal and ocular sero-mucus 364 CHAPTER 5: POST-MORTEM EXAMINATION OF CHICKEN LYMPHOID TISSUES AFTER INFECTION WITH AV. PARAGALLINARUM SEROVAR C-3 INFECTION secretions which transform into caseous deposits around the para-nasal regions and eyes, which is often accompanied by conjunctivitis (Deshmukh et al. 2015). Facial oedema, excessive secretion of tears, swelling of the sinuses, swollen-head syndrome (especially in males), lethargy, anorexia, diarrhoea, poor appetite and water consumption, difficulty in breathing, fetid odour of exudates and poor growth in younger chickens are also observed (Blackall, 1999; Pattison et al. 2008). In the lower air tract, rales may be heard due to infection, and lesions can be present leading to acute catarrh (Blackall and Soriano, 2008). In older layer birds, the reproductive organs such as the ovary and salpinx are affected by IC, resulting in poor egg quality and a significant drop in egg production (Deshmukh et al. 2015). In broiler birds, due to inanition caused by IC as a result of poor appetite and decreased water consumption, there is a drastic decline in the feed conversion efficiency, resulting in a reduction in flesh growth and extreme culling in juvenile birds (Deshmukh et al. 2015). Gross pathological and histological studies have been documented and conducted on chickens infected with Av. paragallinarum (Fujiwara and Konno, 1965; Sawata et al. 1985; Droual et al. 1990; Hoerr et al. 1994; Blackall and Soriano, 2008; Paudel et al. 2017). A study by Fujiwara and Konno (1965), showed histopathological responses of chickens from 12 h to 3 months following intranasal inoculation with Av. paragallinarum. The chronology in histological changes were recorded, whereby at 20 h pathological changes were first observed, consequently by 7–10 days the severity within tissues were seen and finally tissue repairment occurred within 14–21 days (Fujiwara and Konno, 1965). Furthermore, the study revealed sloughing, tissue disintegration and hyperplasia of mucosal and glandular epithelia of the nasal cavity, infraorbital sinuses, and trachea caused by IC (Fujiwara and Konno, 1965). Additionally, in the tunica propria of the mucous membranes, hyperemia (excess of blood being transported and supplied to organs) with heterophil accumulation were also observed (Fujiwara and Konno, 1965). Furthermore, within the nasal cavity, the mucous membrane of the lamina propria showed mast cells during IC invasion, which shows that the innate immune response was at play (Sawata et al. 1985). In contrast, when infections in the lower respiratory 365 CHAPTER 5: POST-MORTEM EXAMINATION OF CHICKEN LYMPHOID TISSUES AFTER INFECTION WITH AV. PARAGALLINARUM SEROVAR C-3 INFECTION tract of birds occurred, acute catarrhal bronchopneumonia, pneumonia or air sacculitis resulted, whereby the lumen of secondary and tertiary bronchi were congested with heterophils and cell debris making it difficult for the bird to breathe (Blackall and Soriano, 2008). Moreover, epithelial cells of capillaries situated in the lungs displayed swelling and hyperplasia (Blackall and Soriano, 2008). In some birds, catarrhal inflammation of air sacs was observed with swelling, hyperplasia and heterophil infiltration (Blackall and Soriano, 2008). Lesions may also be observed, due to acute catarrhal inflammation of the upper respiratory tract (Akter et al. 2013). It is suspected that the immune molecules and responses of mast cells, heterophils, and macrophages may be accountable for the severe vascular alterations and cell damage leading to the clinical signs and symptoms of infectious coryza. Necropsy is a term used to describe a post-mortem examination performed on an animal species, as opposed to autopsy which is used exclusively for human patients (King, 1989). Necropsy is a valuable tool in furthering our knowledge and providing perception for specific diseases (King, 1989). A study by Sandoval et al. (1994), Av. paragallinarum was isolated not only from the infraorbital sinuses, but also liver, kidney, lungs, eye, tarsus, heart and ovary, whereby this was the first report of the bacteria spreading to downstream organs other than the site of primary infection which is the URT. This finding suggests that Av. paragallinarum may be invasive and is also likely to cause septicaemia in organs, resulting in a systemic inflammatory response eventually leading to shock and perhaps even mortality in chickens (Sandoval et al. 1994). Akter et al. (2013) reported on necropsy findings following infection of chickens with Av. paragallinarum, whereby the mucous membranes of nasal passages and trachea showed haemorrhage. Histopathological findings by Akter et al. (2013) of nasal septum collected from samples, showed parakeratosis, congestion of nasal passages, hyperplasia of mucous glandular cells and hyperplasia of nasal sinuses. 366 CHAPTER 5: POST-MORTEM EXAMINATION OF CHICKEN LYMPHOID TISSUES AFTER INFECTION WITH AV. PARAGALLINARUM SEROVAR C-3 INFECTION Immunohistochemistry (IHC) has an important role in pathology and is a powerful diagnostic tool that uses monoclonal and polyclonal antibodies to detect and determine the distribution of specific antigens within tissues (Duraiyan et al. 2012). The availability of biopsies or fragments of tissue is required, which are embedded and then cut with a microtome, whereby the sections are stained and incubated with an appropriate antibody (Duraiyan et al. 2012). The antibody present within the tissue, can then be visualized using a light, fluorescent or electron microscope and in some cases autoradiography, using a marker such as a fluorescent dye, enzyme label, radioactive element or colloidal gold that is directly linked to an appropriate primary or secondary antibody (Coons et al. 1941; Coons and Kalpan, 1950; Nakane and Pierce, 1966, Faulk and Taylor, 1971; Mason and Sammons, 1978 ). Conventional analysis using histology techniques of hematoxylin and eosin, performed after an autopsy is necessary, however differential diagnosis cannot be performed and hence there are limitations, therefore immunohistochemistry can provide better insight (Bernardi et al. 2005; Roulson et al. 2005; de Matos et al. 2010). The main goal of IHC is to conduct staining of tissue sections, by causing minimal damage on the cell or tissue, and by using very small amounts of antibody, thereby allowing pathologists to make a diagnosis and prognosis in health and disease (Duraiyan et al. 2012). Although, IHC is the standard method for detecting proteins in situ on thin sections of formalin-fixed paraffin-embedded (FFPE) tissue followed by an assessment of antibody reactivity using image analysis, it is costly, time-consuming, laborious and prone to human error (Raab, 2000; Prichard, 2014; Kalyazhny, 2016; Guirado, 2018). The aim of this study is to perform a necropsy on birds infected with infectious coryza (IC), specifically the SA-3 strain (serovar C-3), and conduct immunohistochemical staining on the tissues harvested from both healthy (control birds) and infected chickens (experimental birds) presented with score 1, score 2 and score 3. Necropsy will provide some pathological insight of how the disease and bacterial pathogen affects the tissues of the infected chickens that displayed clinical signs and symptoms. Immunohistochemistry using a selection of monoclonal 367 CHAPTER 5: POST-MORTEM EXAMINATION OF CHICKEN LYMPHOID TISSUES AFTER INFECTION WITH AV. PARAGALLINARUM SEROVAR C-3 INFECTION antibodies for the detection of T lymphocyte, B lymphocyte and macrophage lineages were used, to understand the distribution and function of these immune cells during the immunological response at the different stages of IC, as the disease progresses from mild (score 1) to moderate (score 2) to severe (score 3). 5.2. Materials and methods 5.2.1. Study design and ethics approval Ethics approval was obtained as per Section 3.2.1, Chapter 3 and Section 4.2.1, Chapter 4 for conducting necropsy and immunohistochemistry (Chapter 5) as the aftermath of the research study. Following the challenge study conducted in Chapter 4 (Section 4.2.1 – Section 4.2.8) based on the method and scoring described in Section 3.2.4 (Chapter 3), SPF/unvaccinated White Leghorn chickens at 25 weeks were randomly selected from the total population of experimental (NE= 30) and control (NC= 8) subjects, where 2-4 chickens showing the same disease score (0, 1, 2 or 3) as well as re-infected chickens (RI) were chosen for sacrifice. 5.2.2. Necropsy, sample collection and formalin fixation There were no mortalities recorded in the study caused by IC (Chapter 4), however morbidity was high. A total of 12 chickens (Ns=12) were selected for sacrifice from: control (Control 1, Control 2), score 1 (E1, E23), score 2 (E8, E20), score 3 (E11, E19, E22, E24) and RI (E15, E16) chickens. Chickens selected based on the disease score, were then sacrificed by decapitation. Nasal swabs were then collected from the dead chickens, by first making a small and precise incision into the sinus cavity of the heads obtained from both infected, re-infected and control birds using sterile scissors, proceeded by the insertion of a sterile cotton swab into 368 CHAPTER 5: POST-MORTEM EXAMINATION OF CHICKEN LYMPHOID TISSUES AFTER INFECTION WITH AV. PARAGALLINARUM SEROVAR C-3 INFECTION the sinus cavity. The heads were stored at -80°C and sinuses were dissected and stored in 500µl of RNAlater® RNA Stabilization Solution (Thermo Fisher Scientific) at -80°C for future studies. The sacrificed chickens were then necropsied and the relevant lymphoid tissues were harvested such as spleen, liver, intestine, uterus (shell gland) and trachea (Figure 5.1). The trachea of chickens Control 1-healthy, E15 RI-score 3, E19-score 3, were swabbed with a sterile cotton swab. The liver of chicken E20 was also swabbed with a sterile cotton swab. The harvested tissues were fixed in 10% buffered formalin immediately after dissection and stored at 4°C or room temperature until further use. Tissues need to be fixed for 6-72 h in 10% buffered formalin prior to processing (Wolff et al. 2013). Fixation is crucial as it allows for thin sectioning of tissue by hardening of the tissue, it prevents autolysis of cells and infectious agents and improves cell avidity for efficacious dissection, processing and microscopic examination of histopathology specimens (Grizzle, 2009). 369 CHAPTER 5: POST-MORTEM EXAMINATION OF CHICKEN LYMPHOID TISSUES AFTER INFECTION WITH AV. PARAGALLINARUM SEROVAR C-3 INFECTION Trachea Liver Spleen Intestine Chicken carcass Uterus (Shell gland) Figure 5.1: Necropsy and gross post-mortem examination was conducted on a chicken carcass. The lymphoid tissues such as the liver, intestine, trachea, uterus (shell gland) and spleen were removed, harvested and fixed in 10% buffered formalin and stored at 4°C or room temperature until further use. 370 CHAPTER 5: POST-MORTEM EXAMINATION OF CHICKEN LYMPHOID TISSUES AFTER INFECTION WITH AV. PARAGALLINARUM SEROVAR C-3 INFECTION 5.2.3. Microbial cultivation, isolation and identification To investigate which microorganism was the main causative agent of disease and also to prove Koch’s postulates, re-isolation of the pathogen from the site of infection and from other suspected areas was attempted (Falkow, 1988). Cotton swabs collected from the nasal cavity, trachea and liver of chickens mentioned in Section 5.2.2 (Chapter 5) were then spread over the surface of blood tryptose agar (BTA) plates containing cattle blood (Onderstepoort Biological Products, Pretoria) and cross-streaked with Staphylococcus epidermidis for cultivation as well as TSA plates (supplemented with 0.2% (v/v) NAD+), as described in Section 3.2.3.1 (Chapter 3) and Section 4.2.3 (Chapter 4). BTA plates were cultured at 37°C for 24 h in a candle jar (due to microaerophilic nature of Av. paragallinarum) and TSA plates supplemented with 0.2% NAD+ (v/v) were incubated at 37°C for 24 h since it is a non-selective media and a variety of micro-organisms obtained from the nasal swabs wanted to be grown. Single colonies of suspected Av. paragallinarum displaying satellitic behaviour near the S. epidermidis “feeder cultures”, were plated onto fresh BTA plates and cross-streaked with S. epidermidis and were once again placed in a candle jar and incubated at 37°C for 24 h. The bacterial culture was passaged every 2 days on BTA plates to keep the bacterial culture viable. A pre-inoculum was prepared from the samples collected as described in Section 3.2.3.1. (Chapter 3), containing a bacterial culture of less than 24 hours of age, which was then inoculated into a 50 ml tube containing 50 ml of TSB supplemented with 0.04% NAD+ (v/v) and incubated for a further 10-14 h at 37C (Labwit Scientific), grown to an optical density (OD600) of 1.0. The bacterial culture was then centrifuged at 3000 x g for 10 min, to obtain a pellet which was re-suspended in 10 ml of 1X phosphate-buffered saline (PBS) (pH 7.4, Merck) which was kept at 4C until further use. 371 CHAPTER 5: POST-MORTEM EXAMINATION OF CHICKEN LYMPHOID TISSUES AFTER INFECTION WITH AV. PARAGALLINARUM SEROVAR C-3 INFECTION A volume of 1-3 ml of the bacterial culture was used for genomic DNA extraction as described in Section 3.2.3.2 (Chapter 3), a species-specific PCR was then performed following the extraction of genomic DNA as described in Section 3.2.3.3 (Chapter 3) and finally agarose gel electrophoresis and visualisation of the correct DNA fragment size was conducted as per Section 3.2.3.4 (Chapter 3). 5.2.4. Immunohistochemistry (IHC) of samples collected 5.2.4.1. Anatomical grossing, tissue processing and paraffin wax impregnation A B Figure 5.2: Summarized flow diagram of steps followed for IHC staining. (A) Lymphoid tissue fragments embedded in paraffin wax. (B) IHC slide containing sections of FFPE tissues stained with anti-CD20 monoclonal antibody. The lymphoid tissues fixed in 10% buffered formalin were sent to and conducted at the National Health Laboratory Service (NHLS, Department of Histopathology, Universitas, 372 CHAPTER 5: POST-MORTEM EXAMINATION OF CHICKEN LYMPHOID TISSUES AFTER INFECTION WITH AV. PARAGALLINARUM SEROVAR C-3 INFECTION Bloemfontein, South Africa) to obtain FFPE tissues stained with monoclonal antibodies such as such as anti-CD3 (T lymphocytes), anti-CD20 (B lymphocytes) and anti-CD68 (macrophages) to detect and determine the distribution and expression of immune cells within tissues. Haematoxylin and eosin staining of lymphoid tissues were also conducted, however the results were not included in this study. The Department of Histopathology (NHLS, Universitas, Bloemfontein, South Africa) is an accredited laboratory by the South African National Accreditation System (SANA), whereby calibration, quality assurance and controls and standard operating procedures (SOPs) are followed and used on a routine basis. A summarized protocol of the different steps and procedures used in immunohistochemical staining is shown (Figure 5.2). Table 5.1: Reagents and conditions used for processing of tissue samples with Tissue‐Tek® VIP5 automated processor (Sakura Finetek) as per the SOP of the NHLS. Steps Reagent Conditions Time 1 Formalin 10% 2 h 2 Ethanol 50% 15 min 3 Ethanol 70% 1 h 4 Ethanol 96% 1 h 5 Ethanol 100% 30 min 6 Ethanol 100% 1 h 7 Ethanol 100% 1 h 8 Ethanol 100% 1 h 9 Xylene 100% 45 min 10 Xylene 100% 45 min 11 Paraffin Wax 62°C 30 min 12 Paraffin Wax 62°C 1 h 13 Paraffin Wax 62°C 1 h 14 Paraffin Wax 62°C 1 h 373 CHAPTER 5: POST-MORTEM EXAMINATION OF CHICKEN LYMPHOID TISSUES AFTER INFECTION WITH AV. PARAGALLINARUM SEROVAR C-3 INFECTION Once, at the Department of Histopthology at the NHLS, the samples were prepared for tissue processing, all tissues were processed and embedded using Tissue-Tek® (Sakura Finetek) devices and apparatus. Each tissue was trimmed using an ultra-sharp 130 mm feather blade of the Tissue-Tek® Accu-Edge® trimming knife (Sakura Finetek) for trimming the tissue so that it was no larger than 10 mm x 10 mm x 3 mm and placed into a Tissue‐Tek® processing/embedding cassette (Sakura Finetek), whereby details of the specimen on the block was labelled using a pencil. We allowed for 2 samples per block to prevent overcrowding of samples on one block and for cost and time effectiveness. The tissue cassette was then completely submerged in 10% buffered formalin for 16-24 h at room temperature. The tissue cassette was then processed overnight using a Tissue‐Tek® VIP5 automated processor (Sakura Finetek) to impregnate the tissues with paraffin wax, which included steps such as dehydration, clearing, and paraffin embedding, using fresh solutions other than paraffin was performed. The program used for the lymphoid tissues containing the reagents used and respective processing conditions are depicted in Table 5.1. 5.2.4.2. Paraffin embedding and tissue sectioning Prior to embedding the tissues in paraffin wax, the tissues needed to be placed facing downwards and as flat as possible against the mould. Multiple pieces of tissue needed to be placed in the middle of the mould to ensure that adequate wax surrounded the tissue fragments, although overcrowding of the blocks should be avoided. For tubular structures such as the trachea needed to be embedded with the lumen facing downwards, to allow for a cross- section of the lumen could be obtained. Large and hard tissue fragments should be embedded at an angle. The correct orientation of the tissues needs to be conducted prior to embedding, so that there would be an even amount of wax that surrounds the section within the mould as well as to facilitate the sectioning of the tissues. There are different types of moulds, however the most commonly used are metal moulds available in an assorted range of sizes and depths. 374 CHAPTER 5: POST-MORTEM EXAMINATION OF CHICKEN LYMPHOID TISSUES AFTER INFECTION WITH AV. PARAGALLINARUM SEROVAR C-3 INFECTION The tissues were embedded using the Tissue‐Tek® Embedding Center (Sakura Finetek). Before embedding tissues, the temperature indicators of each compartment of the console needed to be checked such as the paraffin chamber (62°C), hot plate (62°C), forceps chamber (65°C) and, the left and right thermal chambers (62°C); the paraffin volume, base mould and paraffin tray. Both cryo console (cold plate) and light key (work light) were switched on. In the first step, heated forceps were used to remove one cassette from the paraffin bath and was placed on either the left or right hot plate. The cassette cover was removed. One of the base moulds was selected from the heated chamber that would best fit the tissues in the cassette. While holding the base mould under the paraffin dispenser, the fingerplate was pressed to dispense just enough paraffin to half-fill the base mould. The base mould was then moved to the cold plate so that the base mould cooled rapidly and a thin layer of paraffin solidified. However, should the tissues not sink to the bottom of the mould, return the base mould back to the hot plate and then once the paraffin has melted use the forceps to lightly push the tissues down into a proper position onto the bottom of the base mould, the base mould should be returned to the cold plate and the procedure repeated until the tissues are properly orientated. During this process the paraffin should never completely solidify. Once the paraffin layer had cooled, a cassette was placed over properly positioned tissues in the base mould, whereby the embedded tissues adhere to the cassette. The base mould was then moved to the hot plate under the paraffin dispenser and while holding the cassette under the paraffin dispenser, the fingerplate was pressed to dispense paraffin into the base mould until the cassette was filled with paraffin, whereby care was taken to not overfill the base mould. The embedded tissues were then placed onto the cold plate. The paraffin block was then checked to ensure that it had completely solidified and was then released from the base mould and stored on the cold plate until sectioning with a microtome. Once the tissues embedded in paraffin were ready, the universal cassette clamp and knife holder were correctly secured. The hand wheel was checked if locked and the blade was 375 CHAPTER 5: POST-MORTEM EXAMINATION OF CHICKEN LYMPHOID TISSUES AFTER INFECTION WITH AV. PARAGALLINARUM SEROVAR C-3 INFECTION inserted and secured. The paraffin embedded block was then placed in the block holder, whereby the block was trimmed to expose the tissue for large tissues at 10-20 µm, 5 µm for small tissues and no trimming was needed for minute biopsies. Once trimming was completed, the blocks were placed on an ice tray to cool. The microtome (Leica Biosystems) was set to 3-4 µm for cutting. Before proceeding to cut, the block was polished and then cut. The cut sections were then stretched up in a floatation bath at a temperature of 45-56°C. The sections were then picked up onto labelled frosted slides. The slides were then placed in an oven (EcoTherm Labotec) at 45-60°C for ± 20 minutes to dissolve the excess wax, for drying and to ensure the cut section adheres to the slide. The slides were then ready to be stained. 5.2.4.3. Antibody staining and microscopy The following monoclonal antibodies of mouse origin: CONFIRM anti-CD20 (L26 clone, 760- 2531) and CONFIRM anti-CD68 (KP-1 clone, 790-2931), and of rabbit origin CONFIRM anti- CD3 (2GV6 clone, 790-4341) were used for IHC staining (Ventana Medical Systems Inc.). CD20 is a non-glycosylated phosphoprotein expressed on the cell surface of all mature B lineage cells, CD3 is a pan T cell marker and CD68 is a glycosylated glycoprotein expressed in macrophage and monocyte lineages (Maloney et al. 1994; Dorfman et al. 2006; Chistiakov et al. 2017). Immunohistochemical studies were performed on paraffin sections using the BenchMark XT fully automated slide staining system (Ventana Medical Systems Inc.) and OptiView DAB IHC detection kit (Ventana Medical Systems Inc.) which is an indirect, biotin- free system and multimer kit based on the chromagen 3,3′-diaminobenzidine (DAB) which in the presence of peroxidase activity is oxidized forming an insoluble brown product which can be visualized for immunohistological staining. The antigen retrieval step was conducted by the BenchMark XT fully automated slide staining system using Cell Conditioning Solution (CC1) at 100°C for 30 min (Ventana Medical Systems Inc.). Anti-CD20 and anti-CD3 were used to stain all lymphoid tissues such as liver, spleen, intestine, uterus (shell gland) and trachea, 376 CHAPTER 5: POST-MORTEM EXAMINATION OF CHICKEN LYMPHOID TISSUES AFTER INFECTION WITH AV. PARAGALLINARUM SEROVAR C-3 INFECTION whereas anti-CD68 was used to stain the trachea for macrophages and monocytes. Tissues known to express anti-CD20, anti-CD3 and anti-CD68 of interest were used as positive controls. The antibody-stained slides were then rinsed with EZ Prep (X1) (Ventana Medical Systems Inc.) working solution. This was followed by a washing step with tap water. The slides were counterstained with Mayer's haematoxylin (for staining of the nucleus and cytoplasm) and washed in Scott's tap water substitute; a blue alkaline solution that helps with colour development of blue tissue stains (Leica Biosystems). Finally, the slides were rinsed several times with 100% ethanol until clear to dehydrate and were then coverslipped. Visualisation of the IHC slides was conducted with the Eclipse 50i microscope, DS-Fi1 digital microscope camera and NIS-Elements F 4.00.06 Build 786 microscope imaging software (Nikon), whereby images were taken at 100X magnification. 5.3. Results and discussion 5.3.1. Necropsy, sample collection and formalin fixation Gross post-mortem examination was conducted on the dead chickens from control and experimental birds. During the dissection, a purulent green fluid in the abdominal cavity was observed most likely due to infection in one of the experimental chickens at score 1 (Figure 5.3A). Splenomegaly (enlargement of the spleen) was observed with chicken E20, attributed to an infection present in the chicken as the spleen has a role in haematopoiesis and immunity by removal of abnormal erythrocytes, clearance of pathogens/antigens and the synthesis of immunoglobulin G (IgG) (Figure 5.3B) (Chapman and Azevedo, 2018). Jaundice of the liver but not hepatomegaly, in 10 out of 12 chickens was observed which was due to high levels of bilirubin formed due to haemolysis of red blood cells (Figure 5.3C). Liver lesions were observed in chicken E20 (Figure 5.3D). The liver lesions might have been due to Av. paragallinarum spreading to other organs other than the upper respiratory tract, as reported 377 CHAPTER 5: POST-MORTEM EXAMINATION OF CHICKEN LYMPHOID TISSUES AFTER INFECTION WITH AV. PARAGALLINARUM SEROVAR C-3 INFECTION (Figure 5.3D) (Sandoval et al. 1994). Additionally, liver lesions may have been caused by the systemic effect caused by the immune response due to IC infection. The liver is the main organ of detoxification; and LPS (lipopolysaccharide) present in the outer membrane of Gram- negative bacteria Av. paragallinarum is an endotoxin, hence liver disease may develop as a result of IC, causing jaundice and in some cases liver lesions. There was mild to moderate inflammation and mucosal secretions in the nasal passages of score 2 and 3 chickens. Moreover, in the nasal passage of chicken E15 RI with score 3, there was haemorrhage (Figure 5.4A). The trachea of E15 RI (Figure 5.4C) and E19, were congested with mucous and also displayed haemorrhage. The trachea of the control chicken was not congested with mucous, nor was there haemorrhage (Figure 5.4 B). A B C D Figure 5.3: Following the dissection, post-mortem examination of internal lymphoid organs. (A) A purulent green fluid in the abdominal cavity was observed most likely due to infection. Unfortunately, samples swabs were not taken of the fluid. (B) Splenomegaly was observed with chicken E20, attributed to an infection present in the chicken. (C) Liver lesions were observed in chicken E20, due to Av. paragallinarum spreading to other organs other than the upper respiratory tract. (D) Jaundice of the liver but not hepatomegaly, in 10 out of 12 chickens was observed which was due to liver disease . 378 CHAPTER 5: POST-MORTEM EXAMINATION OF CHICKEN LYMPHOID TISSUES AFTER INFECTION WITH AV. PARAGALLINARUM SEROVAR C-3 INFECTION A B C Figure 5.4: Necropsy of trachea. (A) The nasal passage of chicken E15 RI with score 3, there was haemorrhage. (B) The trachea of Control 1 was clear without congestion with mucous. (C) The trachea of E15 RI were congested with mucous and also displayed haemorrhage. 5.3.2. Microbial cultivation and identification The present investigation was carried out for the isolation and identification of Av. paragallinarum, the causative agent of IC, from layer chickens by cultural and morphological examinations as well as molecular techniques. For this study, a total of 12 nasal swab samples were collected from the sinus cavity of dead birds (Ns= 12). None of the swab samples cultured on TSA plates supplemented with 0.2% NAD+ (v/v) displayed any growth (Figure 5.5). The colony characteristics of Av. paragallinarum observed on the BTA plate containing S. epidermidis were seen as tiny dewdrops, mucoid, smooth iridescent colonies with no haemolysis, similar to the findings of other authors (Blackall, 1989; Page et al. 1963) (Figure 5.6). It was also observed that as the disease score progressed from score 1 to score 2 to score 3, the microbial growth from the nasal exudates on the BTA plates increased and as such it became more difficult to re-isolate Av. paragallinarum, due to the chickens being 379 CHAPTER 5: POST-MORTEM EXAMINATION OF CHICKEN LYMPHOID TISSUES AFTER INFECTION WITH AV. PARAGALLINARUM SEROVAR C-3 INFECTION immunocompromised during the infection (Figure 5.6). However, it was possible that these microorganisms present in the sinus cavity are commensals, and thus have a role to play in the immunity of the host and can induce T cell regulatory responses similar to the gut which also has a mucosal environment (Belkaid and Hand, 2014.). Unfortunately, we could only successfully culture and re-isolate Av. paragallinarum from 6 out of 12 nasal swab samples from the chickens (E1, E8, E20, E22, E23, E24), as a result of the cultures not surviving too long following the transfer from the host environment and also due to the bacteria not being able to survive or adapt after passaging. Nasal swabs from chicken Control 2 did not display any growth on the BTA plate and Control 1 did display growth, however the microorganism did not have any morphological characteristics similar to Av. paragallinarum (Figure 5.6). Nonetheless, DNA extraction was conducted on Control 1 just to ensure the microorganism did not mask Av. paragallinarum on the BTA plate. Figure 5.5: No growth observed on TSA supplemented with 0.2% NAD+ (v/v) plates for nasal swabs obtained from chickens E1-score 1, E23-score 1, E8-score 2, E20-score 2, E22-score 3 and E24-score 3. 380 CHAPTER 5: POST-MORTEM EXAMINATION OF CHICKEN LYMPHOID TISSUES AFTER INFECTION WITH AV. PARAGALLINARUM SEROVAR C-3 INFECTION Control 1 E23 E23 Control 2 A B E22 E20 E24 E16 C D Figure 5.6: Nasal swab samples cultured on BTA plates. (A) Control chickens at score 0: Control 1 showing growth not pertaining to Av. paragallinarum. No growth was seen for Control 2. (B) E23 at score 1 showing growth of Av. paragallinarum seen as tiny dew drop colonies with no haemolysis. (C) E20 at score 2 showing a mixed culture of Av. paragallinarum and other microorganisms which appear to be haemolytic, colonies of Av. paragallinarum were still clearly visible. (D) Cultures of 48 h of E22, E24 and E16 at score 3 showing an overgrowth of microorganisms which appear to be haemolytic, we could not isolate Av. paragallinarum from E16 even at 24 h growth, but were successful with E22 and E24. The liver of chicken E20 which was also swabbed with a sterile cotton swab, showed no growth on BTA or TSA supplemented with 0.2% NAD+ (v/v) plates (Figure 5.7). The tracheal swabs of chickens (Control 1, E15 RI and E19) were cultured on BTA plates and TSA supplemented with 0.2% NAD+ (v/v) plates. None of the tracheal swabs cultured on TSA supplemented with 0.2% NAD+ (v/v) plates had any growth (Figure 5.8A). Av. paragallinarum was not found in the tracheal swab of the control chicken, as there was no growth on the BTA plate, implying that no cross contamination occurred between the control and experimental groups during the challenge study, as IC is contagious and spreads across cages (Figure 5.8B). The tracheal swabs of both chicken E15 RI (Figure 5.8C) and E19 (both at score 3) were cultured and Av. paragallinarum was successfully isolated. 381 CHAPTER 5: POST-MORTEM EXAMINATION OF CHICKEN LYMPHOID TISSUES AFTER INFECTION WITH AV. PARAGALLINARUM SEROVAR C-3 INFECTION A B Figure 5.7: Liver swabs of chicken E20 showed that there was no growth observed on (A) BTA or (B) TSA supplemented with 0.2% NAD+ (v/v) plates. Indicating that the lesions could be caused as a result of a systemic reaction caused by the avian immune response to IC. E19 E15 A B C Figure 5.8: Tracheal swabs of chickens Control 1, E15 RI and E19. (A) Tracheal swab samples of Control 1, E15 RI and E19 that showed no growth. (B) Tracheal swab from Control 1 showing no growth, indicating there was no cross contamination between Control 1 and experimental birds. (C) Tracheal swab samples of E15 RI and E19 showed growth, however the culture was a mixed culture and aseptic techniques had to be performed before achieving a pure culture of Av. paragallinarum, which was successfully re-isolated. 382 CHAPTER 5: POST-MORTEM EXAMINATION OF CHICKEN LYMPHOID TISSUES AFTER INFECTION WITH AV. PARAGALLINARUM SEROVAR C-3 INFECTION M C1 E1 E23 E8 E20 E22 E24 E15 E19 C2+ C3+ NC M 500 bp Figure 5.9: HPG2-PCR conducted from nasal and tracheal swab samples, whereby amplification was observed for all samples, with an expected band size of 500 bp, confirming the presence of Av. paragallinarum DNA. Lane M- molecular marker O'GeneRuler DNA Ladder; lane 1: Control 1 (C1)- score 0 (control bird) ; lane 2: E1- score 1; lane 3: E23- score 1; lane 4: E8- score 2; lane 5: E20-score 2; lane 6: E22- score 3; lane 7: E24- score 3; lane 8: E15 RI- score 3 (tracheal swab); lane 9: E19- score 3 (tracheal swab); lane 10: Modesto (C-2) positive control; lane 11: SA-3 (C-3) positive control; lane 12: negative control; Lane M molecular marker O'GeneRuler DNA Ladder. The nasal swab sample from Control 1 was also cultured and DNA extraction was performed, to verify if Av. paragallinarum was present, where no colonies from Av. paragallinarum was found. A species-specific/ HPG2-PCR was performed, using the DNA extracted from the re- isolated bacterial culture obtained from nasal swabs of chickens Control 1 (C1), E1, E8, E20, E22, E23, E24 and the tracheal swab samples from E15 RI and E19. Following the species- specific PCR/ HPG2-PCR an expected amplicon size of 500 bp was obtained indicative that Av. paragallinarum was successfully re-isolated and cultured from the nasal and tracheal 383 CHAPTER 5: POST-MORTEM EXAMINATION OF CHICKEN LYMPHOID TISSUES AFTER INFECTION WITH AV. PARAGALLINARUM SEROVAR C-3 INFECTION swabs sampled (Figure 5.9). Re-isolation and identification of Av. paragallinarum from the tracheal swabs was successfully conducted, this suggests that Av. paragallinarum can spread further down the respiratory tract (Droual et al. 1990; Hoerr et al. 1994; Deshmukh et al. 2015). The re-isolation and culture of Av. paragallinarum from the nasal and tracheal samples is in accordance with the four criteria that was established by Robert Koch and is proof of Koch’s postulates in identifying the causative agent of IC, that (1) the pathogen was present in all cases of the disease (2) the pathogen could be isolated from the diseased host and cultivated as a pure culture (3) the pathogen from the pure culture must have been able to cause disease when inoculated into healthy hosts (4) the pathogen must be re-isolated from the current host and shown to be similar as the originally inoculated pathogen. There was no other strain of Av. paragallinarum apart from the SA-3 strain (serovar C-3) inoculated into the infra-orbital cavity of the SPF chickens, hence only that specific strain could be re-isolated and cultured. Moreover, Av. paragallinarum serovar could not be re-isolated from the control chickens, hence we can assume that the strains are similar. This assumption can be validated, as challenge chickens were injected with Av. paragallinarum serovar C-3 (SA-3 strain) shown in previous studies (Chapter 3 and Chapter 4) supported with data from BLASTn that shows that the strain used throughout the experimental studies shares 100% correlation with the Av. paragallinarum strain SA-3. 5.3.3. Immunohistochemistry (IHC) of samples collected Immunohistochemistry (IHC) was conducted and following visualization as described in Section 5.2.4.3 (Chapter 5), analysis of all lymphoid images was performed and the results reported. Initially we had intended to study the bursa of Fabricius and thymus that are the major lymphoid organs in the avian immune system, the sites of B and T cell maturation, that 384 CHAPTER 5: POST-MORTEM EXAMINATION OF CHICKEN LYMPHOID TISSUES AFTER INFECTION WITH AV. PARAGALLINARUM SEROVAR C-3 INFECTION actively play a role in the innate and adaptive immune system. However the chickens used in the study were already mature at 25 weeks of age, hence both organs had undergone involution, had completely disappeared and could no longer be found/obtained. After IHC was performed, a brown precipitate could be observed on the tissue sections when visualized with a light microscope at a magnification of 100X when stained with monoclonal antibody. This gave a qualitative indication of the level of expression of the protein of interest (CD3, CD20 and CD68) within the tissue during disease progression. The intensity or level of expression of each antibody marker that bound to antigen was classified as negative (-), weakly positive (+), moderately positive (++) and strongly positive (+++). The anti-CD3 marker is a pan- T lymphocyte marker, whereby the results for the liver, spleen and intestines were compiled in Figure 5.10 and Table 5.2. The anti-CD20 marker is a pan- B lymphocyte marker, whereby the results for the liver, spleen and intestines were compiled in Figure 5.11 and Table 5.3. The liver is a unique organ that is supplied with antigen-rich blood from the gastrointestinal system which then passes a network of sinusoids and screened by antigen presenting cells and lymphocytes (Crispe, 2003). Moreover, the liver is a non-lymphoid organ, whereby 90% of the reticuloendothelial system (also known as the monocyte phagocyte system) is found in the liver (Baas et al. 1994). However, the liver consists of both conventional and unconventional subpopulations of lymphocytes from the innate immune system, comprising of natural killer cells (NK) and natural killer T cells (NKT), and the B and T lymphocytes involved in adaptive immunity (Racanelli and Rehermann, 2006). T lymphocytes are categorized as conventional (CD4+ and CD8+ cells) and unconventional ( T cells ) (Racanelli and Rehermann, 2006). The liver also has Kupffer cells which are macrophages found specifically in the liver, are primarily involved in phagocytosis and constitute for approximately 20% of parenchymal cells in the liver (Mackay, 2002). 385 CHAPTER 5: POST-MORTEM EXAMINATION OF CHICKEN LYMPHOID TISSUES AFTER INFECTION WITH AV. PARAGALLINARUM SEROVAR C-3 INFECTION Control Score 1 Score 2 Score 3 Liver 100X 100X 100X 100X Control 1 (++) E1 (+) E20 (+++) E22 (++) 100X 100X 100X Spleen 100X Control 1 (+++) E1 (+++) E8 (+++) E22 (+++) 100X Intestine 100X 100X 100X Control 1 (++) E1 (++) E8 (+++) E22 (++) Figure 5.10: Results for immunohistochemical staining with anti-CD3 marker for the liver, spleen and intestine of control and infected birds. Anti-CD3 was used to determine the expression and distribution of T lymphocytes on the cells of lymphoid tissues harvested. 386 CHAPTER 5: POST-MORTEM EXAMINATION OF CHICKEN LYMPHOID TISSUES AFTER INFECTION WITH AV. PARAGALLINARUM SEROVAR C-3 INFECTION Control Score 1 Score 2 Score 3 100X 100X 100X 100X Liver Control 1 (-) E1 (+) E8 (++) E22 (+) Spleen 100X 100X 100X 100X Control 1 (+) E1 (++) E8 (+++) E22 (+++) Intestine 100X 100X 100X 100X Control 1 (+) E1 (++) E8 (+++) E22 (+++) Figure 5.11: Results for immunohistochemical staining with anti-CD20 marker for the liver, spleen and intestine of control and infected birds. Anti-CD20 was used to determine the expression and distribution of B lymphocytes on the cells of lymphoid tissues harvested. 387 CHAPTER 5: POST-MORTEM EXAMINATION OF CHICKEN LYMPHOID TISSUES AFTER INFECTION WITH AV. PARAGALLINARUM SEROVAR C-3 INFECTION Table 5.2: IHC results for the detection of cells expressing CD3, which is the T lymphocyte population in the liver, spleen and intestine for control, score 1, score 2 and score 3 chickens. The liver contains a very small population of CD3+ cells of CD4+ (T helper) cells, CD8+ (cytotoxic) cells and NKT (natural killer T) cells involved in cytolytic activity, as seen in Control 1 bird that was not infected with the Av. paragallinarum SA-3 strain (serovar C-3) (Figure 5.10) (Table 5.2). For immunohistochemical staining of the liver with anti-CD3 we observed an increase in the T lymphocyte population from a score 1 (E1) that was weakly positive (+), to a score 2 (E20) that was strongly positive (+++) and then a decrease at a score 3 (E22) that gave a weakly positive (+) expression of the marker (Figure 5.10) (Table 5.2). At score 1 (E1 and E23), the innate immune system is activated, whereby Kupffer cells are activated by 388 CHAPTER 5: POST-MORTEM EXAMINATION OF CHICKEN LYMPHOID TISSUES AFTER INFECTION WITH AV. PARAGALLINARUM SEROVAR C-3 INFECTION various bacterial stimuli such as lipopolysaccharides (LPS) and in turn secrete cytokines such as IL‐1β, IL‐6 and TNF‐α to promote the infiltration and antimicrobial activity of neutrophils (Figure 5.10) (Table 5.2) (Gregory and Wing, 1998). In addition, the neutrophils eliminate bacteria that are attached to Kupffer cells and hepatocytes via surface phagocytosis, whereby there is stimulation of other innate immune cells by the secretion of cytokines that promotes the influx and activation of CD4+ and CD8+ T cells (Gregory and Wing, 1998), which was why there was an efflux of CD3+ cells at score 2 (E8 and E20) (Figure 5.10) (Table 5.2). However, when the adaptive immune system becomes activated, T cell mediation against bacteria relies solely on the constant supply of activated effector CD8+ T cells that have a role in cytotoxicity for the maintenance of the immune responses and in controlling the spread and expansion of bacterial pathogens (Racanelli and Rehermann, 2006). During bacterial invasion, Kupffer cells, dendritic cells (DC) and liver sinusoidal endothelial cells (LSEC) present antigens to naïve CD4+ T cells whereby IL-4 and IL-10 are secreted leading to immunological consequences, naïve CD8+ T cells whereby they become effector CD8+ T cells involved in cytotoxic activity. The decrease observed at score 3 (E11, E15 RI, E16 RI, E19, E22 and E24) may be due to CD8+ T cells becoming activated and once bound to endocytosed antigens presented by major histocompatibility complex (MHC) class II undergoes apoptosis, leading to a reduction in the T cell population observed (Figure 5.10) (Table 5.2). The results for the IHC staining of the liver with anti-CD20, were similar to that of the anti-CD3, whereby we see an increase in the expression of the B lymphocyte population at a score 2 (E8) giving a moderately positive (++) result (Figure 5.11) (Table 5.3). In Control 1, we observe that there were no B lymphocytes present, as none of the cells were stained positive (Figure 5.11) (Table 5.3). For immunohistochemical staining of the liver with anti-CD20 we observed an increase in the T lymphocyte population from a score 1 (E1) that was weakly positive (+), to a score 2 (E8) that was strongly positive (++) and then a decrease at a score 3 (E22) that gave a weakly positive (+) expression of the marker (Figure 5.11) (Table 5.3). The slight increase in the B cell population at score 2 (E8), was due to the adaptive immunity that “kicks 389 CHAPTER 5: POST-MORTEM EXAMINATION OF CHICKEN LYMPHOID TISSUES AFTER INFECTION WITH AV. PARAGALLINARUM SEROVAR C-3 INFECTION in” from score 1 (E1) to score 2 (E8), hence there was an increase in the peripheral B cell response as the humoral response came into play. At score 3 (E22), there was a decrease in the humoral response due to the feedback mechanism of deterring immune cells and molecules from damaging the host’s tissues (Chapter 2). Although B cells function in the humoral response by secretion of antibodies such as IgA, IgM and IgY, the exact function of B cells in the liver is still unknown, especially since the liver is neither a lymphoid or ectopic organ. Moreover, from the results in Table 5.3 it was observed that there was negative (-) to very low expression (+) of CD20 in the liver, except with chicken E20 whereby there was moderate expression of the B lymphocytes. The liver obtained from E20 had both high CD3 and CD20 expression (+++), implying that both the humoral and cell-mediated responses occurred leading to the release inflammatory cytokines and antibodies leading to the liver lesions that were observed. Hence, from our results obtained it is apparent that the liver has a role to play in B cell proliferation due to high expression (+++) of the CD20 at score 2 (E8 and E20) (Figure 5.11) (Table 5.3). In the spleen, CD3 and CD20 were constantly highly expressed (++/+++) in all scores and in the control bird (Figure 5.10 and Figure 5.11) (Table 5.2 and Table 5.3). Initially, lymphomyeloid tissues develop from either epithelial (bursa of Fabricius and thymus) or mesenchymal (spleen, lymph nodes and bone marrow) anlages which harbour haematopoietic cells. Haematopoietic stem cells enter the bursal or thymic anlages which are the central lymphoid organs and develop to become immunologically competent B and T cells (Sturkie, 1943). However, upon maturation the mature T and B cells enter the circulation and colonize the peripheral lymphoid organs also known as ectopic organs such as the spleen, lymph node and gut-, bronchus- and skin-associated lymphoid tissues (Davison et al. 2011). Thus, in these organs B and T lymphocytes, are compartmentalized into B and T dependent zones (Schat et al. 2014). In the avian spleen, the T dependent zone includes the peri- arteriolar lymphatic sheath (PALS) that surrounds the splenic central artery and the 390 CHAPTER 5: POST-MORTEM EXAMINATION OF CHICKEN LYMPHOID TISSUES AFTER INFECTION WITH AV. PARAGALLINARUM SEROVAR C-3 INFECTION interfollicular region, whereas germinal centres (GC) and the peri-ellipsoidal white pulp (PWP) of the spleen are B dependent zones (Davison et al. 2011). In avian anatomy, the spleen becomes the secondary lymphoid organ after the bursa of Fabricius and thymus undergo involution. Hence, there is an abundance of T and B lymphocytes in the spleen, which is why B and T lymphocytes were constantly and highly expressed (++/+++) throughout all scores and in the control bird with IHC anti-CD3 and anti-CD20 staining of the spleen (Figure 5.10 and Figure 5.11) (Table 5.2 and Table 5.3). In the avian immune system, the spleen has a closed circulatory system and is not a reservoir of erythrocytes for immediate release into the bloodstream (Sturkie, 1943). Moreover, there are capillaries that connect the red pulp of the spleen to the sinuses, whereby the sinuses are drained by collecting veins that will eventually leave the spleen. Hence, it is possible that the injected SA-3 strain of Av. paragallinarum via infra-orbital injection directly into the sinus cavity circulated to the spleen, leading to an increased inflammatory and adaptive responses from the T and B lymphocytes respectively, whereby the T and B lymphocytes also migrated to peripheral tissues leading to systemic immunological responses in the host organism. However, the hypothesis proposed has not been proven. One of the symptoms of IC is diarrhoea, which occurs primarily because some of the exudates containing Av. paragallinarum spreads to the water and feed in cages. Eventually, the chickens ingest the contaminated water and feed, thus causing infection and an inflammatory response in the gut. The gut-associated lymphoid tissue (GALT) consists of an assorted range of cell subsets of unique and representative cell populations from systemic tissues (Davison et al. 2011). The avian GALT commences in the lamina propria of the villus (Schat et al. 2014). Each single villus is composed of connective tissue fibres, smooth muscle fibres, nerves and blood and lymph vessels, whereby macrophages amalgamate with lymphocytes in the lamina propria (Davison et al. 2011). Additionally, B and T cells in the intestine form distinct regions when more lymphocytes infiltrate the villus (Schat et al. 2014). The T cells are found at the 391 CHAPTER 5: POST-MORTEM EXAMINATION OF CHICKEN LYMPHOID TISSUES AFTER INFECTION WITH AV. PARAGALLINARUM SEROVAR C-3 INFECTION centre of the villus, whereas B cells and GC are located in the lymphoid follicles entwined with dendritic cells, CD4+ cells and macrophages, situated at the deep and mid-section of the lamina propria (Hoshi and Mori, 1973; Jeurissen et al. 1994). Furthermore, the avian GALT is populated with heterophils, macrophages, dendritic cells, natural killer (NK) cells, and B and T lymphocytes, however the proportions of each cell type depends on the locality and age of the bird (Davison et al. 2011). The gut epithelial layers also consist of highly specialized lymphocytes called the intraepithelial lymphocytes (IEL) (Davison et al. 2011). Table 5.3: IHC results for the detection of cells expressing CD20, which is the B lymphocyte population in the liver, spleen and intestine for control, score 1, score 2 and score 3 chickens. 392 CHAPTER 5: POST-MORTEM EXAMINATION OF CHICKEN LYMPHOID TISSUES AFTER INFECTION WITH AV. PARAGALLINARUM SEROVAR C-3 INFECTION The IHC results for anti-CD3 for the small intestine showed moderate expression (++) of the T lymphocyte population in control bird (Control 1), score 1 birds (E1 and E23) and score 3 birds (E11, E15 RI, E16 RI, E19, E22 and E24) with strongly positive expression (+++) of T lymphocytes at score 2 in chickens E8 and E20. Initially, the small intestinal IEL population is populated with NK cells and T cells (with γδ or αβ T cell receptor (TCR)), whereby most of the T cells express the CD8 co-receptor with smaller populations of TCRαβ+CD4+ and CD4+CD8+ cells (Vervelde and Jeurissen, 1993; Lillehoj, 1994; Göbel et al. 2001; Lillehoj et al. 2004). In contrast to the IEL population, the T cell population of the lamina propria is sparsely populated with γδ T cells, with the majority of the T population being the αβ T cells dominated by CD4+ cells and a minority of CD8+ cells (Davison et al. 2011). Thus, due to the presence of distinct T cell populations in the IEL and lamina propria, we observed moderate expression (++) of CD3+ cells in control and score 1 birds (Figure 5.10) (Table 5.2). However, during the first few days of infection the innate immune response plays a crucial role with macrophages, NK cells and TCRγδ+ T cells, which are active during the transition from the non-infectious to score 1 phase, which is perhaps the reason we saw small clusters of T cells consisting of CD4+ T helper cells and naïve CD8+ cells (Figure 5.10). During the adaptive immune response, from score 1 to score 2, the naïve CD8+ cells become activated and are involved in cytotoxic activity, whereby CD4+ cells are T helper cells involved in the activation of B cells via the MHC class II (Vainio et al. 1984). The influx in CD3+ effector T cells at score 2 could also be attributed to the migration of T cells from peripheral lymphoid tissues. We then, observed a slight decrease in the T lymphocyte population (Figure 5.10) (Table 5.2), which might be due to CD8+ cytotoxic T cells that underwent apoptosis after antigen presentation and display via macrophages complexed to MHC class I leading to the release of cytotoxins causing cell death of the CD8+ cell-MHC class I- antigen complex formed. The IHC results for anti-CD20 of the small intestine showed low expression (-/+) of the B lymphocyte population in control bird (Control 1) and score 1 birds (E1 and E23), with 393 CHAPTER 5: POST-MORTEM EXAMINATION OF CHICKEN LYMPHOID TISSUES AFTER INFECTION WITH AV. PARAGALLINARUM SEROVAR C-3 INFECTION moderate expression (++) in score 2 birds (E8 and E20) and moderate (++) to high expression (+++) in score 3 birds (E11, E15 RI, E16 RI, E22 and E24) (Figure 5.11) (Table 5.3). Chicken E19 had low expression of the marker. Unlike other tissues, B cells are almost absent in the IEL, which is why we observed very low expression (-/+) of CD20 in the control and score 1 birds. During the early to late adaptive responses as seen with score 2 and 3 chickens (Figure 5.11) (Table 5.3), the B lymphocyte population increases from moderate (++) to high (+++) expression with anti-CD20 marker, due to potential B cell migration from ectopic B cell rich lymphoid tissues to the gut periphery and also due to clonal expansion of B cells when they become activated mainly because B cells have a role in the humoral response in the gut. Following clonal expansion, the B cells become plasma cells that secrete immunoglobulins and memory cells that develop and build immunological memory until the next encounter with the same antigen leading to more rapid immunity against the pathogen. Moreover, B cells secrete IgA antibody at high concentrations in intestinal fluids (Lebacq-Verheyden et al. 1972). Immunoglobulin (Ig) A is mainly found in its monomeric form, however it is secreted in a polymeric configuration (Bienenstock et al. 1973). It has been proposed that bacterial and viral targets coated in IgA followed by ligation with FcαR in humans leads to a cascade of immunological responses such as phagocytosis, respiratory burst and release of cytokines, in the annihilation of invading microorganisms (Monteiro and Van De Winkel, 2003). However, in chickens the function of IgA and its immune mechanisms have yet to be elucidated. For bird Control 2, we observed very high expression (+++) of both CD3 and CD20 in the spleen and intestine. The spleen is a major secondary lymphoid tissue and constantly expresses T and B lymphocytes which was why we observed high expression (+++) of T and B lymphocytes regardless of whether an infection was present or not, however the high expression (+++) of CD3 and CD20 in the GALT indicated infection. This indicated T and B lymphocyte infiltration into these lymphoid organs and increased adaptive immune responses. Therefore, Control 2 could have been exposed to Av. paragallinarum or other pathogens, 394 CHAPTER 5: POST-MORTEM EXAMINATION OF CHICKEN LYMPHOID TISSUES AFTER INFECTION WITH AV. PARAGALLINARUM SEROVAR C-3 INFECTION however the bacterial load as mentioned in Chapter 4 was not sufficient to cause IC. The liver of Control 2 with anti-CD20 stained negative and with anti-CD3 there was moderate expression similar to Control 1, which indicated there were small clusters of naïve B and T lymphocytes present. For the re-infected chickens E15 RI and E16 RI, there was not much of a difference in the IHC staining of the anti-CD3 and anti-CD20 of the liver, spleen and intestine results compared to other infected chickens (Table 5.2 and Table 5.3). This implies that the second injection given when the chickens were at score 2, did not have any significant systemic effect on the tissues of infected chickens as immunological memory had already been established prior to the second injection via infra-orbital injection. Therefore, the chickens E15 RI and E16 RI developed score 3, not as a result of the second injection containing the SA-3 strain (serovar C-3) of Av. paragallinarum , but because of previous exposure from the first injection. From the necropsy and IHC results the immunological and systemic responses from IC infection, there were no severe lesions found nor extensive internal damage caused, as described in the literature for the re-infected chickens (Sawata et al. 1985; Blackall and Soriano, 2008). The sinus responses of re-infected chickens when compared to score 3 chickens had similar symptoms such as haemorrhage and congestion. We performed IHC on the cross-sections (CS) and longitudinal sections(LS) of the trachea using anti-CD3, anti-CD20 and anti-CD68. The results were recorded in Table 5.4 and as Figure 5.12. From the results we observed moderate expression (++) of CD3 from Control 1 and E15 RI- score 3, with low expression (+) in E19- score 3 (Figure 5.12) in the mucosa, submucosa and cartilaginous layers of the trachea. In Section 5.3.2, it was found that Av. paragallinarum was present in E19 and E15 RI both at score 3, hence the decreased expression of CD3 seen in E19 was expected as T cells undergo apoptosis after antigen 395 CHAPTER 5: POST-MORTEM EXAMINATION OF CHICKEN LYMPHOID TISSUES AFTER INFECTION WITH AV. PARAGALLINARUM SEROVAR C-3 INFECTION presentation following the cell-mediated response, which is the reason for the diminished T cell population. However, there was no change in E15 RI, due to T cells still actively playing a role in the cell-mediated response consisting of naïve T cells and effector T cells in the trachea during IC infection. Table 5.4: IHC results for the detection of cells expressing CD3, CD20 and CD68; which represent the T lymphocyte, B lymphocyte and macrophage/monocyte population within the trachea of control, score 3 and score 3 RI birds. Both Control 1 and E19 at score 3 moderately expressed (++) CD20 in tracheal tissue, indicating the presence of B lymphocytes involved in the mucosal humoral response by the release of antibody IgA. There was no expression (-) of CD20 in E15 RI-score 3, which might be because the humoral response had already occurred in the trachea before the tissue was harvested, there was no intrinsic expression of CD20 or the antigen could not be retrieved from the trachea during the antigen retrieval phase of IHC. However, since T and B lymphocytes were expressed in E19 having the same score, this indicated that similar results also had to be obtained for E15. As such, the results for E15 showed that once the adaptive 396 CHAPTER 5: POST-MORTEM EXAMINATION OF CHICKEN LYMPHOID TISSUES AFTER INFECTION WITH AV. PARAGALLINARUM SEROVAR C-3 INFECTION immune responses occurred, it would be difficult to obtain the expressed antigens in the tissues if not harvested at the correct period of expression. CD68 was used to detect the presence of macrophages or monocytes in the trachea. Only Control bird 1 expressed CD68, which was moderately expressed (++). Macrophages and monocytes play a role in innate immunity, as personal scavengers of the host, involved in phagocytosis. At score 3 in chickens E19 and E15 RI, the macrophages were absent due to adaptive immunity that took over the immune response of the host, which was the reason CD68 stained negative in both score 3 chickens. Moreover, macrophages in the presence of Av. paragallinarum would have ingested the antigen leading to the cascade in immunological responses and adaptive immunity, which is perhaps why no macrophages were present even at score 3, as most possibly they were destroyed or cleared during the pathogenic attack from the upper to the lower respiratory tract. There are numerous suicide programmes employed by macrophages to prevent the spread of microbial replication (Chow et al. 2016). Pro-death factors can be activated which can trigger apoptotic cell death, resulting in the release of caspases (caspase-1 and caspase-11 in humans) leading to pyroptosis, which is a fast and lytic form of macrophage suicide (Czabotar et al. 2014; Chow et al. 2016). Pyroptosis is often a highly inflammatory response due to the release of pro-inflammatory cytokines, whereby there is activation of neutrophils that function in the clearance of the intracellularly ingested pathogens by macrophages (Chow et al. 2016). Infectious coryza (IC) causes a decrease in egg production, hence it was investigated to see whether the uterus (shell gland) had a specific type of T or B lymphocyte distribution and expression. Surprisingly, Control 1, E11-score 3 and E15 RI-score 3 expressed low and sparse levels of CD3, related to T lymphocyte expression. Implying that the major adaptive response was the cell-mediated response, involving naïve and effector T lymphocytes within 397 CHAPTER 5: POST-MORTEM EXAMINATION OF CHICKEN LYMPHOID TISSUES AFTER INFECTION WITH AV. PARAGALLINARUM SEROVAR C-3 INFECTION the uterus. There were very few cells that expressed CD3 in the uterus, but nonetheless there was some expression. However, the CD20 antigen was not expressed at all in any of the birds, perhaps this might be because the uterus (shell gland) is not a B lymphocyte-rich tissue. The shell gland has a vital role in egg production as it secretes albumin and the egg shell, during egg formation in the hen. It is also possible that only specific parts of the reproductive tract express B lymphocytes, whereby the uterus is not one of these B lymphocyte-rich tissues. From the results obtained (Table 5.5 and Figure 5.13), it was observed that the uterus of chickens at different clinical scores, had very low numbers B lymphocytes, thus this could be the reason why Av. paragallinarum could easily and systemically affect the reproductive organs as some of these regions have lowered B and T cell populations, resulting in a delayed humoral immune response and more favourable spread of the pathogen within the uterus. However, decreased appetite caused by IC also affects the production and formation of eggs, as there is a decrease in the percentage of calcium needed for egg formation, especially the egg shell. Hence, there is definitely a correlation between the chicken’s diet and egg formation (Surai and Sparks, 2001). However, IHC needs to be conducted in future studies on other tissues of the reproductive tract such as the ovary, magnum and isthmus to determine the distribution of T and B lymphocytes. Table 5.5: IHC results for the detection of cells expressing CD3 and CD20; which represent the T lymphocyte and B lymphocyte population within the uterus (shell gland) of control, score 3 and score 3 RI birds. 398 CHAPTER 5: POST-MORTEM EXAMINATION OF CHICKEN LYMPHOID TISSUES AFTER INFECTION WITH AV. PARAGALLINARUM SEROVAR C-3 INFECTION Control 1 E19- Score 3 E15 RI- Score 3 CD3 100X 100X 100X LS (++) CS (+) CS (++) CD20 100X 100X 100X CS (++) CS (++) CS (-) CD68 100X 100X 100X CS (++) CS (-) CS (-) Figure 5.12: Results for immunohistochemical staining of the trachea with anti-CD3, anti-CD20 and anti-CD68 marker for control, score 3 and score 3 RI birds. We analysed both cross-sections (CS) and longitudinal sections (LS) at a 100X magnification. 399 CHAPTER 5: POST-MORTEM EXAMINATION OF CHICKEN LYMPHOID TISSUES AFTER INFECTION WITH AV. PARAGALLINARUM SEROVAR C-3 INFECTION CD3 CD20 100X 100X Control 1 (+) (-) 100X 100X E11-Score 3 (+) (-) 100X E15 RI- Score 3 100X (+) (-) Figure 5.13: Results for immunohistochemical staining of the uterus (shell gland) with anti-CD3 and anti-CD20 marker for control, score 3 and score 3 RI birds. Surprisingly, it was observed that only CD3 was expressed across all three chickens at a control, score 3 and score 3 RI; and not CD20. 400 CHAPTER 5: POST-MORTEM EXAMINATION OF CHICKEN LYMPHOID TISSUES AFTER INFECTION WITH AV. PARAGALLINARUM SEROVAR C-3 INFECTION 5.4. Conclusions In this chapter (Chapter 5) we tried to conduct necropsy on control, score 1, score 2 and score 3 chickens and perform post-mortem examination of the tissues. Additionally, we tried to determine the distribution and expression of T and B lymphocytes, via the use of IHC and specific antibody markers CD3, CD20 and CD68 respectively. We were successful in determining the expression and distribution of T and B lymphocytes in the liver, spleen and intestine, as well as the trachea and uterus (shell gland). Liver lesions were also obtained, however we were unsuccessful at isolating Av. paragallinarum from the liver. In addition, it is still uncertain whether lesions and internal damage caused by IC, as stated in literature was as a result of secondary infection or solely the result of the Av. paragallinarum. During cultivation of the nasal swabs on the BTA plates, there was growth of other non-Av. paragallinarum microorganisms for infected and control chickens (Figure 5.6), but no growth was observed on TSA plates supplemented with NAD+ from the chickens, hence in future studies these microorganisms should be identified. Perhaps the interactions between the microorganisms growing alongside Av. paragallinarum can provide insight whether there is a symbiotic relationship leading to aggravation of IC or whether these microorganisms have a role in the immunity of the host by providing mucosal protection in the upper respiratory tract. The non-Av. paragallinarum microorganisms growing on the BTA plates but not TSA plates supplemented with NAD+, appeared to be haemolytic in nature due to lysis of BTA plates and could be identified as staphylococci/micrococci which can also grow in basic medium like TSA, however no growth was seen on the TSA plates supplemented with NAD+. A possible explanation for this set-back could have been that the media used was problematic and was not suitable for bacterial cultivation during the study. Thus, in future experiments more suitable media for Av. paragallinarum should be used. Moreover, it was found that Av. paragallinarum does spread to the lower respiratory tract into the trachea, as we were able to isolate the bacteria. It was also found that the trachea expressed T and B lymphocytes, primarily involved in the adaptive immune response as a mechanism to defend against Av. paragallinarum as 401 CHAPTER 5: POST-MORTEM EXAMINATION OF CHICKEN LYMPHOID TISSUES AFTER INFECTION WITH AV. PARAGALLINARUM SEROVAR C-3 INFECTION per IHC results. Furthermore, from the uterus, it was found that CD20 was not expressed in Control 1, score 3 and score 3 RI birds, which suggests that this organ has no humoral defences on standby. Hence during Av. paragallinarum or pathogenic infection the uterus can potentially be vulnerable to systemic infection, since it has been documented that Av. paragallinarum can spread to other organs other than the upper respiratory tract (Sandoval et al. 1994). Therefore, this might have an effect on egg production in hens, leading to the decline in egg production. Finally, it was discovered that T and B lymphocytes are not constantly expressed during an infection in the lymphoid tissue, except if the lymphoid tissue becomes a major secondary lymphoid organ like the spleen. The expression of the T and B lymphocytes depends on the timing of when the immune responses occurred and the time of harvest, therefore if the tissues were harvested after an immune response or after clearance, none of the T or B lymphocytes would be observed. Since we could not isolate Av. paragallinarum from the tissues, we could only conclude that the effect of IC was systemic leading to the inflammatory and adaptive immune responses as well as B and T lymphocyte distributions observed within the tissue sections following IHC. It should be taken into consideration that during an infectious challenge there is an induction of multiple immune responses whereby some of these responses are unsuccessful at controlling infection and at times, these responses can also be damaging to the tissues of the host. 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Moreover, failure of commercial vaccination attempts developed for IC, is becoming a serious problem, with the emergence of new serovar variants. Vaccines can no longer provide cross protection against IC. Since prevention is better than cure, it becomes imperative that we understand host-pathogen interactions, disease progression and the immune mechanisms employed by the host’s immune defence system against Av. paragallinarum strain SA-3 (serovar C-3) as the knowledge obtained from studying the avian immune response is a powerful tool that can be used to combat IC. From the study, bioinformatics tools for the analysis of differentially expressed genes was conducted. The in silico results obtained for the generation of immune signalling pathway maps, results for biological processes through gene enrichment, functional annotations and the generation of functional correlating networks of genes assisted in predicting in vivo immune responses during IC infection, in not only blood but also lymphoid and non-lymphoid organs of the avian host such as humoral immune responses, cell-mediated responses, the effect of IL-8, the functions of innate immune cells and the role of leukocytes, as seen in later chapters. The pathogenesis of IC infection with serovar C-3, was found to be similar to that of influenza A virus, suggesting that the presence of HA, prophages and prophage remnants 410 CHAPTER 6: CONCLUDING REMARKS AND FUTURE STUDIES may have a role to play in virulence. The role of prophages and prophage remnants in the virulence of Av. paragallinarum serogroups needs further investigation. During the pilot study chickens infected with Av. paragallinarum serovar C-3 (SA-3 strain) did not yield any IC related signs or symptoms. This suggested that the SPF chickens used in the pilot study obtained had an already well-established immune system based on existing antibodies present in Day 0 chicken blood. Hence, it should be a prerequisite that antibody testing be conducted prior to animal experimentation, as a precautionary measure, so that the immune effects present in the host organism stimulated by previous exposure to pathogens, vaccinations or drugs be known and appropriate measures be taken that would not jeopardise the study. It is also important that during an infection model, the correct dosage/concentration of bacterial culture is administered to achieve the disease threshold, whereby for this study 108 CFU/ml was found to be the appropriate concentration for IC infection. In the infection model, different cell morphologies from blood smears together with leukocyte, CD4 and CD8 flow cytometry profiles of chicken blood samples at different scores were evaluated, providing insight into the innate, humoral and cell-mediated immune responses involved in pathogen elimination. The Th cell response had a major role to play in both innate and adaptive responses of IC infection. No statistically significant decline in egg production was observed in the study, which might be due to a small sample size in the study. Additionally, IL-8 was found to be highly expressed as the symptoms of IC became more aggressive, which suggests that IL-8 could potentially be used as a biomarker for IC. The study also showed that only a minority of chickens reached a score of 3, whereas the rest of the chickens recovered. Moreover, the flow cytometry profiles showing high immune- related activity and the ELISA results displaying high expression of IL-8, for control birds, 411 CHAPTER 6: CONCLUDING REMARKS AND FUTURE STUDIES indicated that they had an already well-established immune response suspected due to previous exposure to microorganisms before or during the study. However, this did not lead to pathology as the chickens were asymptomatic for IC, thus the study was not jeopardised. The expression and distribution of T and B lymphocytes in the liver, spleen, intestine, trachea and uterus (shell gland), was determined, via the use of necropsy, IHC and specific antibody markers CD3, CD20 and CD68 respectively. Liver lesions were also observed, however there was no success at isolating Av. paragallinarum or any other microorganism from the liver. The role of commensals growing alongside Av. paragallinarum can provide insight whether there is a symbiotic relationship leading to aggravation of IC or whether these microorganisms have a role in the immunity of the host by providing mucosal protection in the upper respiratory tract. Av. paragallinarum serovar C-3 (SA-3 strain) does spread to the lower respiratory tract into the trachea, as we were able to isolate the bacteria. Furthermore, CD20 was not found to be expressed in the uterus of Control 1, score 3 and score 3 RI birds, which suggests that this organ has no active humoral defences. Hence the uterus can potentially be vulnerable to systemic infection by pathogens, which could have an effect on egg production. Av. paragallinarum serovar C-3 (SA-3) could not be isolated from the tissues, we could only conclude that the effect of IC was systemic leading to the inflammatory and adaptive immune responses as well as B and T lymphocyte distributions observed within the tissue sections following IHC. In the disease profiles, flow cytometry profiles and IHC results, it was observed that following the adaptive immune responses and during score 3, towards more severe symptoms, there was a decline in immune activity, which might have resulted due to the inhibition of immune cells and molecules from damaging the host’s tissues, which acts as a protective mechanism for the host. This assumption, was also found using bioinformatics tools, whereby it was shown that a Th2 shift occurs which provides protection to the host from hyperinflammation by countering the tissue-damaging effects of macrophages, Th1 cells and proinflammatory cytokines. 412 CHAPTER 6: CONCLUDING REMARKS AND FUTURE STUDIES This research was novel and very unique since pathogenesis of IC and the monitoring of the avian immune responses was conducted, encompassing disease progression at a genetic, cellular, tissue, and clinical level as a whole. Focus on functional pathways and interaction networks was conducted to gain comprehensive insights into biological processes relevant to protection and pathogenesis in IC infection, which in future can be harnessed for relevant biomarker signatures. The in vivo results obtained from the experimental studies in combination with the in silico results obtained from bioinformatics tools may provide powerful insight and a birds-eye view into the immune mechanisms between host-pathogen interactions for this disease. Finally, results from this project will also further improve diagnostic tests and vaccination practices for Av. paragallinarum serovar C-3 (SA-3 strain) in the veterinary field, through the knowledge gained and the development of diagnostic products such as IC specific ELISA kits, as well as point-of-care testing and haematological avian devices. Future Research The next measures from the knowledge gained from this study, would be to conduct RNA- Seq (RNA sequencing) using next-generation sequencing (NGS) on tissue and sinus samples collected, to uncover the presence and quantity of RNA in the biological samples at continuously shifting time intervals and stages of disease progression of IC infection. Hence, the transitioning genes that are up- or downregulated during the progressive disease scores will be unravelled, which would enable more in depth understanding of immune regulatory and immune signalling pathways of IC. The metabolic regulation of immune responses in the avian host during IC infection, is also an area that needs attention. In our study, the gut was found to be an important lymphoid organ in mucosal immunity. In future studies, the interactions of microorganisms in the gut during Av. paragallinarum infection needs to be investigated, as the gut microbiota impacts many areas of animal health from innate 413 CHAPTER 6: CONCLUDING REMARKS AND FUTURE STUDIES immunity to appetite and energy metabolism. Furthermore, challenge studies with other serovars or serogroups needs to be conducted for comparative purposes of immune-related responses and in disease monitoring to Av. paragallinarum serovar C-3 (SA-3 strain) infection, for standardisation purposes. The role of prophages and prophage remnants in the virulence of Av. paragallinarum needs to be studied, as well as similar pathogenesis to influenza A virus. From the ELISA results obtained from antibody screening and sandwich ELISA of IL-8, the development of an IC-based ELISA to assist in the diagnosis and prognosis of IC, should be conducted from all serogroups and serovars. 414