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Control and Management of Avian Tuberculosis in a Captive Collection of Wildfowl at the Wildfowl and Wetlands Trust Centre in Llanelli, Using an Enzyme-Linked Immunosorbent

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Control and Management of Avian Tuberculosis in a Captive Collection of Wildfowl at the Wildfowl and Wetlands Trust Centre in Llanelli, Using an Enzyme-Linked Immunosorbent Aus der Klinik für Geflügel der Tierärztlichen Hochschule Hannover und dem Research Department des Wildfowl and Wetlands Trust in Slimbridge, United Kingdom Control and Management of Avian Tuberculosis in a Captive Collection of Wildfowl at the Wildfowl and Wetlands Trust Centre in Llanelli, using an Enzyme-linked Immunosorbent Assay as Diagnostic Aid INAUGURAL – DISSERTATION Zur Erlangung des Grades einer Doktorin der Veterinärmedizin (Dr. vet. med.) durch die Tierärztliche Hochschule Hannover Vorgelegt von Harriet Petra Zsivanovits aus Hermannstadt Hannover 2001 Wissenschaftliche Betreuung: Univ. – Prof. Dr. U. Neumann Dr. R. Cromie PhD 1. Gutachter: Univ. – Prof. Dr. U. Neumann 2. Gutachter: Priv. – Doz. Dr. M. Böer Tag der mündlichen Prüfung: 23.11.2001 Für meine Eltern und meine Schwester Table of contents Abbreviations A. Introduction 13 B. Literature review 17 1. Avian immune system 17 1.1. Non specific defence 17 1.1.1. Non-cellular components 17 1.1.2. Myeloid system or cellular system 18 1.1.2.1. Leucocytes 18 1.1.2.2. Thrombocytes, macrophages and natural killer cells 19 1.2. Specific defence 20 1.2.1. Humoral system 21 1.2.1.1. Immunoglobulins 21 1.2.1.2. Comparison of the avian immunoglobulins 22 1.2.2. Lymphocyte activity 24 1.2.2.1. B-lymphocytes 24 1.2.2.2. T-lymphocytes 25 1.3. Influence of host and environmental factors on the immune response 26 2. Characterisation of mycobacteria with particular reference to 27 Mycobacterium avium 2.1. Classification of mycobacteria 27 2.1.1. Identification and conventional typing 27 2.1.2. The Mycobacterium avium-Mycobacterium intracellulare complex 29 2.1.3. Serotyping of mycobacteria 30 2.1.3.1. Agglutination serotypes 30 2.1.3.2. Immunodiffusion serotypes 30 Table of contents 2.1.4. Distribution in the environment 32 2.2. Cultivation of mycobacteria 33 2.2.1. Colony morphology 33 2.2.2. Colony variation and virulence 33 2.3. Genetic characteristics 35 2.3.1. DNA and plasmids 35 2.3.2. Cell wall and envelope 36 2.4. Pathogenesis 37 2.4.1. Mechanism of infection 37 2.4.2. Interaction with mononuclear phagocytes 38 2.5. Immune response to mycobacteria 39 2.5.1. Cellular immunity 39 2.5.2. Role of lymphocytes and natural killer cells 40 2.5.3. Role of cytokines 40 3. Manifestation of avian tuberculosis 43 3.1. Epidemiological aspects 43 3.1.1. Importance for collections with captive birds 43 3.1.2. Distribution and species susceptibility to Mycobacterium avium 43 3.2. Clinical findings 46 3.3. Route of infection and localisation 47 3.4. Pathology 49 3.4.1. Macroscopic findings 49 3.4.2. Microscopic findings 50 3.4.3. Amyloidosis in avian tuberculosis 51 3.5. Avian tuberculosis as zoonosis 51 Table of contents 4. Treatment of avian tuberculosis 54 4.1. Drug resistance of mycobacteria 54 4.2. Antimycobacterial drugs 56 4.3. Recommendations for treatment of mycobacterial infections in pet birds 59 4.4. Reported cases of treatment of avian tuberculosis 61 5. Diagnosis of avian tuberculosis 63 5.1. Diagnosis based on genetic structures 63 5.1.1. Rapid culture methods 63 5.1.2. Rapid assays for identification 64 5.1.2.1. DNA probes 64 5.1.2.2. Polymerase chain reaction 64 5.1.2.3. High-performance liquid chromatography 65 5.2. Lymphocyte tranformation test 66 5.3. Clinically relevant diagnostical techniques 66 5.3.1. Avian tuberculin skin test 66 5.3.2. Radiology and endoscopy 67 5.3.2.1. Radiology 67 5.3.2.2. Endoscopy 67 5.3.3. Direct examination by staining and microscopy 68 5.3.4. Culture 69 5.3.5. Agglutination test 70 5.3.6. Haematological analysis and blood chemistry 71 Table of contents 6. Diagnosis by using an enzyme-linked immonusorbent assay (ELISA) 73 6.1. General consideration of an ELISA 73 6.2. ELISA principles 74 6.3. Components of an ELISA 75 6.4. Interpretation of the ELISA results 76 6.4.1. Determination of the cut-off point 76 6.4.2. Sensitivity and specificity 77 6.5. Preliminary use of the ELISA 79 6.6. Comparison of ELISAs for detecting antibodies to infectious diseases in birds 81 7. Summary and assignment 84 C. Materials and Methods 85 1. Description of the birds used in the experiment 85 2. Blood sampling 88 3. Identification of the birds’ enclosures 95 4. Principles of the enzyme-linked immunosorbent assay used in this study 96 4.1 ELISA method 98 4.2. Determination of conjugated antibody dilution 100 4.3. Antigens used in the ELISA 100 4.4. Interpretation of the ELISA results 102 5. Post mortem and histopathological examination and PCR of birds testing positive for 103 avian tuberculosis 6. Epidemiological considerations 105 7. Statistical analysis 106 Table of contents D. Results 107 1. Preliminary investigations 107 2. ELISA results 110 3. Post mortem examination and comparison of post mortem findings 115 with ELISA results 3.1. Post mortem and histopathological examination and PCR 117 3.1.1. Individual gross post mortem findings 117 3.1.2. Findings of histopathological examination and PCR 122 3.2. Validation of sensitivity and specificity of the ELISA 125 4. Physical environment and husbandry details 128 5. Analysis of post mortem data from 1989 to 1999 132 5.1. Incidence of avian tuberculosis over a ten year study period (1989 to 1999) 132 5.2. Analysis of the data by pens 135 5.3. Analysis of the data according to different tribes 137 5.4. Analysis of the data according to sex and season 142 E. Discussion 145 1. Evaluation of the enzyme-linked immunosorbent assay (ELISA) 145 1.1. Antigens used in the ELISA 145 1.2. Sensitivity and specificity of the ELISA 146 1.3. Gross post mortem findings referred to histopathology and PCR results 149 as basis of the determination of the cut-off point 1.3.1. Determination of the cut-off point 152 1.4. Conjugate used in the ELISA 153 1.5. Antibody related reactions in the ELISA 155 1.6. Evaluation of the ELISA as diagnostic tool in a screening programme 158 2. Discussion of the post mortem results from 1989 to 1999 161 2.1. Annual incidence of avian tuberculosis, from 1989 to 1999 161 2.2. Incidence of avian tuberculosis according to sex and season 163 2.3. Incidence of avian tuberculosis according to taxonomic tribes 164 Table of contents 2.4. Incidence of avian tuberculosis according to pen with regard to 171 environment and husbandry practices 3. Recommendations for management and control of avian tuberculosis 175 3.1. Disinfection 175 3.2. Sanitation 176 3.3. Husbandry and modification of enclosures 179 3.4. Recommendations for management 183 F. Summary 191 G. Erweiterte Zusammenfassung 195 H. References 203 I. Appendices 233 1. Tables with comprehensive data regarding the graphics in the chapter 'Results' 233 2. Recipes for buffers used in the ELISA (PAINTER, 1995) 238 3. Detailed ELISA results of all birds which were tested in this study 240 4. Detailed ELISA and post mortem results of birds which were tested and have 251 died during this study 5. Detailed post mortem results of all birds which died from 1989 to 1999 253 Abbreviations AIDS Acquired Immunodeficiency Syndrome BACTEC Becton and Dickinson diagnostic instrument system B-cell Bursa Fabricii derived lymphocyte b.i.d. twice a day °C degree Celsius CD3/CD4+/CD8+ cluster of differentiation (antigens on the surface of immune cells) DNA desoxyribonuclein acid EDTA ethylene diamine tetracetate ELISA enzyme-linked immunosorbent assay GM-CSF granulocyte-macrophage colony-stimulating factor GPL glycopeptidolipid HIV Human Immunodeficiency Virus HPLC high-performance liquid chromatography HRP horseradish peroxidase IFNγ interferon-gamma Ig immunoglobulin IL interleukin kDa kilodalton µl microliter LAM lipoarabinomannan LPS lipopolysaccharide M molar MAI complex Mycobacterium avium - Mycobacterium intracellulare complex MD megadaltons MOTT mycobacteria other than tuberculous mycobacteria ml millilitre mm millimeter nm nanometer µm micrometer NK natural killer cell NTM non-tuberculous mycobacteria OPD o-phenylenediamine. pH pondus hydrogenii (measurement for hydrogen ion concentration) PCR polymerase chain reaction PPEM potential pathogenic environmental mycobacteria RFLP restriction fragment length polymorphism RNA ribonucleic acid rRNA ribosomal ribonucleic acid S sedimentation coefficient (Svedberg) s.i.d once a day T-cell thymus-lymphocyte TGF transforming growth factor TMB 3,3',5,5'-tetramethylbenzidine TNF tumor necrosis factor WWT Wildfowl and Wetlands Trust Introduction 13 A. Introduction The Wildfowl and Wetlands Trust (WWT) was founded in 1946 by Sir Peter Scott. The aim is to conserve wetlands for wildlife and people via education, research, direct conservation actions and the use of wildfowl in captive breeding and reintroduction programmes. To these ends the Trust maintains several captive collections of wildfowl and manages protected areas of wetlands as reserves to provide refuges for wild birds. The first reserve and captive collection were established at Slimbridge in Gloucestershire, where the headquarters of the Trust remain. Eight other Trust centres have opened since, of which six also hold captive wildfowl, and two providing refuges for wild birds only. Wild birds are encouraged within the grounds of each collection as well as on the surrounding reserves. Captive birds are pinioned and kept in open pens. WWT programmes have successfully bred and reintroduced a number of threatened species to the wild. The WWT centre at Llanelli, Carmarthenshire, South Wales, is situated on the estuary of the river Loughor. The reserve comprises approximately 300 acres, of which 45 acres are enclosed by a ground-predator-proofed fence and houses a collection of some 1200 wildfowl.
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