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ISOLATION and IDENTIFICATION of Avibacterium Paragallinarum from LAYER CHICKENS

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ISOLATION and IDENTIFICATION of Avibacterium Paragallinarum from LAYER CHICKENS ISOLATION AND IDENTIFICATION OF Avibacterium paragallinarum FROM LAYER CHICKENS A THESIS Submitted to Bangladesh Agricultural University, Mymensingh In partial Fulfillment of the Requirements for the Degree of Master of Science in Pathology BY SALEHA AKTER Roll No.: 11Vet Path JJ 14 M Registration No.: 38019, Session: 20011-12 Department of Pathology Faculty of Veterinary Science Bangladesh Agricultural University Mymensingh May 2012 ISOLATION AND IDENTIFICATION OF Avibacterium paragallinarum FROM LAYER CHICKENS A THESIS Submitted to Bangladesh Agricultural University, Mymensingh In partial Fulfillment of the Requirements for the Degree of Master of Science in Pathology By SALEHA AKTER Approved as to style and content by Prof. Dr. Priya Mohan Das Prof. Dr. Md. Mokbul Hossain Co- Supervisor Supervisor Prof. Dr. Priya Mohan Das Chairman, BOS & Head Department of Pathology May, 2012 ACKNOWLEDGEMENTS The author is grateful and indebted to the almighty Allah without whose grace she would have ever been able to pursue her higher studies in this field of science and to complete her thesis for the degree of Master of Science (MS) in Pathology. The author expresses her ever indebtedness, deepest sense of gratitude, sincere appreciation and profound regards to her reverend and beloved teacher and research Supervisor, Professor Dr. Md.Mokbul Hossian, Department of Pathology Bangladesh Bangladesh Agricultural University, (BAU) Mymensingh for his scholastic guidance, uncompromising principles, sympathetic supervision, valuable advice, constant inspiration, affectionate feeling, radical investigation and constructive criticism in all phases of this study and preparing the manuscript. The author finds it a great pleasure in expressing her heartfelt gratitude and immense indebtedness to her honourable and respected Research Co –Supervisor Dr. Priya Mohan Das, Professor and Head, Department of Pathology, BAU, Mymensingh for his sympathy, kind co-operation, inspiration, scholastic supervision and suggestions, constructive criticism and valuable advice for the completion of the research work and preparation of the thesis. The author wishes to express cordial respect and profound gratitude to her respected teachers Prof. Dr.Md. Iqbal Hossain, Prof. Dr. Md. Abdul Baki, Prof. Dr. Md. Rafiqul Islam, Prof. Dr. Md. Habibur Rahman, Prof. Dr. Emdadul Haque Chowdhury, Prof. Dr. Md. Abu Hadi Noor Ali Khan, Dr. Mrs. Jahanara Begum, Dr. Mrs. Munmun Parvin, and Dr. Md. Nuruzzaman lecturers Department of Pathology, BAU, Mymensingh for their valuable advice and constant inspiration throughout the entire period of study. The author explicit gratefulness and cordial thanks to Dr. Abu Sufiun, and all other Ph. iv D. students of Department of Pathology, BAU, Mymensingh for their valuable suggestions, practical help and co-operation in every step of the research work and in writing the thesis. The author is also thankful to all office staffs, Department of Pathology, BAU, Mymensingh for their technical assistance during the research work. The author expresses her thanks to Ali, Solaiman, Mamun, Sankar, Arif, Sharif, Shuvonkar, Mehedi, Poly, Luna and Sumi for their continuous help in conducting the research work. Dictions not enough to express the author’s immense gratitude and endless love to her beloved parents for their heartiest blessing and encouragement for her higher education and inspiration throughout her academic life. Last but not the least the author expresses her deeper sense of gratitude and sincere thanks to all her well wishers kith and kin for their constant inspiration and blessing throughout the entire period of her academic life. May, 2012 The Author v ABSTRACT The present research work was conducted for the isolation and identification of Avibacterium paragallinarum, the etiological agent of infectious coryza (IC) along with pathological study of the affected organs from layer chickens. A total of 30 nasal swab samples collected aseptically from the dead chickens from SK Veterinary Diagnostic Center, Mymensingh and 4 live samples: 2 from Gazipur, and 2 from Tangail. The used methods were culture of organisms in different media, staining, sugar fermentation test, biochemical tests of the isolated causal agent and histopathology of the affected tissues. A total of 30 swabs from nasal passage of dead birds died of other diseases, no A. paragallinarum was isolated and identified. Two out of 4 suspected clinical cases of IC confirmed by isolation of A. paragallinarum. The organism A. paragallinarum was canfirmed on the basis of colony morphology, staining characters, sugar fermentation test, MR test, VP test, indole test and catalase test. The clinical signs of all 4 cases were nasal discharge; conjunctivitis with swelling of the sinuses, face and wattles; decreased feed and water consumption and reduced egg production. At necropsy, the gross lesions noted were mucous in nasal passage and tracheal hemorrhage. Histopathology of the nose showed acanthosis, congestion, mucous glandular cell hyperplasia, hyperplasia of nasal sinus and parakeratosis. From Bangladesh, this is the first time A. paragallinarum was isolated and identified from clinical cases. Further study is essential involving the other areas of serological and molecular identification of the etiological agent of infectious coryza since the disease has great economic importance. vi CONTENTS CHAPTER LIST OF CONTENTS PAGE NO. ACKNOWLEDGEMENTS iv ABSTRACT vi LIST OF FIGURES xii LIST OF TABLES xiv LIST OF ABBREVIATION AND SYMBOLS xv 1 INTRODUCTION 01 2 REVIEW OF LITERATURE 04 2.1 Avibacterium paragallinarum 04 2.1.1 History of A. paragallinarum 04 2.1.2 Etiology of infectious coryza 05 2.1.3 Host of infectious coryza 05 2.1.4 Clinical signs of the disease 06 2.1.5 Cultural properties of A. paragallinaru 08 2.1.6 Morphology and staining characteristics of 13 A. paragallinarum 2.1.7 Isolation and identification of A. paragallinarum 14 2.1.8 Biochemical properties of A. paragallinarum 18 2.1.9 Antibiotic sensitivity of A. paragallinarum 19 2.1.10 Pathogenicity of A. paragallinarum 20 2.1.11 Prevalence of infectious coryza 22 2.2. Staphylococcus aureus 26 2.2.1 Colonies 26 vii CONTENTS(COND.) CHAPTER LIST OF CONTENTS PAGE NO. 2.2.2 Staining characters 26 2.2.3 Biochemical tests of S. aureus 27 2.2.4 Pathology and pathogenesis of S. aureus 27 3 MATERIALS AND METHODS 28 3.1 Materials 28 3.1.1. Study area 28 3.1.2 Cotton swab 28 3.1.3 Bacteriological media for culture 29 3.1.3.1 Solid media 29 3.1.3.2 Liquid media (broth) 29 3.1.3.3 Chemicals, reagents and solutions 29 3.1.3.4 Sugars 29 3.1.3.5 Reagents for biochemical test 29 3.1.4 Glassware and other necessary instruments 30 3.1.5 Hexisol hand rub 30 3.1.6 20% sterile buffered glycerin 30 3.1.7 Preparation of various bacteriological culture 30 media and different liquid solution 3.1.7.1 Nutrient broth 30 3.1.7.2 Nutrient agar 31 3.1.7.3 Blood agar 31 3.1.7.4 Bacteriological peptone 32 3.1.7.5 MR-VP medium 32 3.1.7.6 Phosphate buffer solution 33 viii CONTENTS(COND.) CHAPTER LIST OF CONTENTS PAGE NO. 3.1.7.6 Phosphate buffer solution 33 3.1.7.7 10% buffer formalin 33 3.2 METHODS 33 3.2.1 Cleaning and sterilization of glassware 33 and plastic ware 3.2.2 Brief description of the experimental design 34 3.2.3 Collection and transportation of samples 37 3.2.4 Isolation and identification of organisms 37 3.2.4.1 Isolation and identification of S. aureus 37 3.2.4.1. Primary culture of S. aureus 37 3.2.4.1.2 Isolation of S. aureus in pure culture 37 3.2.4.2 Isolation and identification of pure culture 37 3.2.4.2.1 Primary culture of A. paragallinarum 37 3.2.4.2.2 Isolation of A. paragallinarum in pure culture 37 3.2.4.3 Identification of the isolates 38 3.2.4.3.1 Study of colony morphology for identification 38 3.2.4.3.2 Morphological characterization by Gram’s 38 staining method 3.2.4.3.2.1 Preparation of Gram staining solution 38 3.2.4.3.2 Microscopic study of the suspected colonies 39 3.2.4.4 Biochemical studies for the identification 40 of organism 3.2.4.4.1 Carbohydrate fermentation test 40 3.2.4.4.2 Indole test 42 ix CONTENTS(COND.) CHAPTER LIST OF CONTENTS PAGE NO. 3 .2.4.4.3 Methyl-Red & Voges-Proskauer (MR-VR) test 42 3.2.4.4.4 Alpha- napthanol solution 43 3.2.4.4.5 Potassium hydroxide solution 43 3.2.4. Enzyme activity test 43 3.2.4.5.1 Catalase test 43 3.2.5 Maintenance of the stock culture 44 3.2.5.1 Phosphate buffered saline solution 44 3.2.5.2 20% sterile buffered glycerin method 44 3.2.6 Pathological studies 44 3.2.6.1 Gross pathology 44 3.2.6.2 Histopathology 44 3.2.6.2.1 Processing of nasal passage tissue 45 3.2.6.2.2 Preparation of decalcifying solution 45 3.2.6.2.3 Preparation of stains 46 3.2.6..2.4 Routine hematoxyrlin eosin staining procedure 47 3.2.7 Photomicrography 47 4 RESULTS 48 4.1 Results of isolation and identification of S. aureus 48 4.1.1 Results of cultural examination 48 4.1.1.1 Culture on nutrient broth 48 4.1.1.2 Culture on mannitol salt agar 48 4.1.1.3 Culture on nutrient agar 48 4.1.1.4 Culture on blood agar 49 4.1.2 Results of Gram's stain 49 x CONTENTS(COND.) CHAPTER LIST OF CONTENTS PAGE NO. 4.1.3 Enzymatic activity test 49 4.1.3.1 Catalase activity test 49 4.2 Results of isolation and identification of 49 etiological agent, A.
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