MOLECULAR AND IMMUNOLOGICAL SD0100025 CHARACTERIZATION OF MYCOBACTERIA ASSOCIATED WITH BOVINE FARCY. Thesis submitted in accordance with requirements of the University of Khartoum for the Degree of Doctor of Philosophy (Ph.D). By Victor Loku Kwajok B.V.Med. 1978, University of Cairo/Egypt. M.V.Sc.1992, University of Khartoum/Sudan. Supervisor: Dr. Maowia M. Mukhtar. Institute of Endemic Diseases, University of Khartoum. Department of Preventive Medicine and Veterinary Public Health. Faculty of Veterinary Science, University of Khartoum. 2000. 32/27 SOME PAGES ARE MISSING IN THE ORIGINAL DOCUMENT LIST OF CONTENTS DEDICATION. * ii v ACKNOWLEDGEMENTS vi ABBREVIATIONS viii ABSTRACT. xi CHAPTER ONE. REVIEW OF LITERATURE. 1. General Introduction. 1. 1.1. Molecular Systematics of genus Mycobacterium. 2. 1.2. Molecular taxonomy of M. farcinogenese and M. senegalense 11. 1.3. Immunology of bovine farcy agents. 36. CHAPTER TWO. MATERIALS AND METHODOLOGY 41. Isolation, identification and characterization of M. farcinogenes. 2.1 Phenotypic characterization. 41. 2.1.1. Morphological and biochemical tests. 2.1.2. Degradation tests. 2.1.3. Rapid fluorogenic enzyme tests. 2.1.4. Nutritional tests. 2.1.5. Physiological tests. 2.2. Molecular characterization. 52. 2.2.1. DNA extraction and purification 2.2.2. PCR amplification and application. 2.2.3. DNA sequencing of 16SrDNA. 2.2.4. PCR-based restriction fragment length polymorphism. 2.3 Imrmmological analyses of bovine farcy agents. 71. 2.3.1 .EL1SA technique for sera diagnosis. 2.3.2 Animal pathogenicity Tests. 2.3.3 Protein antigen profiles determination using. CHAPTER THREE: RESULTS. 78. CHAPTER. FOUR: DISCUSSION. 124. REFERENCES. 133. APPENDIX 180. iif LIST OF TABLES Substrates used in Rapid fluorogenic enzyme tests. 48. Oliginucleotide primers used in the PCR amplification and sequence of 16SrDNA. 61. The clinical isolates. 79. Test Strains and their sources. 79. Levels of nucleotide similarity based on 16S rDNA sequences of M. farcinogenese, M. Senegal ense and related taxa. 93. ELISA results. 118. Protein antigens profiles (molecular weights) 121. IV LIST OF FIGURES. Sources of taxonomic information used to characterize actinomycetes. 4. Protocol for PCR amplification and 16SrDNA sequencing. 53. Protocol for ribotyping experiments labeled rDNA probes. 68. Sj and UPGMA. 82. Ssm and UPGMA. • 83. DNA bands of clinical isolates. 85. PCR amplification of DNA of clinical isolates. 86. Restriction fragment length polymorphism patterns. 88. Dendrograms showing similarity values. 89. DNA sequences of clinical isolates. 94. Protein antigen bands. 122. Protein antigen profiles. 123. ABBREVIATIONS. AIDS Aquired immunodeficiecy syndrome. AMC Aminomefhyl coumarin (fluorogenic indicator). Apal Restriction enzyme isolated from Acetobacter pasieurianus. API Analytical profile Index. ASCII Accesibilily of sequence data in computer. ATCC American collection of type culture. ATPase Adenosinc triphosphate. Bp Base pair. BSA Bovine serum albumen. BstEIl Restrictiuon enzyme isolated from Baccilus stearolhermphilusE. DAB diaminobenzidine. DDBJ DNA databank of Japan. DMT Delayed hypersensitivity type. DM SO Dimethyl sulphoxide. DNA Deoxyribonucleic acid. DNTP Deoxynucleotide triphosphate. EBI European Bioinformatic Institute. EcoRI Restriction enzyme isolated from Escherichia coli IIY13. EDTA Ethylenditetra acetic acid. E1A Enzyme immunoassay. ELISA Enzyme linked immunosorbant assay. EMBL European Molecular Biology Laboratory. FAMES Fatty acid methyl esters. IIP File transfer protocol. G+C Guanine plus cytosine ratio of the DNA. GPL Glycopeptidolipids. GYEA Glucose yeast extract agar. HIV Human immunodeficiency virus. IIPLC High performance liquid chromatography. MSP I leal shocked protein. VIII ICSB International Committee on Systematic Bacteriology. IS Insertion sequence (Insertion element). 1WGMT . International Working Group on Mycobacterial Taxonomy. Kb Kilobase (molecular size measure). LRF Large subunit fragment LSUrRNA Large subunit rllNA. MAMBS Mycolic acid methyl esters. Md Megadallon (DNA molecular size measure). MK Menac|iiinoneK with isoprene units. • MU Methylumbelliferone(fluorogenicindicator). NCTC National Collection of Type Cultures. Notl Restriction enzyme isolated from Nocardia otitidis-caviarum. NrampI Natural resistance associated macrophage Proteinl. OD Optimal density. 32P Probed labelled with a high-energy radio isotope. PAGE Polyacrylamide gel electrophoresis (ulse agar gel electrophoresis). PBS Phosphate buffer saline. PCR Polymerase chain reaction. PFGH Pulse field gel electrophoresis. PstI Restriction enzyme isolated from Providencia stuartii. PyMS Pyrolysis Mass spectrometry. RDP Ribosomal Database Project. RFLP Restriction fragment length polymorphism. JIN A Ribonucleic acid. SDS Sodium dodecyl sulphate. Sill Restriction enzyme isolated from Streptomyces fimbriatus. Sml Restriction enzyme isolated from Serratia marcescens SB. Sj/UPGMA Unweighted pair group method of arithmetic algorithm using Sj Ssm/UPG.MA. Unweighted pair group method of arithmetic algorithm using Ssm. TAIi Tris-acelate ethylditetra acetic. TBE Tris-borate EDTA. TBS Tris-buffer saline. TLC Thin layer chromatography. „ IX Tm Thermal denaturatuion method. TPE Tri-phosphate EDTA. UPGMA, Unweighted pair group method with arithmetic averages algorithm. UV Ultra-Violet transilluminator. WHO World Health Organization. WWW World wide web. ABSTRACT. The ability to delineate taxo-species has had a profound influence in bacteriology, notably the identification and classification of pathogenic bacteria. However, improved methods and automated data acquisition system can be expected to facilitate the generation of high-quality databases for variety of purposes. The aim of the study was to: i, Isolate and identify Mycobaclerium jarcinogenese from the clinical samples (lymph nods and sreum), ii.characterization of these species including Mycobaclerium senegalense and the related taxa (Mycobaclerium chelonae, Mycobacleriuni forluitum, Mycobacierium peregrinum and Nocardia farcinica) using Molecular biology methods (DNA extraction, PCR amplification, restriction fi-agment length polymorphism determination using restriction enzymes and DNA sequencing) and Hi. Jinmunological analysis of the species (animal pathogenicily tests, ELISA using sera samples from the clinical cases, protein antigen bands determination using SDS-PAGE method, and antigen-antibodies immunoassay using Western blotting and iinnumodiniision tests). Seventeen clinical isolates identified as Mycobaclerium farcinogenese were obtained fromfive hundred and seventy eight (578) lymph nodes and 36 positive sera samples of the two hundred and sixty nine (269) sera samples which were tested. Molecular characterization of the test strains was carried out using independent taxonomic criteria derived from the application of morphological, enzymatic and chemotaxonomic methods. DNA extraction method gave clearly resolved bands on agarose gel electrophoresis with clear common bands of 1500 base pairs. The extracted DNA was used as template for PCR amplification with universal primer 27f (5' AGAGTTTGATCCGGCTCAG-3') and primer 1525r (5'AAGGAGGTGATCGAGCC-3') with appended restriction sites being ideal primers lor amplification. No significant difference in the DNA fingerprints was found, indicating that DNA fingerprints of the farcy agents were reproducible over successive generations and were in line with their placement in the genus A/ycobiu feriiiin. XI PCR-DNA fingerprinting using BamHI restriction enzymes for restriction fragment length polymorphism analysis as a means for differentiating between Mycobaclerium farcinogemse and Mycobacterium senegalense. •. The I6S1DNA sequencing of Mycobacterium farcinogense and Mycobacterium senegalense the farcy sole agents, gave data of variable signals wilh 1482 nucleotides with 65 corresponding almost complete nucleotide sequences in 1404 positions. Manual alignment of the sequenced DNA using computer, was clear and showed l6SrDNA similarity values of 99.8-99.9%, which corresponded, to 7 and 12 nucleotide differences. Immunoassay of the protein antigens of the test strains of 'Mycobaclerium farcinogenese and the anti sera produced from the laboratory animals-guinea pigs gave positive results. Mycobacterium farcinogenese isolates produced approximately similar profiles of protein bands. VII CHAPTER ONE: LITERATURE REVIEW GENERAL INTRODUCTION. Bovine farcy is a chronic disease of African bovines caused by Mycohacterium fare-hingenes and Mycohacterium senegaiense (Chamoseau. 1969. 1972). These species have a long and torlous history since the time of a French veterinarian lidward Nocard(1888). Bacterial systematics began as a largely intuitive science but has become increasingly objective due to the development and application of chemotaxonomic. molecular systematic and numerical phenetic methods. The new advances, especially in molecular systematics promoted to compare older and more recent approaches to bacterial classification (Wayne el. al. 1987. Murray et.al.. 1990). This exercise led to the view that bacterial classification at all levels in the taxonomic hicrachy should be based on the integrated use of genotypic and phenotypic data (O'Donnell et.al.. 1993). Polyphasic taxonomy, was introduced by Cohvell (1970) to signify successive or simultaneous studies on groups of organisms using a set of taxonomic procedures designed to yield good quality genotypic and phenotypic data (Goodfellow et.al.. 1997b).Genotypic .infemiation