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Epidemiological Investigations on the Occurrence of Mycobacterium

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Epidemiological Investigations on the Occurrence of Mycobacterium Epidemiological investigations on the occurrence of Mycobacterium avium subspecies paratuberculosis in different matrices from cattle and zoo animals by IS900 polymerase chain reaction assays Dissertation to obtain the Ph. D. degree in the Ph. D. Program for Agricultural Sciences in Goettingen (PAG) at the Faculty of Agricultural Sciences, Georg-August-University Göttingen, Germany presented by Pia Münster born in Düsseldorf, Germany Göttingen, March 2012 D 7 1. Name of supervisor: Prof. Dr. Dr. Claus-Peter Czerny 2. Name of co-supervisor: Prof. Dr. Dr. Matthias Gauly Date of dissertation: 31st May 2012 Contents Contents 1 GENERAL INTRODUCTION .................................................................................... 1 2 LITERATURE REVIEW ............................................................................................ 4 2.1 THE PATHOGEN ..................................................................................................................... 4 2.1.1 TAXONOMY .......................................................................................................................... 5 2.1.2 THE GENOME ........................................................................................................................ 7 2.1.2.1 GENOTYPES ....................................................................................................................... 8 2.1.2.2 INSERTION SEQUENCE (IS900) .......................................................................................... 9 2.1.3 PATHOGENESIS ................................................................................................................... 12 2.1.4 TRANSMISSION ................................................................................................................... 14 2.1.5 PREVALENCE ...................................................................................................................... 19 2.1.6 HOST RANGE ....................................................................................................................... 25 2.1.7 ZOONOTIC ASPECT .............................................................................................................. 29 2.2 DIAGNOSTIC METHODS ........................................................................................................ 31 2.2.1 INDIRECT DIAGNOSTIC METHODS ....................................................................................... 31 2.2.1.1 CELL MEDIATED IMMUNITY (CMI) ................................................................................. 31 2.2.1.1.1 SKIN TESTING (ST) ....................................................................................................... 32 2.2.1.1.2 INTERFERON-GAMMA DETECTION (IFN-) ................................................................... 32 2.2.1.2 HUMORAL IMMUNE RESPONSE ........................................................................................ 32 2.2.1.2.1 AGAR GEL IMMUNODIFFUSION (AGID) ........................................................................ 33 2.2.1.2.2 COMPLEMENT FIXATION TEST (CFT) .......................................................................... 33 2.2.1.2.3 ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA) .................................................. 33 2.2.2 DIRECT DIAGNOSTIC METHODS .......................................................................................... 35 2.2.2.1 ZIEHL-NEELSEN STAINING (ZN) ...................................................................................... 35 2.2.2.2 BACTERIOLOGY ............................................................................................................... 35 2.2.2.2.1 CONVENTIONAL CULTURE ............................................................................................ 35 2.2.2.2.2 RADIOMETRIC CULTURE (BACTEC) ............................................................................ 38 2.2.2.3 POLYMERASE CHAIN REACTION (PCR) ........................................................................... 39 2.2.2.3.1 CONVENTIONAL PCR ................................................................................................... 39 2.2.2.3.2 REAL-TIME PCR ........................................................................................................... 42 2.3 CONTROL MEASURES .......................................................................................................... 46 Contents 3 STUDIES PERFORMED ........................................................................................... 48 CHAPTER I .................................................................................................................................. 49 SHORT COMMUNICATION: DETECTION OF MYCOBACTERIUM AVIUM SSP. PARATUBERCULOSIS IN ILEOCAECAL LYMPH NODES COLLECTED FROM ELDERLY SLAUGHTER COWS USING A SEMI- NESTED IS900 POLYMERASE CHAIN REACTION ......................................................................... 49 CHAPTER II ................................................................................................................................ 60 A LONGITUDINAL STUDY TO CHARACTERIZE THE DISTRIBUTION PATTERNS OF MYCOBACTERIUM AVIUM SSP. PARATUBERCULOSIS IN SEMEN, BLOOD AND FECES OF A NATURALLY INFECTED BULL BY IS900 SEMI-NESTED AND QUANTITATIVE REAL-TIME PCR ................................................... 60 CHAPTER III ............................................................................................................................... 82 BRIEF COMMUNICATION: DETECTION OF MYCOBACTERIUM AVIUM SUBSPECIES PARATUBERCULOSIS BY IS900-BASED PCR ASSAYS FROM AN ALPACA (VICUGNA PACOS) KEPT IN A GERMAN ZOOLOGICAL GARDEN............................................................................................... 82 CHAPTER IV ............................................................................................................................... 90 DISTRIBUTION OF MYCOBACTERIUM AVIUM SSP. PARATUBERCULOSIS IN A GERMAN ZOOLOGICAL GARDEN DETERMINED BY IS900 SEMI-NESTED AND QUANTITATIVE REAL-TIME PCR ............... 90 AUTHOR’S CONTRIBUTION ...................................................................................................... 108 4 GENERAL DISCUSSION ....................................................................................... 109 4.1 CONCLUSION ...................................................................................................................... 115 4.2 FUTURE PROSPECTS ........................................................................................................... 117 5 SUMMARY ............................................................................................................... 118 6 ZUSAMMENFASSUNG .......................................................................................... 120 7 REFERENCES .......................................................................................................... 122 8 APPENDIX ................................................................................................................ 141 LIST OF PUBLICATIONS ........................................................................................................... 142 LIST OF PRESENTATIONS......................................................................................................... 143 LIST OF POSTER ....................................................................................................................... 144 CURRICULUM VITAE ............................................................................................................... 145 ACKNOWLEDGEMENTS ............................................................................................................ 146 DECLARATION .......................................................................................................................... 147 General Introduction 1 General Introduction Mycobacterium avium ssp. paratuberculosis (MAP) is the infectious agent of Johne‟s disease, a degenerative wasting disorder in cattle and other ruminants. Paratuberculosis is responsible for considerable economic impacts in dairy and beef industry and is of increasing international importance (Harris and Barletta, 2001). Since the first description of the pathogen MAP in 1895 (Johne, 1895), the cattle industry has been trying to stop or to decrease the spread of paratuberculosis. Due to its long incubation time of up to ten years, MAP has a complex epidemiologic profile. MAP can spread unnoticed until the first clinical case is identified because of the existence of asymptomatic carriers and subclinically infected animals in a herd. Besides suitable molecular biological methods for the early diagnosis of MAP infected cattle, the generation of effective paratuberculosis control programs requires reliable data on MAP prevalence. MAP occurs in most countries of the world with an increasing prevalence. It is assumed that for every cow showing disease manifestation at least 25 other animals are infected (Whitlock and Buergelt, 1996). Current diagnostic methods detect only 15% to 25% of those subclinically infected animals. Therefore, the true MAP prevalence might well exceed current estimates. In the past, serological methods were commonly used to estimate MAP prevalences (Nielsen and Toft, 2009). However, since subclinical paratuberculosis infections are characterized by low-level stimulation of the humoral immune response probably resulting in negative antibody detection,
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