Epidemiology and Diagnosis of Brucellosis in Mongolian Bactrian Camels INAUGURALDISSERTATION zur Erlangung der Würde eines Doktors der Philosophie vorgelegt der Philosophisch-Naturwissenschaftlichen Fakultät der Universität Basel von Chimedtseren Bayasgalan aus der Mongolei Basel, 2019 Originaldokument gespeichert auf dem Dokumentenserver der Universität Basel edoc.unibas.ch 1 Genehmigt von der Philosophisch-Naturwissenschaftlichen Fakultät auf Antrag von Prof. Marcel Tanner, PD Dr. Esther Schelling und Prof. Gertraud Schüpbach Basel, den 21. Februar 2017 Prof. Dr. Martin Spiess, Dekan Dedicated to my beloved father for his measureless love and his appreciation from Heaven. I also want to dedicate this PhD to my mother, my sister, my brother and my husband for their eternal love and support. Table of Contents Table of Contents Acknowledgements ...................................................................................................................... v Summary ...................................................................................................................................... ix Zusammenfassung ...................................................................................................................... xi List of Tables .............................................................................................................................. xiv List of Figures ............................................................................................................................. xv List of Abbreviations ................................................................................................................. xvi 1. Introduction ............................................................................................................................... 1 1.1. History of brucellosis in Mongolia ................................................................................. 2 1.2. Current control efforts and epidemiological knowledge .............................................. 3 1.3. A review of camels in Mongolia ..................................................................................... 5 1.4. Brucellosis in Mongolian camels ................................................................................... 6 1.5. Goal, Objectives and Hypotheses .................................................................................. 7 2. General Methodology ............................................................................................................... 9 2.1. Epidemiology of camel brucellosis in Mongolia ........................................................... 9 2.1.1. Target population ....................................................................................................... 9 2.1.2. Study design .............................................................................................................. 9 2.1.3. Sample size calculation ........................................................................................... 11 2.1.4. Random multi-stage cluster sampling ...................................................................... 12 2.1.5. Field team ................................................................................................................ 15 2.1.6. Data management and analysis ............................................................................... 16 2.1.7. Ethical considerations .............................................................................................. 16 2.2. Assessment of serological diagnostic tests for camels in Mongolia ........................ 16 2.2.1. Serological tests ...................................................................................................... 16 2.2.1.1. Rose Bengal Test ................................................................................... 17 2.2.1.2. Complement Fixation Test ...................................................................... 18 2.2.1.3. Indirect Enzyme Linked Immunosorbent Assays .................................... 19 2.2.1.4. Competative Enzyme Linked Immunosorbent Assays ............................ 20 2.2.1.5. Fluorescence polarization assay ............................................................ 20 2.2.2. Assessment of serological tests for camels and test comparisons ........................... 21 i Table of Contents 2.2.2.1. ROC curve and Likelihood ratio .............................................................. 23 2.2.2.2. Kappa statistic ........................................................................................ 23 2.2.2.3. Classification of positive and negative sera ............................................ 25 2.3. Identification of the agent of camel brucellosis in Mongolia ..................................... 25 2.3.1. Samples ................................................................................................................... 25 2.3.2. Brucella spp. characterization of the culture ............................................................. 26 2.3.3. Molecular methods................................................................................................... 27 2.3.3.1. Samples ................................................................................................. 27 2.3.3.2. DNA extractions ..................................................................................... 27 2.3.3.3. Bruce-ladder multiplex PCR ................................................................... 28 2.3.3.4. Quantitative real-time PCR (qPCR) ........................................................ 28 2.4. The role of camels in the ongoing ruminant mass vaccination in Mongolia............. 29 2.4.1. Data collection ......................................................................................................... 29 2.4.2. Model descriptions ................................................................................................... 30 3. Collaborations and Responsibilities ...................................................................................... 31 4. Risk factors of brucellosis seroposotivity in Bactrian camels of Mongolia ....................... 33 4.1. Abstract ......................................................................................................................... 34 4.2. Background ................................................................................................................... 35 4.3. Results ........................................................................................................................... 37 4.4. Discussion ..................................................................................................................... 43 4.5. Conclusions ................................................................................................................... 44 4.6. Materials and methods.................................................................................................. 45 4.6.1. Study design and selection of herds ........................................................................ 45 4.6.2. Sample size ............................................................................................................. 47 4.6.3. Selection of animals and sampling ........................................................................... 47 4.6.4. Serological testing ................................................................................................... 48 4.6.5. Bacteriological examination ..................................................................................... 48 4.6.6. Questionnaires ......................................................................................................... 49 4.6.7. Data management and analysis ............................................................................... 49 4.7. Declarations ................................................................................................................... 50 4.8. Acknowledgements ....................................................................................................... 51 5. Serological tests for brucellosis in Mongolian Bactrian camels ......................................... 55 5.1. Abstract ......................................................................................................................... 56 5.2. Introduction ................................................................................................................... 56 ii Table of Contents 5.3. Materials and Methods .................................................................................................. 59 5.3.1. Samples ................................................................................................................... 59 5.3.2. Serological testing ................................................................................................... 60 5.3.3. Bacteriology ............................................................................................................. 61 5.4. Data analyses ................................................................................................................ 62 5.5. Results ........................................................................................................................... 63 5.6. Discussion ..................................................................................................................... 68 5.7. Conclusions ..................................................................................................................