1 Institute of Animal Science University of Hohenheim Prof. Dr. Ludwig Hölzle Transcriptomic analyses during infectious anemia in pigs DISSERTATION submitted in fulfillment of the requirements for the degree “Doktor der Agrarwissenschaften” (Dr. sc. agr. / PhD in Agricultural Science) to the Faculty of Agricultural Sciences presented by Sarah-Lena Mack born in Stuttgart, Germany 2018 2 Die vorliegende Arbeit wurde am 03.04.2019 von der Fakultät Agrarwissenschaften der Universität Hohenheim als "Dissertation zur Erlangung des Grades eines Doktors der Agrarwissenschaften" angenommen (Datum: vgl. Schreiben des Vorsitzenden des Pro- motionsausschusses an den/die Doktoranden/in). Tag der mündlichen Prüfung: 20.05.2019 Leiter/in der Prüfung: Prof. Dr. Jörn Bennewitz Berichterstatter/in 1. Prüfer/in: Prof. Dr. Ludwig Hölzle Mitberichterstatter/in, 2. Prüfer/in: Prof. Dr. Holger Stefanski ggf. weitere Berichter/in bzw. Prüfer/in: Prof. Dr. Mathias Ritzmann < Hohenheim, 18.12.2018, Sarah-Lena Mack > 3 Für meine Eltern 4 Table of contents Table of contents ......................................................................................................... 4 Table of figures ............................................................................................................ 7 List of tables ................................................................................................................ 8 List of abbreviations ................................................................................................... 9 Overview .................................................................................................................... 10 1 Introduction .................................................................................................... 12 1.1 Historical perspective ....................................................................................... 12 1.2 Mycoplasma suis .............................................................................................. 12 1.2.1 Microbiology and taxonomy .............................................................................. 12 1.3 Infectious anemia of pigs .................................................................................. 13 1.3.1 Acute infection .................................................................................................. 14 1.3.2 Chronic infection ............................................................................................... 15 1.4 Epidemiology .................................................................................................... 15 1.5 Pathogenesis and immunology of IAP .............................................................. 16 1.6 Diagnosis ......................................................................................................... 17 1.6.1 Clinical and hematological alterations ............................................................... 17 1.6.2 Bacteriological diagnosis .................................................................................. 18 1.6.3 Microscopy ....................................................................................................... 18 1.6.4 Serology ........................................................................................................... 18 1.6.5 Molecular diagnosis .......................................................................................... 19 1.7 Therapy and prophylaxis .................................................................................. 19 1.8 Transcriptome .................................................................................................. 19 1.8.1 Microarrays....................................................................................................... 20 1.8.2 RNA-Sequencing .............................................................................................. 21 2 Objective ......................................................................................................... 26 2.1 Establishment of an acute IAP model under experiment conditions .................. 27 2.2 Mechanisms used by M. suis to cause disease and to avoid the immune system and to persist in the host, respectively .............................................................. 28 2.3 Transcriptional map of M. suis and analysis of differentially expressed genes during the course of acute infection .................................................................. 29 3 Material and Methods ..................................................................................... 30 3.1 Materials ........................................................................................................... 30 3.1.1 Chemicals ........................................................................................................ 30 5 3.1.2 Drugs................................................................................................................ 30 3.1.3 Buffer and solutions .......................................................................................... 31 3.1.4 Kits ................................................................................................................... 31 3.1.5 Instruments....................................................................................................... 31 3.1.6 Consumabels ................................................................................................... 32 3.2 Animal experiment procedure ........................................................................... 32 3.2.1 M. suis strain .................................................................................................... 32 3.2.2 Animals, experimental design and sampling ..................................................... 32 3.2.3 Splenectomy ..................................................................................................... 33 3.2.4 Detection of M suis via RT-PCR ....................................................................... 34 3.2.5 RNA preparation ............................................................................................... 34 3.2.6 Agarose-gel electrophoresis ............................................................................. 35 3.2.7 Agilent 2100 Bioanalyzer .................................................................................. 35 3.3 RNA-Seq and transcriptome analysis ............................................................... 37 3.3.1 Analysis of gene expression level ..................................................................... 37 3.3.2 Functional analyses of expressed genes and proteins ...................................... 37 3.3.3 Quantitative real-time RT-PCR (qRT-PCR) validation ....................................... 38 3.4 Microarray hybridizations and data analysis ..................................................... 41 3.4.1 Statistical analysis ............................................................................................ 41 4 Results ............................................................................................................ 43 4.1 M. suis infection ................................................................................................ 43 4.2 RNA- Seq-results.............................................................................................. 44 4.2.1 RNA-Seq analysis during acute infection .......................................................... 44 4.2.2 Quantitative and functional analyses of M. suis transcripts expressed during acute infection ............................................................................................................ 46 4.2.3 Characterization of highly expressed and differentially expressed hypothetical proteins 62 4.3 Microarray results ............................................................................................. 62 4.3.1 Transcriptome profile changes between M. suis infected and non-infected animals 62 4.3.2 Differential gene expression analysis identifies most affected biological functions and gene clusters in M. suis-infected animals ......................................................... 65 4.3.3 Pathway analyses............................................................................................. 67 6 4.3.4 Differential expression of candidate genes involved in anemia and endothelial cell damage ............................................................................................................ 68 4.3.5 Differential expression of candidate genes involved in psoriasis in pigs ............ 69 4.3.6 DE genes and immune system related pathway analysis in M. suis infected animals 69 4.3.7 Candidate genes involved in pigs’ immune response on M. suis ...................... 73 5 Discussion ...................................................................................................... 79 5.1 RNA-Seq .......................................................................................................... 80 5.1.1 Hypothetical genes ........................................................................................... 82 5.1.2 DE genes ......................................................................................................... 82 5.1.3 Association between expression level and gene function ................................. 83 5.1.4 M. suis- host interaction .................................................................................... 84 5.1.5 Expressed genes