Promotor: Prof. dr. Daisy Vanrompay Laboratory of Immunology and Animal Biotechnology Department of Animal Production Faculty of Bioscience Engineering Ghent University Belgium Dean: Prof. dr. ir. Guido Van Huylenbroeck Rector: Prof. dr. Anne De Paepe Kristien DE PUYSSELEYR Development and implementation of novel diagnostic tests for epidemiological and zoonotic research on Chlamydia suis Thesis submitted in fulfillment of the requirements for the degree of Doctor (PhD) in Applied Biological Sciences Nederlandse vertaling titel: Ontwikkeling en implementatie van diagnostische testen voor epidemiologisch en zoönotisch onderzoek naar Chlamydia suis. Refer to this thesis: De Puysseleyr, K. (2015). Development and implementation of novel diagnostic tests for epidemiological and zoonotic research on Chlamydia suis. PhD thesis, Ghent University, Ghent, Belgium ISBN-nummer: 978-90-5989-796-0 The author and the promotor give the authorization to consult and to copy parts of this work for personal use only. Every other use is subject to copyright laws. Permission to reproduce any material contained in this work should be obtained from the author. Members of the examination committee Prof. dr. Anita Van Landschoot (Chairman) Department of Applied Biosciences Faculty of Bioscience Engineering Ghent University Prof. dr. Nico Boon (Secretary) Department of Biochemical and Microbial Technology Faculty of Bioscience Engineering Ghent University Prof. dr. Daisy Vanrompay(Promotor) Department of Animal Production Faculty of Bioscience Engineering Ghent University Prof. dr. Eric Cox (Co-promotor) Department of Virology, Parasitology and Immunology Faculty of Veterinary Medicine Ghent University Prof. dr. Frank Pasmans Department of Pathology, Bacteriology and Poultry Disease Faculty of Veterinary Medicine Ghent University Prof. dr. Bruno Goddeeris Department of Biosystems Faculty of Bioscience Engineering KU Leuven Department of Virology, Parasitology and Immunology Faculty of Veterinary Medicine Ghent University Dr. ir. Joris Michiels Department of Applied Biosciences Faculty of Bioscience Engineering Ghent University Dr. ir. Hein Imberechts Centrum voor onderzoek in Diergeneeskunde en Agrochemie CODA-CERVA Brussel Table of contents I Table of Contents Table of Contents ........................................................................................................................ I Abbreviations ............................................................................................................................ V Study Objectives ........................................................................................................................ 1 Chapter I. Chlamydial infection biology ................................................................................ 3 1. Introduction ........................................................................................................................ 5 2. History ................................................................................................................................ 5 3. Taxonomy ........................................................................................................................... 6 4. Developmental cycle ........................................................................................................ 10 5. Chlamydial effector proteins ............................................................................................ 12 6. The Chlamydial Outer Membrane Complex (COMC) .................................................... 12 7. Chlamydia suis infections in pigs ..................................................................................... 17 7.1. Chlamydiaceae species ............................................................................................ 17 7.2. Diagnosis of C. suis ................................................................................................. 18 7.2.1. Culture ....................................................................................................... 18 7.2.2. Immunoassays ........................................................................................... 19 7.2.3. Serology ..................................................................................................... 19 7.2.4. Immunohistochemistry .............................................................................. 20 7.2.5. Molecular diagnosis ................................................................................... 21 7.3. Epidemiology ........................................................................................................... 22 7.3.1. Seroprevalence ........................................................................................... 22 7.3.2. Molecular diagnosis ................................................................................... 23 7.4. Pathogenesis ............................................................................................................ 24 7.4.1. Experimental infections ............................................................................. 24 7.4.2. Field studies ............................................................................................... 26 7.5. Antibiotic resistance ................................................................................................ 27 7.5.1. Persistence ................................................................................................. 29 7.5.2. Diagnostic considerations .......................................................................... 29 7.5.3. Probiotics and vaccination ......................................................................... 30 7.6. Zoonosis ................................................................................................................... 31 II Table of contents Chapter II. Development and validation of a real-time PCR for Chlamydia suis diagnosis in swine and humans .............................................................................................................. 33 Abstract .................................................................................................................................... 34 1. Introduction ...................................................................................................................... 35 2. Materials and methods ..................................................................................................... 36 2.1. Bacterial cultures ..................................................................................................... 36 2.2. DNA extraction ........................................................................................................ 36 2.3. Primers and probes .................................................................................................. 36 2.4. Inhibition control plasmid ........................................................................................ 38 2.5. Species-specific real-time PCR ............................................................................... 38 2.6. Detection limit and specificity ................................................................................. 39 2.7. Case studies ............................................................................................................. 40 2.7.1. Pig farms .................................................................................................... 40 2.7.2. Slaughterhouse employees ........................................................................ 41 3. Results .............................................................................................................................. 42 3.1. Primers and probes .................................................................................................. 42 3.2. Internal inhibition control ........................................................................................ 42 3.3. Species-specific real-time PCR ............................................................................... 43 3.4. Case studies ............................................................................................................. 44 3.4.1. Pig farms .................................................................................................... 44 3.4.2. Slaughterhouse employees ........................................................................ 44 4. Discussion ........................................................................................................................ 45 Chapter III. Evaluation of the presence and zoonotic transmission of Chlamydia suis in a pig slaughterhouse .................................................................................................................. 49 Abstract .................................................................................................................................... 50 1. Introduction ...................................................................................................................... 51 2. Materials and Methods ..................................................................................................... 52 2.1. Sampling of pigs and humans .................................................................................. 52 2.2. Sampling of air and contact surfaces ....................................................................... 52 2.3. DNA extraction .......................................................................................................