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Aeromonas Salmonicida - Epidemiology, Whole Genome Sequencing, Detection and in Vivo Imaging

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Aeromonas Salmonicida - Epidemiology, Whole Genome Sequencing, Detection and in Vivo Imaging Downloaded from orbit.dtu.dk on: Oct 03, 2021 Aeromonas salmonicida - Epidemiology, whole genome sequencing, detection and in vivo imaging Bartkova, Simona Publication date: 2016 Document Version Publisher's PDF, also known as Version of record Link back to DTU Orbit Citation (APA): Bartkova, S. (2016). Aeromonas salmonicida - Epidemiology, whole genome sequencing, detection and in vivo imaging. National Veterinary Institute, Technical University of Denmark. General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. Users may download and print one copy of any publication from the public portal for the purpose of private study or research. You may not further distribute the material or use it for any profit-making activity or commercial gain You may freely distribute the URL identifying the publication in the public portal If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Aeromonas salmonicida Epidemiology, whole genome sequencing, detection and in vivo imaging Simona Bartkova PhD Thesis 2016 National Veterinary Institute Section for Bacteriology and Pathology Technical University of Denmark, Frederiksberg C Supervisors: Associate professor Inger Dalsgaard Technical University of Denmark Senior researcher Branko Kokotovic Technical University of Denmark Assessment committee: Professor Karl Pedersen Technical University of Denmark Professor Anders Miki Bojesen University of Copenhagen Senior researcher Duncan John Colquhoun Norwegian Veterinary Institute Table of contents Acknowledgements 3 List of manuscripts 4 List of Abbreviations 5 Summary 6 Sammendrag (summary in Danish) 8 Introduction 10 Chapter 1: Aeromonas salmonicida subsp. salmonicida 12 1.1 Background and taxonomy 12 1.2 Biochemical and morphological characteristics 12 1.3 Cultivation 14 1.4 Virulence 14 Chapter 2: Furunculosis 18 2.1 Historical background 18 2.2 Clinical signs of disease 18 2.3 Antibiotic treatment 19 2.4 Vaccination 20 2.5 Transmission 21 2.6 Route of entry and colonization site(s) 23 2.7 Susceptibility in fish species 24 2.8 In vivo imaging 25 2.9 Manuscript I 26 Chapter 3: Detection 28 3.1 Detection of carrier fish 28 3.2 PCR 29 3.3 Real-time PCR 30 3.4 Manuscript II 31 Chapter 4: Epidemiology 33 4.1 Epidemiology and genetic variation 33 4.2 MLST-v 34 4.3 Whole genome sequencing 37 4.4 Manuscript III 39 Chapter 5: Methodological considerations 40 5.1 Materials and methods for MLST-v 40 5.2 In vivo imaging (Manuscript I) 41 5.3 Real-time PCR (Manuscript II) 42 5.4 Whole genome sequencing (Manuscript III) 43 Chapter 6: Discussion and future perspectives 44 6.1 Discussion 44 6.2 Conclusion and future perspective 46 References 48 Accompanying manuscripts 68 2 Acknowledgements This research was financed by the Danish Council for Strategic Research under the ProFish project (Grant no. DSF: 11-116252) and the National Veterinary Institute (DTU). I would like to acknowledge all the ProFish project partners for their work and commitment to the ProFish project and for all the informative discussions we have had. It has been a pleasure collaborating with all of you. First and foremost, I am truly thankful for all the guidance I have received throughout my PhD from my main supervisor Inger Dalsgaard and co-supervisor Branko Kokotovic. Over the last three years (or a little more to be correct) I have gained a lot of valuable knowledge from you both, which I will take with me wherever my future career takes me. I know it has not always been easy with me as a student, but you have somehow managed to keep me grounded and helped me whenever needed. Although especially the last couple of months were intense and stressful for all of us (not to mention full of unexpected events), in the end we managed to put it all together. Finally, I would like to say that I will always be grateful to you Inger Dalsgaard for giving me the opportunity to do a PhD at the National Veterinary Institute with you as my supervisor and supporting me all the way to the end. I would also like to give a special thank you to the laboratory technicians Lisbeth Schade Hansen, Lene Gertman, Katja Ann Kristensen and Margrethe Carlsen as well as other technicians that have helped me in the laboratory along the way. This project would not have been possible without you. Especially Lene Gertman who patiently introduced me to the laboratory world at the National Veterinary Institute and Lisbeth Schade Hansen who took over the difficult task of keeping an eye on me and helping me with the project. This thesis would also have not been accomplished without the help of all the manuscript co-authors and especially without the whole genome sequencing analysis and guidance of “Shinny” Pimlapas Leekitcharoenphon. Through my time as a PhD student there have also been several other PhD students at this section, whom I have had the pleasure of getting to know. We have all had our ups and downs during our PhDs, though luckily we always had each other for support and most importantly entertainment. Some of you have already finished your PhD and have moved on in the science world, while others still have some time before finishing and I wish everyone all the best! Finally, I wish to express my gratitude to my family, friends and basketball teammates and coaches. You have supported me the whole way and especially during the last couple of stressful months when I needed it the most; particularly my parents who I could always turn to for extra guidance, inspiration, support and of course comfort and homemade food. 3 List of manuscripts This thesis includes the following original manuscripts that have either been submitted, accepted or are published online. Manuscript I (accepted in Journal of Fish Diseases) Title: Infection routes of Aeromonas salmonicida in rainbow trout monitored in vivo by real-time bioluminescence imaging Authors: Simona Bartkova, Branko Kokotovic, Inger Dalsgaard Manuscript II (published online in Journal of Fish Diseases) Title: Detection and quantification of Aeromonas salmonicida in fish tissue by real-time PCR Authors: Simona Bartkova, Branko Kokotovic, Helle Frank Skall, Niels Lorenzen, Inger Dalsgaard Manuscript III (submitted to Frontiers in Microbiology) Title: Epidemiology and genetics of Aeromonas salmonicida using whole genome sequencing Authors: Simona Bartkova, Pimlapas Leekitcharoenphon, Frank Møller Aarestrup, Inger Dalsgaard 4 List of Abbreviations AFLP Amplified fragment length polymorphism A-layer Virulence associated surface protein array ARGs Antibiotic resistance genes A. salmonicida Aeromonas salmonicida subsp. salmonicida BHI Brain heart infusion BLI Bioluminescence imaging bp Base pairs β Beta CBB Coomassie brilliant blue CC Clonal complex CFU Colony-forming units ECPs Extracellular products ELISA Enzyme-linked immunosorbent assay FMNH2 Reduced riboflavin phosphate GCAT Glycerophospholipid:cholesterol acyltransferase GFP Green fluorescence protein IROMPs Iron-regulated outer membrane proteins LB Luria Bertani LPS Lipopolysaccharide LUX Light upon eXtension M-CGH Microarray-based comparative genomic hybridization MLST Multilocus sequence typing MLST-v MLST with housekeeping genes and virulence associated genes NGS Next-generation sequencing PCR Polymerase chain reaction PFGE Pulsed-field gel electrophoresis RAPD Randomly amplified DNA polymorphism fingerprinting analysis Real-time PCR Quantitative real-time polymerase chain reaction R plasmids Plasmids carrying antibiotic resistance genes SIF Stress-inducible furunculosis SLVs Single-locus variants SNP Single nucleotide polymorphism ST Sequence type TSA Tryptic soy agar T2SS Type II secretion system T3SS Type III secretion system T6SS Type VI secretion system VBNC Viable but non-culturable cells VIB Veal infusion broth WGS Whole genome sequencing 5 Summary Aeromonas salmonicida subsp. salmonicida is a bacterial fish pathogen that is the causative agent of furunculosis, a septicemic infection responsible for great losses in aquaculture around the world. In Denmark furunculosis was first seen in freshwater in the 1950s, though currently the infection causes problems in sea reared rainbow trout (Oncorhynchus mykiss) production. Outbreaks occur repeatedly during stressful conditions such as elevated temperatures, in spite of commercial vaccines being applied. Besides seemingly lacking adequate protection, the vaccines also produce undesirable side effects. Antibiotics are therefore used as treatment, which due to the possibility of developing resistance is neither a favorable nor sustainable solution. To complicate things further, it is possible that fish can be carriers of A. salmonicida and transfer the bacterium from freshwater to the sea where they develop septicemia when exposed to stressful sea-rearing conditions and high temperatures. By use of traditional bacteriological methods, continuous investigation of bacterial diagnostics on samples from different rainbow trout farms in Denmark was done, while studying the following three aspects of the concerns regarding A. salmonicida. First, we focused on investigation of the route of entry and initial dissemination of A. salmonicida in fish. This was done by tracing the bacterium using in vivo bioluminescence imaging.
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