Characterization of Coxiella burnetii outbreak strains Runa Kuley Characterization of Coxiella burnetii outbreak strains Thesis committee Promotors Prof. Dr M.A. Smits Professor of Intestinal Health of Animals Runa Kuley Wageningen University & Research Prof. Dr J.M. Wells Professor of Host-Microbe Interactomics Wageningen University & Research Co-promotor Dr A. Bossers Senior Scientist, Department of Infection Biology Thesis Wageningen University & Research submitted in fulfilment of the requirements for the degree of doctor at Wageningen University Other members by the authority of the Rector Magnificus Prof. Dr. H.F.J. Savelkoul, Wageningen University & Research Prof. Dr A.P.J. Mol, Prof. Dr J.P.M. van Putten, Utrecht University in the presence of the Dr A.L. Zomer, Utrecht University Thesis Committee appointed by the Academic Board Dr C.A.W. Klaassen, Erasmus MC Rotterdam to be defended in public on Monday May 8, 2017 This research was conducted under the auspices of the Graduate School of Wageningen Institute of at 4 p.m. in the Aula. Animal Sciences (WIAS). Characterization of Coxiella burnetii outbreak strains Thesis committee Promotors Prof. Dr M.A. Smits Professor of Intestinal Health of Animals Runa Kuley Wageningen University & Research Prof. Dr J.M. Wells Professor of Host-Microbe Interactomics Wageningen University & Research Co-promotor Dr A. Bossers Senior Scientist, Department of Infection Biology Thesis Wageningen University & Research submitted in fulfilment of the requirements for the degree of doctor at Wageningen University Other members by the authority of the Rector Magnificus Prof. Dr. H.F.J. Savelkoul, Wageningen University & Research Prof. Dr A.P.J. Mol, Prof. Dr J.P.M. van Putten, Utrecht University in the presence of the Dr A.L. Zomer, Utrecht University Thesis Committee appointed by the Academic Board Dr C.A.W. Klaassen, Erasmus MC Rotterdam to be defended in public on Monday May 8, 2017 This research was conducted under the auspices of the Graduate School of Wageningen Institute of at 4 p.m. in the Aula. Animal Sciences (WIAS). Runa Kuley Characterization of Coxiella burnetii outbreak strains, 226 pages PhD thesis, Wageningen University, Wageningen, NL (2017) With references, with summary in English and Dutch ISBN 978-94-6343-151-4 DOI 10.18174/410829 Runa Kuley Characterization of Coxiella burnetii outbreak strains, 226 pages PhD thesis, Wageningen University, Wageningen, NL (2017) With references, with summary in English and Dutch ISBN 978-94-6343-151-4 DOI 10.18174/410829 Contents Chapter 1: General introduction and thesis outline 9 Chapter 2: Cell-free propagation of Coxiella burnetii does not affect its relative virulence. 37 Chapter 3: Major differential gene regulation in Coxiella burnetii between in vivo and in 59 vitro cultivation models Chapter 4: First complete genome sequence of the Dutch veterinary Coxiella burnetii 85 strain NL3262, originating from the largest global Q fever outbreak, and draft genome sequence of its epidemiologically linked chronic human isolate NLhu3345937 Chapter 5: Plasticity and polymorphisms in critical genes correlate with increased 93 virulence of Dutch outbreak-related Coxiella burnetii strains Chapter 6: Coxiella burnetii strain specific immune responses are more depending on host 131 origin than on MLVA genotype classification. Chapter 7: Coxiella burnetii isolates originating from infected cattle induce a more 155 pronounced pro-inflammatory cytokine response compared to isolates from infected goats and sheep. Chapter 8: General discussion 177 Summary 197 Samenvatting 203 Curriculum vitae 211 Acknowledgements 217 Training and supervision plan 223 Colophon 226 Contents Chapter 1: General introduction and thesis outline 9 Chapter 2: Cell-free propagation of Coxiella burnetii does not affect its relative virulence. 37 Chapter 3: Major differential gene regulation in Coxiella burnetii between in vivo and in 59 vitro cultivation models Chapter 4: First complete genome sequence of the Dutch veterinary Coxiella burnetii 85 strain NL3262, originating from the largest global Q fever outbreak, and draft genome sequence of its epidemiologically linked chronic human isolate NLhu3345937 Chapter 5: Plasticity and polymorphisms in critical genes correlate with increased 93 virulence of Dutch outbreak-related Coxiella burnetii strains Chapter 6: Coxiella burnetii strain specific immune responses are more depending on host 131 origin than on MLVA genotype classification. Chapter 7: Coxiella burnetii isolates originating from infected cattle induce a more 155 pronounced pro-inflammatory cytokine response compared to isolates from infected goats and sheep. Chapter 8: General discussion 177 Summary 197 Samenvatting 203 Curriculum vitae 211 Acknowledgements 217 Training and supervision plan 223 Colophon 226 Chapter 1 General introduction and thesis outline Chapter 1 General introduction and thesis outline Chapter 1 General Introduction Q fever disease Coxiella burnetii is the etiological agent of Q fever; a worldwide zoonotic disease that can result in C. burnetii infects a broad range of hosts, but infections are wide-spread in domestic ruminants [8,9]. large outbreaks in humans. In the recent years, there has been an immense improvement in our Among these, goats, sheep and cattle are the primary reservoirs of the pathogen and represent the most understanding of both the biology and pathogenicity of C. burnetii. However very little is known frequent source of human infection [10]. Infections in animals are assumed to occur mostly by about the molecular mechanisms involved in its interaction with hosts. In addition, only a few inhalation of the pathogen from the environment and are often asymptomatic [11]. The disease virulence factors have been identified and biologically validated. Virulence factors of this pathogen symptoms are usually manifested in pregnant animals and widely differ between hosts [8,10,12]. In most likely include determinants promoting its survival and replication in hostile intracellular pregnant goats and sheep, the main clinical manifestations are abortions at final gestation stage. environments and facilitating manipulation or evasion of host immune responses. Hence, the Infected cattle usually do not show symptoms although abortions, subfertility and metritis are seen in molecular characterization of C. burnetii isolates is essential for virulence factor analysis, some cases [13]. Abortions in infected animals result in excretion of large numbers of C. burnetii 9 epidemiological knowledge, outbreak investigations, development of diagnostic tools, therapeutic (10 /gram placenta) into the environment. Inhalation of contaminated aerosols from the environment interventions and most importantly for vaccine development. In this chapter, we present an overview are the main risk factor for infection in humans [8,9,13,14]. Following infection, 60% of infected of the current knowledge on C. burnetii biology as well as the approaches used to investigate this humans are usually asymptomatic. In symptomatic patients, the clinical signs of acute infections vary pathogen and identify virulence factors, which is crucial for development of an effective and safe greatly but generally manifest as flu-like illness and pneumonia. Following acute Q fever, a persistent vaccine for long-term control of Q fever. fatigue condition causing significant disabilities in the daily life of the individual is referred to as Q fever fatigue syndrome (QFS). QFS has been described and well documented in many countries Discovery of the Q fever agent following an outbreak, but the cause of development of this condition and proper treatment course are Q fever stands for “Query fever” as very little was known about the causative agent and the disease still unknown [15,16]. Around 1-5% of the clinical cases can result in chronic infections after months when it was first discovered in the 1930s. Q fever was first described in abattoir workers in Brisbane, or years of contracting the disease. This occurs mainly in immune deficient individuals or in Queensland, Australia by Edward Holbrook Derrick [1,2]. Simultaneously, the pathogen was isolated individuals with pre-existing cardiac valvular disease and often leads to life-threatening endocarditis by Frank Macfarlane Burnet and Mavis Freeman from samples sent by Derrick, who hypothesized Q [8,13,17–19]. fever was of rickettsial origin [3]. Around the same time Herald Cox and Gordon Davis at the Rocky Mountain Laboratory in Hamilton, independently isolated a new infectious agent from ticks collected Q fever outbreaks in the Netherlands and source identification at Nine Mile Creek, Montana with rickettsia-like properties [4]. A laboratory worker who became Q fever outbreaks are reported frequently worldwide and have a major public health impact. The accidently infected by this new agent displayed symptoms remarkably similar to the Q fever agent outbreak in the Netherlands during 2007-2010 increased the attention to Q fever as more than 4000 isolated from abattoir workers in Australia, suggesting a common infectious agent. Further cross- human cases were registered. It is by far the largest Q fever outbreak reported in the literature protection studies in mice confirmed these two newly discovered infectious agents to be the same [17,19,20]. The factors leading to the outbreak are not fully understood but have been connected with pathogen [5]. The pathogen was initially designated as Rickettsia burneti, on account of its rickettsia- an increase