Zurich Open Repository and Archive University of Zurich Main Library Strickhofstrasse 39 CH-8057 Zurich www.zora.uzh.ch Year: 2017 Parasite factors, prognostic markers and epidemiological aspects in babesia canis babesiosis Eichenberger, Ramon Marc Posted at the Zurich Open Repository and Archive, University of Zurich ZORA URL: https://doi.org/10.5167/uzh-151591 Dissertation Published Version Originally published at: Eichenberger, Ramon Marc. Parasite factors, prognostic markers and epidemiological aspects in babesia canis babesiosis. 2017, University of Zurich, Vetsuisse Faculty. Parasite Factors, Prognostic Markers and Epidemiological Aspects in Babesia canis Babesiosis ___________________________________________________________________________ Dissertation zur Erlangung der naturwissenschaftlichen Doktorwürde (Dr. sc. nat.) vorgelegt der Mathematisch-naturwissenschaftlichen Fakultät der Universität Zürich von Ramon M. Eichenberger aus Beinwil a.S., AG Promotionskomitee Prof. Dr. Peter Deplazes (Leiter der Dissertation) Prof. Dr. Adrian B. Hehl (Vorsitz der Dissertation) Prof. Dr. Regina Hofmann-Lehmann Prof. Dr. Hanspeter Nägeli Prof. Dr. Hans-Peter Beck Zürich, 2017 Parasite Factors, Prognostic Markers and Epidemiological Aspects in Babesia canis Babesiosis ___________________________________________________________________________ Faculty of Science University of Zurich Life Science Graduate School Microbiology and Immunology PhD Program PhD thesis Submitted by Ramon M. Eichenberger from Beinwil a.S., AG Thesis supervisor: Prof. Dr. Peter Deplazes Responsible thesis advisor: Prof. Dr. Adrian B. Hehl Thesis committee members: Prof. Dr. Regina Hofmann-Lehmann Prof. Dr. Hanspeter Nägeli Prof. Dr. Hans-Peter Beck Zürich, 2017 Table of contents PART I SUMMARY..................................................................................................................... 4 Summary ...........................................................................................................................................4 Zusammenfassung ............................................................................................................................6 PART II INTRODUCTION ............................................................................................................. 8 The biology of Babesia canis ............................................................................................................8 1.1. Babesia and the phylum Apicomplexa......................................................................................8 1.2. Babesia in dogs; development and epidemiology of canine babesiosis ..................................9 1.3. Pathophysiology of B. canis infections. ................................................................................. 11 1.4. Diagnosis of B. canis infections .............................................................................................. 13 1.5. Genetic characterization and proteomic background of B. canis .......................................... 13 Parasite-host interaction – insights from related apicomplexan organisms ................................. 15 2.1. Secretory organelles in apicomplexan parasites ................................................................... 15 2.2. Protein trafficking in the intra-erythrocytic apicomplexans Plasmodium and Babesia ........ 17 2.3. Immune evasion and antigenic variation – strategies to escape host defense ..................... 18 The framework of apicomplexan genomes ................................................................................... 19 Aims of the thesis .......................................................................................................................... 21 References ..................................................................................................................................... 22 PART III MANUSCRIPTS ......................................................................................................... 33 General remarks ............................................................................................................................ 33 Manuscripts ................................................................................................................................... 34 2.1. Combined analysis of the Babesia canis genome, transcriptome and secreted proteome during virulent infection identifies potential pathogenicity factors .............................................. 34 2.2. Prognostic markers in acute Babesia canis Infections ........................................................... 64 2.3. Ticks on dogs and cats: a pet owner-based survey in a rural town in northeastern Switzerland ..................................................................................................................................... 83 PART IV DISCUSSION & PERSPECTIVES ................................................................................... 94 Discussion ...................................................................................................................................... 94 1.1. General ................................................................................................................................... 94 1.2. Establishment of indigenous foci of canine babesiosis is dependent on the vector distribution ..................................................................................................................................... 95 1.3. Clinical pathophysiology and prognostic markers: Are clinical signs host- or parasite induced? ......................................................................................................................................... 96 1.4. Models for alternative secretory pathways in Babesia canis ................................................ 97 Perspectives ................................................................................................................................... 99 2.1. Screening for diagnostic and vaccine candidates ................................................................ 100 References ................................................................................................................................... 101 Acknowledgements ................................................................................................................... 105 Curriculum Vitae ....................................................................................................................... 106 PART I SUMMARY PART I SUMMARY Summary The triad of the phylogenetically related protozoan blood parasites Babesia, Plasmodium and Theileria are the causative agents of important human and veterinary diseases worldwide. Canine babesiosis, a persistent endemic disease in diverse dog populations, is caused by apicomplexan hemo- parasites of the genus Babesia. Parasite host transmission is mediated by ticks and is facilitated by the international and domestic transportation of dogs as well as the focal availability of tick vectors. The presence of chronic subclinical carrier states in dogs, the inability to completely eliminate all infections, and the transovarial transmission of the parasite within the vector tick population further lead to a spreading of new infections. Babesia canis is the predominant and clinically relevant canine Babesia species in Europe. Although the pathophysiology of canine babesiosis has been extensively studied, parasite-host interactions of the parasite are poorly understood. In common, the peripheral parasitemia remains usually low which however does not prevent severe clinical signs and high fatality rates. Hence, the pathogenic capacities of B. canis lie within the secreted factors, all of which have been poorly characterized. This PhD project aimed to characterize key factors involved in B. canis infections, such as the B. canis secretome which affects infected erythrocyte biology (Subproject 1), the description of clinical factors and pathophysiologic mechanisms of babesiosis in dogs (Subproject 2), and the analysis of tick-vector dissemination after a locally restricted outbreak of B. canis (Subproject 3). Like other apicomplexan parasites, B. canis has developed a repertoire of organelles, pathways and strategies to survive, subsist and replicate within the infected host-cell. In a first subproject, we assembled and annotated the B. canis reference genome from a virulent Hungarian parasite strain to generate an understanding of B. canis virulence and pathogenicity factors. Out of the 3467 predicted gene models we identified 509 potential secreted parasite proteins. To identify specific parasite protein candidates involved during acute crisis of the disease, we analyzed the B. canis blood stage proteome and transcriptome using parasites isolated from experimentally infected dogs. Our data indicate the presence of parasite factors involved in cytoadherence, immune evasion, and infected red blood cell remodeling. These combined analyses provide a predicted and partially validated set of exported factors from B. canis blood stages potentially involved in the direct parasite-host interplay during the acute phase of the disease. In a second subproject, we focused on host factors during acute B. canis infections which are associated with severe and fatal clinical outcomes. By examining 28 different laboratory parameters in 15 naturally infected animals at the time of first admission and before