Aus dem Institut für Parasitologie und Tropenveterinärmedizin des Fachbereichs Veterinärmedizin der Freien Universität Berlin lnaugural-Dissertation zur Erlangung des Grades eines PhD of Biomedical Sciences an der Freien Universität Berlin vorgelegt von Christina Maria Bredtmann Tierärztin aus Recklinghausen Berlin 2019 Journal-Nr.: 4146 Freie Universität Berlin Aus dem Institut für Parasitologie und Tropenveterinärmedizin des Fachbereichs Veterinärmedizin der Freien Universität Berlin Molecular and Proteomic Species Characterisation of Cyathostomins Inaugural-Dissertation zur Erlangung des Grades eines PhD of Biomedical Sciences an der Freien Universität Berlin vorgelegt von Christina Maria Bredtmann Tierärztin aus Recklinghausen Berlin 201 9 Journal-Nr.: 4146 Gedruckt mit Genehmigung des Fachbereichs Veterinärmedizin der Freien Universität Berlin Dekan: Univ.- Prof. Dr. Jürgen Zentek Erster Gutachter: Univ.- Prof. Dr. Georg von Samson-Himmelstjerna Zweiter Gutachter: Univ.-Prof. Dr. Uwe Rösler Dritter Gutachter: Prof. Dr. Benedikt Kaufer Deskriptoren (nach CAB-Thesaurus): Coronocyclus coronatus, Cylicostephanus calicatus, Cylicostephanus longibursatus, Cylicostephanus minutus, MALDI-TOF, Polymerase chain reaction Tag der Promotion: 5. September 2019 Table of Contents List of Tables .............................................................................................................................. III List of Figures ............................................................................................................................. III List of Abbreviations ................................................................................................................... V 1 Preface ................................................................................................................................ 1 2 Literature review ................................................................................................................. 3 2.1 Overview on cyathostomins ........................................................................................ 3 2.1.1 Systematics ........................................................................................................... 3 2.1.2 Morphology .......................................................................................................... 7 General morphology ..................................................................................... 7 Morphological features of selected species ................................................. 9 2.1.3 Occurrence of cyathostomins ............................................................................ 13 2.1.4 Life cyle and epidemiology ................................................................................. 15 Life cycle in general ..................................................................................... 15 Species-specific knowledge on the life cycle .............................................. 18 2.1.5 Cyathostomin associated disease ...................................................................... 18 2.1.6 Anthelmintic management ................................................................................ 21 Pasture/stable management ...................................................................... 21 Chemotherapeutic management ................................................................ 21 Anthelmintic compounds ............................................................................ 23 2.1.7 Anthelmintic resistance ...................................................................................... 24 Anthelmintic resistance in general ............................................................. 24 Species-specific knowledge on resistance .................................................. 25 2.2 Molecular approaches ............................................................................................... 27 2.2.1 Molecular diagnostics ........................................................................................ 27 Ribosomal marker ....................................................................................... 28 Mitochondrial marker ................................................................................. 29 2.2.2 Molecular phylogenetics .................................................................................... 29 2.3 Proteomics ................................................................................................................. 31 2.3.1 Matrix-Assisted Laser Desorption/Ionisation Time-Of-Flight Mass Spectrometry (MALDI-TOF MS) ............................................................................................................... 31 Species identification with MALDI-TOF MS ................................................ 31 I The principle of MALDI-TOF MS .................................................................. 32 2.4 Study Objectives ........................................................................................................ 35 3 Nematode Species Identification - Current Status, Challenges and Future Perspectives for Cyathostomins .......................................................................................................................... 36 4 Concurrent Proteomic Fingerprinting and Molecular Analysis of Cyathostomins ........... 56 4.1 Publication ................................................................................................................. 57 4.2 Supporting Information ............................................................................................. 79 4.2.1 Supporting information, Text 1 .......................................................................... 79 4.2.2 Supporting figures .............................................................................................. 81 5 Nuclear and mitochondrial marker sequences reveal close relationship between Coronocyclus coronatus and a potential Cylicostephanus calicatus cryptic species complex . 85 5.1 Publication ................................................................................................................. 85 5.2 Supporting Information ............................................................................................. 96 5.2.1 Supporting figures .............................................................................................. 96 5.2.2 Supporting tables ............................................................................................... 98 6 Discussion ........................................................................................................................ 101 6.1 The publications and how they interlink ................................................................. 101 6.2 Research purposes ................................................................................................... 102 6.2.1 Validity of species and genera and implications on phylogeny ....................... 102 6.2.2 Species identification ....................................................................................... 106 6.2.3 Clinical impact .................................................................................................. 109 6.2.4 Anthelmintic treatment and anthelmintic resistance ...................................... 110 6.3 Outlook .................................................................................................................... 110 7 Summary ......................................................................................................................... 112 8 Zusammenfassung .......................................................................................................... 114 9 References ...................................................................................................................... 117 10 Publications ..................................................................................................................... 129 10.1 Publications in peer-reviewed journals ................................................................... 129 10.2 Abstracts of oral or poster presentations in conference proceedings.................... 130 11 Disclosure of the own share in the body of work ........................................................... 131 12 Acknowledgements ......................................................................................................... 132 13 Funding sources .............................................................................................................. 133 14 Statement of Authorship ................................................................................................ 134 II List of Tables Table 2-1: Overview of cyathostomin species (based on Lichtenfels et al. 2008) ..................... 4 Table 2-2: Most common cyathostomin species (Traversa et al., 2007) ................................. 13 Table 3-1: Comparison of methods for cyathostomin species identification. ......................... 48 Table 5-1: Description of parasite specimen ............................................................................ 98 Table 5-2: Primer and PCR conditions .................................................................................... 100 List of Figures Figure 2-1: Buccal capsule and part of oesophagus of Coronocyclus coronatus. ...................... 9 Figure 2-2: Buccal capsule (“Mouth open”) and anterior end of oesophagus of Cylicostephanus calicatus.