NOVEL APPROACHES TO THE CONTROL OF CYATHOSTOMINS IN EQUIDS THESIS SUBMITTED IN ACCORDANCE WITH THE REQUIREMENTS OF THE UNIVERSITY OF LIVERPOOL FOR THE DEGREE OF DOCTOR OF PHILOSOPHY BY LAURA ELIZABETH PEACHEY APRIL 2016 I ABSTRACT Novel approaches to the control of cyathostomins in equids Laura Elizabeth Peachey Cyathostomins, are clade 5 gastrointestinal (GI) nematodes infecting equids. They are associated with a range of pathologies, the most serious of which is larval cyathostominosis, a protein losing enteropathy with a 50 % mortality rate. Importantly, they are the most abundant GI nematode of equids in the developed world and, as they do not induce protective immunity, equids remain at risk of infection throughout their lives. The effective control of cyathostomins is currently threatened by the development of anthelmintic resistance (AR) to all three major classes of anthelmintic licenced for use in equids. Of primary concern is the emerging resistance to the macrocyclic lactones (MLs), which are the mainstay of cyathostomin control. It is therefore important that novel options for controlling cyathostomins are explored. In this study in vitro tests were used to explore a range of options for developing novel treatments against AR cyathostomins and improving the efficacy of the widely used ML, ivermectin (IVM). First the potential use of ethnoveterinary medicines for treatment of cyathostomins in equids in Ethiopia and the UK was explored (Chapters 3, 4 and 5). A participatory rural appraisal was performed in the Oromia region of Ethiopia to identify plants currently used as anthelmintics in equids and other livestock. A total of 37 species of plant were identified, data on dosing and side effects were also recorded. These data were triangulated with a literature review to identify five plants for in vitro screening for anthelmintic activity against cyathostomins. A literature review was used to identify plants for in vitro screening in the UK. A total of 138 publications, reporting the anthelmintic activity of plant extracts against nematodes, were found. The data collected was used to design a ranking system to provide a shortlist of five plant candidates. Shortlisted plants were collected, dried and chemically extracted to give crude extracts, which were screened against cyathostomins in the egg hatch test (EHT) and larval migration inhibition test (LMIT). A total of 7/9 plants screened in this way showed anthelmintic activity in either the EHT and/or the LMIT with median effective concentrations (EC-50s) in the range 0.18- 8.90 mg/ml. For some of the most efficacious extracts there were reports of in vivo efficacy against GI nematodes of other veterinary parasitic nematodes, making these good candidates for future in vivo trials. One of the plants shortlisted for the UK, for which there was extensive evidence of anthelmintic activity in the literature, was the cysteine proteinase (CP) containing Carica papaya. Extract from this species, papaya latex supernatant (PLS), could not be processed and tested using the same protocols as the other plant species, as the active compound is an enzyme. The EHT and LMIT were optimised for use with PLS and its efficacy evaluated in chapter 5. It was found that PLS had a potent anthelmintic effect against cyathostomins in the EHT, which was attributable to the action of CPs, with EC-50 values in repeats ranging between 0.12-0.22 µM. PLS also showed efficacy in the LMIT, although this appeared due to another active compound. To the authors knowledge this is the first report of efficacy of PLS against the free living stages of any parasitic nematode, which may indicate that cyathostomins are particularly susceptible to CPs. In chapter 6 the role of P-glycoproteins (P-gps) in IVM resistance in cyathostomins was investigated, with the more specific aim of assessing whether P-gp inhibitors could potentially be used in combination with IVM to improve its efficacy against AR parasites. The transcription of pgp-9, which is putatively associated with IVM resistance in Teladorsagia II circumcincta and Haemonchus contortus, was measured after exposure to IVM in in ML resistant (AR) versus anthelmintic naïve (AN) cyathostomin third stage larvae (L3). Pgp-9 expression was significantly increased after incubation with IVM in the AR, but not the AN, parasite population. Two in vitro tests, the LMIT and the larval development test (LDT), were used to assess the effect of a range of P-gp inhibitors (ketoconazole, pluronic 85 and ivermectin aglycone) on IVM efficacy in ML resistant (AR) versus anthelmintic naïve (AN) cyathostomins. In the LMIT ketoconazole and ivermectin aglycone conferred a significant increase in IVM efficacy in the AR parasites, but had less effect on the AN parasites. Pluronic 85 also had a profound effect on IVM efficacy which depended on IVM concentration, however there was no differential effect dependent on parasite population. In the LDT, ketoconazole and pluronic 85 both significantly increased IVM efficacy in both AR and AN parasites. These results suggest that there is a lifecycle stage-specific association of P-gps with AR cyathostomins, and that P-gp inhibitors could potentially be used to improve IVM efficacy in AR cyathostomin populations. In summary in this study has identified a range of potential novel treatments for cyathostomins. The data also suggest a role for P-gps in the emerging resistance to MLs, which could potentially be exploited to improve ML efficacy in AR cyathostomin populations. Importantly, this work will underpin the financial and ethical justification for future in vivo trials on the extracts/compounds tested here. III ACKNOWLEDGEMENTS I would like to thank my primary supervisor Dr Jane Hodgkinson for her endless support and encouragement, her belief in my ability and her persistence in obtaining further funding so that I could complete my PhD. In addition her support through some difficult personal circumstances was invaluable and I will appreciate this for many years to come. I would also like to thank my supervisor Dr Gina Pinchbeck, firstly for inspiring me to do this project, and secondly for her continued support and willingness to help in any way possible. I am also indebted to my supervisor Professor Jacqui Matthews, for providing me with the in vitro tools to perform this project, and for her thorough critique of my thesis and publications. I have learnt a huge amount from her rigorous approach, and I hope to take these skills forward with me in my research career. There are many other staff and students at the University of Liverpool and collaborating institutions without whom this project would not have been possible, and whom I would like to thank; Dr. Claire Scantlebury at the University of Liverpool who performed the participatory rural appraisal in Ethiopia; Mairi Mitchell, Valerie Relf, and Claire McArthur at Moredun Research Institute for their guidance with in vitro tests; Professor Sebsebe Demissew at the University of Addis Ababa for his assistance with plant identification; Dr Faith Burden, Dr Getachew Mulugeta, Nikki Stradling and Liz Hazell-Smith at the Donkey Sanctuary for their support throughout the project and assistance with plant and parasite sample collection; Gebre Tefera from SPANA, Ethiopia for his assistance with plant collection; Dr Etana Debela at the University of Hawassa, Ethiopia, for his assistance with plant extractions; Timothy Baxter at Ness Gardens, University of Liverpool for his help with sourcing UK plants; Ann Leyden at the University of Liverpool Chemistry Department for allowing me to perform plant extractions in her laboratory; Dr Buttle at the University of Sheffield for his assistance with the cysteine proteinase enzyme titrations; Dr Jerzy Behnke and Fadlul Mansur at the University of Nottingham for donating papaya latex supernatant and providing advice; Dr Anne Lespine at Institut national de la recherche agronomique, Toulouse, for donating ivermectin aglycone and providing advice; Professor Georg von Samson-Himmelstjerna and Dr Jurgen Krucken at the Free University of Berlin for providing sequence data; Dr Ben Makepeace at the University of Liverpool for his advice regarding real time PCR; Catherine Hartley and Jenna Dawson at the University of Liverpool for their support in the laboratory and help with larval cultures; and last but not least the Veterinary Parasitology research group for their fabulous, moral boosting support. Finally, and in many ways most importantly, special thanks must go to some of my family. I would like to take this opportunity to dedicate this thesis to my late father, Tony Peachey, who sadly passed away during my PhD. He has been my inspiration throughout my scientific career, and has instilled in me a deep love of science and truth, which will remain with me forever. This PhD is his achievement, as much as it is mine. I would also like to dedicate this thesis to my darling daughter, Ada, who came into this world shortly after my Father left it. Much of the time that went into this PhD was rightfully hers. She has put up with an absent Mummy IV brilliantly, and therefore deserves much of the credit. She is a beautiful inspiration in everything I do. Next I would like to thank my Mother enormously for her strength, support with childcare, thesis proof reading and for believing in me against all odds. Huge thanks must also go to my parents in law Jon and Julie Fairhurst, whose help with childcare in the vital last stages of thesis writing has been absolutely