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Downloaded for Personal Non-Commercial Research Or Study, Without Prior Permission Or Charge https://theses.gla.ac.uk/ Theses Digitisation: https://www.gla.ac.uk/myglasgow/research/enlighten/theses/digitisation/ This is a digitised version of the original print thesis. Copyright and moral rights for this work are retained by the author A copy can be downloaded for personal non-commercial research or study, without prior permission or charge This work cannot be reproduced or quoted extensively from without first obtaining permission in writing from the author The content must not be changed in any way or sold commercially in any format or medium without the formal permission of the author When referring to this work, full bibliographic details including the author, title, awarding institution and date of the thesis must be given Enlighten: Theses https://theses.gla.ac.uk/ [email protected] Development of microsatellites and the population genetic analysis of the parasitic nematode Teladorsagia circumcincta Victoria Louise Grillo A thesis submitted for degree of Doctor of Philosophy Supervisor: Dr John Gilleard Division of Infection and Immunity Veterinary Faculty University of Glasgow December 2005 ProQuest Number: 10390671 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a com plete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest ProQuest 10390671 Published by ProQuest LLO (2017). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States C ode Microform Edition © ProQuest LLO. ProQuest LLO. 789 East Eisenhower Parkway P.Q. Box 1346 Ann Arbor, Ml 48106- 1346 GLASGOW UNIVERSITY .LIBRARY: ^ Declaration I declare that this thesis consists entirely of my own work, unless specifically indicated and has been composed by myself. This thesis has not been accepted in any previous application for a degree in this or any other university. Victoria Grillo Abstract Gastrointestinal parasitic nematodes of sheep and goats are of major economic importance worldwide. The control of parasitic nematodes, in both domestic livestock and man, is dependent upon the strategic use of anthelmintic drugs such as the benzimidazoles, levamisoles and ivermectins. However, for parasitic nematodes of sheep and goats, resistance to these drugs is becoming increasingly common. Anthelmintic resistance in T. circumcincta, a parasitic gastrointestinal nematode of sheep and goats, is of major economic importance especially in countries of the northern hemisphere, such as the U.K. However, relatively little is understood regarding how resistance develops and spreads in parasitic nematode populations, including r. circumcincta. In order to address these issues, an understanding of the population genetic structure is required but as yet very few studies have investigated this area. The main aim of my thesis was to investigate the population genetic structure of T. circumcincta using multilocus genomic markers. However there are very few molecular tools available for this parasite species and very little genomic sequence available from which to design markers. Therefore, a key first step of this project was to isolate microsatellite markers from T. circumcincta that were suitable for population genetic analysis. Three approaches were used to isolate micro satellite mai'kers from T. circumcincta. Hybridisation screening of small insert genomic libraries, amplification of conserved loci with previously known loci in Haemonchus contortus and EST database searching, yielded 45, 19 and 14 microsatellites respectively. Five of these micro satellite markers were found to be sufficiently robust and polymorphic to be used in population genetic studies. These were used to genotype single adults, L3 stages or eggs from eighteen T. circumcincta populations from Scotland, France and New Zealand. Overall, the level of polymorphism for all populations investigated was high, which corresponds with previous studies of trichostrongylid nematodes which have been predominantly performed using mitochondrial DNA markers. The majority of diversity seen was within populations rather than between populations. However, population genetic analysis with the microsatellites markers supports the existence of a ‘cryptic’ species of T. circumcincta in French goats, as originally suggested by Leignel et al. (2002). In the FrMe population, the same individuals genetically differentiated with multilocus genotyping where found to be typed as ‘cryptic’ when using the previously described markers; ITS-2 and (3-tubulin (Leignel et al. 2002). The micro satellites markers used in this study, demonstrated independent evidence that this ‘cryptic’ species is potentially a new species of T. circumcincta. In addition, moderate levels of ii differentiation are seen between the New Zealand population and all the other populations in this study. From the populations analysed from the U.K., the data suggest that there is very little differentiation seen between populations regardless of the host species, geographical location or source of origin as a field or laboratory maintained isolate. Population genetic analysis using the micro satellites detected no genetic differentiation between T. davtiani, T. circumcincta and T. trifurcata supporting the suggestion that these may be morphological variants of the same species. In addition to investigating the population genetic structure of T. circumcincta, a survey of U.K. goat farms was conducted, as the current prevalence of anthelmintic resistance in U.K. goats is unknown. For many parts of the world, drug resistance in sheep nematodes has been preceded by drug resistance in goat nematodes, suggesting that goats may be a source of resistant parasites for sheep. Therefore the survey was conducted to investigate the efficacy of parasitic control methods and as well as to identify anthelmintic resistant populations of T. circumcincta for use in further population genetic studies. The survey found not only farmers with poor drug efficacies, but also highlighted the need to improve the tests used to investigate and quantify parasitic nematode control. Acknowledgements I would like to thank John Gilleard, my supervisor, for continuous advice, discussion and enthusiasm in addition to the various useful debates. John has provided me with the knowledge and skills to allow me to become a confident scientist in my own right. I would also like to thank the University of Glasgow for funding this project. In addition, I would like to thank my assessors, Frank Jackson and Andy Tait for providing advice and discussion when required. I would also like to thank everyone in the Veterinary Parasitology Depaitment (Division of Infection and Immunity), past and present for their help throughout and in particular Judith Smith, Pamela McGarr, Annabelle Couthier, and Stevie Robertson for their endless laughs and practical advice in the lab, James McGolderick for his advice regarding my goat survey and allowing me to bring smelly samples into his lab, as well as doing some FEC for me. I would also like to acknowledge Willie Weir and Erica Packard for their continuous sense of humour and optimism throughout my writing up, which I believe kept me sane! The collaboration with the Moredun Institute during this project has been highly productive not only for the endless numbers of T. circumcincta but providing me with the parasitology skills required for my survey. In particular, Frank and Liz Jackson, Dave Bartley and Lindsay Stenhouse for advice, discussion, and always welcoming me as one of the extended lab family, especially at tea break! 1 would like to acknowledge everyone who provided T. circumcincta populations, including Dr Warwick Grant for the New Zealand sample, Dr Jacques Cabaret for the French populations prepared at INRA, Barbara Craig, Dr BarbaraWimmer and Dr Josephine Pemberton for the samples from Hirta and Dr Sian Mitchell for the Welsh sample. I would also like to thank all the goat farmers who took part in the survey. I would like to thank all my friends and family for endless jokes regarding my (worm) project, in particular Babs and Roddy for support, chats, coffee and nights out when necessary and Rich for proof reading. It has been a long stint here in Glasgow, but the love and emotional support from my parents and in particular my soon to be husband, Jason has been unfailing along the way. I would like to thank my parents in particular for their encouragement and support of their daughter’s seemingly endless education! I would like to dedicate this thesis to Jason, as he was the one who encouraged me to go after what I want, and has been so patient with the time it has taken to complete. Without him, 1 wouldn’t have accomplished this! IV Table of Contents Title page................................................................... i Declaration............................................................................ i Abstract................................................................................................ ii Acknowledgements...................................................................................................... iv Table of Contents......................................................................................................... v List of Tables................................................................................................................ix
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