McIntyre, Jennifer Ruth (2020) Genetic markers of anthelmintic resistance in gastrointestinal parasites of ruminants. PhD thesis. https://theses.gla.ac.uk/79021/ 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] Genetic Markers of Anthelmintic Resistance in Gastrointestinal Parasites of Ruminants Jennifer Ruth McIntyre BVM&S BVetSci(Hons) MRCVS Submitted in fulfilment of the requirements for the degree of Doctor of Philosophy Institute of Biodiversity, Animal Health and Comparative Medicine College of Medical, Veterinary and Life Sciences February 2020 © Jennifer Ruth McIntyre, 2020 2 Abstract Parasitic gastroenteritis is the primary production limiting disease of sheep in the UK and is a considerable welfare concern. A global problem, it is caused by nematode parasites and mixed species infections can be common. In the UK, the primary pathogen in growing lambs is Teladorsagia circumcincta, an abomasal parasite of small ruminants, causing severe pathology and reduced weight gain. T. circumcincta is expertly adapted to both the host and the farming year and control is extremely difficult. The majority of UK farmers will use anthelmintics to manage parasitic gastroenteritis. Nevertheless, anthelmintic resistance is increasing, reducing control options. Many farmers will now dose sheep with a macrocyclic lactone (e.g. ivermectin) to treat T. circumcincta as this species has developed resistance to multiple anthelmintic classes. Unfortunately, over fifty percent of farms in recent UK studies had detectable ivermectin resistance. There is a pressing need to conserve anthelmintics for future use. However, the mechanism of ivermectin resistance is unknown, and the lack of a sensitive test for ivermectin resistance limits research into resistance spread and development. Many excellent studies have investigated ivermectin resistance in nematode parasites, however mutations responsible for ivermectin resistance remain elusive. The purpose of this PhD was to perform a genome wide association study to identify genomic regions under ivermectin selection within UK T. circumcincta field populations. L3 progeny were sequenced pre- and post- ivermectin treatment using next generation sequencing techniques (ddRAD-Seq and Pool-Seq) and population genetics analyses were performed. Multiple loci were genetically differentiated between pre- and post-ivermectin populations. However, the reference genomes used were highly fragmented and the number of loci under selection cannot be concluded. Genes identified included those with neuronal functions, metabolic and regulatory genes. Many genes had associations with pharyngeal structures and chemosensory behaviour. Nevertheless, multiple copies of genes expected to be single copy were detected in both reference genomes and these may have affected read alignment and results. The work performed here provides an important basis for future studies, and has generated high quality next generation sequenced resources from two UK field populations of T. circumcincta. 3 Table of Contents List of Tables ................................................................................ 8 List of Figures .............................................................................. 10 List of Accompanying Material ........................................................... 12 Acknowledgement ......................................................................... 13 Author’s Declaration ...................................................................... 14 Abbreviations .............................................................................. 15 1 Introduction ........................................................................... 19 1.1 Sheep Farming Systems ......................................................... 19 1.2 Parasitic Gastroenteritis ........................................................ 21 1.3 Teladorsagiosis ................................................................... 22 1.4 Management of Parasitic Gastroenteritis .................................... 24 1.5 Anthelmintic Resistance ........................................................ 28 1.6 In vitro tests of anthelmintic sensitivity ..................................... 29 1.7 Prevalence of Anthelmintic Resistance ...................................... 33 1.8 Ivermectin Resistance .......................................................... 35 1.8.1 IVM resistance is inherited as a fully or partially dominant trait .... 35 1.8.2 IVM affects motility, fecundity and reproduction ..................... 36 1.8.3 IVM interacts with ligand gated ion channels, but the cause of resistance is unknown ............................................................... 37 1.9 Benzimidazole Resistance ...................................................... 44 1.10 Levamisole Resistance ........................................................ 45 1.11 Monepantel Resistance ....................................................... 48 1.12 What factors affect the genetic footprint of resistance? ............... 50 1.13 Why might anthelmintic resistance have arisen so rapidly? ............ 51 1.14 What are the concerns with anthelmintic resistance? ................... 52 1.15 Is there hope in the face of anthelmintic resistance? ................... 53 1.16 Investigating the genetic basis of drug resistance using next generation sequencing ............................................................................... 55 2 Materials and Methods ............................................................... 61 2.1 Parasite material ................................................................ 61 2.1.1 Laboratory isolates ......................................................... 61 MTci2 (Moredun T. circumcincta 2) ...................................................................................... 61 MTci5 (Moredun T. circumcincta 5) ...................................................................................... 61 2.1.2 Farm 1 ........................................................................ 61 2.1.3 Farm 2 ........................................................................ 61 2.1.4 Farm 3 ........................................................................ 62 2.2 Sample Collection and Processing ............................................. 62 2.2.1 Collection of faecal samples .............................................. 62 4 2.2.2 Faecal egg counts........................................................... 62 2.2.3 Coprocultures and Baermannisation of L3 .............................. 63 2.2.4 Morphological examination of L3 ......................................... 64 2.2.5 Faecal egg count reduction test .......................................... 64 2.3 Bioassays .......................................................................... 64 2.3.1 Egg hatch test ............................................................... 65 2.3.2 Larval development test ................................................... 66 2.4 Molecular Biology ................................................................ 67 2.4.1 Lysate protocol ............................................................. 67 2.4.2 Speciation of strongyles by PCR .......................................... 67 2.4.3 Microsatellite PCR and Analysis .......................................... 69 2.4.4 Pyrosequencing ............................................................. 72 2.4.5 hprt-1 allele specific PCR ................................................. 74 2.5 Genomic DNA Library Preparation for Next Generation Sequencing: ddRAD-Seq and Pool-Seq ............................................................... 76 2.5.1 Whole Genome Amplification of DNA for ddRAD-Seq .................. 76 2.5.2 Double digest of WGA DNA ................................................ 77 2.5.3 Adaptor ligation ............................................................. 77 2.5.4 Size selection, Illumina Library Prep and DNA Sequencing ........... 78 2.5.5 Preparation of Genomic DNA Libraries for Pool-Seq ................... 78 2.6 Bioinformatics: Reference genomes .......................................... 79 2.7 Bioinformatics: ddRAD-Seq ..................................................... 80 2.7.1 Cleaning and demultiplexing of ddRAD-Seq reads ..................... 80 2.7.2 Alignment of ddRAD-Seq reads to Tci2 genome ........................ 80 2.7.3 Formation and filtering of RADloci ....................................... 81 2.7.4 Population structure and Kinship analysis .............................. 81 2.7.5 Hardy Weinberg analysis ................................................... 82 2.7.6 Linkage disequilibrium analysis ........................................... 82 2.7.7 SNP density across the genome ........................................... 83 2.7.8 ddRAD-Seq genetic differentiation analysis ............................ 83 2.8 Bioinformatics: Preliminary annotation of Tci2