Studies of the hindgut and faecal volatile organic compound metabolome and microbiome of the horse Thesis submitted in accordance with the requirements of the University of Liverpool for the degree of Doctor in Philosophy by Rachael Siân Slater November 2019 ‘Most large animals, in fact are not the single individuals they seem to be. They are walking menageries, whole communities of different species which, in their various ways, are committed by evolution, for better or for worse, in sickness and in health, to live together'. D. Attenborough, (1990) Contents List of Figures ................................................................................................................... viii List of Tables ..................................................................................................................... xiv List of Abbreviations ........................................................................................................ xvii Abstract ............................................................................................................................ xix Publications arising from this thesis .................................................................................. xx Acknowledgements .......................................................................................................... xxi Chapter 1 Introduction ....................................................................................................... 1 1.1 Anatomy and physiology of the equine gastrointestinal tract ...................................... 1 1.1.1 The Mouth.............................................................................................................. 1 1.1.2 The Oesophagus and Stomach ................................................................................ 2 1.1.3 Small intestine ........................................................................................................ 2 1.1.4 The hindgut ............................................................................................................ 3 1.2 Microbiology of the gastrointestinal tract .................................................................... 4 1.2.1 Nutritional contribution .......................................................................................... 4 1.2.2 Competitive pathogenicity ...................................................................................... 5 1.2.3 Stimulation of the immune system ......................................................................... 6 1.3 The microorganisms of the equine gastrointestinal tract ............................................. 7 1.3.1 Bacteria .................................................................................................................. 8 1.3.2 Protozoa ................................................................................................................12 1.3.3 Fungi .....................................................................................................................13 1.3.4 Archaea .................................................................................................................14 1.4 Factors correlated with changes in the equine microbiome and metabolome ............14 1.4.1 Age ........................................................................................................................15 1.4.2 Sex ........................................................................................................................15 1.4.3 Diet .......................................................................................................................15 1.4.4 Season ...................................................................................................................18 1.4.5 Host genetics and breed ........................................................................................18 1.4.6 Body condition ......................................................................................................19 1.4.7 Stress, transport and exercise ................................................................................20 1.4.8 Inter-individual variation .......................................................................................21 1.4.9 Antimicrobials .......................................................................................................21 1.4.10 Probiotics ............................................................................................................22 1.5 Gastrointestinal disease related to the hindgut microbiota ........................................22 i 1.5.1. Colic .....................................................................................................................22 1.5.2 Gastrointestinal parasites ......................................................................................24 1.6 Introduction to tapeworm infection ............................................................................26 1.6.1 Morphology and lifecycle .......................................................................................26 1.6.2 Prevalence .............................................................................................................27 1.6.3 Pathogenesis .........................................................................................................28 1.6.4 Current diagnosis ...................................................................................................29 1.6.5 Treatment .............................................................................................................31 1.6.6 Parasite resistance to anthelmintic drugs...............................................................32 1.7 Introduction to volatile organic compounds ................................................................32 1.7.1 Volatile organic compounds as biomarkers for gastrointestinal disease ..................34 1.8 Hypothesis, aims and objectives ..................................................................................35 Chapter 2 Methodology: general methods and method optimisation ...............................37 2.1 Introduction .................................................................................................................37 2.2 Methods to sample the equine hindgut.......................................................................38 2.2.1 Surgical procedure .................................................................................................38 2.2.2 Cannulated animals ...............................................................................................38 2.2.3 Direct removal of faeces from rectum....................................................................38 2.2.4 Naturally-voided faeces .........................................................................................38 2.2.5 Intestinal contents post mortem ............................................................................39 2.2.6 Chosen sampling methods .....................................................................................39 2.3 Methods to extract volatile organic compounds from faeces ......................................39 2.4 Methods to characterise the hindgut metabolome .....................................................42 2.5 Selected methods to characterise faecal the metabolome: Headspace solid phase micro-extraction gas chromatography mass spectrometry (HS-SPME-GCMS) ...................43 2.6 Methods to characterise the hindgut microbiota ........................................................44 2.7 Selected methods to characterise the microbiome: targeted 16S rRNA MiSeq next generation sequencing ......................................................................................................47 2.8 General metabolomics methods used in this thesis .....................................................48 2.8.1 Headspace-solid phase micro extraction (HS-SPME) .................................................48 2.8.2 Perkin Elmer Clarus 500 GCMS analysis conditions ...................................................48 2.8.3 Reference solution ....................................................................................................49 2.8.4 Metabolomics data analysis .....................................................................................49 2.8.4.1 Data processing and VOC identification ..............................................................49 2.8.4.2 Metabolome statistical analysis ..........................................................................51 2.8.5 Determination of parasite burden ............................................................................53 ii 2.9. Method development of the preparation steps of equine faecal samples for HS- SPME-GCMS ......................................................................................................................54 2.9.1 Introduction ..........................................................................................................54 2.9.2 Materials and Methods..........................................................................................54 2.9.2.1 Sample collection................................................................................................54 2.9.2.2 Mass optimisation ..............................................................................................55 2.9.2.3 SPME fibre coating ..............................................................................................55