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I the UNIVERSITY of NOTTINGHAM SCHOOL of BIOLOGY THE UNIVERSITY OF NOTTINGHAM SCHOOL OF BIOLOGY INVESTIGATIONS INTO THE EFFECTS OF PLANT DERIVED CYSTEINE PROTEINASES ON TAPEWORMS (CESTODA) by FADLUL AZIM FAUZI BIN MANSUR B.Sc M.D. M.Sc Thesis submitted to the University of Nottingham for the degree of Doctor of Philosophy November 2012 i Dedication: This thesis is affectionately dedicated to my father, my mother, my wife and my children; Emil and Emily. ii ABSTRACT Gastrointestinal (GI) helminths pose a significant threat to the livestock industry and are a recognized cause of global morbidity in humans. Control relies principally on chemotherapy but in the case of nematodes is rapidly losing efficacy through widespread development and spread of resistance to conventional anthelmintics and hence the urgent need for novel classes of anthelmintics. Cysteine proteinases (CPs) from papaya latex have been shown to be effective against three murine nematodes Heligmosomoides bakeri, Protospirura muricola and Trichuris muris in vitro and in vivo and against the economically important nematode parasite of sheep Haemonchus contortus. Preliminary evidence suggests an even broader spectrum of activity with efficacy against the canine hookworm Ancylostoma ceylanicum, juvenile stages of parasitic plant nematodes of the genera Meloidogyne and Globodera and a murine cestode Hymenolepis microstoma in vitro. This project focused on tapeworms. Using 2 different rodent cestodes Hymenolepis diminuta and Hymenolepis microstoma and 1 equine cestode Anoplocephala perfoliata I have been able to show that CPs do indeed affect cestodes whether young newly hatched scoleces in vitro (by causing a significant reduction in motility leading to death of the worms) or mature adult worms in vitro (by causing a significant reduction in motility leading to death of the worms) and in vivo (resulting in a significant, but relatively small, reduction in worm burden and biomass), despite no effects on worm fecundity. Although only minimally efficacious against Hymenolepis microstoma and moderately efficacious against Hymenolepis diminuta in vivo, efficacy was enhanced by the synergistic effects of the immune system demonstrated against Hymenolepis diminuta in the non-permissive host. The results offer the possibility that with further refinement, CPs may be developed into broad spectrum anthelmintics that in addition to their marked effects on nematodes also remove any concurrently residing tapeworms. iii ACKNOWLEDGEMENTS Special thanks are due to my supervisor, Prof. Jerzy M. Behnke for all his help, support, guidance and encouragement throughout the course of this project. I would also like to thank Dr. David Buttle, Dr. Ian Duce, Dr. Ian Mellor and Dr. Hany Elsheikha for their invaluable help and advice they provided. Especial thanks are due to Ann Lowe and Tim Smith for their help and laboratory support. Special thanks to Wenceslaus Luoga for his immeasurable help and friendship. I acknowledge, with gratitude, the good friends I have made during this project at the school of Biology (Andrew Phiri, Charumathi Anbalagan, Amy Hall, Yadong Zheng, Fawzia Shawesh, Hamid Kachel and Omer Jaff). Finally, very special thanks are due to my father, my mother, my wife and my children who have supported me throughout my life through their patience, prayers and support. iv ABBREVIATIONS µm Micrometer µM Micromolar µmol Micromol ANOVA Analysis of variance CP Cysteine proteinase CPL Crude papaya latex DER Diglycidyl ether of polypropylene glycol DMAE Dimethylaminoethanol E-64 L-trans-epoxysuccinyl-leucylamido (4-guanidino)-butane ED50 Effective dose 50 ED100 Effective dose 100 ELISA Enzyme-linked immunosorbent assay EPG Eggs per gram faeces ERL 3,4 Epoxy Cyclohexyl Methyl 3,4 epoxy cyclohexyl carboxylate g gravity GI Gastrointestinal HC Hanks’ cysteine IC50 Half maximal inhibitory concentration kg kilogram Log10 logarithm base 10 mg miligram ml mililitre mmol milimol MW Molecular weight nmol nanomol NSA Nonenyl succinic anhydride p.i. Post infection PLS Papaya latex supernatant rmGLM Repeated measures general linear model SEM Scanning electron microscopy TEM Transmission electron microscopy v TABLE OF CONTENTS ABSTRACT .................................................................................................................. iii ACKNOWLEDGEMENTS .......................................................................................... iv ABBREVIATIONS ....................................................................................................... v LIST OF FIGURES ...................................................................................................... xi LIST OF TABLES ...................................................................................................... xiii CHAPTER 1 .................................................................................................................. 1 1.0 Summary ......................................................................................................... 1 1.1 Background ..................................................................................................... 1 1.1.2 Cestodes as a global problem ................................................................... 1 1.1.3 Taeniasis ................................................................................................. 2 1.1.4 Neurocysticercosis .................................................................................. 2 1.1.5 Cystic echinococcosis ............................................................................. 3 1.1.6 Alveolar echinococcosis ......................................................................... 3 1.1.7 Diphyllobothriasis ................................................................................... 3 1.1.8 Rare cases of cestode infections ............................................................. 4 1.1.9 Cestodes of veterinary importance ........................................................... 5 1.1.10 Cestodes in companion animals .............................................................. 6 1.1.11 Controlling cestode infections ..................................................................... 6 1.1.11.1 Surgical intervention ............................................................................ 6 1.1.11.2 Arthropod control ................................................................................. 7 1.1.11.3 Immunotherapy ................................................................................... 7 1.1.12 Problems associated with synthetic chemotherapy ................................. 8 1.1.13 Usefulness of plant extracts as cestocidals .............................................. 9 1.1.14 Cysteine proteinase as anthelmintics? .................................................. 10 1.1.15 Animal models used in this project ....................................................... 12 1.1.16 Cestode biology ..................................................................................... 13 1.1.17 Murine cestode life cycle ...................................................................... 13 1.1.18 Equine cestode lifecycle ....................................................................... 15 1.2 Aims and objectives ...................................................................................... 16 CHAPTER 2 ................................................................................................................ 18 2.0 Summary ....................................................................................................... 18 2.1 Enzyme preparations ..................................................................................... 18 2.1.1 Crude Papaya Latex ............................................................................... 18 vi 2.1.2 Papaya Latex Supernatant ...................................................................... 18 2.1.3 Pineapple extract .................................................................................... 19 2.1.4 Stem bromelain ...................................................................................... 19 2.1.5 Papain ..................................................................................................... 19 2.1.6 CP active-site titration ............................................................................ 20 2.1.7 Measurement of enzyme activity in the rodent GI tract ....................... 20 2.2 In vitro experiments ...................................................................................... 20 2.2.1 Animals .................................................................................................. 20 2.2.1 Parasites ................................................................................................. 21 2.2.2 H. diminuta parasite production ............................................................. 21 2.2.3 H. microstoma production ..................................................................... 21 2.2.4 Collection of A perfoliata ..................................................................... 21 2.2.5 Production of rodent cestode cysticercoids ............................................ 22 2.2.6 Artificial excystation of cysticercoids ................................................... 22 2.2.6 In vitro motility assays ........................................................................... 22 2.2.7 In vitro motility grading for adult worms .............................................. 22 2.2.8 In vitro motility grading
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