FACULTY OF SCIENCE UNIVERSITY OF COPENHAGEN Master Thesis Mohamed Bayoumi Fahmy Hawash – TQS650 Population genetic and phylogenetic studies on Trichuris spp. recovered from pigs, humans and baboons in different geographical regions Supervisor: Peter Nejsum Section of Parasitology and Aquatic diseases Department of Veterinary Disease Biology Submitted : 31/08/2014 Copenhagen University Dyrlægevej 100, 1871 Frederiksberg C, I Denmark Mohamed Bayoumi Fahmy Hawash ــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ Cover Photo from: www.tvblogs.nationalgeographic.com/blog/big-baboon-house/ II Contents CONTENTS ................................................................................................................... I SUMMARY ................................................................................................................. III PREFACE ..................................................................................................................... V ACKNOWLEDGMENT............................................................................................ VI BACKGROUND ........................................................................................................... 1 Parasitology ............................................................................................................................. 1 Phylogeny ........................................................................................................................................................................ 1 Trichuris spp. and their hosts ........................................................................................................................................ 1 Morphology .................................................................................................................................................................... 1 Life Cycle ........................................................................................................................................................................ 2 Pathology and clinical symptoms .................................................................................................................................. 3 Epidemiology of T. suis and T. trichiura....................................................................................................................... 4 Economical and public health importance of T. trichiura and T. suis ....................................................................... 5 Molecular Biology ................................................................................................................... 5 Phylogenetics and Population Genetics. ..................................................................................... 5 1. DNA marker ......................................................................................................................................................... 7 Nuclear DNA markers .............................................................................................................................................. 8 Mitochondrial DNA (mtDNA) markers .................................................................................................................. 8 2. Molecular Biological Methodologies ................................................................................................................... 9 Polymerase Chain Reaction (PCR) and PCR linked techniques ........................................................................... 9 3. Computational analysis ........................................................................................................................................ 9 3.1. Phylogenetic Tree ........................................................................................................................................ 9 3.1.1. Distance based Trees .............................................................................................................................. 9 Neighbor Joining (NJ) ................................................................................................................................... 10 3.1.2. Character based Trees ......................................................................................................................... 10 Maximum Parsimony (MP) .......................................................................................................................... 10 Maximum Likelihood (ML) .......................................................................................................................... 11 Bayesian Analysis........................................................................................................................................... 11 3.1.3. Bootstrapping ....................................................................................................................................... 12 3.2. Phylogenetic network ................................................................................................................................ 12 3.3. Coalescent analysis.................................................................................................................................... 12 Next generation sequencing (NGS) technologies and parasite genomics ......................... 13 Parasite genomes .......................................................................................................................................................... 14 Parasite mitochondrial genome .................................................................................................................................. 14 NGS workflow for mitochondrial genome ................................................................................................................. 15 THESIS'S STUDIES INTRODUCTION .................................................................. 17 Brief introduction and major findings ................................................................................ 17 I Limitations ............................................................................................................................. 19 Conclusions and Perspectives .............................................................................................. 20 REFERNECES ............................................................................................................ 21 MANUSCRIPT 1 ......................................................................................................... 27 SUPPLEMENTARY DATA OF MANUSCRIPT 1 ................................................ 53 MANUSCRIPT 2 ......................................................................................................... 57 SUPPLEMENTARY DATA OF MANUSCRIPT 2 ................................................ 80 APPENDIX OF RAW DATA .................................................................................... 85 II Summary Trichuris suis and T. trichiura are two different whipworms that infect pigs and humans respectively. Trichuris suis is found in pigs in almost all over the world while T. trichiura is found in humans mainly in areas with poor sanitation and is responsible for nearly 460 million infections. The taxonomic status of whipworms in non-human primates is unsettled but they are normally designated as T. trichiura as well. The pattern and nature of transmission of Trichuris spp. infecting human, non human primates and pigs is poorly understood. Population genetics can provide inferences about the direction of parasite transmission on different levels (between individuals, populations and geographical locations) beside the natural and demographic history of the parasite. In the first study, I applied different population genetic and phylogenetic approaches to investigate the pattern of transmission and the demographic history of Truchuris spp. in pigs, humans and baboons. I tested a hypothesis regarding the transmission and history of Trichuris spp. between humans and pigs which is the Trichuris sp. was found in either humans or pigs before it was transmitted to the other host in the Neolithic era upon domestication of animals when the two hosts were in sympatry nearly 10,000 years ago. Moreover I investigated the evolutionary and phylogenetic relationship of the Trichuris infecting humans and a non human primate (baboon). Coalescent analysis, Analysis of Molecular Variance (AMOVA) and phylogenetic analysis were applied on two mitochondrial markers namely the nad1 and rrnL genes on T. suis populations from domestic pigs from Denmark, USA, Uganda and China; T. trichiura from humans from China and Uganda; Trichuris from olive and yellow baboons from Denmark (Copenhagen Zoo and Knuttenborg Park) and from USA (Southwest National Primate Research Center (SNPRC), Texas). The demographic history reconstructed through coalescent analysis of the different populations of Trichuris spp. from pigs and humans revealed independent demographic history of the parasite in the two hosts indicating that the speciation events happened much older than the Neolithic era 10,000 years ago and hence the hypothesis was rejected. Moreover, the demographic history of T. suis in different areas underlined the major role of the human trading activity in transmitting the parasite through pig transport. No genetic differentiation between Trichuris obtained from baboons and humans in Uganda emphasizing on the possible zoonotic