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Molecular Characterization of Wuchereria Bancrofti In MOLECULAR CHARACTERIZATION OF WUCHERERIA BANCROFTI IN MOSQUITOES OF PANGANI DISTRICT, NORTH EASTERN TANZANIA GODLISTEN MATERU A DISSERTATION SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE IN ONE HEALTH MOLECULAR BIOLOGY OF THE SOKOINE UNIVERSITY OF AGRICULTURE. MOROGORO, TANZANIA. 2015 ii ABSTRACT Wuchereria bancrofti is the most widely distributed of the three nematodes known to cause lymphatic filariasis, the other two being Brugia malayi and B. timori. The present study was carried out to investigate strains of W. bancrofti in mosquito vectors responsible for lymphatic filariasis transmission in Pangani district, north-eastern Tanzania. In addition, the vector abundance and vector infection rates were investigated. The presence of W. bancrofti in mosquitoes was determined by polymerase chain reaction (PCR) using primers NV1 and NV2 while Poolscreen2 software was used to determine W. bancrofti infection rate in mosquitoes. A total of 951 mosquitoes were collected in five randomly selected villages of Pangani district including Bweni, Madanga, Meka, Msaraza and Pangani West. Out of 951 collected mosquitoes, the majority were Culex quinquefasciatus (99.36%) followed by Anopheles gambiae (0.32%) and other Culex species (0.32%). The W. bancrofti vector infection rate in the present study was found to be 35.1%, indicating that there may be positive individuals in Pangani district. Phylogenetic analysis of Ssp 1repeat region sequence of W. bancrofti obtained in the study clustered the parasite into a distinct group compared with other W. bancrofti. In addition, the W. bancrofti sequences in Pangani district were not, 100% identical but genetically related. Further studies, using alternate typing methods, are however required for an in-depth understanding of strains that respond more slowly to drugs or strains that demonstrate greater fecundity. The information would enhance strategy development regarding the impact of mass drug administration (MDA), such as how long to run an MDA program and the optimal size of the human population treatment unit. iii DECLARATION I, Godlisten Materu, do hereby declare to the Senate of Sokoine University of Agriculture that this dissertation is my own original work done within the period of registration and that it has neither been submitted nor being concurrently submitted in any other institution. ___________________________ ___________________ Godlisten Materu Date (MSc. One Health Molecular Biology candidate) The above declaration is hereby confirmed; ___________________________ ___________________ Prof. Gerald Misinzo Date (Supervisor) iv COPYRIGHT No part of this dissertation may be reproduced, stored in any retrieval system or transmitted in any form or by any means without prior permission of the author or Sokoine University of Agriculture in that behalf. v AKNOWLEDGEMENTS My genuine appreciation goes to my supervisor Prof. Gerald Misinzo, for his constructive comments, guidance and encouragement. Without his contribution, this work would not have been in its present complete form. I certify his role and I will cherish, honour and keep in memory his kindness and mentorship. I am grateful to Tanzania Commission of Science and Technology (COSTECH) for providing the scholarship and funds for this study. I extend my sincere thanks to Pangani District Medical Officer, Dr. Yusuf Makange and Mr. Bernard Malongo for their assistance during mosquito collection. I want to also express my heartfelt gratitude and appreciation to Miss Mariam Makange for her laboratory assistance. I would like to express my sincere thanks to my employer, National Institute for Medical Research (NIMR) for granting me with a study leave to perform this study. Finally, much thanks to Emson Mshiu and my family in their generic sense for their kindness, compassion, encouragement and cooperation in all the time I have been away for studies. I will keep on praying for every individual who in one way or another smoothened the progress and accomplishment of this dissertation. vi DEDICATION This work is dedicated to all those people who have made this course a success both directly and indirectly. Special thanks to my family and friends for standing with me throughout the entire course as a sign of appreciation I would like to say, may our Almighty God bless you. vii TABLE OF CONTENTS ABSTRACT ........................................................................................................................ ii DECLARATION ............................................................................................................... iii COPYRIGHT .....................................................................................................................iv AKNOWLEDGEMENTS ................................................................................................... v DEDICATION ....................................................................................................................vi TABLE OF CONTENTS ................................................................................................. vii LIST OF TABLES .............................................................................................................. x LIST OF FIGURES ...........................................................................................................xi LIST OF APPENDICES .................................................................................................. xii LIST OF ABBREVIATIONS AND ACRONYMS ..................................................... xiii CHAPTER ONE .................................................................................................................. 1 1.0 INTRODUCTION ................................................................................................... 1 1.1 Background Information ........................................................................................... 1 1.2 Problem Statement and Justification ......................................................................... 2 1.3 Objectives .................................................................................................................. 3 1.3.1 Main objective ............................................................................................. 3 1.3.2 Specific objectives ....................................................................................... 3 CHAPTER TWO ................................................................................................................. 4 2.0 LITERATURE REVIEW ....................................................................................... 4 2.1 Lymphatic Filariasis .................................................................................................. 4 2.1.1 Aetiology ..................................................................................................... 4 2.1.2 Transmission ................................................................................................ 4 2.1.3 Pathogenesis................................................................................................. 5 2.1.4 Epidemiology ............................................................................................... 6 viii 2.1.5 Pathology and clinical symptoms ................................................................ 7 2.1.6 Diagnosis ..................................................................................................... 7 2.1.7 Control ......................................................................................................... 8 2.1.7.1 Chemotherapy ............................................................................. 8 2.1.7.2 Vector control .............................................................................. 8 2.1.8 Geographical distribution ............................................................................ 9 2.2 Wuchereria Bancrofti .............................................................................................. 12 2.2.1 Structure of W. bancrofti ........................................................................... 12 2.2.2 Genomic structure of W. bancrofti ............................................................ 12 2.2.3 Life cycle of W. bancrofti .......................................................................... 13 CHAPTER THREE .......................................................................................................... 15 3.0 MATERIALS AND METHODS .......................................................................... 15 3.1 Description of the Study Area ................................................................................. 15 3.1.1 Study design ................................................................................................... 16 3.1.2 Sample size and sampling strategy ............................................................ 16 3.1.3 Mosquito storage and identification .......................................................... 16 3.2 Laboratory Analyses ............................................................................................... 16 3.2.1 DNA extraction from mosquitoes .............................................................. 16 3.2.2 Detection of W. bancrofti using PCR ....................................................... 17 3.2.3 Determination of infection rate of W. bancrofti in mosquito vectors ........ 18 3.2.4 DNA purification and sequencing ............................................................
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