Epidemiological and Biomolecular Studies on Echinococcus granulosus in Sudan By Rihab Ali Omer Abdalla Hamid B.V.SC., M.V.SC A thesis submitted in partial fulfillment of the requirements of the degree of PhD University of Khartoum Supervisor Prof. Imad Eldain Elamin Eltahir Aradaib April, 2006 Dedication To the soul of my father who has dreamed a lot to see the day I get this title To my mother who has supported me and gave me her blessing to overcome all the difficulties I faced during my work To my sisters and brother To my husband Ayman, the one who was always behind me providing all the support encouragemen, power and strength To my son Monzir, the beautiful smile which shone and enlightened my life during the last period of my work List of contents Dedication i List of contents ii List of Tables iii List of Figures iv Acknowledgement v Summary vi Arabic summary vii Chapter One: General Introduction and Literature Review 1 1.1 Introduction 1 Objectives of the study 2 1.2 Classification 3 1.3 Life Cycle (Fig 1) 4 1.4 Distribution of cystic echinococcosis (Fig 2) 7 1.5 Cystic echinococcosis in Africa 9 1.6 Cystic echinococcosis in Sudan 10 1.7 Molecular epidemiology of cystic echinococcosis 12 1.8 Techniques of strain characterization 13 1.8.1 General morphology 14 1.8.2 Biochemical Methods 15 1.8.3 Developmental method in vivo 15 1.8.4 Developmental method in vitro 16 1.8.5 Genetic Characterization 16 1.8.5.a Sequencing of partial mitochondrial cytochrome coxidase 17 subunit 1 (cox1). (Bowles et al., 1992). 1.8.5.b Analysis of ribosomal DNA regions ITS1 by polymerase 17 chain reaction-restriction fragment length polymorphism (PCR-RFLP) (Bowles and McManus, 1993) 1.8.5.c Analysis of ribosomal DNA region ITS2 by polymerase 18 chain reaction-restriction fragment length polymorphism (PCR-RFLP) (Gasser and Chilton, 1995) 1.8.5.d A PCR system for detection of species and genotypes of 18 Echinococcus granulosus complex (Dinkel et al, 2004) 1.9 Diagnosis of Echinococcus in definitive and intermediate host 19 1.9.1 Diagnosis of Echinococcus in definitive host 19 1.9.1.a Detection of serum antibodies 19 1.9.1.b Detection of coproantigens 20 1.9.1.c Detection of copro-DNA 21 1.9.2 Diagnosis of cystic hydatidosis in intermediate host 21 1.9.2.a Serodiagnosis of hydatid disease in animals 22 1.9.3 Diagnosis of cystic hydatidosis in humans 23 1.9.3.a Imaging Techniques 23 1.9.3.b Immunodiagnosis of human hydatidosis 23 Chapter Two: General Material and Methods 27 2.1 Epidemiological survey 27 2.2 Hydatid cysts 27 2.3 Collection of hydatid cysts or cyst material from human 30 patients 2.4 Parasitological studies 30 2.5 Preservation of hydatid and adult E.granulosus samples 30 2.6 Preservation of the faecal samples 30 2.7 Preparation of hydatid cyst material from intact cysts for DNA 31 extraction 2.8 DNA extraction 31 2.9 DNA extraction from the faecal samples 32 2.10 Determination of the DNA concentration in samples 33 2.11 Polymerase Chain Reactions 33 2.11.1 Cestode specific PCR (cs PCR) 33 2.11.2 PCR assay specific for E.granulosus G1 (g1 PCR) 33 2.11.3 PCR assay specific for E.granulosus G6/7 and E. ortleppi 34 (g5/6/7 PCR, g6/7 PCR, g5 PCR 2.11.4 Semi nested PCRs specific for E. ortleppi and E. granulosus 34 (G6/7) 2.12 Detection of the PCR products (Agarose gel electrophoresis) 35 2.12.1 Preparation of the gel 35 2.12.2 Preparation of the sample and running of the gel 35 2.12.3 Visualization of the PCR products 35 2.13 Mitochondrial gene sequencing 34 2.14 Used Chemicals 36 2.15 Puffers and solutions 36 2.16 Enzymes 37 2.17 Oligonucleotides 37 2.18 2/7 Disposables 38 2.19 2/8 Instruments 38 Chapter Three : Epidemiological and Biomolecular Study of Cystic 40 Echinococcosis in Humans and Animals in Sudan Abstract 40 Introduction 41 Materials and Methods 43 1. Epidemiological survey Epidemiological survey 43 2. Parasitological studies 43 3. DNA extraction 44 4. Polymerase chain reaction (PCR ) and molecular 44 characterization 5. Visualization of the PCR products 45 6. Mitochondrial gene sequencing 45 Results 46 1. Epidemiological Survey 46 2. Strain characterization 47 Discussion 60 Chapter Four: Strain Characterization and Coprodiagnostic PCR of 63 E.granulosus in Central Sudan Abstract 63 Introduction 64 Materials and Methods 66 1. Stray dogs 66 2. Scraping 66 3. Preservation of the faecal samples and harvested worms 66 4. DNA extraction from E.granulosus adult worms 67 5. DNA extraction from the faecal samples 67 6. Polymerase Chain Reactions (PCRs) 68 7. Visualization of the PCR products 69 8. Control for inhibition 69 9. Mitochondrial gene sequencing 69 Results 71 1. Necropsy 71 2. PCR of the E.granulosus worms 71 3. Copro PCR 71 Discussion 79 Chapter Five: Experimental Infection of Dogs with Protoscoleces 81 of Camel Origin and Molecular Characterization of the Maturing Adult Worms Using PCR Abstract 81 Introduction 83 Materials and Methods 85 1. Experimental Animals 85 2. Infective Materials 85 3. Parasitological Parameters 85 4. Necropsy 86 5. Molecular characterization of the adult worms 86 Results 88 Parasitological findings 88 Necropsy 88 Molecular Characterization of the adult worms. 88 Discussion 92 Chapter Six : General Discussion 94 References 102 List of Tables Chapter one Table 1 : Species and strains of Echinococcosis complex 26 Chapter two Table 2 28 Numbers of examined camels, cattle, sheep and goats in different study areas Chapter three Table 1: Prevalence of cystic echinococcosis in livestock in 48 Central Sudan Table 2: Prevalence of cystic echinococcosis in livestock in 50 Western Sudan Table 3 52 Prevalence of cystic echinococcosis in livestock in Southern Sudan Table 4 55 Strain characterization of hydatid cyst samples from camel, cattle, sheep, and goats from different parts of Sudan Chapter four Table 1 72 Necropsy and Copro-PCR results of the 42 dogs shot in central Sudan Chapter five Table 1 89 Results of the experimental infection of dogs in groups A and B with protoscolices of camel origin List of Figures Chapter one Figure 1: Life cycle of Echinococcus granulosus 6 Figure 2: World Distribution of cystic echinococcosis 8 Chapter two Figure 1: Study area 29 Chapter three Figures 1 and 2: Prevalence and fertility of hydatid cysts from camel, cattle, 49 sheep and goats in central Sudan Figures 3 and 4: Prevalence and fertility of hydatid cysts from camel, cattle, 51 sheep and goats in Western Sudan Figures 5 and 6: Prevalence and fertility of hydatid cysts from camel, cattle, 53 sheep and goats in southern Sudan Figure 7: Predilection sites of hydatid cysts in camel, cattle, sheep and goats in 54 different parts of Sudan Figure 8: Visualization of the first amplified 254 bp PCR product from 56 differnt cyst samples. Figure 9: Semicamel PCR amplification of the 171 bp specific PCR product of 57 the camel (G6) strain from the first 254 bp PCR product for the above gel. Figure 10: Semicattle PCR amplification of the 171 bp specific PCR product 58 of the cattle (E.ortleppi) strain from the first 254 bp PCR product for the G5/6/7 PCR. Figure 11: Visualization of the 254 bp G1 (sheep strain) PCR product from 59 differnt cyst samples . Chapter four Figure 1: Visualization of the first amplified 254 bp PCR product from 73 E.granulosus worms. Figure 2: Semicamel PCR amplification of the 171 bp specific PCR products 74 of the camel (G6) strain from the first 254bp Figure 3: Visualization of the first amplified 373bp PCR product from copro 75 DNA Figure 4: Visualization of the 254bp G5/6/7 nested PCR from the first cestode 76 1 specific P3CR 4 Figure 5: Semicamel PCR amplification of the 171bp specific PCR products 77 of the camel (G6) str a5in fro m6 th e7 G5/6 /78 PCR 9 10 11 12 NC PC Figure 6: Visualization of the amplified 254 bp G1 (sheep strain) PCR 78 product from copro DNA. Chapter five Figure 1: Visualization of the first amplified 254 bp PCR products from 90 differnt worm samples. Figure 2, Seminested PCR amplification of the 171 bp specific PCR product 87 of the camel (G6) strain from the first 254 bp PCR product for the above gel. Acknowledgement First, many thanks to Allah, my God who insert the desire of work in my sides, and give me the support to work and power to continue. Many thanks to Prof. Imad Aradaib, my supervisor who helped in designing the research protocol and looked after me with their kind supervision and advices. Thanks to the members of the Department of Parasitology, University of Hohenehim, Germany for helping me during the course of the study and for making my stay in Germany easy and enjoyable. Special thanks are due to Prof Ute Mackenstedt for her valuable supervision as well as official and personal helps. Many thank Dr. Thomas Romig for helping me in planning my work, and for his valuable advice and suggestions and for revising the manuscript. Special thanks to Dr. Anke Dinkel for her supervision and for helping me in performing the molecular biology techniques. May thanks are also due to Dr. Michael Merli for his useful guidance during my work in Hohenheim. May thanks are also extended to Dr. Mohamed Elamin and International Red Cross Commission (ICRC), Khartoum for providing me with the human samples My thanks are also due the family of the Central Veterinary Research laboratories, Soba, specially Prof. Mohamed Salih Algablabi, and Prof. Salah Mukhtar. Special thanks to the staff of Parasitology, Soba, specially Mr. Nasir, Khalil, Ahmed Elshik for technical help. Thanks to my colleges, Dr. Osman Mukhtar, Dr. Yahia Hassan Beik, Dr. Wegdan Hassan Ali and Dr. Selma Osman. and all other friends in the Central Veterinary Research Laboratories (CVRL), Soba. Thanks are also extended to the German Academic Exchange Service (DAAD) for financial support of this work and for being a real family during my stay in Germany. I also acknowledge the important support offered from my mother, sisters and brother. My thanks are also due to my husband Dr. Ayman Elnahas for being very helpful during the work and also for support and encouragement. Thanks Monzir for sharing this time with me.