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Molecular Study of Some Blood Parasites in Camels in Al-Diwaniyah Province

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Molecular Study of Some Blood Parasites in Camels in Al-Diwaniyah Province Republic of Iraq Ministry of Higher Education & Scientific Research University of Al-Qadissiyiah College of Veterinary Medicine Molecular study of some blood parasites in Camels in Al-Diwaniyah province [ A Thesis Submitted to the Council of the College of Veterinary Medicine, University of Al-Qadissiyiah in Partial Fulfillment of the Requirements for the Degree of Master of Science in Veterinary Medicine / Parasitology By Zainab Hussein Chyad Al-Naily B.V.M.S. 2008 D.V.M.&S. 2014 Supervised by Prof. Dr. Ghaidaa Abbas Jasim 2018 AD September 1440 AH Muharram بســــم هللا الرحــمن الرحــيم ْ )وق ل ر ب زدني علما ( صدق هللا العلي العظيم )سورة طه اﻵية 114( Certification I certify that this thesis entitled “Molecular study of some blood parasites in camels in Al-Diwaniyah province” was prepared under my supervision at the College of Veterinary Medicine/Al-Qadissiyah University in partial fulfillment of the requirements for the degree of Master Science / Parasitology. Prof. Dr. Ghaidaa Abbas Jasim (advisor) / / 2018 The recommendation of the Department in the view of the available recommendation, I forward this thesis for debate by the examining committee. Prof. Dr. Alaa Abdul Aziz Abed Assis Dean For Scientific Affairs and Postgraduate Studies/ College of Veterinary Medicine/ Al-Qadisiyah University / / 2018 Certification of Examination Committee We, the examining committee, after reading this thesis entitled (Molecular study of some blood parasites in Camels in Al-Diwaniyah province ) and examining the Student Zainab Hussein Chyad Al-Naily in its contents, find it adequate as thesis, for the Degree of Master Science / Parasitology. Prof. Dr. Abdulsadah A. Rahi Al-Ghnaimawi Chairman Assist Prof. Assist Prof. Dr. Ahmed Jasim Neamah Dr. Azhar Chafat Karawan Member Member Prof. Dr. Ghaidaa Abbas Jasim Member and Supervisor Approved by the council of the College of Veterinary Medicine University of Al- Qadisiyah . Prof . Assist Prof. Dr. Jabbar A. A. AL-Sa'aidi Dr. Monyer Abdluameir Alftlawi Dean of the College Head of Department of Microbiology Date of examination 27 / 9 / 2018 Dedication To the great Prophet and his family infallible. To the spring tenderness and perfume My mother To my lover who made my life happy , accompanied me and giving me a helping hand My husband . To my dear and my strength in my life My brothers and sisters . To my big home / Iraq I dedicate this work. Zainab Acknowledgement Firstly I would like to thank the kind merciful ALLAH , the messenger of ALLAH MUHAMMED and AHL AL-BAIT for helping me in completion this work. I am deeply appreciated to my supervisors Professor Dr. Ghaidaa Abbas Jasim for her following the work step by step for advises , the guidance ,assistance and the long patience throughout the study , and the thank worthy spending time and knowledge for the completion of this study. I would like to express my great thank to Prof. Dr. Ahmed Jasim Neamah for help me in molecular practical and completion of this study. I would like to express my great thank to the staff of the Department of Veterinary Medicine/ Al- Qadissiya University. I would like to thank Assist. Prof. Ali Mohammed Ghazi, Dr. Kazem Hassan Dr. Mataa Ghaleb Atshan work in slaughterhouse , Essam Al Zayadi and Riyadh Ali for their help me in collection of samples and Dr.Haydar Kamel help me in my practical steps Finally the end I would like express appreciation to my beloved husband (Akeel) who helped me and was always my support. Zainab رقم اﻻيداع في دار الكتب والوثائق الوطنية لسنة List of contents No. Tittle Page Chapter one Introduction 1 1 2 Chapter two Literature review 4 2-1 The Trypanosoma spp. 4 2-1-1 Classification 4 2-1-2 Morphology 5 2-1-3 Modes of transmission 6 2-1-4 Life cycle 7 2-1-5 Clinical finding and Pathogenesis 8 2-1-6 Diagnosis 9 2-1-6-1 parasitological diagnosis 9 2-1-6-2 Serological diagnosis 10 2-1-6-3 Molecular diagnosis 11 2-1-7 Immune response 12 2-1-8 control 14 2-2 Babesia spp 15 2-2-1 Classification 16 2-2-2 Morphology 16 2-2-3 Modes of transmission 17 2-2-4 Life cycle 18 2-2-5 Clinical finding and pathogenesis 19 2-2-6 Diagnosis 21 2-2-6-1 Parasitological diagnosis 21 2-2-6-2 Serological diagnosis 21 2-2-6-3 Molecular diagnosis 22 2-2-7 Immune response 23 2-2-8 Control 24 2-3 Theileria spp. 25 2-3-1 Classification 26 2-3-2 Morphology 26 2-3-3 Modes of transmission 27 2-3-4 Life cycle 27 2-3-5 Clinical finding and Pathogenesis 28 2-3-6 Diagnosis 29 2-3-6-1 parasitological diagnosis 29 2-3-6-2 Serological diagnosis 30 2-3-6-3 Molecular diagnosis 30 2-3-7 Immune response 31 2-3-8 control 31 2-4 Anaplasma spp 32 2-4-1 Classification 32 2-4-2 Morphology 33 2-4-3 Modes of transmission 33 2-4-4 Life cycle 34 2-4-5 Clinical finding and pathogenesis 35 2-4-6 Diagnosis 36 2-4-6-1 Parasitological diagnosis 37 2-4-6-2 Serological diagnosis 37 2-4-6-3 Molecular diagnosis 38 2-4-7 Immune response 38 2-4-8 Control 39 Chapter three 3 40 Materials and Methods 3-1 Materials 40 3-1-1 Equipment and instruments 40 3-1-2 Chemical and Reagents 41 3-1-3 Molecular identification 42 3-1-4 Primers 42 3-2 The methods 44 3-2-1 Blood samples collection 44 3-2-2 Microscopic examination 44 3-2-3 Polymerase Chain Reaction 44 3-2-3-1 Genomic DNA Extraction 45 3-2-3-2 Genome DNA profile 46 3-2-3-3 Agarose gel electrophoresis 46 A Casting of the Agarose Gel 46 B Loading and Running DNA in Gel Agarose 47 3-2-3-4 Protocol 47 3-2-3-5 PCR Thermo cycler Conditions 48 3-2-3-6 PCR product analysis 49 3-2-4 DNA sequence 49 3-2-4-1 DNA sequencing Analysis 49 3-2-4-2 Nucleotide sequences analysis and gene bank 49 submissions 3-2-5 Phylogenetic analyses 50 3-2-6 Statistical analysis 50 Chapter Four 4 51 Results and Discussion 4-1-1 Trypanosoma spp 51 4-1-2 Babesia spp 54 4-1-3 Theileria spp 57 4-1-4 Anaplasma spp 60 4-1-5 Mix infection 63 4-1-6 The prevalence of blood parasites according to the 64 months of study 4-2 Result of Molecular Diagnosis by using Polymerase 65 Chain Reaction (PCR) technique 4-2-1 Diagnosis Trypanosoma spp by PCR 66 4-2-2 Diagnosis Babesia spp by PCR 68 4-2-3 Diagnosis Theileria spp by PCR 70 4-2-4 Diagnosis Anaplasma spp by PCR 71 4-3 Results of DNA Sequencing 72 4-4- Result of phylogenic analysis 73 4-4-1 The phylogenetic tree of Trypanosoma spp 74 4-4-2 The phylogenetic tree of Theileria spp 75 4-4-3 The phylogenetic tree of Anaplasma spp 76 Chapter five 5 77 Conclusions and Recommendations 5-1 Conclusions 77 5-2 Recommendations 78 6 References 79 List of Tables No. Title Page 3-1 Instruments and Equipment 40 3-2 Chemicals and Reagents 41 3-3 Chemicals and reagents for DNA extraction and 42 polymerase chain reaction ( Kits) 3-4 Primers 43 3-5 Protocol of monoplex PCR reaction mixture volume 47 3-6 Protocol of Multiplex PCR reaction mixture volume 48 3-7 PCR amplification program 48 4-1 show the infection camels with some blood parasites 51 4-2 The prevalence of Trypanosoma infection in camel 52 according to the microscopic examination and sex 4-3 The prevalence of Trypanosome infection in camel 53 according to the microscopic examination and age 4-4 The prevalence of Babesia infection in camel according 55 to the microscopic examination and sex 4-5 The prevalence of Babesia infection in camel according 56 to the microscopic examination and age 4-6 The prevalence of Theileria spp. infection in camel 58 according to the microscopic examination and sex 4-7 The prevalence of Theileria spp. infection in camel 59 according to the microscopic examination and sex 4-8 The prevalence of Anaplasma spp. infection in camel 61 according to the microscopic examination and sex 4-9 - The prevalence of Anaplasma spp. infection in camel 62 according to the microscopic examination and age 4-10 show mix infection 63 4-11 show the prevalence of some blood parasites according to 65 the months of study 4-12 show the diagnosis blood parasites by PCR 66 4-13 Summarized the sequencing of blood parasites by NCBI- 73 Blast List of Figures No. Title Page 2-1 Trypanosoma spp life cycle 7 2-2 Babesia spp life cycle 19 2-3 Theileria spp life cycle 28 2-4 Anaplasma spp life cycle 35 4-1 Show Trypanosoma spp between camel red blood cells 51 4-2 Show Babesia spp inside a camel red blood cells 55 4-3 Show Theileria spp. inside a camel red blood cells 57 4-4 Show Anaplasma spp. inside camel red blood 60 4-5 show mix infection 63 4-6 Agarose gel electrophoresis image Trypanosoma spp. 66 4-7 Agarose gel electrophoresis image T.evansi 67 4-8 Agarose gel electrophoresis image Babesia bovis 68 4-9 Agarose gel electrophoresis image Babesia begimna 69 4-10 Agarose gel electrophoresis image Theileria spp and T 70 annulata 4-11 Agarose gel electrophoresis image A .marginale 71 4-12 Agarose gel electrophoresis image A.
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