Prevalence, Risk Factors and Genetic Diversity of Equine Piroplasmosis in Kelantan, Malaysia
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PREVALENCE, RISK FACTORS AND GENETIC DIVERSITY OF EQUINE PIROPLASMOSIS IN KELANTAN, MALAYSIA QAES TALB SHUKUR ALSARHAN DOCTOR OF PHILOSOPHY 2017 Prevalence, Risk Factors and Genetic Diversity of Equine Piroplasmosis in Kelantan, Malaysia by QAES TALB SHUKUR ALSARHAN A thesis submitted in fulfillment of the requirements for the degree of Doctor of Philosophy Faculty of Veterinary Medicine UNIVERSITI MALAYSIA KELANTAN 2017 THESIS DECLARATION I hereby certify that the work embodied in this thesis is the result of the original research and has not been submitted for a higher degree to any other University or Institution. I agree that my thesis is to be made immediately available OPEN ACCESS as hardcopy or on-line open access (full text). EMBARGOES I agree that my thesis is to be made available as hardcopy or on-line (full text) for a period approved by the Post Graduate Committee. Dated from ___________ until ___________ (Contains confidential information under the office CONFIDENTIAL Official Secret Act 1972)* (Contains restricted information as specified by the RESTRICTED organization where research was done) * I acknowledge that Universiti Malaysia Kelantan reserves the right as follows. 1. The thesis is the property of Universiti Malaysia Kelantan. 2. The library of Universiti Malaysia Kelantan has the right to make copies for the purpose of research only. 3. The library has the right to make copies of the thesis for academic exchange. _____________________ __________________________ SIGNATURE SIGNATURE OF SUPERVISOR _______________________ _________________________ IC/ PASSPORT NO. NAME OF SUPERVISOR Date: Date: i ACKNOWLEDGMENT First of all, my thanks are to my God. I would like to express my sincere thanks to my supervisor, Associated Professor Dr. Mohd Mokhtar Arshad for his advice, patience and enthusiasm throughout the years of work. I would also like to express my gratitude and thanks to my co-supervisors, Professor Dr. Imad Ibrahim Al-sultan, Professor Dr. Mohd Azam Khan Goriman Khan, Universiti Malaysia Kelantan (UMK) and Dr. Azlinda Abu Bakar, Universiti Sanis Malaysia (USM), for helpful discussions and comments on my thesis. I would also like to extend my gratitude and appreciation to Dr. Maizan Mohammed for helping and advising me on molecular work. I am indebted to the Faculty of Veterinary Medicine, UMK, for making this possible by providing all necessary chemicals and equipment in the laboratory. I am deeply and extremely grateful to all of the UMK laboratory assistants, especially to Mr. Nor Faizull, Mr. Badrul Hisham, Mrs Eizzati, and Miss Nani Izreen for their support in so many ways. I would also like to thank the UMK veterinary clinic staff, especially Dr. Mimi, Mr. Hamid, and Mr. Nizam for their help in the sample collections. Special acknowledgment goest to my friend, Dr. Omer Khazaal, Dr. Ali Saeed, Dr. Ahmad Mahmood, Dr. Maher Mohammed for their direct and indirect assistance during my PhD study. Qaes ii TABLE OF CONTENTS PAGE THESIS DECLARATION i ACKNOWLEDGEMENTS ii TABLE OF CONTENTS iii LIST OF TABLES xi LIST OF FIGURES xv LIST OF ABBREIVATIONS xx LIST OF SYMBOLS xxiii LIST OF EQUATIONS xxiv ABSTRAK xxv ABSTRACT xxvi CHAPTER 1 INTRODUCTION 1.1 General introduction 1 1.2 Problem statement 5 1.3 Research questions 6 1.4 Hypothesis 6 1.5 The objectives of study 7 CHAPTER 2 LITERATURE REVIEW 2.1 History of equine piroplasmosis (EP) 8 2.2 Etiological agents and taxonomy 10 iii 2.2.1 Theileria equi 12 2.2.1.1 Morphology 12 2.2.1.2 Life cycle 14 2.2.2 Babesia caballi 17 2.2.2.1 Morphology 17 2.2.2.2 Life cycle 18 2.3 The genes commonly targeted in the T. equi and B. caballi 21 2.4 Sequencing and genetic diversity for T. equi and B. caballi 22 2.5 Epidemiology of equine piroplasmosis 25 2.5.1 Geographic distribution 25 2.5.2 Susceptibility to the disease 33 2.5.2.1 The susceptibility related to equids factors 33 2.5.2.2 The susceptibility related to environmental and stables 36 factors 2.6 Transmission of the causative agents 38 2.6.1 Biological transmission 38 2.6.2 Iatrogenic or mechanical transmission 40 2.6.3 Intrauterine or transplacental transmission 41 2.7 The tick vectors for equine piroplasms infections 42 2.7.1 Taxonomy of Ixodid ticks 46 2.7.2 Morphology of Ixodid ticks 47 2.7.2.1 Tick family identification 48 2.7.2.2 Tick genus identification 52 iv 2.7.2.3 Tick species identification 53 2.7.3 Tick life cycle 53 2.7.4 Overview of the identified Ixodid ticks genera in this study 55 2.7.4.1 Genus Rhipicephalus Koch, 1844 (Soulsby, 1982) 55 2.7.4.2 Genus Haemaphysalis Koch, 1844 (Soulsby, 1982) 58 2.7.4.3 Genus Dermacentor Koch, 1844 (Soulsby, 1982) 59 2.8 Ixodid ticks in Malaysia 60 2.9 Pathogenesis of equine piroplasmosis 62 2.10 Clinical signs of equine piroplasmosis 66 2.11 Clinical pathology of equine piroplasmosis 69 2.11.1 Changes in hematological parameters 69 2.11.2 Changes in serum biochemistry parameters 73 2.11.2.1 Aspartate aminotransferase (AST) 74 2.11.2.2 Alanine aminotransferase (ALT) 74 2.11.2.3 Alkaline phosphatase (ALKP) 75 2.11.2.4 Total bilirubin 75 2.11.2.5 Total protein 76 2.11.2.6 Blood urea nitrogen (BUN) 76 2.11.2.7 Calcium 77 2.11.2.8 Glucose 78 2.11.2.9 Phosphorus 78 2.11.2.10 Creatinine 79 2.11.3 Pathological changes of equine piroplasmosis 79 v 2.11.3.1 Macroscopic findings 80 2.11.3.2 Microscopic findings 80 2.12 Immunity to equine piroplasmosis 81 2.13 Public health of significance of equine piroplasmosis 85 2.14 Diagnosis of equine piroplasmosis 86 2.14.1 Overview of the methods used for diagnosis EP in this study 87 2.14.1.1 Microscopic examination of stained blood smears 87 2.14.1.2 Competitive enzyme linked immunosorbent assay 89 2.14.1.3 Conventional and multiplex polymerase chain reaction 91 2.14.2 Other methods for diagnosis of equine piroplasmosis 94 2.14.2.1 Biological tests 94 2.14.2.2 In vitro culture technique 95 2.14.2.3 Other serological tests 96 2.14.2.4 Other molecular techniques 97 2.15 Differential Diagnosis of equine piroplasmosis 99 2.16 Prognosis of equine piroplasmosis 99 2.17 Treatment of equine piroplasmosis 100 2.18 Control of of equine piroplasmosis 103 2.18.1 Vaccination 103 2.18.2 Control of ticks 104 CHAPTER 3 DIAGNOSIS, PREVALENCE, RISK FACTORS AND VECTOR OF EQUINE PIROPLASMOSIS IN EQUIDS IN KELANTAN 3.1 Introduction 105 vi 3.2 Materials and Methods 108 3.2.1 Study area 108 3.2.2 Determining the number of equids 109 3.2.3 Animals and sample collection 109 3.2.4 Epidemiological data collection 110 3.2.5 Climatic data 113 3.2.6 Laboratory analysis 113 3.2.6.1 Microscopic examination of blood smears 113 3.2.6.2 Competitive enzyme linked immunosorbent assay 115 3.2.6.3 Polymerase chain reaction techniques 120 3.2.6.3.1 DNA extraction from equids blood 120 3.2.6.3.2 Determination of DNA concentration and purity 123 3.2.6.3.3 PCR amplification of piroplasms DNA from equids 124 blood samples 3.2.7 Evaluation the efficiency of different methods for diagnosis 129 the disease 3.2.8 Statistical analysis 130 3.3 Results 131 3.3.1 Morphological and biometerical finding with parasitemia 131 3.3.2 Prevalence of EP by different tests 135 3.3.3 Evaluation of cELISA and multiplex PCR for detecting T. equi and B. caballi infections 137 3.3.4 Prevalence of T. equi, B. caballi and both protozoa infections 143 by regions vii 3.3.5 Risk factors associated with seroprevalence of EP causative 150 agents 3.3.6 Ixodid ticks: identification and infestation rate 163 3.4 Discussion 171 3.4.1 Morphological and biometerical finding with parasitemia 172 3.4.2 Determing the prevalence of EP infections using different 173 methods 3.4.3 Evaluating of cELISA and multiplex PCR for detecting T. equi 177 and B. caballi infections 3.4.4 Equids factors associated with T. equi, B. caballi and both 179 protozoa 3.4.5 Stables factors associated with T. equi, B. caballi and both 182 protozoa 3.4.6 Climatic factors associated with T. equi, B. caballi and both 185 protozoa 3.4.7 Ixodid ticks: identification and infestation rate 186 3.5 Conclusions 189 CHAPTER 4 EVALUATION OF HEMATOLOGY, BIOCHEMISTRY AND ANTIBODY TITER BETWEEN EQUIDS CLINICALLY AND SUBCLINICALLY INFECTED WITH EQUINE PIROPLASMOSIS 4.1 Introduction 192 4.2 Materials and methods 194 4.2.1 Animals and study area 194 4.2.2 Recording clinical sings 194 4.2.3 Fecal samples collection 194 4.2.4 Determination of antibody titers against T. equi and B. caballi 195 4.2.5 Hematological analysis 195 viii 4.2.6 Serum biochemistry analysis 196 4.2.7 Statistical analysis 197 4.3 Results 198 4.3.1 Seroprevalence of clinical and subclinical forms in equids 198 with the relative risk 4.3.2 Level of antibodies titration against piroplasms in equids with 198 clinical and subclinical forms of equine piroplasmosis 4.3.3 Hematological and serum biochemistry parameters in equids 199 with clinical and subclinical form of equine piroplasmosis 4.4 Discussion 206 4.5 Conclusions 212 CHAPTER 5 GENETIC DIVERSITY AND PHYLOGENIC ANALYSES OF THEILERIA EQUI AND BABESIA CABALLI DETECTED IN EQUIDS AND IXODID TICKS IN KELANTAN 5.1 Introduction 214 5.2 Materials and Methods 217 5.2.1 Ticks collection from equids 217 5.2.2 DNA extraction from Ixodid ticks 217 5.2.3 PCR amplification of piroplasms DNA extracted Ixodid ticks 219 5.2.4 DNA sequencing and phylogeny analyses 219 5.2.5 Statistical analyses 221 5.3 Results 221 5.3.1 Detection rate of T.