IDENTIFICATION AND MOLECULAR CHARACTERISATION OF MALARIA PARASITES OF MACAQUES IN KAPIT, SARAWAK PAUL CLIFF SIMON DIVIS

Transcription

1 IDENTIFICATION AND MOLECULAR CHARACTERISATION OF MALARIA PARASITES OF MACAQUES IN KAPIT, SARAWAK PAUL CLIFF SIMON DIVIS A thesis submitted in fulfillment of the requirements for the degree of Master of Science Faculty of Medicine and Health Sciences UNIVERSITI MALAYSIA SARAWAK 2007

2 ACKNOWLEDGEMENT The writing of this thesis has been one of the most significant academic challenges I have ever had to face. This would not have been possible without the blessings of the Heavenly Father. I would like to acknowledge my appreciation to the following individuals for my success. First and foremost, my supervisor, Professor Dr. Balbir Singh who has been graciously helpful, and has assisted me in numerous ways. I was driven and inspired by his wisdom, knowledge and commitment towards assisting me with the thesis. Also, Professor Dr. Janet Cox-Singh, my co-supervisor, for giving me guidance, advice, suggestions and encouragement in various ways. I would also like to extend my appreciation to my dearest laboratory mates: Sunita Sara Gill Shamsul, David Lee Kim Sung, Cynthia Patricia Nicholas, Peter Yee Han Chung, Lau Hui Chong, Grace Siaw Ee Lu, Angela Siner, Wilson Tan Cheong Huat, Siti Khatijah Zakaria, Roynston Albert, Wu Chen Wei, Boon Siaw Shi, Cyrus Daneshvar, Sophia Lau Tick Ying and Monica Yau Swee Eng, including those who did their attachment in the laboratory even for a few weeks. Thank you for sharing your knowledge, joy and tears. I would also like to thank Universiti Malaysia Sarawak (UNIMAS) for the Student Fellowship, which has supported me during my two years of research. I acknowledge UNIMAS [Short Term Grant numbers: 01(82)/422/2004(159) and 01(129)/518/2005(17)] and the Wellcome Trust (grant number L18403/F05/00/078538/2/05/07) of the United 11

3 Kingdom for their financial support for this project. I also would like to thank Dr. Alan Thomas of the Biomedical Primate Research Centre, Rijswijk, The Netherlands for providing DNA samples of monkey malarias. I would like to acknowledge the permission to access and collect macaque blood samples and conduct research from Sarawak Biodiversity Centre (permit no. SBC-RP-0081(i)-BS) and Sarawak Forestry Department (permit no. NPW (1)-98). Last but not least, my family members, especially my parents for supporting and encouraging me to pursue this degree. Without their encouragement, this degree would not have been realised. It is to them that I dedicate this work. May the Almighty God bless all of us and may His love always pour onto us wherever we go, forever and ever. Amen. iii

4 TABLE OF CONTENTS ACKNOWLEDGEMENTS TABLE OF CONTENTS LIST OF FIGURES LIST OF TABLES ABBREVIATIONS ABSTRACT ABSTRAK 11 iv vii ix X11 X111 xv Chapter One: Introduction 1.1 Introduction to malaria Life cycle of the malaria parasites Non-human Detection primate of malaria malaria parasites parasites Microscopic analysis Polymerase chain reaction (PCR) based assays The SSU rrna genes of Plasmodium species The circumsporozoite protein (csp) genes of Plasmodium species Objectives of this study 21 Chapter Two: Development of species-specific PCR primers for the identification of non-human primate Plasmodium species 2.1 Introduction Materials and methods Plasmodium DNA template Amplification of the SSU rrna genes Cloning of PCR products Extraction of plasmid DNA EcoR I digestion Sequencing of the SSU rrna genes DNA sequence and phylogenetic analysis Designing of PCR primers Determination of optimum annealing temperatures (T, ) and 32 sensitivity of detection Specificity of primers Preparation of 2.5% Nusieve 3: 1 agarose Results The isoforms of SSU rrna genes of P. coatneyi, P. fieldi and P. inui Phylogenetic analysis Development of species-specific PCR primers Determination of optimum annealing temperatures and 43 sensitivity of primers Specificity of primers Final PCR parameters for non-human primate 48 Plasmodium species 2.4 Discussion 48 iv

5 Chapter Three: Morphological and molecular identification of malaria parasites from non-human primates 3.1 Introduction Materials and methods Study area and trapping of wild macaques Blood samples Thick and thin blood films Estimation of parasitaemia DNA extraction from blood spots on filter paper DNA extraction from whole blood Nested PCR assays Results Detection of malaria parasites by PCR assay Examination of blood films by microscopy Examination of thick blood films Macaque LT Macaque LT Macaque LT Macaque LT Macaque LT Macaque LT Macaque LT Examination of thin blood films Macaque LT Macaque LT Macaque LT Macaque LT Macaque LT Macaque LT Macaque LT Discussion 80 Chapter Four: Characterisation of the small subunit ribosomal RNA and circumsporozoite protein genes of Plasmodium species from macaques in Sarawak 4.1 Introduction Materials and methods Macaque isolates DNA extraction from whole blood Amplification of the SSU rrna genes Amplification of the csp genes Cloning of the PCR products Preparation of glycerol stocks Extraction of plasmid DNA EcoR I digestion Sequencing of the SSU rrna genes Sequencing of the csp genes Construction of phylogenetic trees Determination of percentage pairwise divergence 90 V

6 4.3 Results Analyses of the SSU rrna genes Amplification, cloning and sequencing of the SSU rrna 93 genes Phylogenetic analysis Polymorphisms within the SSU rrna genes among 103 Plasmodium isolates P. knowlesi and P. knowlesi-like clones P. coatneyi clones P. fieldi and P. fieldi-like clones P. cynomolgi clones P. inui and P. inui-like clones Analyses of the csp genes Amplification, cloning and sequencing of the csp genes Phylogenetic analysis Polymorphisms of the non-repetitive regions of the csp 124 genes P. knowlesi clones P. coatneyi clones P. fieldi clones P. cynomolgi clones P. inui and P. inui-like clones Region I and region II-plus Diversity of tandem repeat regions within the csp genes P. knowlesi clones P. coatneyi clones P. fieldi clones P. cynomolgi clones P. inui and P. inui-like clones Discussion 140 Chapter Five: Summary and indications for future research 5.1 Summary Indications for future research 149 REFERENCES 150 APPENDICES A: Gel electrophoresis 166 B: DNA sequences of the SSU rrna genes 168 C: Percentage divergence of the SSU rrna gene sequences between 190 Plasmodium species and clones isolated from long-tailed macaques of the Kapit Division calculated with the Kimura 2-parameter method using transitions and transversions D: DNA sequences of the csp genes 193 E: Percentages divergence of partial csp gene sequences between Plasmodium 213 species and clones isolated from long-tailed macaques of the Kapit Division calculated with the Kimura 2-parameter method using transitions and transversions vi

7 LIST OF FIGURES Figure 1.1 Life cycle of malaria parasite (Source: Anon, 2006) 3 Figure 1.2 Transcription of distinct rrna genes during P. vivax 15 development (Source: Li et al., 1997) Figure 1.3 Schematic diagram of the circumsporozoite protein (csp) gene of 19 Plasmodium species Figure 2.1 EcoR I digestions of plasmid DNA of selected colonies from P. 38 coatneyi (Pct), P. fieldi (Pfi) and P. inui (Pin). Figure 2.2 Phylogenetic tree of the SSU rrna gene sequences of primate 40 Plasmodium species constructed according to the NJ method with 1,000 replicates. Figure 2.3 Phylogenetic tree based on the SSU rrna gene sequences of 41 primate Plasmodium species produced by ML method. Figure 2.4 Phylogenetic tree based on the SSU rrna gene sequences of 42 primate Plasmodium species produced by MP with branch and bound search method. Figure 2.5 Gel electrophoresis shows the specificity of each primer pair. 47 Figure 3.1 Morphology of malaria parasites taken from thick blood films of 65 long-tailed macaques LT3, LT4, LT7 and LT15. Figure 3.2 Morphology of malaria parasites taken from thick blood films of 68 long-tailed macaques LT20, LT22 and LT33. Figure 3.3 Morphology of malaria parasites taken from thin blood films of 70 long-tailed macaque LT3. Figure 3.4 Morphology of malaria parasites taken from thin blood films of 71 long-tailed macaque LT4. Figure 3.5 Morphology of malaria parasites taken from thin blood films of 73 long-tailed macaque LT7. Figure 3.6 Morphology of malaria parasites taken from thin blood films of 74 long-tailed macaque LT15. Figure 3.7 Morphology of malaria parasites taken from thin blood films of 76 long-tailed macaque LT20. Figure 3.8 Morphology of malaria parasites taken from thin blood films of 78 long-tailed macaque LT22. vii

8 Figure 3.9 Morphology of malaria parasites taken from thin blood films of 79 long-tailed macaque LT33. Figure 4.1 Size variation in the SSU rrna gene of Plasmodium species different clones derived from LT33 isolate. in 94 Figure 4.2 Figure 4.3 Figure 4.4 Figure 4.5 Figure 4.5 Figure 4.6 Figure 4.7 Figure 4.8 Phylogenetic tree based on the SSU rrna genes of Plasmodium 97 species produced by the NJ method. Part of the NJ phylogenetic tree, of the asexually transcribed 98 form of the SSU rrna genes of Plasmodium species, derived from Figure 4.2. Phylogenetic tree based on the SSU rrna genes of Plasmodium 101 species produced by the ML method. Phylogenetic tree based on the SSU rrna genes of Plasmodium 102 species produced by the MP with heuristic search method. Size variation in the csp gene of Plasmodium species in different 117 clones derived from isolate LT33. Phylogenetic tree based on the csp genes of Plasmodium species 120 produced by the NJ method. Phylogenetic tree based on the csp genes of Plasmodium species 122 produced by the ML method. Phylogenetic tree based on the csp genes of Plasmodium species 123 produced by the MP with heuristic search method. viii

9 LIST OF TABLES Table 1.1 Periodicity, geographic range and natural hosts for the non-human 6 primate Plasmodium species Table 1.2 Morphological characteristics of the malaria parasites in Old World 10 monkeys of Southeast Asia (simplified from Coatney et al., 1971) Table 2.1 Forward and reverse internal primers for sequencing the SSU rrna 29 genes. Table 2.2 The SSU rrna genes of primate Plasmodium species obtained from 30 GenBank. Table 2.3 Nucleotide sequences of forward and reverse oligonucleotides 33 designed for identification of respective Plasmodium species. Table 2.4 Range of annealing temperature (T1) for primer pairs of each 35 Plasmodium species. Table 2.5 Comparison of sizes of the SSU rrna gene isoforms sequences 38 derived from P. coatneyi, P. fieldi and P. inui. Table 2.6 Sensitivity of different primer pairs in detection of P. coatneyi, P. 44 cynomolgi, P. fieldi, P. inui, P. fragile and P. knowlesi DNA with different annealing temperatures. Table 2.7 Specificity of different primer pairs in detection of DNA of various 46 species of Plasmodium with selected annealing temperatures (T1). Table 2.8 Summary of PCR parameters for each species-specific primer pairs in 49 nest 2 PCR amplifications. Table 3.1 Blood samples collected from long-tailed (Macaca fascicularis) and 54 pig-tailed (Macaca nemestrina) macaques of the Kapit Division, Sarawak. Table 3.2 Species-specific PCR primers used for the identification of non-human 59 primate Plasmodium species in nest 2 PCR assays. Table 3.3 Summary of PCR assay for the identification of malaria parasites 60 from long-tailed and pig-tailed macaques. Table 3.4 Summary of the identification of Plasmodium species by nested PCR 62 assays on 30 Plasmodium-positive macaques from the Kapit Division of Sarawak. Table 3.5 Estimation of parasitaemia in seven long-tailed macaques of Kapit 64 Division based on examination of Giemsa-stained thick blood films. ix

10 Table 4.1 Forward and reverse internal primers for sequencing the csp genes. 89 Table 4.2 Sequences of the csp genes of Plasmodium species used in the 91 phylogenetic analyses. Table 4.3 Sequences of the SSU rrna genes of P. knowlesi from Kapit Division, 92 Malaysian Borneo and Thailand. Table 4.4 Summary of colony-pcr, approximate insert sizes and codes for 95 selected complete sequences of Plasmodium SSU rrna clones derived from seven macaques of the Kapit Division. Table 4.5 Polymorphisms in the asexually transcribed form of the SSU rrna 104 genes of P. knowlesi and P. knowlesi-like parasites. Table 4.6 Polymorphims in the sexually transcribed form of the SSU rrna 107 genes of P. knowlesi, clone from macaque (LT) and human (KH) isolate from Kapit. Table 4.7 Polymorphisms in the asexually transcribed form of the SSU rrna 108 gene of P. coatneyi and clones from macaque (LT) from Kapit. Table 4.8 Polymorphisms in the asexually transcribed form of the SSU rrna 110 gene of P. fieldi and P. fieldi-like clones from macaque (LT) isolates from Kapit. Table 4.9 Polymorphisms in the sexually transcribed form of the SSU rrna 111 gene of P. fieldi and P. fieldi-like clones from macaque (LT) isolates from Kapit. Table 4.10 Polymorphisms in the asexually transcribed form of the SSU rrna 113 gene of P. cynomolgi (Pcy) strain Ceylon and Vietnam, clones from macaque (LT) isolates from Kapit. Table 4.11 Polymorphisms in the asexually transcribed form of the SSU rrna 114 gene of P. inui and P. inui-like clones from macaque (LT) isolates from Kapit. Table 4.12 Polymorphisms in the sexually transcribed form of the SSU rrna 116 gene of P. inui from macaque (LT) isolates from Kapit. Table 4.13 Summary of complete csp gene sequences for P. inui, P. fieldi and P. 116 coatneyi. Table 4.14 Summary of number of transformants analysed by colony-pcr, 119 approximate insert sizes and codes for selected complete sequences of Plasmodium csp clones derived from seven macaques of the Kapit Division. X

11 Table 4.15 Polymorphisms in the non-repetitive region of the csp genes of P. 125 knowlesi (Pk) strains H and Nuri, human isolates (KH) and clones from long-tailed macaques (LT in bold) of the Kapit Division. Table 4.16 Polymorphisms in the non-repetitive region of the csp genes of P. 127 coatneyi (Pct) strain Hackeri and newly sequenced P. coatneyi clone, and clones from long-tailed macaques (LT in bold) of the Kapit Division. Table 4.17 Polymorphisms in the non-repetitive region of the csp genes of P. 127 fieldi (Pfi), and a clone from long-tailed macaque LT4 of the Kapit Division. Table 4.18 Table 4.19 Table 4.20 Polymorphisms in the non-repetitive region of the csp genes of P. 128 cynomolgi (Pcy) strains Ceylon and Berok, and clones from long-tailed macaques (LT in bold) of the Kapit Division. Polymorphisms in the non-repetitive region of the csp genes of newly 130 sequenced P. inui (Pin), and clones from long-tailed macaques (LT in bold) of the Kapit Division. Comparison of region I, region II-plus, amino acid motifs and sizes of 131 tandem repeat regions and total length of the csp genes for P. knowlesi isolates from macaques (LT), humans (KH), strain H and Nuri. Table 4.21 Comparison of region I, region 11-plus, amino acid motifs and sizes of 133 tandem repeat regions and total length of the csp genes for P. coatneyi strain Hackeri, newly sequenced P. coatneyi and clones from macaque (LT). Table 4.22 Table 4.23 Table 4.24 Comparison of region I, region II-plus, amino acid motifs and sizes of 133 tandem repeat regions and total length of the csp genes for newly sequenced P. fieldi and clone from macaque (LT). Comparison of region I, region II-plus, amino acid motifs and sizes of 134 tandem repeat regions and total length of the csp genes for P. cynomolgi strain Berok, Ceylon and clone from macaque (LT). Comparison of region I, region II-plus, amino acid motifs and sizes of 135 tandem repeat regions and total length of the csp genes for P. inui and P. inui-like clones from macaques (LT). Table 4.25 Comparison of malaria parasites in seven macaques of Kapit Division 142 identified by nested PCR examinations and characterisations of the SSU rrna and csp genes. X1

12 ABBREVIATIONS bp base pair csp circumsporozoite protein DNA deoxyribonucleic acid dntp deoxynucleotide triphosphate EDTA ethylenediaminetetraacetic H2O water acid IRBC infected red blood cell MgCl2 magnesium chloride min minute ml millilitre ML maximum likelihood method mm milimolar MP maximum parsimony method Mya million years ago NJ neighbour-joining method PCR polymerase chain reaction Pct Plasmodium coatneyi Pcy Plasmodium cynomolgi Pf Plasmodium falciparum Pfi Plasmodium fieldi Pfrg Plasmodium fragile Pin Plasmodium inui Pk Plasmodium knowlesi Pm Plasmodium malariae Po Plasmodium ovale Pv Plasmodium vivax RBC red blood cell RNA ribonucleic acid rpm revolutions per minute S Svedberg unit for ribosome; rate of sedimentation of a particle in a centrifuge, an indicator of size sec second SSU rrna small subunit ribosomal TBE Tris-borate EDTA ribonucleic acid WBC white blood cell WHO World Health Organisation PM micromolar xii

13 ABSTRACT Plasmodium knowlesi, a malaria parasite naturally found in long-tailed (Macaca fascicularis) and pig-tailed (M. nemestrina) macaques, was found in a large proportion of malaria patients at Kapit Hospital in Sarawak. P. knowlesi and other malaria parasites such as P. cynomolgi, P. coatneyi, P. fieldi and P. inui have been described in long-tailed and pig-tailed macaques in Southeast Asia but not in Sarawak. Identification of these monkey malaria parasites has been primarily based on morphological characteristics. However, accurate identification is difficult when mixed species infections occur or low parasite counts are encountered. The objectives of this study were to develop species- specific polymerase chain reaction (PCR) primers for molecular detection of monkey malaria parasites and to use molecular methods to identify and characterise Plasmodium species from macaques. Since P. knowlesi is potentially of zoonotic origin, these methods would assist in determining whether macaques are the natural reservoir of P. knowlesi in the Kapit Division. In this study, species-specific PCR primers for the identification of P. knowlesi, P. cynomolgi, P. coatneyi, P. fieldi, P. inui and P. fragile were developed based on the variable regions in the small subunit ribosomal RNA (SSU rrna) genes. Since complete SSU rrna gene sequences were not available for P. inui, P. coatneyi and P. fieldi, these genes were initially sequenced. Nested PCR assays parameters were optimised and the assays were validated for specificity using DNA from P. knowlesi, P. cynomolgi, P. coatneyi, P. fieldi, P. inui, P. fragile and the human malaria parasites. These primers were subsequently used in nested PCR assays for the examination of malaria parasites from 31 long-tailed and seven pig-tailed macaques from the Kapit Division. Thirty (78.9%) of these were positive for malaria parasites, consisting of 27 wild long-tailed, one captive long-tailed and two captive pig-tailed macaques. X111

14 Sixteen (59.3%) of the Plasmodium-positive wild long-tailed macaques examined were infected with P. knowlesi, and all were mixed infections with other Plasmodium species such as P. cynomolgi, P. coatneyi, P. fieldi or P. inui. Nine (33.3%) wild and one captive long-tailed macaques were infected with either single or mixed species of P. cynomolgi, P. coatneyi, P. fieldi and P. inui. Malaria parasites from the remaining two (7.4%) wild long-tailed and two (100%) captive pig-tailed macaques could not be identified by PCR assays. Malaria parasites from seven long-tailed macaques were further characterised by cloning and sequencing the SSU rrna and circumsporozoite protein (csp) genes. The phylogenetic analyses based on these two genes revealed that P. knowlesi isolated from macaques of the Kapit Division were indistinguishable from P. knowlesi isolated from humans. These results, together with entomological and epidemiological data strongly suggest that human P. knowlesi infections are zoonotic and that long-tailed macaques in Kapit Division are one of the reservoir hosts. In addition, other malaria parasites that were phylogenetically indistinguishable from P. coatneyi, P. inui, P. cynomolgi and P. fieldi were detected together with four species that may represent novel Plasmodium species in these macaques. However, to confirm novel Plasmodium species further studies involving more genes and samples are necessary together with isolation of single strains and more detailed molecular analyses. Future studies on human samples for the presence of monkey malaria parasites are necessary to determine whether simian malaria parasites, other than P. knowlesi, are being transmitted to humans in nature. x1v

15 ABSTRAK Plasmodium knowlesi, parasit malaria kera (Macaca fascicularis) dan beruk (M. nemestrina), telah dilaporkan menjangkiti sejumlah besar pesakit malaria di Hospital Kapit di Sarawak. P. knowlesi dan parasit malaria yang lain seperti P. cynomolgi, P. coatneyi, P. fieldi dan P. inui telah ditemui pada kera dan beruk di Asia Tenggara tetapi belum lagi di Sarawak. Langkah utama pengenalpastian parasit malaria monyet adalah berdasarkan kepada ciri-ciri morfologi. Akan tetapi, pengenalpastian secara tepat adalah sukar apabila jangkitan spesies campuran berlaku atau kiraan parasit yang rendah ditemui. Objektif utama dalam kajian ini adalah untuk menghasilkan primer tindakbalas rantai polymerase (PCR) spesies-spesifik bagi pengesanan parasit malaria monyet secara molekular dan mengaplikasikan kaedah molekular untuk mengenalpasti dan mencirikan spesies Plasmodium daripada monyet. Memandangkan P. knowlesi berpotensi menjadi zoonosis, kaedah ini berupaya membantu dalam menentukan sama ada monyet adalah pembawa semulajadi P. knowlesi di Bahagian Kapit. Dalam kajian im, primer PCR spesies-spesifik untuk pengenalpastian P. knowlesi, P. cynomolgi, P. fieldi, P. inui dan P. fragile telah dihasilkan berdasarkan kepada bahagian variasi dalam jujukan gen-gen subunit kecil RNA ribosom (SSU rrna). Memandangkan tiada jujukan lengkap gen SSU rrna untuk P. inui, P. coatneyi dan P. fieldi, gen-gen ini pada mulanya dijujuk. Parameter-parameter asai PCR tersarang telah dioptimumkan dan spesifisiti asai-asai tersebut telah disahkan dengan menggunakan DNA daripada P. knowlesi, P. cynomolgi, P. coatneyi, P. fieldi, P. fragile dan parasit-parasit malaria manusia. Seterusnya, primer-primer ini digunakan dalam asai PCR tersarang bagi pemeriksaan parasit malaria daripada 31 ekor kera dan tujuh ekor beruk dari Bahagian Kapit. Tiga puluh (78.9%) daripada jumlah ekor monyet ini didapati positif untuk xv

16 parasit malaria, yang terdiri daripada 27 ekor kera liar, seekor kera peliharaan dan dua ekor beruk peliharaan. Enam belas (59.3%) daripada jumlah kera liar yang didapati positif dengan Plasmodium telah dijangkiti dengan P. knowlesi, dan kesemuanya adalah jangkitan campuran dengan species Plasmodium yang lain seperti P. cynomolgi, P. coatneyi, P. fieldi atau P. inui. Sembilan (33.3%) ekor kera liar and seekor kera peliharaan telah dijangkiti dengan sama ada satu atau campuran spesies P. cynomolgi, P. coatneyi, P. fieldi dan P. inui. Spesies parasit malaria daripada lebihan dua (7.4%) ekor kera liar dan dua (100%) ekor beruk peliharaan tidak dapat dikenalpasti oleh asaiasai PCR. Parasit malaria daripada tujuh ekor kera tersebut telah dicirikan dengan mengklon dan menjujuk gen SSU rrna dan gen protein circumsporozoite (csp). Analisis filogenetik berdasarkan kepada kedua-dua gen tersebut menunjukkan tiada perbezaan yang ketara antara P. knowlesi daripada monyet dan manusia dari Bahagian Kapit. Keputusan ini, bersamaan dengan data-data entomologi dan epidemiologi mencadangkan bahawa jangkitan P. knowlesi ke atas manusia adalah bersifat zoonosis dan kera merupakan salah satu punca perumah di Bahagian Kapit. Tambahan lagi, parasit malaria lain yang menunjukkan tiada perbezaan yang ketara secara filogenetik dengan P. coatneyi, P. inui, P. cynomolgi dan P. inui serta empat spesies yang mungkin mewakili Plasmodium baru turut ditemui daripada monyet-monyet ini. Walau bagaimanapun, untuk pengesahan Plasmodium species yang baru, kajian lanjutan melibatkan lebih banyak gen and sampel adalah perlu bersamaan dengan pemencilan strain-strain tunggal dan analisis-analisis molekular yang terperinci. Kajian lanjutan terhadap sampel manusia untuk pengenalpastian parasit malaria monyet adalah perlu bagi menentukan sama ada parasit malaria tersebut, selain P. knowlesi, berupaya menjangkiti manusia secara semulajadi. xvi

17 Chapter One Introduction 1.1 Introduction to malaria Malaria is a serious problem in over half of the world's countries. It is widely spread in the tropical and subtropical regions and causes fatalities in approximately one to three million children and pregnant women, especially in Sub-Saharan Africa (WHO, 2005). Unfortunately, to date, no successful vaccine against malaria has been developed. Instead, preventive drugs are taken continuously to reduce the risk of infection. However, these prophylactic drug treatments are too expensive for most of the people living in endemic areas. Most importantly, in certain regions, drug resistance is becoming increasingly common. Malaria is commonly associated with poverty and is a major interference to economic development in malaria-endemic countries. The economic impact includes increase in cost of health care, lost of working and schooling days lost due to sickness, loss of investment and a decline in tourism (Greenwood et al., 2005). Many efforts have been initiated to combat malaria globally (WHO, 2005); the Millennium Development Goals aims to have malaria halted by 2015 while the Roll Back Malaria Partnership was launched in 1998 with the aim to halve the burden of malaria by 2010 (Anon, 2007a). Malaria is caused by protozoan parasites of the genus Plasmodium. The most serious forms of the disease in humans are caused by P. falciparum and P. vivax, but other species such as P. ovale, P. malariae and P. knowlesi can also cause human malaria (Knell, 1991; Singh et al., 2004; WHO, 2005). Clinical symptoms include fever, shivering, I

18 headache, nausea, vomiting, muscle aches and fatigue. These can rapidly progress to include organ failure, delirium, convulsion, coma and death particularly for P. falciparum (WHO, 2005). Besides humans, Plasmodium species also infect birds, reptiles, monkeys, apes and rodents, and approximately 170 Plasmodium species have been discovered (Garnham, 1966). 1.2 Life cycle of malaria parasites Briefly, the malaria parasite life cycle involves two hosts: vertebrates and invertebrates (Knell, 1991). When a malaria-infected female Anopheles mosquito takes its blood meal from a vertebrate host, the sporozoites are injected with the mosquito's saliva into the bloodstream and travel to the liver (Figure 1.1). The Kupffer cells of the liver destroy many sporozoites in the bloodstream and only fractions of them manage to escape from the destruction by penetrating the hepatocytes (Pradel et at., 2004). Later, the parasites grow and multiply into schizonts in the hepatocytes (pre-erythrocytic schizogony). A single schizont can produce thousands of new infective forms called merozoites. Mature schizonts in the hepatocytes release the merozoites into the bloodstream where they invade circulating erythrocytes (erythrocytic schizogony). For P. vivax and P. ovale, parasites of humans and P. cynomolgi and P. simiovale, parasites of non-human primates, a dormant stage called `hypnozoite' can persist in the liver and cause relapse by invading the bloodstream weeks or years later (Cogswell, 1992; Sherman, 1998). In the erythrocytes, the merozoites undergo asexual multiplication by developing into ring forms, or early trophozoites. These trophozoites consume haemoglobin in erythrocytes and mature into schizonts and produce new merozoites inside the 2

19 EaNsgensang male ob"10cvle aý _ Femate gamobocyde Opirq / lefusaö by male garnets IN ANOPHELINE MOSQUITO IN MAN Sexual +rmvture gametocytes! } rnrnarr I ý. xoaerytenocytlc X A cyc11. Erymrocytic (asexual` cycle MBrOtOrt@S º ', ý Q.,.. 1! MA'ü2'4QRf3i'. ý y " I toxrrn i _-' Figure 1.1 Life cycle of malaria parasite (Source: Anon, 2006) 3

20 erythrocytes. The erythrocytes subsequently lyse and release merozoites that can invade new erythrocytes and restart the erythrocytic cycle. Clinical symptoms result when the erythrocytes rupture and release cellular debris from infected erythrocytes into the bloodstream (Knell, 1991). The host response to these toxins produces classic paroxysms of fever and chills. The timing of the erythrocytic cycles differs depending on the species of the parasites. For example, the length of time required for completion of an erythrocytic cycle is 24 hours (quotidian periodicity) for P. knowlesi, 48 hours (tertian periodicity) for P. falciparum and P. uivax and 72 hours (quartan periodicity) for P. malariae and P. brasilianum (Coatney et al., 1971). Some of the trophozoites in the erythrocytes take an alternative pathway by developing into sexual forms called gametocytes, which are macrogametocytes (female) and microgametocytes (male). The gametocytes do not lyse the erythrocytes and the Anopheles mosquito ingests the erythrocytes that contain gametocytes during a blood meal. During the sexual phase, the gametocytes continue their development in the midgut of the mosquito. Before fertilisation occurs, macrogametocytes escape from the erythrocytes and form macrogametes. Meanwhile, the microgametocyte exflagellates and develops into eight haploid motile microgametes. The macrogamete and microgamate then fuse and form a diploid zygote. This transforms into an ookinete that penetrates the midgut epithelium and resolves in the extracellular space between the midgut epithelium and the basal lamina. The ookinete consequently matures into an oocyst and divides asexually into numerous sporozoites that burst into the hemocoel and reach the salivary glands of the mosquito. The life cycle of the malaria parasites repeats when the mosquito bites the vertebrate host during a blood meal. 4

21 1.3 Non-human primate malaria parasites A total of 29 malaria parasites are naturally found in the non-human primate hosts (Garnham, 1966; Coatney et al., 1971; Gysin, 1998). The primates range from Madagascar lemurs of the prosimians, the New World and Old World monkeys, gibbons of the Hylobatidae, the African great apes (Pan sp. and Gorilla sp. ) and Asian great ape or the orang-utan (Pongo spp. ) (Martin, 1990; Whitten, 1998). A summary of the life cycle, periodicity, geographical range and natural hosts for each of these 29 non-human primate Plasmodium species are shown in Table 1.1. In understanding the taxonomy of the human malarias, many of the non-human primate Plasmodium species were studied morphologically, molecularly or clinically. For example, P. brasilianum falls under the malariae-type parasites because it has erythrocytic stage periodicity of 72 hours, as exhibited by P. malariae of the humans, and these two species cannot be distinguished morphologically (Taliaferro, 1932) and molecularly (Cochrane et at., 1985; Lal et at., 1988; Leclerc et at., 2004a). These two species should therefore be considered as a single species (Fandeur et at., 2000). P. simium, however, falls under the vivax-type parasites due to similar morphological characteristics, exhibits tertian erythrocytic stage periodicity and is phylogenetically indistinguishable from P. vivax (Escalante et at., 1995; McCutchan et at., 1996). The relationship between P. simium and P. vivax has given rise to speculation regarding the origin of P. vivax (Escalante et at., 2005). Recent studies indicate that P. vivax was brought to the Americas by human migrations from Asia during the pre-columbian times and had adapted to transmission in monkeys of South American forests as the parasite called P. simium (Carter & Mendis, 2002; Carter, 2003). Meanwhile, P. reichenowi was classified as falciparum-type parasite because its morphological 5

22 Table 1.1: Periodicity, geographic range and natural hosts for the non-human primate Plasmodium species Plasmodium Periodicity Geographic range species knowlesi* coatneyi* cynomolgi* Quotidian Tertian Tertian Peninsular Malaysia, Philippines Peninsular Malaysia, Philippines Peninsular Malaysia, India, Indonesia, Sri Lanka fieldi ' Tertian Peninsular Malaysia inui* Quartan Peninsular Malaysia, India, Borneo, Indonesia, Taiwan, Sri Lanka fragile' Tertian Sri Lanka, India simiouale* Tertian Sri Lanka shortii* Quartan Sri Lanka, India gonderi" Tertian Cameroon, Zaire petersi* No data georgesi ' No data brasilianum*'= Quartan Africa Africa Brazil, Venezuela, Colombia, Peru, Panama Natural hosts Macaca fascicularis, M. nemestrina, Presbytis melalophos M. fascicularis M. fascicularis, M. nemestrina, M. radiata, M. mulatta, M. sinica, M. cyclopis, P. cristatus, P. entellus M. fascicularis, M. nemestrina M. fascicularis, M. nemestrina, M. mulatta, M. radiata, M. sinica, M. cyclopis, P. cristatus, P. obscurus, Cynophithecus niger M. sinica, M. radiata M. sinica M. fascicularis umbrosa Cercocebus atys, C. atterimus, C. galeritus agilus, Mandrillus leucophaeus C. albigena C. albigena, C. galeritus Many species of Alouatta, Ateles, Brachyteles, Callicebus, Cebus, Chiropotes, Lagothrix, Saimiri simium** Tertian Brazil Alouatta fuscus, Brachyteles arachnoides eylesiý Tertian Peninsular Malaysia Hylobates lar hylobao Tertian Malaysian Borneo, Hylobates moloch Indonesia jefferiý Tertian Peninsular Malaysia H. lar youngi'ý Tertian Peninsular Malaysia H. lar pithecil Tertian Malaysian Borneo Pongo pygmaeus siluaticuml Tertian Malaysian Borneo P. pygmaeus reichenowiv Tertian Cameroon, Sierra Leone, Pan troglodytes, Gorilla gorilla Zaire, Congo schwetziv Tertian Cameroon, Sierra Leone, P. troglodytes, G. gorilla Zaire, Congo rodhainiv Quartan Sierra Leona, Angola, Congo P. troglodytes, G. gorilla girardi$ No data Madagascar Lemur fuluus ficluus, L. ficluus rufus Toleyi$ No data Madagascar L. fuluus fuluus, L. fuluus rufus lemuris$ No data Madagascar L. collaris coulangesi± No data Madagascar Lemur macaco macaco percygarnhami$ No data Madagascar L. macaco macaco uilenbergi$ No data Madagascar L. fuluus fulvus bucki$ No data Madagascar L. macaco macaco indicates malaria parasites of the Old World monkeys, '**' of the New World monkeys, of the gibbons, `91' of the Asian great apes, `Y' of the African great apes and of the Madagascar lemurs. All data were summarised from Coatney et al. (1971), Garnham (1966) and Gysin (1998). 6

23 characteristics are reminiscent of P. falciparum and it is genetically related to P. falciparum (Lal & Goldman, 1991; Escalante et al., 1995, 1998; McCutchan et al., 1996; Qari et al., 1996). From the clinical aspect, P. coatneyi was used for studies on the pathophysiology and immunology of cerebral malaria and maternal malaria caused by P. falciparum since both exhibit sequestration characteristics (Collins et al., 2001). A total of eight species of the non-human primate malaria have been reported as transmissible to man either by natural means, that is by mosquito bite, or experimentally by blood passage. These include P. knowlesi (Knowles & Das Gupta, 1932; Chin et at., 1965), P. inui (Das Gupta, 1938; Coatney et at., 1966), P. cynomolgi (Eyles et at., 1960; Beye et al., 1961; Contacos et at., 1962; Bennett & Warren, 1965), P. brasilianum (Clark & Dunn, 1931a, 1931b; Contacos et at., 1963), P. simium (Deane et at., 1966), P. eylesi (Contacos, 1970; Coatney et at., 1971), P. schwetzi (Rodhain & Dellaert, 1955a, 1955b; Contacos et at., 1970; Coatney et at., 1971) and P. rodhaini (Coatney et al., 1971; Meder, 1994). Three of these, P. knowlesi, P. cynomolgi and P. inui, occur in macaques in Southeast Asia. P. knowlesi was first discovered in the blood of a long-tailed macaque imported from Singapore by Napier and Campbell in 1932 working in India (Garnham, 1966; Coatney et at., 1971). Knowles and Das Gupta (1932) found that this parasite caused chronic infections in long-tailed macaques but lethal infections in rhesus macaques. They also described that the blood forms of the parasite could be transmitted to man by inoculation of infected blood. P. knowlesi was subsequently used as a pyretic agent for the treatment of neurosyphilis (Chopra & Das Gupta, 1936; Ciuca et at., 1955) but it was only in 1965 that the first report appeared of a human naturally infected by P. knowlesi. Chin and co- 7

24 workers (1965) described the case of an American surveyor, working in Pahang, who became ill upon return to the United States. His infection was initially diagnosed as P. falciparum, as only ring forms were observed. However, subsequent inoculations of his blood into volunteers at the United States Penitentiary, Atlanta, Georgia and rhesus macaques later confirmed that the infection was P. knowlesi. This was followed by a second human case, a report of a presumptive case of a field worker, who acquired P. knowlesi in Johore, West Malaysia (Fong et at., 1971). Human infections were thought to be rare until a large number of humans naturally infected with P. knowlesi were discovered in the Kapit Division of Sarawak, Malaysian Borneo (Singh et at., 2004). Recently, there have also been reports of single human P. knowlesi infections in Thailand (Jongwutiwes et al., 2004) and Myanmar (Zheng et at., 2006). P. cynomolgi was first described in long-tailed macaques from Java, Indonesia by Martin Mayer in 1907 (Coatney et at., 1971). An accidental infection of humans by mosquito bites of P. cynomolgi in a laboratory in the United States was reported by Eyles and colleagues (1960). Subsequent experiments were carried out whereby volunteers were bitten by mosquitoes heavily infected with P. cynomolgi (Eyles et al., 1960; Contacos et al., 1962) and it was proven that P. cynomolgi could be successfully transmitted to humans by mosquito bites. Besides P. knowlesi and P. cynomolgi, another of the macaque parasite, P. inui is infectious to humans, as shown by Das Gupta (1938), whereby a volunteer developed a patent parasitaemia and febrile symptoms after he was given blood directly from an infected long-tailed macaque by intramuscular injection. In addition, volunteers also became infected following exposure to the bites of P. inui infected mosquitoes (Coatney et at., 1966). However, no natural human infections by P. 8