Giardia: A Model Organism

Cover......Page 1
Frontmatter......Page 2
Title Page\r......Page 3
Copyright Page\r......Page 4
Preface/Introduction......Page 8
Table of Contents\r......Page 10
Section I: Taxonomy and Epidemiology of Giardia......Page 16
1.1.2 Generic Names......Page 18
1.1.5 The Three Species......Page 19
1.2.1 Inter- and Intraspecific MorphologicalVariation......Page 20
1.2.3 In vitro and In vivo Studies......Page 21
1.2.4 Infectivity and Clinical Disease......Page 22
1.3 Phylogenetic Relationships......Page 23
1.4 Molecular Epidemiology and Host Specifi city......Page 24
1.5 Taxonomic Certainty......Page 25
1.6 In Conclusion – Sex and Stability......Page 26
References......Page 27
2.1 Introduction......Page 32
2.3 Seasonality......Page 33
2.5 Giardiasis in Immunosuppressed Individuals......Page 34
2.7 Correlation Between Assemblages and Symptoms......Page 35
2.8 Tools for Molecular Genotyping......Page 37
2.9 Molecular Epidemiology of Assemblage A......Page 38
2.11 Recombination and Molecular Epidemiology......Page 40
References......Page 41
3. 1.1 The Importance of Waterborne/Environmental Transmission......Page 44
3.1.2 How Does Giardia Lend Itselfto Transmission by the Waterborne Routeor Environmental Transmission?......Page 45
3.1.3 The Relative Importance of Different Environmental Transmission Vehicles......Page 47
3.2.1 Drinking Water Outbreaks......Page 48
3.2.3 Foodborne Outbreaks......Page 55
3.3.1 Standard Methods for Analysis of Water......Page 56
3.3.2 Regulatory Procedures: the Value of Monitoring......Page 58
3.3.3 Standard Methods for Fruit/Vegetables......Page 60
3.3.5 Standard Methods for Other Environmental Samples......Page 61
3.3.6 Novel/State-of-the-art Methods and Future Approaches......Page 62
3.3.7 Risk Assessment and Risk Management......Page 63
3.4.1 Water Matrices......Page 65
3.4.2 Soil......Page 66
3.5.1 In Water......Page 69
3.5.3 In Beverages......Page 72
3.6.1 Millenium Development Goals......Page 73
3.6.2 Water Scarcity......Page 74
3.6.4 Climate Change......Page 75
References......Page 76
4.1 Introduction......Page 86
4.3 Prevalence in Farm Animals......Page 87
4.4 Prevalence in Companion Animals......Page 88
4.5 Epidemiology......Page 92
4.6 Pathogenesis......Page 93
4.8 Diagnosis......Page 94
4.8.3 Polymerase Chain Reaction (PCR)......Page 95
4.9.1.1 Benzimidazoles......Page 96
4.9.2 Alternative Approaches......Page 97
4.9.3.1 Measures to Support Curative Treatment......Page 98
4.10 Molecular Epidemiology......Page 99
References......Page 100
Seciton II: Molecular Biology of Giardia......Page 108
5.2 Available Giardia Data......Page 110
5.3 Resources for Giardia Genomics......Page 112
5.4 Comparison of Assemblage A, B, and E Isolates......Page 113
5.5 The Future of Giardia Comparative Genomics......Page 114
References......Page 115
6.2.1 Giardia Produces a Severely TruncatedAsn-linked Glycan (N-glycan)......Page 118
6.2.2 Giardia is Missing N-glycan-dependent Quality Control of Protein Foldingin the ER Lumen......Page 119
6.2.3 The PredictedGlycosylphosphatidylinositol (GPI) Anchorof Giardia Contains Just Two Mannose Residues......Page 120
6.2.6 Use of the Plant Lectin Wheat Germ Agglutinin (WGA) to Enrich Giardia Glycoproteins......Page 121
6.3.1 Giardia N-glycans are DramaticallySimplifi ed Relative to Those of the Hostand Most Other Parasites......Page 122
References......Page 123
7.1 Introduction......Page 126
7.2 Mass Spectrometric Analysis of Phospholipids, Sterols, and FattyAcids......Page 128
7.2.1 Results of Phospholipid Analyses......Page 129
7.2.2 Results of Fatty-Acid Analyses by GC-MS......Page 131
7.3 Lipid Metabolic Genes Present in the Database of WBC6 Isolate......Page 133
7.4 The Proposed Pathway......Page 135
7.4.1 Compartment 1......Page 136
7.5 Conclusion and Future Direction......Page 138
References......Page 139
8.1 Carbohydrate (Glucose) Catabolism......Page 142
8.2 Glycolysis and the Pentose Phosphate Pathway......Page 143
8.3.5 Aldehyde Dehydrogenase(-CoA Acetylating)......Page 144
8.5.3 Phospho N-acetylglucosamineMutase (Pgm)......Page 145
8.5.4 UDP N-acetyl GlucosaminePyrophosphorylase (Uap)......Page 146
8.6 Regulation and Inhibition......Page 147
8.8 Comparative Biochemistry and Metabolism......Page 148
8.9 Uridine/Thymidine Phosphorylase Activity (URTPase)......Page 149
8.10 Metabolomics......Page 150
References......Page 151
Section III: Cellular Biology of Giardia......Page 154
9.1 Introduction......Page 156
9.2 The Cell Surface......Page 159
9.3 The Cytoskeleton......Page 162
9.4 The Flagella......Page 163
9.6 The Median Body......Page 164
9.9 The Endocytic System......Page 165
9.10 The Secretory System......Page 166
9.13 The Interphasic Nuclei......Page 167
9.15 The Fine Structure of the Encystation Process......Page 168
9.17 The Fine Structure of the Excystation Process......Page 171
References......Page 172
10.2 The Cell Cycle Throughout Giardia’s Life Cycle......Page 176
10.3 Cell Division of Giardia Trophozoites......Page 177
10.4.1 Mechanism of Chromosome Segregation and Mitosis......Page 178
10.6 Division of the Flagellar Apparatus......Page 183
10.6.2 Transformation of Parent Flagella during Division......Page 185
10.6.3 De Novo Assembly of Daughter Flagella......Page 187
10.6.5 Developmental Asymmetry of Microtubular Roots of Caudal Flagella......Page 188
10.7 Ventral Disc......Page 189
10.7.1 Parent Ventral Disc Disassembly......Page 190
10.7.2 De Novo Assembly of Daughter Ventral Discs......Page 191
10.8 Cytokinesis......Page 193
References......Page 195
11.1 Introduction......Page 200
11.3.1 Protein Targeting......Page 202
11.3.2 Mitosomal Processing Peptidase......Page 203
11.3.3 Protein Import Machinery......Page 204
11.4 FeS Cluster Assembly Machinery......Page 208
11.5 Energy Metabolism and Membrane Potential......Page 209
11.6 Interaction with Other Cellular Structures......Page 210
11.7 Perspectives......Page 211
References......Page 212
12.2 Giardia Phosphatases and Kinases......Page 216
12.3 Signaling in the Cell Cycle......Page 219
12.4.1 Encystation......Page 220
12.5 Conclusion......Page 222
References......Page 223
13.1.2 General Transcription Factors......Page 226
13.1.3 RNA Polymerases......Page 227
13.1.6 Modifi cation of mRNAs......Page 228
13.2 Genome Structure......Page 229
13.3 DNA Replication and Recombination......Page 230
References......Page 232
14.1 Introduction......Page 234
14.2.1 Endoplasmatic Reticulum and Golgi Apparatus......Page 235
14.2.2 Peripheral Vesicles and Endocytic Transport......Page 237
14.3.1 Proliferating Trophozoites......Page 238
14.3.2 Encysting Trophozoites......Page 240
References......Page 243
15.1 Introduction......Page 248
15.2.1 The Mechanism of RNAi: General Features......Page 249
15.2.2.1 Dicer......Page 250
15.2.2.2 Argonaute Proteins......Page 251
15.2.2.3 RNA-dependent RNA-polymerase (RdRP)......Page 252
15.2.3 Small RNA Molecules in Giardia and Their Putative Biological Functions......Page 253
15.3.1 Is This Short Enough?......Page 254
15.3.3 Bridging the Gap......Page 255
References......Page 257
16.1 Introduction......Page 260
16.2 Switching Characteristics......Page 261
16.5 Spatial Organization, Antigenicity, and Motifs......Page 262
16.7 Immune Responses to VSP and Immune Selection......Page 264
16.8 Biological Selection......Page 267
16.9 VSP Secretion......Page 268
16.10 Control of Antigenic Variation......Page 269
References......Page 270
Section IV: Pathology, Treatment and Diagnostics of Giardia and the Host Immune Response......Page 274
17.1 Introduction......Page 276
17.2 Trophozite Adhesion: What is In,What is Out......Page 277
17.3 Ancestral Structural Proteinsin a Very Evolved Adhesion Apparatus......Page 279
17.4 Molecular Factors Involvedin Giardia Adhesion to Host Cells......Page 280
17.5 Consequences of Giardia-Host Cell Interactions......Page 283
References......Page 285
18.1 Introduction......Page 290
18.2 Molecular Components of the Cytoskeleton......Page 292
18.2.1 The Role of the Ventral Disc in Giardial Attachment......Page 302
18.2.2 The Structure and Putative Function of the Median Body......Page 305
18.2.3 Flagellar Structure and Motility......Page 307
18.2.4 Structure and Putative Function of Axoneme-Associated Elements......Page 308
18.3 Flagellar Assembly and Interphase Flagellar Length Maintenance......Page 309
18.5 The Cytoskeleton and Encystation/Excystation......Page 310
References......Page 311
19.1 Introduction......Page 316
19.2 Clinical Manifestations of Giardiasis......Page 317
19.3 Chronic Gastrointestinal Disorders Associated with Giardiasis......Page 318
19.4 Pathophysiological Processes Causing Symptoms in Giardiasis......Page 320
19.4.1 Giardia Promotes Excessive Enterocyte Apoptosis......Page 321
19.4.2 Giardia Disrupts Intestinal Barrier Function......Page 322
19.4.3 Giardia Induces a Diffuse Shortening of Brush Border Microvilli and Causes Electrolyte Transport Abnormalities......Page 323
19.5 Role of Parasitic Factorsin the Pathogenesis of Giardiasis......Page 324
References......Page 327
20.1 Infections in Humans......Page 334
20.3 The Antibody Response......Page 335
20.4 The Cellular Immune Response......Page 337
20.6 Innate Immunity......Page 338
20.7.1 Defensins......Page 340
20.7.3 Mast Cells......Page 341
References......Page 343
21.1 Introduction......Page 348
21.2.1 Factors to Consider Before Developing Anti-Giardia Vaccine for Developing Countries......Page 349
21.3 Candidate Antigens for a Vaccine Against Giardia Pathology......Page 350
21.3.5 Variant Surface Proteins (VSPs)......Page 351
21.4.2 Hurdles in Constructing a VaccineUsing Giardia Trophozoite Proteins......Page 352
21.4.4 Success or Failure of Vaccine in Reducing the Pathology......Page 353
21.5 Transmission-blocking Vaccines Against Giardia Using Cyst Wall Protein 2......Page 354
21.5.1 Biochemical Composition of the Cyst Wall......Page 355
21.5.2 Local Immune Response to CWPs......Page 356
21.5.3 Use of rPro-CWP2 as an Oral Vaccine......Page 357
21.5.4 Lactic Acid Bacteria (LAB) as a LiveTBV Delivery Vehicle......Page 358
21.5.5 Effi cacy of the TBV Using a DNA Vaccine Strategy......Page 363
References......Page 364
22.1 Introduction......Page 368
22.2.1 Giardia and Human Giardiasis......Page 369
22.2.2 Symptoms and Basis or Laboratory Investigations......Page 370
22.2.3 Giardia Species and Assemblages......Page 371
22.3.1 Micrometry......Page 372
22.3.2 Trophozoite Morphometry and Morphology......Page 373
22.4 Rationale for Laboratory Diagnosis of Infection......Page 374
22.6.1 Biophysical Methods......Page 376
22.6.1.1.1.2 Method......Page 377
22.6.2 Centrifugal Flotation......Page 378
22.7.1 Triple Faeces Test (TFT)......Page 379
22.7.1.1.1 Chlorazol Black Staining of SAF-Preserved Specimens......Page 380
22.8 Microscopical Examination of Samples......Page 381
22.10 Immunomagnetic Separation(IMS) for Giardia Cysts......Page 383
22.11.1 Method......Page 384
22.12.2.2 Lateral Flow Immunochromatographic(Dipstick) Assays......Page 385
22.14 Antibody Detection......Page 386
22.16 Molecular Diagnosis – Nucleic Acid Detection Methods......Page 387
22.16.2 Primer, Gene Locus Selection, PCR and Mixed Infections......Page 388
References......Page 389
Section V: Methods for Giardia Research......Page 394
23.1 Introduction......Page 396
23.2.2 Growth Medium and Cultivation of Giardia Trophozoites In Vitro......Page 398
23.2.3 Cryopreservation of GiardiaTrophozoites Grown In Vitro......Page 400
23.2.4 Encystation of Giardia In Vitro......Page 401
23.2.6 Excystation of Giardia In Vitro......Page 406
References......Page 408
24.1 Introduction......Page 410
24.2.2 Whole Culture Synchronization......Page 412
24.3.3 Whole Culture Synchronization......Page 413
24.3.5 Wash and DNA Labeling......Page 414
References......Page 415
25.1.1 DNA versus RNA Constructs......Page 416
25.1.2 Transient versus Stable Transfection......Page 417
25.2.1 Preparation of Giardia Nuclear Extracts......Page 419
25.2.2 Preparation of Probes (for Radioactiveand Non-radioactive Detection of Signals)......Page 421
25.2.3 Preparation of Membrane for Non-radioactive Detection of Gel-shifts......Page 422
25.3.1 Primer Extension, S1 Nuclease Protection, and 5\' RACE......Page 423
25.3.2 Nuclear Run-on......Page 424
References......Page 425
26.2 The BEI Research Resources Repository......Page 428
References......Page 431
List of Contributors......Page 432