Giardia: A Model Organism

W

Hugo D. Luján • Staffan Svärd Editors

Giardia A Model Organism

SpringerWienNewYork

Hugo D. Luján, PhD Laboratory of Biochemistry and Molecular Biology, School of Medicine, Catholic University of Córdoba, Córdoba, Argentina Staffan Svärd, PhD Department of Cell and Molecular Biology, Uppsala University, Uppsala, Sweden

This work is subject to copyright. All rights are reserved, whether the whole or part of the material is concerned, specifically those of translation, reprinting, re-use of illustrations, broadcasting, reproduction by photocopying machines or similar means, and storage in data banks. Product Liability: The publisher can give no guarantee for all the information contained in this book. This does also refer to information about drug dosage and application thereof. In every individual case the respective user must check its accuracy by consulting other pharmaceutical literature. The use of registered names, trademarks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. © 2011 Springer-Verlag/Wien Printed in Austria SpringerWienNewYork is part of Springer Science + Business Media springer.at

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Library of Congress Control Number: 2011923664

ISBN 978-3-7091-0197-1 SpringerWienNewYork

This book is dedicated to Huw Smith Hugo D. Luján and Staffan Svärd

Preface/Introduction

Giardia as a parasite has fascinated scientists for more than 300 years. This flagellated protozoan parasite of the order Diplomonadida was observed already in 1681 by Antony van Leeuwenhoek, using a homemade microscope. Research on Giardia and giardiasis, the disease caused by the parasite, has advanced considerably since the first observation. More than half of the 5500 Giardia publications in PubMed (August, 2010) have been published since 1998 and the last 5 years have been very productive. Giardia intestinalis (syn. G. duodenalis and G. lamblia) is important since it causes diarrheal disease in humans and young animals. Transmission occurs by cysts discharged in the feces of infected hosts. The parasite is distributed worldwide and symptomatic infections occur in developing and developed countries. Infections by water or food are the main modes of transmission. The parasite is found in most mammals and it is a potential zoonosis. Molecular analyses have identified seven distinct G. intestinalis genotypes or assemblages and two, A and B, are found in humans. The epidemiology and taxonomy of G. intestinalis are discussed further in Section I. The progress in Giardia research has been slowed down due to the lack of genetic systems. However, the “omics” age has contributed enormously to the research field and the publication of the first Giardia genome has been followed by several genomics, transcriptomics, glycomics, proteomics and lipidomics projects. This has and will contribute to the understanding of the Giardia metabolome and Section II deals with the molecular biology of Giardia. Besides its significance as a pathogen, Giardia has always attracted the attention of cell biologists. This

is not surprising considering its unique organelles such as the adhesive disc, median body, the mitosome and the two nuclei, combined with unique variants of processes such as RNA interference, cell signaling, protein transport, antigenic variation and cell differentiation. Furthermore, in the light of the current strategies dealing with the development of the new field of Synthetic Biology, the knowledge of the simplified machineries used by Giardia to undertake all basic, eukaryotic cellular processes makes this organism an exceptional model that will, undoubtedly, facilitate the development of the simplest eukaryotic synthetic cell. Thus, Giardia is a gold-mine for cell biologists and this is described in Section III, Cell biology of Giardia. Clinical manifestations of human infection range from asymptomatic infections to acute diarrhea and malabsorption. The disease mechanism has for a long time been elusive but the recent progress in understanding of the disease is reviewed in Section IV. In the end of the book basic methods for Giardia research are presented. These protocols are useful for long-time “giardiaologists” and for researchers that have just discovered this wonderful parasite. The book “Giardia-a model organism” is the most extensive book dealing with Giardia published so far and all the current leaders in their respective Giardia research field have contributed to the book. This makes this book a must for everybody interested in molecular parasitology and Giardia research. Hugo D. Luján and Staffan Svärd

Contents

Preface/Introduction

VII

Hugo D. Luján and Staffan Svärd

Section I Taxonomy and Epidemiology of Giardia

1

1

3

2.11 Recombination and Molecular Epidemiology 2.12 Conclusions References

3

25 26 26

Waterborne and Environmentally-Borne Giardiasis 29 Lucy J. Robertson and Yvonne Ai Lian Lim

Taxonomy of Giardia Species R. C. Andrew Thompson and Paul T. Monis

Abstract 1.1 Taxonomic Recognition 1.1.1 Introduction 1.1.2 Generic Names 1.1.3 Species Names 1.1.4 Taxonomic Uncertainty 1.1.5 The Three Species 1.1.6 Additional Species 1.2 Phenotypic Variation 1.2.1 Inter- and Intraspecific Morphological Variation 1.2.2 Host Specificity 1.2.3 In vitro and In vivo Studies 1.2.4 Infectivity and Clinical Disease 1.3 Phylogenetic Relationships 1.4 Molecular Epidemiology and Host Specificity 1.5 Taxonomic Certainty 1.6 In conclusion – Sex and Stability References

2

Epidemiology of Giardiasis in Humans

3 3 3 3 4 4 4 5 5 5 6 6 7 8 9 10 11 12

17

Simone M. Cacciò and Hein Sprong Abstract 2.1 Introduction 2.2 Prevalence of the Infection 2.3 Seasonality 2.4 Giardiasis in Children 2.5 Giardiasis in Immunosuppressed Individuals 2.6 Risk Factors 2.7 Correlation Between Assemblages and Symptoms 2.8 Tools for Molecular Genotyping 2.9 Molecular Epidemiology of Assemblage A 2.10 Molecular Epidemiology of Assemblage B

17 17 18 18 19 19 20 20 22 23 25

Abstract 29 3.1 Introduction 29 3.1.1 The Importance of Waterborne/Environmental Transmission 29 3.1.2 How Does Giardia Lend Itself to Transmission by the Waterborne Route or Environmental Transmission? 30 3.1.3 The Relative Importance of Different Environmental Transmission Vehicles 32 3.2 Waterborne and Foodborne Giardiasis Outbreaks 33 3.2.1 Drinking Water Outbreaks 33 3.2.2 Recreational Water Outbreaks 40 3.2.3 Foodborne Outbreaks 40 3.3 Detection of Giardia Cysts in Water and Environmental Matrices 41 3.3.1 Standard Methods for Analysis of Water 41 3.3.2 Regulatory Procedures: the Value of Monitoring 43 3.3.3 Standard Methods for Fruit/Vegetables 45 3.3.4 Standard Methods for Shellfish 46 3.3.5 Standard Methods for Other Environmental Samples 46 3.3.6 Novel/State-of-the-art Methods and Future Approaches 47 3.3.7 Risk Assessment and Risk Management 48 3.4 Occurrence of Giardia in Water and Environmental Matrices: A Global Perspective 50 3.4.1 Water Matrices 50 3.4.2 Soil 51 3.4.3 Food Products 54 3.5 Approaches to Removal and Inactivation of Giardia Cysts in Water and Food 54 3.5.1 In Water 54 3.5.2 In Food 57 3.5.3 In Beverages 57 3.6 Conclusion and Future Challenges in Environmentally-Borne Giardiasis 58 3.6.1 Millenium Development Goals 58

X

Contents

3.6.2 3.6.3 3.6.4 3.6.5 References

4

Water Scarcity Wastewater and Water Re-use for Irrigation Climate Change Conclusion

Giardia in Pets and Farm Animals, and Their Zoonotic Potential

59 60 60 61 61

71

Thomas Geurden and Merle Olson Abstract 4.1 Introduction 4.2 Life Cycle 4.3 Prevalence in Farm Animals 4.4 Prevalence in Companion Animals 4.5 Epidemiology 4.6 Pathogenesis 4.7 Clinical Signs 4.8 Diagnosis 4.8.1 Microscopical Examination 4.8.2 Antigen Detection 4.8.3 Polymerase Chain Reaction (PCR) 4.9 Treatment and Control 4.9.1 Chemotherapeutic Treatment 4.9.1.1 Benzimidazoles 4.9.1.2 Pyrantel-febantel-praziquantel Combo 4.9.1.3 Paromomycin 4.9.2 Alternative Approaches 4.9.3 Control 4.9.3.1 Measures to Support Curative Treatment 4.9.3.2 Measures to Prevent Infection 4.10 Molecular Epidemiology References

71 71 72 72 73 77 78 79 79 80 80 80 81 81 81 82 82 82 83 83 84 84 85

Section II Molecular Biology of Giardia

93

5

95

Genomics of Giardia Hilary G. Morrison and Staffan Svärd

Abstract 5.1 Genomics and Comparative Genomics 5.2 Available Giardia Data 5.3 Resources for Giardia Genomics 5.4 Comparison of Assemblage A, B, and E Isolates 5.5 The Future of Giardia Comparative Genomics References

6

The Glycoproteins of Giardia

95 95 95 97 98 99 100

103

John Samuelson and Phillips W. Robbins Abstract 6.1 Introduction 6.2 Results 6.2.1 Giardia Produces a Severely Truncated Asn-linked Glycan (N-glycan)

103 103 103 103

6.2.2

Giardia is Missing N-glycan-dependent Quality Control of Protein Folding in the ER Lumen 6.2.3 The Predicted Glycosylphosphatidylinositol (GPI) Anchor of Giardia Contains Just Two Mannose Residues 6.2.4 Giardia has a Single Nucleotide Sugar Transporter (NST) 6.2.5 Giardia is a Rare Protist that has an O-GlcNAc Transferase (OGT) that Modifies Nucleocytosolic Proteins 6.2.6 Use of the Plant Lectin Wheat Germ Agglutinin (WGA) to Enrich Giardia Glycoproteins 6.2.7 Use of Multidimensional Protein Identification Technology (MudPIT) to Identify Total Proteins of Giardia Trophozoites 6.3 Discussion 6.3.1 Giardia N-glycans are Dramatically Simplified Relative to Those of the Host and Most Other Parasites 6.3.2 Future Research Acknowledgments References

7

Mass Spectrometric Analysis of Phospholipids and Fatty Acids in Giardia lamblia

104

105 106

106

106

107 107

107 108 108 108

111

Mayte Yichoy, Ernesto S. Nakayasu, Atasi De Chatterjee, Stephen B. Aley, Igor C. Almeida and Siddhartha Das Abstract 7.1 Introduction 7.2 Mass Spectrometric Analysis of Phospholipids, Sterols, and Fatty Acids 7.2.1 Results of Phospholipid Analyses 7.2.2 Results of Fatty-Acid Analyses by GC-MS 7.3 Lipid Metabolic Genes Present in the Database of WBC6 Isolate 7.4 The Proposed Pathway 7.4.1 Compartment 1 7.4.2 Compartments 2 and 3 7.5 Conclusion and Future Direction Acknowledgments References

8

Giardia Metabolism

111 111 113 114 116 118 120 121 123 123 123 124

127

Edward L. Jarroll, Harry van Keulen, Timothy A. Paget and Donald G. Lindmark Abstract 8.1 Carbohydrate (Glucose) Catabolism 8.2 Glycolysis and the Pentose Phosphate Pathway 8.3 Pyruvate Metabolism 8.3.1 Effects of O2 and Glucose Concentration on Pyruvate Metabolism

127 127 128 129 129

Contents Pyruvate: Ferredoxin Oxidoreductase (PFOR) 8.3.3 Hydrogenase 8.3.4 Acetyl CoA Synthetase (Nucleoside Diphosphate – Forming) 8.3.5 Aldehyde Dehydrogenase (-CoA Acetylating) 8.4 Arginine Dihydrolase Pathway (ADiHP) 8.5 Synthesis of N-acetylgalactosamine from Glucose 8.5.1 Glucosamine 6-P Deaminase (Gnp) 8.5.2 Glucosamine 6-P N-acetylase (Gna) 8.5.3 Phospho N-acetylglucosamine Mutase (Pgm) 8.5.4 UDP N-acetyl Glucosamine Pyrophosphorylase (Uap) 8.5.5 UDP-N-acetylglucosamine 4′-epimerase (Uae) 8.5.6 Cyst Wall Synthase (Cws) 8.6 Regulation and Inhibition 8.7 Metabolism and Drugs 8.8 Comparative Biochemistry and Metabolism 8.9 Uridine/Thymidine Phosphorylase Activity (URTPase) 8.10 Metabolomics References

XI

10

8.3.2

Section III Cellular Biology of Giardia 9

The Ultrastructure of Giardia During Growth and Differentiation

129 129

130 130 130 130 131 132 132 132 133 133 134 135 136

139

Abstract 9.1 Introduction 9.2 The Cell Surface 9.3 The Cytoskeleton 9.4 The Flagella 9.5 The Ventral or Adhesive Disc 9.6 The Median Body 9.7 The Funis 9.8 Microfilaments 9.9 The Endocytic System 9.10 The Secretory System 9.11 Glycogen Particles 9.12 Mitosomes 9.13 The Interphasic Nuclei 9.13.1 The Two Nuclei Present Slight Differences 9.13.2 The Nuclei in Division 9.14 Karyokinesis and Disc Participation 9.15 The Fine Structure of the Encystation Process 9.16 The Cyst 9.17 The Fine Structure of the Excystation Process Acknowledgments References

Abstract 10.1 Introduction 10.2 The Cell Cycle Throughout Giardia’s Life Cycle 10.3 Cell Division of Giardia Trophozoites 10.4 Mitosis 10.4.1 Mechanism of Chromosome Segregation and Mitosis 10.4.2 Implications of Mode of Mitosis on Nuclear Inheritance and Heterozygosity 10.5 Division of Microtubule Cytoskeleton 10.6 Division of the Flagellar Apparatus 10.6.1 Parent Flagella Distribution 10.6.2 Transformation of Parent Flagella during Division 10.6.3 De Novo Assembly of Daughter Flagella 10.6.4 Maturation of Flagella 10.6.5 Developmental Asymmetry of Microtubular Roots of Caudal Flagella 10.7 Ventral Disc 10.7.1 Parent Ventral Disc Disassembly 10.7.2 De Novo Assembly of Daughter Ventral Discs 10.8 Cytokinesis 10.9 Asymmetry and Aging in Giardia Division 10.10 Conclusions References

11

141 141 144 147 148 149 149 150 150 150 151 152 152 152

The Giardia Mitosomes

161 161 162 162 163 163 168 168 168 170 170 172 173 173 174 175 176 178 180 180 180

185

Jan Tachezy and Pavel Doležal

141

Marlene Benchimol and Wanderley De Souza

161

Scott C. Dawson, Eva Nohýnková and Michael Cipriano

129 129 130

Cell Cycle Regulation and Cell Division in Giardia

Abstract 11.1 Introduction 11.2 Morphology and Cellular Distribution 11.3 Protein Targeting, Translocation, and Maturation 11.3.1 Protein Targeting 11.3.2 Mitosomal Processing Peptidase 11.3.3 Protein Import Machinery 11.4 FeS Cluster Assembly Machinery 11.5 Energy Metabolism and Membrane Potential 11.6 Interaction with Other Cellular Structures 11.7 Perspectives Acknowledgements References

12

185 185 187 187 187 188 189 193 194 195 196 197 197

Signaling Pathways in Giardia lamblia 201 Tineke Lauwaet and Frances D. Gillin

153 153 153 153 156 156 157 157

Abstract 12.1 Introduction 12.2 Giardia Phosphatases and Kinases 12.3 Signaling in the Cell Cycle 12.4 Signaling in the Life Cycle 12.4.1 Encystation 11.4.2 Excystation

201 201 201 204 205 205 207

XII

Contents

12.5 Conclusion Acknowledgements References

13

Transcription and Recombination in Giardia

207 208 208

16

14

Intracellular Protein Trafficking

211 211 211 211 212 213 213 213 214 215 217

219

Adrian B. Hehl Abstract 14.1 Introduction 14.2 Organelles and Machineries of the Membrane Transport System 14.2.1 Endoplasmatic Reticulum and Golgi Apparatus 14.2.2 Peripheral Vesicles and Endocytic Transport 14.3 Secretory Transport During Growth and Differentiation 14.3.1 Proliferating Trophozoites 14.3.2 Encysting Trophozoites 14.3.3 Excysting Parasites 14.4 Summary References

15

Post-transcriptional Gene Silencing and Translation in Giardia

219 219

223 223 225 228 228 228

233

234 235 235 236 237 238 239 239

245 245 246 247 247 247 249 249 252 253 254 255

259

17

261

Interaction of Giardia with Host Cells

Guadalupe Ortega-Pierres, Maria Luisa Bazán-Tejeda, Rocio Fonseca-Liñán, Rosa María Bermúdez-Cruz and Raúl Argüello-García Abstract 17.1 Introduction 17.2 Trophozite Adhesion: What is In, What is Out 17.3 Ancestral Structural Proteins in a Very Evolved Adhesion Apparatus 17.4 Molecular Factors Involved in Giardia Adhesion to Host Cells 17.5 Consequences of Giardia-Host Cell Interactions 17.6 Conclusions 17.6 Acknowledgments References

18 233 233 234

245

Section IV Pathology, Treatment and Diagnostics of Giardia and the Host Immune Response

220

Pablo R. Gargantini, César G. Prucca, and Hugo D. Luján Abstract 15.1 Introduction 15.2 The RNAi Pathway in Giardia 15.2.1 The Mechanism of RNAi: General Features 15.2.2 The RNAi Machinery 15.2.2.1 Dicer 15.2.2.2 Argonaute Proteins 15.2.2.3 RNA-dependent RNA-polymerase (RdRP) 15.2.3 Small RNA Molecules in Giardia and Their Putative Biological Functions 15.3 The Translational Machinery 15.3.1 Is This Short Enough?

Abstract 16.1 Introduction 16.2 Switching Characteristics 16.3 Basic Description of VSPs 16.4 Genomic Organization 16.5 Spatial Organization, Antigenicity, and Motifs 16.6 Differences Among VSPs 16.7 Immune Responses to VSP and Immune Selection 16.8 Biological Selection 16.9 VSP Secretion 16.10 Control of Antigenic Variation References

220

222

Antigenic Variation in Giardia

240 240 242

Theodore E. Nash

211

Rodney D. Adam Abstract 13.1 Transcription 13.1.1 Overview 13.1.2 General Transcription Factors 13.1.3 RNA Polymerases 13.1.4 Specific Transcription Factors and Regulated Transcription 13.1.5 Promoters 13.1.6 Modification of mRNAs 13.2 Genome Structure 13.3 DNA Replication and Recombination References

15.3.2 A Prokaryotic Resemblance 15.3.3 Bridging the Gap References

Primary Microtubule Structures in Giardia

261 261 262 264 265 268 270 270 270

275

Scott C. Dawson Abstract 18.1 Introduction 18.2 Molecular Components of the Cytoskeleton 18.2.1 The Role of the Ventral Disc in Giardial Attachment 18.2.2 The Structure and Putative Function of the Median Body 18.2.3 Flagellar Structure and Motility 18.2.4 Structure and Putative Function of Axoneme-Associated Elements 18.3 Flagellar Assembly and Interphase Flagellar Length Maintenance

275 275 277 287 290 292 293 294

Contents Duplication and Division of Cytoskeletal Structures 18.5 The Cytoskeleton and Encystation/Excystation 18.6 Perspectives References

XIII

18.4

19

295 295 296 296

Pathophysiological Processes and Clinical Manifestations of Giardiasis 301 Andre G. Buret and James Cotton

Abstract 19.1 Introduction 19.2 Clinical Manifestations of Giardiasis 19.3 Chronic Gastrointestinal Disorders Associated with Giardiasis 19.4 Pathophysiological Processes Causing Symptoms in Giardiasis 19.4.1 Giardia Promotes Excessive Enterocyte Apoptosis 19.4.2 Giardia Disrupts Intestinal Barrier Function 19.4.3 Giardia Induces a Diffuse Shortening of Brush Border Microvilli and Causes Electrolyte Transport Abnormalities 19.5 Role of Parasitic Factors in the Pathogenesis of Giardiasis 19.6 Role of Host Factors in Pathogenesis 19.7 Conclusion References

20

Immunology of Giardiasis

301 301 302 303 305 306 307

308 309 312 312 312

319

Steven M. Singer Abstract 20.1 Infections in Humans 20.2 Infections in Animals 20.3 The Antibody Response 20.4 The Cellular Immune Response 20.5 Cytokines in Giardiasis 20.6 Innate Immunity 20.7 Anti-parasite Effector Mechanisms 20.7.1 Defensins 20.7.2 Nitric Oxide 20.7.3 Mast Cells 20.8 Gut Ecology 20.9 Summary References

21

Vaccination Against Giardia

319 319 320 320 322 323 323 325 325 326 326 328 328 328

333

Peter Lee, Aws Abdul-Wahid and Gaétan Faubert Abstract 21.1 Introduction 21.2 Targetting Transmission versus Pathology 21.2.1 Factors to Consider Before Developing Anti-Giardia Vaccine for Developing Countries 21.2.2 Factors to Consider Before Developing Anti-Giardia Vaccine for Developed Countries

333 333 334

334

335

21.3 Candidate Antigens for a Vaccine Against Giardia Pathology 335 21.3.1 The Heat Shock Proteins (HSPs) 336 21.3.2 The Lectins 336 21.3.3 The Giardins 336 21.3.4 The Tubulins 336 21.3.5 Variant Surface Proteins (VSPs) 336 21.4 Vaccines Designed to Reduce Pathology 337 21.4.1 Introduction 337 21.4.2 Hurdles in Constructing a Vaccine Using Giardia Trophozoite Proteins 337 21.4.3 Immune Responses Required for Reducing the Pathology 338 21.4.4 Success or Failure of Vaccine in Reducing the Pathology 338 21.5 Transmission-blocking Vaccines Against Giardia Using Cyst Wall Protein 2 339 21.5.1 Biochemical Composition of the Cyst Wall 340 21.5.2 Local Immune Response to CWPs 341 21.5.3 Use of rPro-CWP2 as an Oral Vaccine 342 21.5.4 Lactic Acid Bacteria (LAB) as a Live TBV Delivery Vehicle 343 21.5.5 Efficacy of the TBV Using a DNA Vaccine Strategy 348 References 349

22

Diagnosis of Human Giardiasis

353

Huw V. Smith and Theo G. Mank Abstract 22.1 Introduction 22.1.1 Early Studies and Their Impact on Diagnosis 22.2 Giardia Diagnosis 22.2.1 Giardia and Human Giardiasis 22.2.2 Symptoms and Basis for Laboratory Investigations 22.2.3 Giardia Species and Assemblages 22.3 Brightfield, Phase Contrast (PC) and Differential Interference Contrast (DIC) Microscopy 22.3.1 Micrometry 22.3.2 Trophozoite Morphometry and Morphology 22.3.3 Cyst Morphometry and Morphology 22.4 Rationale for Laboratory Diagnosis of Infection 22.5 Examination for Trophozoites and Cysts in Un-Concentrated (Direct) Stool Samples 22.6 Concentration of Cysts From Faeces 22.6.1 Biophysical Methods 22.6.1.1 Formol-Ether (Ethyl Acetate) Concentration 22.6.2 Centrifugal Flotation 22.7 Giardia Requests as Part of an Enteropathogenic Parasite Screen 22.7.1 Triple Faeces Test (TFT) 22.7.1.1 Preparation of Chlorazol Black Stain 22.8 Microscopical Examination of Samples

353 353 354 353 354 355 356 357 357 358 359 359 361 361 361 362 363 364 364 365 366

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Contents

22.9 Infection in the Absence of Detectable Cysts 22.10 Immunomagnetic Separation (IMS) for Giardia Cysts 22.11 Permanent Staining – Detection of Giardia Trophozoites and Cysts in Faecal Smears by Giemsa Staining 22.11.1 Method 22.12 Immunological Methods 22.12.1 Antigen Detection Using Antibodies Labelled with Fluorescent Reporters 22.12.2 Antigen Detection Using Antibodies Labelled with Enzyme and Other Reporters 22.12.2.1 Enzyme Immunoassays 22.12.2.2 Lateral Flow Immunochromatographic (Dipstick) Assays 22.13 Sensitivity of Detection in Faeces 22.14 Antibody Detection 22.15 Biopsy 22.16 Molecular Diagnosis – Nucleic Acid Detection Methods 22.16.1 Extraction of G. duodenalis DNA from Stools 22.16.2 Primer, Gene Locus Selection, PCR and Mixed Infections 22.16.2.1 Molecular Diagnosis in Routine Clinical Practice 22.16.3 Reporting Results of PCR-RFLP/ Sequencing Examination 22.17 Shipping of Cysts and Cyst DNA for Quality Assurance and Round Robin Testing References

Section V Methods for Giardia Research 23

Methods for Giardia Culture, Cryopreservation, Encystation, and Excystation In Vitro

368

Synchronization of Giardia

395

Karin Troell and Staffan Svärd 368

369 369 370 370

370 370

370 371 371 372

Abstract 24.1 Introduction 24.2 Materials 24.2.1 Cell Culture 24.2.2 Whole Culture Synchronization 24.2.3 Flow Cytometry 24.2.3.1 Fixing Cells 24.2.3.2 Wash and DNA Labeling 24.3 Methods 24.3.1 Cell Culture 24.3.2 Measure of Generation Time 24.3.3 Whole Culture Synchronization 24.3.4 Flow Cytometry 24.3.4.1 Fixing Cells 24.3.5 Wash and DNA Labeling 24.4 Notes References

395 395 397 397 397 398 398 398 398 398 398 398 399 399 399 400 400

372

25 373

Methods for Giardia Transfection and Gene Expression

401

Janet Yee and Joella Joseph 373 374 374 374 374

279

381

Barbara J. Davids and Frances D. Gillin Abstract 23.1 Introduction 23.2 Materials 23.2.1 General Considerations 23.2.2 Growth Medium and Cultivation of Giardia Trophozoites In Vitro 23.2.3 Cryopreservation of Giardia Trophozoites Grown In Vitro 23.2.4 Encystation of Giardia In Vitro 23.2.5 Ideas to Optimize Encystation Efficiency, if Needed 23.2.6 Excystation of Giardia In Vitro Acknowledgements References

24

381 381 383 383 383 385 386 391 391 393 393

Abstract 25.1 Giardia Transfection 25.1.1 DNA versus RNA Constructs 25.1.2 Transient versus Stable Transfection 25.1.3 Puromycin versus Neomycin for Drug Selection for Stable Transfection 25.2 Gel-shift Assays 25.2.1 Preparation of Giardia Nuclear Extracts 25.2.2 Preparation of Probes (for Radioactive and Non-radioactive Detection of Signals) 25.2.3 Preparation of Membrane for Non-radioactive Detection of Gel-shifts 25.3 Identification of Transcription Initiation Sites 25.3.1 Primer Extension, S1 Nuclease Protection, and 5′ RACE 25.3.2 Nuclear Run-on References

26

Biological Resource Centers for Giardia Research

401 401 401 402 404 404 404

406 407 408 408 409 410

413

Robert Molestina and Hugo D. Luján Abstract 26.1 Collection of Giardia Strains at the ATCC 26.2 The BEI Research Resources Repository References

List of Contributors

413 413 413 416

417

Section I Taxonomy and Epidemiology of Giardia

Taxonomy of Giardia Species R. C. Andrew Thompson and Paul T. Monis

Abstract The taxonomy of Giardia has been controversial for well over 100 years, resulting in a confusing nomenclature with different names often being used for the same species. This has led to uncertainty in our understanding of the epidemiology of Giardia infections, and particularly the question of host specificity and zoonotic transmission. The lack of morphological characters on which to base a species level taxonomy for the forms of Giardia that infect mammals has not allowed these issues to be resolved. It is only recently that PCR-based tools have been developed and applied directly to isolates of Giardia from a range of mammalian species. As a consequence, the taxonomy of Giardia can now be revised providing a more effective platform for epidemiological studies and importantly, improving communication between researchers in the field.

1.1 Taxonomic Recognition

1

1981, Class Trepomonadea (Cavalier Smith, 1993), order Diplomonadida (Wenyon, 1926 emend Brugerolle, 1975) and family Hexamitidae. Members of this family are diplozoic-flagellated protozoa that possess paired organelles, including two similar, transcriptionally active diploid nuclei, the absence of mitochondria and peroxisomes and a unique attachment organelle – the ventral (“adhesive”) disc (Kabnick and Peattie, 1990; Morrison et al., 2007). It is this ventral disc that serves as the principle distinguishing character that separates Giardia from other members of the Hexamitidae. It is a structure supported by a cytoskeleton of microtubules, microfilaments and associated fibrous structures. The ventral disc of Giardia has been shown to be composed of a variety of cytoskeletal proteins, principally tubulin (both D and E subunits) and closely related proteins called giardins (Ankarklev et al., 2010). Surrounding the disc is a marginal groove and ventrolateral flange, although not all isolates of Giardia possess a complete flange, e.g., G. psittaci from the budgerigar, Meliopsittacus undulatus.

1.1.1 Introduction 1.1.2 Generic Names The discovery of the parasite we now know as Giardia by Antony van Leeuwenhoek in 1681 provided biologists and clinicians with a truly unique organism for study. Perhaps the most controversial area of research has been that of taxonomy. Although possessing an unusual and distinctive set of morphological features that separate this parasite from all other protozoa, Giardia’s wide host range and lack of morphological features to measure host specificity have resulted in years of debate and confusion for which molecular tools are only now helping to resolve. Giardia belongs to the Phylum Metamonada Grassé, 1952 stat. nov. et emend. Cavalier-Smith,

H. D. Luján et al. (eds.), Giardia © Springer-Verlag/Wien 2011

The generic name Giardia was first established by Kunstler in 1882 for a flagellate found in the intestine of tadpoles of anuran amphibians. Six years later, Blanchard (1888) put forward the suggestion that Lamblia should be the generic name in commemoration of the parasite’s first accurate description by Lambl (1859). Unfortunately this suggestion was not challenged until 55 years, later when Alexeieff (1914) argued that there was no taxonomic rationale for Lambl’s suggestion and synonymised Lamblia Blanchard, 1888 and Giardia Kunstler, 1882, a proposal accepted by the majority of early workers (Kofoid

4

and Christiansen, 1915; Kofoid, 1920; Hegner, 1922; Dobell, 1940). However, the fact that many years elapsed before Alexeieff re-established Giardia as the correct generic name meant that “Lambl” became somewhat entrenched in the field at the time, and has “lingered” ever since as discussed below.

1.1.3 Species Names The first detailed description of Giardia was given by Lambl (1859) for a flagellate in the human intestine which he named Cercomonas intestinalis. However, as pointed out by Filice (1952), this name was preempted in toto by the transfer, nine years earlier, of Bodo intestinalis Ehrenberg into the genus Cercomonas Dujardin by Diesing (1850). Thus, according to the International Code of Zoological Nomenclature (prior to 1961), both the generic and specific names given by Lambl fall into homonymy (Filice, 1952). Workers obviously accepted that Cercomonas was not the correct generic name, as illustrated by the subsequent description of the same flagellate in tadpoles by Kunstler (1882), Giardia agilis. Seven years before Kunstler’s description, Davaine (1875) described a form of Giardia in the rabbit which he called Hexamita duodenalis. Although the generic name ascribed to this parasite was clearly not correct, Filice (1952) proposed that the specific name used by Davaine should be retained as a valid name for the form of Giardia in the rabbit. This is a very important observation since if a single specific name is to be used for forms of Giardia in humans and other mammals, then duodenalis has priority over intestinalis according to the Rules of Zoological Nomenclature. Indeed, Stiles (quoted by Filice, 1952) stated that “If you look upon the form in the rabbit as identical with that in man, duodenalis would be the correct name. If you consider the various forms in man, rabbits, rats, etc. as distinct, then in all probability a new name should be suggested for the form that occurs in man”. This quote by Stiles refers to the various forms of Giardia occurring in different hosts. Indeed, since 1859, a total of 51 species of Giardia have been described including two in humans and 28 in other mammals, one in fish, 14 in birds, four in amphibians and two in reptiles (Thompson and Monis, 2004).

R. C. A. Thompson and P. T. Monis

1.1.4 Taxonomic Uncertainty In a seminal and influential contribution to the field, Filice (1952) re-evaluated the species-level taxonomy of Giardia, and in particular critically examined available differential criteria, host occurrence and morphology, and concluded that with the data available at that time “it would be valueless to name species on the basis of host differences”. After rejecting host specificity because of the lack of any reliable experimental evidence, he undertook a thorough reappraisal of which morphological characters could be used as reliable means for differentiating species. He concluded that described species of Giardia could be divided into only three morphologically distinct groups. Differentiation was based primarily on the shape of the characteristic internal median bodies, as well as body shape and length. Filice (1952) concluded that within these three groups, there might well be morphologically similar forms exhibiting distinct physiological characteristics, but that their taxonomic status awaited the advent of more refined and discriminatory methodology. The soundly based, reproducible and logical scheme proposed by Filice (1952) found widespread favour and forms the basis of a widely accepted taxonomy that has provided stability until molecular data have provided the basis for another taxonomic appraisal (see below).

1.1.5 The Three Species Filice (1952) concluded that only consistent morphological differences should be used as the basis for defining species; these being the overall shape of the trophozoite and the median bodies, of which the latter are considered the most valuable (Bemrick, 1962, 1984; Meyer and Radulescu, 1979; Bertram et al., 1984). Using these criteria, Filice split Giardia into three morphologically distinct species, or groups, two of which infect mammals. The first group of organisms, G. duodenalis, have pyriform-shaped trophozoites which possess a distinctive “claw-hammer” median body, infect a variety of mammals, including humans. Although on the grounds of zoological nomenclature the specific name duodenalis would appear to be correct, the names intestinalis and even lamblia are often used as syn-

Chap. 1 Taxonomy of Giardia Species

5

onyms, particularly for isolates of human origin. This appears to be on the basis of preference rather than taxonomic grounds (see above) and has created unnecessary confusion and controversy. Many authors have emphasised, most notably Meyer (1985) that there is no taxonomic justification other than personal preference, for using the names intestinalis or lamblia and the use of these other names for the “duodenalis” group suggests that there is something unique about these parasites, compared to G. duodenalis, which is clearly not the case. Members of the second G. muris group, have rounded median bodies, with a rounder trophozoite shape, and primarily infect rodents, whereas trophozoites of G. agilis have long, narrow bodies, relatively short adhesive discs, long, club-shaped median bodies, and have only been isolated from amphibians. Filice placed only one described species in the “agilis” group, G. agilis, a parasite of amphibians. It has also been suggested that some forms described from birds, including G. sanguinis, G. ardeae and G. hydebaradensis, probably should be included in the muris group (Filice, 1952; Kulda and Nohýnková, 1978). Other described forms, including at least 20 described species from mammals and some from birds, were placed into the “duodenalis” group, but the status of forms from reptiles and fish remains unclear.

size of the ventral disc, a notch in the caudal region of the ventral disc, and a single caudal flagellum, are similar to those of G. muris (Erlandsen et al., 1990; McRoberts et al., 1996). The nuclei of G. ardeae are long, slender and tear-drop in shape, unlike those of either G. duodenalis or G. muris, which are rounded (Erlandsen et al., 1990). The mixture of morphological features suggests an interesting origin for G. ardeae. Molecular data from the 18S rRNA and trios phosphate isomerase genes show incongruence in both the placement of G. ardeae relative to G. muris and the branch lengths separating them (Monis et al., 1999), suggesting that there has been gene transfer. An additional species from mammals, Giardia microti, was described on the basis of cyst morphology (Feely, 1988). The cysts of G. microti contain two differentiated trophozoites with mature ventral discs, whereas the cysts of G. duodenalis contain a single trophozoite which has four nuclei and lacks a ventral disc. Although originally isolated from Microtus ochrogaster, G. microti has also been recovered from a variety of other rodents (e.g., Erlandsen et al., 1988).

1.1.6 Additional Species

The occurrence of morphological variation within G. duodenalis has been reported on numerous occasions (Bertram et al., 1984; Monis and Andrews, 1998). This variation has usually involved differences in body dimensions and/or shape. The value of such variation as discriminatory and taxonomic criteria has been extensively reviewed (Thompson et al., 1990; Thompson and Monis, 2004). Apart from the characters of size and shape which appear to be of limited value for differentiating species, the major issue has been one of the isolate variability and questionable statistical analyses (reviewed in Thompson et al., 1990). As described above, morphological variation at the ultrastructural level has proved useful as a taxonomic tool in Giardia, particularly with respect to the ventral disc and the caudal flagella. Erlandsen and Bemrick (1987) reported differences in a discrete morphological character associated with the ventral disc, between isolates of G. duodenalis. However, a study by Binz

In addition to the three species, or morphological groupings, that Filice (1952) proposed, subsequent ultrastructural characterisation of Giardia isolates from birds has demonstrated morphological features that are considered to warrant species recognition for two forms: Giardia psittaci (Erlandsen and Bemrick, 1987) and Giardia ardeae (Erlandsen et al., 1990). Giardia psittaci described from budgerigars are morphologically similar to G. duodenalis, possessing claw-hammer median bodies and pyriform trophozoite shape, but they lack a complete ventrolateral flange and marginal groove (Erlandsen and Bemrick, 1987). Giardia ardeae share morphological features with G. duodenalis and G. muris (Erlandsen et al., 1990). The median bodies in trophozoites of G. ardeae, described from herons and ibis, vary in shape and orientation compared to G. duodenalis whereas the

1.2 Phenotypic Variation 1.2.1 Inter- and Intraspecific Morphological Variation

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(1996) compared 12 isolates of Giardia from humans that all conformed to the G. duodenalis morphological group on the basis of Filice’s (1952) system but which exhibited both genotypic and phenotypic differences. Using scanning electron microscopy, trophozoite length was shown to vary from 13.5 mm to 18.4 mm but was found to be variable with time. In contrast, caudal flagellar length remained constant varying from 4.6 mm to 16.1 mm between isolates (Binz, 1996).

1.2.2 Host Specificity Host occurrence was a major factor supporting the description of most species during the 1900s. However, the lack of reliable supportive morphological data, and problems with the interpretation of crossinfection experiments, has meant that host specificity could not be regarded, until recently, as a reliable phenotypic trait. This has had an important influence not only on Giardia taxonomy but also on the ongoing debate as to whether giardiasis is a zoonosis. Although the results of experimental cross-infection experiments questioned the notion of host adapted species as a tenable criterion for species recognition, it is now clear that some species may be host specific whereas others are capable of infecting a broad range of host species (see below). To this end, numerous cross-transmission experiments have been undertaken for both taxonomic and epidemiological reasons to determine whether G. duodenalis is strictly host specific and to elucidate whether humans may be susceptible to infection with isolates of G. duodenalis from other animals. The majority of experiments have involved trying to establish infection with human isolates of Giardia in a variety of animal species and very few experiments have involved the attempted infection of humans with isolates from other animals (reviewed in Monis and Thompson, 2003; Thompson and Monis 2004). There has been great variability in results among different laboratories, and the accurate interpretation of data has been difficult due largely to procedural factors (for example, differences in the number and age of cysts dosed; the use of isolates that have not been genetically characterised) and the unknown contribution of host and/or parasite factors to the results.

R. C. A. Thompson and P. T. Monis

1.2.3 In vitro and In vivo Studies Following the development of axenic culture techniques, workers reported variability in success in being able to establish isolates of Giardia of human origin. This not only suggested differences between isolates in their ability to multiply in defined culture media but also led to concern that those isolates that could be amplified and studied genetically would not be representative of the gene pool. Apart from humans, there have been no reports of the successful axenisation of dog-derived G. duodenalis which appear to be refractory to culture in vitro, and there has been limited success with isolates from livestock (Meloni et al., 1987; Ey et al., 1997). Once established in culture, isolates of human origin show variation in growth rates. Binz demonstrated that the mean generation times obtained for 12 isolates in vitro varied from 6.6 h to 24.5 h (Binz, 1996). These 12 isolates also varied in their DNA content, from 0.060 pg per trophozoite to 0.165 pg per trophozoite, representing a 2.75-fold difference. These results may help to explain why workers have been unable to establish some isolates of G. duodenalis in in vitro culture. They are also potentially very significant for the epidemiology and clinical outcomes of Giardia infection. There is some evidence to suggest that the so-called “slow growing” isolates of G. duodenalis may be more persistent in vivo in terms of their involvement in chronic infections and association with nutritional and allergic disorders, as well as being more refractory to chemotherapy (Thompson, 2002; Thompson and Lymbery, 1996; Homan and Mank, 2001). Differences in growth rates and intestinal distribution are also likely to influence the outcome of competitive interactions in situations where infection levels and the frequency of transmission are high and multiple genotypes are likely to coexist in endemic foci (Thompson and Lymbery, 1996; Thompson et al., 1996; Monis et al., 1998). Following the delineation of genetic groupings, or assemblages, within G. duodenalis (see below) it became clear that different culture conditions selected for a particular genotype from a mixture of genotypes. In particular, Assemblage A isolates appear to have a selective advantage under axenic in vitro culture conditions compared with Assemblage

Chap. 1 Taxonomy of Giardia Species

B isolates and vice versa for passage in suckling mice (Andrews et al., 1992a; Binz et al., 1992; Thompson and Lymbery, 1996). All of the recognised genetic groups of G. duodenalis can be propagated by experimental infection of suckling mice and differences in the growth patterns of isolates have been observed. Assemblage A, B and F isolates rapidly adapt to growth in suckling mice, but Assemblage C and D isolates grow erratically (Mayrhofer et al., 1992; Ey et al., 1997; Monis et al., 1998). The differences in the growth of different genotypes in vitro and in suckling mice have important implications for the culture of samples containing a mixture of genotypes, as selection of specific genotypes can occur (Andrews et al., 1992a; Thompson et al., 1996). The metabolic and culture requirement differences that exist between the genetic groups are likely to reflect the host-preference that some of these groups exhibit. Differences in metabolism and biochemistry, drug sensitivity, predilection site in vivo and duration of infection, pH preference and susceptibility to infection with a dsRNA virus have been found to correlate with genetic differences (Andrews et al., 1992b; Binz et al., 1992; Hall et al., 1992; Farbey et al., 1995; Binz, 1996; Monis et al., 1996; Thompson et al., 1996; Reynoldson, 2002).

1.2.4 Infectivity and Clinical Disease A variety of studies have compared the infectivity of different isolates of Giardia and have demonstrated marked differences (Visvesvara et al., 1988; Andrews et al., 1992a; Williamson et al., 2000). Similarly, there have been a number of reports describing differences in laboratory infections established in rodents between isolates recovered from humans exhibiting variable symptomatology (Aggarwal and Nash, 1987; Astiazaran-Garcia et al., 2000). Unfortunately, in most cases the observed phenotypic differences have not been shown to have a genetic basis or the isolates were not genetically characterised or the methods used did not allow correlation of the genotype with the major genetic assemblages currently recognised. More recent studies have demonstrated conflicting results for the correlation between disease and genotype in humans. A longitudinal study in day-

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care centres in Perth, Western Australia, found that children infected with isolates of Giardia belonging to Assemblage A were 26 times more likely to have diarrhoea than children infected with Assemblage B isolates (Read et al., 2002). In contrast, a survey conducted in Ethiopia found a significant correlation between symptomatic infection and the presence of Assemblage B (Gelanew et al., 2007). A similar correlation was reported by Homan and Mank (2001) with Assemblage B isolates associated with persistent diarrhoea, whereas Assemblage A infections were associated with intermittent diarrhoea. However, in a case-control study in Bangladesh, Haque et al. (2005) reported that, although Assemblage B was the most prevalent and had the highest parasite burden, patients infected with Assemblage A (genotype A2) had the highest probability of developing diarrhoea. Similarly, Sahagun et al. (2008) also found a strong correlation between symptomatic infection and Assemblage A2 in patients from Spain. Interestingly, the proportion of asymptomatic: symptomatic infections with Assemblage A was similar for all three of these latter studies (62% Gelanew et al. (2007); 57% Haque et al. (2005); 67% Sahagun et al. (2008) symptomatic). The key difference was that all detected Assemblage B infections in Gelanew et al. (2007) were associated with diarrhoea, compared with 16% of infections resulting in diarrhoea in Haque et al. (2005) and 42% in Sahagun et al. (2008). One factor that was not considered was the degree of genetic variation within Assemblage B, which could possibly account for the differences between the studies. It is also likely that the outcome of infection is a complex phenotype and that host factors will also affect the development of disease. In non-human hosts, an avian isolate of G. duodenalis exhibiting aggressive pathogenesis has also been shown experimentally to establish infections in domestic kittens and lambs (McDonnell et al., 2003) but the isolate has yet to be characterised genetically with respect to the known assemblages. A study by Geurden et al. (2008), found the prevalence of Assemblages A and E in dairy calves (59% and 41% respectively) to be different to that in beef calves (16% and 84%, respectively). Assemblage E was more frequently detected (74% of cases) in calves with clinical disease compared to Assemblage A (26% of cases).

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1.3 Phylogenetic Relationships Giardia has long been of interest to evolutionary biologists, being a member of a lineage of organisms that has provided insight into the evolution of eukaryotic cells. The earliest work examined the phylogenetic position of Giardia using ribosomal RNA sequences, showing that Giardia possess an unusual ribosomal RNA and belong to an early branching lineage of eukaryotes (Edlind and Chakraborty, 1987; Sogin et al., 1989). Phylogenetic studies were extended to the Diplomonadida (van Keulen et al., 1993) and the early branching of Giardia confirmed using other conserved loci such as the elongation factors (Hashimoto et al., 1994, 1995) and cyclin-dependent kinases (Riley and Krieger, 1995). This placement led to the assumption that Giardia is a primitive organism with a pivotal position in the evolution of eukaryotes. However, this view is now less well supported. Phylogenetic analysis using morphological characters suggests that Giardia is one of the most highly adapted members of the Diplomonadida (Siddall et al., 1992). More recently, molecular studies have demonstrated that supposed primitive features, such as the absence of mitochondria, are due to secondary loss rather than divergence from an ancestral eukaryote prior to the acquisition of such organelles (Embley and Hirt, 1998; Hashimoto et al., 1998; Roger et al., 1998). The loss of mitochondria has been proposed to predate the divergence between diplomands and parabasalids (Roger et al., 1999). Giardia and possibly diplomands in general appear to have acquired genes via lateral gene transfer from a variety of sources, including bacteria (Morrison et al., 2001; Nixon et al., 2002; Andersson et al., 2003) and archaea (Suguri et al., 2001) complicating the placement of Giardia within the “tree of life” and requiring the use of multiple loci for phylogenetic analyses. While a great deal of attention has been given to determining the evolutionary history of the genus Giardia, the phylogenetic relationships within G. duodenalis were not examined until the late 1990s, when Monis et al. (1999) used four loci (amplified regions of the genes encoding glutamate dehydrogenase, triose phosphate isomerase, elongation factor 1 D and small subunit ribosomal RNA) to examine the phylogeny of the major assemblages. In addition to DNA sequence data, this study also used genetic data

R. C. A. Thompson and P. T. Monis

generated by enzyme electrophoretic analysis of 23 loci. This study demonstrated general agreement between the relationships inferred from the enzyme data and the DNA sequence data. The distances separating some of the assemblages (e.g. A versus B) were greater than those separating some genera of bacteria. Neighbour Joining analysis of enzyme electrophoretic data from isolates from diverse hosts has provided evidence of further sub-structuring within the recognised assemblages, some of which appear to correlate with host origin (Monis et al., 2003). The cluster of Assemblage A isolates from non-human mammalian hosts identified in this study that were external to the known AI and AII groups may be equivalent to the novel Assemblage A subtype described from deer (van der Giessen et al., 2006; Lalle et al., 2007), where in both cases the novel genotypes are external to the cluster containing AI and AII. The study by Monis et al. (1999), was based on a relatively small set of isolates, so it is important to note that the host-association exhibited by some of the assemblages has also been supported by additional independent molecular typing studies (e.g. Assemblage E and livestock (Sedinova et al., 2003; Trout et al., 2006), Assemblage F and cats (Souza et al., 2007), Assemblages C/D and dogs (Souza et al., 2007), Assemblages A and B and humans (Souza et al., 2007; Yason and Rivera, 2007; Lebbad et al., 2008). One area of interest regarding Giardia evolution has been the possible mechanisms of speciation. The available phylogenetic data provide no evidence for cospeciation of any Giardia species or assemblage with its particular hosts. In the case of mammals, dogs and cats are more closely related to each other than to artiodactylids, and all three are more closely related to each other than to rodents or primates. If cospeciation had occurred then a similar pattern would be expected among the assemblages. This suggests that host switching or host adaptation (or a combination of both) rather than co-evolution has been the basis for host specificity. It is fortunate that the initial genetic studies of G. duodenalis utilised multi-locus analysis. Recent studies have demonstrated genetic recombination between different isolates of G. duodenalis (reviewed by Caccio et al., 2005; and see below), which will confuse the interpretation of results based on a single locus. This means that future studies of the phylogeny of G. duodenalis will continue to

Chap. 1 Taxonomy of Giardia Species

require the use of multiple loci. Genotyping studies for epidemiology will also likely require the use of more than one locus to ensure correct identification.

1.4 Molecular Epidemiology and Host Specificity Of the 51 species of Giardia that have been described only six can be distinguished on the basis of morphological characters. The remaining species were described principally on the basis of host occurrence. However, demonstrating host specificity has continued, and continues to be one of the most problematic and controversial areas in the Giardia field. In particular, the question of zoonotic potential has been a major focus of much research. As emphasised above, cross-infection experiments have contributed little to elucidating this question or taxonomic recognition, although they questioned the notion of presumed host specificity as a tenable criterion for species recognition. We have had to wait many years to be close to resolving these issues with the advent of molecular tools that can reliably characterise isolates of Giardia genetically. In particular, it has been the ability to apply PCR-based tools directly to faecal or environmental samples, without a reliance on subsequent in vitro or in vivo laboratory amplification that has helped to address the question of host specificity between isolates of Giardia (Monis and Thompson, 2003; Thompson and Monis, 2004). Such an approach has demonstrated considerable genetic heterogeneity within the G. duodenalis morphological group, and epidemiologically has demonstrated that there are four main cycles of transmission in which host-specific and zoonotic assemblages of Giardia can be maintained in nature. Assemblages A and B can be maintained by direct transmission between humans; Assemblage E between livestock; Assemblage C/D between dogs; Assemblage F between cats; and novel wildlife genotypes between various wildlife species. However, Assemblage A and, to a lesser extent, Assemblage B, can also infect companion animals, livestock and wildlife and it is demonstrating the frequency of transmission of these two zoonotic assemblages between different hosts, including humans, which remains the biggest challenge in understanding the epidemiology of Giardia infections.

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The occurrence of G. duodenalis in wildlife was the most important factor initially demonstrating Giardia as a zoonotic agent. The link between infected wildlife such as beavers and waterborne outbreaks in people led the World Health Organization (WHO, 1979) to categorise Giardia infection as a zoonosis. It is therefore surprising that there is little evidence to support the role of wildlife as the original contaminating source of waterborne disease in humans (Thompson, 2004; Appelbee et al., 2005; Kutz et al., 2009; Thompson et al., 2009a). In such cases, epidemiological evidence suggests that Giardia infections in wildlife are more likely to be contracted through environmental contamination of human origin, or less likely, domestic animal origin, for example beavers and coyotes in North America, primates in Africa, muskoxen in the Arctic, house mice on remote islands, marsupials in Australia and marine cetaceans in various parts of the world (Graczyk et al., 2002; Moro et al., 2003; Sulaiman et al., 2003; Appelbee et al., 2005, 2010; Dixon et al., 2008; Kutz et al., 2008; Teichroeb et al., 2009; Thompson et al., 2009b, 2010). Some wildlife, particularly aquatic species, will thus serve to amplify the numbers of the originally contaminating isolate (Monzingo and Hibbler, 1987; Bemrick and Erlandsen, 1988; Thompson, 2004; Appelbee et al., 2005; Thompson et al., 2009a). G. muris from mice and isolates characterised from rats (G. simondi), microtine rodents (G. microti) and bandicoots are all genetically distinct and believed to be host specific. However, there is only limited information on host range, prevalence of infections and geographical distribution. In all cases the hosts of these species of Giardia are also susceptible to zoonotic genotypes. More recently, a novel genotype of Giardia was described in an Australian marsupial, a bandicoot known as the quenda (Isoodon obesulus), and on the basis of genetic characteristics would appear to represent a distinct species that may be endemic within Australian native fauna (Adams et al., 2004; Thompson and Monis, 2004; Thompson et al., 2010). The issue of whether G. duodenalis in companion animals can infect humans is perhaps the most controversial and emotive aspect of the zoonosis debate. Numerous prevalence surveys which have genotyped Giardia in different parts of the world have demonstrated that dogs and cats may be infected with either,

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or both, zoonotic and host-specific assemblages of Giardia (Leonhard et al., 2007; Thompson et al., 2007). These results emphasise the potential public health risk from domestic dogs and cats, but data on the frequency of zoonotic transmission are lacking (Thompson 2004; Leonhard et al., 2007). Molecular epidemiological studies in localised endemic foci of transmission have provided evidence in support of the role of dogs in cycles of zoonotic Giardia transmission involving humans and domestic dogs from communities in tea growing areas of Assam, India, temple communities in Bangkok, Thailand and northern Canadian aboriginal communities (Traub et al., 2004; Inpankaew et al., 2007; Salb et al., 2008). These studies demonstrated isolates of G. duodenalis from the same assemblage infecting dogs and their owners sharing the same living area. A limitation of these studies is that the molecular tools used for most studies have relatively poor genetic resolution and are unlikely to reliably detect genetic diversity or substructuring with the assemblages. As with companion animals, livestock can be infected with Giardia from zoonotic and host-specific assemblages and thus there is potential for giardial contamination of ground and surface waters from livestock operations. However, there is no epidemiological evidence that cattle represent a significant public health risk (Olson et al., 2004; Thompson, 2004; Hunter and Thompson, 2005). Molecular epidemiological studies in several countries have shown that cattle are most commonly infected with the non-zoonotic livestock Assemblage E (G. bovis), and although the zoonotic genotype Assemblage A (G. duodenalis) has been reported, studies in Australia suggest that zoonotic genotypes may only be present transiently in cattle under conditions where the frequency of transmission with G. bovis is high and competition is thus likely to occur (Becher et al., 2004; Thompson, 2004). However, a study in a localised area of Uganda showed that humans were likely to have introduced Giardia into a remote national park and to have been the source of Giardia infection in a small number of cohabiting dairy cattle, as well as gorillas (Graczyk et al., 2002). Genetic studies using enzyme electrophoresis have shown that Assemblages B and E have a large amount of genetic diversity, greater than that detected using the current suite of genes in PCR-based assays

R. C. A. Thompson and P. T. Monis

(Mayrhofer et al., 1995; Monis et al., 1999, 2003). Molecular tools with genetic resolution equivalent to the enzyme electrophoretic studies will be required to definitively demonstrate zoonotic transmission within Assemblage B, or to determine whether there are genetic groups within the assemblages that have different host preferences.

1.5 Taxonomic Certainty In 1952, Filice emphasised that his rationalisation of the species taxonomy of Giardia was only a temporary solution in the absence of valid discriminatory criteria other than morphology. He acknowledged that there was phenotypic evidence supporting the existence of different forms within the G. duodenalis morphological group. We now have appropriate, genetic discriminatory tools, and molecular characterisation of Giardia isolated from different host species has revealed the existence of a number of distinct genotypic assemblages (evolutionary lineages), some of which appear to have distinct host preferences (e.g. Assemblages C, F and G for dogs, cats and rats, respectively) or have a limited host range (e.g. Assemblage E for hoofed livestock, particularly cattle). As such, there is now sufficient information to revise the taxonomy of Giardia so that it reflects the biological and evolutionary differences within G duodenalis, particularly host specificity. Early workers on Giardia recognised such host specificity as reflected in the largely host-related nomenclature they proposed that now provides the basis for formalising a revised taxonomic nomenclature (Table 1.1), which is essential for effective communication at all levels. Thus the choice of species names reflects those originally proposed, in many cases 60–70 years ago. Although the descriptions provided varied in their detail, it is of little consequence given the lack of any useful morphological features to discriminate between variants of the G. duodenalis morphological group (reviewed in Thompson and Monis, 2004). In the case of Assemblages A and B, we proposed that they be referred to as G. duodenalis and G. enterica (a name proposed in 1920 by Kofoid for an isolate of Giardia in humans subsequent to Lambl’s description of Giardia in humans that was eventually named G. duodenalis). The genetic distance separating Assemblages A and

Chap. 1 Taxonomy of Giardia Species

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Table 1.1 Species in the genus Giardia Species

Hosts

Morphological characteristics

Trophozoite dimensions length/width (μm)

G. duodenalis (=Assemblage A)

Wide range of domestic and wild mammals including humans

Pear-shaped trophozoites with claw-shaped median bodies.

12–15/6–8

G. agilis

Amphibians

Long, narrow trophozoites with club-shaped median bodies.

20–30/4–5

G. muris

Rodents

Rounded trophozoites with small round median bodies.

9–12/5–7

G. ardeae

Birds

Rounded trophozoites, with prominent ~10/~6.5 notch in ventral disc and rudimentary caudal flagellum. Median bodies round-oval to claw shaped.

G. psittaci

Birds

Pear-shaped trophozoites, with no ventrolateral flange. Claw-shaped median bodies.

~14/~6

G. microti

Rodents

Trophozoites similar to G. duodenalis. Mature cysts contain fully differentiated trophozoites.

12–15/6–8

G. enterica (=Assemblage B)

Humans and other primates, dogs, some species of wild mammals

Pear-shaped trophozoites with claw-shaped median bodies.

12–15/6–8

G. canis (=Assemblage C/D)

Dogs, other canids

Pear-shaped trophozoites with claw-shaped median bodies.

12–15/6–8

G. cati (=Assemblage F)

Cats

Pear-shaped trophozoites with claw-shaped median bodies.

12–15/6–8

G. bovis (=Assemblage E)

Cattle and other hoofed livestock

Pear-shaped trophozoites with claw-shaped median bodies.

12–15/6–8

G. simondi (=Assemblage G)

Rats

Pear-shaped trophozoites with claw-shaped median bodies.

12–15/6–8

B is at the same level as that separating the other proposed species strongly suggesting that separate species names for each of these assemblages is warranted (Monis et al., 2009). This case is further strengthened considering the differences in in vitro and in vivo growth rates (see above). The fact that the genetic characteristics of the assemblages are maintained in sympatry in endemic areas where the cycles of transmission may overlap reinforces the argument that the assemblages represent separate species. However, it is unlikely that the revised taxonomy summarised in Table 1.1 will provide a definitive species level classification for Giardia. There is increasing recognition of genetic subgroupings within assemblages/species and this will be a focus of future research. In particular, it is

likely that some of the underlying substructure within Assemblages A and B will account for the apparently conflicting reports of different assemblages with different clinical outcomes.

1.6 In Conclusion – Sex and Stability Over a decade ago, population genetic studies of Giardia in endemic communities where the frequency of transmission is very high, found evidence of occasional bouts of genetic exchange in the parasite (Meloni et al., 1995). These authors demonstrated multiple banding patterns in a number of isolates of Giardia by allozyme electrophoresis which if a true reflection of the underlying genotypes of the isolates,

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would seem to indicate that G. duodenalis is functionally diploid, and that recombination or sexual reproduction must have occurred at some stage to produce the apparent heterozygotes (Meloni et al., 1995). These observations have been supported by more recent population genetic studies (Cooper et al., 2007) and molecular analyses which further challenge the idea that G. duodenalis is a strictly clonal, asexual organism by providing evidence of recombination between homologous chromosomes within and between assemblages (Lasek-Nesselquist et al., 2009). It has also been demonstrated that Giardia has maintained some meiotic machinery, the ability of chromosomes to cross-over and some recombination sites (Ramesh et al., 2005; Poxleitner et al., 2008). It has been suggested that the identification of recombination between assemblages suggests a shared gene pool and calls into question whether it is appropriate to divide the genetically distinct assemblages of G. duodenalis into a species complex. However, sexual reproduction has never been observed in Giardia which could be explained by extremely infrequent sexual reproduction (Ankarlev et al., 2010), as suggested by the population genetic data (Meloni et al., 1995; Cooper et al., 2007). The evolutionary advantage that genetic exchange gives to Giardia is the capacity to respond to adversity, for example selection pressures imposed by regular exposure to antigiardial drugs or competition with co-habiting “strains” in circumstances where the likelihood of mixed infections is common (Hopkins et al., 1999). Thus it may well be a relatively rare event and further population genetic studies are required in foci of infection where the frequency of infection is high. The fact that available data indicates that the genetic assemblages of Giardia are conserved in terms of geographic location and host occurrence suggests that any recombination is not reflected at the assemblage and species level.

References Adams PJ, Monis PT, Elliot AD, and Thompson RCA (2004) Cyst morphology and sequence analysis of the small subunit rDNA and ef1_identifies a novel Giardia genotype in a quenda (Isoodon obesulus) from Western Australia. Infect Genet Evol 4: 365–370 Aggarwal A and Nash TE (1987) Comparison of two antigenically distinct Giardia lamblia isolates in gerbils. Am J Trop Med Hyg 36: 325–332

R. C. A. Thompson and P. T. Monis Alexeieff A (1914) Notes protistologiques. Zoologischer Anzeiger 44: 193–213 Andersson JO, Sjogren AM, Davis LA, Embley TM, and Roger AJ (2003) Phylogenetic analyses of diplomonad genes reveal frequent lateral gene transfers affecting eukaryotes. Current Biol 13: 94–104 Andrews RH, Chilton NB, and Mayrhofer G (1992a) Selection of specific genotypes of Giardia intestinalis by growth in vitro and in vivo. Parasitology 105: 375–386 Andrews RH, Mayrhofer G, Chilton NB, Boreham PF, and Grimmond TR (1992b) Changes in allozyme pattern of the protozoan parasite Giardia intestinalis. Int J Parasitol 22: 403–406 Ankarklev J, Jerlstrom-Hultqvist J, Ringqvist E, Troell K, and Svard SG (2010) Behind the smile: cell biology and disease mechanisms of Giardia species. Nat Rev Microbiol 8: 413–422 Appelbee AJ, Thompson RCA, and Olson ME (2005) Giardia and Cryptosporidium in mammalian wildlife – current status and future needs. Trends Parasitol 21: 370–376 Appelbee AJ, Thompson RCA, Measures LM, and Olson ME (2010) Giardia and Cryptosporidium in harp and hooded seals from the Gulf of St. Lawrence, Canada. Vet Parasitol (in press) Astiazaran-Garcia H, Espinosa-Cantellano M, Castanon G, Chavez-Munguia B, and Martinez-Palomo A (2000) Giardia lamblia: effect of infection with symptomatic and asymptomatic isolates on the growth of gerbils (Meriones unguiculatus). Exp Parasitol 95: 128–135 Becher KA, Robertson ID, Fraser DM, Palmer DG, and Thompson RCA (2004) Molecular epidemiology of Giardia and Cryptosporidium infections in dairy calves originating from three sources in Western Australia. Vet Parasitol 123: 1–9 Bemrick WJ (1962) The host specificity of Giardia from laboratory strains of Mus musculus and Rattus norvegicus. J Parasitol 48: 287–290 Bemrick WJ (1984) Some perspectives on the transmission of giardiasis. In: Giardia and Giardiasis (S.L. Erlandsen and E.A. Meyer, eds.), Plenum Press, New York, pp 379–400 Bemrick WJ and Erlandsen SL (1988) Giardiasis is it really a zoonosis? Parasitol Today 4: 69–71 Bertram MA, Meyer EA, Anderson DL, and Jones CT (1984) A morphometric comparison of five axenic Giardia isolates. J Parasitol 70: 530–535 Binz N (1996) Phenotypic characteristics of differing genetic groups of Giardia duodenalis and their implications for species identification. PhD Thesis, Murdoch University, Western Australia Binz N, Thompson RCA, Lymbery AJ, and Hobbs RP (1992) Comparative studies on the growth dynamics of two genetically distinct isolates of Giardia duodenalis in vitro. Int J Parasitol 22: 195–202 Blanchard R (1888) Remarques sur le megastome intestinal. Bull Soc Zool France 30: 18–19 Brugerolle G (1975) Ultrastructure of the genus Enteromonas da Fonseca (Zoomastigophorea) and revision of the order of Diplomonadida Wenyon. J Protozool 22: 468–475 Caccio SM, Thompson RCA, McLauchlin J, and Smith HV (2005) Unravelling Cryptosporidium and Giardia epidemiology. Trends Parasitol 21: 430–437

Chap. 1 Taxonomy of Giardia Species Cavalier Smith T (1993) Kingdom Protozoa and its 18 phyla. Microbiol Rev 57: 953–994 Cooper MA, Adam RD, Worobey M, and Sterling CR (2007) Population genetics provides evidence for recombination in Giardia. Curr Biol 17: 1984–1988 Davaine C (1875) Monadiens. In: Dictionnaires encyclopedique des sciences medicales (P. Asselin and G. Masson, eds), Ser. 2, Vol. 9. Place de l’Ecole-de-Medecine, Paris Diesing CM (1850) Systema Helminthium. Sumptibus Academiae Caesareae Scientiarum. Vindobonae. Gerald’s Sohn, Vienna Dixon BR, Parrington LJ, Parenteau M, Leclair D, Santín M, and Fayer R (2008) Giardia duodenalis and Cryptosporidium spp. in the intestinal contents of ringed seals (Phoca hispida) and bearded seals (Erignathus barbatus) in Nunavik, Quebec, Canada. J Parasitol 94: 1161–1163 Dobell C (1940) Vilem Lambl (1824–1895) – A portrait and a biographical note. Parasitology 32: 122–125 Edlind TD and Chakraborty PR (1987) Unusual ribosomal RNA of the intestinal parasite Giardia lamblia. Nucleic Acids Res 15: 7889–7901 Embley TM and Hirt RP (1998) Early branching eukaryotes? Curr Opin Genet Dev 8: 624–629 Erlandsen SL and Bemrick WL (1987) SEM evidence for a new species, Giardia psittaci. J Parasitol 73: 623–629 Erlandsen SL, Sherlock LA, Januschka M, Schupp DG, Schaefer FW, III, Jakubowski W, and Bemrick WJ (1988) Cross-species transmission of Giardia spp.: inoculation of beavers and muskrats with cysts of human, beaver, mouse, and muskrat origin. App Env Microbiol 54: 2777–2785 Erlandsen SL, Bemrick WJ, Wells CL, Feely DE, Knudson L, Campbell SR, Van Keulen H, and Jarroll EL (1990) Axenic culture and characterization of Giardia ardeae from the great blue heron (Ardea herodias). J Parasitol 76: 717–724 Ey PL, Mansouri M, Kulda J, Nohynkova E, Monis PT, Andrews RH, and Mayrhofer G (1997) Genetic analysis of Giardia from hoofed farm animals reveals artiodactyl-specific and potentially zoonotic genotypes. J Euk Microbiol 44: 626–635 Farbey MD, Reynoldson JA, and Thompson RCA (1995) In vitro drug susceptibility of 29 isolates of Giardia duodenalis from humans as assessed by an adhesion assay. Int J Parasitol 25: 593–599 Feely DE (1988) Morphology of the cyst of Giardia microti by light and electron microscopy. J Protozool 35: 52–54 Filice FP (1952) Studies on the cytology and life history of a Giardia from the laboratory rat. Univ California Publns Zool 57: 53–146 Gelanew T, Lalle M, Hailu A, Pozio E, and Caccio SM (2007) Molecular characterization of human isolates of Giardia duodenalis from Ethiopia. Acta Trop 102: 92–99 Geurden T, Geldhof P, Levecke B, Mertens C, Berkvens D, Casaert S, Vercruysse J, and Claerebout E (2008) Mixed Giardia duodenalis assemblage A and E infections in calves. Int J Parasitol 38: 259–264 Graczyk TK, Bozso-Nizeyi JB, Ssebide B, Thompson RCA, Read C, and Cranfield MR (2002) Anthropozoonotic Giardia duodenalis genotype (assemblage) A infections in habitats of free-ranging human-habituated gorillas, Uganda. J Parasitol 88: 905–909

13 Hall, ML, Costa ND, Thompson RCA, Lymbery AJ, Meloni BP, and Wales RG (1992) Genetic variants of Giardia duodenalis differ in their metabolism. Parasitol Res 78: 712–714 Haque R, Roy S, Kabir M, Stroup SE, Mondal D, and Houpt ER (2005) Giardia assemblage A infection and diarrhea in Bangladesh. J Infect Dis 192: 2171–2173 Hashimoto T, Nakamura Y, Nakamura F, Shirakura T, Adachi J, Goto N, Okamoto K, and Hasegawa M (1994) Protein phylogeny gives a robust estimation for early divergences of eukaryotes: phylogenetic place of a mitochondrialacking protozoan, Giardia lamblia. Mol Biol Evoln 11: 65–71 Hashimoto T, Nakamura Y, Kamaishi T, Nakamura F, Adachi J, Okamoto K, and Hasegawa M (1995) Phylogenetic place of mitochondrion-lacking protozoan, Giardia lamblia, inferred from amino acid sequences of elongation factor 2. Mol Biol Evoln 12: 782–793 Hashimoto T, Sanchez LB, Shirakura T, Muller M, and Hasegawa M (1998) Secondary absence of mitochondria in Giardia lamblia and Trichomonas vaginalis revealed by valyl-tRNA synthetase phylogeny. Proc Nat Acad Sci USA 95: 6860–6865 Hegner RW (1922) A comparative study of the Giardia living in man, rabbit and dog. Am J Hyg 2: 442–454 Homan WL and Mank TG (2001) Human giardiasis: genotype linked differences in clinical symptomatology. Int J Parasitol 31: 822–826 Hopkins RM, Constantine CC, Groth DA, Wetherall JD, Reynoldson JA, and Thompson RCA (1999) DNA fingerprinting of Giardia duodenalis isolates using the intergenic rDNA spacer. Parasitology 118: 531–539 Hunter PR and Thompson RCA (2005) The zoonotic transmission of Giardia and Cryptosporidium. Int J Parasitol 35: 1181–1190 Inpankaew T, Traub R, Thompson RCA, and Sukthana Y (2007) Canine parasitic zoonoses and temple communities in Thailand. SE Asian J Trop Med Pub Hlth 38: 247–255 Kabnick KS and Peattie DA (1990) In situ analyses reveal that the two nuclei of Giardia lamblia are equivalent. J Cell Sci 95: 353–360 Kofoid CA (1920) A critical review of the nomenclature of human intestinal flagellates, Cercomonas, Chilomastix, Trichomonas, Tetratrichomonas and Giardia. Univ California Publns Zool 20: 145–168 Kofoid CA and Christiansen EB (1915) On binary and multiple fission in Giardia muris (Grassi). Univ California Publns Zool 16: 30–54 Kulda J and Nohýnková E (1978) Flagellates of the human intestine and of intestines of other species. In: Parasitic protozoa (P.P. Kreier ed.), Academic Press, New York, pp 83–89 Kunstler J (1882) Sur cinq protozoaires parasites nouveaus. C R Séances Soc Biol Filiales 95: 347–349 Kutz SJ, Thompson RA, Polley L, Kandola K, Nagy J, Wielinga CM, and Elkin BT (2008) Giardia assemblage A: human genotype in muskoxen in the Canadian Arctic. Parasites Vectors 1: 32 Kutz SJ, Thompson RCA, and Polley L (2009) Wildlife with Giardia: villain or victim and vector? In: Giardia and cryptosporidium: from molecules to disease (G. Ortega-Pierres,

14 S. Caccio, R. Fayer, T.G. Mank, H.V. Smith, and R.C.A. Thompson, eds.), CABi Wallingford, UK, pp 94–106 Lalle M, Franipane di Regalbono A, Poppi L, Nobili G, Tonzani D, Pozio E, and Caccio SM (2007) A novel Giardia duodenalis assemblage A subtype in fallow deer. J Parasitol 93: 426–428 Lambl W (1859) Mikroskopische untersuchungen der darmexcrete. Vierteljahtsschrift Prak Heikunde (Prag) 61: 1–58 Lasek-Nesselquist E, Welch DM, Thompson RCA, Steuart RF, and Sogin ML (2009) Genetic exchange within and between assemblages of Giardia duodenalis. J Euk Microbiol 56: 504–518 Lebbad M, Ankarklev J, Tellez A, Leiva B, Anderson JO, and Svard S (2008) Dominance of Giardia assemblage B in Leon, Nicaragua. Acta Trop 106: 44–53 Leonhard S, Pfister K, Beelitz P, Wielinga C, and Thompson RCA (2007) The molecular characterisation of Giardia from dogs in Southern Germany. Vet Parasitol 150: 33–38 Mayrhofer G, Andrews RH, Ey PL, Albert MJ, Grimmond TR, and Merry DJ (1992) The use of suckling mice to isolate and grow Giardia from mammalian faecal specimens for genetic analysis. Parasitology 105: 255–263 Mayrhofer G, Andrews RH, Ey PL, and Chilton NB (1995) Division of Giardia isolates from humans into two genetically distinct assemblages by electrophoretic analysis of enzymes encoded at 27 loci and comparison with Giardia muris. Parasitology 111: 11–17 McDonnell PA, Scott KG, Teoh DA, Olson ME, Upcroft JA, Upcroft P, and Buret G (2003) Giardia duodenalis trophozoites isolated from a parrot (Cacatua galerita) colonize the small intestinal tracts of domestic kittens and lambs. Vet Parasitol 111: 31–46 McRoberts KM, Meloni BP, Morgan UM, Marano R, Binz N, Eriandsen SL, Halse SA, and Thompson RCA (1996) Morphological and molecular characterization of Giardia isolated from the straw-necked ibis (Threskiornis spinicollis) in Western Australia. J Parasitol 82: 711–718 Meloni BP and Thompson RCA (1987) Comparative studies on the axenic in vitro cultivation of Giardia of human and canine origin: evidence for intraspecific variation. Trans Roy Soc Trop Med Hyg 81: 637–640 Meloni BP, Lymbery AJ, and Thompson RCA (1995) Genetic characterization of isolates of Giardia duodenalis by enzyme electrophoresis: implications for reproductive biology, population structure, taxonomy and epidemiology. J Parasitol 81: 368–383 Meyer EA (1985) The epidemiology of giardiasis. Parasitol Today 1: 101–105 Meyer EA and Radulescu S (1979) Giardia and giardiasis. Adv Parasitol 17: 1–47 Monis PT and Andrews RH (1998) Molecular epidemiology – assumptions and limitations of commonly applied methods. Int J Parasitol 28: 981–987 Monis PT and Thompson RCA (2003) Cryptosporidium and Giardia zoonoses: fact or fiction? Inf Gen Evoln 3: 233–244 Monis PT, Mayrhofer G, Andrews RH, Homan WL, Limper L, and Ey PL (1996) Molecular genetic analysis of Giardia intestinalis isolates at the glutamate dehydrogenase locus. Parasitology 112: 1–12 Monis PT, Andrews RH, Mayrhofer G, Mackrill J, Kulda J, Isaac-Renton JL, and Ey PL (1998) Novel lineages of Gi-

R. C. A. Thompson and P. T. Monis ardia intestinalis identified by genetic analysis of organisms isolated from dogs in Australia. Parasitology 116: 7–19 Monis PT, Andrews RH, Mayrhofer G, and Ey PL (1999) Molecular systematics of the parasitic protozoan Giardia intestinalis. Mol Biol Evoln 16: 1135–1144 Monis PT, Andrews RH, Mayrhofer G, and Ey PL (2003) Genetic diversity within the morphological species Giardia intestinalis and its relationship to host origin. Infect Genet Evoln 3: 29–38 Monis PT, Caccio SM, and Thompson RCA (2009) Variation in Giardia: towards a taxonomic revision of the genus. Trends Parasitol 25: 93–100 Monzingo DL Jr and Hibler CP (1987) Prevalence of Giardia sp. in a beaver colony and the resulting environmental contamination. J Wild Dis 23: 576–585 Moro D, Lawson MA, Hobbs RP, and Thompson RCA (2003) Pathogens of house mice on arid Boullanger Island and subantartic Macquarie Island, Australia. J Wildl Dis 39: 762–771 Morrison HG, Roger AJ, Nystul TG, Gillin FD, and Sogin ML (2001) Giardia lamblia expresses a proteobacterial-like DnaK homolog. Mol Biol Evol 18: 530–541 Morrison HG, McArthur AG, Gillin FD, Aley SB, Adam RD, Olsen GJ, Best AA, Cande WZ, Chen F, Cipriano MJ, Davids BJ, Dawson SC, Elmendorf HG, Hehl AB, Holder ME, Huse SM, Kim UU, Lasek-Nesselquist E, Manning G, Nigam A, Nixon JE, Palm D, Passamaneck NE, Prabhu A, Reich CI, Reiner DS, Samuelson J, Svard SG, and Sogin ML (2007) Genomic minimialism in the early diverging intestinal parasite Giardia lamblia. Science 317: 1921–1926 Nixon JE, Wang A, Field J, Morrison HG, McArthur AG, Sogin ML, Loftus BJ, and Samuelson J (2002) Evidence for lateral transfer of genes encoding ferredoxins, nitroreductases, NADH oxidase, and alcohol dehydrogenase 3 from anaerobic prokaryotes to Giardia lamblia and Entamoeba histolytica. Euk Cell 1: 181–190 Olson ME, O’Handley RM, Ralston BJ, McAllister TA, and Thompson RCA (2004) Update on Cryptosporidium and Giardia infections in cattle. Trends Parasitol 20: 185–191 Poxleitner MK, Carpenter ML, Mancuso JJ, Wang CJ, Dawson SC, and Cande WZ (2008) Evidence for karyogamy and exchange of genetic material in the binucleate intestinal parasite Giardia intestinalis. Science 319: 1530–1533 Ramesh MA, Malik SB, and Logsdon JM Jr (2005) A phylogenomic inventory of meiotic genes; evidence for sex in Giardia and an early eukaryotic origin of meiosis. Curr Biol 15: 185–191 Read C, Walters J, Robertson ID, and Thompson RCA (2002) Correlation between genotype of Giardia duodenalis and diarrhoea. Int J Parasitol 32: 229–231 Reynoldson JA (2002) Therapeutics and new drug targets for giardiasis. In: Giardia: the cosmopolitan parasite (B.E. Olson, M.E. Olson, and P.M. Wallis eds.), Wallingford, UK, CAB International, pp 159–175 Riley DE and Krieger JN (1995) Molecular and phylogenetic analysis of PCR-amplified cyclin-dependent kinase (CDK) family sequences from representatives of the earliest available lineages of eukaryotes. J Mol Evoln 41: 407–413 Roger AJ, Svard SG, Tovar J, Clark CG, Smith MW, Gillin FD, and Sogin ML (1998) A mitochondrial-like chaperonin 60

Chap. 1 Taxonomy of Giardia Species gene in Giardia lamblia: evidence that diplomonads once harbored an endosymbiont related to the progenitor of mitochondria. Proc Nat Acad Sci USA 95: 229–234 Roger AJ, Morrison HG, and Sogin ML (1999) Primary structure and phylogenetic relationships of a malate dehydrogenase gene from Giardia lamblia. J Mol Evoln 48: 750–755 Sahagun J, Clavel A, Goni P, Seral C, Llorente MT, Castillo FJ, Capilla S, Arias A, and Gomez-Lus R (2008) Correlation between the presence of symptoms and the Giardia duodenalis genotype. Eur J Clin Microbiol Infect Dis 27: 81–83 Salb AL, Barkeman WB, Elkin BT, Thompson RCA, Whiteside RCA, Black SR, Dubey JP, and Kutz SJ (2008) Parasites in dogs in two northern Canadian communities: implications for human, dog, and wildlife health. Emerg Infect Dis 14: 60–63 Sedinova J, Flegr J, Ey PL, and Kulda J (2003) Use of random amplified polymorphic DNA (RAPD) analysis for the identification of Giardia intestinalis subtypes and phylogenetic tree construction. J Euk Microbiol 50: 198–203 Siddall ME, Hong H, and Desser SS (1992) Phylogenetic analysis of the Diplomonadida (Wenyon, 1926) Brugerolle, 1975: evidence for heterochrony in protozoa and against Giardia lamblia as a “missing link”. J Protozool 39: 361–367 Sogin ML, Gunderson JH, Elwood HJ, Alonso RA, and Peattie DA (1989) Phylogenetic meaning of the kingdom concept: an unusual ribosomal RNA from Giardia lamblia. Science 243: 75–77 Souza SL, Gennari SM, Richtzenhain LJ, Pena HF, Funada MR, Cortez A, Gregori F, and Soares RM (2007) Molecular identification of Giardia duodenalis isolates from humans, dogs, cats and cattle from the state of Sao Paulo, Brazil, by sequence analysis of fragments of glutamate dehydrogenase (gdh) coding gene. Vet Parasitol 149: 258–264 Suguri S, Henze K, Sanchez LB, Moore DV, and Muller M (2001) Archaebacterial relationships of the phosphoenolpyruvate carboxykinase gene reveal mosaicism of Giardia intestinalis core metabolism. J Eukaryot Microbiol 48: 493–497 Sulaiman IM, Fayer R, Bern C, Gilman RH, Trout JM, Schantz PM, Das P, Lal AA, and Xiao L (2003) Triosephosphate isomerase gene characterization and potential zoonotic transmission of Giardia duodenalis. Emerg Infect Dis 9: 1444–1452 Teichroeb JA, Kutz SJ, Parkar U, Thompson RCA, and Sicotte P (2009) Ecology of the Gastrointestinal Parasites of Colobus vellerosus at Boabeng-Fiema, Ghana: possible anthropozoonotic transmission. Am J Phys Anthropol 140: 498–507 Thompson RCA (2002) Towards a better understanding of host specificity and the transmission of Giardia: The impact of molecular epidemiology. In: Giardia: the cosmopolitan parasite (B.E. Olson, M.E. Olson, and P.M. Wallis eds.), CAB International, Wallingford, UK, pp 55–69 Thmpson RCA (2004) The zoonotic significance and molecular epidemiology of Giardia and giardiasis. Vet Parasito 126: 15–35 Thompson RCA and Lymbery AJ (1996) Genetic variability in parasites and host-parasite interactions. Parasitology 112(Suppl): S7–S22

15 Thompson RCA and Monis PT (2004) Variation in Giardia: implications for taxonomy and epidemiology. Adv Parasitol 58: 69–137 Thompson RCA, Lymbery AJ, and Meloni BP (1990) Genetic variation in Giardia Kunstler, 1882: taxonomic and epidemiological significance. Protozool Abs 14: 1–28 Thompson RCA, Lymbery AJ, Pearce DA, Finn KC, Reynoldson JA, and Meloni BP (1996) Giardia duodenalis: exposure to metronidazole inhibits competitive interactions between isolates of the parasite in vitro. J Parasitol 82: 679–683 Thompson RCA, Traub RJ, and Parameswaran N (2007) Molecular epidemiology of foodborne parasitic zoonoses. In: Food-borne parasitic zoonoses (K.D. Murrell and B. Fried eds.), Spinger, pp 383–415 Thompson RCA, Kutz SJ, and Smith A (2009a) Parasite zoonoses and wildlife: emerging issues. Int J Env Res Pub Hlth 6: 678–693 Thompson RCA, Colwell DD, Shury T, Appelbee AJ, Read C, Njiru Z, and Olson ME (2009b) The molecular epidemiology of Cryptosporidium and Giardia infections in coyotes from Alberta, Canada, and observations on some cohabiting parasites. Vet Parasitol 159: 167–170 Thompson RCA, Smith A, Lymbery AJ, Averis S, Morris KD, and Wayne AF (2010) Giardia in Western Australian wildlife. Vet Parasitol (in press) Traub RJ, Monis PT, Robertson I, Irwin P, Mencke N, and Thompson RCA (2004) Epidemiological and molecular evidence supports the zoonotic transmission of Giardia among humans and dogs living in the same community. Parasitology 128: 253–262 Trout JM, Santin M, Greiner EC, and Fayer R (2006) Prevalence and genotypes of Giardia duodenalis in 1–2 year old dairy cattle. Vet Parasitol 140: 217–222 van der Giessen JW, de Vries A, Roos M, Wielinga P, Kortbeek LM, and Mank TG (2006) Genotyping of Giardia in Dutch patients and animals: a phylogenetic analysis of human and animal isolates. Int J Parasitol 36: 849–858 van Keulen H, Gutell RR, Gates MA, Campbell SR, Erlandsen SL, Jarroll EL, Kulda J, and Meyer EA (1993) Unique phylogenetic position of Diplomonadida based on the complete small subunit ribosomal RNA sequence of Giardia ardeae, G. muris, G. duodenalis and Hexamita sp. Faseb J 7: 223–231 Visvesvara GS, Dickerson JW, and Healy GR (1988) Variable infectivity of human-derived Giardia lamblia cysts for Mongolian gerbils (Meriones unguiculatus). J Clin Microbiol 26: 837–841 WHO (1979) Parasitic Zoonoses. Report of a WHO Expert Committee with the participation of FAO. Technical Report Series No. 637. World Health Organization, Geneva Williamson AL, O’Donoghue PJ, Upcroft JA, and Upcroft P (2000) Immune and pathophysiological responses to different strains of Giardia duodenalis in neonatal mice. Int J Parasitol 30: 129–136 Yason JA and Rivera WL (2007) Genotyping of Giardia duodenalis isolates among residents of slum area in Manila, Philippines. Parasitol Res 101: 681–687

Epidemiology of Giardiasis in Humans Simone M. Cacciò and Hein Sprong

Abstract Giardia lamblia is a widespread flagellated parasite of mammalian species, including humans, and is regarded as the most common cause of protozoan diarrhea worldwide. Owing to its invariant morphology, investigation on aspects such as host specificity and transmission patterns requires a direct genetic characterization of cysts and trophozoites from host and environmental samples. A number of molecular assays have been developed to help unravel the complex epidemiology of this infection. A coherent picture has emerged from those studies, indicating the existence of seven genetic groups (or assemblages), two of which (A and B) are found in both humans and animals, whereas the remaining five (C–G) are relatively host-specific. With the rapid accumulation of sequence data and the refinement of the assays, the elucidation of many epidemiologic aspects seemed only a matter of time and proper study design. However, the occurrence of mixed infections, the allelic sequence heterozygosity between the nuclei, and the reported occurrence of several distinct recombinational events indicate a level of biological complexity that affects the molecular typing, particularly for assemblage B. Under these circumstances, caution must be applied in the interpretation of molecular data in epidemiology of giardiasis. A specific database for the storage and analysis of sequence and epidemiologic data has been recently developed, and represents an important tool for future studies.

2.1 Introduction Giardia is an intestinal flagellate that infects a wide range of vertebrate hosts. The genus currently comprises six species, namely: Giardia agilis, Giardia

H. D. Luján et al. (eds.), Giardia © Springer-Verlag/Wien 2011

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ardeae, Giardia duodenalis, Giardia microti, Giardia muris, and Giardia psittaci, which are distinguished on the basis of the morphology and ultrastructure of their trophozoites (Adam, 2001). For a detailed account of the taxonomy of G. duodenalis, see Chapter 1. Giardia duodenalis (syn. G. intestinalis, G. lamblia) is the only species found in humans; it has a global distribution causing an estimated 280 million cases yearly (Lane and Lloyd, 2002), and is the most common intestinal protozoan parasite of humans in both developing and developed countries. In Asia, Africa, and Latin America, about 200 million people have symptomatic giardiasis with some 500,000 new cases reported each year (WHO, 1996). It is also a frequently encountered parasite of domestic animals, especially livestock, dogs and cats, and numerous species of wild mammals and birds, and even fish have been documented as hosts of Giardia. Several characteristics of G. duodenalis life cycle influence the epidemiology of infection. Cysts are immediately infectious when excreted in feces and can be transmitted by person-to-person or animal-toanimal contact. Cysts are remarkably stable and can survive for weeks to months in the environment. The environmental contamination can lead to the contamination of drinking water and food or recreational water or areas, such as playground and sandpits (Cacciò et al., 2005; Smith et al., 2006). Infection exclusively occurs by the ingestion of cysts, either via drinks or food, by accidental swallowing of recreational water by direct contact during insufficient hygiene or sexual activities. In humans, the infective dose for a symptomatic infection is about 10–100 cysts. The relative long period between infection and disease complicates the identification of the source of infection. In this chapter, we will summarize the knowledge emerged from descriptive epidemiologic studies as well as that obtained using molecular genotyping

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2.3 Seasonality In the last report of giardiasis in the USA (period 2003–2005), a marked seasonality in the onset of illness was observed in early summer through early autumn (Yoder and Beach, 2007). A twofold increase in the transmission of giardiasis occurred during the summer, coinciding with increased outdoor activities (e.g., swimming and camping). Similarly, a study in New Zealand (period 1996–2000) showed a signifi-

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Estimates of the prevalence of giardiasis vary greatly because the disease is reportable only in some countries, the diagnostic methods used differ in sensitivity and, in endemic areas, many infected persons are not symptomatic, have no access to medical care or do not seek medical treatment. Nevertheless, Giardia is considered the most common cause of protozoan diarrhea worldwide. In the United States, the incidence of giardiasis per 100,000 population ranged from 1.4 cases in Louisiana to 30 cases in Vermont. Two states (Vermont and Minnesota) reported the greatest number of cases per 100,000 population in the last reporting period (2003–2005). Northern states reported more cases annually per 100,000 population than southern states (Yoder and Beach, 2007). In Europe, data on giardiasis are collected by 23 countries and are made available by the European Center for Disease Control (http://ecdc.europa.eu/). In 2006, Romania reported the highest rates of infection (816.9 per 100,000, sixteen times the EU average), followed by Estonia (34.9 per 100,000) and then Bulgaria (28.7 per 100,000), and Sweden (14.2 per 100,000). The overall notification rate of giardiasis was 58.1 per 100,000, which is extremely high compared to the two major foodborne pathogens Campylobacteriosis (39.5 per 100,000) and Salmonellosis (33.9 per 100,000). In New Zealand, giardiasis is notifiable since 1996, and represents the third most commonly notified communicable disease after campylobacteriosis and salmonellosis. In a recent survey in New Zealand, it was confirmed that giardiasis has one of the highest incidence rates (49.4 per 100,000 population) compared with other developed countries, and this may be related to environmental or social factors (Hoque et al., 2004). In a recent review of giardiasis in Asia (Dib et al., 2008), based on 33 studies published in the period 2002–2007, it has been shown that the prevalence varied markedly between studies being higher in urban than in rural areas, among poor communities, slightly higher in males than in females, among university

M

2.2 Prevalence of the Infection

students, old-aged people, HIV-positive patients, and gastric carcinoma patients. High prevalence rates were observed in children in Nepal (73.4%), in Thailand (37.7%) and in Malaysia (24.9%), and multiple infections with other parasites were also frequently recorded (Dib et al., 2008). The true incidence of giardiasis is certainly underestimated. In a metaanalysis of giardiasis and cryptosporidiosis in Nordic countries (Denmark, Finland, Norway, and Sweden), it has been estimated that the actual prevalence of Giardia is 2.97% in the asymptomatic population and 5.84% in the symptomatic population (Horman et al., 2004). A case-control study in general practices found an incidence of 3.3% in the asymptomatic population symptoms compared to 5.4% in the symptomatic population systems (de Wit et al., 2001a, b). From these data, it has been calculated that, for each registered case of giardiasis, around 250 symptomatic cases are expected to occur.

Cases

of G. duodenalis isolates. For a more detailed account of animal giardiasis and of environmental issues, see Chapters 3 and 4, respectively.

S. M. Cacciò and H. Sprong

p. ct. ov. ec. g. O N Au Se D

Fig. 2.1 Seasonal distribution of giardiasis cases in EU and EEA/EFTA countries in 2006 (n = 12,460). These data are based on data from 16 countries and are from European Centre for Disease Prevention and Control: Annual Epidemiological Report on Communicable Diseases in Europe 2008. Stockholm, European Centre for Disease Prevention and Control, 2008

Chap. 2 Epidemiology of Giardiasis in Humans

19

cant seasonal variation of Giardia notification, with peaks in late summer and early autumn (Hoque et al., 2004). Data from European countries show an average monthly incidence of ~950 cases with an autumn peak in September to November of ~1350 cases (European Centre for Disease Prevention and Control: Annual Epidemiological Report on Communicable Diseases in Europe 2008. Stockholm, European Centre for Disease Prevention and Control, 2008) (Fig. 2.1). Bearing in mind the delay between infection, development of symptoms and submission of specimen which may amount to 5 weeks or more, this late summer/autumn peak probably represents an increase in infection during the mid to late summer months. Infections associated with travel and out-door recreation may at least partly explain such trend.

2.4 Giardiasis in Children The highest cases of giardiasis are consistently reported among children aged 1–4 and 5–9 years and adults aged 35–39 years, usually the parents of the children, in particular their mothers (Fig. 2.2). Giardia and Cryptosporidium were recently included in the “Neglected Disease Initiative” to underline their link with poverty-related issues (Savioli et al., 2006). Not surprisingly, a number of studies have been devoted to understand whether the health effects of Giardia in children in developing countries are transient or if they result in long-term health consequences,

Cases/100,000

15

10

5

0 0–4

5–14

15–24

25–44

45–64

>64

Fig. 2.2 Age-specific notification rates of giardiasis cases in EU and EEA/EFTA countries in 2006 (n = 7264). These data reflect the age distribution of human giardiasis. Data are from European Centre for Disease Prevention and Control: Annual Epidemiological Report on Communicable Diseases in Europe 2008. Stockholm, European Centre for Disease Prevention and Control, 2008

such as poor cognitive function and failure to thrive. To date, results have been controversial. A study in Peru has followed 220 children and found that the risk of Giardia infection did not vary with weight for age or height for age, and that the parasite was not associated with an increased risk of diarrhea at any age interval (Hollm-Delgado et al., 2008). On the other hand, a longitudinal study in Guatemala reported a reduced growth in 2-year-old Giardia-infected children compared to non-infected children of the same age (Farthing et al., 1986). The authors pointed out at the confounding effect of concomitant infections with other enteropathogens, yet were able to conclude that Giardia has independent deleterious effects on children’s growth. Outbreaks of giardiasis in day-care centers in developed countries are frequently reported as well.

2.5 Giardiasis in Immunosuppressed Individuals Globally, the number of immunosuppressed people increases each year, not only due to the continuous spread of the human immunodeficiency virus (HIV) pandemic, but also due to malnutrition, chemotherapy for malignancy, and immunosuppressive therapy. All individuals affected by immunosuppression are at risk of infection by opportunistic parasites (reviewed by Stark et al., 2009). Giardiasis is not considered a major cause of enteritis in HIV-infected patients, and it is not listed among the opportunistic parasitic infections because it does not cause prolonged symptoms and therapy is independent from the patient’s immune status. The observed prevalence varies between 1.5% and 17.7% in the few reports published (reviewed by Stark et al., 2009). The symptoms of giardiasis in HIV-infected individuals appear to be similar to, and no more severe than, those of giardiasis in HIVnegative individuals, with asymptomatic infection occurring commonly in the presence of HIV (reviewed by Faubert, 2000). However, when CD4+ counts are reduced and cause progressive immunosuppression, the risk of symptomatic Giardia infections increases, with a tendency towards chronic diarrhea (Dwivedi et al., 2007). Although little is known about Giardia infections in other immunosuppressed individuals, a number

20

of studies have shown that the parasite is more prevalent in the stools of hypogammaglobulinemic patients than in those of immunocompetent hosts (Faubert, 2000). It has also been shown that the vast majority of hypogammaglobulinemic patients that shed Giardia cysts are symptomatic, presenting with chronic diarrhea. Symptomatic giardiasis has been observed in X-linked infantile congenital hypogammaglobulinemia (Bruton’s syndrome) and also in the common variable (late-onset) acquired hypogammaglobulinemia.

2.6 Risk Factors Human giardiasis arises through in two broad settings: outbreaks and endemic transmissions, the latter leading to sporadic infections. Much of our knowledge on risk factors is derived from outbreak investigations, and few studies have addressed the situation for endemic or sporadic giardiasis. Outbreaks are most frequently waterborne, are caused by drinking or recreational water contamination, although other transmission routes have also been implicated (Eisenberg et al., 2002; Thompson and Chalmers, 2002; Adam, 2001, Thompson et al., 2000). The routes of transmission for sporadic cases are largely unknown but recent case-control studies identified the importance of person-to-person spread, travel, contact with livestock, and potable and recreational water as risk factors for sporadic disease. A retrospective case-control study in rural New England looked at 171 patients and 684 age- and sex-matched control, and identified the household use of shallow water sources as the main risk factor, followed by foreign travel, daycare center exposure, and household case contact (Chute et al., 1987). A matched case-control study in the United Kingdom (232 cases and 574 controls) identified swallowing water while swimming, recreational fresh water contact, drinking treated tap water, and eating lettuce as positively and independently associated with infection (Stuart et al., 2003). A casecontrol study in Germany included 120 laboratoryconfirmed autochthonous Giardia cases with clinical manifestations (diarrhea, cramps, and bloating) and 240 randomly selected controls from the local population registry matched by county of residence and age-group (Espelage et al., 2010). Cases were more

S. M. Cacciò and H. Sprong

likely to be male, immunocompromised, and daily consumers of green salad. Remarkably, contact with animals (pets/farm animals) and exposure to surface water (swimming/water sports) were not associated with symptomatic disease. A case-control study in Italy, performed during the Catholic Jubilee of year 2000 to look for the effect of mass gathering on transmission of giardiasis, enrolled 52 cases and 72 controls, all residents of Rome (Faustini et al., 2006). Multivariate analysis showed that traveling abroad, exposure to surface water, and high educational level were the main risk factors associated with giardiasis. Having a maid from a high-prevalence country was independently associated, although not statistically significant. A study in Auckland, New Zealand, explored the risk of nappy changing by comparing 183 patients with Giardia-positive stools with 336 age-matched controls identified randomly from the telephone book (Hoque et al., 2001). The risk of infection was significantly higher for housewives and nursing mothers compared with other occupational groups. Physical contact with children wearing nappies showed a significant association with giardiasis. Nappy changing was associated with a fourfold increased risk, and giardiasis was associated strongly with childcare center attendance. Of these two factors, child-care center attendance and nappy changing, only nappy changing remained a significant risk for infection after logistic regression (Hoque et al., 2001).

2.7 Correlation Between Assemblages and Symptoms It was only in the late 70s that Giardia was recognized as a human pathogen, based on symptoms such as malabsorption and the pathology observed in the upper part of the small intestine in patients from whom the organism was isolated (Koulda and Nohynkova, 1978). In 1981, the World Health Organization added Giardia to its list of parasitic pathogens (WHO, 1981). The clinical effects of the infection in humans are highly variable, and range from the asymptomatic carrier stage to a severe malabsorption syndrome. It is well known that human giardiasis can be divided into two disease phases: acute and chronic. The acute phase is usually short-lived, characterized by flatu-

Primary school children, mean age 9 years

Patients admitted to the Dhaka Hospital (all ages)

Children from urban slums in South India

Dutch patients who consulted their GP

Stool samples from outpatient clinics, from asymptomatic patients, and from familiars of a patient with enteric parasite infection Symptomatic cases of giardiasis in south-west London (all ages)

Cuba

Bangladesh

India

The Netherlands

Spain

Samples from two Hospitals, a day care center, a primary school, and two rural communities Children hospitalized for acute gastroenteritis

Ethiopia

Albania

Brazil

Stool and duodenal aspirate samples from individuals referred by outpatient specialists Children from an urban shanty town

Turkey

United Kingdom

Type of study Aboriginal community, mean age 10 years

Location Malaysia

Ass. A 1

All symptomatic

No symptoms Diarrhea, abdominal pain, and nausea

No symptoms Diarrhea

Diarrhea, vomiting, nausea, headache, weight loss, fever, and abdominal pain No symptoms Diarrhea

Intermittent diarrhea and moderate symptoms Persistent diarrhea and severe symptoms No symptoms Diarrhea, abdominal pain, rapid weight loss, abdominal cramps, flatulence, and nausea

No symptoms Diarrhea, vomiting, and fever

12

12

19 10

0

14 29

22 3

31

12

2 7

2 17

14

26 35

9

0 29 14

0

48 40

174 108

1 10

Ass. B 41

9

2 5

No symptoms 4 Diarrhea plus at least two other 4 symptoms (nausea, vomiting, loss of appetite, weight loss, and abdominal pain) No symptoms (control group) 10 Acute diarrhea 29

Symptoms Diarrhea, vomiting, nausea, fever, and abdominal pain

Mixed A + B infection in 10% of samples. Higher number of cyst shed after mixed infection or infection with assemblage B High number of mixed infection (25%), mostly associated with symptoms (83%)

Assemblage A statistically associated with fever; 3% of infections were mixed A + B Results confirmed by typing duodenal aspirates

Correlation between ass. A and diarrhea found also in children

Inverse correlation between parasite load and diarrhea; mixed A + B infections in both cases (4.2%) and controls (7.5%) Infection with ass. A and mixed A + B infection (10%) associated with diarrhea (p = 0.07) No concomitant infections

PCR-RFLP and sequencing of the beta-giardin gene Sequencing of Highest infection rates in the 18S rDNA gene warm season

Real-time PCR on 18S rDNA

Sequencing of tpi and 18S rDNA genes PCR-RFLP at the tpi gene

PCR-RFLP at the tpi gene

PCR-RFLP at the gdh gene

PCR-RFLP at the tpi gene

Real-time PCR on 18S rDNA

Methodology Other relevant findings Sequencing of Risk factors included age 18S rDNA gene (<12 years), gender (female), and eating fresh fruits Sequencing of Correlation between parasite the gdh and load and symptoms beta-giardin genes

Table 2.1 Correlation between assemblage A and B and symptoms from 11 independent studies. Bold values indicate statistically significant associations

Berrilli et al. (2006)

Gelanew et al. (2007)

Kohli et al. (2008)

Aydin et al. (2004)

Breathnach et al. (2010)

Sahagun et al. (2007)

Homan and Mank (2001)

Ajjampur et al. (2009)

Haque et al. (2009)

Pelayo et al. (2008)

Reference Madhy et al. (2009)

Chap. 2 Epidemiology of Giardiasis in Humans 21

22

lence and abdominal distension with cramps. Diarrhea is initially frequent and watery but later becomes bulky, greasy, and typically offensive. In chronic giardiasis, malaise, weight loss, and other features of malabsorption become prominent and stools are usually pale or yellow, frequent, and of small volume. Malabsorption of vitamins and D-xylose can occur, while disaccharidase deficiencies (most commonly lactase) are frequently detected in chronic cases (Faubert, 2000). Many possible factors contribute to variation in clinical manifestations, including the virulence of the parasite strain, the number of cysts ingested, the age of the host, and the state of the immune system at the time of infection. Understanding the relative contribution of the parasite’s genetic variability and host factors in the establishment of clinical giardiasis has been the subject of a number of studies in both developed and developing countries. So far, these studies have reached controversial conclusions. Table 2.1 displays an updated summary of those studies. Assemblage A was associated with diarrhea (and other symptoms) in studies in India, Spain, and Turkey, whereas an association with assemblage B was reported in Malaysia, The Netherlands, and Ethiopia. No association with either assemblage was found in Cuba, The United Kingdom, Brazil, and Albania. Whereas those conflicting results can be explained by differences in the study design, in the population considered (adults versus children), in the definition of symptoms, is presently unknown. Larger studies with asymptomatic and symptomatic cases followed up longitudinally could lead to better knowledge of the association of these assemblages with diarrhea. However, the complexity in the determination of Giardia assemblages and genotypes, and the frequent occurrence of mixed infections (see the following sections), indicate that care should also be given to the genotyping techniques applied.

2.8 Tools for Molecular Genotyping DNA sequence analysis has nowadays largely replaced protein analysis, mainly because in vitro amplification of specific targets by PCR is applicable to small amounts of material, and does not require culturing of the parasite. The availability of the complete

S. M. Cacciò and H. Sprong

genome sequence of WB, an assemblage A, subgroup AI, representative (Morrison et al., 2007) and of the genome draft of GS, an assemblage B, subgroup BIV, representative (Franzen et al., 2009), allow to select candidate markers on a rationale basis (e.g., amount and pattern of sequence variability, syntenic conservation, and gene function). Table 2.2 summarizes the markers that have been used so far to genotype Giardia isolates. For some of them, information is available for all G. duodenalis assemblages and for other Giardia species, whereas others have been applied only to genotype G. duodenalis assemblages A and B (Table 2.2). The various markers are not physically linked in the genome, a feature that is useful for genetic studies, including those aimed at tracing recombination (Lasek-Nesselquist et al., 2009). The use of a multilocus typing scheme has been proposed as an informative approach for genotyping (Cacciò et al., 2008; Lebbad et al., 2009). Current MLST-schemes are predominantly based on household genes, and the variation found in these markers is sufficient for genotyping. Highly variable molecular markers for subtyping, such as microsatellites, which are necessary for biotracing the detection of diffuse outbreaks, local transmission routes, have not been identified so far. Furthermore, with the extensive application of these PCR assays, it is evident that genotyping Giardia isolates is less than obvious. Researchers are confronted to the unreliability of PCR assays, resulting in amplification or lack of amplification of a certain locus from a given isolate, or in the detection of different assemblages (or species) when isolates are typed at more than one locus. As part of this finding may be due to the heterogeneous nature of the sample (i.e., to the presence of more than one type of cysts in a fecal or an environmental sample), PCR assays based on the use of assemblage-specific primers have been developed (Geurden et al., 2007, 2009) to show the frequent occurrence of mixed infections. Similarly, real-time PCR assays that detect specifically assemblages A and B in fecal samples and purified cysts, have shown frequent mixed infections in human isolates (Almeida et al., 2010). Other researchers, by sequencing multiple plasmids containing cloned PCR products, have shown that several haplotypes (i.e., different sequences) can be identified in the amplification product obtained from single isolates (Lasek-Nesselquist et al., 2008; Kosuwin et al., 2010).

Chap. 2 Epidemiology of Giardiasis in Humans

23

Table 2.2 Genes that are frequently used for molecular typing of Giardia. A large database with genomic and proteomic information on Giardia duodenalis is available at http://giardiadb.org/giardiadb/ Target genes

Function

Location

Sequences availability

Mlh1

Involved in DNA repair

CH991767

G. duodenalis assemblage A and B

Glutamate dehydrogenase

Housekeeping enzyme

CH991814

All G. duodenalis assemblages, G. muris, G. ardeae

Triose phosphate isomerase

Housekeeping enzyme

CH991767

All G. duodenalis assemblages, G. microti, G. muris, G. ardeae

Beta-giardin

Structural protein

CH991793

All G. duodenalis assemblages, G. muris

Elongation factor 1-D

Component of the translational apparatus

CH991798

All G. duodenalis assemblages, G. muris, G. ardeae

Ferredoxin

Mediates electron transfer

CH991769

G. duodenalis assemblage A and B

Histone H2B

Nucleosomal protein

CH991767

G. duodenalis assemblage A and B

Histone H4

Nucleosomal protein

CH991767

G. duodenalis assemblage A and B

Actin

Structural protein

CH991776

G. duodenalis assemblage A and B, G. ardeae

D-tubulin

Structural protein

CH991767

G. duodenalis assemblage A and B

Chaperonin 60

Heat shock protein

CH991769

G. duodenalis assemblage A and B

Open reading frame C4

Hypothetical heat shock protein

CH991763

G. duodenalis assemblage A and B

18S rDNA

Small subunit of the ribosome

Multiple chromosomes

All G. duodenalis assemblages, G. muris, G. microti, G. ardeae, G. agilis

Intergenic ribosomal spacer

Non-coding ribosomal sequence

Multiple chromosomes

G. duodenalis assemblage A and B

ITS regions and 5.8S rDNA

Ribosomal

Multiple chromosomes

All G. duodenalis assemblages, G. muris, G. microti, G. ardeae

Ribosomal protein L7a

Ribosomal

CH991782

G. duodenalis assemblage A and B

2.9 Molecular Epidemiology of Assemblage A For the sake of clarity, the data are presented and discussed separately for assemblages A and B. Protein polymorphisms, which have been studied quite extensively in the early 90s, already demonstrated genetic variability between and among human and animal isolates of assemblage A (Mayrhofer et al., 1995). In the most recently published study (Monis et al., 2003), four subgroups (AI, AII, AIII, and AIV) were described by the analysis of 10 isolates at 23 genetic loci, and the host distribution indicated that human isolates belong to subgroups AI and AII, while animal isolates belong to subgroups AI, AIII, and AIV. Therefore, only subgroup AI seems to have zoonotic potential, whereas subgroup AII seems to be human-specific.

The correlation between subgroups defined by protein analysis and those identified by DNA analysis was first demonstrated by Monis et al. (1999) using two reference strains, Ad-1 for subgroup AI and Ad-2 for subgroup AII, and four genetic loci. Surprisingly, as no DNA information has been generated from the isolates that defined subgroups AIII (cats) and AIV (cat, alpaca, and guinea pig), the existence of those subgroups remained based only on protein polymorphisms. A number of studies have focused on the comparative analysis of genetic polymorphisms of household genes (mostly at single loci) between human and animal isolates, with the aim of defining transmission routes and zoonotic potential of various animals, mainly livestock and pets (reviewed in Cacciò and Ryan, 2008). This has led to the description of new variants (sometimes referred to as sub-

24

S. M. Cacciò and H. Sprong

genotypes), some of which were found in different hosts, including humans. This finding was interpreted as an indirect evidence for zoonotic potential. The amount of DNA sequences has accumulated rapidly over time, allowing extensive evaluation of the genetic variability between and within G. duodenalis assemblages (Wielinga and Thompson, 2007). Recently, a database containing DNA sequence and epidemiologic data has been constructed (Sprong et al., 2009) and currently store information on 978 human and 1440 animal isolates, which together comprises 3886 sequences from four genetic loci markers (18S rDNA, tpi, bg, and gdh). Analysis of the data for assemblage A revealed a number of interesting aspects. At least three subgroups exist in this assemblage, where the subgroup AI is predominantly found in livestock and appears to be zoonotic. MLGs of subgroup AII are mostly found in humans and only a single cat isolate had an MLG found in humans. Therefore, when inferred only using genetic data, zoonotic transmission appears to be a rare event. Admittedly, this is a rather “dirty” approach, as the lack of epidemiologic links between the isolates should not be underestimated. Furthermore, full MLGs of subgroup AII from animals are rare: often they are incomplete or mixed with host-specific assemblages (see below). The newly proposed subgroup AIII is es-

sentially restricted to wild animals, particularly wild ruminants. How subgroup AIII is related to the AIII or AIV subgroups defined by Monis et al. (2003) remains to be determined. Indeed, a few studies have combined a designed sampling strategy with molecular genotyping of the isolates. A study in a remote tea growing community in northern India, where humans and dogs live in close contact, have provided epidemiologic evidence supporting the role of dogs in the transmission to humans, albeit the molecular data were less convincing (Traub et al., 2004). Another study in Temple communities in Bangkok (Thailand) has provided more epidemiologic and molecular evidence to support the role of dogs as reservoirs for human infection (Traub et al., 2009). Thus, the role of animals in the transmission of G. duodenalis assemblage A needs to be studied in the context of sub-assemblages. Zoonotic transmission of assemblage AI appears to be minimal in developed countries, but the situation may be different in endemic regions, e.g., in localized foci where humans and animals live promiscuously (Table 2.3). Transmission of assemblage AII mostly occurs between humans, and may also take place from humans to animals. Zoonotic transmission of assemblage AII seems possible, but how often this happens is uncertain.

Table 2.3 Host distribution of sub-assemblages of A. All sequences of BG, GDH, and TPI in the ZOOPNET-database belonging to Assemblage A were subdivided into sub-assemblages AI, AII, and AIII bases on SNPs (Caccio et al., 2008, Sprong et al., 2009). Distribution of sub-assemblages within each source is calculated as their percentage of occurrence in the three cumulative markers. Bold numbers indicate the highest percentage per column (source). A selection of only Western European isolates is shown in the lower table Cat

Cattle

Dog

Goat/sheep

Human

Wildlife

AI

69%

62%

73%

78%

25%

AII

25%

35%

27%

22%

75%

3%

AIII

5%

4%

0%

0%

0%

52%

Global

59

113

120

36

AI

29%

34%

46%

33

AII

71%

66%

54%

67%

Total (n)

594

44%

86

Western Europe

AIII Total (n)

0% 39

0% 50

0% 41

0% 12

4%

40%

96%

0%

0% 165

60% 69

Chap. 2 Epidemiology of Giardiasis in Humans

2.10 Molecular Epidemiology of Assemblage B As reported above for assemblage A, the analysis of protein polymorphisms has been at the basis of the recognition of genetic heterogeneity of G. duodenalis isolates of assemblage B. Andrews et al. (1989) were the first to propose the existence of subgroups III and IV in assemblage B. Mayrhofer et al. (1995) demonstrated that assemblage B isolates were more heterogeneous than assemblage A isolates, to the point that assemblage B isolates were all different when compared at 27 genetic loci. The authors arguably commented that the “true magnitude of genetic diversity within assemblage B remains unknown”. In the more recent analysis published by Monis et al. (2003), ten isolates were characterized at 23 genetic loci to support the existence of four subgroups (BI, BII, BIII, and BIV). As was the case for assemblage A, human isolates appear to form two clusters (subgroups BIII and BIV), whereas animal isolates (monkeys and a dog) belonged to subgroups BI and BII. The single human isolate (BAH12) in subgroup BIII was, however, closer to subgroups BI and BII. Therefore, zoonotic potential appears to be minimal, if any. DNA sequence polymorphism was investigated in a number of studies, mainly using gdh and tpi sequences in initial reports (Baruch et al., 1996; Sulaiman et al., 2003) and beta-giardin in more recent reports (Lalle et al., 2005; Lebbad et al., 2009). Extensive polymorphism was observed at all loci (reviewed by Wielinga and Thompson, 2007). More recently, by direct sequencing of PCR products generated from assemblage B isolates, several groups reported the presence of “mixed positions” or “double peaks”, i.e., of two overlapping signals at specific positions in the sequencing profiles (e.g., Gelanew et al., 2007; Lebbad et al., 2008). This was interpreted as due to mixed infections and/or to allelic sequence heterogeneity, albeit it was impossible to distinguish between the two mechanisms. When the presence of different sequences was analyzed by sequencing multiple plasmids containing cloned PCR products, a greater number of haplotypes were found in single isolates. This was demonstrated not only for field (fecal) isolates from marine mammals (LasekNesselquist et al., 2008) and humans (Kosuwin et al.,

25

2010), but also for axenic isolates of human origin (Lasek-Nesselquist et al., 2009). Screening of assemblage B sequences in the Giardia database revealed that 21% of 1151 sequences (from four different loci) contained “mixed” profiles, as compared to 5% in 1250 assemblage A sequences (Sprong et al., 2009). A large-scale analysis of the genome of GS, an assemblage B isolate, revealed an overall level of allelic sequence divergence of 0.53%, compared to less than 0.01% in the WB genome (Franzen et al., 2009). While more allelic sequence divergence was observed in non-coding than in coding sequences (1.25% versus 0.3%), as expected, an important fraction (38%) of the polymorphism changes encoded proteins and involves 1962 genes. Therefore, almost 2000 proteins with a slightly altered primary structure are found in the GS genome, with potential implications in the biology and virulence of the parasite (Franzen et al., 2009). Clearly, our understanding of the genetics, both at the level of single isolates and at the level of the population, is incomplete, even if important progresses have been achieved. Under these circumstances, the application of molecular tools to the epidemiology needs careful evaluation.

2.11 Recombination and Molecular Epidemiology Traditionally, species within the Giardia genus have been considered as eukaryotic organisms that show an absence of sexual reproduction in their simple life cycles (Adam, 2001). This view has been challenged by a number of recent studies (reviewed by Cacciò and Sprong, 2010). Besides the interest in evolutionary biology (Logsdon, 2008), there are important implications of recombination for the population genetics, taxonomy, and epidemiology of Giardia. Accumulating evidences support the occurrence of genetic exchanges within assemblages (at the individual level or between individual of the same assemblage) and even between assemblages. This has been observed among human field isolates of assemblage A, subgroup AII (Cooper et al., 2007), not only among axenic isolates of assemblages A, subgroups AI and B (Teodorovic et al., 2007), but also among other G. duodenalis as-

26

S. M. Cacciò and H. Sprong

Table 2.4 Recombination events in Giardia duodenalis: Facts or artifacts? Incomplete insight in the genetic exchange of Giardia complicates molecular typing. Potential mechanisms with consequences are depicted (Ass.: Assemblage, ASH: Allelic Sequence Heterogeneity) Genetic exchange between two

Frequent

Rare

1

Nuclei of one genotype

Low ASH Ass. A–G

High ASH Ass. A–G

2

Genotypes from one (sub)-assemblage

High ASH No (sub) Ass. structure exists

ASH depends on frequency of 1 (Sub)Ass structure is stably expanding

3

Genotypes from different (sub)-assemblages

High ASH No Ass. structure exists

ASH depends on frequencies of 1 and 2 Ass. structure is stably expanding

4

Mixed infections Without genetic exchange With genetic exchange T1 With genetic exchange T2/3

Variable frequency of PCR/sequencing artifacts and MLST artifacts High frequency of PCR/sequencing artifacts and MLST artifacts Complete chaos

semblages by comparative and phylogenetic analyses of Genbank sequences (Lasek-Nesselquist et al., 2009). It is rather obvious that the distinction between mixed infections and true recombinants is crucial. This will require analysis of single cells (i.e., single cysts) to be formally undisputable. Since genotyping of single cysts is technically feasible (Miller and Sterling, 2007), and assemblage-specific PCR-based assays are available (Geurden et al., 2009; Almeida et al., 2010), research in this direction will be of paramount importance. A prerequisite for inter- and intra-assemblage recombination is that mixed Giardia infections occur in individual host. This seems to be the case, especially in humans and dogs (Sprong et al., 2009), where in MLG analysis ~20% and ~30% of the isolates were inter-assemblage mixtures. The presence of more than one Giardia genotype has important implications for the etiology of giardiasis: it is unclear how or when humans and animals become infected with two or more genotypes. Either infection with different Giardia genotypes occurs simultaneously, because of environmental mixing, for example in water, or, alternatively, subjects are asymptomatically infected with one Giardia assemblage, but become ill/symptomatic from a second infection with another Giardia assemblage. The latter hypothesis is supported by the finding of asymptomatic subjects. The occurrence of mixed infections is important for molecular typing of Giardia. Using only one marker for the assignment of isolates to specific (sub)-assemblages is not always reliable, as different markers can give different re-

sults. For example, isolates can be typed as ‘‘potentially zoonotic’’ with one marker, but as ‘‘host-adapted’’ with another. More reliable results are obtained when multiple markers are used for typing.

2.12 Conclusions Our knowledge on the molecular and cellular biology of Giardia duodenalis has increased dramatically over the past years, and has impacted on all related fields, including taxonomy and epidemiology. There is a need to continue the research into the population genetics and epidemiology of G. duodenalis to obtain a more accurate picture of the distribution, interactions, and transmission of this parasite. Significant advances can also be anticipated from genome sequencing of representative isolates from assemblages C–G, which can be rapidly obtained by modern pyrosequencing techniques. Until this picture remains blurred, however, we believe that caution must be applied in the interpretation of genotyping data in molecular epidemiologic studies.

References Adam RD (2001) Biology of Giardia lamblia. Clin Microbiol Rev 14: 447–475 Ajjampur SS, Sankaran P, Kannan A, et al. (2009) Giardia duodenalis assemblages associated with diarrhea in children in South India identified by PCR-RFLP. Am J Trop Med Hyg 80: 16–19

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27 Espelage W, an der Heiden M, Stark K, and Alpers K (2010) Prevalence and clinical correlations of genetic subtypes of Giardia lamblia in an urban setting. BMC Public Health (In press) Farthing MJ, Mata L, Urrutia JJ, and Kronmal RA (1986) Natural history of Giardia infection of infants and children in rural Guatemala and its impact on physical growth. Am J Clin Nutr 43: 395–405 Faubert G (2000) Immune response to Giardia duodenalis. Clin Microbiol Rev 13: 35–54 Faustini A, Marinacci C, Fabrizi E, et al. (2006) The impact of the Catholic Jubilee in 2000 on infectious diseases. A case-control study of giardiasis, Rome, Italy 2000–2001. Epidemiol Infect 134: 649–658 Franzen O, Jerlström-Hultqvist J, Castro E, et al. (2009) Draft genome sequencing of Giardia intestinalis assemblage B isolate GS: is human giardiasis caused by two different species? PLOS Pathog (In press) Gelanew T, Lalle M, Hailu A, Pozio E, and Cacciò SM (2007) Molecular characterization of human isolates of Giardia duodenalis from Ethiopia. Acta Trop 102: 92–99 Geurden T, Geldhof P, Levecke B, et al. (2007) Mixed Giardia duodenalis assemblage A and E infections in calves. Int J Parasitol 38: 259–264 Geurden T, Levecke B, Cacciò SM, et al. (2009) Multilocus genotyping of Cryptosporidium and Giardia in nonoutbreak related cases of diarrhoea in human patients in Belgium. Parasitology 136: 1161–1168 Haque R, Roy S, Kabir M, Stroup SE, Mondal D, and Houpt ER (2005) Giardia assemblage A infection and diarrhea in Bangladesh. J Infect Dis 192: 2171–2173 Haque R, Mondal D, Karim A, et al. (2009) Prospective casecontrol study of the association between common enteric protozoal parasites and diarrhea in Bangladesh. Clin Infect Dis 48: 1191–1197 Hollm-Delgado MG, Gilman RH, Bern C, Cabrera L, Sterling CR, Black RE, and Checkley W (2008) Lack of an adverse effect of Giardia intestinalis infection on the health of Peruvian children. Am J Epidemiol 168: 647–655 Homan WL and Mank TG (2001) Human giardiasis: genotype linked differences in clinical symptomatology. Int J Parasitol 31: 822–826 Hopkins RM, Meloni BP, Groth DM, Wetherall JD, Reynoldson JA, and Thompson RC (1997) Ribosomal RNA sequencing reveals differences between the genotypes of Giardia isolates recovered from humans and dogs living in the same locality. J Parasitol 83: 44–51 Hoque ME, Hope VT, Scragg R, Kjellström T, and Lay-Yee R (2001) Nappy handling and risk of giardiasis. Lancet 357: 1017–1018 Hoque E, Hope V, Scragg R, Baker M, and Shrestha R (2004) A descriptive epidemiology of giardiasis in New Zealand and gaps in surveillance data. N Z J Med 117: U1149 Hörman A, Korpela H, Sutinen J, Wedel H, and Hanninen ML (2004) Meta-analysis in assessment of the prevalence and annual incidence of Giardia spp. and Cryptosporidium spp. infections in humans in the Nordic countries. Int J Parasitol 34: 1337–1346 Kohli A, Bushen OY, Pinkerton RC, et al. (2008) Giardia duodenalis assemblage, clinical presentation and markers of

28 intestinal inflammation in Brazilian children. Trans R Soc Trop Med Hyg 102: 718–725 Kosuwin R, Putaporntip C, Pattanawong U, and Jongwutiwes S (2010) Clonal diversity in Giardia duodenalis isolates from Thailand: evidences for intragenic recombination and purifying selection at the beta giardin locus. Gene 449: 1–8 Koulda J and Nohynkova E (1978) Flagellates of the human intestine and of intestines of other species. In: Protozoa of veterinary and medical interest 1978 (J.P. Kreier, ed.), New York, N.Y., Academic Press Inc., pp 69–104 Lalle M, Pozio E, Capelli G, Bruschi F, Crotti D, Cacciò SM (2005) Genetic heterogeneity at the E-giardin locus among human and animal isolates of Giardia duodenalis and identification of potentially zoonotic sub-genotypes. Int J Parasitol 35: 207–213 Lane S and Lloyd D (2002) Current trends in research into the waterborne parasite Giardia. Crit Rev Microbiol 28: 123–147 Lasek-Nesselquist E, Bogomolni AL, Gast RJ, et al. (2008) Molecular characterization of Giardia intestinalis haplotypes in marine animals: variation and zoonotic potential. Dis Aquat Organ 81: 39–51 Lasek-Nesselquist E, Welch DM, Thompson RC, Steuart RF, and Sogin ML (2009) Genetic exchange within and between assemblages of Giardia duodenalis. J Euk Microbiol 56: 504–518 Lebbad M, Ankarklev J, Tellez A, Leiva B, Andersson JO, and Svärd S (2008) Dominance of Giardia assemblage B in Leon, Nicarauga Acta Trop 106: 44–53 Logsdon JM Jr (2008) Evolutionary genetics: sex happens in Giardia. Curr Biol 18: R66–R68 Mahdy AK, Surin J, Mohd-Adnan A, Wan KL, and Lim YA (2009) Molecular characterization of Giardia duodenalis isolated from Semai Pahang Orang Asli (Peninsular Malaysia aborigines). Parasitology 136: 1237–1241 Mayrhofer G, Andrews RH, Ey PL, and Chilton NB (1995) Division of Giardia isolates from humans into two genetically distinct assemblages by electrophoretic analysis of enzymes encoded at 27 loci and comparison with Giardia muris. Parasitology 111: 11–17 Miller KM and Sterling CR (2007) Sensitivity of nested PCR in the detection of low numbers of Giardia lamblia cysts. Appl Environ Microbiol 73: 5949–5950 Monis PT, Andrews RH, Mayrhofer G, and Ey PL (1999) Molecular systematics of the parasitic protozoan Giardia intestinalis. Mol Biol Evol 16: 1135–1144 Monis PT, Andrews RH, Mayrhofer G, and Ey PL (2003) Genetic diversity within the morphological species Giardia intestinalis and its relationship to host origin. Infect Genet Evol 3: 29–38 Morrison HG, McArthur AG, Gillin FD, et al. (2007) Genomic minimalism in the early diverging intestinal parasite Giardia lamblia. Science 317: 1921–1926

S. M. Cacciò and H. Sprong Paintlia AS, Descoteaux S, Spencer B, et al. (1998) Giardia lamblia groups A and B among young adults in India. Clin Infect Dis 26: 190–191 Pelayo L, Nuñez FA, Rojas L, et al. (2008) Giardia infections in Cuban children: the genotypes circulating in a rural population. Ann Trop Med Parasitol 102: 585–595 Sahagun J, Clavel A, Goni P, et al. (2007) Correlation between the presence of symptoms and the Giardia duodenalis genotype. Eur J Clin Microbiol Infect Dis 27: 81–83 Savioli L, Smith H, and Thompson A (2006) Giardia and Cryptosporidfium join the “Neglected Diseases Initiative”. Trends Parasitol 22: 203–208 Smith H, Cacciò SM, Tait A, McLauchlin J, and Thompson RCA (2006) Tools for investigating the environmental transmission of Cryptosporidfium and Giardia infections in humans. Trends Parasitol 22: 160–167 Sprong H, Cacciò SM, and van der Giessen J (2009) Identification of zoonotic genotypes of Giatrdia duodenalis. PLOS Neg Trop Dis 3: e558 Stark D, Barratt JNL, van Hal S, Marriot D, Harkness J, and Ellis JT (2009) Clinical Significance of enteric protozoa in the immunosuppressed human population. Clin Microbiol Rev 22: 634–650 Stuart JM, Orr HJ, Warburton FG, et al. (2004) Risk Factors for sporadic giardiasis: a case-control study in Southwestern England. Emerg Infect Dis 9: 229–233 Sulaiman IM, Fayer R, Bern C, et al. (2003) Triosephosphate isomerase gene characterization and potential zoonotic transmission of Giardia duodenalis. Emerg Infect Dis 9: 1444–1452 Teodorovic S, Braverman JM, and Elmendorf HG (2007) Unusually low levels of genetic variation among Giardia lamblia isolates. Eukaryot Cell 6: 1421–1430 Thompson RC (2000) Giardiasis as a re-emerging infectious disease and its zoonotic potential. Int J Parasitol 30: 1259–1267 Traub RJ, Monis P, Robertson I, Irwin P, Mencke N, and Thompson RCA (2004) Epidemiological and molecular evidence support the zoonotic transmission of Giardia among humans and dogs living in the same community. Parasitology 128: 53–262 Traub RJ, Inpankaew T, Reid SA, et al. (2009) Transmission cycles of Giardia duodenalis in dogs and humans in Temple communities in Bangkok – a critical evaluation of its prevalence using three diagnostic tests in the field in the absence of a gold standard. Acta Trop 111: 125–132 WHO Expert Committee (1981) Intestinal protozoan and helminthic infections. WHO Tech Rep Ser 58: 666–671 WHO, The World Health Report (1996) Fighting Disease Fostering Development. World Health Organization, Geneva. Wielinga CM and Thompson RC (2007) Comparative evaluation of Giardia duodenalis sequence data. Parasitology 134: 1795–1821 Yoder JS and Beach MJ (2007) Giardiasis surveillance – United States, 2003–2005. MMWR Surveill Summ 56: 11–18

Waterborne and Environmentally-Borne Giardiasis Lucy J. Robertson and Yvonne Ai Lian Lim

Abstract Even in industrialised countries, giardiasis may be transmitted via the waterborne/foodborne/environmentally-borne route, resulting in sporadic cases or community-wide outbreaks. In countries where giardiasis is endemic, the risk of environmentally borne transmission is even greater. In this chapter our knowledge regarding transmission of Giardia via these routes is reviewed, with consideration of the relative importance of transmission via these routes, outbreaks that have occurred, detection methodologies (including standard procedures and novel approaches), regulation, risk assessment and risk management, occurrence of Giardia cysts in different matrices and approaches to removal and inactivation. The chapter concludes by discussing future challenges that may impact on transmission of giardiasis by the waterborne route, including water scarcity, wastewater reuse, globalisation, demographic alterations and climate change. We suggest that addressing these issues requires not only technological advances but also, from a global perspective, a multidisciplinary, integrated approach built on shared skills and resources. This is particularly important for countries that are most financially constrained. Establishment of communication channels between all relevant sectors, including meteorologists, risk assessors, public health personnel, water and sanitation engineers, epidemiologists, veterinarians and emergency planners, remains a vital asset.

3.1 Introduction 3. 1.1 The Importance of Waterborne/ Environmental Transmission The public health importance of waterborne or foodborne transmission of infection lies in the potenH. D. Luján et al. (eds.), Giardia © Springer-Verlag/Wien 2011

3

tial for a large population becoming infected. Commonsense, backed up by hard data, suggests that, for giardiasis, the waterborne route is particularly important in comparison with foodborne transmission (Escobedo et al., 2010). A large water body, such as a river, reservoir or lake, may have multiple users and inputs making it vulnerable to contamination, and may serve as a water supply for a large community. If a contamination event of sufficient size occurs at a central water source, such as a reservoir, water containing a large number of infectious units in excess of the infectious dose may be distributed to thousands of individuals. Assuming that the infectious agent is of sufficient virulence and viability to cause infection if ingested by a susceptible host, the potential for infection occurring is dependent upon the volume of water consumed and other host-related factors such as immunity. On the whole, in developed countries at least, such events rarely occur, and generally large municipally run reservoirs or other water supplies are more likely than smaller water supplies to have effective and well-maintained water treatments. Additionally they are more likely to participate in monitoring or surveillance to ensure that such threats are minimised, and that, if they do occur, these incidents might be contained. Nevertheless, such events are not impossible, and the outbreak of waterborne cryptosporidiosis that occurred in Milwaukee in 1993, resulting in an estimate of over 400,000 symptomatic infections (Mac Kenzie et al., 1994), is a testament to this possibility. With respect to Giardia, although waterborne outbreaks affecting hundreds of thousands of individuals have not been reported, there have been several that are considered to have caused giardiasis in thousands of the population (see next section). In general, waterborne outbreaks of giardiasis are considered to occur as point-source outbreaks. That is, infection is caused by direct exposure to contaminated water, the outbreak resulting from exposure of

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many people, and secondary transmission having a relatively minor role. In point-source outbreaks, transmission is terminated when the contamination is removed. However, outbreaks might also occur due to the propagated process, in which secondary transmission, potentially via the environment, may result in an outbreak situation. The relative contribution of the different potential transmission pathways (point– source, environment–person; propagation via secondary infection, person–person; propagation via the environment, person–environment–person) has been investigated for the Milwaukee outbreak of cryptosporidiosis (Eisenberg et al., 2005), but has not been modelled for giardiasis outbreaks. Nevertheless, because of the ability of Giardia to exploit different transmission pathways, it is clear that although waterborne outbreaks of giardiasis may be initially point–source, a proportion of cases may be the result of point–source and propagated processes.

3.1.2 How Does Giardia Lend Itself to Transmission by the Waterborne Route or Environmental Transmission? There are various factors in the biology of Giardia that ensure that waterborne or foodborne transmission is likely to be successful. These have been previously listed in earlier reviews (e.g. Smith et al., 1995, 2006, 2007; Escobedo et al., 2010), and are outlined below. The first of these factors is that large numbers of cysts are excreted into the environment from an infected human being or animal. The cyst excretion rate is highly changeable, and depends on many variables including parasite isolate and period post infection, and a range of host factors including diet, age, sex and immunological status. Smith et al. (2007) state that human infections may result in excretion of up to 2 × 106 cysts per gram of faeces, and data presented by Geurden et al. (2010) demonstrate that experimentally cattle may also excrete faeces with concentrations of approximately 1.5 × 105 cysts per gram of faeces, although the variation from the same cow within a matter of days can be several orders of magnitude. Thus, the high excretion rate coupled with a prolonged excretion period of several days or even weeks (albeit with variations in rate of excretion dur-

L.J. Robertson and Y.A.L. Lim

ing this period) provides the potential for significant environmental contamination, despite the dilution factor being large. That environmental contamination is significant is borne out by almost all surveys of environmental waters for Giardia cysts reporting positive results, although obviously such survey results are dependent not only on the water itself, the catchment area and inputs into the water, but also on the recovery efficiency of the analytical method in the particular water type, or other matrix, being investigated. There are a large number of such surveys published in the literature; a summary of some of these surveys is provided in a later section of this chapter and can also be found at www.waterbornepathogens. org. The second factor that assists in waterborne/environmental transmission of Giardia is that the infectious dose is relatively low. Theoretically, ingestion of a single infectious cyst by a susceptible host should be sufficient to cause infection. In Rendtorff’s (1954) classic infection study, doses ranging from 1 to 106 were ingested by volunteers, and a dose of 10 cysts was reported to result in infection in 100% (2 out of 2) volunteers. However, it is worth noting that although 53% of the volunteers became infected, and changes in bowel motions were observed, none of the volunteers in this study developed symptoms of giardiasis. Thus, although the individuals were infected, they did not have classical clinical giardiasis. The infection-to-illness ratio varies between isolates, as shown by the different response of volunteers subject to two different isolates from symptomatic human infections in a study by Nash et al. (1987). The probability of infection has since been described (Rose et al., 1991b) by two exponential models: Psingle = 1 – exp(–rN) and Pannual = 1 – (1 – Psingle)EF. The variables in these models are defined as follows: Psingle = probability of infection for a single event, Pannual = annualised probability of infection, r = fraction of organisms ingested that initiate infection, and N = average number of ingested organisms. The value for r developed by Rose et al. (1991b) was 0.01982 (95% confidence interval,: 0.009798– 0.03582). It was estimated that an exposure to an annual geometric mean of 0.0007 cysts per 100 L would result in a 1/10,000 annual risk of infection assuming that 2 L of water are consumed daily. However, as not all cysts in water are infectious, not all genotypes in-

Chap. 3 Waterborne and Environmentally-Borne Giardiasis

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fect humans and a water consumption of 2 L daily might not generally apply, it should be noted that this slightly overestimates the risk. On the other hand, underestimation of risk might occur due to detection method inefficiencies, peak contamination levels and prolonged duration of exposure. Additionally, this exponential model assumes that the microorganisms are distributed randomly in a given environmental medium and follow the Poisson distribution. It also assumes that the probability of infection per ingested organism does not vary. However, with the overestimation and underestimation taken together, it is suggested that the model may be useful for estimating the probability of infection (Rose et al., 1991b). Thus, the high excretion rate and the low infectious dose are two complementary factors that, in concert, provide the opportunity for Giardia cysts to occur in the environment in sufficient concentrations for ingestion to result in infections. The third factor that assists in waterborne/environmental transmission of Giardia is the robustness of the cyst transmission stage. Despite being immediately infectious upon excretion, the cysts are also able to survive for prolonged periods in the environment, provided that they are not desiccated or exposed to freeze-thaw fracture cycles, and do not experience elevated temperatures. Studies of survival of Giardia cysts in the environment suggest that at low temperatures, and in the absence of freeze-thaw cycles, Giardia cysts may remain viable or infective for at least one month (De Regnier et al., 1989; Robertson and Gjerde, 2006). Additionally, the small size of Giardia cysts (11–15 μm in length and 7–10 μm width) may enable them to penetrate sand filters, whilst their robustness confers some resistance to many of the commonly used water treatment procedures (see later section). Thus, waterborne outbreaks of giardiasis may occur from Giardia cysts in raw water evading removal or inactivation in fully functional water treatment plants, and the prolonged survival of Giardia cysts in the environment increases their possibilities of being ingested by a susceptible host. The robustness of Giardia cysts is considered to be due to the filamentous cyst wall. This has been shown to contain carbohydrate and protein in the ratio 3:2, with the carbohydrate component of the filaments a unique (β1– 3)-linked N-acetylgalactosamine (GalNAc) homopolymer (Gerwig et al., 2002) for which degradative

enzymes have not been found, although presumably they must exist as there is no apparent irreversible accumulation. Conformational studies have demonstrated that the highly insoluble nature of the cyst wall is not due to the conformational properties of a single GalNAc polysaccharide chain, but is a result of strong interchain interactions to which the potential covalent linkages between GalNAc polymers and the wall protein might contribute. The fourth factor that may enhance the potential for waterborne or environmental transmission of Giardia is the zoonotic nature of this parasite, and the low host specificity of those genotypes that are infectious to humans. Although molecular methods have demonstrated that the majority of domestic and wild animal species are not usually hosts to those genotypes of Giardia that are infectious to humans (assemblages A and B), many mammals can be infected by Giardia belonging to these assemblages. This increases the potential for environmental spread and contamination, as well as for amplification of cyst numbers by infection of semi-aquatic animals (e.g. beavers) living in a watershed or water catchment area. The fifth factor that may facilitate waterborne or environmental transmission of Giardia is the potential for onward contamination by transport hosts. Animals as diverse as different species of birds (Plutzer and Tomor, 2009) and insects (Graczyk et al., 2005; Conn et al., 2007) may contribute to the dissemination of Giardia cysts in the environment, with the potential to spread contamination to otherwise pristine environments. Aquatic birds, in particular, have been suggested to represent an important epidemiologic link in water-associated transmission cycles of Giardia, with a significant role in environmental contamination of aquatic habitats (Graczyk et al., 2008b; Majewska et al., 2009), whilst promiscuous-landing synanthropic flies are particularly associated with the carriage of protozoan parasites to food (Graczyk et al., 2005). Filter-feeding molluscs also have the potential to act as transport hosts of Giardia (Robertson, 2007); accumulation and concentration of Giardia cysts within different species of molluscs that are often consumed by people have been documented in a range of studies; however, it is unclear whether the cysts remain viable, and some evidence suggests that they are inactivated within these shellfish.

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3.1.3 The Relative Importance of Different Environmental Transmission Vehicles Contamination of the environment with infective Giardia cysts enables different substances to act as transmission vehicles, including drinking water, recreational water, different foodstuffs and other environmental matrices that may be ingested. The relative importance of these is summarised in Table 3.1, and discussed below. Transmission via contamination of drinking water is currently the most important route of transmission for Giardia. Not only is there the potential for a large population to become infected, but also consumers, in developed countries at least, tend to “trust” water from the tap and assume that it is a safe product to consume. Indeed drinking large volumes of tap water is often considered to be healthy and to promote a healthy self-image. In the outbreak of waterborne giardiasis in Bergen, Norway in 2004, people who drank large volumes of water had a significantly higher risk of illness, with over 60% of the 83 cases interviewed drinking more than 5 glasses of water daily and many reporting drinking several litres daily (Nygård et al., 2006); many of these interviewees mentioned this as being a part of dieting, healthy living or exercising. Recreational water, in contrast, is not considered as safe to drink, with recognised sources of contamination, from “faecal accidents” in swimming pools to sewage outflow in recreational lakes. Indeed, recreational water, in industrialised countries at least, is infrequently treated as a drinking water source. Instead, ingestion is usually accidental, particularly for adults, and unlikely to be more than a couple of mouthfuls. Additionally, for swimming pools, the levels of disinfection used are generally high compared with that used in drinking water, although for recreational lakes and rivers, disinfection is non-existent. Thus the increased likelihood of contamination may be outweighed by the reduced consumption and, in some

instances, the elevated levels of disinfection, in the likelihood of resulting in infection. Nevertheless, outbreaks of giardiasis in which recreational water has been the vehicle of transmission have been reported on many occasions, with over 10% of the reported outbreaks of giardiasis between 1955 and 2003 associated with contaminated recreational water (Karanis et al., 2007). Foodborne transmission of Giardia is likely to result in fewer cases of infection than waterborne transmission, and occurs in a defined setting. Due to the lower numbers of infections, it is possible that not all foodborne outbreaks are identified, and indeed two reviews published 12 years apart provide identical tables listing foodborne outbreaks of giardiasis (Smith et al., 1995, 2007). It has been suggested that whilst this repetition may indicate an absence of cases of foodborne giardiasis, it is more likely to be a reflection of the difficulty in associating cases or outbreaks of giardiasis with food because there is relatively little awareness amongst physicians about the possible role of foods in transmission of parasitic infections such as giardiasis (Escobedo et al., 2010). Further information on foodborne giardiasis can be found elsewhere in this chapter. Ingestion of mud or sand may be voluntary or accidental. The voluntary consumption of this type of matrix (geophagia or pica) is particularly common amongst small children and individuals with mental problems, and in both these groups, ingestion of soil may contribute to the transmission of pathogens, including Giardia (e.g. Giacometti et al., 1997; Mumtaz et al., 2009). Involuntary ingestion of mud/sand and similar matrices may occur when food materials are not properly washed, or may occur in association with particular activities such as sporting events. A crosscountry cycle race between Rena and Lillehammer in Norway in August 2009 resulted in at least 3018 participants developing diarrhoea within 12 days (23% of the 13,026 responders to a questionnaire that was dis-

Table 3.1 Relative importance of different potential environmental transmission vehicles Drinking water

Most important

Recreational water

Sprouted seeds

Leafy vegetables and herbs eaten raw/soft fruit

Shellfish Mud/sand

Least important

Chap. 3 Waterborne and Environmentally-Borne Giardiasis

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tributed to the 15,312 participants) (http://www.fhi. no/eway/default.aspx?pid=233&trg=MainLeft_5565 &MainArea_5661=5565:0:15,4640:1:0:0:::0:0&Mai nLeft_5565=5544:81695::1:5569:1:::0:0). Aetiological agents were reported for only 4 people (3 with Campylobacter and 1 with Cryptosporidium) and transmission was considered to have resulted from mud splashes into the face/hands over a 94-km course that had been made extremely muddy by adverse weather conditions. Indeed, those participants who reported having mud splashed into their faces during the race were approximately 6 times more likely to be sick than those who did not, and use of a bike with a screen also had a protective effect. Although, in this instance, this was not a giardiasis outbreak, the potential for such an outbreak occurring obviously exists, particularly if grazing animals in the region are infected with an isolate that is also infectious to humans.

outbreaks each. Subsequently the number of outbreaks has declined, and in the last decade since 2000, there have been only 11 reported outbreaks. The largest giardiasis outbreak since 2000 occurred in Bergen, Norway in 2004 where 48,000 people were exposed to contaminated drinking water, with 2500 persons receiving medical treatment and 1300 laboratory-confirmed giardiasis cases (Nygård et al., 2006; Robertson et al., 2006b; http://bgrg.uib.no/). Based on the numbers of outbreaks according to countries, USA accounted for the majority (77.4%; 82 of 106), followed by Canada 13.2% (14), United Kingdom (UK) 2.8% (3), Sweden and Germany1.9% (2) each and Finland, New Zealand and Norway 0.9% (1) each (Table 3.3). It is interesting to note that these outbreaks of waterborne giardiasis were all reported from developed countries where detection and monitoring systems are more likely to be in place. As in many less developed countries giardiasis is endemic, and infrastructures related to water supply, sewage disposal and public health are often lacking, it is likely that these countries are at even greater risk of waterborne giardiasis. However, currently it is unknown whether waterborne giardiasis outbreaks have occurred in Asia, Africa and South America, as no reports are available internationally. The decrease in outbreaks associated with drinking water since the mid-1980s has been postulated by Escobedo et al. (2010) to be related to the introduction of regulations (discussed in the next section), notably in USA and UK, concerning monitoring of protozoan parasites such as Cryptosporidium and Giardia in raw water sources and implementation of water treatments appropriate to the water source. Thus, establishment of a surveillance system, as has been in place in USA for at least a decade, is imperative for generation of comprehensive baseline data to provide useful information for the implementation of effective control measures for minimising contamination and identifying treatment deficiencies. For most outbreaks related to drinking water, the main cause identified was deficiencies in water treatment processes (Table 3.2), including insufficient barriers and inadequate or poorly operated treatment and disinfection systems (over 75% of outbreaks). Another relatively common contributor is distribution system deficiencies (noted in over 12% of outbreaks). When treatment or distribution deficiencies happen in

3.2 Waterborne and Foodborne Giardiasis Outbreaks The acquisition of human giardiasis via ingestion of contaminated matrices such as water and food has gained much global attention. Given that waterborne giardiasis is more common than foodborne giardiasis, it is understandable that greater interest has been directed towards waterborne transmission. In a comprehensive global review from World War I until 2003, G. duodenalis was reported to be responsible for 132 (41%) of 325 waterborne outbreaks associated with protozoan parasites (Karanis et al., 2007).

3.2.1 Drinking Water Outbreaks Of these 132 giardiasis outbreaks, 103 (78%) were associated with contaminated drinking water systems. Since the review by Karanis et al. (2007), another 3 outbreaks have been reported from Norway, Finland and USA, bringing the current worldwide total of documented drinking water outbreaks to 106 (Table 3.2). The earliest incident was reported in 1954 in Oregon, USA, where an estimated 50,000 people were infected. Thereafter, reports of outbreaks continued to rise, peaking in the late 1970s and early 1980s, with each decade documenting more than 30

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L.J. Robertson and Y.A.L. Lim

Table 3.2 Reported outbreaks of waterborne giardiasis due to contaminated drinking water Year

Country

Estimated cases

Suspected cause(s)

References

1954– 1955

Oregon, USA

50000

Mixed aetiology. Surface water source treated only by chlorination.

Veazie, 1969; Meyer, 1973

1965– 1966

Colorado, USA

123

Sewage contamination of two wells serving local communities. Possible contamination of mountain creek serving as additional water source.

Barwick et al., 2000; CDR, 1997

1969

Colorado

19

Contaminated private water system.

Moore et al., 1969; Taylor et al., 1972

1970

California, USA

34

Treatment deficiencies. Surface water source. Water treated only by filtration and disinfection. However, filters were used intermittently.

Craun, 1979

1972

Colorado, USA

28

Treatment deficiencies. Surface water source. Water treated only with disinfection. However, chlorinator was defective.

Craun, 1979

1972

Colorado, USA

24

Treatment deficiencies. Surface water source. Water treated only with disinfection. However, chlorinator was defective.

Craun, 1979

1972

Colorado, USA

297

Treatment deficiencies. Surface water source. One source bypassed the filtration system. Use of alum before filtration discontinued prior to the outbreak.

Vernon, 1973

1972

Utah, USA

60

Untreated surface water source.

Craun, 1979

1972– 1973

Colorado, USA

12

Sewage contamination of untreated well water source from septic tank seepage.

Craun, 1979

1973

Colorado, USA

16

Untreated surface water source.

Craun, 1979

1973

Tennessee, USA

5

Contamination of untreated ground water supply. Nearby outhouse possible source of contamination.

Brady and Wolfe, 1974

1973– 1974

Vermont, USA

32

Surface water source. Treated only by disinfection.

Craun, 1979

1973– 1974

Vermont, USA

20

Surface water source. Treated only by disinfection.

Craun, 1979

1974

Colorado, USA

18

Surface water source. Treated only by disinfection.

Craun, 1979

1974

New York, USA

78

Surface water source. Treated only by disinfection.

Craun, 1979

1974

Utah, USA

34

Untreated surface water source; remote mountain stream. Active beaver ponds and grazing sheep noted in the area.

Barbour et al., 1976

1974– 1975

New York, USA

4800–5300 Mixed aetiology. Municipal water supply where Giardia cyst detected only treated with chlorination. Human settlements within watershed area used chloramines for disinfection.

CDC, 1975; Shaw et al., 1977

1975

Idaho, USA

9

Untreated surface water source

Craun, 1979

1976 – 1979

California, USA

42

Source of contamination unidentified. However, 2 of 3 beavers trapped in watershed were Giardia-positive.

Keifer et al., 1980

1976

Colorado, USA

12

Untreated surface water source

Craun, 1979

1976

Washington, USA

600

Treatment deficiencies. Chlorination equipment failure. Numerous deficiencies in condition and operation of pressure filters prior to the outbreak. Ineffective chemical pretreatment. First outbreak involving filtered water supply. Three infected beavers found within foraging distance of water intake points.

CDC, 1977b; Kirner et al., 1978

(Continued)

Chap. 3 Waterborne and Environmentally-Borne Giardiasis

35

Table 3.2 (Continued) Year

Country

Estimated cases

Suspected cause(s)

References

1976

Colorado, USA

27

Mixed aetiology. Surface water source treated only by disinfection. Giardia cysts found in water sample from beaver pond located upstream from water reservoir.

CDC, 1977a,b

1977

New Hampshire, USA

7000

Mixed aetiology. Evidence for two simultaneous outbreaks. Drinking water originated from two rivers and two largely independent water systems. Detection of Giardia cysts in raw and finished water from both systems. For first river, 30-year-old physical treatment plant had numerous deficiencies. For second river, new treatment plant in operation; lack of experience; faulty construction of common wall separating filtered and unfiltered water. Spring thawing may also have caused simultaneous runoff of contaminated ground material into rivers. Possible human faecal contamination of streams due to recreational activities. Direct sewage discharges along upstream portion of one of water systems. Giardia-positive beaver also found.

CDC, 1977b; Lippy, 1978

1977

Utah, USA

7

Untreated well water influenced by surface water (stream). Beaver dam half a mile upstream from well.

CDC, 1977a

1977

Montana, USA

55

Untreated surface water source.

Craun, 1979

1977

Montana, USA

246

Treatment deficiencies of surface water source. Treated only with disinfection.

Craun, 1979

1978

Colorado, USA

5000

Contaminated water source. Treatment deficiencies of water source. Sewer line obstruction and leakage of sewage into water source. No chemical pretreatment. Filter breakthrough and interruption of chlorination prior to outbreak.

CDC, 1978

1978

Utah, USA

18

Contaminated water source.

Haley et al., 1980

1978

Washington, USA

23

Contaminated non-community water system

Haley et al., 1980

1978

New York, USA

130

Contaminated community water system

Haley et al., 1980

1979

Colorado, USA

53

Contaminated source. Treatment deficiencies. Alum did not provide good floc.

CDC, 1980

1979

Oregon, USA

ND

Contaminated municipal supply. Giardia cysts detected in beaver faeces in watershed.

Keifer et al., 1980

1979

Pennsylvania, USA

3500

Contaminated water source. Treatment deficiencies. Interrupted and ineffective chlorination. Infected beavers within watershed.

CDC, 1980; Lippy, 1981

1980

Arizona, USA

2000

Distribution system deficiency. Direct cross-connection between potable water pipes and a pipe carrying sewage effluent for irrigation.

Starko et al., 1980

1980

Montana, USA

780

Mixed aetiology. Very heavy water run-off resulted from warm sunny weather and snow darkened by ash fall from Mt St Helens volcanic eruption. Unfiltered, inadequately chlorinated surface water. Distribution system deficiency. Antiquated water system. No distribution system storage. Individual service water meters or barriers.

Weniger et al., 1983

1981

Colorado, USA

100

Unfiltered surface water source.

Hopkins et al., 1985 (Continued)

36

L.J. Robertson and Y.A.L. Lim

Table 3.2 (Continued) Year

Country

1981

Estimated cases

Suspected cause(s)

References

British Columbia, 60 Canada

Animal faecal contamination of municipal water system. Surface water source only treated by chlorination. Beavers and muskrats suspected source of contamination.

Bryck et al., 1988

1981

Colorado, USA

29

Treatment deficiencies of surface water source; filter offline.

Hopkins et al., 1985

1981

Colorado, USA

40

Contaminated raw water and water in distribution system. Istre et al., 1984; Treatment deficiencies. Short chlorine contact time due to Hopkins et al., 1985 pump failure. Inadequate filter. Giardia cysts found in water samples both before and after treatment.

1981

Colorado, USA

85

Treatment deficiencies. Backup of unfiltered beaver pond water into springhouse.

Hopkins et al., 1985

1981

Colorado, USA

135

Treatment deficiencies. Surface water. Filtration with inadequate pretreatment.

Hopkins et al., 1985

1982

Colorado, USA

NA

Treatment deficiencies of ground water source (i.e. shallow well, adjacent to a river). No pretreatment before filtration.

Hopkins et al., 1985

1982

Nevada, USA

324

Contaminated supply reservoir. Only treated with Navin et al., 1985 chlorination. City supplied in part by surface water chemically coagulated, settled and chlorinated, but not filtered. Giardia cysts detected in the water supply. Giardiainfected beaver found in one of the reservoirs.

1982

Alberta, Canada

>150

Town water supply. Treated only with chlorination. Infected beaver swimming in town reservoir.

McClure and McKenzie, 1988

1982

Colorado, USA

28

Unfiltered surface water source. Heavy run-off.

Hopkins et al., 1985

1982

Colorado, USA

28

Contaminated unfiltered surface water.

Hopkins et al., 1985

1982

Mjovik, Sweden

56

Distribution system deficiency. Construction deficiencies. 17-year-old water distribution system damaged by tree roots. Sewage related incident. Faulty sewer construction. Possibly defective sand-filter.

Neringer et al., 1987

1982– 1983

Alberta, Canada

>895

Deficiencies in municipal water treatment facilities.

McClure and McKenzie, 1988; Harley, 1988

1983

Colorado, USA

50

Treatment deficiencies. Surface water source. Filtration with inadequate pretreatment.

Hopkins et al., 1985

1983

Montana, USA

100

Contaminated surface water source due to increased melt run-off from volcanic eruption. Animals and human usage within the watershed.

Erlandsen and Bemrick, 1988

1983– 1984

Pennsylvania, USA

347

Mixed aetiology. Human sewage contamination of surface water supplies. Treatment deficiencies. Distribution system deficiency. Numerous waterline breaks due to low temperatures.

Erlandsen and Bemrick, 1988; Sykora et al., 1988

1983

Pennsylvania, USA

ND

Contaminated water source.

Sykora et al., 1988; Rose et al., 1991a

1984

Pennsylvania, USA

ND

Unknown.

Sykora et al., 1988

1984

Pennsylvania, USA

ND

Contaminated water source.

Sykora et al., 1988; Rose et al., 1991a (Continued)

Chap. 3 Waterborne and Environmentally-Borne Giardiasis

37

Table 3.2 (Continued) Year

Country

Estimated cases

Suspected cause(s)

References

1985

Bristol, UK

108

Contaminated water system. Distribution system deficiency. Possible contamination during engineering work on the water main serving affected area of city.

Jephcott et al., 1986; Galbraith et al., 1987

1985– 1986

Massachusetts, USA

703

Mixed aetiology. Contaminated water in reservoirs. Infected Kent et al., 1988 animals within watershed. Signs of human recreational use. Chlorination only treatment. Community water reservoir, out of service for 3 years and put in use again just prior to outbreak.

1986

Vermont, USA

37

Contaminated park water (stream as water source). Treatment deficiencies. Contact times for disinfection estimated as only a few minutes during periods of peak water use. Two cysts found in filtered water. Beaver dam and numerous homes with septic field sewage systems near park. Recent release of large volume of water into stream following destruction of a beaver dam.

Birkhead et al., 1989

1986

British Columbia, 362 Canada

Contaminated surface water source. Water treated only with chlorination. A reservoir pond containing Giardia-infected beaver suspected source of contamination.

Moorehead et al., 1990

1986

British Columbia, ND Canada

Contaminated surface water source. Second outbreak in the area. Despite reservoir improvements, second outbreak occurred when implicated water source re-instituted.

Moorehead et al., 1990

1986

Salen, Sweden

>1400

Distribution system deficiency. Sewage-contaminated drinking water. Simultaneous outbreak of giardiasis and amoebiasis. Overflow of sewage water into the drinking water system.

Andersson and de Yong, 1989

1989

Colorado, USA

19

Treatment deficiency. River water.

Herwaldt et al., 1992

1989

New York, USA

308

Treatment deficiency. Reservoir water.

Herwaldt et al., 1992

1989

New York, USA

152

Treatment deficiency. Reservoir water.

Herwaldt et al., 1992

1989

New York, USA

53

Treatment deficiency. Lake water

Herwaldt et al., 1992

1990

Alaska, USA

18

Untreated river water used as water supply as well water frozen.

Herwaldt et al., 1992

1990

Vermont, USA

24

Treatment deficiency. Lake water.

Herwaldt et al., 1992

1990

Edinburgh, UK

9

Faecal contamination of water tank, probably deliberate.

Ramsay and Marsh 1990; Bell et al., 1991

1990

Colorado, USA

123

Treatment deficiency. Spring water. Spring vulnerable to contamination above ground due to land erosion.

Herwaldt et al., 1992

1991

California, USA

15

Distribution system deficiency. Cross-connection at storage tanks resulted in contaminated surface water entering distribution system using spring water source.

Moore et al., 1993

1991

Pennsylvania, USA

13

Contaminated water source (i.e. well and underground storage tanks). Treatment deficiency. Coliforms detected in water sample. Undetermined source of contamination of either well or underground storage tanks.

Moore et al., 1993

1991

West Midlands, UK

31

Contaminated water reservoir. Treatment deficiencies. Irregular chlorination. Village water abstracted originally from groundwater supply. Livestock grazing in area; suspected source of a reservoir contamination.

Furtado et al., 1998

(Continued)

38

L.J. Robertson and Y.A.L. Lim

Table 3.2 (Continued) Year

Country

Estimated cases

Suspected cause(s)

References

1992

Idaho, USA

15

Consumption of chlorinated, unfiltered groundwater (well).

Moore et al., 1993

1992

Nevada, USA

80

Contaminated surface water source (lake). Treatment deficiency. Chlorination of finished water not consistently maintained. Low levels of Giardia cysts detected in unfiltered surface water.

Moore et al., 1993

1993

Pennsylvania, USA

20

Treatment deficiency of well water. Sewage contamination of filtered and chlorinated well water. Giardia cysts and E. coli detected in tap water.

Kramer et al., 1996

1993

South Dakota, USA

7

Consumption of untreated groundwater (well), contaminated by nearby creek. Giardia cysts detected in well water. Faecal coliforms in well and tap water.

Kramer et al., 1996

1994

Ontario, Canada

300

High concentrations of Giardia cysts in treated water. Two separate surface water supplies. Leakage from storm and sanitary sewage systems aggravated by surface run-off following a winter thaw suspected source of contamination of one supply.

Wallis et al., 2001

1994

Tennessee, USA

304

Distribution system deficiency. Cross-connection between potable and wastewater lines. Potable water used to cool seals of wastewater pump. Pressure fall in potable water system probably caused backflow of wastewater into line for potable water. High concentrations of Giardia cysts in tap water.

Kramer et al., 1996

1994

New Hampshire, USA

18

Contaminated reservoir. Unfiltered, chlorinated surface water. Treatment deficiencies. Suspicions of inadequate chlorine contact times.

Kramer et al., 1996

1994

New Hampshire, USA

36

Unfiltered, chlorinated surface water (lake). Sewagecontaminated finished water. Treatment deficiencies. Suspicions of inadequate chlorine contact times.

Kramer et al., 1996

1995

Washington, USA

87

Distribution system deficiency. Well water. Contamination Lee et al., 2002 of multiple community wells due to illegal cross-connection between domestic water supply and irrigation system at plant nursery.

1995

British Columbia, ND Canada

Source unknown. Simultaneous outbreak of giardiasis and campylobacteriosis. Five cases of cryptosporidiosis also reported.

Ong et al., 1999

1995

Alaska, USA

10

Untreated spring water.

Levy et al., 1998

1995

New York, USA

1449

Treatment deficiency. Lake water. Although no identified interruptions in chlorination at treatment plant, post-filter water turbidity readings, serving as index of effectiveness of filtration, exceeded regulated limit before and during outbreak.

Levy et al., 1998

1997

Oregon, USA

100

Distribution system deficiency. Well/spring water. Noncommunity system combined untreated groundwater and chlorinated spring water. Rodents suspected cause of contamination of a storage reservoir. No data on Giardia in rodents.

Barwick et al., 2000

1997

New York, USA

50

Mixed aetiology. Chlorinated, unfiltered lake water. Beaver found in valve box near reservoir: No data on Giardia in beaver. Treatment deficiency.

Barwick et al., 2000

(Continued)

Chap. 3 Waterborne and Environmentally-Borne Giardiasis

39

Table 3.2 (Continued) Year

Country

Estimated cases

Suspected cause(s)

References

1998– 1999

Florida, USA

2

Untreated well water. Recent rainfall and possible flooding suspected causes of contamination.

Barwick et al., 2000; Lee et al., 2002

2000

Rheinland-Pfalz, Germany

8

No filtration.

Gornik et al., 2000

2000

Minnesota, USA

12

Untreated well water. Possible contamination of well by animal faeces.

Lee et al., 2002

2000

New Mexico, USA

4

Unknown.

Lee et al., 2002

2000

Colorado, USA

27

Treatment deficiency. River water. Pump failure and defective filter cartridge resulted in river water entering drinking water holding tank without filtration. Giardia cysts in sample from water holding tank. No information regarding chlorine levels in water samples.

Lee et al., 2002

2000

New Hampshire, USA

5

Treatment deficiency. River water.

Lee et al., 2002

2000

Florida, USA

2

Distribution system deficiency. Well water.

Lee et al., 2002

2000

Neuwied, Germany

ND

Contamination. Treatment only with chlorination.

Messner, 2001

2001

Manawatu, New Zealand

14

Treatment deficiency. Creek water. Poor maintenance of treatment at a farm. Removal of course filter at creek due to ongoing clogging prior to outbreak. Replacement of undersink filter cartridge with one of unknown specifications from door-to-door salesman. Subsequent person-to-person transmission.

Webber, 2002

NA

British Columbia, 83 Canada

Unknown.

Isaac-Renton et al., 1994

NA

British Columbia, 124 Canada

Contaminated community drinking water supply. Infected beaver found above drinking water intake. No change in water source or introduction of water treatment of any kind after first outbreak in area.

Wallis, 1987; IsaacRenton et al., 1994

NA

British Columbia, NA Canada

Surface water source. Chlorination only treatment.

Wallis, 1987

NA

Newfoundland, Canada

NA

Treatment deficiencies. Chlorination only treatment. Minimal contact time before water reached first customer.

Wallis et al., 1996

2004

Bergen, Norway

1300 lab confirmed

Heavy rainfall. Break in sewage pipe with leakage into reservoir close to intake pipe. Chlorination was the only treatment. Two cysts detected in raw water and 5 cysts in treated water.

Nygård et al., 2006; Robertson et al., 2006b

2007

Nokia, Finland

250

Distribution system deficiency. Sewage-contaminated drinking water. Cross-connection between potable water pipes and a pipe carrying sewage effluent for 2 days.

Rimhanen-Finne et al., submitted.

2007

New Hamsphire, USA

31

Faecal contamination of well water. Presumptive Giardia cyst identified in a home water filter. Coliforms within distribution system. Well contaminated by surface water located 12.5 m from Giardia-contaminated brook where beavers resided (no Giardia detected in beavers). Both Giardia in water and human specimens were Assemblage B, but differed from each other at five nucleotide positions.

Daly et al., 2010

NA not available; ND no data. Updated from Karanis et al. (2007).

40

L.J. Robertson and Y.A.L. Lim

Table 3.3 Country distribution of documented outbreaks of waterborne giardiasis associated with drinking water. Country

Number of outbreaks

Percentage

USA

82

77.4

Canada

14

13.2

UK

3

2.8

Sweden

2

1.9

Germany

2

1.9

Finland

1

0.9

New Zealand

1

0.9

Norway

1

0.9

Total

106

100

concert with sufficient viable human infectious cysts in the water source, massive waterborne outbreaks can occur following the en masse dissemination of viable cysts of Giardia in the drinking water.

3.2.2 Recreational Water Outbreaks Recreational waters encompass swimming and wading pools, thermal and other natural springs, fresh and marine waters, water parks, interactive fountains, and other venues where water contact or activities may take place. Giardia has been identified as the aetiological agent in at least 18 (13.6% of 132) outbreaks associated with contaminated recreational water (Table 3.4), most reported from USA (78.8%; 14 of 18), with 16.7% (3) from UK and 5.6% (1) from Canada. The largest recreational water outbreak occurred in 1995 at a water park in Georgia causing an estimated 5449 cases after a probable faecal accident in the children’s pool. Several stool specimens were found to be positive for both Cryptosporidium and Giardia (Levy et al., 1998). In another outbreak in 1996, in which Giardia was also implicated, an estimated 3000 persons acquired cryptosporidiosis after being exposed to untreated water at a swimming pool and water from a jet-ski spray while watching a water show at a water park in California (Levy et al., 1998). As with the outbreak in Georgia, some stool specimens were also found to be positive for Giardia, although these may simply have been concomitant infections acquired by another transmission route and

not associated with the Cryptosporidium outbreak per se. Contamination of natural bodies of recreational water may be due to urban and non-urban run-off, industrial pollution, storm waters, and human or animal wastes (Smith et al., 1995; Kramer et al., 1998), whereas contamination in swimming pools is often associated with accidental faecal contamination, particularly by toddlers in paddling pools, but can also be caused by poorly constructed and/or maintained plumbing, poor filtration systems and insufficient use of disinfectants (Joce et al., 1991).

3.2.3 Foodborne Outbreaks Although waterborne transmission of giardiasis is well recognised, relatively little attention has been given to foodborne transmission. Thus far, there have been nine documented foodborne giardiasis outbreaks (Table 3.5), mostly associated with infected food handlers with inadequate personal hygiene. However, the possibility of direct contamination of food products during their cultivation, harvesting and subsequent handling and transport of these products from production site to consumer cannot be excluded. Foodstuffs implicated in giardiasis outbreaks have generally been eaten raw or were inadequately cooked. Of these reported outbreaks, two highlighted the potential role of zoonotic transmission, namely the consumption of a Christmas pudding contaminated with rodent faeces and tripe soup made from the offal of an infected sheep. It is interesting to note that there have been no documented reports of foodborne outbreaks since the 1990s. It is probable that the absence of outbreaks of foodborne giardiasis is a manifestation of the complexity in associating cases or outbreaks of giardiasis with contaminated food. In addition, not all countries have a system for reporting foodborne diseases, and even in those countries which do, there is severe under-reporting largely due to the ignorance of either victim or physician in the possible aetiological role of foods, particularly for parasitic infections such as giardiasis. Furthermore, once suspected contaminated food has been eaten or discarded, it is unavailable for analysis, making confirmation of foodborne transmission impossible. Reporting of foodborne outbreaks is often complicated and obscure. For example, as noted by Escobedo et al. (2010), an outbreak of giardiasis

Chap. 3 Waterborne and Environmentally-Borne Giardiasis

41

Table 3.4 Waterborne outbreaks of giardiasis due to contaminated recreational water Year

Country

Estimated cases

Comments

Reference

1982

USA

70

Contaminated swimming pool. Reports of turbid water and low free chlorine residuals in one pool. Possible faecal accidents.

Harter et al., 1984

1985

USA

9

Contaminated swimming pool. Faecal accident caused by handicapped child whilst in pool. Chlorine levels not recorded that day. Zero chlorine level the following day.

Porter et al., 1988

1986

Canada

59

Contaminated water-slide pool, probably through emptying of adjacent toddlers’ wading pool into the implicated water-slide pool.

Greensmith et al., 1988

1991

USA

14

Contaminated swimming pool.

Moore et al., 1993

1991

USA

9

Contaminated wading pool.

Moore et al., 1993

1991

USA

7

Contaminated wading pool.

Moore et al., 1993

1991

USA

4

Contaminated lake.

Moore et al., 1993

1993

USA

12

Unintentional ingestion of untreated lake water.

Kramer et al., 1996

1993

USA

6

Unintentional ingestion of untreated river water.

Kramer et al., 1996

1993

USA

43

Unintentional ingestion of untreated lake water.

Kramer et al., 1996

1994

USA

80

Contaminated swimming pool and wading pool. Intermittent breakdown of swimming pool’s filter. Lack of filtration in wading pool.

Kramer et al., 1996

1995

USA

5449

Also Cryptosporidium parvum. Contaminated children’s pool. Possible faecal accident.

Levy et al., 1998

1996

USA

77

Also Cryptosporidium parvum. Contaminated children’s wading pool. Wading pool supplied by municipal well water coagulated, settled, filtered and chlorine-disinfected.

Levy et al., 1998

1996

USA

3000

Also Cryptosporidium parvum. Contaminated swimming pool. Park patrons exposed to untreated water, at swimming pool and when water from jet ski sprayed show audience.

Levy et al., 1998

1999

USA

18

Contaminated pond.

Lee et al., 2002

1999

UK

54

Also Cryptosporidium parvum. Contaminated CDR, 2000 swimming pool. Cryptosporidium oocysts and Giardialike cysts detected in filter samples.

2000

UK

17

Contaminated water play.

CDR, 2001

Adapted from: Karanis et al. (2007).

associated with consumption of oysters in Washington State, USA, in April 1998, affecting 3 individuals, has been reported (Smith De Waal et al., 2001; Robertson, 2007), but it was not included in the CDC reports covering this period (Lynch et al., 2006), nor in most reviews particularly targeting foodborne giardiasis (Dawson, 2005).

3.3 Detection of Giardia Cysts in Water and Environmental Matrices 3.3.1 Standard Methods for Analysis of Water Detection of Giardia cysts in water samples requires that the cysts be isolated, concentrated and identified.

42

L.J. Robertson and Y.A.L. Lim

Table 3.5 Documented outbreaks of foodborne giardiasis Estimated cases

Food type

Probable source of contamination

Reference

3

Christmas pudding

Rodent faeces

Conroy, 1960

29

Home–canned salmon

Food handler

Osterholm et al., 1981

13

Noodle salad

Food handler

Petersen et al., 1988

88

Sandwiches

–

White et al., 1989

10

Fruit salad

Food handler

Porter et al., 1990

–

Tripe soup

Infected sheep

Karabiber and Aktas, 1991

27

Ice

Food handler

Quick et al., 1992

26

Raw sliced vegetables

Food handler

Mintz et al., 1993

3

Oysters

–

Smith De Waal et al., 2001

Additionally, it may be important or useful to identify the genotype of the Giardia cysts detected (or, possibly, if there are several cysts, the predominant genotype), and their viability or infectivity of the cysts assessed. Although, as previously stated, Giardia cyst excretion by an infected individual may be high, the environmental dilution effect means that the concentration of Giardia cysts in most water types, particularly drinking water, is likely to be low. Unlike bacteria, Giardia cysts cannot be cultivated up to an easily identifiable level, and usually the task of the analyst is to find the individual cysts within the water sample and identify them correctly. Waterborne outbreaks, and the perceived requirement for being able to analyse water samples for these parasites as accurately as possible, both for routine monitoring and for investigating waterborne outbreaks, have resulted in years of method improvement, spiking studies and interlaboratory trials. These have led to the development of standard procedures for detecting Giardia cysts in water samples. These are performance-based procedures in which alternative procedures may be used provided that the necessary quality control tests are performed and the quality control acceptance criteria are achieved. The most commonly used of these are the US EPA Method 1623: Cryptosporidium and Giardia in water by filtration/IMS/FA (http://www.epa.gov/nerlcwww/ 1623de05.pdf), current version published in 2005 and ISO Method 15553 Isolation and identification of Cryptosporidium oocysts and Giardia cysts from water (http://www.iso.org/iso/iso_catalogue/catalogue_

tc/catalogue_detail.htm?csnumber=39804&commid =52834), current version published in 2006. For both these standard methods the principle is the same. First, a relatively large volume of water (10 L–1000 L) is concentrated by filtration (although ISO Method 15553 states that flocculation methods (calcium carbonate or iron III sulphate) may be used for smaller volumes (10 L), provided that performance criteria are met). The concentrated particulates are eluated from the filter, and Giardia cysts are purified using immunomagnetic separation (IMS). Following dissociation of the parasites from the beads, a labelling procedure is performed using a monoclonal antibody (mAb) conjugated to a fluorochrome (usually fluorescein isothiocyanate; FITC) along with a nucleic acid stain as an identification aid. The sample is then examined by fluorescence microscopy for the presence of labelled cysts, which are then confirmed using differential interference microscopy. Probably the most important advance in the development of this method was the use of fluorescently labelled mAbs in the detection step (Sauch, 1985; Rose et al., 1989), commonly known as immunofluorescent-antibody testing (IFAT), which significantly enhanced the ability to detect Giardia cysts in water sample concentrates. Another significant improvement was the development of IMS for Giardia cysts; this lagged slightly behind the development of an equivalent procedure for Cryptosporidium oocysts, and considering that more than 10 years after the initial IMS product was developed there is still no competitor commercially available, this is

Chap. 3 Waterborne and Environmentally-Borne Giardiasis

43

indicated to be difficult. Other advances have included the development of different filter types, with improvements in both filtration and recovery, as well as ease of use, use of flow cytometer-sorted parasite suspensions for accurate calculation of recovery efficiencies and attempts towards automation of the procedure. Despite improvements, these widely used, standard methods are recognised as being far from perfect, and in US EPA Method 1623, the quality control acceptance criteria for percentage recovery efficiency from matrix spikes are listed as between 15 and 118%. Additionally, a table presenting the distribution of matrix spike recoveries from multiple samples collected from 87 source waters shows that for over 25% of 270 samples recovery efficiencies were less than 40% (including more than 5%, or 14 samples, having recovery efficiencies of less than 10%), whilst only 10% (27) of the samples had recovery efficiencies of over 80%. In ISO Method 15553, it is stated that whilst the methods described are the most commonly used and best validated, newer methods or improvements to existing methods will be developed. For some particularly dirty water samples, it is obvious that standard methods may need to be adapted to cope with the extra burden of different contaminants. For example, filter backwash samples from swimming pools may not be possible to filter and may require extra washing procedures, and IMS may need to be repeated (Greinert et al., 2004).

ium oocysts per 10 L of water supplied from the works is one or more. In such cases, appropriate treatment has to be installed and the treated water has to be analysed by a prescribed method, to ensure that these limits are not breached. Even though the direct costs associated with this monitoring have been extremely high, it has been suggested that in itself it has resulted in a decrease in the number of cases of cryptosporidiosis in the UK. As a result of these regulations, water companies closed some treatment works, upgraded others, and were generally attentive to their operation and maintenance activities. Not only did this result in an apparent disappearance of the “spring peak” in cryptosporidiosis in northwest England, but also an annual reduction of 905 reported cases has been estimated, which can be extrapolated to 6770 cases in total (Lake et al., 2007). However, using a Systems-Actions-Management framework to investigate the risk reduction offered by routine monitoring of a water supply for Cryptosporidium it has been suggested that infrequent direct monitoring of pathogens probably provides a negligible risk barrier, whereas “event-driven” monitoring with barrier performance-based treatment verification methods may reduce the probability of undetected pathogen passage through a water treatment plant (Signor and Ashbolt, 2006). Why the UK authorities have introduced regulatory monitoring for Cryptosporidium, but not for Giardia, finds its origins mostly in historical information regarding outbreaks in the UK and perceived need. However, the UK Drinking Water Inspectorate enabled a study to be undertaken in 2002 in which efforts were made to determine whether the regulatory method for Cryptosporidium could be modified for simultaneous monitoring of Giardia, without compromising the Cryptosporidium results (http://www.dwi.gov.uk/research/reports/DWI70-2155_giardia.pdf). However, whilst laboratory trials indicated that this was possible, although recovery efficiencies for Giardia cysts were relatively low, when the study was brought into the field, the recovery efficiencies of Giardia cysts were so low that it was considered that it would have to be adapted, and therefore might impact on the regulatory analysis for Cryptosporidium. These unusual results were discussed in the report with respect to the theory that the less robust Giardia cysts might have been destroyed/ made unrecognisable by the field conditions. How-

3.3.2 Regulatory Procedures: the Value of Monitoring Monitoring, or analysis of water samples for contamination with protozoan parasites, on a regular basis has been the subject of considerable discussion for different regulatory authorities. In the UK, there is regulatory monitoring of treated drinking water for Cryptosporidium oocysts under the Water Supply (Water Quality) Regulations 1999, SI No. 1524. Under these regulations, water undertakers are obliged to carry out a risk assessment for each of their treatment works to establish whether there is a significant risk from Cryptosporidium oocysts in water supplied from the works. It is considered that there is a significant risk if the average number of Cryptosporid-

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ever, as Giardia cysts have frequently been detected in research surveys, these results seem to be rather unusual. A more likely explanation would be that an interaction between the field matrix and the IMS buffers affected the isolation procedure, rather than a physical destruction of the Giardia cysts themselves. In USA, where there have been more waterborne outbreaks of giardiasis reported than elsewhere in the world, monitoring of source water for Giardia is conducted as part of water management, although both the long-term enhanced surface water treatment rule and the interim surface water treatment rule are directed towards Cryptosporidium. The Safe Drinking Water Act is the umbrella legislation covering monitoring of water supplies for all contaminants in drinking water, including Giardia, and within this Act monitoring of source water for Giardia was included to provide baseline information on microbial occurrence and human exposure on which the development of national estimates of the impacts (costs and benefits) of various regulatory options could be based. Monitoring for Giardia (and Cryptosporidium) was first performed under the information collection rule (ICR) and ICR supplemental surveys (ICRSS). In the ICR, raw water for surface and ground water public water systems serving at least 10,000 people was monitored for at least 18 months, and if 10 or more oocysts or cysts per litre were detected in the raw water during any of the first 12 months, then treated water might also be monitored. However, when ICR was introduced and implemented (July 1997–December 1998), US EPA Method 1623 had not been developed, and partly because of the relative inefficiency of the analytical method, the ICRSS was then introduced using US EPA Method 1623 to provide complementary data from selected large and medium-sized water systems, and involved two samples being analysed per month at the participating sites for one year (March 1999–February 2000). The information obtained from these surveys has provided useful input for models and has been the basis of assessments for later regulations such as the Final Long Term 2 Enhanced Surface Water Treatment Rule (EPA, 2005). It is perhaps worth noting that in the period 2005–2006, only two drinking water-associated disease outbreaks were recorded by CDC in USA (Yoder et al., 2008), one with Cryptosporidium and one Giardia (in California, in 2005,

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involving 41 persons and associated with a point-ofuse contamination at a gymnasium). No parasitic outbreaks associated with contaminated surface water were recorded in the 2005–2006 surveillance period and it was suggested that the dramatic decrease in the number of outbreaks caused by parasites might be attributable to enhanced EPA regulation of surface water sources. However, most other countries, including Canada, have not undertaken such extensive monitoring procedures as in USA and the UK, presumably because it is expensive and not considered a priority, and thus Canadian guidelines on protozoa in water (Health Canada, 2004) include statements such as “until better monitoring data and information on the viability and infectivity of cysts and oocysts present in drinking water are available, measures should be implemented to reduce the risk of illness as much as possible”, whilst also recommending that “periodic monitoring of source waters for changes in cyst and oocyst concentrations should be used to adjust treatment processes and to confirm cyst and oocyst concentrations and the adequacy of current treatment processes” (Health Canada, 2004). However, we suggest that such statements do not provide water undertakers with sufficiently precise guidelines. In the process of revision of the WHO Guidelines for Drinking Water Quality, a preventive, risk-based approach has been identified as providing the necessary expansion of the current approach to protect the consumer against health effects from drinking water (Medema et al., 2009). This is because the reactive system, in which there is end-product monitoring, has resulted in outbreaks because by the time that the warning signal is received, the consumers’ health is already at risk. In the risk assessment developed, in this context associated with Cryptosporidium, it is noted that, despite methodological difficulties, monitoring is necessary in order to understand occurrence in catchment and source waters (Medema et al., 2009). However, the authors point out that most regulatory monitoring programmes depend upon a regular (e.g. monthly) sampling scheme, which may miss important peak events. Peak events that may result in contamination surface and groundwater may be weather-related (e.g. heavy rainfall, snowmelt), or may be associated with other causes such as farming practices, accidental spills and water management

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practices. As peak events are often catchment specific, a site-specific pre-sampling survey may identify likely events, and the effects of such events on water quality may be deduced from relatively simple indicators, such as turbidity or faecal indicator bacteria, that can be used as a guide for more complex and expensive monitoring programmes (Medema et al., 2009). Although this document is specifically directed towards Cryptosporidium, one of the most critical pathogens for the water industry due to its ubiquitous occurrence in the environment, its robustness and its resistance to disinfection, and that has resulted in a wealth of information becoming available, the conclusions regarding monitoring in water are also largely relevant for Giardia. Thus, regulatory, event-driven monitoring of source water for Giardia contamination, using a site-specific monitoring programme with relatively simple indicators included to determine the optimum sampling scheme, may provide important, site-specific data for risk assessment for a particular individual water source. In such analyses, use of standard methods, with appropriate quality control, is obviously essential. Thus, knowledge of pathogens in the catchment and prevention of contamination by human and animal waste are essential as part of the WHO Water Safety Plan. However, ensuring that the control measures in place are sufficiently effective to prevent pathogen transmission cannot be determined by source water monitoring (Smith and Nichols, 2010). To this end, monitoring of performance indicators is important (e.g. turbidity or particle removal, pressure in distribution system), although the applicability of such indicators should be validated by appropriate research.

Robertson and Gjerde (2000) used seeded samples of lettuce, salad mix, bean sprouts and strawberries to develop a method based upon elution (by washing in a detergent buffer), concentration by centrifugation, isolation by IMS and finally screening by IFAT as for water samples. The mean recovery (±SD) efficiency of Giardia cysts from all the matrices, apart from bean sprouts, was 67 (±4), with sample sizes ranging from approximately 80 to 110 g and inoculum sizes ranging from 56 to 233 cysts. Bean sprouts, however, were found to be a difficult matrix to work with due to interference from material washed from this vegetable and later speculated to be mucopolysaccharides from the sprout or seed cell walls or bacterial exopolysaccharides (Robertson and Gjerde, 2001a). Further work refining this method suggested that sample age was an important parameter for optimising recovery and choosing the most suitable sample weight – sufficient to enable detection of low-level contamination, but low enough to minimise interference from constituents of the sample itself, as well as minimising the volume of elution buffer necessary and thus reducing the potential for losses during centrifugation and other manipulation processes (Robertson and Gjerde, 2001a). It was also suggested that improved immunomagnetic techniques may have the potential to increase recovery efficiency; however as per today, the Giardia IMS market is still dominated by a single product. A similar approach was used by Cook et al. (2007) to develop a method for analysis of lettuce for Giardia cysts, in which stomaching for 30 seconds into a 1 M glycine buffer (pH 5.5) was used for the initial elution stage, followed by concentration of eluate by centrifugation, IMS and IFAT. This method, which is very similar to that of Robertson and Gjerde (2000), both being based on standard water methods, gave mean recovery (±SD) efficiencies of approximately 46% (±19). The relatively high standard deviation, compared with that obtained by Robertson and Gjerde (2000), is slightly concerning, but it may be due to characteristics of the Giardia isolate used in the original inocula. However, although both these methods are based on standard methods for water, neither of them has been validated by interlaboratory trials to date. The development of a standardised ISO method (ISO/TC 34/SC 9 – standardisation of a process for detection of parasites in food) should be useful in this respect.

3.3.3 Standard Methods for Fruit/Vegetables Despite the potential for transmission of protozoan parasites via ingestion of contaminated fruit/vegetables, there are currently few standardised methods for detecting the transmissive stages of protozoan parasites on/in foods (Smith and Nichols, 2010). Methods were considered generally inefficient, with low and variable recovery efficiency (Rose and Slifko, 1999), until a method for detecting Cryptosporidium and Giardia on fruit/vegetable surfaces based upon the standard methods for water analysis was developed.

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3.3.4 Standard Methods for Shellfish Analysis of shellfish for protozoan parasites has been the focus of a number of research projects from approximately 2000 onwards, despite the lack of any reported outbreaks of these infections associated with shellfish consumption, with particular focus on Cryptosporidium. However, a widely accepted, optimised method for analysis has yet to be described (Robertson, 2007). Different research groups have sometimes reported different efficacies of very similar methods. In general, the methods used involve a tissue homogenisation step (although some research group have used gill-washing or haemolymph), followed by a concentration procedure (usually centrifugation) and a purification procedure. This may be flotation or lipid extraction or IMS; although IMS has been considered useful by some groups, others have found that its performance is so severely affected by the nature of the matrix that it is not worth using (Schets et al., 2007). The very different biochemical nature of shellfish, compared with water concentrates or washings from fruits and vegetables, suggested to Robertson and Gjerde (2008) that another approach should be attempted rather than washing and flotation as a first step, and considering the relatively high protein content of shellfish (8–20% depending on species), they developed a pepsin-digestion method based on the methodology usually used for the detection of Trichinella spp. larvae in meat, or for recovering Ostertagia ostertagi larvae from the abomasal mucosa of cattle, with further isolation. Although this resulted in relatively high recovery efficiencies (70–80%) from 3 different types of shellfish homogenate, the viability of the parasites was reduced. Interlaboratory comparisons of this method and others are essential before a standard method is selected.

3.3.5 Standard Methods for Other Environmental Samples Apart from water of various types and food products, the other samples that may be of interest for analysis (other than for diagnostic purposes) are probably sewage, sludge/slurry, soil, filter backwash and sediments. Analysis of sewage may be conducted to research the occurrence of Giardia infections/genotypes

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in human populations, or effects of sewage treatment regimes on Giardia concentrations and viabilities, whilst analysis of soil, filter debris and sediments may be of particular importance in some outbreak situations. Analysis of sludge/slurry may be important for risk analysis should the sludge be destined for use on agricultural land. Among the various publications reporting on Giardia in sewage, analytical techniques have included filtration and flotation (e.g. Kistemann et al., 2008), centrifugation and IMS (Sulaiman et al., 2004; Robertson et al., 2006a; Robertson et al., 2008), repeat centrifugation and flotation (Robertson et al., 2000; Lim et al., 2007), direct analysis of 50 μL native samples (Robertson et al., 2006a), filtration and IMS (Briancesco and Bonadonna, 2005). Generally, detection has been reliant on IFAT, although various research groups have also investigated the use of molecular methods (e.g. Guy et al., 2003; Bertrand et al., 2004). However, sewage and sludge are difficult matrices to work with in terms of potential inhibitors, and molecular detection methodologies are currently not recommended, although when applied on purified samples they may provide useful information regarding genotype. In slurry/sludge analyses, various approaches have been used, including sucrose/sucrose-phenol flotation (Santos et al., 2004; Graczyk et al., 2007; Reinoso and Becares, 2008), ether clarification (Santos et al., 2004), centrifugation with or without either sucrose flotation or ether clarification (Rondello Bonatti et al., 2007), direct analysis of native samples (RimhanenFinne et al., 2004), sedimentation and IMS (MassanetNicolau, 2003). Again, IFAT has been the primary detection technique, although fluorescent in situ hybridisation (FISH) has also been used, not least to provide a handle on cyst viability (Graczyk et al., 2007, 2008a). In the polymicrobial waterborne outbreak that occurred in Nokia, Finland, in association with accidental coupling between a pipe handling processed wastewater and a drinking water distribution pipe, soft deposits were analysed for Giardia cysts by filtration followed by IMS and one was found to be positive (Rimhanen-Finne et al., submitted). In the waterborne outbreak of giardiasis that occurred in Bergen, Norway, during the outbreak investigation, a septic tank at a particular tourist site was

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considered as a possible source of contamination (Robertson et al., 2006b). Not only were samples of the septic tank analysed for Giardia cysts, but also 12 soil samples taken from the vicinity of the septic tank (Robertson et al., 2006b). Whilst the septic tank samples were analysed by repeated centrifugations, followed by IMS, the soil samples were subjected to a series of mixing, settling and decanting procedures followed by either salt flotation and IMS, or simply IMS. Giardia cysts were detected in four of the soil samples and also in the septic tank samples. Molecular characterisation of the septic tank samples in comparison with molecular characterisation of patient samples was used as evidence to suggest that the septic tank was unlikely to be the source of contamination (Robertson et al., 2006b). However, despite having a standard technique for analysing matrices such as soil and sediments for Giardia cysts, a standardised method has yet to be developed and validated.

DNA extraction methods for Giardia cysts in environmental matrices have been conducted, particularly without the use of IMS, which is an expensive step in the procedure. A combination of freeze-thaw, sonication, and kits has been recommended (Guy et al., 2003), as well as the use of Chelex 100 chelating resin post-lysis (Anceno et al., 2007a; Yu et al., 2009). Whilst the publication by Rochelle et al (1997) provided the first real information on the potential for using PCR to detect waterborne Giardia cysts, the authors were careful to emphasise both the requirement for refinement of the technique (primers, PCR optimisation) and the need for parallel use of conventional techniques. Since then, PCR has been used in a variety of studies for detection and identification of Giardia cysts in water and environmental samples. Rimhanen-Finne et al. (2002) published a paper in which the detection limit for Giardia cysts by PCR (coupled with IMS) was 50 cysts in 2 L of surface water, and used this method to detect Giardia contamination in one out of 54 surface waters. However, it is perhaps revealing that when an outbreak situation occurred in Finland, the investigation led by the same author apparently did not attempt to use molecular techniques, but instead relied on standard methods (Rimhanen-Finne et al., submitted). As cysts are often in low concentrations, detection sensitivity must be maximised. Use of a repetitive sampling/multi-tube approach should be considered in order to account for stochastic pipetting events in setting up a PCR from template with low concentrations of DNA (Taberlet et al., 1996). Additionally, use of nested PCR may increase both sensitivity and specificity. A study on detection of Giardia cysts using a nested PCR targeting the triosephosphate isomerase gene demonstrated that for pure isolates, in which the cysts were transferred directly into the PCR tubes, 80% of 50 single cyst samples gave a positive result, whilst 100% of 50 replicates containing 10 cysts gave a positive result (Miller and Sterling, 2007). Reverse transcription polymerase chain reaction (RT-PCR) targeting the heat shock protein gene was proposed over 12 years ago as a method for detecting only viable Giardia cysts in water samples (Abbaszadegan et al., 1997). However, it had low sensitivity (103 cysts/100 μL). More promising results were achieved by Kaucner and Stinear (1998), but the method was not widely adopted by other laboratories.

3.3.6 Novel/State-of-the-art Methods and Future Approaches Detection and identification of low numbers of Giardia cysts on or within matrices of varying complexity remains a challenge, despite significant advances over the past couple of decades, the most important of these being use of IFAT for detection and IMS for separation. The use of flow cytometry and automated methods for assessing labelling has also been an interesting advance, although it lies beyond the scope of the majority of laboratories. The use of 4,6-diamidino-2-phenylindole as an adjunct for identification by characteristic labelling of the cyst nuclei has also been important. The application of molecular techniques for identification of genotypes has been a key development, particularly for identifying whether cysts are of public health significance and the probable origin of contaminating cysts. Molecular techniques have also been directed towards detection. One of the challenges of using molecular methods for identifying Giardia cysts in environmental samples is the efficiency of the DNA extraction method, particularly as the number of organisms is often very low. Additionally, the presence of PCR inhibitors, such as humic acids, fulvic acids and phenols, within the matrix may also pose a problem. Comparisons of

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A yet more recent publication (Lee et al., 2009), using a new primer set against the same gene, has suggested that a sensitivity of 1 cyst per 100 μL water concentrate can be achieved when heat shock treatment is applied. However, as with all methods, extensive interlaboratory validation is necessary before this apparently encouraging result can be considered beyond a research tool. Real-time PCR (qPCR) for detection of Giardia in water/environmental samples was first described by Guy et al. (2003), using primers targeting the β-giardin gene. It was considered to be sensitive down to a single cyst, but in environmental samples the quantitative evaluation was dependent on inhibitors. A later publication (Bertrand et al., 2004) sought to improve the specificity; it was directed towards detection and quantification of Giardia cysts in wastewater, and used probes targeted to the elongation factor 1A gene. A sensitivity of 18 cysts in 200 μL purified suspension (or 180 cysts/L of wastewater assuming that the PCR efficiency is not decreased in environmental samples) was achieved. However, when applied to 6 wastewater samples, only 5 produced amplification curves, and quantification by IFA always gave higher results than quantification by qPCR. Whilst the authors were of the opinion that the qPCR assay provided a good indication of the level of Giardia contamination, this method clearly needs further optimisation before it can be used for standard monitoring. One methodology that has recently gained attention for analysis of Giardia in environmental samples is loop-mediated isothermal amplification (LAMP). This methodology is based on auto-cycling strand displacement DNA synthesis by Bst polymerase. In testing the use of this technology on water and environmental samples (Plutzer and Karanis, 2009), of 10 surface water samples that were positive by IFAT, 7 were positive using LAMP, with primers targeting the Elongation Factor 1 alpha (EF-1α) gene, and of 15 sewage water samples that were positive by IFAT, 9 were positive by LAMP. Some samples that were positive by LAMP were considered to contain relatively few cysts (1 in 10 L), but other samples that were negative by LAMP had relatively high concentrations of cysts (400 cysts in 0.5 L). Use of standard PCR on the same samples found 4 of the surface water samples positive and 10 of the sewage water sam-

L.J. Robertson and Y.A.L. Lim

ples positive using primers targeting the SSU rRNA gene, and 3 of the surface water samples positive and 9 of the sewage water samples positive using primers targeting the glutamate dehydrogenase (GDH) gene. Samples positive by LAMP were not always those that were positive by PCR, and vice versa. The authors considered LAMP to be superior to PCR due to its relative ease and cheapness, and also because it is not affected by inhibitors in the sample. The lack of quantification and the occurrence of false negatives are problems that suggest that this technology is not yet sufficiently developed for routine analysis. However, a later publication (Plutzer et al., 2010) using spiked drinking water samples did not apparently have this problem; cyst concentrations down to 1 cyst in 10 L were detected, even in high-turbidity water samples. The authors emphasise that the lack of requirement for IMS means that this is a cost-effective method, as is the lack of necessity for a fluorescence microscope (need for IFAT) or a thermal cycler (required for PCR). However, the reagents for extraction of DNA are necessary, as well as those for the LAMP reaction. A positive reaction is detected as a white turbidity, due to the formation of magnesium pyrophosphate, a by-product of the amplification reaction produced in proportion to the amount of amplified products. The authors suggest that a turbidimeter can be used to quantify the results. Although the published research to date regarding this technology is exciting, again this tool remains in the realm of research until further groups have tested and validated it on different matrices. Real-time, online monitoring for waterborne pathogens has also been the goal of some scientists, with the intention that action can be taken before the pathogen is in the distribution network. Such technologies have, to-date, focussed upon Cryptosporidium (Quist et al., 2008; Shaw, 2009), and are currently in their infancy. Whether they become valuable aids in water monitoring has yet to be determined, but if so, they can be potentially developed to include Giardia.

3.3.7 Risk Assessment and Risk Management Apart from during an outbreak situation, probably the most important reason for analysing environ-

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mental matrices for Giardia cysts is to obtain credible information that can be used for risk assessment and, subsequently, risk management. In risk assessment, an objective, preferably quantitative, evaluation of the risk associated with a particular matrix is performed, taking into account as much as possible the associated assumptions and uncertainties. Risk management is the logical subsequent step after risk assessment, and provides the framework by which the risk can be monitored (if it is likely to fluctuate under different conditions), minimised and controlled. In the context of waterborne or environmentally transmitted giardiasis, the risk assessment requires information on the occurrence of infectious Giardia cysts in the matrix of interest, the dose response, pathogenicity and exposure of the population at risk. This information not only varies according to the isolate of Giardia in a given environment, but will also vary according to the matrix under consideration and the population at risk (for example dietary habits, water consumption rate, immunological status). Various models have been developed into which this information can be included, and also the impacts of different risk mitigation approaches. One model which is commonly used is the Monte Carlo model, which is based on repeated random sampling to compute results and is therefore useful for modelling a phenomenon that has significant associated uncertainty. Using a Monte Carlo model developed to assess the relative risks of infection associated with the presence of Giardia in drinking water, the impact has been assessed of various approaches for modelling the initial parameters of the model on the final risk assessment (Jaidi et al., 2009). In the Monte Carlo simulations performed in this work, it was shown that when the concentrations of parasites in raw water were below the detection limit, a uniform distribution provided the best description, but above detection limits, a mixed distribution was preferable. Additionally, the selection of process performance distributions for modelling the performance of treatment has a significant effect on the estimated risks (Jaidi et al., 2009). Risk assessments can also be used to provide answers to specific, relatively narrow questions, and as long as the assumptions included in the risk assessment are understood, it may provide the foundations for developing appropriate guidelines by which the

risk may be minimised, if deemed unacceptably high. For example, a risk assessment used to address the human health impact, arising from consumption of particular salad vegetables with contaminated water, calculated that the annual risk of infection with Giardia derived from lettuce irrigated with water containing the highest detected concentration of Giardia cysts (1633 cysts per 100 L) was 1.96 × 10–1 (Mota et al., 2009). This suggests that addressing potential points of contamination, both pre and post harvest, for fruits and vegetables that are consumed raw, should be a food industry priority (Mota et al., 2009). In Norway, in the wake of the Bergen giardiasis outbreak, the Norwegian Scientific Committee for Food Safety undertook a risk assessment associated with parasites in water, to address specific questions posed by the Norwegian Food Safety Authority, and provided various suggestions on how the risk may be minimised (VKM, 2009). Whilst it is important that all assumptions used are noted in such risk analyses, and the resultant risk management directives, it is also important to minimise the effects of such assumptions as far as possible. For example, it is known that the recovery efficiency of the methods for analysing environmental samples can be low, and vary between matrices and catchments. In one study (Petterson et al., 2007), the impact of incorporating method recovery data on concentration estimates was investigated using a dataset of 99 points with paired recovery estimates. Stochastic concentrations were estimated using either (a) no consideration of recovery efficiency; (b) limited recovery data, with sample recovery considered as an independent random variable; and (c) each result adjusted for a concurrently derived recovery estimate. Whilst the second two approaches provided similar results, the first approach underestimated by approximately 100%, indicating that when using such data to infer health risks to consumers, recovery data should be incorporated into source water concentration estimates, and, if such data are unavailable, conservatively low estimates should be assumed. Thus, such assessments can inform monitoring programmes, indicating that recovery data should be collected for particular sites such that sitespecific relationships can be incorporated into source water concentration estimates. In general, more effort has been directed to date towards risk assessment and risk management con-

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cerned with Cryptosporidium in drinking water (e.g. Medema et al., 2009) and other potential environmental transmission vehicles; lessons learned here should be of utility and application to similar considerations for Giardia.

3.4 Occurrence of Giardia in Water and Environmental Matrices: A Global Perspective 3.4.1 Water Matrices The occurrence and concentrations of Giardia cysts in different water types (raw water, drinking water and recreational water) have been well documented. A selected example of studies on the occurrence, concentrations and genotypes (where investigated) in different enviornmental matrices from various countries is presented in Table 3.6. Giardia cysts excreted by infected hosts (human and/or animal) may contaminate the environment via faeces, sewage effluent, slurry discharges or run-off from land. Substantial amounts of data on the occurrence of Giardia cysts in water are available from North America and the UK. However, since the turn of the century, more data have been published from other countries especially Europe, South America and Asia. Data of occurrence of Giardia cysts in water matrices accrued from North America, South America, Europe and Asia have shown high occurrence rates, with most being above 30% and a few reaching almost 100% of sampled water (Anceno et al., 2007b; Schets et al., 2008). Concentrations of cysts per litre of surface water are generally between <0.01 to <100 cysts; however, concentrations are higher in developing countries, especially in Asia. For example, in Malaysia the highest level of cysts detected in one litre of surface water was 12,780 cysts (Lim et al., 2008), and in a canal in Thailand, concentrations of up to 32,400 cysts/L were detected (Anceno et al., 2007b). This is not surprising as the water was sampled from rivers that had been categorised as highly polluted. However, in Japan, one of the most developed and wealthy countries in Asia, concentrations as high as 580 cysts/L have also been detected (Hashimoto et al., 2001). This river was also reportedly polluted with Giardia cysts and Cryptosporidium oocysts at the level of 100/L in

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an earlier study by the same authors. In recent years, numerous studies have also employed advanced techniques such as PCR-based methodology to evaluate the genotypes of Giardia found in water. Molecular approaches have detected zoonotic genotype, G. duodenalis assemblage A, in surface water sources in Hungary (Plutzer et al., 2008), Portugal (Lobo et al., 2009) and Spain (Castro-Hermida et al., 2009). The presence of cysts in groundwater is alarming as it is considered to be at a relatively low risk of being contaminated, particularly in comparison with surface water. From a total of 18 well-water samples taken in Bulgaria, 22.2% were found contaminated with cysts with concentrations ranging from 0.016 to 127.5. These cysts were detected in well water used for drinking and cleaning purposes. The massive contamination with Giardia cysts of well water in one village suggested the probable run-off of faecal contaminated surface water (Karanis et al., 2006). In Malaysia, Giardia cysts were detected in 18% of 28 samples from 2 vertical wells with a concentration of up to 0.25 cysts/L (Lim et al., 2008). One of the contributing factors was the inappropriate location of the toilet (i.e. upstream of the well). Although no outbreak associated with groundwater contamination has been reported in Malaysia, this finding is of concern for local communities and warrants further study, especially in rural communities that are dependent on wells as their main water source (Lim et al., 2008). These results emphasise the need to protect the groundwater resources from sewage and surface water contamination. Vulnerable groundwater resources that cannot be protected should be considered to be at the same risk for contamination as surface water resources (Karanis et al., 2006). Occurrence of cysts in recreational water has also been well evaluated in various countries, mainly in Europe where recreational water-based activities are very popular. Although the level of contamination is generally low (e.g., <1 to <20 cysts/L), more recent studies have highlighted higher levels of contamination, especially in recreational streams in Switzerland (Wicki et al., 2009) and a recreational lake in Malaysia (Lim et al., 2009). One of the main reasons implicated is discharge of sewage effluent into the recreational water. In both these studies, more than 90% samples were found to be positive, indicating the ubiquitous nature of this protozoan parasite in recreational water.

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Molecular analysis of Giardia cysts from the recreational lake water in Malaysia demonstrated occurrence of G. duodenalis assemblage A. The finding of a zoonotic assemblage indicated a potential risk to the lake users, and a clearer assessment of the source and route of transmission is imperative to determine the dynamics of Giardia transmission between human and animal hosts living in the same geographical area (Lim et al., 2009). Besides recreational fresh water, Giardia cysts have also been found in estuarine and coastal marine waters. Naranjo et al. (1989) found that marine sewage outfall effluents contained an average concentration of 0.199 cyst/L, while Ho and Tam (1998) noted that cysts were detected more frequently in water samples of lower-grade beach water compared with higher-grade beach water in Hong Kong. Although cyst concentrations are generally less than 10 cysts/L, the potential health risk faced by swimmers bathing in these waters warrants further investigation. Furthermore, as large quantities of sewage are often discharged into coastal water after primary treatment, the actual local health risk due to the presence of Giardia in sewage also needs further evaluation (Ho and Tam, 1998). Studies that analysed final or treated water have shown that Giardia cysts occur in potable waters, although in most instances there has not been a detectable increase in the number of cases of giardiasis in the communities to which the water is supplied. Generally the concentrations of cysts are very low (e.g., <1 cysts/L) in treated water. Although concentrations of cysts were reported to be high in surface water in Asia, the concentration of cysts in treated water is low based on two studies carried out in Japan (Hashimoto et al., 2001) and in Taiwan indicating high efficiencies of drinking water treatment plant processes in these countries. An interesting study was carried out in Bulgaria investigating bottled water for the presence of Giardia cysts (Karanis et al., 2006). Among eight samples analysed, one was positive with 0.5 cysts/L. This is a cause for public health concern, especially with the recent rise in preference for drinking bottled water rather than treated tap water, due to convenience or lack of confidence in tap water quality. Currently, regulations regarding monitoring for Giardia cysts in bottled water are not well established and contamina-

tion of bottled water may result in widespread outbreaks of diseases. As shown in numerous studies, sewage treatment plants can contribute cysts to receiving waters used for the abstraction of drinking water. Findings from Bulgaria, Norway, Kenya and Malaysia demonstrate concentrations as high as 51,333 cysts/L being discharged in sewage treatment plant effluent (Robertson et al., 2006a; Lim et al., 2007). Sewage effluent from inland treatment plants is usually discharged into rivers. In developing countries in Asia, these effluents can contaminate downstream water sources used by rural communities that are dependent on river water as their main source of drinking water and services such as drinking water treatment works, livestock farms and food producers.

3.4.2 Soil Besides water, Giardia cysts have also been detected in soil (Table 3.6). A study conducted in a semirural community in Malaysia demonstrated Giardia cysts in 0.7% of 138 soil samples with a concentration of 2.9 cysts/g (Lim et al., 2008). Because of the high prevalence of parasitic infections in this community and the habit of defaecating indiscriminately, the most likely source of contamination was human excreta. The low concentrations of cysts detected might be due to (a) low levels of defaecation in house compounds, (b) rapid transport of cysts across the surface of the soils by rain and/or (c) percolation of cysts into the sandy soil. Sandy soil does not entrap (oo)cysts as efficiently as clay soil because it is more porous and does not retain water well. The occurrence of cysts in soil within the house compound, especially in the vicinity of children who regularly play with soil, should be of concern to public health officials (Lim et al., 2008). Although little is known about the fate of protozoan parasites in the soil environment, a study assessing the effects of drying and temperature on Cryptosporidium oocysts placed in semi-permeable membranes on pastures showed that they were susceptible to drying (Svoboda et al., 1997). Viability decreased to undetectable levels after 2–4 weeks in summer, whilst in winter, the combined effects of drying and freezing appeared to kill oocysts rapidly within a

52

L.J. Robertson and Y.A.L. Lim

Table 3.6 Selected examples of the occurrence of Giardia cysts in water and environmental matrices Country

Number Occurrence; Concentration of samples (% samples range (cysts/litre) positive)

Genotype

Reference

Surface water USA

181

USA Canada Spain

16

0.02–1.4

No information

Rose et al., 1990

85

81.2

0.04–66

No information

LeChevallier et al., 1991

22

32

–

No information

Roach et al., 1993

8

63

<0.01–0.21

No information

DeLeon et al., 1993

UK

53

33

0.01–1.05

No information

Smith et al., 1993

Japan

13

92

40–580

No information

Hashimoto et al., 2001

Norway

408

11.8

<0.1

No information

Robertson and Gjerde, 2001c

Taiwan

26

46.2

<0.029–10.3

No information

Hsu et al., 2001

Russia

87

29.9

0.02a

No information

Egorov et al., 2002

Canada

249

100

0.005–0.34

No information

Cited by Smith and Grimason, 2003

Canada

1760

21

–

No information

Cited by Smith and Grimason, 2003

Czech Republic

–

–

0–4.85

No information

Cited by Smith and Grimason, 2003

Honduras

–

–

3.8–21

No information

Cited by Smith and Grimason, 2003

Venezuala

12

33

0.008

No information

Cited by Smith and Grimason, 2003

Mexico

58

50

0.17–16.33

No information

Chaidez et al., 2005

85.7

0.4–29.3

No information

Gómez-Couso et al., 2005

Spain

7

Bulgaria

41

26.8

0.004–116

No information

Karanis et al., 2006

France

36

83.3

0.1–16.5

No information

Coupe et al., 2006

Russia

16

18.8

7–35.7

No information

Karanis et al., 2006

Hungary

34

48.4

<10.3

No information

Plutzer et al., 2007

Thailand

120

98.3

<32,400

No information

Anceno et al., 2007b

Hungary

16

62.5

0.1–17.4

Assemblage A and B

Plutzer et al., 2008

Malaysia

174

39

0.7–12,780

No information

Lim et al., 2008

Netherlands

32

96.9

0.1–16.7

No information

Schets et al., 2008

USA

192

24.5

–

No information

Ryu and 2008

Brazil

15

26.6

0.2

No information

Nishi et al., 2009

France

162

93.8

0.05–51.1

No information

Mons et al., 2009

7

57.1

0.01–0.08

No information

Vernile et al., 2009

58

–

Assemblage A

Lobo et al., 2009

Italy Portugal

69

Spain

116

67.2

2–722

Assemblage A and E

Castro-Hermida et al., 2009

Brazil

13

46.1

<3.4

No information

Razzolini et al., 2010

France

21

38.1

0.1–12

No information

Coupe et al., 2006

Italy

21

38.1

–

No information

Oliveri et al., 2006

Recreational water

(Continued)

Chap. 3 Waterborne and Environmentally-Borne Giardiasis

53

Table 3.6 (Continued) Country

Netherlands Hungary Switzerland Malaysia

Number Occurrence; Concentration of samples (% samples range (cysts/litre) positive)

Genotype

Reference

55

36.4

0.04–0.25

No information

Schets et al., 2008

3

33.3

0.1–17.4

No information

Plutzer et al., 2008

40

97.5

1–216

No information

Wicki et al., 2009

9

77.8

0.17–1.1

Assemblage A

Lim et al., 2009

Groundwater Bulgaria

18

22.2

0.016–127.5

No information

Karanis et al., 2006

Malaysia

28

83.3

<0.25

No information

Lim et al., 2008

Treated water USA

36

0

–

No information

Rose et al., 1991a

USA

82

16.9

–

No information

LeChevallier et al., 1991

Spain

9

22

<0.01–0.03

No information

DeLeon et al., 1993

106

19

0.01–1.67

No information

Smith et al., 1993

11.5

0.5–2

No information

Hashimoto et al., 2001

No information

Egorov et al., 2002

UK (Scotland) Japan Russia

26 70

7.1

0.00016

a

Canada

42

17

–

No information

Cited by Smith and Grimason, 2003

Canada

249

98.5

0.045–1.72

No information

Cited by Smith and Grimason, 2003

Canada

1760

18.2

–

No information

Cited by Smith and Grimason, 2003

Germany

12

83.3

0.02–1.03

No information

Cited by Smith and Grimason, 2003

Venezuela

11

36

0.013a

No information

Cited by Smith and Grimason, 2003

Taiwan

31

77

–

No information

Cited by Smith and Grimason, 2003

Bulgaria

46

13

0.004–0.5

No information

Karanis et al., 2006

Hungary

45

26.7

<0.63

No information

Plutzer et al., 2007

Portugal

106

25.6

–

No information

Lobo et al., 2009

12

41.7

<0.06

No information

Razzolini et al., 2010

8

12.5

0.5

No information

Karanis et al., 2006

Kenya

26

65.4

0.1–90

No information

Grimason et al., 1996

Spain

16

87.5

7–2541

No information

Gómez-Couso et al., 2005 Karanis et al., 2006

Brazil Bottled water Bulgaria Sewage effluent

Bulgaria

7

42.9

139–604

No information

Norway

72

53

100–51333

Both Assemblages Robertson et al., 2006a A and B detected in sewage influent

Malaysia

28

17.9

1–1462

No information

Lim et al., 2007

138

0.7

<2.9

No information

Lim et al., 2008

Soil Malaysia

54

few days. Viability assay using fluorogenic vital dyes has shown that Cryptosporidium oocysts can survive between 4 and 8 weeks in tropical soil where moisture content is high (Lim et al., 1999; Farizawati et al., 2005).

3.4.3 Food Products Foodborne giardiasis is associated with food products that are usually eaten raw or cooked inadequately. Although there have not been many reports of occurrence of cysts in food, the presence of Giardia cysts in food does have significant implications for public health and global food safety. For example, it has recently been found that consumption of green salad was an important risk factor for acquiring Giardia infection in Germany (Espelage et al., 2010). This finding suggests a significant risk to public health, especially as Giardia cysts have been detected in a variety of vegetables such as water spinach, lettuce, various herbs, strawberries, sprouted seeds, potatoes, carrots and cilantro (Monge and Arias, 1996; Amahmid et al., 1999; Takayanagui et al., 2000; Robertson and Gjerde, 2001b; Vuong et al., 2007). Giardia cysts may be introduced into these food items during cultivation via the use of livestock wastes as fertiliser, run-off from slurry heaps, contaminated water for washing or keeping the produce moist and contaminated irrigation water. Studies which analysed irrigation water and wash water used in the fresh produce industry have demonstrated the presence of Giardia cysts, highlighting the possible role of irrigation water in contaminating fruit or vegetables (Robertson and Gjerde, 2001b; Robertson et al., 2002; Thurston-Enriquez et al., 2002; Chaidez et al., 2005; Lonigro et al., 2006; Vuong et al., 2007). This was evident from a study in Cambodia that reported the occurrence of Giardia in water spinach (56% of 35; 6.6 cysts/g) that had been grown in untreated wastewater ponds (Vuong et al., 2007). Contamination may occur at any point of time between production and consumption, which includes harvesting, packing and selling procedures, as well as the procedures associated with preparation of the product into a final meal. In addition, the health risks posed by occupational exposure of farmers during the harvest

L.J. Robertson and Y.A.L. Lim

and subsequent handling and transport of vegetables from production sites to the markets (e.g. through splashing of vegetables with contaminated water to keep them moist and fresh as well as through unhygienic practices linked to the reuse of contaminated containers or baskets) are expected to be high (Vuong et al., 2007). Besides food products derived from plants, shellfish also have the potential to transmit Giardia infection to consumers (Robertson, 2007). The filterfeeding mechanism of shellfish allows concentration of pathogenic microorganisms within their digestive glands/tracts. Given that shellfish are commonly eaten raw, or only very lightly cooked, they have the potential of transmitting foodborne giardiasis. In Netherlands, commercial and non-commercial oysters (Crassostrea gigas) were examined for the presence of Giardia cysts. Four of 179 (2.2%) oysters harboured Giardia cysts. Viable cysts were detected in surface waters that enter the oyster harvesting areas. The detection of Giardia in oysters suggests that consumption of raw oysters has the potential to cause gastrointestinal illness (Schets et al., 2007). In addition, Giardia cysts have also been identified in samples of oysters, blue mussels and Mediterranean mussels (Gómez-Couso et al., 2004, 2005; Schets et al., 2007; Robertson and Gjerde, 2008).

3.5 Approaches to Removal and Inactivation of Giardia Cysts in Water and Food 3.5.1 In Water One of the most crucial biological features of Giardia in environmentally borne transmission is the recalcitrant nature of its infective cyst stage. Giardia cysts are very resistant to environmental pressures and to many of the disinfectant regimes commonly used in the water industry. This robustness is attributed to the composition of the cyst wall described in a previous section. Other cyst characteristics, including small size, specific gravity (ca. 1.05) and surface charge (i.e., zeta potential), also contribute significantly to the physical treatment processes required, as does the low concentrations at which cysts generally occur in surface waters (Smith et al., 1995).

Chap. 3 Waterborne and Environmentally-Borne Giardiasis

55

Removal of Giardia cysts by water treatment plants is achieved by physical removal and disinfection processes. The multiple-barrier approach is generally accepted as the guiding principle for providing safe drinking water, and processes such as catchment protection, filtration, flocculation and sedimentation may all be important contributors to the reduction of viable, infectious Giardia cysts in drinking water supplies. As there are a number of disinfectant regimes available, knowledge of the effects of individual disinfection procedures on Giardia cysts is crucial. Disinfectants used include chlorine, chloramine, chlorine dioxide, ozone, ultraviolet (UV) irradiation and photocatalytic disinfection. Numerous studies have evaluated the effects of various types of disinfection procedures. However, it has to be borne in mind that the differing methods and experimental procedures to evaluate inactivation treatments have implications for interpretation of results, often making accurate interstudy comparisons difficult. Factors which may influence the outcome of disinfection studies include the species of cysts used (e.g., G. duodenalis, G. muris), their sources (e.g. human or animal), cyst viability and the methods used to determine the viability/infectivity (e.g. in vitro excystation, fluorogenic vital dyes, cell culture, animal infectivity). Many studies that have employed vital dye stains and in vitro excystation have produced underestimations of the effectiveness of disinfection treatments (Erickson and Ortega, 2006). The most common type of disinfectant is chlorine. Doses of chlorine typically applied in water treatment range from 5 to 15 mg/L, with a recommended exposure time of 30 min to 2 h (Mujeriego and Asano, 1999). Chlorine readily dissolves in water at room temperature to generate hypochlorous acid (HOCl), or as a salt of hypochlorite (OCl–). HOCl plus OCl– are called free chlorine. HOCl is a more effective biocide than OCl–, but dissociates into OCl– at pH values above 7 (Len et al., 2000). The main features of cyst injury elicited by chlorine include cyst wall damage, plasma lemma breakage, lysis of peripheral vacuoles and nuclear degradation (Li et al., 2004). Although experiments have demonstrated that Giardia cysts could be inactivated by chlorine if temperatures were sufficiently high, they have been demonstrated to be resistant to chlorination at low temperatures and high pH (Jarroll et al., 1981; Rice et al., 1982; Hoff et al.,

1985; Leahy et al., 1987; Hibler et al., 1987). Studies have shown that Ct’ values required for 99% inactivation of Giardia cysts were many orders of magnitude higher than those necessary for 99% inactivation of bacterial and viral pathogens or indicators. These results pinpoint that chlorine is only able to inactivate Giardia cysts at concentrations well above those generally employed in routine water treatment practices. In addition, these results also confirmed that the conservative procedure of evaluating water quality based on presence/absence of coliforms is unreliable and inaccurate. Although disheartening, these results explain why waterborne giardiasis outbreaks still occur, even when drinking water has been treated by chlorination or in the absence of faecal indicators. The most recent example due to inadequate chlorination treatment is the extensive outbreak in Bergen, Norway (Nygård et al., 2006; Robertson et al., 2006b). Chlorine has limited potency against protozoan parasites, and it is clearly insufficient as the only disinfectant treatment option to inactivate Giardia cysts in water. The effectiveness of chloramine and chlorine dioxide has also been evaluated. Chloramine such as monochloramines (NH2Cl), dichloramines (NHCl2) or trichloramines (NCl3) are formed when ammonia is added to chlorine. These compounds are more persistent in the water system (Pontius, 1997). Investigation into the use of chloramines as an alternative to chlorine demonstrated that the compound 3-chloro-4,4-dimethyl-2-oxazolidinone reduced excystation of Giardia cysts derived from dogs at lower concentrations or after relatively shorter contact times compared with chlorine (Kong et al., 1988). However, chloramine apparently lacks an oocysticidal effect on Cryptosporidium oocysts (Korich et al., 1990). Chlorine dioxide has been generally considered to have greater biocidal efficiency than that of free chlorine or monochloramine (Hoff, 1986), but chlorine dioxide also has limited ability to inactivate Giardia cysts, as assessed by vital dye staining and excystation (Winiecka-Krusnell and Linder, 1998). Ozone has been considered as an alternative disinfectant due to the relative ineffectiveness of chlorine and its derivatives. Exposure of G. duodenalis cysts to ozone causes extensive protein degradation and profound structural modifications to the cyst wall. The initial investigations on the use of ozone to inac-

56

tivate Giardia cysts demonstrated that Giardia cysts were more susceptible to ozonisation than chlorination, requiring a Ct’ value much lower compared with that needed for chlorination. In addition, the Ct’ value was only twice or three times that required for inactivation of bacteria and virus (Wickramanayake et al., 1984). Inactivation of G. muris by 1.52–2.70 log units required ozone concentrations of only 0.28– 1.04 mg·min/L (Owens et al., 2000). However, as with chlorination, the efficacy of ozonisation was also apparently reduced at lower temperatures, although the temperature effect was apparently not as high as with chlorination. Nevertheless, using infectivity to assess the susceptibility of both G. muris cysts and G. duodenalis cysts to inactivation by ozone demonstrated that both species were of similar sensitivity to ozonisation, and Ct’ value necessary for 3 log unit inactivation was over twice that recommended by the Surface Water Treatment Rule (Finch et al., 1993). Similar values were also obtained in the study by Widmer et al. (2002). The requirement for higher activation energy for protozoa compared with bacteria, especially at low temperatures, requires significantly higher ozone exposures, and these may result in the formation of disinfection by-products, of which bromate is considered to be of most concern due to its potentially carcinogenic effects, and occurs during ozonisation of bromide-containing waters (von Gunten, 2003). Treatment options to remedy this problem include addition of ammonia, pH reduction, scavenging of hydroxyl radicals and scavenging or reduction of HOBr (hypobromous acid). Another aspect that has been indicated for consideration regarding water disinfection and Giardia cyst inactivation by ozonisation is that the disinfection efficacy apparently reduces as cyst concentration decreases (Haas and Kaymak, 2003). Given that most bench-scale trials use large numbers of cysts, such studies do not represent the natural occurrence of Giardia cyst concentrations in water. The crucial implication is that in real water treatment situations, the efficacy of disinfection with ozone against Giardia may be less than those assumed from the results of laboratory studies. UV light has been used to inactivate microorganisms since the early 1890s (Bess et al., 2004). Although UV light may not kill the organism directly, it renders the organism unable to reproduce and establish in the host. The use of UV irradiation has several

L.J. Robertson and Y.A.L. Lim

advantages as a disinfection process: (a) it does not rely on the use of chemical additions; (b) it requires relatively short contact times and (c) UV disinfection by-products have not been identified to date (Betancourt and Rose, 2004). The potential use of UV irradiation technology for inactivation of Cryptosporidium oocysts in drinking water was first identified in 1995 (Campbell et al., 1995), subsequently confirmed by Clancy et al. (1998), and since then there have been various studies on the inactivation of Giardia cysts by UV. These studies have generally demonstrated that Giardia cysts are susceptible to UV disinfection at doses that are applicable to water (Craik et al., 2000; Belosevic et al., 2001; Campbell and Wallis, 2002; Linden et al., 2002; Mofidi et al., 2002; Shin et al., 2009). Experiments investigating whether DNA repair occur in Giardia cysts have produced conflicting results. Craik et al. (2000) and Linden et al. (2002) concluded that G. muris and G. duodenalis cysts lose their infective ability permanently as a result of UV damage. However, Belosevic et al. (2001) demonstrated some DNA repair after UV radiation. In addition, Kruithof et al. (2005) showed in vivo reactivation of G. muris cysts, and Shin et al (2005) also discovered some repair of G. duodenalis DNA following UV exposure. These results indicated that there is a possibility that some isolates of Giardia may be more or less sensitive to UV irradiation and not others (Li et al., 2007). However, UV inactivation of C. parvum and C. hominis oocysts is apparently irreversible, even if UV repair genes are present (Rochelle et al., 2004). Oocyst DNA is damaged when UV light is absorbed by the double bond in the thymine base in a DNA molecule, resulting in the opening of the bond and allowing it to react with the adjacent thymine base, forming a tight four-member ring called thymine dimer (TD). Based on previous observations that accumulation of TDs occurs in the genome of oocysts following UV inactivation (Rochelle et al., 2005), Al-Adhami et al. (2007) developed an anti-TD immunofluorescence assay to demonstrate DNA damage in C. parvum and C. hominis oocysts after exposure to UV irradiation. Similar investigations on Giardia could provide useful insights on the effect of UV inactivation. Wastewater is often also treated by UV, but infection studies using UV-treated wastewater treatment plant samples (Neto et al., 2006) indicated that the infectivity of

Chap. 3 Waterborne and Environmentally-Borne Giardiasis

57

naturally occurring Giardia cysts was not completely abrogated by this treatment, possibly because of the relatively high turbidity and quantities of particulate matter. In a similar study in Canada, Li et al. (2009) demonstrated that Giardia cysts in secondary effluent both upstream and downstream of UV reactors in wastewater treatment plants could cause infections in gerbils, indicating that the inactivation provided by the UV systems was less than anticipated from the UV dose responses published in the literature. Photocatalytic disinfection is a nanotechnology system of disinfection based on the interaction between light and solid semiconductor particles. First suggested as a promising source of hydroxyl radicals for disinfection of water (Matsunaga et al., 1985), photocatalysis using titanium dioxide (TiO2) as a sensitiser was initially investigated for inactivation of Cryptosporidium oocysts in 2002, with promising results (Curtis et al., 2002). Experiments by Lee et al. (2004) suggested this technology could also be applicable to inactivation of Giardia cysts, and Yu and Kim (2004) proposed that a photoreactor involving TiO2-immobilised optic fibre reactor could be usefully applied to Giardia inactivation. A further study using TiO2 and modified catalyst silver-loaded TiO2 (Ag-TiO2) (Sökmen et al., 2008) indicated not only that photocatalytic disinfection might be an environmentally friendly technology for water disinfection, but that TiO2 thin film coated materials (glass or PET) may be more promising for total inactivation of Giardia, and that the photocatalytic action of TiO2 could be improved by catalyst modification. The authors recommend that feasible photoreactor design should be seriously investigated for online water treatment. A more recent study (Navalon et al., 2009) demonstrated the efficacy of this technique in inactivating Giardia cysts (and Cryptosporidium oocysts) using a commercial fibrous ceramic TiO2 photocatalyst. The efficiency was enhanced by addition of a small concentration of chlorine and the authors consider that the photocatalytic process is suitable for safe and complete water disinfection.

foods is the difficulty in isolating cysts from the food matrix. Although there are more studies on the inactivation of Cryptosporidium oocysts than Giardia cysts in food, results for Cryptosporidium are of relevance for Giardia as oocysts are generally more resistant than cysts. Some of the studies on the effects of inactivation on Cryptosporidium oocysts in foodstuffs are summarised below: Using vital dyes for the assessment of oocyst viability, it was observed that survival of C. parvum oocysts varied when inoculated onto various types of salad leaf. Survival was greater in products with a textured leaf compared with products with a smoother leaf surface, and this was attributed to desiccation being slower on the rougher surfaces. On parsley, which withers very quickly and has a very short shelf life, oocysts were inactivated within 24 h (Warnes and Keevil, 2003). In yogurt, fermentation and cold storage resulted in very little inactivation of C. parvum oocysts with controls (pasteurised milk without the addition of live yogurt) having similar levels of inactivation as those of the yogurt samples (Deng and Cliver, 1999). The maximum doses of radiation (10 kGy) recommended for food preservation by the Food and Agriculture Organization and the World Health Organization (World Health Organization, 1998) are not high enough to inactivate C. parvum oocysts. In an animal infectivity study, C. parvum oocysts suspended in water and irradiated with less than 10 kGy induced infections in mice, whereas no oocysts were produced in mice infected with oocysts irradiated at 50 kGy (Yu and Park, 2003). In contrast, another study showed that lower radiation doses are sufficient to inactivate C. parvum oocysts in food matrices as no infectious oocysts were detected in unshucked and shucked oysters irradiated at 2 kGy (Collins et al., 2005).

3.5.2 In Food One of the main reasons why there are few studies investigating the survival of protozoan parasites in

3.5.3 In Beverages The survival of protozoan parasites in beverages has also been investigated. Both carbonation and low pH were considered major factors in decreasing viability of C. parvum oocysts held in a carbonated soft drink, whereas low pH and alcohol content were the factors ascribed to decreased viability in beers (Friedman et al., 1997). In contrast to these beverages, no sig-

58

L.J. Robertson and Y.A.L. Lim

nificant differences in C. parvum oocyst survival were noted among water, orange juice and milk after 153 days, when approximately 90% of the oocysts had been inactivated (Enemark et al., 2003). Variable survival of C. parvum oocysts also has been found in natural mineral waters enriched with minerals to different degrees (dependent on the type of rock strata through which the water has percolated). Although the mineral content did not affect C. parvum oocyst inactivation at 4oC, waters with higher mineral contents had higher inactivation rates at 20oC (Nichols et al., 2004). Another disinfection process that has been tested for inactivation of pathogens in a variety of foods is high pressure. Following 30 s of treatment at 80,000 psi, C. parvum oocysts were inactivated by 3.4 and >4.1 log units in apple and orange juices, respectively (Slifko et al., 2000). Inactivation was >4.2 log units for both juices after 60 s of treatment. Freezing may also not be considered a safeguard for fruit juices or milk because C. parvum oocysts were able to survive in both these matrices for up to 3 weeks at –20oC (Enemark et al., 2003). In contrast, C. parvum oocysts did not survive freezing and hardening in an ice cream freezer and could be attributed to rapid freezing through continuous blade movement of the freezer (Deng and Cliver, 1999). There is growing interest in using UV technology for food preservation. A 6-log inactivation of C. parvum oocysts was achieved in fresh apple cider exposed to UV light at 14.32 mJ/cm2 (Hanes et al., 2002).

of the elderly rising. In the most advanced countries, the ageing of the population is already underway, but it is predicted that a number of east and central European countries, as well as countries in east and southeast Asia, will experience significant ageing from about 2020 (Batini et al., 2006). Such population changes may indeed affect the dynamics of giardiasis and result in greater severity from infection or increased vulnerability to infection. Data from a study from Scotland concerned with hospitalisation due to giardiasis suggested that the >70 years age group may be of importance, perhaps due to immunosenescence or the presence of other problems that may be exacerbated by concurrent giardiasis (Robertson, 1996). In addition to these factors, we believe that it is likely to be our use, and misuse, of water in the years ahead that is likely to have most impact on waterborne giardiasis. Water is abundant globally, and is basically a renewable resource, given that the total quantity of water is essentially constant and unaffected by human activities. However, the distribution of water varies considerably, driven by natural cycles including freezing, thawing, fluctuations in precipitation, water runoff and evapotranspiration, as well as human activities, which in many cases have become the primary “drivers” affecting the planet’s water systems (UN Water, 2009). Thus, water adequacy has emerged as one of the primary resource issues of the industrialised world, and for many developing countries, the problem is critical.

3.6 Conclusion and Future Challenges in Environmentally-Borne Giardiasis

3.6.1 Millenium Development Goals

The waterborne route of transmission continues to be of relevance in giardiasis, and despite our improving technologies for water treatment, many factors suggest that this is likely to continue. Additionally, other environmental matrices may become of increasing importance. Our demand for year-round fresh fruit and vegetables impacts on trade and import, and with increasing requirements for “food sovereignty”, the potential for the distribution of pathogens must not be neglected. Additionally, the global population itself is changing, in both distribution and demographics; the world is considered to be in a period of demographic transition, with the share of the young falling and that

One of the UN millennium development goals (MDG) was to halve by 2015 the proportion of people without sustainable access to safe drinking water and basic sanitation; although this is on track for drinking water, with 87% of the global population using improved drinking water compared with 77% in 1990 (UNICEF and World Health Organization, 2008), sanitation has been less successful, and at its current rate of progress, the world will miss the MDG sanitation target by over 700 million people (UNICEF and World Health Organization, 2008). With respect to control of giardiasis, both satisfactory water supply and adequate sanitation are essential. However, although having an improved drinking water supply

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obviously decreases the chance of giardiasis, it should be noted that in the MDG definition, water supply is divided into a 3-tier ladder, with an unimproved drinking water source described as an unprotected well or spring, surface water (e.g. river, lake, pond, stream, canal, irrigation channels), tanker truck and bottled water. Improved drinking water is described as including public taps or standpipes, tube wells or boreholes, protected springs and wells and rainwater collection. Finally, piped water on premises, the highest tier in the MDG safe drinking water definition, means that there is water connection inside the user’s dwelling, plot or yard. Whilst the goals are clearly commendable, in themselves they are insufficient to protect against waterborne giardiasis, which also requires that the water supply is adequately treated or protected, and that the infrastructure of delivery is maintained to avoid post-treatment contamination. Without doubt, the population of Bergen, Norway, received water in 2004 that fulfilled the MDG definitions of safe drinking water, but this alone did not prevent the widespread outbreak of waterborne giardiasis that occurred there. The intention of the improved sanitation facilities, as described in the MDG, is to ensure hygienic separation of human excreta from human contact, and therefore includes flush or pour-flush toilet to piped sewer system, septic tank or pit latrine, ventilated improved pit latrine, pit latrine with slab and composting toilet. Interestingly, whilst the world’s urban sanitation coverage had risen to 79% in 2008, rural coverage was only 45% (UNICEF and World Health Organization, 2008). However, sanitation facilities that are shared among houses are not considered as “improved” according to the MDG definitions, and the limited data available support the perception that shared facilities often fail to ensure the target of hygienic separation of excreta and contact. Estimates from 2006, when the world’s population was almost equally divided between urban and rural dwellers, suggested that most shared sanitation users are urban dwellers. Although more than 7 out of 10 people without improved sanitation were rural inhabitants, the rapid population growth in urban areas poses its own challenge, as the increase in the number of urban dwellers using improved sanitation has not kept pace with urban population growth, and more than two thirds of shared sanitation users are urban dwellers (UNICEF

and World Health Organization, 2008). For those without any form of available sanitation, the only recourse is open defecation. This is recognised as being of fundamental importance to development due to the health hazard posed to anyone in the neighbourhood from a range of diarrhoeal and other infections, including giardiasis. Whilst the proportion of people tolerating open defecation as their only form of sanitation decreased from 31% in 1990 to 23% in 2006 (UNICEF and World Health Organization, 2008), population growth means that the number of people (778 million) has changed little from 1990. Although sewage discharge into water courses that may be later used for water supply obviously impacts on water quality, it is widespread and concentrated (i.e. practiced by a large number of people within a limited area) open defecation that is most likely to result in contamination of water supplies, and thus impact on the spread of diseases such as giardiasis.

3.6.2 Water Scarcity Of the approximately 1.4 billion km3 of water on earth, approximately 2.5% (35 million km3) is freshwater, of which about 70% is in the form of ice and permanent snow cover in the Arctic and Antarctic, and mountainous regions (http://www.unwater.org/statistics_res.html). The total volume of usable fresh water supply for humans and ecosystems is approximately 200,000 km3, less than 1% of all fresh water resources and less than 0.025% of water on earth. However, although the amount of water in the world exceeds the minimum threshold (1700 m3 per person) conventionally quoted as being necessary to grow food, support industries and maintain the environment, the uneven distribution of water, along with the uneven population distribution, is the basis of the problem. For example, almost 25% of the world’s supply is found in Lake Baikal in sparsely populated Siberia. Furthermore, Latin America has 12 times more water per person than South Asia, and whilst Canada and Iceland, for example, can enjoy an excess of water (90,000 m3 per person and 510,000 m3 per person, respectively), Yemen has only 198 m3 per person (United Nations Development Programme, 2006). Over the past century, water use has been growing at more than twice the rate of population increase, and by 2025 it has

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been estimated that 1800 million people will be living in countries with absolute water scarcity, and two thirds of the world’s population could be under stress conditions (UN Water, 2006). Rapidly expanding urban areas will place pressure on neighbouring water resources, thereby exacerbating the situation. It has been suggested that 20 L of clean water per day per person is sufficient, but in Europe and USA, average water use per person is between 200 and 600 litres per day, whilst, to add to the unequal burden, those in developing countries typically pay 5–10 times more per unit of water than people with access to piped water (UNDP, 2006). Whilst intensive use of groundwater over many years has opened debate on the sustainability of this approach, surface freshwater bodies have only a limited capacity for processing pollutant charges, and the resultant degradation in water quality can be a major cause of water scarcity. Water scarcity, local, regional and international, has a potentially huge range of impacts, including conflicts arising because of the difficulties associated with sharing a limited but essential resource. Amongst these potential problems, transmission of giardiasis should rightly be ranked as of relatively diminutive importance, but is nevertheless of relevance to this chapter. Water scarcity means that the water availability for all human processes, including domestic (drinking, cooking, washing, sanitation), industrial (manufacturing) and agricultural (irrigation, drinking water for animals), as well as a natural resource, is limited. When water is limited, compromises must be made, and more people and animals, both domesticated and wild, are forced to share the same supply. This means not only that the risk for waterborne transmission between people is elevated, but also that the risk of maintaining the zoonotic cycle is increased. Water must be recycled, reclaimed and conserved, but if such initiatives compromise health and maintain infection transmission routes, then the benefits of recycling have been bought very dear.

3.6.3 Wastewater and Water Re-use for Irrigation One approach to the conservation and recycling of water has been the use of wastewater and water reuse for agriculture. This is not a new concept, and in re-

L.J. Robertson and Y.A.L. Lim

gions where water supplies are restricted, it is an obvious measure to adopt. It has been noted that appropriate treatments of sewage may reduce the concentration of infective pathogens, including Giardia cysts, from 99% to 99.9999%, but in order for there to be better access to scientific information for decision making, a global database for biological contaminant loading from wastewater should be developed, as well as definitions on public health protection with respect to water reuse and reclamation (Rose, 2007). However, in many places where such reuse is already practiced, such treatments are not employed and may result in considerable infection transmission: in a study in the suburbs of Asmara, Eritrea demonstrated that agricultural reuse of wastewater was the major cause of an elevated prevalence of giardiasis (Srikanth and Naik, 2004). Clearly water recirculation can have an important positive impact in regions where supplies are limited, but it is essential that treatment is appropriate to ensure that pathogens, including Giardia, as well as other contaminants, are prevented from entering the food chain.

3.6.4 Climate Change In the scientific community, the fact that the global climate is changing is no longer in doubt, although the extent of change, the speed and various other factors continue to be a matter of debate. Nevertheless, global air and ocean temperatures have been demonstrated to have risen, widespread melting of snow and ice has been documented, significant changes in amounts of precipitation have been recorded, and the global average sea level is rising. Additionally, the severity and frequency of extreme weather events, including heat waves and unusually intense precipitation, have increased. The impact on the environment has included longer growing seasons, retreating glaciers and alterations in species ranges. These changes are consistent with those predicted for the continuing climate change trend. However, it is also anticipated that regionally climate changes may differ, posing different risks and challenges; in Northern Europe, for example, longer, warmer summers, milder winters and more extreme weather events (excess precipitation and high winds) are predicted. Anticipated effects include increased growth of vegetation, more frequent

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winter flooding, greater ground instability and erosion, glacier retreat and reduced snow cover, endangerment of some ecosystems and extensive species loss. The intergovernmental panel on climate change (IPCC) believes that the burden of water-related disease is likely to be affected by climate change-related alterations in rainfall, surface water availability and water quality (Confalonieri et al., 2007). Various studies have already demonstrated that transmission of enteric pathogens increases during heavy rain, sometimes resulting in outbreaks; indeed regarding outbreaks of cryptosporidiosis and giardiasis in the UK, it has been shown that years in which outbreaks have occurred are associated with cumulative rainfall exceedances (Nichols et al., 2009). As well as direct effects of the weather, it has been suggested that transmission of waterborne parasites, including Giardia, may be affected by the effect of climate change on host populations, including the distribution of reservoir hosts due to changes in environmental conditions, and behavioural and intrinsic alterations in human populations that may increase vulnerability (Robertson, 2010). In general, IPCC suggests that adaptation to climate change is likely to benefit from experiences gained in reaction to extreme climate events, by specific implementation of proactive climate change risk management adaptation plans (Alcamo et al., 2007). With regards to waterborne pathogens, including Giardia, it has been pointed out that knowledge of the efficacy and the weakness in our current safeguards against their transmission is essential for predicting whether they will be sufficient under conditions of climate change (Robertson, 2010).

paramount, the use of standardised validated methods, the auditing of laboratory performance for determining the occurrence of cysts in water, food and soil, and the creation of a systematic and coordinated national surveillance system should be encouraged. Recognition of the impact that waterborne transmission of pathogens, including Giardia, has on public health and national economies would benefit such systems. Global key players would also gain from the utilisation of standardised validated methods, enabling reliable data comparisons and also being useful for identifying risk factors and for developing national and international policies to reduce the chance of future outbreaks. Whilst our knowledge of Giardia and its waterborne transmission potential increases, including our understanding concerning occurrence, detection, survival, disinfection and risk, new challenges are being posed associated with a changing globe in which water shortage, climate change, trade patterns and demographic alterations all have a role. Facing these challenges requires that communication channels are kept open between all relevant sectors, including meteorologists, risk assessors, public health personnel, water and sanitation engineers, epidemiologists, veterinarians and emergency planners. Thus, an enhanced understanding of the transmission of giardiasis and the significance of environmental contamination requires a multidisciplinary approach built on shared skills, expertise and resources, especially in countries that are financially constrained. Establishment of such a multidisciplinary and integrated approach could underpin stable and powerful partnerships in efforts to prevent and control giardiasis.

3.6.5 Conclusion The potential for transmission of waterborne/ foodborne/environmentally borne giardiasis, as sporadic cases or as communitywide outbreaks still exists, even in industrialised countries, as evidenced by the 3 outbreaks reported since 2004, and that occurred in USA, Norway, and Finland. Obviously, even greater risks exist in countries where giardiasis is endemic, especially in Asia, Africa and South America. Countries in these regions currently do not have national surveillance systems in place for giardiasis. Given that the availability of accurate and timely data is

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Wallis PM ( 1987) Development of criteria for Giardia in drinking water. Unpublished report to Health and Welfare Canada. Wallis PM, Erlandsen SL, Isaac-Renton JL, Olson ME, Robertson WJ, and van Keulen H (1996) Prevalence of Giardia cysts and Cryptosporidium oocysts and characterization of Giardia spp. isolated from drinking water in Canada. Appl Environ Microbiol 62: 2789–2797 Wallis PM, Matson D, Jones M, and Jamieson J (2001) Application of monitoring data for Giardia and Cryptosporidium to boil water advisories. Risk Anal 21: 1077–1085 Warnes S and Keevil CW (2003) Survival of Cryptosporidium parvum in faecal wastes and salad crops. Available at: http://www.teagasc.ie/publications/2003/conferences/ cryptosporidiumparvum/paper02.html Webber C ( 2002) Outbreak of giardiasis in Bay of Plenty and Manawatu. In: Annual summary of outbreaks in New Zealand 2001, Report for the Ministry of Health, April 2002. pp 38–39 Weniger BG, Blaser MJ, Gedrose J, Lippy EC, and Juranek DD (1983) An outbreak of waterborne giardiasis associated with heavy water runoff due to warm weather and volcanic ashfall. Am J Public Health 73: 868–872 White KE, Hedberg CW, Edmonson LM, Jones DBW, Osterholme MT, and MacDonald KL (1989) An outbreak of giardiasis in a nursing home with evidence for multiple modes of transmission. J Infect Dis 160: 298–304 Wicki M, Svoboda P, and Tanner M (2009) Occurrence of Giardia lamblia in recreational streams in Basel-Landschaft, Switzerland. Environ Res 109: 524–527 Wickramanayake GB, Rubin AJ, and Sproul OJ (1984) Inactivation of Giardia lamblia cysts with ozone. Appl Environ Microbiol 48: 671–672 Widmer G, Clancy T, Ward HD, Miller D, Batzer GM, Pearson CB, and Bukhari Z (2002) Structural and biochemical alterations in Giardia lamblia cysts exposed to ozone. J Parasitol 88: 1100–1106 Winiecka-Krusnell J and Linder E (1998) Cysticidal effect of chlorine dioxide on Giardia intestinalis cysts. Acta Trop 70(3): 369–372 World Health Organization (1998) Codex general standard for irradiated foods. In: Food irradiation: a technique for preserving and improving the safety of food. World Health Organization, Geneva, pp 72–74 Yoder J, Roberts V, Craun GF, Hill V, Hicks LA, Alexander NT, Radke V, Calderon RL, Hlavsa MC, Beach MJ, and Roy SL (2008) Surveillance for waterborne disease and outbreaks associated with drinking water and water not intended for drinking – United States, 2005–2006. MMWR Surveill Summ 57: 39–62 Yu JR and Park WY (2003) The effect of γ-irradiation on the viability of Cryptosporidium parvum. J Parasitol 89: 639– 642 Yu M-J and Kim B-W (2004) Photocatalytic cell disruption of Giardia lamblia in a UV/TiO2 immobilized optical-fiber reactor. J Microbiol Biotechnol 14: 1105–1113 Yu X, Van Dyke MI, Portt A, and Huck PM (2009) Development of a direct DNA extraction protocol for real-time PCR detection of Giardia lamblia from surface water. Ecotoxicology 18: 661–668

Giardia in Pets and Farm Animals, and Their Zoonotic Potential Thomas Geurden and Merle Olson

Abstract Although the protozoan parasite Giardia duodenalis is worldwide recognized as an important cause of gastro-intestinal disease in human patients, the relevance as a pathogen in production and pet animals and the zoonotic potential of animals were prone to more debate. Since long, clinical disease has been associated with giardiasis in companion animals, but over the last few years, an increasing amount of data also confirmed the clinical and subclinical relevance of infection in production animals. Next to the clinical relevance, animal giardiasis was studied from a public health point of view, as the parasite was implicated in a large number of waterborne outbreaks, and at least part of these outbreaks were thought to be due to animal contamination of water supplies. Especially livestock and to a lesser extent wildlife have been considered as potential reservoirs for this waterborne transmission. Furthermore, pet animals have been associated with direct transmission of giardiasis to human patients, especially in endemic settings. Molecular epidemiological research over the last two decades did provide a better insight in the zoonotic transmission pathways, although many questions still remain. In this chapter, the current knowledge on clinical relevance of giardiasis in production and pet animals is reviewed, along with the diagnosis, the treatment, the control of infection and the zoonotic potential of animal giardiasis.

4.1 Introduction Although the protozoan parasite Giardia has been known since Antonie van Leeuwenhoek’s first de-

H. D. Luján et al. (eds.), Giardia © Springer-Verlag/Wien 2011

4

scription in 1681, the clinical and public health relevance of giardiasis was not fully acknowledged until late 20th century. Nowadays Giardia is recognized as the most common parasitological cause of diarrhea in human patients, with an estimated 280 million infections per year, and is a major concern to drinking water authorities, as it is a frequently diagnosed waterborne infection. Similarly, giardiasis is the most common parasitic infection in companion animals (Thompson et al., 1993). Because of the impact on socio-economic development, especially in developing countries, Giardia is included in the “Neglected Disease Initiative” of the World Health Organization (Lane and Lloyd, 2002; Savioli et al., 2006). In laboratory animals, such as mice and gerbils, the pathology caused by infection has been described and is widely accepted (Buret et al., 1991, 1992), whereas in companion animals and food animals the pathological changes have not been well documented (O’Handley et al., 1999). In both humans and animals, the clinical outcome of a Giardia infection in animals is highly variable and infection can result in either acute or chronic diarrhea, nausea, weight loss, and hypersensitivity but asymptomatic infections are also known to occur frequently. Research into the parasite’s epidemiology has mainly focused on prevalence and on molecular characterization of isolates from different animals and from human hosts in order to elucidate the zoonotic hazard. The development of molecular markers facilitated the identification of common and specific (sub)genotypes or species in both animal hosts and human patients. Since, production, companion, and wild animals have been considered as a potential zoonotic reservoir for human Giardia infections, although recent data seem to provide a more detailed insight into the transmission cycles.

72

4.2 Life Cycle The life cycle of G. duodenalis comprises two main stages: a trophozoite stage which colonizes the intestinal epithelium of the host and causes disease, and an infectious cyst stage which is resistant in the environment. After oral ingestion, the cyst wall disrupts and trophozoites are released in the upper part of the small intestine. For the colonization of the small intestine attachment to epithelial cells is essential, through the trophozoite’s ventral adhesive disk. The trophozoites multiply by binary fission in the lumen of the small intestine, although sexual reproduction has been suggested (Meloni et al., 1989). Finally, exposure to biliary salts leads to encystation of trophozoites, and cysts are immediately infectious upon excretion are passed in the feces, allowing completion of the life cycle as soon as 72 h after infection (Thompson et al., 1993). Early experimental studies suggested a somewhat longer prepatent period varying from 6 to 21 days (Taminelli et al., 1989; Koudela and Vitovec, 1998), but this was probably due to the method used for diagnosis as other studies indicated a prepatent period around 3–10 days (Xiao and Herd, 1994; Geurden et al., 2006a).

4.3 Prevalence in Farm Animals Giardia has been reported worldwide in farm animals, although prevalence data are mainly available for cattle, and to a lesser extent for other ruminants. For horses and pigs, prevalence data are fewer. Overall, both the animal and farm prevalence vary considerably between studies in all animal species. Although management, geographical, and climatological parameters do partially account for this variation, differences in study design also need to be taken into consideration, including the number of animals or farms included in the study. In horses for example, most studies have been conducted on a limited number of animals or sites, which will certainly bias the estimation of both animal and farm prevalence. The assay used to diagnose infection is another important factor to consider. Since there is no gold standard reference test for the diagnosis of Giardia, the use of diagnostic techniques with different and unknown sensitivity and specificity, might seriously impact prevalence estimation and

T. Geurden and M. Olson

trouble study comparison. In calves for example, the prevalence estimate using three different diagnostic assays resulted in a different estimate for each assay (Geurden et al., 2004). Similarly, PCR provided a higher prevalence estimate in post-weaned calves compared to immunofluorescence assay (Trout et al., 2005). Another important factor influencing the prevalence estimation, is the age of the animals included in the study (see Table 4.1). In calves for example, both longitudinal and cross-sectional prevalence studies indicate a peak prevalence in animals aged between 1 and 6 months, and a decrease in prevalence from the age of 6 months onwards (Xiao et al., 1994; Nydam et al., 2001; Ralston et al., 2003; Becher et al., 2004). Although there are few data on age-related prevalence in other farm animals, most authors seem to consider a similar infection pattern. In Table 4.1, an overview of the most recent and large-scale prevalence studies in cattle around the world is provided. In contrast to Cryptosporidium (Ralston et al., 2003), Giardia seems to be equally prevalent in dairy and beef calves. In these cattle studies, both the animal and the farm prevalence vary considerably, with animal prevalence ranging from 9 to 73% and the farm prevalence from 45 to 100%. In Table 4.2, an overview of the prevalence in other farm animals is provided, and similarly to cattle the prevalence is highly variable. Despite the high variability of prevalences reported in farm animals, some conclusions can still be drawn form the data presented in Tables 4.1 and 4.2. As the cyst excretion is highly variable between animals, especially in chronic infections, and as most studies are performed as a cross-sectional prevalence study, a potential underestimation of the prevalence is at hand when only considering the animal prevalence. Therefore, the farm prevalence is probably more informative than the animal prevalence to study the prevalence of infection in a cross-sectional study design. In cattle for example, the cumulative incidence on a farm where Giardia has been diagnosed, is known to be 100% (O’Handley et al., 1999) implying that every animal on that farm will get infected at some point. As the farm prevalence varies between 45 and 100%, we can conclude that the infection is widespread in the cattle population worldwide. Similarly, the cumulative prevalence was found to be 100% in goats (Xiao, 1994; Castro-Hermida et al., 2005),

Chap. 4 Giardia in Pets and Farm Animals, and Their Zoonotic Potential

73

Table 4.1 The animal prevalence (PA) and farm prevalence (PF) of Giardia in cattle in different countries. The age of the animals, in months (m), the number of animals (#A) and farms (#F) is presented along with the diagnostic assay (Diag) used in the study (IFA Immunofluorescence microscopy, PCR polymerase chain reaction or ME microscopical examination; – not known) Country

Diag

#A

#F

Age (m)

PA

PF

100

<2.5

22

48

Reference

Dairy < 6 m Belgium

IFA

499

Canada

IFA

Canada

ME

Denmark

IFA

New Zealand

IFA

New Zealand

IFA

–

Norway

IFA

1386

Spain

IFA

USA

ME

USA

PCR

Vietnam

386

Geurden et al. (2008b)

20

<6

73

100

Olson et al. (1997a)

505

<6

–

45

Ruest et al. (1998)

377

50

<1

24

82

Maddox-Hyttel et al. (2006)

715

12

<2

41

100

10

<2

–

31

Winkworth et al. (2008)

136

<6

49

93

Hamnes et al. (2006)

734

60

<6

29–57

67

Castro-Hermida et al. (2006a)

2943

109

<6

20

70

Wade et al. (2000b)

407

14

<2

40

100

Trout et al. (2004)

IFA

68

8

<3

50

88

IFA

518

50

1–12

43

100

–

Hunt et al. (2000)

Geurden et al. (2008c)

Dairy > 6 m Denmark

Maddox-Hyttel et al. (2006)

Denmark

IFA

255

50

>12

40

60

Maddox-Hyttel et al. (2006)

Spain

IFA

734

60

>6

25–40

67

Castro-Hermida et al. (2006a)

Spain

IFA

379

60

>36

27

97

Castro-Hermida et al. (2007)

Spain

ME

199

30

<24

26

53

Quilez et al. (1996)

USA

PCR

456

14

3–11

52

100

Trout et al. (2005)

USA

PCR

571

14

12–24

36

100

Trout et al. (2006)

USA

PCR

541

14

>24

27

100

Trout et al. (2006)

Belgium

IFA

333

50

<2.5

45

64

Geurden et al. (2008b)

Canada

IFA

193

10

<2.5

36

100

McAllister et al. (2005)

Canada

IFA

495

9

<3

34

100

Appelbee et al. (2003)

Canada

IFA

605

100

<6

23

48

Gow and Waldner (2006)

Canada

IFA

605

100

>24

17

69

Gow and Waldner (2006)

Canada

IFA

669

39

>24

9

64

McAllister et al. (2005)

Beef

close to 100% in sheep (Xiao and Herd, 1994), and around 71% in horses. Given the high farm prevalence reported in these studies, a high proportion of farm animals is at risk of infection. In pigs, there are no data on cumulative incidence, but the high farm prevalences also suggest a widespread occurrence of infection.

4.4 Prevalence in Companion Animals In Tables 4.3 and 4.4 an overview of the prevalence of Giardia in dogs and cats from different countries and backgrounds is provided. Similar to farm animals, the prevalence has been reported with considerable variation, and part of the variability can also be attributed

74

T. Geurden and M. Olson

Table 4.2 The animal prevalence (PA) and farm prevalence (PF) of Giardia duodenalis in other production animals in different countries. The number of animals (#A) and farms (#F) is presented along with the diagnostic assay (Diag) used in the study (IFA Immunofluorescence microscopy, PCR polymerase chain reaction or ME microscopical examination, cELISA copro-antigen ELISA; – not known) Diag

#A

#F

PA

PF

Reference

Australia

ME

1647

–

9

–

Ryan et al. (2005)

Belgium

IFA

137

Geurden et al. (2008d)

Country Sheep

10

36

100

38

100

Canada

IFA

89

6

Italy

ME

325

20

Spain

IFA

446

38

Spain

IFA

575

Belgium

IFA

148

Brazil

ME

Spain

IFA

Spain

ME

574

Spain

ME/ELISA

315

40

42

95

Ruiz et al. (2008)

Canada

IFA

236

6

9

66

Olson et al. (1997b)

Canada

IFA

piglets

312

43

4

23

weaners

309

43

10

58

1.5

Olson et al. (1997b)

10

Giangaspero et al. (2005)

19

92

Castro-Hermida et al. (2006a)

68

33

97

Castro-Hermida et al. (2006b)

10

53

80

Geurden et al. (2008d)

105

6

14

66

Bomfim et al. (2005)

116

20

20

90

Castro-Hermida et al. (2006a)

Goats

–

4

–

Díaz et al. (1996)

Pigs

Guselle and Olson (2000)

growers

346

46

11

77

finishers

357

46

15

75

54

38

6

46

224

45

4

52

boars sows Denmark

IFA

Maddox-Hyttel et al. (2006)

weaners

504

50

38

84

piglets

488

50

3

22

sows

245

50

4

18

684

100

1.5

10

Hamnes et al. (2007)

Norway

IFA

Croatia

cELISA

–

38

–

66

Bilic and Bilkei (2006)

18

30

Rinaldi et al. (2007)

Water buffalo Italy

cELISA

347

90

Italy

IFA

150

5

13.33

–

Veronesi et al. (2009)

Brazil

ME

396

3

0.5

–

De Souza et al. (2009)

USA

IFA

305

17

4.6

–

Atwill et al. (2000)

USA

IFA

91

0

–

Johnson et al. (1997)

Canada

IFA

35

100

Olson et al. (1997b)

Horses

– 4

20

Chap. 4 Giardia in Pets and Farm Animals, and Their Zoonotic Potential

75

Table 4.3 The animal prevalence (PA) of Giardia in dogs in different countries, and from different backgrounds. The number of animals (#A) is presented along with the diagnostic assay (Diag) used in the study (IFA Immunofluorescence microscopy; ELISA; ICG Immunochromatography; PCR polymerase chain reaction or ME microscopical examination; – not known) Country

Diag

PA

#A

Reference

Household dogs Argentina

ME

2193

9.0

Fontanarrosa et al. (2006)

Brazil

ME

72

12.3

Brazil

ME

122

4.1

Mundim et al. (2007)

Huber et al. (2005)

Brazil

ME

100

9.0

Meireles et al. (2008)

Belgium

IFA

451

9.3

Claerebout et al. (2009)

Czech Republic

ME

699

1.9

Borkovacova (2003)

France

ME

93

12.9

Beugnet et al. (2000)

Greece

ME

281

4.3

Papazahariadou et al. (2007)

Italy

ME

156

16.7

Capelli et al. (2006)

Italy

ME + IFA

616

21.3

Capelli et al. (2003)

Japan

ME

1035

14.6

Itoh et al. (2001)

Netherlands

ME + Elisa + PCR

152

15.2

Overgaauw et al. (2009)

South Korea

ICG

430

7.6

UK

ME

326

13.0

Guest et al. (2007)

USA

ME

79

34.0

Hahn et al. (1988)

Australia

ME

590

14.4

Palmer et al. (2008)

Brazil

ME

94

45.0

Huber et al. (2005)

Brazil

ME

288

39.6

Mundim et al. (2007)

Liu et al. (2008)

Kennel/shelter dogs

Brazil

ME

100

24.0

Meireles et al. (2008)

Belgium

IFA

357

43.9

Claerebout et al. (2009)

Czech Republic

ME

524

9.5

Dubná et al. (2007)

Italy

ELISA

183

55.2

Papini et al. (2005)

Italy

ME

64

20.3

Capelli et al. (2006)

Italy

PCR

127

20.5

Scaramozzino et al. (2009)

Japan

ELISA

361

37.4

Itoh et al. (2005)

Japan

ME

906

0.9

Yamamoto et al. (2009)

Slovak Republic

–

164

4.3

Szabová et al. (2007)

South Korea

ICG

42

47.6

Liu et al. (2008)

Spain

ME

1800

1.0

Martínez-Moreno et al. (2007)

Spain

ME

1161

7.0

Miró et al. (2007)

Thailand

ME

229

7.9

Inpankaew et al. (2007)

UK

ME

117

3.0

Guest et al. (2007)

USA

ME

38

39.0

Hahn et al. (1988) (Continued)

76

T. Geurden and M. Olson

Table 4.3 (Continued) Country

Diag

PA

#A

Reference

Clinically affected dogs Australia

ME

810

5.5

Belgium

IFA

351

18.1

Palmer et al. (2008) Claerebout et al. (2009)

Canada

ICG

1871

13

Olson et al. (2010)

Germany

ELISA

8438

16.6

Barutzki and Schaper (2003)

Germany

ME

1281

2.3

Epe et al. (2004)

Spain

ME

37

8.1

UK

ME

59

10.0

Guest et al. (2007)

Causapé et al. (1996)

USA

ICG

16,064

15.6

Carlin et al. (2006)

USA

ME

1,119,293

4.0

Little et al. (2009)

Stray dogs Italy

ME

87

16.1

Capelli et al. (2006)

Spain

ME

44

2.3

Causapé et al. (1996)

Mexico

ME

200

46.5

Ponce-Macotela et al. (2005)

Table 4.4 The animal prevalence (PA) of Giardia in cats in different countries. The number of animals (#A) is presented along with the diagnostic assay (Diag) used in the study (IFA Immunofluorescence microscopy; Elisa; ICG Immunochromatography; PCR polymerase chain reaction or ME microscopical examination; – not known) Country

Diag

PA

#A

Reference

Household cats Australia

ME

418

0*

McGlade et al. (2003)

France

ME

34

8.8

Beugnet et al. (2000)

Italy

Elisa

86

Japan

Elisa

600

Netherlands

ME

60

USA

ME

211,105

19.7

Papini et al. (2007)

40

Itoh et al. (2006)

13.6

Overgaauw et al. (2009)

0.58

De Santis-Kerr et al. (2006)

Kennel/shelter cats Australia

ME

491

2.6

Palmer et al. (2008)

Australia

ICG

120

9.2

Bissett et al. (2009)

Japan

ME

1079

0

Yamamoto et al. (2009)

Clinically affected cats Australia

ME

572

1.4

Palmer et al. (2008)

Canada

ICG

389

4.3

Olson et al. (2010)

Germany

Elisa

3167

51.6

UK

Elisa

1355

9.0

Barutzki and Schaper (2003) Tzannes et al. (2008)

Stray cats Brazil

ME

51

5.9

Coelho et al. (2009)

Iran

ME

113

0.9

Mohsen and Hssein (2009)

Italy

Elisa

180

13.8

*Some of these samples were found Giardia positive using PCR.

Papini et al. (2007)

Chap. 4 Giardia in Pets and Farm Animals, and Their Zoonotic Potential

to the study design. The majority of the studies in kennels or shelters for example were conducted on a limited number of locations, both for cats and dogs, which will certainly influence our understanding of the prevalence obtained in these studies. Overall, the prevalence in companion animals seems somewhat lower compared to farm animals. Although obvious differences in animal rearing, such as housing and stocking density, will certainly account for this alleged difference, the more frequent use of microscopy as diagnostic technique in companion animal studies compared to farm animal studies needs to be taken into account. Previously, flotation and microscopic examination was indeed found to be less sensitive for epidemiological studies compared to immunological assays in dogs (Geurden et al., 2008a; Olson et al., 2010) and in cattle (Geurden et al., 2004). In another study in cats, a high Giardia prevalence was found using PCR, whereas most of the samples were negative using microscopy (McGlade et al., 2003). Next to species the background of the animals is of importance: in household dogs, prevalences up to 21.3% have been reported, whereas in kennel and stray dogs prevalences up to 55.2 and 46.5%, respectively, were observed. In clinically affected dogs the highest prevalence reported was 18.1%. There are fewer prevalence studies in cats compared to dogs, however with similar variation in prevalences due to the animal background. As for farm animals, the

77

prevalence seems to be higher in young animals, both in dogs (Itoh et al., 2001; Claerebout et al., 2009; Gates and Nolan, 2009; Little et al., 2009) and cats (De Santis-Kerr et al., 2006; Gates and Nolan, 2009). Other factors also influence the prevalence estimation, including housing: dogs kept indoors or coming from breeding kennels also had a higher prevalence than dogs kept outdoors (Itoh et al., 2001, 2009).

4.5 Epidemiology Several parasite characteristics facilitate infection with Giardia, such as the high excretion of cysts by infected animals and the low dose needed for infection. Furthermore, Giardia cysts are immediately infectious upon excretion and do not need to sporulate in the environment. Cysts are also very resistant and able to survive for several weeks in the environment, resulting in a gradual increase in environmental infection pressure (Xiao et al., 1993; Olson et al., 1999; Wade et al., 2000a). An overview of the relevant parasite characteristics facilitating transmission of infection, is presented in Table 4.5. A novel host is infected by oral intake of infectious cysts, and as soon as 3 days after infection cysts can be recovered from the feces. The cyst excretion peaks in young animals, possibly due to the slow development of adaptive immunity by the host (Yanke et al.,

Table 4.5 Parasite characteristics of G. duodenalis, the epidemiological consequence and the appropriate preventive measures Parasite characteristic

Epidemiological consequence

Preventive measure

High excretion of cysts in the feces, which are immediately infective

Infection pressure can increase in a short period of time

– avoid crowding – isolation of excreting animals – hygiene*

Cysts are resistent to environmental conditions

Excreted cysts can survive for several weeks in the environment

– hygiene*

Cysts are resistant to chemical disinfection

Disinfection with common disinfectants are not reliable

Products based on ammonia, chlorine dioxide, hydrogen dioxide or ozone

Cysts are sensitive for heat and desiccation

Infection is mostly seen indoors

– disinfections using steam – avoid overpopulation – hygiene*

*hygiene:

– frequent removal of feces – thorough cleaning, preferably using a high pressure device – additional vacancy during a prolonged period – or alternate use of housing compartments.

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1998; O’Handley et al., 2003). Young animals should therefore be considered as a major source of environmental contamination, and as a principal source for direct transmission. The development of adaptive immunity results in an intermittent and/or reduced cyst excretion (Xiao et al., 1994; Nydam et al., 2001), and decreased contribution to the environmental contamination. Older and adult hosts can however not be excluded as source of infection, since a limited number of cysts (1–100) are sufficient to establish a novel infection in susceptible animals (Schaefer et al., 1991; Cacciò et al., 2005). In sheep, goats, pigs, and cattle, a periparturient rise of the cyst excretion has been suggested (Xiao and Herd, 1994; Wade et al., 2000a; Castro-Hermida et al., 2005), in which case the dam or sow needs to be considered as source of infection. In horses, cats, and dogs there are at present no indications for an increased cyst excretion around the partus. The predatory behavior of some animal species such as cats and dogs allows the foodborne route of infection to be a potentially significant, and cats have been infected with an avian and rodent strain of Giardia (McDonnell et al., 2003). Direct contact with an infected host is a potential route of infection, and especially young animals are of importance, as the prevalence and cyst excretion peaks in this age category (Xiao et al., 1994; Itoh et al., 2001; Nydam et al., 2001; Ralston et al., 2003; Becher et al., 2004; De Santis-Kerr et al., 2006; Claerebout et al., 2009; Gates and Nolan, 2009; Little et al., 2009). Infection can also spread indirectly through a contaminated environment, such as the animal housing. Animals reared indoors are more likely to acquire infection than outside (Ruest et al., 1998; Itoh et al., 2001, 2009), and particularly larger group housing seems to favor transmission. However, infections are known to spread in individual or small housing facilities too, due to the subsequent use of the facilities without proper disinfection. Furthermore, intensive rearing systems, such as dairy farms or kennels, favor transmission of infection, due to the concentration of animals able to excrete high numbers of infective cysts and the continuous introduction or presence of susceptible animals. It is thus important to discriminate between high and low transmission environments, in order to understand the epidemiology of Giardia in farm and companion animals.

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4.6 Pathogenesis Although clinical signs associated with a Giardia infection have been documented in a variety of animals, the pathogenesis is not yet fully understood. Studies in human epithelial cell lines (Buret et al., 1990a), in laboratory animals (Buret et al., 1990b, 2002; Scott et al., 2002), in goat kids (Koudela and Vitovec, 1998) and in calves (Taminelli et al., 1989; Ruest et al., 1997; O’Handley et al., 2001) indicate that giardiasis essentially leads to villus and microvillus alterations, including a decreased crypt to villus ratio, shortening of the microvillus brush border and brush border enzyme deficiencies. As suggested by the absence of these particular alterations in response to a Giardia infection in T-cell deficient mice (Scott et al., 2000), they are not only a direct consequence of the interaction between trophozoites and epithelium, but are also mediated by the host’s immune response. The pathogenesis of giardiasis can therefore be considered as a multifactorial process, involving both parasite characteristics and the host immune response. An increase in epithelial permeability has been described, which appears to result from enterocyte apoptosis (Cevallos et al., 1995; Chin et al., 2002) and from cytoskeletal reorganization induced by trophozoite toxic products (Buret et al., 2002; Scott et al., 2002). This leads to local disruption of tight-junctional proteins and an increase in intestinal permeability. Whether Giardia secretory-excretory products induce direct proteolytic degradation of tight-junctional proteins is uncertain (Buret, 2007), but the increased epithelial permeability leads to a higher number of intraepithelial lymphocytes (IEL) and to activation of T-lymphocytes. Trophozoite toxins and T-cell activation also initiate a diffuse shortening of brush border microvilli and a decreased activity of the small intestinal brush border enzymes, especially lipase, some proteases and the disaccharidases lactase, maltase, and sucrase (Buret et al., 1990a; Scott et al., 2000). The increased intestinal permeability also permits macromolecular transport that can lead to hypersensitivity to food proteins (Hardin et al., 1997). Malabsorption due to an infection with Giardia has been associated with an increased number of IEL and a decreased villus to crypt ratio (Farthing, 1997; Ruest et al., 1997; Koudela and Vitovec, 1998). The diffuse microvillus shortening leads to a decrease in overall

Chap. 4 Giardia in Pets and Farm Animals, and Their Zoonotic Potential

absorptive area in the small intestine and an impaired intake of water, electrolytes, and nutrients (Buret, 2007). The combined effect of this decreased resorption and the brush border enzyme deficiencies results in malabsorptive diarrhea and lower weight gain, both in murine models (Buret et al., 1990a) and in ruminant experimental models (Olson et al., 1995; Ruest et al., 1997; Koudela and Vitovec, 1998). The reduced activity of lipase and the increased production of mucine by goblet cells may explain the steatorrhea and mucous diarrhea which has been described in Giardia infected hosts (Zajac, 1992; Moncada et al., 2003). Giardiasis can lead to decreased transit time of food in the gut and an increase in gut contractility. The reduced time the food is exposed to digestive enzymes and the reduced absorption of nutrients and water can contribute to the diarrhea. The increased contractility may also explain the abdominal cramps frequently reported in giardiasis (Deselliers et al., 1997).

4.7 Clinical Signs Since the pathogenesis of giardiasis is a combination of both parasite and host factors, the subsequent clinical signs vary considerably between animals and animal species. This lack of consistency in clinical signs has resulted in the perception that Giardia is not a major cause of clinical disease but the wide variation in severity of clinical signs of giardiasis within and among animal species is common to most intestinal bacterial and parasitic disease such as Salmonella and Campylobacter. Clinical infection results in diarrhea which does not respond to antibiotic or coccidiostatic treatment. The excretion of pasty to fluid feces with a mucoid appearance may be indicative for giardiasis, especially when the diarrhea occurs in young animals. Based on observations in experimental infections or in clinical studies, a variety of clinical symptoms have been described in most animal species, including an acute to chronic diarrhea, vomiting, ill thrift, anorexia, flatulence and urticaria (St. Jean, 1987; Zajac, 1992; Olson et al., 1995; Hardin et al., 1997; Koudela and Vitovec, 1998; Geurden et al., 2006a, b). In pigs and horses, a significant association between Giardia infection and the occurrence of clinical signs has not yet been demonstrated (Hamnes et al., 2007), although an experimental infection to study the

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detrimental effects of Giardia in these animal species is still to be performed. Next to obvious clinical symptoms, there is a potential impact on production associated with giardiasis in farm animals. In goat kids and in lambs an experimental infection resulted in a decreased feed efficiency and subsequently a decreased weight gain (Olson et al., 1995; Koudela and Vitovec, 1998). In calves, an increased weight gain was observed in fenbendazole-treated calves compared to infected calves over a four-week period (Geurden et al., 2010a). There are at present however no conclusive data on the long-term effect of infection and the associated economical impact, but Giardia infections have certainly the potential to have a significant impact on animal production as infections in food animals are very common and persist for months. In human patients, infections with assemblage A are more likely to result in clinical signs than infections with assemblage B (Read et al., 2002), although once established infections with assemblage B seem to result in more persistent diarrhea (Homan and Mank, 2001). Whether a similar difference in clinical outcome occurs between the host-specific assemblages and the zoonotic assemblage A and B in farm and companion animals, is not known and should be further studied. In humans, allergic skin disease and increased visceral hypersensitivity (food allergy) are thought to be associated with giardiasis (Giacometti et al., 2003, Dizdar et al., 2007).

4.8 Diagnosis Due to the vagueness of the symptoms, the clinical diagnosis of giardiasis is not straightforward, and is mainly based on clinical history, data on environmental management and the exclusion of other infectious diseases. The clinical diagnosis needs to be confirmed by the detection of the parasite in a fecal sample, either by floatation and microscopical examination, fecal antigen detection or polymerase chain reaction (PCR). Given the intermittent excretion of cysts, especially in the chronic phase of infection, multiple samplings can be necessary, either from the same animal for three consecutive days (O’Handley et al., 1999) or from several animals within the same housing facility. If possible, young animals should be included in

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the sampling since the peak excretion is observed in animals around 2–4 weeks of age, even if they do not yet display any symptomatology yet. The diagnosis of a Giardia infection by serum antibody detection is not readily performed, as the antibody titers are not significantly increased after infection (Yanke et al., 1998; O’Handley et al., 2003).

4.8.1 Microscopical Examination Both the trophozoites and the cysts of Giardia can be detected by microscopic examination, either directly (fecal smear) or after concentration with sucrose, zinc sulfate, or sodium nitrate. The correct floatation procedure also significantly improves the diagnostic sensitivity (Dryden et al., 2006). Steathorrhea, which is observed in giardiasis, can interfere with sucrose flotation (Xiao and Herd, 1993) but can be addressed by treating samples with chloroform. Trophozoites can sometimes be detected in fecal samples with diarrhea due to the increased peristalsis. Given the characteristic movement of the trophozoites, they are preferably visualized in a native smear using recently obtained feces. More frequently, the detection of cysts in the feces is preferred for diagnosis. Prior to examination cysts can be stained. Frequently used stains are iodine (Zajac, 1992) and trichrome (Addiss et al., 1991). The major advantage of microscopical examination is the limited cost of consumables. The major disadvantage is the need for a skilled and experienced microscopist and the lower sensitivity compared to immunological assays (Geurden et al., 2004), and the time lost by transport to and analysis in the laboratory. The small size of the Giardia cysts increases the incidence of false positives by identifying pseudoparasites (yeast, particles) as cysts (Dryden et al., 2006).

4.8.2 Antigen Detection For the detection of parasite antigen immunofluoresence assays (IFA) (Xiao and Herd, 1993), enzymelinked immunosorbent assays (ELISA) (Boone et al., 1999) and rapid solid-phase qualitative immunochromatography assays (Garcia et al., 2003) are commercially available. Most tests were developed and

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evaluated for use in human stool samples. IFA and copro-antigen ELISA use monoclonal antibodies against cyst wall proteins. IFA can be used as a quantitative test, with a detection limit estimated around 1000 cysts per gram of feces (CPG) (Xiao and Herd, 1993). In calves, both IFA and ELISA were found to be sensitive and specific assays for the diagnosis of infection, compared to microscopical examination (Geurden et al., 2004). Similarly in dogs, IFA was found to be the most sensitive technique for clinical diagnosis (Geurden et al., 2008a). Partially due to the requirement of laboratory equipment and trained personnel, the main disadvantage of IFA and ELISA is the elevated cost and the time lost by transport to and analysis in the laboratory. This could be circumvented by the use of immunochromatographic assays enabling on-site diagnosis within 15 min. Immunochromatography uses monoclonal antibodies directed against specific trophozoite or cyst wall proteins. In human medicine several assays are commercialized, including dip-sticks and rapid membrane assays. Similarly, in veterinary medicine the SNAP® Giardia test (IDEXX Laboratories Inc., Westbrook, Maine, USA) is commercialized for use in dogs and has recently been proven to be a valuable alternative in the clinical diagnosis of giardiasis (Geurden et al., 2008a). In production animals, an immunochromatographic assay is available for calves (Speed® Giardia, BioVetoTest, La Seyne-surMer, France), but was found to be poorly sensitive for clinical diagnosis (Carlin et al 2006; Geurden et al., 2010b; Olson et al., 2010). Commercial fecal ELISA kits are effective for dogs and cats but most seem to be less effective for cattle, sheep, pigs, and goats.

4.8.3 Polymerase Chain Reaction (PCR) PCR is primarily used for the identification of different species and genotypes of Giardia for taxonomical and epidemiological research, although there is potential for diagnostic use. Several genes are commonly used for genotyping, and for clinical diagnosis the 18S rDNA (Read et al., 2004) seems most suitable. In theory, the detection limit of PCR is 1 cyst (Amar et al., 2002), which improves considerably diagnostic sensitivity. However, several factors can interfere with PCR such as inhibition, which is known to occur

Chap. 4 Giardia in Pets and Farm Animals, and Their Zoonotic Potential

frequently in DNA extracted from fecal samples. Furthermore, the extraction of parasite DNA from feces needs to be standardized for diagnostic use. At present PCR is considered as too expensive and cumbersome for use in veterinary diagnostics (da Silva et al., 1999), and has yet to be evaluated as diagnostic assay in production animals. In epidemiological research, PCR proved to result in increased prevalence estimates (Tables 4.1–4.4), but similar problems as for clinical diagnosis seem to impede its widespread use.

4.9 Treatment and Control Several compounds have a known efficacy against Giardia, both in vitro, in vivo and in laboratory animals. For sheep, goats, and pigs, there are no published results available on the efficacy of treatment against Giardia. Pigs treated with fenbendazole eliminated the parasite within a week and animals in treated and cleaned pens remained free of the parasite for 8 weeks (M.E. Olson, unpublished results). In calves, a number of studies evaluated the efficacy of different compounds, both in experimental and natural conditions. At present, no drug is however licensed for the treatment of giardiasis in ruminants. In cats and dogs, several compounds have been evaluated, and were found to be efficacious.

4.9.1 Chemotherapeutic Treatment Nitro-imidazoles (NZs) such as metronidazole and tinidazole, quinacrine or furazolidine are frequently used to treat giardiasis in human patients. Although therapy with these compounds is effective, considerable side-effects can occur. Metronidazole is even considered to be carcinogenic (Morgan et al., 1993; Harris et al., 2001). Furthermore, resistance to treatment has been described both for metronidazole and furazolidine (Upcroft et al., 1990). In veterinary medicine metronidazole (St. Jean, 1987; Xiao et al., 1993) and dimetridazole (St. Jean, 1987) have been used in companion animals and in calves, achieving symptomatic improvement. In cats, a total reduction in cyst excretion for at least 15 days was achieved (Scorza and Lappin, 2004). Furthermore, in several countries the NZs are no longer approved for use in livestock.

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More recently, nitazoxanide has been shown in vitro to be a promising new drug against Giardia (CedilloRivera et al., 2002), but no data on in vivo activity in farm and companion animals are available. Azythromicin was found to be efficacious in the treatment of Giardia, yet only one dog was involved in the experiment (Zygner et al., 2008), so further data are needed to confirm these findings.

4.9.1.1 Benzimidazoles An option for treatment of giardiasis is the benzimidazole compounds (BZs), which are well-known broad-spectrum anthelmintics. BZs are believed to have a high safety margin and a selective toxicity (Xiao et al., 1996). In vitro studies indicated that BZs are more efficacious against Giardia than either metronidazole or tinidazole (Edlind et al., 1990; Meloni et al., 1990; Morgan et al., 1993) and that this antiGiardia effect is irreversible (Morgan et al., 1993). The BZs interfere with the polymerization of tubulin, which is a major component of the Giardia trophozoite cytoskeletal structures. Therefore all functional activities of tubulin-dependent structures, such as the median body and the ventral disk, are inhibited. As a result BZs interfere with trophozoite attachment to the intestinal mucosa and prevent intestinal colonization. BZ seems not to affect flagellar tubulin, which has a different tubulin subunit structure (Clark and Holberton, 1988). The BZ mode of action might also include binding to giardins, which are Giardia-specific proteins restricted to the ventral disk (Edlind et al., 1990; Meloni et al., 1990). In calves data on reduction in cyst excretion are available for treatment with fenbendazole (Xiao et al., 1996; O’Handley et al., 1997) and albendazole (Xiao et al., 1996). Fenbendazole has been shown to significantly reduce the peak and the duration of cyst excretion and to result in a clinical benefit (O’Handley et al., 2000), although the total dosage of both BZs needed for Giardia treatment (5–20 mg per kg bodyweight per day during three consecutive days) is higher compared to helminth treatment. Furthermore, the cyst-suppressing effect of BZ treatment was either not complete or short lasting in field conditions, despite the high in vitro efficacy of both drugs. This might be either due to a high environmental infection pressure, which counters the effect of treatment or to the lack of

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persistent efficacy of BZs against Giardia in calves, resulting in a rapid reinfection shortly after the end of the treatment. To prevent reinfection from the environment and to improve long-term production parameters, it has been hypothesized that calves should be treated with a continuous low dosage of BZs (O’Handley et al., 2000), since the treatment duration seems to be more important than treatment dosage (O’Handley et al., 1997). However, there might be a risk for developing BZs resistant Giardia field strains. In dogs, oxfendazole (Villeneuve et al., 2000), albendazole (Chon and Lim, 2005) and fenbendazole (Barr et al., 1994; Zajac et al., 1998; Chon and Lim, 2005) proved to be effective in order to eliminating cyst excretion and improve clinical symptoms. Again a treatment during three consecutive days is advocated along with appropriate hygienic measures (Zajac et al., 1998; Villeneuve et al., 2000). Albendazole has been reported to cause bone marrow depletion in dogs and it is teratogenic in pregnant bitches (Barr et al., 1994). In cats, fenbendazole failed to completely eliminate, but successfully reduced the fecal cyst excretion after treatment, in cats which had a Cryptosporidium co-infection (Keith et al., 2003).

4.9.1.2 Pyrantel-febantel-praziquantel Combo Both in cats and dogs the combination pyrantel-febantel-praziquantel has been tested and found to be efficacious against Giardia, with a significant reduction of the cyst excretion after treatment (Barr et al., 1998; Payne et al., 2002; Scorza et al., 2006; Montoya et al., 2008; Bowmann et al., 2009). A synergistic effect between pyrantel and febantel was demonstrated in an animal model suggesting that the combination product may be preferred over the febantel alone (Olson and Heine, 2009).

4.9.1.3 Paromomycin Paromomycin or aminosidin is a broad-spectrum amino-glycoside antibiotic, with well-known efficacy against several protozoan parasites, such as Cryptosporidium in calves (Fayer and Ellis, 1993; Mancassola et al., 1995; Chartier et al., 1996; Viu et al., 2000; Grinberg et al., 2002), Histomonas meleagridis in chickens (Hu and McDougald, 2004) and Giardia in rats (Awadalla et al., 1995) and humans

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(Wright et al., 2003). Paromomycin binds to the small subunit rRNA and inhibits protein synthesis, which has either a direct effect on Giardia or an indirect effect of nutrient withdrawal caused by the inhibition of bacterial protein synthesis and destruction of the bacterial flora (Edlind et al., 1990; Harris et al., 2001). Paromomycin is poorly absorbed from the gastro-intestinal tract and is therefore well tolerated by calves (Grinberg et al., 2002). In calves, paromomycin was shown to effectively reduce the Giardia cyst excretion in experimentally infected calves during at least 2 weeks, when administered at 50 and 75 mg/kg/day during five consecutive days (Geurden et al., 2006a). In other animals, there are no experimental data on the efficacy of paromomycin.

4.9.2 Alternative Approaches Several compounds were shown to affect parasite growth, adhesion capacity and morphology in vitro, including curcumin (Pérez-Arriaga et al., 2006) and peppermint (Vidal et al., 2007). In mice, extracts from medicinal plants used in Mexican traditional medicine were shown to effectively kill Giardia trophozoites (Barbosa et al., 2006). Similarly, yucca extracts were shown to kill giardia in vitro and in a gerbil model but this product has not been commercialized (McAllister et al., 2001). However, none have so far been evaluated in farm or companion animals reared under natural conditions, with a continuous exposure of animals to infection. The most promising alternative approach is probably vaccination. Since both paromomycin and BZs do not have a persistent efficacy, the effect of treatment is often short lasting in natural conditions due to reinfection from a contaminated environment (O’Handley et al., 2000; Villeneuve et al., 2000; Geurden et al., 2006a, b). Vaccination has the potential to provide an alternative for chemotherapeutic treatment, achieving a prolonged protection against infection and preventing the excretion of cysts in order to break the transmission by reducing environmental contamination. In the United States, a Giardia vaccine is commercially available for use in cats and dogs (Fel-O-Vax Giardia or Giardia Vax, Fort Dodge Animal Health), but the efficacy of a preventive (Olson et al., 1996) or curative (Olson et al., 2001; Payne et al., 2002; Stein et al.,

Chap. 4 Giardia in Pets and Farm Animals, and Their Zoonotic Potential

2003; Anderson et al., 2004) vaccination seems to be variable. In calves, vaccination did result in a humoral immune response, which was however not protective as the cyst excretion and the number of intestinal trophozoites were not reduced, and clinical signs could not be prevented (Uehlinger et al., 2007). Several issues need to be considered as far as vaccination against Giardia is concerned. As the current vaccine consists of sonicated trophozoites, the product consists of multiple antigens and the production is cumbersome (Olson et al., 2001). The use of a recombinant vaccine containing one or more protective antigens has been advocated. Some Giardia proteins seem however to be assemblage-specific while others are common for all assemblages (Steuart et al., 2009). As the Giardia vaccine consists of sonicated trophozoites from an axenic reference strain (Assemblage A), the vaccine might not reflect the specific G. duodenalis assemblages infecting animals, resulting in a poor vaccine efficacy. However, injection of whole trophozoites from one particular strain did induce protection when challenged by a different strain in dogs (Olson et al., 2000). Although several Giardia antigens have been proposed as potential vaccine candidates (Olson et al., 2000; Abdul-Wahid and Faubert, 2007), more research is needed to identify proteins which are both commonly expressed in all assemblages and are known to induce a specific and protective immune response in production animals. As an increase in serological antibodies is not well correlated with protection against infection (Yanke et al., 1998; O’Handley et al., 2003), a local immune response as well as class shifting from IgM to IgG appears to be essential to prevent colonization of the gut by Giardia trophozoites, along with cellular mechanisms (Müller and von Allmen, 2005). Vaccination through subcutaneous injection of an antigen (Uehlinger et al., 2007) might therefore not be a suitable route to induce a localized immune response in the lumen of the small intestine, and an appropriate method to deliver the protective antigen to the mucosa of small intestine is needed. In mice, oral immunization with a live vector expressing Giardia cyst wall protein resulted in a significant reduction of the cyst excretion (Lee and Faubert, 2006). However, oral vaccination implies the passage of the hostile gastrointestinal environment, which might alter the antigenic structure of the protective protein.

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Furthermore, the optimal time of vaccination needs to be considered. As the peak in cyst excretion and clinical signs are mainly observed in young animals, an early vaccination is needed both to decrease the cyst excretion and hence the environmental contamination and to prevent clinical signs. However, maternal antibodies might interfere with immunity development in neonatal animals (Chase et al., 2008), and it is therefore uncertain if a protective immune response can be achieved by active immunization from birth onwards. Passive immunization by injecting the mother with a protective antigen and by transfer of protective antibodies through the milk is another option, as high anti-Giardia antibody titers have been found in colostrum from dairy cattle, which in vitro displayed the ability to prevent adhesion of Giardia throphozoites. Whether the short-lasting effect of passive immunity is sufficient to prevent a Giardia infection later in life is uncertain (O’Handley et al., 2003).

4.9.3 Control 4.9.3.1 Measures to Support Curative Treatment Although compounds such as fenbendazole, albendazole, febantel-pyrantel combo and paromomycin are effective against Giardia, most animals are re-excreting cysts within 2–3 weeks after treatment when maintained in a contaminated environment (Xiao et al., 1996; O’Handley et al., 1997; Villeneuve et al., 2000; Geurden et al., 2006b). Since Giardia cysts can survive for 1 week in feces and up to 7 weeks in soil (Olson et al., 1999), the effective treatment period of most protocols (3–5 days) may be too short to prevent reinfection from a contaminated environment shortly after treatment. The short-term cyst-suppressing effect of treatment in a contaminated environment (Xiao et al., 1996; O’Handley et al., 2000) emphasizes the need for an integrated control program combining both treatment and cleaning-disinfection of the environment at the end of the treatment period to minimize the risk of reinfection after treatment. Giardia cysts are known to be resistant to commonly used disinfectants, such as chlorine. Alternative disinfectants, including chlorine dioxide, ozone, and ultra violet irradiation have been the focus of research in drinking water treatment processes, although there are practical objections against most of these disinfection procedures for use in calf fa-

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cilities. Alternatively, heat or desiccation (Olson et al., 1999) and disinfection with quaternary ammonium (Xiao et al., 1996; O’Handley et al., 1997) can be used in housing facilities. In a recent study, the efficacy of a combination of calf treatment with fenbendazole and environmental cleaning and disinfection with ammonia 10% or relocation was evaluated on commercial dairy farms and found to achieve a significant reduction both in the number of calves excreting cysts and in the number of cysts excreted. The environmental infection pressure was thus decreased by effectively breaking the transmission cycle of Giardia, resulting in a long-term efficacy of treatment (Geurden et al., 2006b). Similarly in dogs, the importance of introducing treated animals into a clean environment was emphasized (Villeneuve et al., 2000). Although in some efficacy trials the housing was clean and disinfected, animals were re-excreting cysts after treatment, suggesting reinfection through fecal material on limbs or in the fur (Xiao et al., 1996; O’Handley et al., 1997, 2000). Animals should therefore be washed after treatment (Zajac et al., 1998; Payne et al., 2002; Uehlinger et al., 2007), although in practice this is only feasible for companion animals.

4.9.3.2 Measures to Prevent Infection As discussed above, management measures are essential to achieve clinical improvement in a curative treatment regimen. Similarly for the prevention of infection, proper management may contribute to a decreased infection rate, and aims to break the parasite’s transmission cycle through specific measures countering the parasite’s ability to spread easily (see also Table 4.5). These management measures include a limited number of animals per housing (Wade et al., 2000a), combined with regular cleaning and disinfection of the housing facilities (Bomfin et al., 2005; Maddox-Hyttel et al., 2006). A well thoughtover housing is also important, as animals reared indoors are more likely to acquire infection than outside (Ruest et al., 1998; Itoh et al., 2001, 2009). As Giardia cysts can persist in moist areas for months and even years and can be washed from manure storage sites by heavy rains, composing of manure appears to inactivate cysts and provides a practical method for manure management (van Herk et al., 2004), both on a farm level and form a public health point of view.

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4.10 Molecular Epidemiology More than 50 different Giardia species have been described, primarily based on host specificity, but this host-specific taxonomy has been replaced by a taxonomy based on morphological characteristics such as shape and length of the trophozoite and median bodies (Filice, 1952). Three distinct groups or Giardia species were described, including G. duodenalis which has a wide mammalian host range. Molecular characterization has since revealed that G. duodenalis is in fact a species complex, comprising seven assemblages (A–G), some of which have distinct host preferences or a limited host range (Thompson and Monis, 2004). In addition to the assemblages A and B which are also prevalent in human patients, several host-specific assemblages have been identified in animals, including assemblages C and D in dogs, assemblage E in hoofed livestock and assemblage F in cats. The genetic structure within the assemblages provides further information in order to understand the potential for inter-species transmission, and assemblage subgroups have been identified on several commonly used loci. Within assemblage A four subgroups (AI–AIV) have been identified, which are robust and identifiable at all loci commonly used for Giardia molecular typing: assemblage AI has been reported in humans and animals, AII in humans, and AIII and IV exclusively in animals. Within assemblage B however, the subgroups are not reproducible at several of these loci (Wielinga et al., 2007), and the usefulness of these subassemblages in terms of molecular epidemiology is at present uncertain. Most cattle, sheep and goats (Ryan et al., 2005; Robertson, 2009), pigs (Langkjaer et al., 2007) and horses (Veronesi et al., 2009) are infected with the livestock-specific assemblage E. Nevertheless, a significant number of cattle, sheep and goats, and pigs are infected with assemblage A, either as a mono- or mixed infection (Ryan et al., 2005; Langkjaer et al., 2007; Geurden et al., 2008b; Xiao and Fayer, 2008; Robertson, 2009). In all ruminant host species, assemblage B has been described, but only in a limited number of cases (Aloisio et al., 2006; Xiao and Fayer, 2008). Similarly in horses, assemblage A and B has been identified (Ey et al., 1997; Traub et al., 2005), but only in a limited number of cases. Dogs can be infected with assemblage A–D, with a dominance of

Chap. 4 Giardia in Pets and Farm Animals, and Their Zoonotic Potential

assemblages C and D in most countries (Xiao and Fayer, 2008; Claerebout et al., 2009). Most studies do report the occurrence of assemblage A in dog samples, especially in dogs living in close contact with humans (Traub et al., 2003; Inpankeaw et al., 2007) or household dogs (Volotão et al., 2007; Claerebout et al., 2009). Infections with assemblage B are less frequently observed in dogs. In cats the host-specific assemblage F is most frequent, but assemblage A is described on a regular basis (Xiao and Fayer, 2008). Because humans are infected only with assemblages A and/or B, studies on zoonotic transmission of giardiasis have focused on these two assemblages. Taking the subgenotyping information into account especially subassemblage AI is of relevance. Although subgenotyping provides valuable additional information to elucidate the zoonotic potential of animal isolates, it is striking that most molecular epidemiological studies in animals do not report the subgenotypes. Yet the available subgenotyping data do not indicate the widespread occurrence of zoonotic transmission. Furthermore, most epidemiological studies do not report statistical associations between animal contact and human infections, in order to support the importance of zoonotic transmission of Giardia from animals to humans (Matthias et al., 1992; Dennis et al., 1993; Gray et al., 1994; Hoque et al., 2003; Stuart, 2003). One study in the UK only found associations with exposure to farm animals and pets (Warburton et al., 1994). On the other hand, circumstantial evidence strongly suggests a zoonotic link between human and animal populations, as the prevalence of giardiasis was found to be higher in rural areas compared to urban areas (Mitchell et al., 1993) and correlated with farm visits (Stuart, 2003). This might suggest that contamination of the environment, for example surface water, contributes the transmission of Giardia, rather than direct contact with infected animals (Heitman et al., 2002). Surface water is indeed an ideal matrix for transmission of infection, as Giardia cysts are able to survive for a long time in water. Surface water is frequently used for the production of drinking water, but in most developed countries, sufficient disinfection and filtration procedures are in place to prevent a continuous infection threat. Occasional outbreaks are however possible, as illustrated by the massive outbreak in Bergen, Norway (Robertson et al., 2006).

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Several G. duodenalis assemblages have also been identified in wildlife species including terrestial (e.g., moose, elk, deer, and coyotes), aquatic (e.g., beavers, muskrats, and otters), and marine (e.g., seals and whales) mammals (Heitman et al., 2002; Appelbee et al., 2005). Transmission within a wildlife species is most common but wildlife can be infected from another mammalian species or by human waste. Wildlife can also infect humans and there are numerous reports of human infection following exposure to water contaminated with beaver feces. Indeed, the name “beaver feaver” was coined following a waterborne Giardia outbreak in Canada. Human activity can also potentially lead to infections in wildlife. An example of this is the seals in the St. Lawrence River, which is heavily polluted, is infected with zoonotic genotypes of G. duodenalis (Measures and Olson, 1999; Appelbee et al. 2005). It is believed that one transmission pathway is humans or domestic animals infecting a wildlife species which in turn infect humans or domestic animals. Wildlife can therefore act as a continuous source of infection that cannot be controlled once it is established in an endemic area. However, most of the studies on the correlation between animal and human infections have been conducted in a limited number of developed countries, and at present it is uncertain to what extent zoonotic transmission is of importance in developing countries, especially in rural areas. To fully understand the potential for and frequency of transmission of Giardia cysts from one host population to another, longitudinal studies in low and high transmission environments are needed, using genotyping tools able to identify the subassemblages involved.

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90 O’Handley RM, Olson ME, McAllister TA, Morck DW, Jelinski M, Royan G, and Cheng KJ (1997) Efficacy of fenbendazole for treatment of giardiosis in calves. Am J Vet Res 58: 384–388 O’Handley RM, Cockwill C, McAllister TA, Jelinski M, Morck DW, and Olson ME (1999) Duration of naturally acquired giardiosis and cryptosporidiosis in dairy calves and their association with diarrhea. J Am Vet Med Ass 214: 391–396 O’Handley RM, Cockwill C, Jelinski M, McAllister TA, and Olson ME (2000) Effects of repeat fenbendazole treatment in dairy calves with giardiosis on cyst excretion, clinical signs and production. Vet Parasitol 89: 209–218 O’Handley RM, Buret AG, McAllister TA, Jelinski M, and Olson ME (2001) Giardiasis in dairy calves: effects of fenbendazole treatment on intestinal structure and function. Int J Parasitol 31: 73–79 O’Handley RM, Ceri H, Anette C, and Olson ME (2003) Passive immunity and serological immune response in dairy calves associated with natural Giardia duodenalis infections. Vet Parasitol 113: 89–98 Olson ME and Heine J (2009) Synergistic effect of febantel and pyrantel embonate in elimination of Giardia in a gerbil model. Parasitol Res (Suppl 1): S135–S140 Olson ME, McAllister TA, Deselliers L, Morck DW, Cheng KJ, Buret AG, and Ceri H (1995) Effects of giardiasis on production in a domestic ruminant (lamb) model. Am J Vet Res 56: 1470–1147 Olson ME, Morck DW, and Ceri H (1996) The efficacy of a Giardia lamblia vaccine in kittens. Can J Vet Res 60: 249– 256 Olson ME, Guselle NJ, O’Handley RM, Swift ML, McAllister TA, Jelinski MD, and Morck DW (1997a) Giardia and Cryptosporidium in dairy calves in British Columbia. Can Vet J 38: 703–706 Olson ME, Thorlakson CL, Deselliers L, Morck DW, and McAllister TA (1997b) Giardia and Cryptosporidium in Canadian farm animals. Vet Parasitol 68: 375–381 Olson ME, Goh J, Phillips M, Guselle N, and McAllister TA (1999) Giardia cyst and Cryptosporidium oocyst survival in water, soil, and cattle feces. J Environ Quality 28: 1991– 1996 Olson ME, Ceri H, and Morck DW (2000) Giardia vaccination. Parasitol Today 16: 213–217 Olson ME, Hannigan CJ, Gaviller PF, and Fulton LA (2001) The use of a Giardia vaccine as an immunotherapeutic agent in dogs. Can Vet J 42: 865–868 Olson ME, Leonard NJ, and Strout J (2010) Prevalence and diagnosis of Giardia infection in dogs and cats using a fecal antigen test and fecal smear. Can Vet J, in press. Overgaauw PAM, van Zutphen L, Hoek D, Yaya FO, Roelfsema J, Pinelli E, van Knapen F, and Kortbeek LM (2009) Zoonotic parasites in fecal samples and fur from dogs and cats in The Netherlands. Vet Parasitol 163: 115–122 Palmer CS, Thompson RCA, Traub RJ, Rees R, and Robertson ID (2008) National study of the gastrointestinal parasites of dogs and cats in Australia. Vet Parasitol 151: 181–190 Papazahariadou M, Founta A, Papadopoulos E, Chliounakis S, Antoniadou-Sotiriadou K, and Theodorides Y (2007) Gastrointestinal parasites of shepherd and hunting dogs in

T. Geurden and M. Olson the Serres Prefecture, Northern Greece. Vet Parasitol 148: 170–173 Papini R, Gorini G, Spaziani A, and Cardini G (2005) Survey on giardiosis in shelter dog populations. Vet Parasitol 128: 333–339 Papini R, Giuliani G, Gorini G, and Cardini G (2007) Survey of feline giardiasis by ELISA test in Italy. Vet Res Commun 31: 297–303 Payne PA, Ridley RK, Dryden MW, Bathgate C, Milliken GA, and Stewart PW (2002) Efficacy of a combination febantelpraziquantel-pyrantel product, with or without vaccination with a commercial Giardia vaccine, for treatment of dogs with naturally occurring giardiosis. J Am Vet Med Ass 220: 330–333 Pérez-Arriaga L, Mendoza-Magaña ML, Cortés-Zárate R, Corona-Rivera A, Bobadilla-Morales L, Troyo-Sanromán R, and Ramírez-Herrera MA (2006) Cytotoxic effect of curcumin on Giardia lamblia trophozoites. Acta Tropica 98: 152–161 Ponce-Macotela M, Peralta-Abarca GE, and Martínez-Gordillo MN (2005) Giardia intestinalis and other zoonotic parasites: prevalence in adult dogs from the southern part of Mexico City. Vet Parasitol 131: 1–4 Quilez J, Sanchez-Acedo C, del Cacho E, Clavel A, and Causape AC (1996) Prevalence of Cryptosporidium and Giardia infections in cattle in Aragon (northeastern Spain). Vet Parasitol 66: 139–146 Ralston BJ, Cockwill C, Guselle N, Van Herk FH, McAllister TA, and Olson ME (2003) Prevalence of Giardia and Cryptosporidium andersoni and their effect on performance in feedlot beef calves. Can J An Sci 83: 153–159 Read C, Walters J, Robertson ID, and Thompson RC (2002) Correlation between genotype of Giardia duodenalis and diarrhoea. Int J Parasitol 32: 229–231 Read CM, Monis PT, and Thompson RCA (2004) Discrimination of all genotypes of Giardia duodenalis at the glutamate dehydrogenase locus using PCR-RFLP. Inf Gen Evol 4: 125–130 Rinaldi L, Condoleo RU, Condoleo R, Saralli G, Bruni G, and Cringoli G (2007) Cryptosporidium and Giardia in water buffaloes (Bubalus bubalis) of the Italian Mediterranean bred. Vet Res Comm (Suppl 1): 253–255 Robertson LJ (2009) Giardia and Cryptosporidium infections in sheep and goats: a review of the potential for transmission to humans via environmental contamination. Epidemiol Infect 137: 913–921 Robertson LJ, Hermansen L, Gjerde BK, Strand E, Alvsvåg JO, and Langeland N (2006) Application of genotyping during an extensive outbreak of waterborne giardiasis in Bergen, Norway, during autumn and winter 2004. Appl Environ Microbiol 72: 2212–2217 Ruest N, Couture Y, Faubert GM, and Girard C (1997) Morphological changes in the jejunum of calves naturally infected with Giardia spp. and Cryptosporidium spp. Vet Parasitol 69: 177–186 Ruest N, Faubert GM, and Couture Y (1998) Prevalence and geographical distribution of Giardia spp. and Cryptosporidium spp. in dairy farms in Quebec. Can Vet J 39: 697–700 Ruiz A, Foronda P, González JF, Guedes A, Abreu-Acosta N, Molina JM, and Valladares B (2008) Occurrence and geno-

Chap. 4 Giardia in Pets and Farm Animals, and Their Zoonotic Potential type characterization of Giardia duodenalis in goat kids from the Canary Islands, Spain. Vet Parasitol 154: 137–141 Ryan UM, Bath C, Robertson I, Read C, Elliot A, McInnes L, Traub R, and Besier B (2005) Sheep may not be an important zoonotic reservoir for Cryptosporidium and Giardia parasites. Appl Environ Microbiol 71: 4992–4997 Savioli L, Smith H, and Thompson A (2006) Giardia and Cryptosporidium join the ‘Neglected Diseases Initiative’. Trends Parasitol 22: 203–208 Scaramozzino P, Di Cave D, Berrilli F, D’Orazi C, Spaziani A, Mazzanti S, Scholl F, and De Liberato C (2009) A study of the prevalence and genotypes of Giardia duodenalis infecting kennelled dogs. Vet J 182: 231–234 Schaefer FW 3rd, Johnson CH, Hsu CH, and Rice EW (1991) Determination of Giardia lamblia cyst infective dose for the Mongolian gerbil (Meriones unguiculatus). Appl Environ Microbiol 57: 2408–2409 Scorza AV and Lappin MR (2004) Metronidazole for the treatment of feline giardiasis. J Feline Med Surg 6: 157–160 Scorza AV, Radecki SV, and Lappin MR (2006) Efficacy of a combination of febantel, pyrantel, and praziquantel for the treatment of kittens experimentally infected with Giardia species. J Feline Med Surg 8: 7–13 Scott KG, Logan MR, Klammer GM, Teoh DA, and Buret AG (2000) Jejunal brush border microvillous alterations in Giardia muris-infected mice: role of T lymphocytes and interleukin-6. Inf Imm 68: 3412–3418 Scott KG, Meddings JB, Kirk DR, Lees-Miller SP, and Buret AG (2002) Intestinal infection with Giardia spp. reduces epithelial barrier function in a myosin light chain kinasedependent fashion. Gastroenterology 123: 1179–1190 St. Jean G (1987) Diagnosis of Giardia infection in 14 calves. J Am Vet Med Ass 191: 831–832 Stein JE, Radecki SV, and Lappin MR (2003) Efficacy of Giardia vaccination in the treatment of giardiosis in cats. J Am Vet Med Ass 222: 1548–1551 Steuart R, O’Handley R, Lipscombe R, and Thompson R (2008) Alpha-2 giardin is an assemblage A specific protein of human infective Giardia duodenalis. Parasitology 135: 1621–1627 Stuart JM, Orr HJ, Warburton FG, Jeyakanth S, Pugh C, Morris I, Sarangi J, and Nichols G (2003) Risk factors for sporadic giardiasis: a case-control study in southwestern England. Emerg Infect Dis 9: 229–233 Szabová E, Juris P, Miterpáková M, Antolová D, Papajová I, and Sefciková H (2007) Prevalence of important zoonotic parasites in dog populations from the Slovak Republic. Helminthologia 44: 170–176 Taminelli V, Eckert J, Sydler T, Gottstein B, Corboz L, and Hofmann M (1989) Experimental infection of calves and sheep with bovine Giardia isolates. Schweiz Arch Tierheilk 131: 551–564 (in German) Thompson RC and Monis PT (2004) Variation in Giardia: implications for taxonomy and epidemiology. Adv Parasitol 58: 69–137 Thompson RC, Reynoldson JA, and Mendis AH (1993) Giardia and giardiosis. Adv Parasitol 32: 71–160 Traub RJ, Robertson ID, Irwin P, Mencke N, Monis P, and Thompson RC (2003) Humans, dogs and parasitic

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zoonoses – unravelling the relationships in a remote endemic community in northeast India using molecular tools. Parasitol Res 90(Suppl 3): S156–S157 Traub R, Wade S, Read C, Thompson A, and Mohammed H (2005) Molecular characterization of potentially zoonotic isolates of Giardia duodenalis in horses. Vet Parasitol 130: 317–321 Trout JM, Santin M, Greiner E, and Fayer R (2004) Prevalence of Giardia duodenalis genotypes in pre-weaned dairy calves. Vet Parasitol 124: 179–186 Trout JM, Santin M, Greiner E, and Fayer R (2005) Prevalence and genotypes of Giardia duodenalis in post-weaned dairy calves. Vet Parasitol 130: 177–183 Trout JM, Santin M, Greiner EC, and Fayer R (2006) Prevalence and genotypes of Giardia duodenalis in 1–2 year old dairy cattle. Vet Parasitol 140: 217–222 Tzannes S, Batchelor DJ, Graham PA, Pinchbeck GL, Wastling J, and German AJ (2008) Prevalence of Cryptosporidium, Giardia and Isospora species infections in pet cats with clinical signs of gastrointestinal disease. J Feline Med Surg 10: 1–8 Uehlinger FD, O’Handley RM, Guselle NJ, Greenwood SJ, and Barkema HW (2007) Efficacy of vaccination in preventing giardiasis in calves. Vet Parasitol 146: 182–188 Upcroft JA, Upcroft P, and Boreham PF (1990) Drug resistance in Giardia intestinalis. Int J Parasitol 20: 489–496 Van Herk FH, McAllister TA, Cockwill CL, Gusselle N, Larney FJ, Miller JJ, and Olson ME (2004) Inactivation of Giardia cysts and Cryptosporidium oocysts in beef feedlot manure by thermophilic windrow composting. Compost Sci Util 12: 235–241 Veronesi F, Passamonti F, Cacciò S, Diaferia M, and Piergili Fioretti D (2009) Epidemiological Survey on Equine Cryptosporidium and Giardia Infections in Italy and Molecular Characterization of Isolates. Zoon Public Health, in press Vidal F, Vidal JC, Gadelha AP, Lopes CS, Coelho MG, and Monteiro-Leal LH (2007) Giardia lamblia: the effects of extracts and fractions from Mentha u piperita Lin. (Lamiaceae) on trophozoites. Exp Parasitol 115: 25–31 Villeneuve V, Beugnet F, and Bourdoiseau G (2000) Efficacy of oxfendazole for the treatment of giardiosis in dogs experiments in dog breeding kennels. Parasite 7: 221–226 Viu M, Quilez J, Sanchez-Acedo C, del Cacho E, and LopezBernad F (2000) Field trial on the therapeutic efficacy of paromomycin on natural Cryptosporidium parvum infections in lambs. Vet Parasitol 90: 163–170 Volotão AC, Costa-Macedo LM, Haddad FS, Brandão A, Peralta JM, and Fernandes O (2007) Genotyping of Giardia duodenalis from human and animal samples from Brazil using beta-giardin gene: a phylogenetic analysis. Acta Trop 102: 10–19 Wade SE, Mohammed HO, and Schaaf SL (2000a) Epidemiologic study of Giardia sp. infection in dairy cattle in southeastern New York State. Vet Parasitol 89: 11–21 Wade SE, Mohammed HO, and Schaaf SL (2000b) Prevalence of Giardia sp. Cryptosporidium parvum and Cryptosporidium andersoni (syn. Cu muris) in 109 dairy herds in five counties of southeastern New York. Vet Parasitol 93: 1–11

92 Warburton AR, Jones PH, and Bruce J (1994) Zoonotic transmission of giardiasis: a case control study. Commun Dis Rep CDR Rev 4: R32–R36 Winkworth CL, Learmonth JJ, Matthaei CD, and Townsend CR (2008) Molecular characterization of Giardia isolates from calves and humans in a region in which dairy farming has recently intensified. Appl Environ Microbiol 74: 5100–5105 Wright JM, Dunn LA, Upcroft P, and Upcroft JA (2003) Efficacy of antigiardial drugs. Exp Opin Drug Safety 2: 529–541 Xiao L (1994) Giardia infection in farm animals. Parasitol Today 10: 436–438 Xiao L and Herd RP (1993) Quantitation of Giardia cysts and Cryptosporidium oocysts in fecal samples by direct immunofluorescence assay. J Clin Microbiol 31: 2944–2946 Xiao L and Herd RP (1994) Infection pattern of Cryptosporidium and Giardia in calves. Vet Parasitol 55: 257–262 Xiao L and Fayer R (2008) Molecular characterisation of species and genotypes of Cryptosporidium and Giardia and assessment of zoonotic transmission. Int J Parasitol 38: 1239–1255 Xiao L, Herd RP, and Rings DM (1993) Concurrent infections of Giardia and Cryptosporidium on two Ohio farms with calf diarrhea. Vet Parasitol 51: 41–48

T. Geurden and M. Olson Xiao L, Herd RP, and McClure KE (1994) Periparturient rise in the excretion of Giardia sp. cysts and Cryptosporidium parvum oocysts as a source of infection for lambs. J Parasitol 80: 55–59 Xiao L, Saeed K, and Herd RP (1996) Efficacy of albendazole and fenbendazole against Giardia infection in cattle. Vet Parasitol 61: 165–170 Yamamoto N, Kon M, Saito T, Maeno N, Koyama M, Sunaoshi K, Yamaguchi M, Morishima Y, and Kawanaka M (2009) Prevalence of intestinal canine and feline parasites in Saitama Prefecture, Japan. Kansenshogaku Zasshi. 83: 223–228 (in Japanese) Yanke SJ, Ceri H, McAllister TA, Morck DW, and Olson ME (1998) Serum immune response to Giardia duodenalis in experimentally infected lambs. Vet Parasitol 75: 9–19 Zajac A (1992) Giardiosis. Comp Contin Ed Practic Vet 14: 604–609 Zygner W, Jaros D, Gójska-Zygner O, and Wedrychowicz H (2008) Azithromycin in the treatment of a dog infected with Giardia intestinalis. Pol J Vet Sci 11: 231–234

Section II Molecular Biology of Giardia

5

Genomics of Giardia Hilary G. Morrison and Staffan Svärd

Abstract Modern sequencing technologies have facilitated comparative parasite genomics, providing insight into the genetic mechanisms responsible for invasion and pathogenicity. Representatives of three distinct Giardia assemblages have been fully sequenced, the most complete being the human isolate WBC6. Comparison of the three genomes (WB, GS, and P15) is under active study in many laboratories. Why high allelic sequence heterozygosity exists in Assemblage B isolates is an intriguing and as yet unsolved question. There is otherwise much similarity between the genomes. The vast majority of genes are homologous in all the three, with only a handful specific to any single genome. This finding elevates many hypothetical open reading frames to true genes. Future Giardia genomics will benefit from draft genomic coverage of more isolates, high-throughput transcriptomics, and refinement of the reference WB assembly.

As genomic sequencing became significantly more affordable in the past decade, researchers quickly moved to add additional, more closely related genomes to the reference strain. The advantages of this comparative genomic approach to understanding parasite biology are exemplified by the growing understanding of the malarial agent, Plasmodium. The first sequence attempted was that of P. falciparum 3D7, and it was the result of the effort of four institutes spanning approximately six years. Before its publication, the efforts to sequence five additional species were underway, along with members of the more distantly related apicomplexans Cryptosporidium and Toxoplasma. 3D7 data were used to predict drug targets and vaccine candidates (Jomaa et al., 1999; Florens et al., 2002). However, it required genomic data from additional species, albeit at “draft” coverage of 3–5u, to recognize gene synteny; to compare location, arrangement, and conservation of antigenic var genes; and to identify well-conserved non-coding regions. The existing Plasmodium genomic datasets continue to be a goldmine of information.

5.1 Genomics and Comparative Genomics 5.2 Available Giardia Data The complete genome sequence of any parasite profoundly enables scientific research. It contains the molecular basis underlying antigenic variation, pathogenicity, host range, and other biological properties. For this reason, there has been an effort toward genome sequencing of at least one member of all groups of medically significant parasites, particularly among the protists. Yet, because these organisms are evolutionarily divergent from each other and from model organisms (e.g. E. coli, Saccharomyces, Drosophila, C. elegans, and Mus musculus), a considerable proportion of any single genome’s coding capacity cannot be interpreted by simple homology to known genetic elements.

H. D. Luján et al. (eds.), Giardia © Springer-Verlag/Wien 2011

Despite its much smaller genome (~12 million base pairs) and comparable medical importance, comparative Giardia genomics has not yet reached a similar level, but indications are promising. Giardia intestinalis isolates are currently divided into seven assemblages, a term of uncertain meaning both within and outside the Giardia research community (Table 5.1). The divisions are largely based on the morphologic features and host range (Adam, 2001); few molecular data exist for most assemblages. A handful of housekeeping genes such as triose phosphate isomerase (tpi), small subunit (SSU) ribosomal RNA, glutamate

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Table 5.1 The hexamitidae Taxonomic group

Known hosts

Genome-scale datasets

Assemblage A

Mammals

WB clone 6 ATCC50803

Assemblage B

Mammals

GS ATCC50581

Assemblage C

Dog

Assemblage D

Dog

Assemblage E

Livestock

Assemblage F

Cat

Assemblage G

Rat

Giardiinae G. intestinalis

G. agilis

Amphibians

G. ardeae

Herons

G. microti

Voles, muskrats

G. muris

Rodents

G. psittaci

Psittacine birds

Octomitis intestinalis

Rodents, amphibians

P15

Hexamitinae Hexamita

Free-living

Spironucleus vortens

Fish

Unfinished genome project, JGI

S. barkhanus

Salmon

EST survey, Dalhousie University

Trepomonas

Free-living

dehydrogenase, ribosomal structural proteins, ferredoxin, and MutL homolog 1 are commonly used for genotyping and phylogenetic studies. Complete genome sequence data from a single isolate, the commonly used WBC6 Assemblage A strain, have been available since 2000. However, almost two thirds of the genome-encoded proteins were annotated as “hypothetical” and had no recognizable homology to any known protein in molecular databases. Before 2000, Genbank contained fewer than 3000 sequences from G. intestinalis, most of these from an early genomic sequence survey of WBC6. A related genome, that of the diplomonad Spironucleus vortens, was attempted at the Joint Genome Institute,

using Sanger sequencing. The genome appears to be highly repetitive, and is unfinished; although there are over 30 MB of assembled reads, the data have not been submitted to Genbank. The WBC6 genome took approximately ten years from the start of sequencing to publication (Morrison et al., 2007), although the data were made available as they were generated. In part, the long time period was due to the changing Sanger sequencing technology, but the project also suffered from the absence of genomic data from a closely related organism. Giardia branches deep in the evolutionary tree of life, and other single-celled protists are only distantly related. Arguably, the closest relative with a sequenced genome may be Trichomonas vaginalis. The genome analysis reflected this deep divergence. Genes were called using a bacterial gene finder, Glimmer (Delcher et al., 1999, 2007); introns are difficult to detect because they do not contain canonical splice sites (Nixon et al., 2002); and gene promoters are merely AT-rich regions upstream of the start codon. Genes are present on both strands of DNA and occasionally overlapped. The major findings were that the genome is compact in structure and content, contains few introns or mitochondrial relics, and has simplified machinery for DNA replication, transcription, RNA processing, and most metabolic pathways. Protein kinases comprise the single largest protein class, likely reflecting the organism’s requirement for a complex signal transduction network for coordinating differentiation. Lateral gene transfer from bacterial and archaeal donors has shaped Giardia’s genome, and previously unknown gene families, for example, cysteine-rich structural proteins, were discovered. The genome showed little evidence of heterozygosity. However, most of the genes were of unknown function and remained unverified by experimental or informatics means. This situation changed with the emergence of “second generation” sequencing technologies in 2006, which increased sequencing throughput to the megabase per day level while simultaneously reducing cost per base by orders of magnitude. Suddenly, sequencing a genome of 10–15 MB could be done at a cost of a few thousand dollars, rather than millions. In 2009, Franzen et al. (2009) published the second full genome sequence of the type strain of Assemblage B. This genome was generated using 454 pyrosequenc-

Chap. 5 Genomics of Giardia

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ing, with only minimal Sanger coverage, and has already dramatically expanded our understanding of Giardia genomics and biology. A third genome, that of the Assemblage E (swine) isolate P15, was completed shortly thereafter using the same strategy. Community support for continued Giardia genome sequencing is high; at a special meeting held during the 2009 Third International Giardia and Cryptosporidium Conference, scientists acknowledged the problems of assemblage vs. species definitions and proposed that draft coverage of additional isolates (both assemblages and species) should be done, possibly through the JGI Community Sequencing Program (http://www.jgi.doe.gov/CSP/index. html). Ideally, genomic survey data would be generated in the near future for multiple human isolates and at least one member of each assemblage and each species, plus a high-quality, completed genome for a diplomonad relative such as Spironucleus, Hexamita, or Trepomonas. Comparisons across these taxonomic levels will indicate what degree of sequence conservation might be used to define a true species and will lead to better gene prediction as Giardia core genes are recognized, as well as assemblage vs. isolate specific variations. Identifying homologous genes in different genomic contexts should improve our ability to accurately define start codons and introns, particularly in-frame introns, and permit prediction of control regions. Ideally, gene content can then be correlated with epidemiology, such as host range and virulence. Comparisons of gene synteny are possible, but will depend upon the quality of the assembly and average contig/ scaffold length. Analysis of single nucleotide polymorphisms (SNPs) is possible using individual gene

sequences from many different isolates (genotyping datasets) as well as the genomic data.

5.3 Resources for Giardia Genomics A second major advance for Giardia genomics was the transition of GiardiaDB from the Marine Biological Laboratory web site to what was then ApiDB, the database resource for apicomplexan genomics. Genomic scale datasets are not easily examined in isolation; they must be combined into a common database in order to best compare genomic capabilities. In 2007, the genomic, EST, and SAGE (serial analysis of gene expression) datasets were integrated into the ApiDB database (Aurrecoechea et al., 2009), now EuPathDB (www.eupathdb.org). The GS and P15 genomes were added to this database, as well as several other genomic-scale datasets from WBC6. This resource provides fully integrated databases that can be queried in sophisticated ways, using Boolean terms. The types of queries available include BLAST searches; keyword searches of annotation, gene IDs, and GO terms; sequence motifs; and other protein characteristics. Functional queries can also be constructed based on transcript and protein expression data, e.g. proteomic datasets. Two of the three genomes are also currently available through NCBI’s Genbank, with the expectation that P15 will also be added. Additionally, genomic datasets can be uploaded to the RAST database (Aziz et al., 2008), which specializes in the analysis of metabolic subsystems and permits comparisons between any of the included genomes. We made a three-way comparison of WB, GS, and 12BC14 using RAST,

Table 5.2 RAST subsystems comparative analysis Pathway (KEGG)

Distinct ECs

Giardia A: WBC6

Giardia B: 12BC14

Giardia B: GS

Plasmodium

Carbon fixation

25

10

11

10

10

Citrate cycle (TCA)

22

3

3

3

12

Pentose phosphate

39

10

11

10

9

Propanoate metabolism

49

1

1

1

3

Pyruvate metabolism

65

9

8

8

10

Starch and sucrose metabolism

73

5

4

6

3

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and sample data are shown in Table 5.2. RAST can be used to make function- or sequence-based comparisons between up to four different organisms, including whole genome alignments. Unlike EuPathDB, which is a curated reference, RAST allows an individual user to annotate genomic datasets privately. Gene synteny can be visualized using EuPathDB and is based on Mercator analyses of the three genomes (Dewey, 2007). Another valuable resource is the identification of gene orthologs and paralogs predicted by OrthoMCL (Li et al., 2003; Chen et al., 2006). Pre-complied gene trees that include Giardia WBC6 and other sequenced eukaryotic genomes and Mercator-calculated gene alignments can be generated and downloaded from EuPathDB for evolutionary analyses.

acid identity in protein coding regions and contain very few genes unique to one or the other genome. Although P15 is a non-human isolate, it too contains a very similar gene complement and the same large gene families. The common multigene families are ankyrin-repeat protein 21.1, NEK kinases, VSPs and other high-cysteine proteins, coiled-coil proteins, spindle pole proteins, and endonucleases. Sampling appears to be robust for each of the three isolates, since the components of metabolic pathways are similar (e.g. Table 5.2) and highly conserved groups such as the tRNA synthetases and ribosomal structural proteins are present in expected numbers. However, their antigenic repertoires are very different, which likely explains the differences in epidemiology. Analyses of gene orthologs (Fig. 5.1) confirm the extensive gene conservation among the assemblages. Very few orthologs are present on only one or two of the genomes. Ortholog groups that are missing in one or more genomes are generally members of multigene families, for example variant-specific surface antigens (VSPs), other high-cysteine proteins, protein 21.1, or NEK kinases. Interestingly, there are more gene orthologs shared between WB (Assemblage A, a human isolate) and P15 (Assemblage E, swine isolate) that between A and B exclusively. This is likely to be an artifact of the more fragmented GS assembly. Some genes that are detected in A and E, but appear to be absent in B are not annotated because they are at the end of a contig. For example, beta tubulin is represented by an open reading frame on scaffold

5.4 Comparison of Assemblage A, B, and E Isolates Analyses of genome content are not dependent on closed genomes or even megabase-sized contigs. If sequencing libraries are random, greater than 95% of the genome is sampled at 3–4u coverage. The genome properties of two Assemblage B strains, GS and 12BC14, and one Assemblage E isolate, P15, are shown in Table 5.3. These three genomes were generated using 454 pyrosequencing to approximately 16u coverage. The major findings reported by Franzen et al. indicated that WB and GS share ~78% amino

Table 5.3 Properties of sequenced Giardia genomes Assemblage

A

B

B

E

Isolate

WB

GS

12BC14

P15

Assembly size (MB)

11.21

11.00

10.7

11.52

Number of contigs

306

2931

2678

820

G+C Content

49%

47%

47%

47%

Average contig size (kbp)

36.6

3.8

4.0

14.1

Total protein-coding genes

5901

4470

n.d.

5064

Giardia orthologs present (4074 ortholog groups)

4374

4204

n.d.

4437

Number of unique orthologs

1190

57

n.d.

83

Percent amino acid homology to WB

n/a

74%

75%

90%

Percent homology within assemblage

n/a

98%

98%

n/a

Chap. 5 Genomics of Giardia

Fig. 5.1 Gene orthologs shared among Assemblages A, B, and E. Numbers represent distinct orthologous groups determined by OrthoMCL

ACGJ01000911 that lacks only the first six amino acid residues. Whole genome alignment using RAST confirms the amino acid conservation: for all bidirectional matches between WB and GS, average amino acid identity is 80.6%. Similarity within the B assemblage (GS vs. 12BC14) is 97.8%, with near perfect gene synteny between the relatively short contigs available for these two genomes. The GS contigs are largely syntenic with WB, although some breaks in synteny were reported. This last observation highlights the importance of having a valid reference genome (or genomes) when carrying out comparative genomic analyses. Reads or contigs can be mapped to the reference, but syntenic breaks may be an indication of assembly error in one or the other genome. A relatively new technology, optical mapping (Latreille et al., 2007), was carried out on the WBC6 isolate (unpublished data). Whole chromosomes are immobilized on a slide and digested with rare-cutting restriction enzymes. The locations of restriction sites are visualized as short gaps and their pattern is compared to the sites predicted from assembled contigs. The results, as well as the results of hybridization analysis of chromosome 3 (Upcroft et al., 2009), indicate misassembled

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scaffolds and even a small number of incorrectly assembled contigs (Fig. 5.2). A corrected WB reference genome will ensure valid comparisons of genome organization. It will also facilitate genomic finishing, by delineating location and size of sequence gaps. A striking discovery by Franzen et al. was the very high level of allelic sequence heterozygosity (ASH) in GS, which was completely lacking in WB. 12BC14 exhibits as much as, if not more than, ASH. The extremely low level of allelic heterozygosity observed in the WB genome was initially unexpected, as it had been commonly believed that the organism reproduced asexually. In 2008, Poxleitner et al. reported fusion between cyst nuclei, suggesting that somatic homologous recombination occurs during the cyst stage. Genomic sequence data from P15 indicate a similarly low level of ASH. However, the genomes of GS and 12BC14 have up to 0.5% heterozygous positions. The cause is unknown; it is possible that the homologous recombination is not occurring in this particular assemblage. The practical effect on genomic studies has been that these two genomes were difficult to assemble accurately and contig consensus sequences do not fully represent the extent of allelic variation. The Roche assembler designed for 454 short read datasets often inserted a sequence gap at a heterozygous position, resulting in frame shifts and artifactually short open reading frames. Clearly, more and better tools are still needed for eukaryotic comparative genomics.

5.5 The Future of Giardia Comparative Genomics A high-quality reference genome is vital for continued work in this area, and the optical mapping data should be used to correct the existing WB assembly. Ideally, existing gaps will be identified and closed by directed polymerase chain reaction amplification and sequencing. It seems clear that draft coverage of multiple additional genomes is preferable to closure in general, however. Gene content, orthology, and synteny can all be assessed from comparing draft genomes to each other and to a high quality reference. As well as additional genomes from novel assemblages and species, it would be valuable to complete the S. vortens genome or generate high coverage data

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H. G. Morrison and S. Svärd

Chromosome 3

CH991771 CH991780 CH991782

CH991767

Chromosome 1

CH991785 CH991769

CH991762

Fig. 5.2 Alignment of WBC6 genomic scaffolds to chromosomes by optical mapping. Blue segments represent MluI restriction enzyme fragments observed (chromosomes) or predicted (scaffolds). Vertical lines indicate matches. Pink area in chromosome 1 indicates an overlap between two mapped scaffolds, thus misassembly of the right end of CH997169. Scaffold CH991767 maps to two different chromosomes and is clearly misassembled

from an alternative non-Giardia diplomonad. We as a research community must reach agreement on the best approaches to analyzing new genomes, particularly in the measurement and representation of allelic sequence heterozygosity. Comparative Giardia genomics will continue to thrive in this era of rapid, inexpensive sequencing technologies and dynamic, well-supported database resources.

References Adam RD (2001) Biology of Giardia lamblia. Clin Microbiol Rev 14(3): 447–475

Aurrecoechea C, Brestelli J, et al. (2009) GiardiaDB and TrichDB: integrated genomic resources for the eukaryotic protist pathogens Giardia lamblia and Trichomonas vaginalis. Nucleic Acids Res 37(Database issue): D526– D530 Aziz RK, Bartels D, et al. (2008) The RAST server: rapid annotations using subsystems technology. BMC Genomics 9: 75 Chen F, Mackey AJ, et al. (2006) OrthoMCL-DB: querying a comprehensive multi-species collection of ortholog groups. Nucleic Acids Res 34(Database issue): D363–D368 Delcher AL, Bratke KA, et al. (2007) Identifying bacterial genes and endosymbiont DNA with Glimmer. Bioinformatics 23(6): 673–679 Delcher AL, Harmon D, et al. (1999) Improved microbial gene identification with GLIMMER. Nucleic Acids Res 27(23): 4636–4641

Chap. 5 Genomics of Giardia Delcher AL, Bratke KA, et al. (2007) Identifying bacterial genes and endosymbiont DNA with Glimmer. Bioinformatics 23(6): 673–679 Dewey CN (2007) Aligning multiple whole genomes with Mercator and MAVID. Methods Mol Biol 395: 221–236 Florens L, Washburn MP, et al. (2002) A proteomic view of the Plasmodium falciparum life cycle. Nature 419(6906): 520–526 Franzen O, Jerlstrom-Hultqvist J, et al. (2009) Draft genome sequencing of Giardia intestinalis assemblage B isolate GS: is human giardiasis caused by two different species? PLoS Pathog 5(8): e1000560 Jomaa H, Wiesner J, et al. (1999) Inhibitors of the nonmevalonate pathway of isoprenoid biosynthesis as antimalarial drugs. Science 285(5433): 1573–1576 Latreille P, Norton S, et al. (2007) Optical mapping as a routine tool for bacterial genome sequence finishing. BMC Genomics 8: 321

101 Li L, Stoeckert CJ Jr, et al. (2003) OrthoMCL: identification of ortholog groups for eukaryotic genomes. Genome Res 13(9): 2178–2189 Morrison HG, McArthur AG, et al. (2007) Genomic minimalism in the early diverging intestinal parasite Giardia lamblia. Science 317(5846): 1921–1926 Nixon JE, Wang A, et al. (2002) A spliceosomal intron in Giardia lamblia. Proc Natl Acad Sci USA 99(6): 3701–3705 Poxleitner MK, Dawson SC, et al. (2008) Cell cycle synchrony in Giardia intestinalis cultures achieved by using nocodazole and aphidicolin. Eukaryot Cell 7(4): 569–574 Upcroft JA, Krauer KG, et al. (2009) Sequence map of the 3-Mb Giardia duodenalis assemblage A chromosome. Chromosome Res 17(8): 1001–1014

The Glycoproteins of Giardia John Samuelson and Phillips W. Robbins

Abstract Glycoprotein structures are remarkably simple in Giardia. This protist produces the shortest Asn-linked glycan (N-glycan) yet described: two N-acetyl-glucosamines (GlcNAc2). The oligosaccharyltransferase (OST) that transfers N-glycans to the peptide has a single catalytic subunit in Giardia but contains four to eight subunits in most eukaryotes. Giardia is missing the ER proteins involved in N-glycan-dependent quality conrol (QC) of protein folding and degradation. There is Darwinian selection for the sites of N-glycan in secreted proteins of eukaryotes with N-glycan-dependent QC, but there is no such selection in Giardia and other protists lacking N-glycan-dependent QC. The glycosylphosphatidylinositol (GPI) anchor of Giardia is predicted to be the simplest of any eukaryote. UDP-GlcNAc is the only nucleotide sugar transported from the cytosol to the lumen of the ER. By contrast, Giardia is one of the rare protists that use GlcNAc to modify Ser and Thr residues on nucleocytosolic proteins. WGA affinity dramatically enriches glycoproteins of Giardia, many of which are unique or are encystation specific. In summary, GlcNAc is the major sugar added to Giardia glycoproteins, which are much less complex than those of the host.

6.1 Introduction The focus of this chapter is on the glycoprotein synthesis by Giardia. In particular, we describe here the carbohydrate modifications of secreted and membrane proteins of Giardia, as well as the use of lectin affinity chromatography to better define trophozoitespecific and cyst-specific glycoproteins. A central idea that in part motivated whole genome sequencing of Giardia was that this early branching

H. D. Luján et al. (eds.), Giardia © Springer-Verlag/Wien 2011

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protist is simpler than higher eukaryotes and so might reveal qualities of ancestral eukaryotes (Morrison et al., 2007). While the studies reviewed here suggest that the glycoproteins of Giardia are indeed much simpler than those of higher eukaryotes, this simplicity likely derives from the secondary loss of glycosyltransferases rather than from their primary absence (Samuelson et al., 2005).

6.2 Results 6.2.1 Giardia Produces a Severely Truncated Asn-linked Glycan (N-glycan) Fungi, plants, and animals synthesize N-glycans by means of a polyisoprenoid-linked precursor: dolichol-PP-Glc3Man9GlcNAc2 (Fig. 6.1A) (Sato et al., 1999). Dolichols, which contain 16–19 isoprene units in fungi and metazoans, are produced by cisprenyltransferases (Helenius and Aebi, 2004). Each of the 14 sugars of the N-glycan precursor is transferred by means of a specific glycosyltransferase. An oligosaccharyltransferase (OST) that transfers the dolichol-linked glycan to Asn residues on nascent peptides is composed of a single catalytic subunit and three to seven non-catalytic subunits in most eukaryotes (Kelleher and Gilmore, 2006). While sites of N-linked glycosylation may contain Thr (Asn-XaaThr, where Xaa cannot be Pro) or Ser (Asn-Xaa-Ser), the sites with Thr are preferred by the OST (Breuer et al., 2001; Kornfeld and Kornfeld, 1985). Because Asn is encoded by AAT or AAC, the density of Nlinked glycosylation sites increases with AT content of the genome (see Sect. 6.2.2) (Cui et al., 2009). Giardia cis-prenyltransferases, which resemble those of bacteria rather than those of most other eukaryotes, produce dolichols containing 11 isoprene

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Fig. 6.1 The N-glycan precursor of the human host A contains 14 sugars (Glc3Man9GlcNAc2). By contrast, the N-glycan precursor of Giardia B contains only two sugars (GlcNAc2)

units (also like bacteria) (Grabin´ska et al., 2010). Giardia lacks glycosyltransferases that add mannose and glucose to N-glycan precursors and so produces severely truncated precursors containing two N-acetylglucosamines (GlcNAc2) (Fig. 6.1B) (Samuelson et al., 2005). Plasmodium, which contains the same set of glycosyltransferases as Giardia, produces an N-glycan precursor that is predominantly a single

J. Samuelson and P. W. Robbins

GlcNAc (Bushkin et al., 2010). Giardia N-glycans, which are not modified in the Golgi apparatus, are predominantly GlcNAc2. Because closely related organisms (e.g. apicomplexans, kinetoplastids, or fungi) have different sets of glycosyltransferases, the diversity of N-glycans in extant eukaryotes is best explained by secondary loss (Samuelson et al., 2005; Bushkin et al., 2010). The OST of Giardia and (Leishmania) is composed of a single catalytic subunit rather than eight subunits in metazoans, fungi, and plants (Kelleher and Gilmore, 2006; Nasab et al., 2008). However, the OST of Giardia still shows the same preference for N-glycan sites with Thr, as has been shown for OSTs of higher eukaryotes (Breuer et al., 2001; Ratner et al., 2008).

6.2.2 Giardia is Missing N-glycan-dependent Quality Control of Protein Folding in the ER Lumen N-glycans play important roles in the quality control (QC) of protein folding in the ER lumen and in the ER-associated degradation (ERAD) of proteins by

Fig. 6.2 A Metazoans, fungi, and plants have an N-glycan-dependent quality control system for glycoprotein folding in the lumen of ER that involves a UDP-Glc:glycoprotein glucosyltransferase (UGGT), glucosidases (Gls), calreticulin (CRT) and/or calnexin (CNX). The N-glycan-dependent quality control system for ER-associated degradation (ERAD) (marked in grey) includes a mannosidase (Mns1), a mannosidase-like lectin (EDEM), and a cytosolic peptide:N-glycanase (PNGase). B The N-glycan-dependent QC for both glycoprotein folding and ERAD is absent in Giardia. This figure is redrawn using data from Banerjee et al. (2007)

Chap. 6 The Glycoproteins of Giardia

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Organisms without N-glycan dependent QC

Organisms with N-glycan dependent QC

A

3.5

Expected

4.0

Actual

3.5

Density of N -glycan sites

Density of N -glycan sites

4.0

3.0 2.5 2.0 1.5 1.0 0.5

B

3.0 2.5 2.0 1.5 1.0 0.5

35

40

45

50

55

60

65

70

75

AT content

35

40

45

50

55

60

65

70

75

AT content

Fig. 6.3 Darwinian selection for sites of N-glycans with Thr in secreted proteins of eukaryotes with N-glycan-dependent QC A is shown by the difference between the actual density of N-glycan sites (black) and the predicted density of N-glycan sites based upon amino acid composition (grey). There is no selection for sites of N-glycan in secreted proteins of Giardia and other protists that lack N-glycan-dependent QC B, so the actual density of N-glycan sites (black) and the predicted density of N-glycan sites (grey) overlap. This figure is drawn using the data from Cui et al. (2009)

cytosolic proteasomes (Fig. 6.2A) (Trombetta and Parodi, 2003; Helenius and Aebi, 2004). A UDPGlc:glycoprotein glucosyltransferase (UGGT) glucosylates mannosylated N-glycans of misfolded proteins, which are refolded by calreticulin (CRT) and/or calnexin (CNX) in association with a protein disulfide isomerase. Alternatively, a mannosidase (Mns1) and a mannosidase-like lectin (EDEM) recognize N-glycans of permanently misfolded glycoproteins that are dislocated to the cytosol and degraded (Wang and Hebert, 2003). There is a positive selection for N-glycan sites in the secreted proteins of diverse eukaryotes with N-glycan-dependent QC of protein folding, so the actual density of N-linked glycosylation sites is twice the expected density (Fig. 6.3A) (Cui et al., 2009). In addition, the density of N-glycan site increases with the increasing AT-content. Because the truncated N-glycans of Giardia do not have the mannosylated arm that is glucosylated by the UGGT, Giardia lacks N-glycan-dependent QC of glycoprotein folding (Fig. 6.2B) (Banerjee et al., 2007). Subsequently, there is no positive selection for N-glycan sites in Giardia and other protists (e.g. Plasmodium) that lack N-glycan-dependent

QC of glycoprotein folding (Fig. 6.3B) (Cui et al., 2009). For these organisms, N-glycan site density in secreted proteins is directly predicted by the AT content of the genome. In addition, there appears to be selection against N-glycan sites in nucleus-encoded apicoplast proteins of Toxoplasma (Bushkin et al., 2010).

6.2.3 The Predicted Glycosylphosphatidylinositol (GPI) Anchor of Giardia Contains Just Two Mannose Residues GPI anchors, which are composed of glycans attached to phosphatidyl inositol, are C-terminal modifications of proteins that anchor the proteins to the membrane. The first complete GPI structure and description of its biosynthesis were for the variant surface glycoprotein (VSG) of Trypanosoma brucei (Ferguson et al., 1988; Orlean and Menon, 2007). Glycosyltransferases in the cytosol and in the ER lumen produce the core glycan (Man3–4GlcN). The final step, which involves cleavage of a C-terminal sequence and addition of the GPI anchor via a phosphodiester bond with ethanolamine,

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uses a transamidase composed of multiple subunits (Orlean and Menon, 2007). A 45-kDa surface antigen of Giardia (Gp45) is modified by a GPI anchor (Das et al., 1991). Bioinformatic predictions, using methods similar to those used to predict N-glycans of diverse protists, suggest that Giardia has the fewest predicted Gpi enzymes (six) of any eukaryote examined (Mitra, Robbins, and Samuelson, unpublished data). The Giardia transamidase appears to have a single catalytic subunit, and the predicted GPI anchor contains just two mannose residues.

6.2.4 Giardia has a Single Nucleotide Sugar Transporter (NST) ER and Golgi mannosidases trim human N-glycans to five sugars (Man3GlcNAc2), and a series of Golgi glycosyltransferases produce complex N-glycans containing additonal GlcNAc, galactose, sialic acid, and fucose (Stanley et al., 2008). Human O-linked glycans include those with a single GalNAc, as well as chains of galactose and GlcNAc (polylactosamine) to which blood group antigens may be attached (North et al., 2009). To produce O-glycans and complex N-glycans, NSTs must transport nucleotide-diphosphate-sugars (e.g. UDP-GlcNAc, UDP-Gal, CMP-sialic acid, and GDP-Fuc) from the cytosol to the lumen of the ER or Golgi apparatus (Caffaro and Hirschberg, 2006). The specificity of numerous NSTs of metazoans, fungi, and protists has been determined by isolating the membranes from transformed Saccharomyces. However, Saccharomyces cannot be used to demonstrate UDP-Glc transport, because the yeast has its own endogenous NST(s) for UDP-Glc. Giardia has a single nucleotide sugar transporter for UDP-GlcNAc, the fewest of any eukaryote (Banerjee et al., 2008). For the most part, UDP-Glc appears to be used for the synthesis of glycolipid rather than for the synthesis of O-linked glycans on Ser or Thr residues of glycoproteins. Transfected Giardia was used to characterize the UDP-Glc transporters of Entamoeba and Caenorhabditis (Banerjee et al., 2008; Caffaro et al., 2008). UDP-Glc transporters provide the activated Glc used in N-glycan-dependent QC of glycoprotein folding in the ER lumen (Fig. 6.2A) (Banerjee et al., 2007).

J. Samuelson and P. W. Robbins

6.2.5 Giardia is a Rare Protist that has an O-GlcNAc Transferase (OGT) that Modifies Nucleocytosolic Proteins The OGT is a nucleocytoplasmic glycosyltransferase that catalyzes the addition of a single O-linked GlcNAc to the Ser or Thr of a polypeptide chain (Love and Hanover, 2005; Hart et al., 2007). The metazoan OGT modifies transcription factors, nuclear pore proteins, and kinases. Disturbances in OGT and OGlcNAcase activities have been implicated in human type 2 diabetes and in a Caenorhabditis elegans model of diabetes (Forsythe et al., 2006; Yang et al., 2008). By contrast, OGTs are absent from Saccharomyces and many other fungi. Giardia has an active OGT that transfers O-linked GlcNAc to nucleocytosolic proteins (Banerjee et al., 2009). OGTs are also predicted in Cryptosporidium, Toxoplasma, and Dictyostelium but are absent in other apicomplexans, kinetoplastids, Entamoeba, and Trichomonas.

6.2.6 Use of the Plant Lectin Wheat Germ Agglutinin (WGA) to Enrich Giardia Glycoproteins Secreted and membrane proteins make up a minority of the total proteins of Giardia and other eukaryotes. By contrast, Giardia glycoproteins are dramatically enriched with WGA affinity chromatography that binds to GlcNAc2 present in N-glycans (Ratner et al., 2008). Among the 194 trophozoite glycoproteins identified by mass spectroscopy are lysosomal enzymes, folding-associated proteins, and 42 unique transmembrane proteins with Cys-, Leu-, or Gly-rich repeats. Cyst glycoproteins are enriched in Gly-rich repeat transmembrane (GRREAT) proteins, cyst wall proteins, and unique membrane proteins. By contrast, cysts have fewer Leu-rich repeat proteins, foldingassociated proteins, and unique secreted proteins. Candidate proteins for the synthase that makes the E-1,3-linked GalNAc homopolymer present in the cyst wall were also identified (Gerwig et al., 2002; Karr and Jarroll, 2004). Distinct compositions of the glycoproteins of Giardia trophozoites and cysts are consistent with marked differences in the appearance of trophozoites and cysts labeled with Alexafluor-conjugated

Chap. 6 The Glycoproteins of Giardia

A

107

B

C

Fig. 6.4 WGA is a plant lectin that binds to the very short N-glycans of Giardia. A WGA (red) stains perinuclear regions and small vesicles of permeabilized trophozoites, the surface of which is stained green with Alexafluor. Nuclei are stained with DAPI. B WGA binds in a punctate manner to the surface of a non-permeabilized trophozoite. C In cysts, WGA binds in a perinuclear pattern and densely labels membranes (arrowheads), which are closely apposed to the walls (arrows), which are stained green with anti-CWP1 antibodies. A and B are 3D reconstructions, while C is a cross-section. All figures are derived using data from Ratner et al. (2008)

WGA (Fig. 6.4). WGA predominantly labels ER and small vesicles in the permeabilized trophozoites, while WGA binds in a punctate pattern to the surface of Giardia with less dense labeling of flagellae. By contrast, WGA predominantly labels the membranes closely apposed to the walls of Giardia cysts, which are labeled with anti-CWP1 antibodies. Other encystation-specific proteins (e.g. Cys proteases and Cys-rich proteins resembling VSPs) (Ward et al., 1997; Touz et al., 2002; Davids et al., 2006) are described in Chapter 12. Encystation-specific vesicles and Golgi function during encystation (Luján et al., 1995; Stefanic et al., 2006) are described in Chapter 14.

6.2.7 Use of Multidimensional Protein Identification Technology (MudPIT) to Identify Total Proteins of Giardia Trophozoites Two-dimensional liquid chromatography was used to separate tryptic peptides of Giardia trophozoites, and 1371 proteins of the WB strain (a representative of Assemblage A) were identified and deposited in GiardiaDB (Liu et al., 2002; Morrison et al.,

2007; Aurrecoechea et al., 2009; Ratner, Steffen, and Samuelson, unpublished data). In doing so many hypothetical proteins were converted to the status of unique Giardia proteins. All but five of these 1371 WB proteins are also present in the predicted proteins of Koch’s postulate strain GS (a representative of Assemblage B) (Franzén et al., 2009). These results suggest that the proteomes of the two assemblages are very similar. By contrast, the majority of predicted proteins of WB <100 amino acids are absent from GS, suggesting that they may not be real genes.

6.3 Discussion 6.3.1 Giardia N-glycans are Dramatically Simplified Relative to Those of the Host and Most Other Parasites When we first examined N-glycans of Giardia and other protists a half dozen years ago, it was assumed that N-glycan precursors of all eukaryotes, with the exception of kinetoplastids, are composed of 14 sugars (Helenius and Aebi, 2004; Samuelson et al., 2005). Indeed Trypanosoma cruzi that is missing Glc in its N-glycan precursors was used by Armando Parodi

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and colleagues to discover the role of glucosylation in the N-glycan-dependent QC control of glycoprotein folding in the ER lumen (Trombetta and Parodi, 2003; Helenius and Aebi, 2004). Similarly, it was assumed that all eukaryotes had N-glycan-dependent QC of glycoprotein folding (Trombetta and Parodi, 2003; Helenius and Aebi, 2004; Banerjee et al., 2007). While it had been demonstrated for a handful of glycoproteins (e.g. hemagglutin [HA] of influenza virus) that their folding was dependent on the N-glycan-dependent QC system, the near doubling of the density of N-glycans sites in eukaryotes with N-glycan-dependent QC was not appreciated (Daniels et al., 2003; Cui et al., 2009). Giardia, by the absence of N-glycan-associated glycosylatransferases and N-glycan-dependent QC systems, has taught us much about the diversity of Asn-linked glycosylation in eukaryotes. Further, Giardia N-glycans serve as a model for those of Plasmodium that has the same set of glycosyltransferases as Giardia’s (Bushkin et al., 2010). As shown in Fig. 6.1, the N-glycan precursor and final product (GlcNAc2) of Giardia are much less complex than the N-glycan precursors and final Nglycans (Helenius and Aebi, 2004; Samuelson et al., 2005; Stanley et al., 2008) of humans. Trypanosoma brucei builds its N-glycans on a pair of precursors containing seven and 11 sugars (Man5GlcNAc2 and Man9GlcNAc2), which results in a mixture of unprocessed high mannose N-glycans and complex N-glycans (Manthri et al., 2008). Toxoplasma gondii builds its N-glycans on two precursors, one comes from the protist and contains 10 sugars (Glc3Man5GlcNAc2) (Samuelson et al., 2005; Garenaux et al., 2008; Bushkin et al., 2010). Remarkably, the other Toxoplasma N-glycan precursor contains 14 sugars and derives from the host (Glc3Man9GlcNAc2). Finally, two other lumenal protists, Entamoeba histolytica and Trichomonas vaginalis, each build their N-glycans on precursors with seven sugars (Man5GlcNAc2) and have N-glycan-dependent QC of glycoprotein folding (Samuelson et al., 2005; Banerjee et al., 2007; Magnelli et al., 2008).

6.3.2 Future Research Unresolved questions concerning protein glycosylation in Giardia primarily concern the function of

J. Samuelson and P. W. Robbins

these modifications. What is the use of the very short N-glycans that modify secreted and membrane glycoproteins? How does the addition of O-linked GlcNAc to nucleocytosolic proteins affect signal transduction? Answers to these questions will require the use of new technologies for knocking down Giardia gene expression using anti-sense methods, morpholinos, or dominant negative technologies (Dawson et al., 2007; Prucca et al., 2008; Carpenter and Cande, 2009). Conversely, Giardia may be used to identify heterologous NSTs that cannot be tested in Saccharomyces (Banerjee et al., 2008). In addition, transformed Giardia may be used to characterize early steps in the synthesis of N-glycans and later steps in the synthesis of GPI anchors. In addition, transformed Giardia may be used to produce Plasmodium antigens that have the same N-glycans as the native glycoproteins (Mitra, Templeton, and Samuelson, unpublished data).

Acknowledgments This work was supported in part by NIH grants. We thank members of the Samuelson and Robbins labs, who performed most of the work. In alphabetical order, these include Aparajita Chatterjee, Sulagna Banerjee, Guy Bushkin, Jike Cui, Kariona Grabin´ska, Paula Magnelli, Sangha Mitra, and Daniel Ratner.

References Aurrecoechea C, Brestelli J, Brunk BP, Carlton JM, Dommer J, Fischer S, et al. (2009) GiardiaDB and TrichDB: integrated genomic resources for the eukaryotic protist pathogens Giardia lamblia and Trichomonas vaginalis. Nucleic Acids Res 37: D526–D530 Banerjee S, Vishwanath P, Cui J, Kelleher DJ, Gilmore R, Robbins PW, and Samuelson J (2007) Evolution of quality control of protein-folding in the ER lumen. Proc Natl Acad Sci USA 104: 11676–11681 Banerjee S, Cui J, Robbins PW, and Samuelson J (2008) Use of Giardia, which appears to have a single nucleotide-sugar transporter for UDP-GlcNAc, to identify the UDP-Glc transporter of Entamoeba. Mol Biochem Parasitol 159: 44–53 Banerjee S, Robbins PW, and Samuelson J (2009) Molecular characterization of nucleocytosolic O-GlcNAc transferases of Giardia lamblia and Cryptosporidium parvum. Glycobiology 19: 331–336 Breuer W, Klein RA, Hardt B, Bartoschek A, and Bause E (2001) Oligosaccharyltransferase is highly specific for the

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109 Gerwig GJ, van Kuik JA, Leeflang, BR, Kamerling JP, Vliegenthart, JF, Karr CD, and Jarroll EL (2002) The Giardia intestinalis filamentous cyst wall contains a novel beta (1-3)-N-acetyl-D-galactosamine polymer: a structural and conformational study. Glycobiology 12: 499–505 Grabin´ska KA, Cui J, Chatterjee A, Guan Z, Raetz CRH, Robbins PW, and Samuelson J (2010) Molecular characterization of the cis-prenyltransferase of Giardia. Glycobiology, in press Hart GW, Housley MP, and Slawson C (2007) Cycling of O-linked beta-N-acetylglucosamine on nucleocytoplasmic proteins. Nature 446: 1017–1022 Helenius A and Aebi M (2004) Roles of N-linked glycans in the endoplasmic reticulum. Annu Rev Biochem 73: 1019– 1049 Karr CD and Jarroll EL (2004) Cyst wall synthase: N-acetylgalactosaminyltransferase activity is induced to form the novel N-acetylgalactosamine polysaccharide in the Giardia cyst wall. Microbiology 150: 1237–1243 Kelleher DJ and Gilmore R (2006) An evolving view of the eukaryotic oligosaccharyltransferase. Glycobiology 16: 47R– 62R Kornfeld R and Kornfeld S (1985) Assembly of aparaginelinked oligosaccharides. Annu Rev Biochem 54: 631–664 Liu H, Lin D, and Yates JR 3rd (2002) Multidimensional separations for protein/peptide analysis in the post-genomic era. Biotechniques 32: 898 Love DC and Hanover JA (2005) The hexosamine signaling pathway: deciphering the “O-GlcNAc code”. Sci STKE. 2005: re13 Luján HD, Marotta A, Mowatt MR, Sciaky N, LippincottSchwartz J, and Nash TE (1995) Developmental induction of Golgi structure and function in the primitive eukaryote Giardia lamblia. J Biol Chem 270: 4612–4618 Magnelli P, Cipollo JF, Ratner DM, Cui J, Kelleher D, Gilmore R, Costello CE, Robbins PW, and Samuelson J (2008) Unique Asn-linked oligosaccharides of the human pathogen Entamoeba histolytica. J Biol Chem 283: 18355–18364 Manthri S, Guther ML, Izquierdo L, Acosta-Serrano A, and Ferguson MA (2008) Deletion of the TbALG3 gene demonstrates site-specific N-glycosylation and N-glycan processing in Trypanosoma brucei. Glycobiology 18: 367–383 Morrison HG, McArthur AG, Gillin FD, Aley SB, Adam RD, Olsen GL, et al. (2007) Genomic minimalism in the early diverging intestinal parasite Giardia lamblia. Science 317: 1921–1926 Nasab FP, Schulz BL, Gamarro F, Parodi AJ, and Aebi M (2008) All in one: Leishmania major STT3 proteins substitute for the whole oligosaccharyltransferase complex in Saccharomyces cerevisiae. Mol Biol Cell 19: 3758–3768 North SJ, Hitchen PG, Haslam SM, and Dell A (2009) Mass spectrometry in the analysis of N-linked and O-linked glycans. Curr Opin Struct Biol 19: 498–506 Orlean P and Menon AK (2007) Thematic review series: lipid posttranslational modifications. GPI anchoring of protein in yeast and mammalian cells, or: how we learned to stop worrying and love glycophospholipids. J Lipid Res 48: 993–1011

110 Prucca CG, Slavin I, Quiroga R, Elías EV, Rivero FD, Saura A, Carranza PG, and Luján HD (2008) Antigenic variation in Giardia lamblia is regulated by RNA interference. Nature 456: 750–754 Ratner DM, Cui J, Steffen M, Moore LL, Robbins PW, and Samuelson J (2008) Changes in the N-glycome, glycoproteins with Asn-linked glycans, of Giardia lamblia with differentiation from trophozoites to cysts. Eukaryot Cell 7: 1930–1940 Samuelson J, Banerjee S, Magnelli P, Cui J, Kelleher DJ, Gilmore R, and Robbins PW (2005) The diversity of dolichol-linked precursors to Asn-linked glycans likely results from secondary loss of sets of glycosyltransferases. Proc Natl Acad Sci USA 102: 1548–1553 Sato M, Sato K, Nishikawa S, Hirata A, Kato J, and Nakano A (1999) The yeast RER2 gene, identified by endoplasmic reticulum protein localization mutations, encodes cisprenyltransferase, a key enzyme in dolichol synthesis. Mol Cell Biol 19: 471–483 Stanley P, Schachter H, and Taniguchi N (2008) N-glycans. In: Essentials of glcyobiology (A. Varki, R.D. Cummings, J.D. Esko, H.H. Freeze, P. Stanley, C.R. Bertozzi, G.W. Hart, and M.E. Etzler, eds.), 2nd Edition. New York, Cold Spring Harbor Laboratory Press, pp 101–114

J. Samuelson and P. W. Robbins Stefanic S, Palm D, Svard SG, and Hehl AB (2006) Organelle proteomics reveals cargo maturation mechanisms associated with Golgi-like encystation vesicles in the early-diverged protozoan Giardia lamblia. J Biol Chem 281: 7595–7604 Touz MC, Nores MJ, Slavin I, Carmona C, Conrad JT, Mowatt MR, Nash TE, Coronel CE, and Lujan HD (2002) The activity of a developmentally regulated cysteine proteinase is required for cyst wall formation in the primitive eukaryote Giardia lamblia. J Biol Chem 277: 8474–8481 Trombetta ES and Parodi AJ (2003) Quality control and protein folding in the secretory pathway. Annu Rev Cell Dev Biol 19: 649–676 Wang T and Hebert DN (2003) EDEM an ER quality control receptor. Nat Struct Biol 10: 319–321 Ward W, Alvarado L, Rawlings ND, Engel JC, Franklin C, and McKerrow JH (1997) A primitive enzyme for a primitive cell: the protease required for excystation of Giardia. Cell 89: 437–444 Yang X, Ongusaha PP, Miles PD, Havstad JC, Zhang F, So WV, Kudlow JE, Michell RH, Olefsky JM, Field SJ, and Evans RM (2008) Phosphoinositide signalling links O-GlcNAc transferase to insulin resistance. Nature 451: 964–969

Mass Spectrometric Analysis of Phospholipids and Fatty Acids in Giardia lamblia Mayte Yichoy, Ernesto S. Nakayasu, Atasi De Chatterjee, Stephen B. Aley, Igor C. Almeida and Siddhartha Das

7

Abstract

7.1 Introduction

In addition to plasma membrane, Giardia lamblia contains numerous membrane-enveloped, primitive organelles, which house a variety of metabolic processes. It has been proposed earlier that this intestinal pathogen lacks the ability to synthesize the majority of its own lipids de novo and depends on supplies from outside sources. Therefore, the questions as to how this ancient eukaryote utilizes exogenous lipids and synthesizes membranes and organelles are extremely important. Does this parasite depend predominantly on remodeling pathways, in which exogenous phospholipids undergo fatty acid and headgroup replacement reactions to generate new phospholipids? To answer this, and to better understand the overall pathway, we carried out a complete lipidomic analysis using electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-QTOF-MS). The results suggest that Giardia has the ability to generate new phospholipids de novo, most likely via the remodeling pathways. Among the newly synthesized lipids, phosphatidylglycerol (PG) is the major phospholipid followed by phosphatidylethanolamine (PE). Gas chromatography-mass spectrometry (GC-MS) analyses indicated that Giardia also has the ability to remodel fatty acids by chain elongation and desaturation reactions. Thus, mass spectrometric analyses provided valuable information about lipid biosynthesis by Giardia and opened the possibility of investigating in greater detail the uptake and utilization of exogenous lipids for the synthesis of membranes and organelles.

Giardia trophozoites colonize the luminal surface of the human small intestine, below the bile duct and are exposed to dietary fatty acids and lipids, intestinal immunoglobulins, digestive enzymes, and newly synthesized bile acids (Gillin et al., 1986, 1987; Stevens et al., 1997; Subramanian et al., 2000). Because bile acid concentrations are particularly high in this site, it can be anticipated that bile and biliary lipids must regulate the growth and differentiation of G. lamblia (Das et al., 2002). Hegner and Eskridge (1938) proposed that bile might favor the growth of Giardia species in vivo. Furthermore, it was shown that bile from a number of mammals stimulated the in vitro growth of trophozoites (Farthing et al., 1983, 1985; Keister, 1983). Gillin et al. (1986) reported that the growth of G. lamblia on serum-free medium supplemented with phosphatidylcholine (PC), cholesterol, and a mixture of six bile salts can support the growth of the parasite in culture. Moreover, Das et al. (1988) found that conjugated bile acids, when present above their critical micellar concentrations, protected trophozoites from lysis by free fatty acids that were produced during the digestion of the host’s dietary triglycerides. In addition, the presence of bile salts in the growth medium has been proposed as being involved in transporting exogenous lipids to the trophozoite by forming mixed micelles (Das et al., 1997). Initial studies on the lipid biochemistry of Giardia concluded that this unicellular parasite lacked de novo lipid synthesis capacity and thus must obtain them from its microenvironment. This conclusion was built upon the observation that Giardia trophozoites failed

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to incorporate the radiolabeled lipid precursors – i.e., glucose, threonine, acetate, and glycerol – into cellular phospholipids, fatty acids, and cholesterol when they were added in the growth medium (Jarroll et al., 1981). In culture, the lipid acquisition by trophozoites appears to be supplied by serum lipoproteins (Jarroll et al., 1989). Several reports suggest that in the small intestine lipid requirements may be met by the biliary lipids (i.e., phosphatidylcholine and cholesterol) (Farthing et al., 1985; Gillin et al., 1986). Moreover, the depletion of fatty acids from serum-containing media and the similarity in fatty acid composition between G. lamblia trophozoites and the culture medium (Jarroll et al., 1981; Kaneda and Goutsu, 1988; Mohareb, et al., 1991) suggest that G. lamblia might incorporate exogenous fatty acids directly from the growth medium. Numerous experiments from this and other laboratories have demonstrated that Giardia is able to assemble complex lipids when it is supplied with exogenous phospholipids or fatty acids. Blair and Weller (1987) documented the incorporation of polyunsaturated and saturated fatty acids into different phospholipids. We observed (Das et al., 1991) that exogenously acquired palmitic and myristic acids were metabolically incorporated into GP49, an invariant surface antigen of G. lamblia. Moreover, Ellis et al. (1996) reported that long-chain fatty acids (e.g., palmitic acid, C16:0) undergo chain elongation and/or desaturation by Giardia, suggesting that elongase and desaturase activities are present in this parasite. We have also reported that exogenously supplied radiolabeled free and conjugated fatty acids were incorporated into phosphatidylglycerol (PG) and other cellular phospholipids (Gibson et al., 1999). Similarly, the production of phosphatidylinositol (PI) and glycosylphosphatidylinositol (GPI)-anchored molecules by base (headgroup) exchange reactions was observed (Subramanian et al., 2000). These observations had led us to hypothesize that Giardia is capable of remodeling exogenous phospholipids and fatty acids by deacylation/reacylation reactions through a process also known as Lands cycle, or by phospholipid headgroup exchange, effectively bypassing the de novo synthesis (i.e., CDP-DAG-based) of entire new phospholipid molecules (Das et al., 2001). Although these studies provide some insight into the metabolism of exogenously

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acquired lipids, the fundamental mechanisms of lipid uptake, intracellular transport, and recycling in Giardia trophozoites are not well understood. Since G. lamblia has limited lipid synthesis ability, it is plausible that the majority of lipids are taken up by this parasite from its small intestinal environment. It is well established that unicellular and multicellular organisms can internalize lipid molecules from the cell exterior primarily by three major mechanisms: (1) receptor-mediated endocytosis (Mukherjee et al., 1997), (2) fluid-phase endocytosis (Nichols and Lippincott-Schwartz 2001, or (3) trans-membrane transport proteins (flippases) (Dolis et al., 1997), followed by diffusion as monomers. It has also been established that they then may be facilitated by lipid transfer proteins (van Meer and Op den Kamp, 1982; Pagano and Sleight, 1985). Using commercially available fluorescent lipid analogues, our laboratory has demonstrated earlier (Stevens et al., 1997; Gibson et al., 1999; Hernandez et al., 2007) that G. lamblia trophozoites selectively incorporate and distribute BODIPY- and NBD-conjugated lipid/fatty acid molecules into several locations. Important organelles involved in the intracellular lipid distribution were the nuclear envelopes that were identified as the endoplasmic reticulum of the parasite (Soltys et al., 1996). Furthermore, the internalization of fluorescent lipids in a time-dependent manner suggested the presence of well-evolved lipid transport pathways in this early branching eukaryote. The presence of actin filaments in Giardia was observed by labeling with TRITC-phalloidin (CastilloRomero et al., 2009) and was shown to regulate the endo- and exocytosis of a fluorescent membrane dye, FM 4-64 (Hernandez et al., 2007). Like actin filaments, the existence of a well-organized microtubule network was observed using monoclonal antibodies to α- and β-tubulins. This microtubule network was sensitive to various microtubule depolymerizing and stabilizing agents (Hernandez et al., 2007). We have also shown how the lipid internalization, intracellular trafficking, and recycling of fluorescent lipid probes were dependent upon the presence of anti-actin and anti-microtubule disrupting/stabilizing agents. We found that BODIPY-ceramide and BODIPY-sphingomyelin (BODIPY-SM) crossed lipid bilayers of giardial cell membranes through actin-regulated endocytic processes and that their intracellular move-

Chap. 7 Mass Spectrometric Analysis of Phospholipids and Fatty Acids in G. lamblia

ments are dependent upon intact microtubule filaments but not completely on their dynamic instability. More recently, we have reported that clathrin- but not caveolae-dependent pathways are involved in internalizing and targeting ceramide and SM in this protozoan parasite (Hernandez et al., 2007). In contrast, the uptake of phosphatidylcholine (PC), which is abundant in the growth medium as well as in the small intestine of humans, is not dependent on cytoskeletons, because cytochalasin-D and other microtubule-depolymerizing drugs neither altered nor reduced the localization of BODIPY-PC (unpublished). Additional experiments suggest that flippases or other phospholipid transport proteins containing exposed thiol groups may regulate the uptake of BODIPY-PC (Fig. 7.1). Like PC, the internalization of BODIPYconjugated palmitic acid and NBD-phosphatidylglycerol (NBD-PG) was not affected by anti-actin agents. However, the intracellular movement of palmitic acid and PG was affected by anti-actin as well as anti-microtubule agents. It is possible that these two lipid analogs, once internalized into the cytoplasm, interact with other lipid and protein molecules to form lipid bodies or vesicle-like structures and use the actin/ microtubule cytoskeleton for intracellular targeting (Castillo-Romero et al., 2009). These studies, although preliminary, support the hypothesis that Giardia has evolved well-regulated machinery that allows A

B

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it to import lipids from its environment for energy production and membrane/organelle biosynthesis.

7.2 Mass Spectrometric Analysis of Phospholipids, Sterols, and Fatty Acids Because lipids and fatty acids are key to giardial growth and encystation, and because a large amount of lipids in Giardia are obtained from the growth medium, we used Electrospray Ionization-Quadrupole Time-of-Flight Mass Spectrometry (ESI-QTOF-MS) and Gas Chromatography-Mass Spectrometry (GCMS) to analyze phospholipids and fatty acids, respectively. Individual lipids and fatty acids were measured using internal standards. G. lamblia trophozoites (WBC6) were cultured axenically (Diamond et al., 1978; Keister, 1983), and in vitro encystation was carried out following the method described previously (Gillin et al., 1989). Water-resistant mature cysts were generated by cultivating trophozoites in high-bile medium (Kane et al., 1991). Prior to mass-spectrometric analysis, lipids were extracted from giardial cell pellets (vegetative and encysting trophozoites), cysts, and bovine serum, and bile was extracted in CHCl3:CH3OH:H2O (1:2:0.8, v/v/v) followed by CHCl3:CH3OH (2:1, v/v). The sample was dried unC

Fig. 7.1 N-ethylmaleimide (NEM) inhibits the internalization of BODIPY-PC by Giardia. Attached trophozoites were labeled with BODIPY-PC (200 μM) for one hour at 37 °C in the presence and absence of 50-μM and 200-μM NEM. Control (panel A) shows the characteristic localization of BODIPY-PC in the plasma membrane. Treatment (panel B) with 50-μM NEM changes the localization pattern of BODIPY-PC from the plasma membranes to cytoplasm. Treatment (panel C) with NEM (200-μM) causes the internalization of more BODIPY-PC to localize throughout the cytoplasm as small vesicle-like structures. N nucleus; F flagella; arrow, plasma membrane; arrowheads small vesicle-like structures. Bar: 5 μM

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der a highly pure nitrogen stream after the last extraction in each step (Almeida et al., 2000). The extracted lipids were fractionated in a silica gel column and eluted with CHCl3, acetone, and CH3OH as described previously (Pernet et al., 2006), and MS spectra for fractionated lipids were acquired in an ESI-QTOFMS (Micromass QTOF1, Waters). Samples were analyzed in both positive- and negative-ion modes, the full-scan spectra were collected in the 200–2000 m/z range, and MS/MS spectra were automatically collected for each parent ion with abundance higher than 20 counts using ramp-collision energy (20–65 eV) according to the mass range. For quantitative analysis, samples were normalized to 5000 cells/μl and spiked with an internal standard at a final concentration of 2.5 μM C11:0/C11:0-PC for positive-ion mode, or 5 μM C12:0/C12:0-PE for negative-ion mode. Peak height of the standards was normalized to 30% of the total peak height (for detailed procedures and methodologies, please see Yichoy et al., 2009). For the analysis of fatty acids by GC-MS, total lipids were isolated as described above, and alkaline hydrolysis and esterification of total fatty acids were carried out following a modified method from Maldonado et al. (2006). The analysis of the sterol fraction by GC-MS was carried out as described by Fridberg et al. (2008).

7.2.1 Results of Phospholipid Analyses Analyses in both positive- and negative-ion modes revealed that PC, SM, PE, PG, and PI were the major phospholipids present in Giardia cells, and that their relative concentrations did not alter dramatically in encysting cells and mature water-resistant cysts (Yichoy et al., 2009). Table 7.1 shows that multiple species of SM and PC are present in this organism. Giardial PCs were composed mostly of palmitic (C16:0), palmitoleic (C16:1), stearic (C18:0), oleic (C18:1), and linoleic (C18:2) acids. However, it has not been determined yet whether these are cis or trans isomers. A few lipid species had the highest peak heights, which were present in both vegetative and encysting trophozoites, as well as in cysts – i.e., C16:0/d18:1-SM, C16:0/C18:1-PC, C18:1/C18:2-PC, C18:1/C18:1-PC, and C18:0/C18:1-PC. In particular,

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C18:1/C18:1-PC appeared to be the most abundant of all these. Figure 7.2 (panels A and B) shows the fragmentation of the major PC (C18:1/C18:1-PC) and SM (C16:0/18:1-SM), respectively. Negative-ionmode spectra revealed the presence of 17 different species of PGs in Giardia not only in vegetative trophozoites but also in encysting cells and cysts. Among them, C18:0/C16:0-PG and C18:1/C16:0-PG are predominant (Table 7.1 and Fig. 7.2, panel C). Figure 7.1C shows fragmentation of the parent-ion species at m/z 749.7, tentatively assigned as C18:0/ C16:0-diacyl-PG. The PGs were detected by identifying the daughter ion at m/z 675.5 ([M – glycerol – H]–), which is derived from the neutral loss of the glycerol moiety linked to phosphate at the headgroup. We also observed a fragment-ion at m/z 689.8 that most likely resulted from the internal fragmentation of the double-bond formed between C-2 and C-3 by dehydration of the glycerol backbone at the headgroup. Further fragmentation analysis of m/z 749.7 confirmed the presence of C18:0 (m/z 283.3) and C16:0 (m/z 255.3) at sn-1 and sn-2 positions, respectively. The positions of the sn-1 and sn-2 fatty acids were determined by their relative peak heights as reviewed by Pulfer and Murphy (2003). Other fragments corresponding to the neutral loss of these fatty acids were also observed at m/z 465.4 ([M – C18:0 – H]–), 483.4 ([M – C18:0 + H2O – H]–), 493.5 ([M – C16:0 – H]–), and 511.5 ([M – C16:0 + H2O – H]–). The glycerol headgroup was confirmed by the formation of daughter ions at m/z 391.3 and 419.3, which represent the loss of the sn-1 and sn-2 fatty acids, respectively, plus the glycerol moiety. A similar pattern of fragmentation was observed for other PG species identified in this study (Table 7.1). Other phospholipids identified in negative-ion modes were six species of PEs and PCs, and two species of PIs, respectively. The fatty acids covalently linked to these phospholipids were similar to those found in positive-ion mode, with C16:0, C16:1, C18:0, and C18:1 fatty acids being the predominant ones. Results (Table 7.2) show that serum and bile (the major source of lipids of the growth medium) are rich in PC, lyso-PC, and diacylglycerol (DAG) and other phospholipids were not found within the limits of detection, suggesting that SM, PE, PG, and PI could be newly generated remodeled phospholipids in Giardia.

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Table 7.1 Lipid analysis and composition of the major phospholipids from differentiating Giardia lamblia*, a–c PL

Ion species

Proposed structure sn-1/sn-2b

Relative abundancec

502.4

M + Li

lyso-C16:0

+

526.4

M + Li

lyso-C18:2

+

528.4

M + Li

lyso-C18:1

+

600.5

M + Li

C11:0/C11:0 (IS)

N/A

740.8

M + Li

C16:0/C16:0

+

752.7

M + Li

C16:0/C17:1 and/or C18:1/C15:0

+

754.8

M + Li

C16:0/C17:1

+

764.8

M + Li

C16:1/C18:1

+

766.8

M + Li

C16:0/C18:1

++

768.8

M + Li

C16:0/C18:0

+

780.6

M + Li

C17:0 /C18:1

+

788.6

M + Li

C18:2/C18:2

+

790.7

M + Li

C18:1/C18:2

+

792.7

M + Li

C18:1/C18:1

++

794.6

M + Li

C18:0/C18:1

++++

806.8

M + Na

C18:1/C18:2

+

808.8

M + Na

C18:1/C18:1

++

814.6

M + Li

C20:4/C18:1

++

816.6

M + Li

C20:4/C18:0

+

m/z

Positive-ion mode PC

SM

709.9

M + Li

C16:0/d18:1

++

725.7

M + Na

C16:0/d18:1

+

737.8

M + Li

C18:0/d18:1

+

804.6

M + formate

C18:1/C16:0

+

806.6

M + formate

C18:0/C16:0

+

820.6

M + Cl

C18:1/C18:1

+

828.6

M + formate

C18:2/C18:1

+

830.6

M + formate

C18:1/C18:1

+

832.6

M–H

C18:0/C18:1

+

578.4

M–H

C12:0/C12:0 (IS)

N/A

Negative-ion mode PC

PE

636.4

M + NaCl – H

C12:0/C12:0 (IS)

N/A

646.4

M + Na + formate – H

C12:0/C12:0 (IS)

N/A

714.5

M–H

C16:0/C18:2 and/or C16:1/C18:1

++

716.5

M–H

C18:1/C16:0

+++

834.6

M–H

C22:6/C22:6 (IS)

N/A (Continued)

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Table 7.1 (Continued) PL

m/z

Ion species

Proposed structure sn-1/sn-2b

Relative abundancec

PG

609.4

M–H

C12:0/C12:0 (IS)

N/A

707.5

M–H

C16:0/15:0 and/or C14:0/C17:0

+

719.6

M–H

C16:0/C16:1 and/or C18:1/C14:0

+

721.5

M–H

C16:0/16:0 and/or C18:0/C14:0

+++

733.5

M–H

C16:0/C17:1 and/or C18:1/C15:0

+

735.5

M–H

C16:0/C17:0 and/or C15:0/C18:0

+

745.5

M–H

C18:2/C16:0 and/or C16:1/C18:1

+

747.5

M–H

C18:1/C16:0

++++

749.5

M–H

C18:0/C16:0

++++

761.5

M–H

C18:1/C17:0

+

763.6

M–H

C18:0/C17:0 and/or C19:0/C16:0

+

771.5

M–H

C18:1/C18:2

+

773.5

M–H

C18:1/C18:1

+

775.5

M–H

C18:0/C18:1

+

777.6

M–H

C16:0/20:0 and/or C18:0/C18:0

+

789.5

M + Na + formate – H

C16:0/C16:0, C18:0/C14:0, and/or C15:0/C17:0

+

817.5

M + Na + formate – H

C18:0/C16:0

+

809.5

M–H

C16:0/C16:0

+

835.5

M–H

C18:1/C16:0

+

PI

*

Adapted from Yichoy et al. (2009). Phospholipid (PL) species were identified by MS-MS analysis in positive- and negative-ion modes. b Relative abundance is designated by the peak height: ++++, up to 100%; +++, up to 75%; ++, up to 50%; +, 10% or less. IS internal standard. c Relative abundances for each sample were similar and therefore only the peak heights for trophozoites are shown. N/A not applicable. a

7.2.2 Results of Fatty-Acid Analyses by GC-MS The fatty-acid content in vegetative trophozoites, 0-h, 6-h, 12-h, 24-h, 48-h, water-resistant cysts, bovine serum, and bile is shown in Table 7.3 The results indicated that C16:0, C18:0, and C18:1 are the major fatty acids in trophozoites, encysting cells, and cysts. These three fatty acids are also present in bile and serum, suggesting that they are acquired from the growth medium. However, less-common acyl groups such as C10:0, C12:0, C14:0, C23:0, and C24:0 were also present and remained unchanged in vegetative trophozoites, encysting cells (0–48 h), and water-resistant cysts (Table 7.3). A significant amount of C18:2 is present in bovine serum but not that much is found in Giardia. It is possible that C18:2 fatty acids are used by Giardia to

synthesize longer-chain and additional double-bond containing fatty acids by elongase and desaturase activities. The search of the Giardia genome database (http://giardiadb.org/giardiadb/; Morrison et al., 2007) yielded the presence of fatty acid elongase 1 gene (accession no. XM_00170849.1, E-value 4e-81), suggesting that fatty acid elongation machinery may be present in this pathogen, and the presence of desaturase enzyme was earlier demonstrated by Ellis et al. (1996). As far as sterols are concerned, our GC-MS data revealed that the sterol fractions purified from trophozoites, encysting cells and cysts contain only cholesterol (not shown), and failed to detect any ergosterol (Ellis et al., 1996), suggesting that cholesterol is the main sterol present in Giardia and that is likely be obtained from the growth medium.

Chap. 7 Mass Spectrometric Analysis of Phospholipids and Fatty Acids in G. lamblia

100

C18:1/C18:1-PC ESI+MSMS@ 792.7

2.62e4

Relative abundance (%)

A

117

0 50

100

m/z

100

150

200

250

300

350

400

450

500

550

600

650

700 750

C16:0/d18:1-SM ESI+MSMS@ 709.9

1.21e4

Relative abundance (%)

B

800

0 50

150

200

250

300

350

400

450

500

550

600

650

700

800

7.06e3

C16:0/C18:0-PG ESI-MS/MS@ 749.8

C

750

Relative abundance (%)

100

m/z

100

0 50

m/z

100

150

200

250

300

350

400

450

500

550

600

650

700

750

800

Fig. 7.2 (Continued)

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C18:1/C16:0-PE ESI+MSMS@ 716.7

D

2.14e4

Relative abundance (%)

100

0 50

E

150

200

250

300

350

400

450 500

550

C16:0/C16:0-PI ESI+MSMS@ 809.7

600

650

700

750

800

1.32e3

Relative abundance (%)

100

m/z

100

m/z 0 50 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000

Fig. 7.2 Analyses of phospholipids by ESI-QTOF-MS. The detailed methodologies of analysis have been described in our recent publication (Yichoy et al., 2009). (A and B) positive-ion mode MS-MS spectra of C18:1/C18:1-PC (m/z 792.7) and C16:0/d16:1SM at m/z 709.9, respectively. (C) MS-MS spectrum of C18:0/C16:0-PG parent-ion at m/z 749.5, ionized in negative-ion mode. “×16” indicates that the portion of that spectrum was magnified sixteen times to make the peaks more visible. The number at the top right corner of each spectrum indicates signal strength, measured as ion intensity at 100% relative abundance. m/z, mass-to-charge ratio. (D) Negative-ion mode MS-MS spectra of C18:1/C16:0-PE (m/z 716.7). (E) C16:0/C16:0-PI (m/z 809.7), respectively. The number at the top right corner of each spectrum indicates signal strength, measured as ion intensity at 100% relative abundance. m/z, mass-to-charge ratio (Yichoy et al., 2009) (Reprinted with permission)

7.3 Lipid Metabolic Genes Present in the Database of WBC6 Isolate Table 7.4 lists the genes of lipid synthesis and metabolic pathways that were annotated in the database of WBC6

isolate (http://giardiadb.org/giardiadb/; Morrison et al., 2007). Nine genes of phospholipid synthesis, transport, and metabolism were identified, including the putative homologues of PC synthase (gPCS), phosphatidylinositol synthase (gPIS), PI transfer protein (alpha isoforms,

Chap. 7 Mass Spectrometric Analysis of Phospholipids and Fatty Acids in G. lamblia Table 7.2 Positive-ion mode MS-MS analysis of phospholipids from bile and serum* m/z

Ion species

Proposed structures sn-1/sn-2

526.4

M + Li

lyso-C18:2-PC

528.4

M + Li

lyso-C18:1-PC

530.5

M + Li

lyso-C18:0-PC

552.4

M + Li

lyso-C20:3-PC

653.7

M + Li

C18:0/C20:3-DAG

764.7

M + Li

C16:0/C18:2-PC

766.7

M + Li

C16:0/C18:1-PC

790.7

M + Li

C18:1/C18:2-PC and/or C18:0/C18:3-PC

792.7

M + Li

18:0/18:2-PC

794.7

M + Li

18:0/18:0-PC

816.7

M + Li

18:0/20:4-PC

*

Adapted from Yichoy et al. (2009).

gPITPα), phosphatidylglycerolphosphate synthase (gPGPS), phospholipid-transport (gPLT) ATPase 1A and 2B, phosphatidylserine synthase (gPSS), phosphatidylserine decarboxylase (gPSD), and headgroup (choline/ethanolamine) kinases. The presence of phospholipid-transport ATPase IA and IIB (also known as phospholipid flippases or gFLIP) suggests that Giardia has evolved an efficient mechanism to internalize phospholipid molecules, especially PC from its smallintestinal environment. Among various phospholipid genes, the presence of gPIS, gPGPS, gPSS, and gPSD is also interesting and may signify the capability of Giardia to synthesize limited phospholipids de novo. Earlier, we have demonstrated that genes for gPGPS and gPSD are expressed in Giardia and remain unchanged throughout the life-cycle of the parasite, suggesting that they may function as a house-keeping gene (Yichoy et al., 2009). The Giardia Genome Project predicts the presence of nine fatty-acid (FA) transport, synthesis, and metabolic genes (Table 7.4). Three 1-acyl-sn-glycerol3-phosphate acyltransferases (SLCs) – i.e., gSLC2, gSLC3, and gSLC4 – were annotated in the database, suggesting that Giardia might use these gene products to import fatty acids from its environment. Other FA genes annotated are putative lysophosphatidic

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acid acyltransferase (gLAAT), elongase 1 (gELO) several long-chain fatty-acid (LCFA)-CoA ligases (gLCFA-CoA ligases) (i.e., LCFA-CoA ligase 4 [gLCFLA4] and three different forms of LCFA-CoA ligase 5 [gLCFLA5]), and acetyl-CoA/pyruvate carboxylase (gACPC). The presence of these fatty-acid genes further indicates that very basic and essential fatty-acid metabolism can be carried out by this parasite, such as transferring fatty acids across the membranes, forming reactive fatty-acid species (fatty acyl-CoA), and acylating lysophosphatidic acid (LPA) to form phosphatidic acid (PA), and elongating and ligating fatty-acid chains (Table 7.4). Giardia contains two isoforms of secreted and cytoplasmic phospholipase B enzymes (gplb) that are responsible for removing sn1 and sn2 fatty acids from a phospholipid at the same time (Morgan et al., 2004). Only five Sphingolipid (SL) metabolic genes were annotated in the Giardia database, including the genes that encode SPT 1 and 2, GlcT-1 (gglct1), and two separate ASMases enzymes (ASMase B [gasmase b] and ASMase 3b [gasmase 3b]). It has been reported earlier that all of these five genes are expressed and regulated differentially in two different stages of the life cycle of Giardia, suggesting that SL pathways could be involved in modulating the growth and differentiation of this waterborne pathogen (Hernandez et al., 2008). Various lipid kinase genes were also annotated (Table 7.4). These include the genes for putative target of rapamycin (gTOR); PI-3, -4, -5-trisphosphate; 3-phosphatase (gPIPase); inositol-1, -4, -5-trisphosphate; 5-phosphatase (gITPase); inositol 5-phosphatase; PI-3-kinase class 3 (gPIK); alpha polypeptide of PI-3-kinase catalytic subunit (gPI3K); three isoforms of PI-4-phosphate 5-kinase (gPIPase); PI-4-kinase (gPI4K); and PI-glycan biosynthesis, class O protein (gPIG). The molecular and bioinformatic analyses revealed that giardial TOR is an analogue of the FRAP/TOR of eukaryotes expressed in dividing parasites and may not be inhibited by rapamycin (Morrison et al., 2002). The bioinformatic analysis of three giardial PIKs genes (gPIKs) – two gIPKs (gPI3K-1 and gPI3K-2) and one gPI4K – was also studied (Cox et al., 2006; Hernandez et al., 2007). The identified genes contain catalytic (p110) but not regulatory (p85) subunits. Transcriptional analysis demonstrated that the genes encoding gPIKs are expressed in Giardia and modulated during encystation.

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Table 7.3 Fatty acid analysis by GC-MS* Retention time (min)

Fatty acida

Relative peak area (%)b

Vegetative trophozoites

0-h 6-h encysting encysting

12-h encysting

24-h encysting

48-h In vitro Bovine Bovine encysting cysts serum bile

13.3

C10:0

0.0

0.0

0.0

2.1

0.0

0.0

0.0

0.0

0.0

18.0

C12:0

1.1

1.9

0.0

2.7

0.0

0.7

0.4

1.7

3.2

22.2

C14:0

3.6

3.4

2.7

6.1

0.3

0.8

0.3

2.8

3.2

24.2

C15:0

1.6

2.0

1.3

1.3

0.4

0.6

0.3

2.3

Trace

26.3

C16:0

33.5

25.7

32.2

33.3

51.7

38.2

47.7

27.4

30.2

28.6

C17:0

2.6

3.6

2.7

2.0

1.1

0.9

1.5

2.7

2.1

31.1

C18:0

15.9

22.3

39.1

23.4

19.1

45.0

27.6

30.9

22.2

32.2

C18:1

22.7

12.2

10.8

16.1

23.7

9.2

18.2

7.7

31.4

34.4

C18:2

1.3

1.5

2.0

1.8

2.7

2.3

1.3

24.5

5.7

36.2

C20:0

3.5

6.6

2.7

2.7

0.9

1.3

2.1

Trace

2.0

37.4

C20:1

0.9

1.4

0.0

0.0

0.0

0.1

0.1

0.0

0.0

38.8

C21:0

1.9

2.7

1.1

1.1

0.0

0.0

0.0

0.0

0.0

41.5

C22:0

3.9

5.6

2.6

2.8

0.1

0.5

0.3

0.0

0.0

44.0

C23:0

2.3

6.2

1.1

1.7

0.0

0.2

0.1

0.0

0.0

46.7

C24:0

4.4

4.9

1.7

2.9

0.0

0.2

0.1

0.0

0.0

48.1

C24:1

0.8

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

Total

100

100

100

100

100

100

100

100

100

*

Adapted from (Yichoy et al., 2009). Fatty acids present in trophozoites, encysting cells, in vitro-derived cysts, and bovine bile and serum. b Relative abundance is designated by peak area and is shown as percentage of the total fatty acid content for each sample. a

In addition, two PI3K inhibitors, wortmannin, and LY 294002 inhibited the replication of trophozoites in culture, supporting the notion that PIKs’ activities could be linked to the growth and encystation of Giardia (Cox et al., 2006; Hernandez et al., 2007).

7.4 The Proposed Pathway To elucidate how Giardia operates its lipid and fatty acid metabolic pathways, we used the information from our lipidomic analysis (Yichoy et al., 2009) and

combined with the data previously obtained from the biochemical experiments (for review, see Das et al., 2002) and genome sequencing (Morrison et al., 2007). This hypothetical model (Fig. 7.3) predicts that Giardia obtains PC, one of its major membrane lipids, from the growth medium and uses it to synthesize PG, PE, PS, and PI accordingly, as depicted in metabolic compartment 1. Diaglyceride (DAG) and FAs may also be obtained from the culture medium and undergo conversions and modifications as depicted in compartments 2 and 3. Giardia synthesizes phospholipases A1 and A2 (PLA1 and PLA2) and it is possible

Chap. 7 Mass Spectrometric Analysis of Phospholipids and Fatty Acids in G. lamblia

121

Table 7.4 Open reading frames for lipid metabolic genes in Giardia lamblia, clone WBC6 Classification

Gene annotation

Phospholipid

Choline/ethanolamine kinase PC synthase PGP synthase/CDP-DAG-glycerol-3-phosphate-3-phosphatidyltransferase Phospholipid-transporting ATPase IA, putative Phospholipid-transporting ATPase IIB, putative PI synthase/CDP-DAG-inositol 3-phosphatidyltransferase PI transfer protein alpha isoform PS decarboxylase PS synthase

Fatty acid

1-acyl-sn-glycerol-3-phosphate acyl transferase (AGPAT) 4 AGAPT3 AGAPT2 Acetyl-CoA carboxylase/pyruvate carboxylase fusion protein, putative Fatty acid elongase 1 Long chain fatty acid CoA ligase 4 Long chain fatty acid CoA ligase 5 Long chain fatty acid CoA ligase, putative Lysophosphatidic acid acyltransferase, putative

Sterol

Lecithin-cholesterol acyl transferase, putative

Neutral lipid

CDP-diacylglycerol-inositol 3-phosphatidyltransferase Phosphatidate cytidylyltransferase Phospholipase B

Sphingolipid

Glycosyltransferase-1 Serine palmitoyltransferase-1 Serine palmitoyltransferase-2 Sphingomyelinase 3b Sphingomyelinase B Sphingosine N-acyltransferase

Signaling

Inositol 5-phosphatase 4 Phosphatidylinositol-3,4,5-trisphosphate 3-phosphatase Phosphatidylinositol-4-phosphate 5-kinase, putative Phosphoinositide-3-kinase, class 3 Target of rapamycin (TOR)/Phosphoinositide-3-kinase, catalytic, alpha polypeptide Type II inositol-1,4,5-trisphosphate 5-phosphatase precursor

that these phospholipases facilitate the deacylation/ reacylation of phospholipids (Vargas-Villarreal et al., 2007). The logical arguments of the proposed pathways are discussed below.

7.4.1 Compartment 1 Table 7.2 of our lipidomic data shows that the mixture of bovine serum and bile, which are the major

122

M. Yichoy et al.

Fig. 7.3 Proposed model of lipid and fatty acid metabolism by Giardia. As we found in our lipidomic analyses (Table 7.2), PC and lyso-PC are the major phospholipids in the serum and bile, and Giardia most likely imports these two lipids with the help of phospholipid transport ATPases or flipasses (gPLT/gFLIP), as shown in (A). We propose that PC serves as a major precursor of PS, PE, PG, and PI syntheses, depicted in compartment 1. Because PS and PE syntheses genes are present in Giardia and because gpsd is expressed (Yichoy et al., 2009) during the growth and encystation, it can be argued that this parasite has the ability to convert PC to PS and PE spontaneously. PG can be synthesized from PC via a remodeling pathway (proposed) or directly from CDP-DAG with the help of gPGPS (encoded by gpgps). PI in Giardia, as proposed, can be synthesized from PG through a PG/PI remodeling reaction. PI can also be synthesized from CDP-DAG (compartment 2) by gPIS. The identifications and characterizations of giardial PIKs (Cox, van der Giezen et al. 2006; Hernandez, Zamora et al. 2007) further suggest that signal transducing phosphoinositides (PIP, PIP2, PIP3, etc.) could be synthesized from PIs. In addition to PC and lyso-PC, serum and bile also contain DAG, palmitic acid (Palm A), and other FAs, and may be taken up by the parasite [(B) and (C)] via a career-mediated process (Gibson et al., 1999), which then could be converted into various products shown in compartments 2 and 3. The genes, enzymes and products of many of these reactions have already been identified and characterized (Ellis et al., 1996; Cox et al., 2006; Hernandez et al., 2007; Morrison et al., 2007; Yichoy et al., 2009)

sources of lipids for Giardia, contains four and seven different classes of lyso-PC and PC, respectively. Therefore, it can be presumed that Giardia has evolved a well-regulated machinery to transport both lyso-PC and PC across the membranes. As depicted in step 1 (Fig. 7.3), PC and lyso-PC can be internalized by gPLT/gFLIP-mediated facilitated diffusion and converted to various downstream phospholipids. Our lipidomic analysis suggest that PE is present in Giardia but not in the growth me-

dium (Table 7.1, Fig. 7.2). Therefore, it is possible that an exogenous PC can be transformed to PS by gPSS (encoded by pss gene). In higher eukaryotes, two different PSS are synthesized, PSS1 and PSS2. PSS1 catalyzes PS production from PC and PSS2 facilitates PS synthesis from PE (Kent, 1995). It is possible that Giardia uses its gPSS enzyme to carry out PS synthesis from both PC and PE. The genome database (Table 7.4) identified gPSD, which shows the ability of this organism to synthesize PE from

Chap. 7 Mass Spectrometric Analysis of Phospholipids and Fatty Acids in G. lamblia

PS by headgroup remodeling reaction. Like PE, PG is also a newly generated phospholipid because it is not present in the medium (Tables 7.1 and 7.2, Yichoy et al., 2009) and may be synthesized from PC via a remodeling reaction. It is likely that gPGPS catalyzes PG formation from CDP-DAG (compartment 2) as shown in Fig. 7.3. In the future, it will be interesting to test if radioactive CDP-DAG is converted to PG by giardial extracts. We reported earlier that the gene (gpgps) that is likely to encode gPGPS is expressed by the parasite in both non-encysting and encysting stages (Yichoy et al., 2009). As far as PI synthesis is concerned, it may originate from PG and/or from CDP-DAG. Various phosphoinositides – i.e., PIP, PIP2, and PIP3 – may be synthesized from PIs by the action of gPIKs to generate downstream signals (Cox et al., 2006; Hernandez et al., 2007).

7.4.2 Compartments 2 and 3 As depicted in Table 7.2, DAG is present in the medium and thus can also be internalized by the parasite via transport proteins. As shown in Fig. 7.3, once internalized, DAG can be converted to triacylglycerol (TAG) by gSLCs. It is also likely that DAG is activated by cytidine diphosphate (CDP) to produce CDP-DAG by the giardial homologue of the diacylglycerol acyltransferase pathway (yet to be identified). Newly synthesized DAG could serve as a precursor of PI synthesis (compartment 1) catalyzed by gPIS (Table 7.4). Like DAG, Giardia also takes up palmitic acid (PalmA) and other fatty acids (FAs) via facilitated diffusion (Gibson et al., 1999). In the cytoplasm, FA or Palm A undergoes elongation and desaturation reactions. The presence of a giardial desaturase enzyme was reported earlier by Ellis et al. (1996), and the gene (gelo) that is likely to encode elongase (gELO) was annotated in the database (Table 7.4). As reported by us (Das et al., 2001; Yichoy et al., 2009), Giardia uses its modified (elongated and desaturated) fatty acids to synthesize phospholipids conjugated with various types of fatty acids as depicted in Table 7.1. For example, 17 different classes of PGs, 19 various types of PCs, and 6 separate classes of PEs were identified by lipidomic analysis (Tables 7.1 and 7.2).

123

7.5 Conclusion and Future Direction Although hypothetical, the proposed model (Fig. 7.3) shows how Giardia scavenges lipids and fatty acids from its environment and utilizes them to generate new lipid molecules that are suitable for carrying out various cellular functions. It appears that PC is the major phospholipid recruited from the culture medium, and most likely to be used as a precursor to generate other phospholipids. It is not clear why Giardia produces PG and how PG is synthesized. PG is essentially a bacterial lipid that localizes in the plasma membranes. In our earlier studies (Gibson et al., 1999), we have shown that fluorescently labeled PG is localized in the ER membranes and in the cytoplasm of Giardia, and therefore its actual function is, at this point, perplexing. Thus, it would be interesting to investigate whether Giardia expresses a special enzyme(s) that is responsible for the synthesis of PG from PC by headgroup remodeling reaction. Identifying this enzyme will not only be a new discovery per se, but knowing its possible function in the giardial life-cycle will also be extremely useful for designing anti-giardial therapies. Our lipidomic analysis also suggests that PE is a newly synthesized molecule and that the genes for gPSD and gPSS homologues are present in the database, suggesting that PE/PS interconversions are possible. With regard to PI, it can be synthesized from the CDP-DAG by gPIS and may serve as the precursor of various phosphoinositides for signaling purposes. In addition to phospholipids, the characterization of giardial flippases and fatty acid elongase/desaturase will also be interesting from the metabolic as well as biological aspects of this ubiquitous parasite that infects millions of people each year, worldwide.

Acknowledgements The authors were supported by the grants S06GM008012 (SD), R01AI070655 (ICA), and S06GM 008012 (ICA) from the National Institutes of Health. MY and ESN were supported in part by the Dodson Dissertation Fellowship and the Georges A. Krutilek Memorial Scholarship from UT-El Paso, respectively. Mass spectrometric and confocal microscopic analyses were carried out in Biomolecule Analysis and Analytical Cytology Core Facilities at

124

BBRC/UTEP supported by the 5G12RR008124 grant from NIH/NCRR/RCMI. We thank Tavis L. Mendez for helping us with the art-work.

References Almeida IC, Camargo MM, et al. (2000) Highly purified glycosylphosphatidylinositols from Trypanosoma cruzi are potent proinflammatory agents. EMBO J 19(7): 1476–1485 Blair RJ and Weller PF (1987) Uptake and esterification of arachidonic acid by trophozoites of Giardia lamblia. Mol Biochem Parasitol 25(1): 11–18 Castillo-Romero A, Leon-Avila G, et al. (2009) Participation of actin on Giardia lamblia growth and encystation. PLoS One 4(9): e7156 Cox SS, van der Giezen M, et al. (2006) Evidence from bioinformatics, expression and inhibition studies of phosphoinositide-3 kinase signalling in Giardia intestinalis. BMC Microbiol 6: 45 Das S, Castillo C, et al. (2001) Phospholipid remodeling/ generation in Giardia: the role of the Lands cycle. Trends Parasitol 17(7): 316–319 Das S, Reiner DS, et al. (1988) Killing of Giardia lamblia trophozoites by human intestinal fluid in vitro. J Infect Dis 157(6): 1257–1260 Das S, Schteingart CD, et al. (1997) Giardia lamblia: evidence for carrier-mediated uptake and release of conjugated bile acids. Exp Parasitol 87(2): 133–141 Das S, Stevens TL, et al. (2002) Lipid metabolism in mucousdwelling amitochondriate protozoa. Int J for Parasitol 32(6): 655–675 Das S, Traynor-Kaplan A, et al. (1991) A surface antigen of Giardia lamblia with a glycosylphosphatidylinositol anchor. J Biol Chem 266(31): 21318–21325 Diamond LS, Harlow DR, et al. (1978) A new medium for the axenic cultivation of Entamoeba histolytica and other Entamoeba. Trans R Soc Trop Med Hyg 72(4): 431–432 Dolis D, Moreau C, et al. (1997) Aminophospholipid translocase and proteins involved in transmembrane phospholipid traffic. Biophys Chem 68(1–3): 221–231 Ellis JE, Wyder MA, et al. (1996) Changes in lipid composition during in vitro encystation and fatty acid desaturase activity of Giardia lamblia. Mol Biochem Parasitol 81(1): 13–25 Farthing MJ, Chong SK, et al. (1983) Acute allergic phenomena in giardiasis. Lancet 2(8364): 1428 Farthing MJ, Keusch GT, et al. (1985) Effects of bile and bile salts on growth and membrane lipid uptake by Giardia lamblia. Possible implications for pathogenesis of intestinal disease. J Clin Invest 76(5): 1727–1732 Fridberg A, Olson CL, et al. (2008) Sphingolipid synthesis is necessary for kinetoplast segregation and cytokinesis in Trypanosoma brucei. J Cell Sci 121(Pt 4): 522–535 Gibson GR, Ramirez D, et al. (1999) Giardia lamblia: incorporation of free and conjugated fatty acids into glycerol-based phospholipids. Exp Parasitol 92(1): 1–11 Gillin FD, Boucher SE, et al. (1989) Giardia lamblia: the roles of bile, lactic acid and pH in the completion of the life cycle in vitro. Exp Parasitol 69(2): 164–174

M. Yichoy et al. Gillin FD, Gault MJ, et al. (1986) Biliary lipids support serum-free growth of Giardia lamblia. Infect Immun 53(3): 641–645 Gillin FD, Reiner DS, et al. (1987) Encystation and expression of cyst antigens by Giardia lamblia in vitro. Science 235(4792): 1040–1043 Hegner R and Eskridge L (1938) Localization of Giardia muris in rats. J Parasitol 24(6): 511–515 Hernandez Y, Castillo C, et al. (2007) Clathrin-dependent pathways and the cytoskeleton network are involved in ceramide endocytosis by a parasitic protozoan, Giardia lamblia. IntJ Parasitol 37(1): 21–32 Hernandez Y, Shpak M, et al. (2008) Novel role of sphingolipid synthesis genes in regulating giardial encystation. Infect and Immun 76(7): 2939–2949 Hernandez Y, Zamora G, et al. (2007) Transcriptional analysis of three major putative phosphatidylinositol kinase genes in a parasitic protozoan, Giardia lamblia. J Eukaryot Microbiol 54(1): 29–32 Jarroll EL, Manning P, et al. (1989) Giardia cyst wall-specific carbohydrate: evidence for the presence of galactosamine. Molecular and Biochemical Parasitology 32(2–3): 121–131 Jarroll EL, Muller PJ, et al. (1981) Lipid and carbohydrate metabolism in Giardia lamblia. Mol Biochem Parasitol 2(3–4): 187–196 Kane A, Ward HD, et al. (1991) In vitro encystation of Giardia lamblia: large-scale production of in vitro cysts and strain and clone differences in encystation efficiency. J Parasitol 77(6): 974–981 Kaneda Y and Goutsu T (1988) Lipid analysis of Giardia lamblia and its culture medium. Ann Trop Medi Parasitol 82(1): 83–90 Keister D (1983) Axenic culture of Giardia lamblia in TYI-S33 medium supplemented with bile. Trans R Soc Trop Med Hyg 77(4): 487–488 Kent C (1995) Eukaryotic phospholipid biosynthesis. Annu Rev Biochem. 64: 315–343 Maldonado RA, Kuniyoshi RK, et al. (2006) Trypanosoma cruzi oleate desaturase: molecular characterization and comparative analysis in other trypanosomatids. J Parasitol 92(5): 1064–1074 Mohareb EW, Rogers EJ, et al. (1991) Giardia lamblia: phospholipid analysis of human isolates. Ann Trop Med Parasitol 85(6): 591–597 Morgan CP, Insall R, et al. (2004) Identification of phospholipase B from dictyostelium discoideum reveals a new lipase family present in mammals, flies and nematodes, but not yeast. Biochem J 382(Pt 2): 441–449 Morrison HG, McArthur AG, et al. (2007) Genomic minimalism in the early diverging intestinal parasite Giardia lamblia. Science 317(5846): 1921–1926 Morrison HG, Zamora G, et al. (2002) Inferring protein fuction from genomic sequence: Giardia lamblia expresses a phosphatidylinositol kinase-related kinase similar to yeast and mammalian TOR. Comp Biochem Physiol 133(4): 477–491 Mukherjee S, Ghosh RN, et al. (1997) Endocytosis. Physiol Rev 77(3): 759–803 Nichols BJ and Lippincott-Schwartz J (2001) Endocytosis without clathrin coats. Trends Cell Biol 11(10): 406–412

Chap. 7 Mass Spectrometric Analysis of Phospholipids and Fatty Acids in G. lamblia Pagano RE and Sleight RG (1985) Defining lipid transport pathways in animal cells. Science 229(4718): 1051–1057 Pernet F, Pelletier CJ, et al. (2006) Comparison of three solidphase extraction methods for fatty acid analysis of lipid fractions in tissues of marine bivalves. J Chromatogr A 1137(2): 127–137 Pulfer M and Murphy RC (2003) Electrospray mass spectrometry of phospholipids. Mass Spectrom Rev 22(5): 332– 364 Soltys BJ, Falah M, et al. (1996) Identification of endoplasmic reticulum in the primitive eukaryote Giardia lamblia using cryoelectron microscopy and antibody to Bip. J Cell Sci 109(Pt 7): 1909–1917 Stevens TL, Gibson GR, et al. (1997) Uptake and cellular localization of exogenous lipids by Giardia lamblia, a primitive eukaryote. Exp parasitol 86(2): 133–143

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Subramanian AB, Navarro S, et al. (2000) Role of exogenous inositol and phosphatidylinositol in glycosylphosphatidylinositol anchor synthesis of GP49 by Giardia lamblia. Biochim Biophys Acta 1483(1): 69–80 van Meer G and Op den Kamp JA (1982) Transbilayer movement of various phosphatidylcholine species in intact human erythrocytes. J Cell Biochem 19(2): 193–204 Vargas-Villarreal J, Escobedo-Guajardo BL et al. (2007) Activity of intracellular phospholipase A1 and A2 in Giardia lamblia. J Parasitol 93(5): 979–984 Yichoy M, Nakayasu ES, et al. (2009) Lipidomic analysis reveals that phosphatidylglycerol and phosphatidylethanolamine are newly generated phospholipids in an earlydivergent protozoan, Giardia lamblia. Mol Biochem Parasitol 165(1) 67–78

Giardia Metabolism Edward L. Jarroll, Harry van Keulen, Timothy A. Paget and Donald G. Lindmark

Abstract This chapter describes key aspects of our basic knowledge of Giardia metabolism. It is well known that this organism has minimal biosynthetic capacity: it lacks de novo lipid, de novo purine, and de novo pyrimidine syntheses (relying solely on salvage pathways). Giardia also lacks mitochondria and cytochrome-mediated oxidative phosphorylation and thus trophozoites use glycolysis (from glucose only) and the arginine dihydrolase pathways relying on substrate level phosphorylation for energy production; glucose is also shunted through a pentose phosphate pathway. The enzymes responsible for end product and energy production in Giardia are soluble – not found in subcellular organelles. The end products of glucose fermentation are acetate, ethanol, alanine, carbon dioxide, and hydrogen. Thus, it is clear that Giardia is well adapted to its environment; however, this comes at a cost because this organism must scavenge nearly all of its biosynthetic pre-cursors from an environment containing a thriving microbial flora. Additionally, Giardia’s metabolism seems to be exquisitely balanced – and this would mean that processes such as the formation of the cyst wall carbohydrate have to be tightly controlled and regulated: encysting Giardia slows their catabolism of glucose for energy, and begin converting glucose to the synthesis of a cyst wall specific sugar, N-acetylgalactosamine, for the synthesis of giardan. A complete pathway of enzymes is induced during encystment for this synthesis including a novel enzyme, cyst wall synthase, that synthesized the giardan homopolymer [β-1,3-N-acetylgalactosamine] . At the same time, trophozoites increase their can tabolism of arginine ostensibly to offset the energy lost from slowing glycolysis.

H. D. Luján et al. (eds.), Giardia © Springer-Verlag/Wien 2011

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Also highlighted in this chapter is the fact that most studies on the metabolism, proteomics, and transcriptomics have been performed on a limited number of isolates, and there are few reports detailing comparative studies of metabolism and biochemistry of isolates. Those studies do indicate metabolic differences among isolates and comparative metabolic studies of genetically distinct isolates are vital to understanding how Giardia survives in the gut and responds to changes in this environment. Variation in metabolism may also be responsible for variation in reproduction rates and virulence among isolates.

8.1 Carbohydrate (Glucose) Catabolism The anaerobic protozoan Giardia lacks de novo lipid (Jarroll et al., 1981), purine and pyrimidine syntheses (Wang and Aldritt, 1983; Aldritt et al., 1985) as well as mitochondria and cytochrome-mediated oxidative phosphorylation. Giardia in axenic culture uses only glucose (glc) as its metabolite for energy production from carbohydrates and relies on substrate level phosphorylation for energy production (Lindmark, 1980; Jarroll et al., 1981; Schofield et al., 1991). The enzymes responsible for end product and energy production in Giardia, are not found in subcellular organelles (they do not contain hydrogenosomes as in Trichomonas) (Jarroll et al., 1981; Lindmark and Muller, 1973). The end products of glucose fermentation are acetate, ethanol, alanine, carbon dioxide, and hydrogen (Lindmark 1980; Paget et al., 1990; Schofield et al., 1990, 1991, 1992; Lloyd et al., 2002). End product balance in growth medium is sensitive to oxygen tension and glucose levels (Schofield et al., 1990, 1992). The proposed pathway of glucose fermentation and energy production is as shown in Fig. 8.1.

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Fig. 8.1 Giardia carbohydrate and energy metabolisms. Numbered items represent known enzymes: 1 hexokinase; 2 glucose phosphate isomerase; 3 pyrophosphate-dependent phosphofructokinase; 4 fructose biphosphate aldolase; 5 glyceraldehyde-3-phosphate dehydrogenase; 6 pyruvate kinase; 7 phosphoenolpyruvate carboxyphosphotransferase; 8 aspartate aminotransferase; 9 malate dehydrogenase; 10 malate dehydrogenase (decarboxylating); 11 acetaldehyde dehydrogenase (NAD); 12 primary alcohol dehdrogenase (NAD); 13 acetyl-CoA synthetase; 14 pyruvate: ferredoxin oxidoreductase; 15 pyruvate phosphate dikinase (PPi forming); 16 arginine deiminase; 17 ornithine transcarbamoylase; 18 carbamate kinase; 19 hydrogenase; 20 glucosamine 6-P deaminase; 21 glucosamine 6-P N-acetylase; 22 phospho-N-acetylglucosamine mutase; 23 uridine diphospho-(UDP) N-acetylglucosamine pyrophosphorylase; 24 UDP-N-acetylglucosamine-4′-epimerase; 25 cyst wall synthase

8.2 Glycolysis and the Pentose Phosphate Pathway In most eukaryotes and prokaryotes, conversion of fructose-6-phosphate (fru 6-P) to fru-1,6-bisP is an irreversible and a regulated step catalyzed by an ATPdependent phosphofructokinase (ATP-PFK). However in Giardia, this reaction is catalyzed by a pyrophosphate-dependent phosphofructokinase (PPiPFK) (Mertens, 1990). PPi-PFK catalyzes a reversible reaction and is not a regulated enzyme (Mertens, 1990, 1993; Phillips and Li, 1995). Pyrophosphatedependent pyruvate phosphate dikinase (PPi-PPdK) has been identified (Hrdy et al., 1993; Bruderer et al.,

1996; Hiltpold et al., 1999) in Giardia. Adenylate kinase in combination with PPi-PPdk (Mowatt et al., 1994) converts two ADP molecules into ATP + AMP and PPi-PPdk converts phosphoenolpyruvate (PEP) plus AMP and Pi into pyruvate + ATP, resulting in the generation of two ATP molecules during the conversion of PEP to pyruvate. Based on the evolution of CO2 from [1-14C] glucose, Jarroll et al. (1981) proposed a functional pentose phosphate pathway (PPP). Additionally glucose-6-phosphate dehydrogenase activity and ribose 5-isomerase genes have been detected in Giardia (Lindmark, 1980; Esteve et al., 2007). The PPP needs further investigation.

Chap. 8 Giardia Metabolism

8.3 Pyruvate Metabolism Lindmark (1980) showed that pyruvate from glycolysis is converted to acetyl-CoA and reduced ferredoxin by the oxygen-sensitive pyruvate: ferredoxin oxidoreductase (PFOR). Reduced ferredoxin is oxidized by hydrogenase producing molecular hydrogen (Lloyd et al., 2002). Acetyl-CoA is converted to acetate, ATP, and CoA by acetyl-CoA synthetase (Sánchez et al., 2000), alternatively acetyl-CoA is converted to acetaldehyde and then to ethanol by a bifunctional aldehyde dehydrogenase (Sánchez, 1998). Alanine is produced from pyruvate by the combined action of alanine aminotransferase and glutamate dehydrogenase (Paget et al., 1990; Schofield et al., 1992).

8.3.1 Effects of O2 and Glucose Concentration on Pyruvate Metabolism Small changes in oxygen concentration have an extreme effect on pyruvate metabolism. Under anaerobic conditions, alanine (a) is the major product. Small amounts of H2 (b, c) are also produced. Even with the addition of small amounts of O2 (<0.25 μm), ethanol production is stimulated (d), and alanine and H2 production (b, c) are inhibited. At higher O2 concentrations (>50 μm), alanine production is stopped (a) and acetate and CO2 (e) are the major products. These results can be used to predict possible metabolic pathways of pyruvate metabolism that may occur in the intestine (O2 concentration 0–60 μm). Pyruvate + Glutamate → Alanine + 2-Oxyglutarate and/or b. Pyruvate → Acetyl CoA + reduced Ferredoxin c. Reduced Ferredoxin → H2 + oxidized Ferredoxin d. Acetyl CoA + NAD+ → Acetaldehyde + NADH + CoA-SH → Ethanol + NAD+ and/or e. Acetyl CoA + ADP → Acetate + ATP + CoA-SH.

a.

Most metabolic studies of Giardia are done in a medium containing 50 mM glucose. When the concentration is reduced to below 10 mM, replication

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rates are reduced by 50% and ethanol production (d) is greatly reduced, alanine production is reduced (a), and acetate and CO2 production remains the same. Further experimentation under controlled O2 and glucose concentration should give a further insight into overall pyruvate metabolism in Giardia.

8.3.2 Pyruvate: Ferredoxin Oxidoreductase (PFOR) PFOR is a homodimer with 138 kDa subunits; pyruvate is the preferred substrate. Purified PFOR donates electrons to Giardia ferredoxin (Sánchez, 1998), and even though oxygen sensitive, Giardia’s PFOR is more stable than this same enzyme from other protozoans.

8.3.3 Hydrogenase Hydrogenase and H2 production was first demonstrated by Lloyd et al. (2002). Mass spectrometric analysis showed low level of H2 production under anaerobic conditions, about a 10-fold lower amount than from Trichomonas. Hydrogenase is oxygen sensitive and is typical of an iron-only hydrogenase. It is inhibited by CO and metronidazole. The hydrogenase gene has been sequenced, but unlike the hydrogenase gene in trichomonads, it shows no N-terminus motif that would direct it to an organelle.

8.3.4 Acetyl CoA Synthetase (Nucleoside Diphosphate – Forming) Giardia’s acetyl-coA synthase gene has been expressed in Escherichia coli: acetyl-CoA and adenine nucleotides are preferred substrates. The enzyme is a single polypeptide chain (Edwards et al., 1989).

8.3.5 Aldehyde Dehydrogenase (-CoA Acetylating) Giardia’s aldehyde dehydrogenase is bifunctional. The first activity catalyzes the irreversible interconversion of acetyl-CoA to acetaldehyde and CoA with NAD+ but not with NADP+ as a cofactor. Acetyl-CoA is the primary substrate. In the second activity, a pri-

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mary alcohol dehydrogenase is also NAD+ specific (Sánchez, 1998).

8.4 Arginine Dihydrolase Pathway (ADiHP) The ADiHP is another potential source of energy (Edwards et al., 1989, 1992; Schofield et al., 1990, 1992). Schofield et al. (1990) have shown in growth medium that there is a rapid depletion of the arginine in the medium and the concurrent production of ornithine and ammonia. [Guanidino-14C] arginine is converted to 14 CO2 by extracts of Giardia indicating the presence of the ADiHP. This was confirmed by the detection of arginine deiminase, catabolic ornithine transcarbamylase, carbamate kinase, and ornithine decarboxylase in Giardia extracts. The ADiHP is present in a number of prokaryotic organisms, but among eukaryotes it has been documented only in T. vaginalis (Linstead and Cranshaw, 1983). In the ADiHP, arginine is converted to ornithine by way of citrulline and with carbamoyl-P converted to NH4+ with the generation of ATP from ADP by substrate-level phosphorylation, which could be a significant source of energy for the cell.

8.5 Synthesis of N-acetylgalactosamine from Glucose Giardia’s cyst wall is composed of ~63% polysaccharide (discussed below) and ~37% protein. The polysaccharide component of the cyst wall is a β1,3-N-acetyl-D-galactosamine polymer (2-acetamido-2-deoxy-D-galactan, Fig. 8.2) now named giardan (Gerwig et al., 2002; ener et al., 2009), and is likely synthesized by a β1,3 UDP-GalNAc transferase tentatively named cyst wall synthase (Cws) (Karr and Jarroll, 2004). A covalent linkage between giardan and some cyst wall proteins may play a role in giardan’s insolubility although this is unproven (Gerwig et al., 2002). Extracellular formation of giardan requires intracellular synthesis of UDP-GalNAc (Fig. 8.2). Glucose is incorporated into UDP-GalNAc by inducible, cytosolic enzymes: glucosamine 6-P deaminase (EC 5.3.1.10), glucosamine 6-P N-acetylase (EC 2.3.1.4), phospho N-acetyl glucosamine mutase (EC 2.7.5.2)

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and uridine diphospho (UDP) N-acetyl glucosamine pyrophosphorylase (EC 2.7.7.23), and UDP-N-acetyl glucosamine 4′-epimerase (EC 5.1.3.7) (Macechko et al., 1992).

8.5.1 Glucosamine 6-P Deaminase (Gnp) Giardia’s Gnp has a molecular mass of 29 kDa and a pH optimum of 8.9 (Steimle et al., 1997). van Keulen et al. (1998) identified two genes, gnp1 and gnp2, encoding Gnps in Giardia but only gnp1 was expressed. The transcript for gnp1 appeared no earlier than 6 h after cells were induced with bile. Using a different nomenclature for the same genes, Knodler et al. (1999) showed that these genes have distinct patterns of expression: Gln6PI-A has a short 5’ untranslated region, and is expressed at a low level during vegetative growth and encystment. The other gene, Gln6PI-B, has two transcripts – one of which was expressed constitutively and the other was up-regulated during encystment. The non-regulated transcript has the longest 5′-UTR known for Giardia and is 5′ capped or blocked. The Gln6PI-B up-regulated transcript has a short, non-capped 5′-UTR and a small promoter region (<56 bp upstream from the start codon) was sufficient for the regulated expression of Gln6PI-B, which also has an antisense overlapping transcript expressed constitutively. A shorter antisense transcript was detected during encystment. The Gnp is transcriptionally control; in mature cysts Gnp is removed by an ubiquitin-mediated pathway (Lopez et al., 2002).

8.5.2 Glucosamine 6-P N-acetylase (Gna) Gna has been cloned and sequenced in Giardia (Lopez et al., 2003); it has a predicted pH optimum of 6.45 and molecular mass of 22.8 kDa. Gna has not yet been characterized kinetically though it is transcriptionally regulated (Lopez et al., 2003).

8.5.3 Phospho N-acetylglucosamine Mutase (Pgm) Giardia’s Pgm was partially purified and characterized by Lindmark and Schmidt (1992). The native enzyme increases 12-fold during encystment and re-

Chap. 8 Giardia Metabolism

Ia

Ib

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Ia + lb

Fig. 8.2 Model polysaccharide chains constructed from disaccharide and linkage information randomly selected from appropriate conformational regions. All polysaccharides contain 30 monosaccharide residues. The polymer constructed from region Ia forms a right-handed (clockwise) helix, whereas the polymer built from region Ib has a left-handed helical conformation. The polysaccharide assembled from regions Ia and Ib (4:1) shows a random coil conformation. Gerwig et al. (2002) The Giardia intestinalis filamentous cyst wall contains a novel β(1-3)-N-acetyl-D-galactosamine polymer: a structural and conformational study. Glycobiology, 2002, vol. 12(8): 499–505 by permission of Oxford University Press

quires Mg2+, glucose 1,6-bisP, and diethyldithiocarbamate (hydroxyquinoline may substitute). The recombinant mutase is active but has not been characterized. Lopez et al. (2003) showed that Pgm is also transcriptionally regulated.

8.5.4 UDP N-acetyl Glucosamine Pyrophosphorylase (Uap) Evidence exists that there are two Uap activities in encysting Giardia. Lopez et al. (2003), Mok et al.

(2005), Mok and Edwards (2005), and ener et al. (2009) reported and characterized the inducible pyrophosphatase (iUap). Lopez et al. (2003) showed that iUap regulation is transcriptional. Bulik et al. (2000) reported an Upp activity which is constitutive (cUap) and stimulated by GlcN-6-P anabolically toward UDP-GalNAc synthesis. The gene encoding cUpp has not yet been cloned, and it may not be a typical Uap but rather another enzyme capable of behaving as a Uap. That both enzymes are present was shown in Giardia lysates using the fact that both enzymes have a different pH optimum. A biphasic curve was

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observed after induction with bile, while non-induced trophozoite lysates showed a monophasic activity curve (ener et al., 2009).

8.5.5 UDP-N-acetylglucosamine 4’-epimerase (Uae) Uae exists as a single copy gene in Giardia and is regulated at the level of transcription (Lopez et al., 2003). Recombinant Uae exhibits an open reading frame of 1158 bp (Lopez et al., 2007). Conversion of UDP-GalNAc to UDP-GlcNAc is favored in vitro; an excess of UDP-GlcNAc is required to drive the reaction toward the synthesis of UDP-GalNAc (Lopez et al., 2007). Uae is present in a large variety of organisms and is capable of catalyzing distinct and reversible reactions: UDP-GlcNAc to UDP-GalNAc and UDP-Glc to UDP-Gal (EC 5.1.3.2) depending upon the organism from which it originates. In some, it can catalyze the conversion of UDP-Glc/GlcNAc to UDP-Gal/GalNAc, while in others it can only convert UDP-Gal to UDP-Glc or UDP-GalNAc to UDPGlcNAc (Ishiyama et al., 2004). Macechko et al. (1992) detected UDP-GlcNAc/GalNAc Uae activity in crude Giardia lysates but could not detect UDPGal to Glc activity. Giardia Uae only catalyzes the reversible epimerization of UDP-GlcNAc to UDPGalNAc and so phylogenetically aligns with the Group 3 prokaryotes, rather than eukaryotes (Ishiyama et al., 2004) making it a possible target for chemotherapeutic attack.

8.5.6 Cyst Wall Synthase (Cws) The UDP-GalNAc synthesized in the cytoplasm must be incorporated into the giardan portion of the cyst wall. Apparently, this is accomplished by a novel inducible, particle-associated Cws. Cws activity increases ~1245-fold during the first 24–36 h of encystment (Karr and Jarroll, 2004). The particles with which it associates are different from the lysosome-like organelles; the vesicles are possibly ESVs, but that remains to be demonstrated. Partially purified exhibits an absolute specificity for UDP-GalNAc, and a requirement for divalent cations with Ca2+ and Mg2+ significantly preferred over Co2+ , Mn2+, and Zn2+.

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8.6 Regulation and Inhibition Sugar phosphate intermediate concentrations generated by these synthetic enzymes change during encystment (ener et al., 2004). The largest absolute increase (~5-fold) is in the level of GlcN-6-P. However, the greatest relative increase in the concentration of amino sugars in the pathway (ca. ~9-fold) is in UDP-GlcNAc produced by Upp. The relatively large increase in UDP-GlcNAc could be due to the presence of two Upp activities in Giardia during encystment. Giardia’s Uae has a preference – at least in vitro – for the catabolic reactions with a larger Vmax and smaller Km for UDP-GalNAc than for UDP-GlcNAc. Thus, the productive synthesis of UDP-GalNAc would be difficult if not impossible. Two conditions are needed to drive the reaction toward UDP-GalNAc synthesis during encystment: the high intracellular UDP-GlcNAc levels brought about by the two Uap activities and the UDP-GalNAc produced must be removed by Cws. Together these suggest that Uae is a regulatory step in giardan biosynthesis (Lopez et al., 2007). The measured sugar phosphate intermediates (ener et al., 2004) and the observed Km values of riUpp and rUae support this observation. If a cell volume of 80 fL, the mean cell volume of a red blood cell, is assumed for a Giardia trophozoite, the concentration of GlcNAc 1-P at 0 h (51 amoles/cell) is 6.4 × 10–4 M; this becomes 2.7 × 10–3 M at 24 h (216 amoles/cells). The anabolic Km measured for riUAP is 9.7 × 10–5 M (ener et al., 2009). Thus, the substrate concentration is 6.5 × Km and 27 × Km, respectively, indicating that the enzyme operates at Vmax in 24-h-induced cells. This is different for the Uae reaction: at 0 h the substrate concentration for UDP-GlcNAc (7 amoles/ cell) is 8.75 × 10–5 M and at 24 h is 8.12 × 10–4 M (65 amoles/cell). With a Km of 1.22 × 10–3 for the Uae (Lopez et al., 2007) this means that the substrate concentration is well below the Km values (0.07 and 0.67 × Km, respectively). Even after 24 h of encystment, the enzyme is not operating at even ½ Vmax suggesting that the removal of the end product by Cws is absolutely required to drive giardan synthesis (ener et al., 2009). If the Cws is not present, the entire pathway would stop and intermediates would no longer be shunted from the glycolytic pathway.

Chap. 8 Giardia Metabolism

8.7 Metabolism and Drugs Metronidazole (MTZ) and tinidazole (TTZ) (Medical Letter, 2004) are drugs of choice for treating giardiasis in the USA. These 5-nitroimidazoles are reduced by Giardia’s PFOR apparently forming toxic metabolites. MTZ does not inhibit oxygen uptake by encysting Giardia after about 12 h or in completely formed cysts; whereas, oxygen uptake by Giardia trophozoites is significantly inhibited by MTZ (Paget et al., 1989, 1993a, 1998). Paget et al. (2004) reported that menadionegenerated radicals kill Giardia trophozoites and cysts. Jarroll and ener (2003) speculated that since the trigger for encystment is usually depletion of a vital nutrient and assuming encystment is irreversible at some point, then inhibiting encystment, especially late in the process, could cause the encysting trophozoites to die rather than just stop encysting. Two likely targets are Uae and Cws since both are at the end of the encystment synthetic pathway when the cell is most likely committed. Additionally, the epimerase differs significantly from the human epimerase and Cws is not found in any other animal to date. Inhibition, at the very least, could render encysting cells incapable of surviving osmotic pressures inside or outside of the host. Few inhibitors of these enzymes are known currently. Steimle et al. (1997) demonstrated that 2-amino-2-deoxyglucitol-6-phosphate, a GlcN-6-P analog, inhibits the activity of glucosamine 6-P deaminase while Jarroll (unpublished) observed that this same analog at 1mM reduced encystment in vitro from ~70% to ~2–3%. Inhibition by this analogue in vitro did not appear to cause trophozoite death during the four-day period for which it was observed. Interestingly, Cws requires a divalent cation for activity. EDTA, a chelating agent, inhibits Cws activity in vitro at 1 mM or higher (Karr and Jarroll, 2004).

8.8 Comparative Biochemistry and Metabolism So far this chapter has highlighted our current understanding of aspects Giardia metabolism. Giardia has an energy metabolism based on anaerobic ATP and PPi syntheses and has limited, but in some cases, unique biosynthetic capacity as in the cyst wall. With the advances in proteomics and genomics (Chapters 5

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and 6) additional information is now available. However by its very nature, metabolic and biochemical analyses require approaches that focus on one enzyme or pathway at a time and involve cultivation of the parasite in large quantities. Gene cloning has helped but it also poses problems since post-translational modification can have profound effects of the activity of some of the enzymes and this modification is not achieved easily in bacterial recombinant systems. Genetic studies and genome sequencing have laid an important foundation for understanding Giardia. This approach is being used to dissect the G. duodenalis species and has shown that this species is highly diverse with genetic variation between isolates equivalent to species in other systems. Currently five assemblages (A–E) have been delineated within this species and these exhibit some variation in host specificity and, for the human infective assemblages A and B, variation in pathogenicity. Complete genomes are now available for assemblage A, B and E isolates and now comparative genomics of these can be performed. In tandem with this, many workers have assessed gene expression in Giardia making it is possible to observe changes in response to significant events such as stress and encystment (RoxströmLindquist et al., 2005; Gallego et al., 2007; Lauwaet et al., 2007; Müller et al., 2008). Although genomic and transcriptional analyses of Giardia are vital, it is important to note that mRNA production does not always correlate well with protein expression, especially for proteins produced in low abundance (Gygi et al., 1999). Also, many proteins undergo post-translational modifications and are therefore only accessible through proteomic and biochemical analysis. At the protein level, isoenzyme analysis of Giardia began in the late 80s and early 90s. The analyses were used to study enzyme and protein variation among isolates from various geographic regions, hosts, and individuals with varying clinical symptoms. From these data, it became clear that significant differences exist (Moore et al., 1982; Nash and Keister, 1985; Wenman et al., 1986; Meloni et al., 1988; Moss et al., 1992; Mayrhofer et al., 1995). However, the isolates used for these studies were not genotyped. When genotype information is included, differences between assemblages are significant. With advances in protein analysis using mass spectrometry and genomics, accurate analysis of proteomes and identification of in-

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dividual proteins using mass spectrometry allows protein differences among the assemblages to be annotated and linked to genome data. To date only a few such studies have been undertaken (Iyer et al., 2004; Stefanic et al., 2006; Ringqvist et al., 2008; Steuart et al., 2008; Kim et al., 2009; Steuart, 2010). While being of immense value, proteomics is unable to consider how factors such as cellular redox, adenylate charge, and post-translational modification impact on metabolism and enzyme activity. At the metabolic level, studies have been limited to single Giardia isolates such as Portland-1, WB, and MR-4 and there are few reports detailing comparative studies of metabolism and biochemistry of Giardia isolates. The study by Strandén and Köhler (1991) looked at a number of Swiss Giardia isolates and these authors observed little variation in metabolism among isolates. This is not surprising as all were cultured under similar conditions. Additional metabolic studies are vital if we are to understand how Giardia survives in the gut and responds to changes in this environment, and variation in metabolism may also be responsible for variation in reproduction rates and virulence among isolates. In the following sections we will highlight how such variation can be observed.

8.9 Uridine/Thymidine Phosphorylase Activity (URTPase) Recent studies by Steuart et al. (2008) compared protein profiled between the human infective assemblages A and B. These authors identified a number of proteins that exhibit assemblage-specific variation, including URTPase (EC 2.4.2.2). This enzyme shows a significant and reproducible pI difference between the two assemblages. The significance of this variation to the physiology and virulence of Giardia isolates is not clear; however, the URTPase variation is interesting since Giardia lacks the pathways for the de novo synthesis of purines and pyrimidines and is thus totally dependent on the use of salvage pathways. Giardia URTPase is an immunodominant protein observed during cases of giardiasis (Palm et al., 2003) URTPase has been characterized from the Portland 1 isolate (not genotyped) and is a single protein with a molecular mass of ca. 43,000 kDa and a pI of

E. L. Jarroll et al.

Fig. 8.3 Pathway showing the role of uridine phosphorylase in the conversion of uridine to uracil and then to the production of UTP. The sites of action of two well-known inhibitors of this pathway, 5-benzylacyclouridine (BAU) and 5-flurouracil (5-FU), are included

5.9. URTPase exhibits activity with uridine, deoxyuridine, and thymidine (Lee et al., 1988). The purified protein from assemblage A and B isolates exhibits different physical properties and significantly different kinetics (Table 8.1) (Steuart RFL, Oniku A, Thompson RCA, Paget TA, unpublished). Our data show that URTPase from assemblage B isolates exhibits biphasic kinetics with two distinct rates and affinities. Extrapolating these we calculate apparent Vmax and Km values for these. It would seem that the assemblage B enzyme has both high and low affinity characteristics and thus is more efficient at scavenging uridine over a wider range of concentrations. This is not seen with the cloned enzyme as both assemblage A and B enzymes have similar affinities and activity. We propose that this variation is most likely due to post-translational modification.

Table 8.1 Kinetic values for uridine phosphorylase isolated and purified from different Giardia trophozoites assemblages. (Steuart RFL, Oniku A, Thompson RCA and Paget TA, unpublished) Isolate

Vmax μM min–1 mg protein–1

km (μM)

7c3 (A)

0.049

0.41

15c1 (A)

0.052

0.43

1214 (A)

0.047

0.41

3c3 (B)

0.019 0.038

0.14 0.51

33c7 (B)

0.015 0.028

0.12 0.48

Chap. 8 Giardia Metabolism

135

5

Ratio of new/spent media

4

3

2

1

Inosine Glucose Cholesterol

L-Tryptophan L-Cystine

Ascorbic acid myo-Inositol

Sorbitol L-Tyrosine

Trehalose Tyramine L-Lysine

Fructose

Mannose Galactose

Dehydroascorbic acid dimer

Glycerol 3-Phosphate Hypoxanthine Ornithine Galactofuranose

Arabinose serine

Threonic acid

GABA N-Acetylglutamic acid Erythronic acid

Malic acid

Pipecolic acid L-Threonine L-Homoserine

Glyceric acid L-Alanine Ornithine/Arginine

L-Threonine Succinic acid

Glycerol

L-Isoleucine L-Proline

Lactic acid

Glycolic acid Urea

Pyruvic acid

0

Fig. 8.4 Partial metabolome data set (42/385) from a comparison of fresh culture medium vs. spent medium after 48 h of culture with an assemblage A isolate of G. duodenalis (2c16). Analysis was performed using GC-MS with derivatization to detect nonvolatile components. Confidence limits are shown by the shaded box – peaks outside this are significant

8.10 Metabolomics Previous work on Giardia’s metabolism has focused on specific pathways and although some NMR based analyses of metabolic end products has been performed (Paget et al., 1993b), no comprehensive profile of intra- or extracellular metabolic products or intermediates has been performed. Metabolomics is an approach that can generate a detailed profile of low-molecular-weight metabolites in a biological system (Koal and Deigner, 2010). Separation and identification techniques used for this can include GC-MS, LC-MS, and NMR spectroscopy. GC-MS is not suitable for non-volatile, thermolabile, or highly polar compounds; however, derivatization of metabolites is used to detect volatile and thermostable analytes. The data generated by this approach not only give compound identity but also concentration. This approach can be used to compare similar biological systems such as two isolates of the same organism or can be used to evaluate the effects of change on these systems. Limitations to this approach include signifi-

cant variation within samples that can be overcome with internal standards as well as variation between samples that requires large numbers of replicates to be analyzed. In addition, the approach is only as good as the library of compounds available. Such a study and some preliminary data are shown in Fig. 8.4 (Steuart RFL, Oniku A, Thompson RCA, Paget TA, unpublished). The data shown in this figure compare the profiles of fresh to spent medium (shown as a ratio) and confidence limits are shown by the upper and lower boundaries of the shaded box peaks with values outside those boxes are significantly different. From these findings alone there are some interesting observations such as the production of compounds such as pipecolic acid and hypoxanthine and the utilization of galactose, inosine in addition to those of cholesterol and glucose which were expected. This chapter clearly shows that to arrive at a detailed understanding of Giardia physiology and the organism’s response to change requires a multi-modal approach, using a range of techniques including met-

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abolic and biochemical analysis of pathways and key regulatory enzymes. This approach is also crucial for identifying drug targets that are aimed at vital processes in the Giardia.

References Aldritt SM, Tien P, and Wang CC (1985) Pyrimidine salvage in Giardia lamblia. J Exp Med 161(3): 437–445 Bruderer T, Wehrli C, and Köhler P (1996) Cloning and characterization of the gene encoding pyruvate phosphate dikinase from Giardia duodenalis. Mol Biochem Parasitol 77: 225–233 Bulik D, van Ophem P, Manning J, Shen Z, Newburg D, and Jarroll E (2000) UDP-N-acetylglucosamine pyrophosphorylase: a key enzyme in encysting Giardia is allosterically regulated. J Biol Chem 275: 14722–14728 Edwards MR, Gilroy FV, Jimenez BM, and O’Sullivan WJ (1989) Alanine is a major end product of metabolism by Giardia lamblia: a proton nuclear magnetic resonance study. Mol Biochem Parasitol 37: 19–26 Edwards MR, Schofield PJ, O’Sullivan WJ, and Costello M (1992) Arginine metabolism during culture of Giardia intestinalis. Mol Biochem Parasitol 53: 97–103 Esteve M, Maugeri D, Stern AL, Beluardi P, and Cazzulo JJ (2007) The pentose phosphate pathway in Trypanosoma cruzi: a potential target for the chemotherapy of Chagas’ disease. An Acad Bras Cien 79: 649–663 Gallego E, Alvarado M, and Wasserman M (2007) Identification and expression of the protein ubiquitination system in Giardia intestinalis. Parasitol Res 101: 1–7 Gerwig G, van Kuik J, Leeflang B, Kamerling J, Vliegenthart J, Karr C, and Jarroll E (2002) Conformational studies of the β(1-3)-N-acetyl-D-galactosamine polymer of the Giardia lamblia filamentous cyst wall. Glycobiology 12: 1–7 Gygi S, Rochon Y, Franza B, and Aebersold R (1999) Correlation between protein and mRNA abundance in yeast. Mol Cell Biol 19: 1720–1730 Hiltpold A, Thomas RM and Köhler P (1999) Purification and characterization of recombinant pyruvate phosphate dikinase from Giardia. Mol Biochem Parasitol 104: 157–169 Hrdy I, Mertens E, and Nohynkova E (1993) Giardia intestinalis: detection and characterization of a pyruvate phosphate dikinase. Exp Parasitol 76: 438–441 Ishiyama N, Creuzenet C, Lam J, and Berghuis A (2004) Crystal structure of WbpP, a genuine UDP-N-acetylglucosamine 4-epimerase from Pseudomonas aeruginosa. J Biol Chem 279: 22635–22642 Iyer L, Koonin E, and Aravind L (2004) Novel predicted peptidases with a potential role in the ubiquitin signaling pathway. Cell Cycle 3: 1440–1445 Jarroll E and ener K (2003) Potential drug targets in cyst-wall biosynthesis by intestinal protozoa. Drug Resist Updat 6: 239–246 Jarroll EL, Muller PJ, Meyer EA, and Morse S (1981) Lipid and carbohydrate metabolism of Giardia lamblia. Mol Biochem Parasitol 2: 187–196

E. L. Jarroll et al. Karr C and Jarroll E (2004) Cyst wall synthase: N-acetylgalactosaminyl-transferase activity is induced to form the novel GalNAc polysaccharide in the Giardia cyst wall. Microbiology 150: 1237–1243 Kim J, Bae S, Sung M, Lee K, and Park S (2009) Comparative proteomic analysis of trophozoites versus cysts of Giardia lamblia. Parasitol Res 104: 475–479 Knodler L, Svärd S, Silberman J, Davids B, and Gillin F (1999) Developmental gene regulation in Giardia lamblia: rst evidence for an encystation-specic promoter and differential 5′ mRNA processing. Mol Microbiol 34: 327–340 Koal T and Deigner HP (2010) Challenges in mass spectrometry based targeted metabolomics. Curr Mol Med Mar 3 2010 [Epub ahead of print] Lauwaet T, Davids B, Torres-Escobar A, Birkeland S, Cipriano M, Preheim S, Palm D, Svärd S, McArthur A, and Gillin F (2007) Protein phosphatase 2A plays a crucial role in Giardia lamblia differentiation. Mol Biochem Parasitol 152: 80–89 Lee C, Jiménez B, and O’Sullivan W (1988) Purification and characterization of uridine (thymidine) phosphorylase from Giardia lamblia. Mol Biochem Parasitol 30: 271–277 Lindmark D and Schmidt K (1992) Phosphoacetylglucosamine mutase of Giardia duodenalis. Society of Protozoology Annual Meeting. University of British Columbia, Vancouver, BC, Canada (abstract) Lindmark DG (1980) Energy metabolism of the anaerobic protozoon Giardia lamblia. Mol Biochem Parasitol 1: 1–12 Lindmark DG and Muller M (1973) Hydrogenosome, a cytoplasmic organelle of the anaerobic flagellate, Tritrichomonas foetus and its role in pyruvate metabolism. J Biol Chem 235: 7724–7728 Linstead D and Cranshaw MA (1983) The pathway of arginine catabolism in the parasitic flagellate Trichomonas vaginalis. Mol Biochem Parasitol 8: 241–252 Lloyd D, Ralphs JR, and Harris JC (2002) Hydrogen production in Giardia intestinalis, a eukaryote with no hydrogenosomes. Trends Parasitol 18: 155–156 Lopez A, Hossain M and van Keulen H (2002) Giardia intestinalis glucosamine 6-phosphate isomerase: the key enzyme to encystment appears to be controlled by ubiquitin attachment. J Euk Microbiol 49: 134–136 Lopez A, ener K, Jarroll E, and van Keulen H (2003) Transcription regulation is demonstrated for five key enzymes in Giardia intestinalis cyst wall polysaccharide biosynthesis. Mol Biochem Parasitol 128: 51–57 Lopez A, ener K, Trosien J, Jarroll E, and van Keulen H (2007) UDP-N-acetylglucosamine 4′-epimerase from the intestinal protozoan Giardia intestinalis lacks UDP-glucose 4′-epimerase activity. J Euk Microbiol 54: 154–160 Macechko PT, Steimle P, Lindmark D, Erlandsen S, and Jarroll E (1992) Galactosamine synthesizing enzymes are induced when Giardia encyst. Mol Biochem Parasitol 56: 301–310 Mayrhofer G, Andrews R, Ey P, and Chilton N (1995) Division of Giardia isolates from humans into two genetically distinct assemblages by electrophoretic analysis of enzymes encoded at 27 loci and comparison with Giardia muris. Parasitology 111: 11–17 Medical Letter (2004) Tinidazole (Tindamax) – a new antiprotozoal drug. Med Lett Drugs Ther. 46: 70–72.

Chap. 8 Giardia Metabolism Meloni B, Lymbery A, and Thompson R (1988) Isoenzyme electrophoresis of 30 isolates of Giardia from humans and felines. Am J Trop Med Hyg 38: 65–73 Mertens E (1990) Occurrence of pyrophosphate: fructose 6-phosphate 1-phosphotransferase in Giardia lamblia trophozoites. Mol Biochem Parasitol 40: 147–149 Mertens E (1993) ATP versus pyrophosphate: glycolysis revisited in parasitic protists. Parasitol Today 9: 122–126 Mok M and Edwards M (2005) Kinetic and physical characterization of the inducible UDP-N-acetylglucosamine pyrophosphorylase from Giardia intestinalis. J Biol Chem 280: 39363–39372 Mok M, Tay E, Sekyere E, Glenn W, Bagnara A, and Edwards M (2005) Giardia intestinalis: molecular characterization of UDP-N-acetylglucosamine pyrophosphorylase. Gene 357: 73–82 Moore G, Sogandares-Bernal F, Dennis M, Root D, Beckwith D, and van Voorhis D (1982) Characterization of Giardia lamblia trophozoite antigens using polyacrylamide gel electrophoresis, high-performance liquid chromatography, and enzyme-labeled immunosorbent assay. Vet Parasitol 10: 229–237 Moss D, Visvesvara G, Mathews H, and Ware D (1992) Isoenzyme comparison of axenic Giardia lamblia strains. J Protozool 39: 559–564 Mowatt MR, Weinbach EC, Howard TC, and Nash TE (1994) Complementation of an Escherichia coli glycolysis mutant by Giardia lamblia triosephosphate isomerase. Exp Parasitol 78: 85–92 Müller J, Ley S, Felger I, Hemphill A, and Müller N (2008) Identification of differentially expressed genes in a Giardia lamblia WB C6 clone resistant to nitazoxanide and metronidazole. J Antimicrob Chemother 62: 72–82 Nash T and Keister D (1985) Differences in excretory-secretory products and surface antigens among 19 isolates of Giardia. J Infect Dis 152: 1166–1171 Paget T, Jarroll E, Manning P, Lindmark D, and Lloyd D (1989) Respiration in the cysts and trophozoites of Giardia muris. J Gen Microbiol 135: 145–154 Paget T, Manning P, and Jarroll E (1993a) Oxygen uptake in cysts and trophozoites of Giardia lamblia. J Euk Microbiol 40: 246–250 Paget T, Kelly M, Jarroll E, Lindmark D, and Lloyd D (1993b) The effects of oxygen on fermentation in Giardia lamblia. Mol Biochem Parasitol 57: 65–72 Paget T, Macechko P, and Jarroll E (1998) Giardia intestinalis: metabolic changes during cytodifferentiation. J Parasitol 84: 222–226 Paget T, Maroulis S, Mitchell A, Edwards M, Jarroll E, and Lloyd D (2004) Menadione-generated radicals kill Giardia intestinalis trophozoites and cysts. Microbiology 150: 1231–1236 Paget TA, Raynor MH, Shipp DW, and Lloyd D (1990) Giardia lamblia produces alanine anaerobically but not in the presence of oxygen. Mol Biochem Parasitol 42: 63–67 Palm J, Weiland M, Griffiths W, Ljungström I, and Svärd S (2003) Identification of immunoreactive proteins during acute human giardiasis. J Infect Dis 187: 1849–1859 Phillips NF and Li Z (1995) Kinetic mechanism of pyrophosphate-dependent phosphofructokinase from Giardia lamblia. Mol Biochem Parasitol 73: 43–51

137 Ringqvist E, Palm J, Skarin H, Hehl A, Weiland M, Davids B, Reiner D, Griffiths W, Eckmann L, Gillin F and Svärd S (2008) Release of metabolic enzymes by Giardia in response to interaction with intestinal epithelial cells. Mol Biochem Parasitol 159: 85–91 Roxström-Lindquist K, Ringqvist E, Palm D, and Svärd S (2005) Giardia lamblia-induced changes in gene expression in differentiated Caco-2 human intestinal epithelial cells. Infect Immun 73: 8204–8208 Sánchez LB (1998) Aldehyde dehydrogenase (CoA-acetylating) and the mechanism of ethanol formation in the amitochondriate protist, Giardia lamblia. Arch Biochem Biophys 354: 57–64 Sánchez LB, Galperin YM, and Müller M (2000) Acetyl-CoA synthetase from the amitochondriate eukaryote Giardia lamblia belongs to the newly recognized superfamily of acyl-CoA synthetases (nucleoside diphosphate-forming). J Biol Chem 275: 5794–5803 Schofield PJ, Costello M, Edwards MR, and O’Sullivan WJ (1990) The arginine dihydrolase pathway is present in Giardia intestinalis. Int J Parasitol 20: 697–699 Schofield PJ, Edwards MR, and Kranz P (1991) Glucose metabolism in Giardia intestinalis. Mol Biochem Parasitol 45: 39–47 Schofield PJ, Edwards MR, Matthews J, and Wilson JR (1992) The pathway of arginine catabolism in Giardia intestinalis. Mol Biochem Parasitol 51: 29–36 ener K, Shen Z, Newburg D, and Jarroll E (2004) Amino sugar phosphate levels change during formation of the Giardia cyst wall. Microbiology 150: 1225–1230 ener K, van Keulen H and Jarroll E (2009) Giardan: structure, synthesis, regulation and inhibition. In: Giardia and Cryptosporidium from molecules to disease (G. Ortega-Pierres S. Cacciò, R. Fayer, T. Mank, H. Smith and R.C. Thompson, eds.). CABI Press, Cambridge, pp 382–397 Stefanic S, Palm D, Svärd S, and Hehl A (2006) Organelle proteomics reveals cargo maturation mechanisms associated with Golgi-like encystation vesicles in the early-diverged protozoan Giardia lamblia. J Biol Chem 281: 7595–7604 Steimle P, Lindmark D, and Jarroll E (1997) Purification and characterization of glucosamine 6-phosphate isomerase from encysting Giardia. Mol Biochem Parasitol 84: 149–153 Steuart R (2010) Proteomic analysis of Giardia: studies from the pre- and post-genomic era. Exp Parasitol 124: 26–30 Steuart R, O’Handley R, Lipscombe R, Lock R, and Thompson R (2008) Alpha 2 giardin is an assemblage A-specific protein of human infective Giardia duodenalis. Parasitology 135: 1621-1627 Strandén A and Köhler P (1991) Swiss Giardia isolates of different host origin show great similarities in their metabolism. Parasitol Res 77: 455–457 van Keulen H, Steimle P, Bulik D, Boroviak R, and Jarroll E (1998) Cloning of two putative Giardia lamblia glucosamine 6-phosphate isomerase genes only one of which is transcriptionally active during encystment. J Euk Microbiol 45: 637–642 Wang CC and Aldritt S (1983) Purine salvage networks in Giardia lamblia. J Exp Med 158(5): 1703–1712 Wenman W, Meuser R, and Wallis P (1986) Antigenic analysis of Giardia duodenalis strains isolated in Alberta. Can J Microbiol 32: 926–929

Section III Cellular Biology of Giardia

The Ultrastructure of Giardia During Growth and Differentiation Marlene Benchimol and Wanderley De Souza

9

Abstract

9.1 Introduction

The life cycle of Giardia lamblia is comprised of two developmental stages: trophozoite and cyst. In this chapter, we review the structural organisation of the protozoan during development. The trophozoite displays a pear-shaped appearance, which contains two nuclei, a highly elaborated cytoskeleton made of microtubules and microtubule-associated proteins that assemble in structures, such as the adhesive disc, the median body, the funis and four pairs of flagella. The cytoplasm contains ribosomes, glycogen particles and a network of tubular structures, which are part of the endoplasmic reticulum. This network reaches the more peripheral regions, establishing continuity with peripheral vesicles. The trophozoite cell surface not only contains a coating mainly made of variant surface proteins but also contains some glycoproteins. Two nuclei, containing a central nucleolus, are observed in each trophozoite. Ultrastructural changes take place during protozoan division where an extranuclear spindle is formed, and the adhesive disc participates in the process of karyokinesis. Under certain stimuli, the trophozoites start a process known as encystation, which leads to their transformation into cysts. Significant structural changes take place during this process, involving the appearance of clefts in the endoplasmic reticulum and formation of large encystation vesicles, which migrate towards the cell periphery, fuse with the plasma membrane and release their contents to form the cyst wall. The cyst forms contain two to four nuclei, whilst the disc fragments and the flagella are internalised. During excystation, the parasite leaves the husk and undergoes cytokinesis, forming two new trophozoites.

Giardia lamblia (syn. intestinalis or duodenalis) is the causative agent of giardiasis, an intestinal illness that affects adults and children worldwide. Giardia cycles as two morphological distinct forms: a swimming trophozoite (Fig. 9.1A), which attaches to intestinal epithelial cells leading to the characteristic symptoms of giardiasis and a highly infective cyst, which is responsible for transmission of the disease (Fig. 9.1B). Transformation between these two developmental stages involves significant morphological (Fig. 9.2), biochemical and physiological changes, which take place during a process known as encystation (i.e. trophozoite to cyst transformation) and excystation (i.e. cyst to trophozoite transformation) (Fig. 9.2). In this chapter, we will review basic aspects of the structural organisation of (a) interphasic (Figs. 9.3, 9.4) and dividing trophozoites (Fig. 9.5), (b) the process of synthesising cyst wall components leading to the formation of cysts (encystation process) (Fig. 9.2), (c) the fine structure of the cyst stage (Fig. 9.6) and (d) the transformation of cysts into trophozoites (Excystation process) (Fig. 9.2). For the majority of this chapter, we will focus on data obtained using several microscopic techniques because other chapters of this book deal with the cell biology and biochemistry of the same structures. The trophozoite form of G. lamblia has a characteristic pear-shaped body (Figs. 9.1, 9.4) that is 12–15 Pm long and 5–9 Pm wide. By light microscopy (Fig. 9.7), Giardia looks like a smiling face, where the eyes are the two nuclei and the mouth is the median body line. Several cytoskeletal structures,

H. D. Luján et al. (eds.), Giardia © Springer-Verlag/Wien 2011

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M. Benchimol and W. De Souza

A

B

1 μm

1 μm

Fig. 9.1 Scanning electron microscopy of G. lamblia in a A trophozoite form and B cyst. D Disc; AF anterior flagella; LF posteriorlateral flagella; VF ventral flagella and CF caudal flagella. Bar: 1 Pm

Fig. 9.2 Schematic diagram of a Giardia cell cycle. (1) Trophozoite form presenting eight flagella, median body, disc and two nuclei. (2) Encystation: protusions corresponding to the ESV (encystation-specific vesicles) are noted, which will originate the cyst wall. (3) Cyst: presence of a cyst wall, two to four nuclei, the disc is fragmented in four segments and the flagella are completely internalised. (4) Early excystation. The cyst wall presents an opening from which the flagella begin to externalise and beat. Presence of new vesicles containing enzymes that helps the distention of the cyst opening, from which the parasite emerges. (5) Late excystation. The parasite has already escaped from the cyst. An oblong-shaped cyst husk is left behind. (6) Excyzoites. After the exit, the parasite proceeds with cytokinesis forming two new trophozoites, leaving an empty husk behind

Chap. 9 The Ultrastructure of Giardia During Growth and Differentiation

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Fig. 9.3 TEM routine preparation of Giardia showing the two nuclei (N), the ventral disc (D), peripheral vesicles (P), flagellar axonemes (A), flagella (F) and mitosomes (M). Bar: 1 Pm

Fig. 9.5 Binary division process in G. lamblia. Bar: 1 Pm. From Benchimol (2007)

Fig. 9.4 Schematic diagram showing the arrangement of cell structures in Giardia. Note the disc (D) situated at the ventral region, the two nuclei (N), the flagellar pairs and basal body pairs (BB). The median body (MB) is seen transversely to the axonemes, and the funis (F) is formed by microtubules connecting the central axonemes to the postero-lateral flagella axonemes. AF Anterior flagella; LF posterior-lateral flagella; VF ventral flagella; CF caudal flagella and PV peripheral vesicles. Bar: 1 Pm

mainly made of microtubules, can be seen in the cytoplasm of these organisms (Figs. 9.3, 9.8). Giardia also contain a ventral disc (Figs. 9.3, 9.8), me-

dian body (Figs. 9.7, 9.9), funis (Figs. 9.8, 9.12) and four pairs of flagella (anterior, posterior, caudal and ventral) (Figs. 9.4, 9.8). Other structures include the peripheral vesicles (Figs. 9.3, 9.10, 9.20), ribosomes and glycogen granules (Fig. 9.11). The endoplasmic reticulum (Figs. 9.3, 9.12) is seen around the nuclei (Fig. 9.3) and radiates towards the cell periphery, whilst mitosomes (Figs. 9.3, 9.13), mitochondrial remnant organelles are present mainly between the basal bodies (Figs. 9.3, 9.13). The cysts are oval in shape (Fig. 9.1B) approximately 5 by 7–10 Pm in diameter and contain four nuclei (Fig. 9.6). They are covered with a wall that is 0.3–0.5 Pm thick and composed of a web of 7–20 nm filaments.

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9.6A

M. Benchimol and W. De Souza

9.6B

9.7

Fig. 9.6 A Cyst of G. lamblia by TEM and B a schematic diagram. The flagella (F) are internalised, and the disc is fragmented (FD). The flagella axonemes (Ax), nuclei (N), vesicles (V) and the peritrophic space (PS) are seen. Note the cyst wall (CW). Bar: 500 nm Fig. 9.7 Light microscopy of G. lamblia where a smiling face can be noted: the “eyes” are the two nuclei, and the mouth is the median body. Bar: 1 Pm

A

B

Fig. 9.8 High-resolution field emission scanning electron microscopy of G. lamblia after detergent treatment. The plasma membrane was removed exposing the cell interior, allowing the funis visualisation and the disc (D) microtubules. The funis microtubules (Fn) are clearly seen anchored to the posterior-lateral flagella (P). Bar: 1 Pm. From Benchimol et al. (2004)

9.2 The Cell Surface It is generally thought that the actual surface of G. lamblia trophozoites is relatively smooth, as suggested by the examination of thin sections by transmission electron microscopy and of the whole

cell by conventional scanning electron microscopy. However, the observations by transmission electron microscopy of fracture-flip replicas revealed the presence of numerous globular particles with diameters ranging from 10 to 15 nm and surface undulations localised over the dorsal region (Kattenbach

Chap. 9 The Ultrastructure of Giardia During Growth and Differentiation

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Fig. 9.9 Scanning electron microscopy of G. lamblia after detergent treatment. The plasma membrane was removed exposing the cell interior, allowing the visualisation of median body. Note that the median body presents different bundles of microtubules. Bar: 1 Pm. From Piva and Benchimol (2004)

Fig. 9.10 TEM of a detail of G. lamblia surface by transmission electron microscopy where it is possible to note that the peripheral vesicles may acquire a typical tubular shape (asterisk) due to the continuity between the vesicles. Small vesicles (arrowhead) are also seen close to these tubules. Bar: 300 nm. From Lanfredi-Rangel et al. (1998)

et al., 1991). The surface coat of Giardia trophozoites is compact, mainly due to the presence of Variant Surface Proteins (VSPs), as shown by transmission electron microscopy and fracture labelling (Pimenta et al., 1991; Prucca and Lujan, 2009). Apparently, few glycosylated proteins are exposed on the surface of these trophozoites. Of the thirteen lectins tested, only D-GlcNAc specific lectins

bound to the protozoan surface (Ward et al., 1988). Several membrane proteins have been identified. Some of these proteins display lectin-like activity, such as a 148 kDa protein with specificity for D-mannose (Sreenivs et al., 1995) and a 28/30 kDa protein known as taglin, which has specificity for mannose-6-phosphate (Lev et al., 1986; Ward et al., 1987).

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D

300 nm

FL

Fig. 9.11 G. lamblia after Thiéry’s technique, which reveals carbohydrates. Note the positive reaction seen in all membranes, such as the nuclear envelope. Intense labelling occurs in the glycogen granules (GL) distributed throughout cytoplasm. D Disc, FL flange, Ax flagella axonemes, N nucleus, Nu nucleolus. Bar: 300 nm

It is important to point out that fracture-flip, where frozen cells are initially fractured, a carbon replica obtained, inverted and then coated with a thin layer of platinum, allows high-resolution observation of the actual cell surface. Deep-etching views of the surface also revealed the presence of rugosities, with a mean diameter of 30 nm, especially on the flagellar membranes. It is possible that some of these structures correspond to macromolecules exposed on the protozoan surface, especially variant surface antigens (Pimenta et al., 1991). Freeze-fracture replicas have

been used to examine the inner organisation of the G. lamblia trophozoites plasma membrane. It has been shown that the density of intramembranous particles, which correspond to integral membrane proteins, is higher on the protoplasmic face than on the extracellular fracture face of the membrane lining the cell body. Very low densities of membrane particles were observed on the fracture faces of the flagellar membrane, indicating differences in the structural organisation of the membranes (Kattenbach et al., 1991). At the lateral crest, a specialised region of the ventral disc, which contained a horseshoe-shaped membrane domain characterised by the near absence of intramembranous particles, was observed (Chavez and Martinez-Palomo, 1995). Cells incubated in the presence of filipin, a polyenic antibiotic that binds to cholesterol and forms complexes easily visualised in freeze-fracture replicas, showed the presence of fewer filipin-cholesterol complexes on the fracture faces of the membrane lining the lateral crest when compared with the membranes lining other regions of the protozoan surface. These observations indicate the existence of microdomains within the plasma membrane of G. lamblia. It has been suggested that the low cholesterol content of the plasma membrane lining within the lateral crest may provide greater flexibility to the organism, thus facilitating the contraction of the outer rim of the ventral disc (Chavez and Martinez-Palomo, 1995).

Fig. 9.12 Cytochemical localisation of glucose-6-phosphatase, an enzyme marker of the endoplasmic reticulum (ER). Reaction product is seen in cisternae of the ER and in the nuclear membrane. The nuclear pores are seen (asterisks). Note the funis (Fu) in close proximity to flagella axonemes (Ax). Bar: 300 nm. From Lanfredi-Rangel et al. (1998)

Chap. 9 The Ultrastructure of Giardia During Growth and Differentiation

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Fig. 9.13 Mitosomes. TEM routine preparation of Giardia showing a partial nucleus (N), one of the flagellar axonemes (Ax) and the mitosomes (M). Bar: 200 nm

Fig. 9.14 Protusions. Scanning electron microscopy of G. lamblia after 18 h in the encystantion medium. Note several protusions (asterisks) corresponding to the ESV (encystantion specific vesicles). Bar: 1 Pm. From Bittencourt-Silvestre et al. (2010)

9.3 The Cytoskeleton A simple examination by light microscopy of stained cells (Fig. 9.7) or Nomarski light microscopy (Fig. 9.15) reveals the presence of four pairs of flagel-

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Fig. 9.15 A DIC; B Fluorescence. Giardia stained with antitubulin (green). The nuclei are labelled in blue after DAPI. Bar: 1 Pm

Fig. 9.16 Funis. TEM of a cross-section in the region of the funis (Fu) showing the array of microtubules. Note that it is possible to distinguish protofilaments arrangement in the microtubules. Ax Flagella axonemes. Bar: 30 nm. From De Souza et al. (2004)

la, the nuclei and other dense cytoplasmic structures. When permeabilised cells are incubated in the presence of a wide spectrum of anti-tubulin antibodies

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and then a labelled secondary antibody, immunofluorescence microscopy reveals the presence of additional tubulin-containing structures (Fig. 9.15). This picture is clearer when thin sections of cells, processed using procedures that better preserve cytoskeletal components, are observed by Transmission Electron Microscopy (TEM). One characteristic feature of the Giardia cytoskeleton is that it is composed mainly of stable microtubular structures, as observed in the four pairs of flagella (Fig. 9.1A), in the ventral or adhesive disc (Figs. 9.3, 9.8), the median body (Figs. 9.7, 9.9, 9.15), the funis (Figs. 9.8, 9.12, 9.16) and smaller sheets of closely apposed microtubules resembling a rudimentary axostyle (Soltys and Gupta, 1994; Campanati et al., 2003).

9.4 The Flagella Four pairs of symmetrically disposed flagella (Fig. 9.1A) begin in basal bodies localised in a region between the two nuclei, and are named the anterior or anterolateral, posterior or postero-lateral, ventral and caudal flagella. Three pairs are directed to the posterior region of the cell, whilst the anterolateral pair is anteriorly directed (Erlandsen and Feely, 1984; Campanati et al., 2002). This organisation reflects the forward dislocation of the parasite (Erlandsen and Feely, 1984; Gosh et al., 2001; Campanati et al., 2002), although each pair behaves differently during swimming.

Fig. 9.17 Paraflagellar Rod. Transmission electron microscopy showing the filamentous network which makes the paraflagellar rod (arrows) connected to the flagellum (F). Bar: 200 nm

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The structure of Giardia’s flagella, except the ventral flagella, is similar to other eukaryotic cells, which contain a central pair and nine concentric doublets of microtubules (9 + 2). The ventral flagella displays a membrane projection filled with a dense material of unknown composition (Holberton, 1973), which has also been described as a fin equipped with a paraflagellar rod (Fig. 9.17) connected to the flagellum by thin filaments (Kulda and Nohýnková, 1995). A 30 kDa polypeptide was identified as the main constituent of this structure (Clark and Holberton, 1988). The analysis of flagella isolated by centrifugation in a Percoll gradient showed that about one-fourth of the ventral flagella presented the rods associated with them and that the material disappears following treatment of the flagella with Triton X-100. Purified intact flagella contained axonemal proteins and an additional set of 30 kDa polypeptides, which do not co-migrate with giardins or present the same charge (Crossley et al., 1986). An antibody raised against this protein labelled a portion of the ventral flagella (Crossley et al., 1986). A paraflagellar rod-like structure has also been described in the anterior and posterior flagella. It appears as irregular masses of dense fibres or dense rods, which run along the intracellular portion of the anterior and posterior axonemes. Each rod follows the inner part of the axoneme for three quarters of the distance between the basal body and the beginning of the free flagella. The rods associated with the posterior flagella are shorter and are associated with the axoneme in the region where they run along the disc cytoskeleton (Friend, 1966). Other “forgotten” cytoskeletal structures that are associated with the anterior flagella (but on the opposite side of where the dense rods are located) and as part of the ventro-lateral flange cytoskeleton, include the marginal plates or striated fibres, whose structure and function have not yet been fully described. These plates are more developed in trophozoites of Giardia muris (Sogayar and Gregorio, 1989) and were first described by Friend (1966) who designated them as the paracrystalline structure of the flange cytoplasm. Holberton also described them as being Triton X-100 soluble. In addition to all the classical flagellar proteins, recent studies have pointed towards the presence of giardins in the flagella (Weiland et al., 2005). Alpha 14-giardin (annexin XXI, EI) was associated with fla-

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Fig. 9.18 The disc: microtubules and microribbons. High magnification of a partial view of the disc where microtubules (arrowhead) and microribbons (asterisk) are seen. Bar: 100 nm

gellar microtubules at local slubs near the proximal part and at the ends of flagella (Vahrmann et al., 2008; Pathuri et al., 2009), whilst Alpha 19-giardin was detected only in the ventral flagella (Saric et al., 2009). A kinesin 13 homologue was also localised to the tip and the cytoplasmic portion of all Giardia flagella (Dawson et al., 2007). EB1, a microtubule-associated protein involved in organisation of microtubules, was found in axonemes and in the median body (Kim et al., 2008).

9.5 The Ventral or Adhesive Disc The ventral or adhesive disc (Figs. 9.3, 9.18, 9.20) is composed of a spiral layer of evenly spaced microtubules, which are connected to the adjacent plasma membrane by thin filaments of unknown composition (Holberton, 1973; Benchimol, 2004a; Sant’Anna et al., 2005). Microribbons (Fig. 9.18) extend from the microtubular wall towards the cytoplasm. The ventral disc adhesive proteins have been studied in some detail and will be discussed in another chapter. By working with isolated discs and breaking the cross bridges that connect disc microtubules, Holberton (1981) obtained detailed images of the ribbons. The periodic and optical diffraction patterns showed essentially a paracrystalline structure. Using the quick-freezing, freeze-fracture, deep-etching and rotary replication technique it was shown that the ribbons are arranged in a parallel array with an interval of 50 nm. It appears that 18 nm thick filaments, connected to each other by short bridges, are regularly spaced at intervals of about 40 nm. Regularly spaced filamentous structures were seen connecting the microtubules to a membranous structure (Kattenbach et al., 1996). Major components of the adhesive disc consist of various forms of tubulins and giardins. Vinculin was also seen in

this structure (Narcisi et al., 1994). The biochemical data on the adhesive disc will be described in another chapter.

9.6 The Median Body The median body is transversally located to the major axis of the cell (Figs. 9.4, 9.7, 9.9, 9.15). This structure, essentially made of microtubules, has been used for a long time as a taxonomic tool to distinguish Giardia species (Filice, 1952). Other proteins, such as kinesin 13 (Dawson et al., 2007), EB1, a microtubule-associated protein involved in the organisation of microtubules (Kim et al., 2008) and a 101 kDa coiled coil protein involved in microtubule bundling reminiscent of beta-giardin, are also localised to the median body. The functional role played by the median body has not yet been established. It has been proposed that it represents a site for microtubule nucleation (Holberton and Ward, 1981) and, during interphase, it is a reserve for microtubules that could be used for extranuclear spindle assembly (Meng et al., 1996). Based on the observations of Feely and Erlandsen (1982) and others (Meng et al., 1996; Correa et al., 2004; Piva and Benchimol, 2004), the median bodies are (1) composed of contractile and calcium-binding proteins; (2) are not one or two structures, but vary in number, shape and position (Fig. 9.9); (3) are found both in mitotic and interphasic trophozoites; (4) are present in about 80% of the cells; (5) could be connected either to the plasma membrane, to the adhesive disc or to caudal flagella and are not completely free in the cells; (6) can protrude on the cell surface; and (7) their microtubules react with several anti-tubulin and beta-giardin antibodies.

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9.7 The Funis The Funis (Figs. 9.8, 9.12, 9.16) is composed of sheets of microtubules that follow the axonemes of the caudal flagella (Erlandsen and Feely, 1984; Kulda and Nohýnková, 1995; Campanati et al., 2002, 2003). The term funis was introduced by Kulda and Nohýnková (1978) to describe two longitudinal sheets of extra microtubules, also named paraxonemal fibrils by Cheissin (1964), that partially wrap the caudal flagella axonemes. These sheets start between the two nuclei in a region next to the basal bodies, and proceed until the caudal tip of the cell. Their function is related to the movement of the caudal region of the cell (Cheissin, 1964; Kulda and Nohýnková, 1978; Campanati et al., 2002; Benchimol et al., 2004; Carvalho and Monteiro-Leal, 2004). Recent observations on the structure and function of the funis show that it is composed of microtubules that besides running until the posterior region of the cell, also spread in the direction of the cytoplasm, plasma membrane and the dense rods of the lateral flagella (Carvalho and Monteiro-Leal, 2004; Piva and Benchimol, 2004). No evidence for the presence of actin in this structure has been obtained, suggesting that its participation in cell movements might be due to microtubule polymerisation/depolymerisation (Piva and Benchimol, 2004) or to a yet to be described novel protein.

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confocal laser scanning microscopy (Castillo-Romero et al., 2009) and (c) morphological changes in cells treated with drugs which in other cells have been shown to interfere with filaments, such as cytochalasin D, Latrunculin A and jasplakinolide (Feely and Erlandsen, 1982; Katelaris et al., 1995; Souza et al., 2001; Correa and Benchimol, 2006; Castillo-Romero et al., 2009). These effects included (a) a change in the morphology of the trophozoite, including fragmentation of the ventral disc, formation of large vacuoles in the cytoplasm, flagella alterations, damage of the caudal regions, cytokinesis but not karyokinesis blockage and the presence of membrane undulations and blebs; (b) inhibition of cell growth; (c) inhibition of adhesion; (d) interference with the process of encystation; (e) reduction in the intensity of labelling and changes in the distribution of the labelling pattern in cells labelled with TRITC-phalloidin and (f) interference with ceramide internalisation and intracellular localisation (Hernandez et al., 2007). The experiments using jasplakinolide deserve an additional comment. Even in cells where no actin filaments have been found, jasplakinolide is able to induce the assembly of typical actin filaments when actin oligomers exist. In the case of Giardia, however, jasplakinolide treatment did not induce the assembly of the microfilaments. Immunofluorescence microscopy using heterologous antibodies, which is an indirect approach, showed labelling of actin-associated proteins (Feely and Erlandsen, 1982; Narcisi et al., 1994).

9.8 Microfilaments Typical microfilaments clearly identified by TEM have not been seen in Giardia. However, an actin gene was sequenced and shown to be present as a single copy. The predicted protein sequence shows an average of 58% amino acid identity with the actin of other eukaryote species, indicating a high level of divergence (Drouin et al., 1995). There is indirect evidence for the presence of actin in this organism. Actin observation includes (a) a faint labelling of structures, such as the periphery of the adhesive disc, the median body and between the nuclei, where the basal bodies are located, using anti-actin antibodies and immunofluorescence microscopy (Feely and Erlandsen, 1982; Narcisi et al., 1994), (b) labelling of the same structures and the flagella in cells incubated in the presence of TRITC-phalloidin and observation by

9.9 The Endocytic System One characteristic feature of Giardia trophozoites is the presence of a large number of vesicles, known as the peripheral vesicles (Figs. 9.3, 9.10, 9.20), which are mainly located along the periphery of the protozoan, except at the region where the adhesive disc is located (Friend, 1966; Adam, 1991; De Souza et al., 2009). These vesicles accumulate a large number of endocytic probes, such as horseradish peroxidase, Lucifer Yellow, FITC-labelled albumin, transferrin and LDL and biotin-labelled fluorospheres. Live cell imaging shows that internalisation starts at the sites where the flagella exit the cell in the region caudal to the adhesive disc (Abodeely et al., 2009). These observations show that the peripheral vesicles (Figs. 9.3,

Chap. 9 The Ultrastructure of Giardia During Growth and Differentiation

9.10, 9.20), correspond to endocytic vesicles and confirm previous studies indicating the involvement of receptor-mediated endocytosis in the uptake of lipids (Lujan et al., 1996). In some experiments, the labelling migrates towards the central region of the cell and accumulates at the perinuclear region. Peripheral vesicles labelled with acridine orange indicated that they are acidic (Kattenbach et al., 1991; LanfrediRangel et al., 1998). Further cytochemical characterisation of the peripheral vesicles showed that some, but not all of the peripheral vesicles, were positive for acid phosphatase and glucose-6-phosphatase (Feely and Dyer, 1987; Kattenbach et al., 1991; Lanfredi-Rangel et al., 1998). These observations point to the fact that the peripheral vesicles could also

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be considered as part of the endo-lysosomal system, as revealed by labelling for acid phosphatase, and the endoplasmic reticulum, as revealed by labelling for glucose-6-phosphatase, protein disulphide isomerase, Hsp70-BIP and KDEL, which contains the ER retention signal. Labelling for KDEL predominated at the perinuclear region (Abodeely et al., 2009). Thus, it was proposed that G. lamblia contains an endosomal-lysosomal system concentrated as a single system, and that the peripheral vesicles represent an ancient organellar system that later subdivided into compartments, such as early and late endosomes and lysosomes (Lanfredi-Rangel et al., 1998). A connection between the peripheral vesicles and the endoplasmic reticulum network (Lanfredi-Rangel et al., 1998) (Fig. 9.19) was designated as the tubular vesicular network (TVN) (Abodeely et al., 2009). The existence of such a system explains the diffusion of ingested material from the periphery of the cell towards the perinuclear region (Abodeely et al., 2009). It is important to note that under certain conditions Giardia is also able to ingest bacteria (Sogayar and Gregorio, 1989).

9.10 The Secretory System

Fig. 9.19 Three-dimensional reconstruction based on cryotomograms of G. lamblia trophozoites after cytochemistry for localisation of glucose-6-phosphatase, an enzyme marker of the endoplasmic reticulum. The network of tubules which constitute the TVN is shown in yellow. The ER cisternae are green and the peripheral vacuoles labeled with glucose-6-phosphatase in blue. From Abodeely et al. (2009)

In all eukaryotic cells the secretory system is composed of the endoplasmic reticulum, intermediate vesicles, the Golgi complex and secretory vesicles. These vesicles migrate in conjunction with microtubules and microtubule-associated proteins towards both the cell periphery, where they fuse and release the vesicles content, or to the endocytic pathway fusing with late endosomes and lysosomes. In G. lamblia trophozoites, the endoplasmic reticulum (ER) is well developed forming a complex tubular network extending from the perinuclear region towards the cell periphery (Figs. 9.12, 9.20). There is still some controversy about the presence of a typical Golgi complex in trophozoites of G. lamblia. Using morphological criteria, a typical Golgi complex does not exist in Giardia although the existence of some concentric membranes has been published (LanfrediRangel et al., 1999). However, there are several pieces of evidence pointing to the existence of a Golgi equivalent structure as is discussed in another chapter in this book.

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Fig. 9.20 Freeze-fracture of G. lamblia showing the diversity of the distribution of nuclear pores in the nuclei (N). Notice that the nuclear pore distribution (arrows) is distinct in both nuclei. PV Peripheral vesicles, ER endoplasmic reticulum, D disc. Bar: 1 Pm. From Benchimol (2004a) with kind permission from Springer Publisher

9.11 Glycogen Particles

9.13 The Interphasic Nuclei

Glycogen granules are present in G. lamblia (Fig. 9.11) and act as an energy reserve in trophozoites of this organism (Benchimol, 2004a; Ladeira et al., 2005). In cysts, a large amount of this complex and ramified polysaccharide is also frequently observed in viable cysts, and is probably consumed during the dormant period. The granules are intensely labelled when thin sections of epoxy-embedded cells are treated sequentially with periodic acid, thiosemicarbazide and silver proteinate, a classic technique to reveal carbohydratecontaining structures (Benchimol, 2004a).

Of all the cellular features within the trophozoite, the most intriguing is the presence of two nuclei (Figs. 9.3, 9.4, 9.7, 9.15, 9.20) which are bilaterally symmetrical. Both Giardia nuclei are oval-shaped of about 1 μm in diameter each, located in the anterior half of the cell, at the left and right sides of the longitudinal axis.

9.12 Mitosomes The trophozoite form of this protist lacks organelles found in higher eukaryotes, such as traditional mitochondria and peroxisomes (Gillin et al., 1996), although Tovar and colleagues (2003) demonstrated that Giardia contains mitochondrial remnant organelles (mitosomes) bound by double membranes (Figs. 9.3, 9.13), which function in iron–sulphur protein maturation. This finding indicates that Giardia is not primitively amitochondrial and that it has retained a functional organelle derived from the original mitochondrial endosymbiont. Mitosomes appear as small cellular structures distributed throughout the cytoplasm, especially around the basal bodies. This organelle will be further analysed in another chapter of this book.

Fig. 9.21 Nucleolus in Giardia lamblia seen by TEM: a nucleus is shown in high magnification where an evident nucleolus (n), nuclear pores (p) and nuclear envelope (m) are seen. Bar: 200 nm. From Tian et al. (2010), with permission of Springer

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The nuclei of trophozoites have been studied using different morphological methods, such as fluorescence microscopy (Fig. 9.15), field emission scanning electron microscopy, routine scanning and transmission electron microscopy (Figs. 9.3, 9.8), freeze-fracture (Fig. 9.20), immunocytochemistry and 3-D reconstruction (Fig. 9.19) (Solari et al., 2003; Benchimol, 2004b, 2005, 2007; Nohýnková et al., 2006; Sagolla et al., 2006). Until recently, Giardia has been described as an “anucleolated” organism; however, studies have shown that each of the two nuclei contains a single small, deeply stained granular nucleolus and rDNA, thus demonstrating that Giardia does indeed have nucleoli (Figs. 9.11, 9.21) (Jiménez-García et al., 2008; Tian et al., 2010).

ever, until now the kinetochore-spindle microtubule associations have not been observed. Currently, the origin of the spindle microtubules is still an open question. The Giardia nuclei are connected by filamentous proteinaceous structures to the anterior flagella and the posterior-lateral and ventral flagella on the right and left sides, respectively (Benchimol, 2007). Consequently, nuclei and flagella axonemes can migrate together during mitosis. These links would explain the behaviour of parental nuclei, which can be maintained together during the dividing process, whereas the daughter nuclei are distributed to the new cell.

9.13.1 The Two Nuclei Present Slight Differences

Evidence has been presented showing the participation of the ventral disc in the karyokinesis (Fig. 9.22) (Solari et al., 2003; Benchimol, 2004a, 2007). Other groups (Ghosh et al., 2001; Yu et al., 2002) have discussed whether the two nuclei are partitioned equationally during mitosis and both agree that the division is equational rather than reductional. Nohýnková will present details of Giardia division in another chapter of this book.

Giardia has two diploid nuclei (2n = 10), which are bilaterally located. Important studies have been done on the behaviour of Giardia nuclei (Wiesehahn et al., 1984; Kabnick and Peattie, 1990; Yu et al., 2002). The DNA content of trophozoites and cysts is localised in five chromosome-like bodies within each nucleus (Erlandsen and Rasch, 1994). Morphological studies using different techniques suggested that the two nuclei in Giardia are not functionally equal because (1) both nuclei in the same cell are distinct in nuclear pore number and distribution (Fig. 9.20) (Benchimol, 2005); (2) nuclear pore complexes are frequently clustered in nuclear envelope domains and (3) dividing nuclei display very few nuclear pores (Benchimol, 2004a).

9.13.2 The Nuclei in Division Giardia divides by binary fission (Fig. 9.5). The steps of Giardia mitosis have been difficult to elucidate because of the presence of the two nuclei. Giardia undergoes semi-open mitosis because the nuclear envelope is present throughout mitosis and openings in the nuclear envelope are seen at the nuclei poles, allowing spindle microtubules to enter the nucleus (Nohýnková et al., 2000; Sagolla et al., 2006). How-

9.14 Karyokinesis and Disc Participation

9.15 The Fine Structure of the Encystation Process Modification of the culture medium has permitted the completion of the Giardia life cycle in vitro (Gillin et al., 1989). Since the development of the encystation medium, studies on Giardia biology have progressed. Reiner and co-workers (1990) described the ESV (encystation-specific vesicles) (Fig. 9.23) observed during the process of encystation. According to these authors, the ESV transport cyst proteins to the cell surface for the formation of the cyst wall. These authors demonstrated that when exposed to encystation stimuli (elevated pH and bile), Giardia lamblia developed vesicles (ESV), which are not normally observed in non-encysting trophozoites. Because the process of encystment is asynchronous, it is not possible to accurately pinpoint the time of the appearance of each stage. The Reiner group (Reiner et al., 1990) also

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Fig. 9.22 Karyokinesis in G. lamblia. Note the disc (D) participation in the nucleus (N) division. Ax Flagella axonemes; C chromatin. Bar: 300 nm. From Benchimol (2007)

A

B

Fig. 9.23 ESVs. Transmission electron microscopy of trophozoites in the process of encystation. Electron dense material is seen within the encystation vesicle both in clefts (asterisk in A) and in vesicles that are still in close connection with profiles of the endoplasmic reticulum (ER) in B. ESV encystation vesicle. Bars: 300 nm. From Lanfredi-Rangel et al. (2003)

showed that cyst antigens were concentrated in ESV early in encystation, and were observed in the cyst wall later in differentiation, thus supporting the idea that ESV comprise a regulated pathway for transport of cyst wall components to nascent walls. As in all eukaryotic cells, trophozoites of Giardia lamblia synthesise proteins in the endoplasmic reticulum. Certainly, the more elaborate secretory system in Giardia occurs during the process of transformation of the trophozoites into their cystic forms, when there is formation of a cyst wall (Gillin et al., 1991, 1996; Marti and Hehl, 2003; De Souza et al., 2004). It has been considered that the cyst wall, which is formed by interconnected filaments containing peptides and carbohydrate moieties (Manning et al., 1992), is assembled because of exocytosis of the encystation vesicles (Gillin et al., 1991, 1996; Erlandsen et al.,

1996). Morphological studies of cells incubated for different periods in the presence of encystation medium showed morphological changes, such as a distinct type of vesicle, ESVs (encystation vesicles), first observed 6 h after encystation induction. These vesicles progressively increased in volume, ending with the release of their contents for assembly of the cyst wall. Almost all ER cisternae, where the ESVs are formed, contain amorphous material, are often triangular (Fig. 9.23) and can be considered as nascent ESVs. Some of ESV protruded at the surface of the plasma membrane, with peripheral vesicles between the ESV and the plasma membrane. Cytochemical detection of glucose-6-phosphatase, an ER marker, at different points of encystation, allowed the establishment of a relationship between the ER and nascent ESV. Three-dimensional reconstructions from serial

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1 μm

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B

1 μm

Fig. 9.24 Encystation process. Two steps of G. lamblia encystation. In A, an immature cyst presents a tail, which corresponds to flagella in process of internalisation. In B, two mature cysts. Bar: 1 Pm (Benchimol, unpublished)

sections of cells stained for the localisation of glucose-6-phosphatase activity showed that there was a close association between the ER profiles and the early ESVs. However, such an association was no longer evident once ESVs were formed (Lanfredi-Rangel et al., 2003). Ultrastructural examination of Giardia trophozoites collected at 8 h post-bile stimulation did not show signs of the cyst wall, but alterations on cell shape were clearly observed first on the lateral flanges (Fig. 9.11). At about 14 h post-bile stimulation, Giardia trophozoites underwent a change in shape from the flattened dorso-ventral pear-shape to a more spherical or rounded appearance. Flagella internalisation was gradual, except for the caudal flagella, which formed a tail in immature cysts (Fig. 9.24). The disc is disassembled during encystation, and stored as four fragments in the immobile cyst (Fig. 9.6). Morphometry showed that there was a gradual increase in the area filled by the ER during encystation. In addition, the area occupied by the ER clefts increased 4.5-fold after 6 h. In addition, freeze-fracture has also been used to analyse the encystation process. Randomly distributed protrusions were seen on the protozoan surface (Fig. 9.14). In favourable fractures, it was possible to notice the presence of membrane-bound structures, with a mean diameter of 0.55 Pm, underlying the protrusions. These structures may correspond to encystation vesicles in the process of fusing with the protozoan plasma membrane. The protrusions could be well seen in highresolution scanning electron micrographs (Fig. 9.14) (Bittencourt-Silvestre et al., 2010).

One important step of the encystation process is the assembly of the cyst wall. Scanning electron microscopy of cells labelled with antibodies against cyst wall proteins showed that during the initial phase of cyst wall formation, small 15 nm in diameter protrusions become enlarged to caplike structures of up to 100 nm in diameter, and are observed throughout the protozoan cell surface (Erlandsen et al., 1996). Transmission electron microscopy of cells fixed in the presence of ruthenium red, which binds to anionic sites, showed the presence of reactive sites that appear as dense spots in defined zones on the surface of encysting cells (Arguello-Garcia et al., 2002). Once the fibrillar structure appeared, no labelling with ruthenium red was observed. This labelling pattern was inhibited by the addition of N-acetylgalactosamine and galactosamine to the incubation medium (ArguelloGarcia et al., 2002). Using an antibody against the 26-kDa cyst wall protein (CWP1) immunofluorescence microscopy showed surface labelling as soon as 5.5 h postinfection, whereas ruthenium red staining was observed by 9–10 h postinfection, suggesting that the exposure of the polypeptide occurs before fibril patch assembly. It is important to point out that not all cells exposing the peptide successfully encysted (Arguello-Garcia et al., 2002). For some authors there is a dispersal of ESV into small secretory vesicles before secretion (Marti and Hehl, 2003). More recently, it was suggested that a typical exocytosis does not occur. During membrane fusion some membrane segments appear to be disrupted and released into the extracellular medium where they could be resealed to form empty vesicles

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(Benchimol, 2004b). It is important to point out that during the period of encystation, G. lamblia maintains a constitutive pathway for the synthesis of the variant surface proteins (Lujan et al., 1998). These proteins are not mixed with the ESV proteins; therefore, the protozoan may have sorting mechanisms to distinguish these two export pathways. At the same time that the ESVs are formed other changes take place in the trophozoite, such as modification of the protozoan shape, shortening and internalisation of the flagella, and fragmentation of the adhesive disc (Palm et al., 2005; Midlej and Benchimol, 2009; BittencourtSilvestre et al., 2010). These changes, including the formation of the ESVs, are inhibited by compounds that interfere with the activity of PI3 kinase, tyrosine kinase and protein kinase C (Bittencourt-Silvestre et al., 2010). Distinct stages in the Giardia cyst formation include a distinct change in trophozoite shape and polarisation, from the characteristic flattened dorsal-ventral axis found in motile trophozoites to a rounded appearance, and the appearance of a “taillike” appendage in later stages of cysts formation.

9.16 The Cyst The cyst (Figs. 9.1B, 9.6, 9.24B) has an oval shape, measuring about 8–12 mm in length and 7–10 mm in

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width. It is covered by a cyst wall that is 0.3–0.5 mm thick. It contains four or two nuclei, basal bodies, axonemes located close to the nuclei and fragments of the adhesive disc (Fig. 9.6). Two distinct regions form the cyst wall with the most external being formed by bundles of 7–20 nm thick fibrils connected to each other by short and thin filaments. The inner layer contains two membranes, designated as inner and outer membranes (Erlandsen et al., 1996). It has been shown that the cyst wall is made of glycoproteins of 102, 88, 75 and 29 kDa (Erlandsen et al., 1990). Mass spectrometry and NMR data have shown the presence of E1-3-N-acetyl-D-galactosamine associated with the protein, confirming previous studies on the binding of labelled lectin to the cyst wall, as revealed by fluorescence and scanning electron microscopy (Gerwig et al., 2002).

9.17 The Fine Structure of the Excystation Process Excystation of Giardia lamblia (Figs. 9.2, 9.25) entails differentiation of dormant cysts into parasitic trophozoites, and can be easily induced in vitro. Despite its importance for infection, this transformation is not fully understood at the cellular or molecular levels. The in vitro excystation process in Giardia

Fig. 9.25 Excystation process. Giardia seen in the moment of excystation. The filaments of the cyst wall open, allowing escape of the parasite. The flagella seem to help the cell exit, leaving an empty husk. Bar: 1 Pm. This figure was artificially coloured. Photo by S.L. Erlandsen

Chap. 9 The Ultrastructure of Giardia During Growth and Differentiation

was studied (Buchel et al., 1991) using transmission electron microscopy (TEM) and demonstrated that control cysts had a thick wall made of microfibrils that appeared not to contain any weak areas. The excystation process began by the cyst wall opening at one pole, the cytoplasm retracted from the wall and the peritrophic space became progressively larger. The outer cytoplasmic envelope detached from the cyst wall and numerous small vesicles can be seen lodged between the wall and the organism, whereas the tight arrangement of the wall microfibrils is lost. The organism emerged through the posterior end of the cyst, leaving behind the empty husk (Fig. 9.2). Emergence is followed by cytokinesis where two new cells appear. The internalised flagella, which were maintained in internal vacuoles during encystation and in the cysts, are externalised during the excystation process, protruding rapidly as the parasite emerged progressively as the opening is enlarged, presumably by flagella action. Although flagella emerging from the organism are immediately distinguishable, the cell body does not show all the morphological features of the G. lamblia trophozoite. A radical rearrangement of the organism occurs gradually: initially oval in shape, the parasite becomes round, then elongates, flattens and undergoes cytokinesis. The daughter trophozoites acquire their typical morphological features: the shape, the adhesive disc with the C-shaped structure distinctly visible on the ventral surface, and the definite placement of the flagella. Thus, excystment seems to reproduce, in reverse, the late stages of encystment. A group (Bingham et al., 1979) proposed that the flagella could play a mechanical role in triggering the opening of the cyst. In a report from another group (Midlej and Benchimol, 2009), an operculum was seen during encystation before the complete closing of the Giardia cyst, suggesting that this could be a weak region, which could favour the exit of the trophozoite observed during the excystment. Another group claimed that an associated mucoid-like material is extruded during Giardia muris excystment (Coggins and Schaefer, 1984). The parasite emerges without a functional disc, but the four disc fragments are quickly reassembled into two new discs on the dividing, early excysting form (Palm et al., 2005). Thus, disc proteins are stored within the cyst, ready to be used in the rapid steps of excystation.

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Ward and colleagues (1997) reported that a protease is required for excystation of Giardia. The authors observed that specific cysteine protease inhibitors block excystation. The protease was localised to vesicles that release their contents just prior to excystation, and this protease is a member of the earliest known branch of the cathepsin B family, a primitive enzyme. In addition, Slavin and collaborators (2002) demonstrated that dephosphorylation of cyst wall proteins by a secreted lysosomal acid phosphatase is essential for excystation of Giardia lamblia.

Acknowledgements This work has been supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Financiadora de Estudos e Projetos (FINEP), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), and Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ). We are also thankful for the support from Universidade Santa Úrsula, Universidade Federal do Rio de Janeiro and Instituto Nacional de Metrologia, Normalização e Qualidade Industrial. We also acknowledge Dr. S.Q. Lu for providing with the Fig. 9.21.

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160 Saric M, Vahrmann A, Niebur D, Kluempers V, Hehl AB, and Scholze H (2009) Dual acylation accounts for the localization of {alpha}19-giardin in the ventral flagellum pair of Giardia lamblia. Eukaryot Cell 8: 1567–1574 Slavin I, Saura A, Carranza PG, Touz MC, Nores MJ, and Lújan HD (2002) Dephosphorylation of cyst wall proteins by a secreted lysosomal acid phosphatase is essential for excystation of Giardia lamblia. Mol Biochem Parasitol 122: 95–98 Sogayar MF and Gregorio EA (1989) Uptake of bacteria by trophozoites of Giardia duodenalis (Say). Ann Trop Med Paasitol 83: 63–66 Solari AJ, Rahn MI, Saura A, and Lujan HD (2003) A unique mechanism of nuclear division in Giardia lamblia involves components of the ventral disk and the nuclear envelope. Biocell 27: 329–346 Soltys BJ and Gupta RS (1994) Immunoelectron microscopy of Giardia lamblia cytoskeleton using antibody to acetylated alpha-tubulin. J Euk Microbiol 41: 625–632 Souza MC, Gonçalves CA, Bairos VA, and da Silva JP (2001) Adherence of Giardia lamblia trophozoites to Int-407 human intestinal cells. Clin Diag Lab Immunol 8: 258–265 Sreenivs K, Ganguly NK, Ghosh S, Sehgal R, and Mahajan RC (1995) Identification of a 148 kDa surface lectin from Giardia lamblia with specificity for alpha-methyl-D-mannoside. FEMS Microbiol 134: 33–37 Tian XF, Yang ZH, Shen H, Adam RD, and Lu SQ (2010) Identification of the nucleoli of Giardia lamblia with TEM and CFM. Parasitol Res [Epub ahead of print] Tovar J, Leon-Avila G, Sanchez LB, Sutak R, Tachezy J, van der Giezen M, Hernandez M, Muller M, and Lucocq JM

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Cell Cycle Regulation and Cell Division in Giardia Scott C. Dawson, Eva Nohýnková and Michael Cipriano

Abstract Cell division is a fundamental area of giardial cell biology and pathogenesis that is likely amenable to antiparasitic drugs. The giardial life cycle is characterized by a binucleate swimming trophozoite stage that colonizes the small intestine by undergoing rapid cell division and attachment, and a presumably dormant cyst stage that persists in the environment. Genome replication and cell division occur during the trophozoite stage, and again prior to encystation, although the details of cytoskeletal rearrangements during encystation/excystation remain unclear. During cell division in trophozoites, the two diploid nuclei first undergo mitosis and later the eight flagella and ventral disc are duplicated and partitioned into two daughter cells. Giardia trophozoites possess a semi-open mitosis with two extranuclear spindles which access chromatin through polar openings in the nuclear membranes. The two nuclei migrate to the cell midline in prophase with lateral chromosome segregation in the left-right axis and cytokinesis along the longitudinal plane (perpendicular to the spindles). This ensures that each daughter inherits one copy of each parental nucleus with mirror image symmetry. Before the completion of mitosis, the daughter flagella undergo a maturation process in which the parent flagella migrate and transform to different flagellar types and new flagella are built. During encystation, the flagella are internalized within the cyst, but do not completely resorb. Daughter discs are formed de novo, and likely are neither templated nor built from components of the parental disc. The two new daughter discs are assembled on the dorsal side of the cell in late telophase, and the parental disc is reorganized and disassembled, presumably to maintain attachment during division. The processes of mitosis, flagellar division, and disc division have only recently been investigated at the molecular and

H. D. Luján et al. (eds.), Giardia © Springer-Verlag/Wien 2011

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cellular levels, but each likely involves microtubule motors such as kinesins and dyneins to generate forces for repositioning of organelles during cell division.

10.1 Introduction In this review, we summarize recent progress in our knowledge concerning cell division in Giardia in the context of the cell cycle. We focus on the cell cycle, mitosis, flagellar duplication and division, and ventral disc division. Despite the recent advances in understanding cell division of the Giardia trophozoite at a cytologic level, many unanswered questions remain. Almost nothing is currently known about either the molecular mechanisms behind particular processes or the regulation of the Giardia cell cycle and the molecular mechanisms underlying cell division and mitosis.

10.2 The Cell Cycle Throughout Giardia’s Life Cycle On the basis of the conventional flow cytometric analysis of the DNA content of encysting cells, early and late cysts, and cells after excystment, a model illustrating the relationship between the Giardia cell cycle and life cycle has been proposed (Bernander et al., 2001). During its life cycle, a Giardia cell passes through two periods of genome replication that alternate with two phases of nuclear division. In a binucleate flagellated trophozoite, DNA replication (S phase of the cell cycle) precedes mitotic division and cytokinesis (M phase), and both phases are segregated by gap periods (G1 and G2). The trophozoite thus undergoes a canonical cell cycle that allows for the multiplication of a pathogenic stage of the parasite in the intestine.

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Differentiation into a cyst occurs in cells that have already replicated their DNA and are in G2 phase, the longest period of Giardia cell cycle (Bernander et al., 2001; Hofstetrova et al., 2010). A restriction point for initiating encystation is located in G2, though what determines the readiness of the G2 cell to encyst is unknown (Reiner et al., 2008). During encystation, the two nuclei divide, without intervening cytokinesis, forming four cyst nuclei (a possible variant of endomitosis), and DNA content is then duplicated (Bernander et al., 2001; Poxleitner et al., 2008). Mature Giardia cysts resemble a quadrinucleate syncytium with a replicated genome (Erlandsen and Rasch, 1994). In accordance with other Giardia species, except for G. microti, with daughter individuals fully developed and separated inside the mature cyst (Feely, 1988), cytokinesis proceeds as late as after excystment. Thus, the gap period between DNA replication and cytokinesis can be as long as several weeks or even more, depending on when the cyst has a chance to excyst. Meanwhile, the cyst must be able to survive in different external conditions, including conditions that could damage the genome, e.g., UV light. The ability of cysts to repair UV-damaged DNA has been reported (Li et al., 2008) and might be a reason why putative meiosis genes involved in DNA doublestrand breaks (DMC1A, SPO11 and HOP11) are expressed in cyst nuclei, although a role for these genes in the mitotic recombination of chromosomes in fused Giardia cyst nuclei during diplomyxis has been suggested (Poxleitner et al., 2008). What happens with a quadri nucleate cell during and after excystment is also unclear. One hypothesis, based on a combination of FACS and microscopy data, is that two rapid, consecutive cell divisions produce four trophozoites with a basic level of genome ploidy from a single cyst (Bernander et al., 2001). During the first division, the quadrinucleate cell without an adhesive disc, referred to as an excyzoite, quickly undergoes cytokinesis (within 30 min after excystation) but not nuclear division: the four intact nuclei are segregated between progeny so that each receives two nuclei with already replicated DNA. Each progeny then enters, without intervening DNA replication (in the absence of S phase), into mitotic division to form two trophozoites with basic sets of chromosomes. If one of the functions of meiosis is

S.C. Dawson et al. the reduction of ploidy, which is not clear (Wilkins and Holliday, 2009), reduction of genome content via successive cell divisions resembles meiosis, in principle (Bernander et al., 2001). Whereas the second division after excystment might proceed as a normal mitotic division (see below), the division of the excyzoite remains completely enigmatic. No potential mechanism has been published so far to explain how the four cyst nuclei are distributed between progeny. Consequently, a fundamental question about the identity of the nuclei in segregated doublets, if they are sisters (twins) coming from the same parent nucleus or non-sisters, each coming from a different parent nucleus, remains unanswered. The latter possibility is supported by a karyogamy study showing that the chromosome number in each nucleus of a binucleate trophozoite remains the same before and after encystment (Tumova et al., 2007a). It is important to note that current knowledge regarding cell cycle and differentiation comes exclusively from studies on genotype A, particularly the WB and Portland isolates. In this review we aim to summarize recent progress in our knowledge concerning cell division in Giardia in the context of the cell cycle. Despite the advances in understanding cell division of the Giardia trophozoite at a cytological level, many unanswered questions remain, as almost nothing is currently known about either the molecular mechanisms behind particular processes or the regulation of the Giardia cell cycle.

10.3 Cell Division of Giardia Trophozoites Giardia has a haploid genome size of 12 Mb, with five chromosomes of sizes from 1.4 to 3.4 Mb (Adam, 2000). Each nucleus is thought to be diploid, thus the giardial trophozoite is essentially a “double” diploid (Bernander et al., 2001). A Giardia trophozoite divides asexually by binary fission; this has been well known for more than 50 years (Filice, 1952). Although different models regarding the plane of cytokinesis have been suggested in the past (Kabnick and Peattie, 1990; Yu et al., 2002; Solari et al., 2003; Benchimol, 2004a), it is only recently that a combination of findings regarding the course of mitosis (Kulda

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Nohynkova, 1995; Sagolla et al., 2006; Tumova et al., 2007b) flagellar transformation (Nohynkova et al., 2006) and cytokinesis (Tumova et al., 2007) enabled the true mechanism to become clear. These studies involved monitoring division in live cells and imaging using immunofluorescence, transmission, and scanning electron microscopy. During division, a parent cell splits in the anterior-posterior direction such that the two daughter cells separate ventral-to-ventral in the plane of the daughter adhesive discs. In this way, the left-right asymmetry of the Giardia cell is maintained (Kulda Nohynkova, 1995; Sagolla et al., 2006; Tumova et al., 2007a,b), as exhibited by the counterclockwise (viewing the cell from the ventral side) spiral layer of the disc microtubules. Whether any of the single stages that have been interpreted as various modes of cytokinesis (Solari et al., 2003; Benchimol, 2004b), represent a specific behavior of Giardia cells (e.g., fusion) remains to be elucidated. There is also evidence of differences in ploidy between the two nuclei within one cell, as well as between strains in different assemblages (Tumova et al., 2007a). When dividing, Giardia alternates between attached and free-swimming phases according to the particular phase of division and the adhesive competence of the parental or newly assembled daughter discs. The whole process can be divided into three phases (Tumova et al., 2007b): (1) the initial attachment phase, which proceeds in the adherent parent cell, (2) the free-swimming phase and (3) the terminal attachment phase, which occurs in adherent progeny. During the initial phase, the cell completes the main processes dealing with the duplication of cell structures, namely reorganization of the parent flagella, assembly of new ones, mitosis, and disassembly of the parent ventral disc; the cell also starts to assemble new daughter discs. Cytokinesis is initiated at this phase with the selection of the site of cell division and the start of furrow ingression, which continues in the free-swimming phase. In the terminal attachment phase, abscission occurs and leads to the physical separation of the two cells. Whereas the initial and the free-swimming phases are very short, about 3 and 1 min, respectively, the terminal phase takes up to 50 min and represents the longest phase of the division process (Tumova et al., 2007b). Overall, the description of the basic cellular architecture of the specific phases of Giardia cell division is almost

complete. However, the molecular basis for these processes and the mechanisms behind their regulation and control mostly remain to be uncovered.

10.4 Mitosis Though mitosis and cell division in Giardia has recently been quite a controversial topic, it has been investigated at some level for over 50 years (Felice, 1952). The recent description of mitotic stages in Giardia using cytological markers, however, confirms that the major cytologic events of mitosis proceed in the same manner as described in many organisms. Giardial mitosis begins with condensation of chromatin (prophase), followed by congression of chromosomes to the spindle midzone (metaphase), the movement of chromosomes to the spindle poles (anaphase A), the separation of spindle poles (anaphase B), and the eventual duplication of flagella and the ventral disc prior to cytokinesis (see details below). Both spindles are bipolar arrays of microtubules with attachments to chromosomes at kinetochores. The organization and function of the giardial spindle is highly conserved with respect to these aspects of the metazoan spindle, despite the strikingly different morphology (i.e. two nuclei and eight flagella) and evolutionary distance between Giardia and metazoans. Several aspects of giardial mitosis differ from more commonly studied metazoan or fungal systems including: the “semi-open” structure of the nuclear envelope and spindle; nuclear migration to the cell center (one nucleus is dorsal, and the other ventral); absence of astral microtubules; and an extended spindle structure in telophase that presumably functions to position each pair of daughter nuclei prior to cytokinesis. Flagellar basal bodies also likely function in spindle microtubule nucleation or spindle positioning.

10.4.1 Mechanism of Chromosome Segregation and Mitosis Mitotic spindles in protists have been classified using several cytologic features: the continuity of the nuclear envelope during mitosis (open, semi-open, and closed); the position of the spindle relative to the nuclear envelope (pleuromitosis vs. orthomitosis);

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A

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Fig. 10.1 Mitosis in trophozoites. Panel A depicts a diagram of the stages of mitosis based on 3D analysis of dividing trophozoites with cytologic markers of both spindle poles (green) and centromeres (yellow). Note the mirror image symmetry of nuclei following cytokinesis. Immunostained trophozoites in metaphase seen in B (red = anti-α-tubulin, blue = DAPI). It is unclear if chromosomes align along a metaphase plate in the center of the two mitotic spindles (sp = spindle poles). In panel C, TEM indicates the semi-open mitosis in late anaphase in a single nucleus. Also note the extranuclear microtubules of the spindle on the nuclear envelope, and the presumptive kinetochore microtubules entering at regions at the far left and right poles of the nucleus close to the spindle poles (sp)

and the position of chromosomes relative to the spindle axis (Raikov, 1994). In most plants and animals, a complete breakdown of the nuclear envelope occurs during prophase, allowing microtubules direct access to chromatin. As with some fungi, most protists have

a closed mitosis, retaining an intact nuclear envelope with an intranuclear spindle (Schuster, 1975; Winey et al., 1995; Ogbadoyi et al., 2000; O’Toole et al., 2003). Giardia, in contrast, has a “semi-open” mitosis, a variant of closed mitosis in which kinetochore

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microtubules from the two extra-nuclear spindles penetrate the nucleus through polar openings in the nuclear envelope. Due to the ubiquity of extranuclear spindles in protists, the ancestral state of eukaryotic mitosis may be more that of like Giardia: an extranuclear spindle that interacts with chromatin through a semi-open nucleus or across the nuclear envelope. Trophozoites possess an elaborate microtubule (MT) cytoskeleton consisting of four major structural arrays: the ventral disc, flagellar axonemes with basal bodies, the funis, and the median body (see Chapter 18). The basal bodies of all eight flagella are located between the two adjacent nuclei in the anterior region of the cell (Fig. 10.1). In interphase trophozoites, centrin localizes to two clusters between the two nuclei, co-localizing with the flagellar basal bodies (Belhadri, 1995; Meng et al., 1996; Correa et al., 2004). Remarkably, most of the described division phases (below) occur in several seconds, and completion of cell division likely occurs within 50 min (Cerva and Nohynkova, 1992). Prophase: Chromosome condensation, nuclear repositioning, spindle nucleation, and assembly. Extensive chromosome condensation, spindle nucleation/ assembly, and nuclear repositioning define the prophase stage of mitosis in Giardia trophozoites (Cerva and Nohynkova, 1992; Sagolla et al., 2006). One of the earlier aspects of prophase in Giardia is chromosome condensation. Individual giardial chromosomes can be resolved following chromatin condensation in mitotic prophase. CenH3 is an H3 histone variant that is a universal marker of centromeres. CenH3 plays an essential role in recruiting kinetochore proteins to the centromere by recruiting motors and other structural and regulatory proteins (Van Hooser et al., 2001), and cenH3 is thus the primary determinant in establishing the site of kinetochore assembly (Malik and Henikoff, 2003). Using a GFP-tagged cenH3 strain, cenH3 was found to localize to a discrete focus on each interphase chromosome in each nucleus, indicating that Giardia chromosomes are monocentric (Dawson et al., 2007a) as in they are in the majority of eukaryotic cell types, rather than holocentric (Dernburg, 2001; Maddox et al., 2004). The assembly of the kinetochore on centromeric chromatin and the attachment of the kinetochore to the spindle microtubules are both required for accurate chromosome segregation during mitosis. In other eukaryotes, the centro-

meric foci are required to build the mitotic kinetochore by recruiting motors, checkpoint proteins, and additional structural elements (Van Hooser et al., 2001). Presumably this also happens in giardial prophase, as the centromeres are clearly visible on condensed chromosomes (Sagolla et al., 2006). Spindle nucleation and assembly in Giardia occur shortly after chromosome condensation. During giardial prophase, the nucleation of the two spindles is made apparent by the appearance of MTs between the two nuclei near the flagellar basal bodies. The MTs of the mitotic spindles then extend around each nucleus. Spindle microtubules further elongate during nuclear migration, until each spindle encompasses each nucleus by the end of prophase. Spindle nucleation is mediated by microtubule organizing centers, or MTOCs. When a broad phylogenetic range of microbial eukaryotes is compared, the morphology and the composition of the spindle MTOCs are found to be quite diverse. The giardial mitotic spindles are most likely nucleated by or near the flagellar basal bodies, which are structurally related to centrioles. This style of spindle nucleation implies that spindle nucleation in Giardia is similar to that of flagellated protists like Chlamydomonas that recruit flagellar basal bodies to function as part of the MTOC at the spindle poles following resorption of their two flagella. Unlike other flagellates, however, giardial trophozoites undergoing mitosis have four pairs of basal bodies and four spindle poles associated with the two nuclei. Presumably the one basal body present at each spindle pole acts as the central structural component of the MTOC of the mitotic spindles. Thus, specific combinations of two axonemes and associated basal bodies at each pole could confer a unique positional identity to each of the four spindle poles. A second basal body is also present at the edge of the spindle pole region, peripheral to the focused spindle microtubules, and may play an indirect role in spindle nucleation and organization. The association of specific flagellar basal bodies with specific spindle poles might provide spatial cues for the proper establishment and maintenance of cell polarity in each generation. Following chromosome condensation and spindle nucleation/assembly, the two nuclei reposition to the center of the cell, one above the other along the dorsal-ventral axis. The ventral nucleus corresponding to

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the left nucleus of the interphase cell as viewed from above remains slightly anterior to the dorsal nucleus (right interphase nucleus) (Tumova et al., 2007b). The nuclear envelopes remain intact and do not fuse. Later in cell division the left and right nuclei are inherited with mirror image symmetry in the two daughter cells (Sagolla et al., 2006). This pattern of nuclear inheritance had been predicted by previous studies (Filice, 1952; Cerva and Nohynkova, 1992; Ghosh et al., 2001; Yu, 2002; Benchimol et al., 2004a); however, the identification of nuclear migration in prophase offers a mechanism for how the mirror image symmetry of daughter nuclei is achieved. What is the mechanism of nuclear migration? One candidate factor that could promote nuclear migration is centrin, a calcium binding protein associated with basal bodies and centrosomes of eukaryotic cells (Salisbury, 1995). Duplication of the centrin foci also occurs during prophase. At the onset of prophase and spindle assembly the number of centrin foci increases from two to four as the result of either duplication or separation of the basal bodies. After duplication, centrin foci are present at the sites of spindle nucleation during nuclear migration and move around the periphery of the nucleus as the spindle microtubules elongate. By the end of prophase, the two centrin foci, each with its own complement of microtubules, are positioned on opposite sides of each nucleus. Centrin is a major component of the nuclear basal body connector in flagellated algae, and comprises a fibrous network linking the basal bodies to the nucleus (Wright et al., 1985, 1989; Salisbury et al., 1988; Marshall and Rosenbaum, 2000; Brugerolle and Mignot 2003). During prophase centrin localizes only to basal bodies associated with the four giardial spindle poles. Although centrin is not associated with all eight basal bodies in mitosis, the centrin fibers may connect spindle pole-associated basal bodies with other basal bodies in preparation for segregation. Basal body migration and nuclear migration during mitosis may be coordinated events, facilitated by the centrin-dependent attachment of basal bodies to the nuclear envelope. Alternatively actin or microfilaments may be involved with nuclear positioning during prophase as in other eukaryotes (Starr, 2009). Metaphase: Upon completion of prophase nuclear migration, the microtubules surrounding each nucleus form two complete and independent bipolar spindles,

S.C. Dawson et al. one dorsal and one ventral (Fig. 10.1B and C). The opposing poles of each spindle are oriented along the left-right axis of the cell with the chromatin clustered tightly in the center of each spindle axis. It remains unclear as to whether there is a canonical metaphase alignment of centromeres along a metaphase plate (although this may be present as a short transitory stage). Following the assembly of kinetochores on centromeres in prophase, the giardial mitotic spindle links to kinetochores on chromosomes via kinetochore microtubules that facilitate chromosome segregation. Giardial kinetochore MTs penetrate the nuclear envelopes at polar openings (Sagolla et al., 2006). It is predicted that more than one microtubule is attached per kinetochore in Giardia, which would result in greater than ten kinetochore MTs per nucleus. The number of kinetochore microtubules attached to chromosomes ranges from a single kinetochore microtubule per chromosome in budding yeast to more than twenty per chromosome in some metazoans (McDonald et al., 1992; Winey et al., 1995). Thus the giardial kinetochore MTs may be more akin to that of metazoans. Inhibition of microtubule depolymerization using Taxol causes anaphase B spindle defects such as the loss of interzonal microtubules in the dual giardial spindles (Sagolla et al., 2006). This observation provides further evidence for the role of kinetochore microtubules in chromosome segregation, as well as demonstrates the bipolar organization of spindle microtubules as is found in other eukaryotes (De Brabander et al., 1986; Amin-Hanjani and Wadsworth, 1991; Jordan et al., 1993). Equilibrium metaphase spindle length is controlled by both intrinsic MT dynamics (i.e., dynamic instability) and active regulators of MT dynamics including MT motor proteins and +TIPs (Akhmanova and Hoogenraad, 2005). As in other eukaryotes, MT dynamics governing spindle length and structure in Giardia are affected by microtubule drugs such as nocodazole and Taxol (Hamaguchi et al., 1987; Snyder and Mullins, 1993; Dawson et al., 2007b). The giardial homolog of the depolymerizing kinesin-13 localizes to the kinetochores of the dual giardial spindles and regulates MT dynamics and spindle length. The phenotype of broken spindles and lagging chromosomes is associated with the overexpression of a kinesin-13 dominant negative mutant, and has been

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observed in many diverse eukaryotes (Walczak and Mitchison, 1996). Thus kinesin-13 retains an evolutionarily conserved function in the regulation of spindle dynamics in the semi-open mitosis of Giardia analogous to that in metazoans (Asakawa et al., 2005). The microtubule-associated protein EB1, a + TIP, is also present at giardial kinetochores. Anaphase A and B: Chromosome segregation in both nuclei occurs in two stages: chromatid segregation in anaphase A, followed by spindle elongation along the left-right axis of the cell in anaphase B (Fig. 10.1C). In Giardia, anaphase A initiates first in one nucleus; however, anaphase B occurs simultaneously in the two nuclei. It is unclear which nucleus initiates mitosis or whether initiation always occurs in the same nucleus. During anaphase A, the centromeres localize to the leading edge (near the spindle pole) of the segregating DNA, a behavior that implies kinetochore attachment to microtubules, rather than some novel mechanism of chromosome segregation. Sister chromatids from each nucleus are segregated to opposite sides (L–R) of the cell, as a result of both nuclear migration and lateral chromosome segregation. Spindle elongation is characteristic of anaphase B, and in Giardia this is visualized by the elongation of the nuclei to the extreme L–R sides of the cell, with the nuclear envelopes remaining intact. Centromeric foci remain tightly clustered together at the spindle poles during anaphase B. Centrin foci remain at the spindle poles throughout anaphase A and B (Sagolla et al., 2006). Telophase: Despite differences among diverse eukaryotes in terms of the timing and mechanism of mitosis, the cell division plane is essentially always perpendicular to the axis of chromosome segregation as defined by the mitotic spindle (Balasubramanian et al., 2004). Giardial cytokinesis – occurring in the longitudinal plane perpendicular to the spindle axis – is consistent with patterns of cytokinesis in other eukaryotes. A hallmark of telophase is the presence of a microtubule bundle with unfocused ends that extends between the nuclei, replacing the bipolar spindle arrays. This structure could represent the remaining spindle microtubules following the loss of focused spindle poles, or could arise from de novo microtubule polymerization. Centromeres remained clustered in telophase nuclei, based on observations using

cenH3::GFP localization in the two daughter nuclei. The two centrin foci on each side of the nuclei move from their anaphase position near the cell periphery to their position between each pair of nuclei as seen in interphase. By the onset of cytokinesis, the DNA decondenses and centromeric foci are visible throughout each nucleus. After karyokinesis, nuclei migrate to their interphase positions in the two daughter cells, prior to cytokinesis. Chromosome segregation along the left–right axis is followed by longitudinal cytokinesis (perpendicular to the plane of nuclear division) along the anteriorposterior axis. At the onset of cytokinesis, a furrow forms at the anterior end of the cell creating a “heartshaped” cell, and ensuring that each daughter cell inherits one copy of each parent nucleus. The cleavage furrow progresses from the anterior to posterior in the longitudinal plane, separating the left and right sides of the heart into the two daughter cells. As a result, each daughter cell inherits two nuclei, one derived from the left and one from the right nucleus of the mother cell. Such a longitudinal cytokinesis could result in daughter cells that either have ventral–ventral or dorsal-ventral orientations relative to one another. Earlier models of cytokinesis in Giardia generally assumed that mitosis occurred in the dorsal–ventral plane, and subsequently inferred that cytokinesis occurred in the frontal plane splitting the cell into dorsal and ventral halves. In these models daughter cells also would inherit two nuclei of different parental origins; however, the nuclear symmetry would be either identical or mirror image depending on the orientation of the daughter cells to one another (Filice, 1952; Cerva and Nohynkova et al., 1992; Ghosh et al., 2001; Yu et al., 2002; Benchimol, 2004a). More recent work (Sagolla et al., 2006) indicates that daughter cells have anterior-posterior orientations, resulting in mirror image symmetry of the two inherited nuclei. The mirror image pattern of nuclear inheritance proposed in the nuclear migration model is supported by fluorescence in situ hybridization (FISH) to an episomal plasmid present in only one nucleus. In this study (Sagolla et al., 2006), all interphase trophozoites had either one left or one right nucleus labeled. In mitosis only the two dorsal or two ventral nuclei were labeled. In the “heart-shaped” cytokinesis stage, mirror image symmetry of nuclei with labeled with an episomal plasmid FISH signal was observed (as has

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been previously reported by Ghosh et al. (2001)). Prior attempts to define the pattern of nuclear inheritance have also used FISH of an episomal plasmid to track either right or left nuclei, but did not look at intermediate stages of mitosis, particularly during the prophase nuclear migration or the later four nuclear stage (Ghosh et al., 2001; Yu et al., 2002). In addition, these studies presumed that cytokinesis occurs in the frontal plane, or dorsal-ventral axis. Given a longitudinal division, the only outcome for nuclear identity is mirror image symmetry between the daughter cells. This pattern of FISH in mirror image nuclei supports the observations of Ghosh et al. wherein the two nuclei are inherited with mirror image symmetry. The fact that no cells contained two labeled nuclei in this or prior studies implies that: (1) there is no nuclear fusion during giardial mitosis; and (2) daughters inherit one copy of each parental nucleus in each generation.

10.4.2 Implications of Mode of Mitosis on Nuclear Inheritance and Heterozygosity Beyond establishing the plane of cytokinesis, the mode of Giardia cell division has important implications that bear on the maintenance of a unique genetic identity for each nucleus. The genetic content of the two nuclei is presumed to be identical (Kabnick and Peattie, 1990; Yu et al., 2002), and Giardia intestinalis assemblage A has been shown to possess a low level (<0.1%) of genetic heterozygosity (Baruch et al., 1996; Lu et al., 1998). Other assemblages including assemblage B have higher (Franzen et al., 2009) or similar levels of heterozygosity (JerlstromHultqvist et al., 2010) as compared to assemblage A. Inheritance of both copies of the left or right nucleus by one daughter could explain low levels of heterozygosity by eliminating sequence differences in each generation (Yu et al., 2002), but still does not explain ploidy differences (Tumova et al., 2007a). In contrast, inheritance of one copy of both left and right nuclei would preserve any genetic differences between the two nuclei among the daughter cells. As is the case for the dikaryon of some basidiomycetes (Kues, 2000), the presence of two genetically disparate nuclei could maintain a parasexual condition in Giardia, where during rare points in the life cycle (i.e., encystation) the two nuclei could fuse and

S.C. Dawson et al. homogenize genetic material. The mode of giardial mitosis in trophozoites, however, ensures that the nuclei remain physically and genetically distinct from one another. Asexually dividing Giardia should theoretically accumulate substantial allelic heterozygosity between each nucleus in a short time if neither nuclear fusion nor meiosis occurs. The recent identification of putative meiotic genes in Giardia (Ramesh et al., 2005) has raised the possibility that rare nuclear fusions or meiotic events provide a mechanism for chromosomal recombination and reduction of such heterozygosity. Canonical meiosis, including the presence of characteristic cytologic evidence such as a synaptonemal complex, has not been directly observed in Giardia trophozoites. Alternatively, the inheritance of genetic material from both nuclei may somehow be important for cell survival.

10.5 Division of Microtubule Cytoskeleton Just as the nuclei undergo mitotic division in order to maintain cell identity in the progeny, the cytoskeleton, comprising eight flagella, the funis and the adhesive disc, must also be duplicated. Similar to nuclear division, the division of the complex cytoskeleton remained unclear until recently. Recent studies (Nohynkova et al., 2006; Tumova et al., 2007a, b) have shown that, in a rapid sequence of events that takes less than three minutes, the interphase flagellar apparatus undergoes extensive reorganization, resulting in the transformation of all but the caudal parent flagella to different flagella in the progeny. Simultaneously with the reorganization of the parent flagella, the parent ventral adhesive disc disassembles, and two daughter discs are formed de novo. The median body gradually disappears, reappearing in the G1 phase of the subsequent cell cycle. How the funis duplicates has not yet been determined.

10.6 Division of the Flagellar Apparatus The basic features of the complex flagellar apparatus of diplomonad Giardia are well known and have been reviewed recently (Dawson and House, 2010).

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It comprises four flagella types: anterior, posteriolateral, ventral, and caudal. As seen in diplomonad cell architecture, each type is represented by two bilaterally symmetric flagella and defined by its location, arrangement, associated structures, and likely functions (see Chapter 18). Pairs of flagellar basal bodies form two symmetric clusters (tetrads) localized side by side in between the anterior poles of two nuclei. Together with the pertinent flagella, two microtubular root fibers and some other fibrillar appendages, the tetrad forms a structural unit called a mastigont (Kulda and Nohynkova, 1995). Some details regarding the arrangement of the Giardia basal body pairs are particularly important for understanding the mitotic distribution of parent flagella between the daughters. The pairs are formed from basal bodies of different flagella types as demonstrated unequivocally by negative staining (Feely, 1990) and transmission electron microscopy (Brugerolle, 1974, 1991). The basal bodies of the anterior/ventral, caudal/posteriolateral, caudal/ventral and anterior/posteriolateral flagella are paired; the tetrads consist of different basal body pairs (when viewing the attached cell from above, the left tetrad is formed from pairs of anterior/ventral and caudal/posteriolateral basal bodies, whereas the right tetrad is composed of pairs of caudal/ventral and anterior/posteriolateral basal bodies); the tetrads are symmetric under a 180-degree rotation (twofold rotational symmetry). The highly ordered and heterogeneous flagellar apparatus is essential for Giardia survival, given its involvement in vital cellular processes, i.e., attachment, movement, and excystment (Dawson and House, 2010). Therefore, it must be preserved in progeny. When Giardia divides, eight parent flagella persist. The parental flagella are inherited by daughter cells in a semi-conservative manner, such that each progeny receives four flagella from the parent cell that are supplemented by four newly arisen flagella to form a complete daughter set. Until recently, it was generally accepted that each daughter Giardia inherits the same parent flagella set (one flagellum from each parent pair, i.e., one mastigont). This idea regarding uniform distribution was based on observations showing that the plane of cytokinesis cleaves the dividing cell between segregated mastigonts (Filice, 1952; Cerva and Nohynkova, 1992; Kulda and Nohynkova, 1995). Other models of cytokinesis did

not follow the partitioning of flagella or intuitively expected symmetric segregation. Posttranslational modifications of tubulin as an efficient experimental tool For a long time, the complexity of the Giardia cytoskeleton and its incomprehensibility during division, have hampered attempts to follow the segregation of cytoskeletal components by conventional microscopy techniques. The finding that the parent and de novo arising flagella can be discriminated using posttranslational modifications of tubulins was an important step toward understanding the behavior of flagella during Giardia division (Nohynkova et al., 2006). Although specific associated structures, such as marginal plates (striated lamellae accompanying axonemes of anterior flagella), dense rods (irregular dense structures associated with axonemes of anterior and posteriolateral flagella) or paraflagellar rods (dense structures located in lateral extensions of ventral flagella) (Kulda and Nohynkova, 1995), may be considered the most appropriate markers, most of these disappear at the onset of mitosis, in accordance with the reorientation/migration of the parent flagella, and reappear only after reorganization is finished (Nohynkova, unpublished). Of the known tubulin-specific posttranslational modifications (Hammond et al., 2008), acetylation, tyrosylation, glutamylation, and glycylation had previously been detected in Giardia by mass spectrometry (Weber et al., 1997), and the spatial distribution of two modifications, acetylation and glycylation, was demonstrated in microtubular systems in interphase cells by immunofluorescence staining (Soltys and Gupta, 1994; Campanati et al., 1999). During Giardia division, immunostaining revealed that acetylation is a common tubulin modification found in all flagella, including newly arising daughter axonemes, whereas polyglycylation is limited to the tubulins of “old” (parent) flagella (Nohynkova et al., 2006). The absence of polyglycylated tubulin in newly growing axonemes is consistent with findings in ciliates, where long poly-glycine side chains attached to alpha and beta-tubulins are regarded as morphogenetic markers of fully assembled cilia (Iftode et al., 2000). Mass spectrometry and sequence analysis have shown that, in interphase Giardia, polyglycylation is a major tubulin-specific modification with up to 20 and 15 glycyl residues detected on

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the carboxy-terminal peptides of alpha- and beta-tubulin, respectively (Weber et al., 1997). Therefore, as in ciliates (Wloga et al., 2009), elongation of the glycine chain is likely to be correlated with the development of Giardia flagella.

10.6.1 Parent Flagella Distribution By using monoclonal antibodies for acetylated and polyglycylated tubulins in combination with scanning and transmission electron microscopy, recent work has shown that, before cytokinesis, two parent mastigonts exchange half of their flagella components (Nohynkova et al., 2006). This means that in contrast to the general assumption, each daughter receives half of each of the two mastigonts, not one of the two mastigonts. As a consequence of the arrangement of basal body pairs in tetrads, each of two daughters inherits a different set of four parent flagella. This work has also demonstrated that Giardia flagella undergo a maturation process. Prolonged flagellum development (maturation) extending over more than one cell cycle was first described in the biflagellated heterokont alga Nephroselmis olivacea; it was shown that, during division, the anterior flagellum transforms into the posterior flagellum in one of the daughters (Melkonian, 1987; Heimann et al., 1989). Later, mitotic transformation from one type of flagellum to another type was found in many distinct groups of flagellated protists (Moestrup, 2000). It is now evident that flagellar transformation is a universal, evolutionarily conserved ontogenetic process, which guarantees the continuity of the flagellar apparatus in the progeny and enables the determination of the mature state of any flagellum, provided the cell undergoes cell division (Moestrup, 2000). In octoflagellated Giardia, each newly assembled flagellum needs three consecutive cell cycles to assume its final, mature position within the cell.

10.6.2 Transformation of Parent Flagella during Division During Giardia division, flagella of the parent anterior and caudal pairs are segregated equally between

S.C. Dawson et al. progeny: these flagella are distributed one-to-one per daughter (Fig. 10.2). The flagella of the parent ventral and posteriolateral pairs, on the other hand, are segregated unequally; one daughter receives both ventral flagella, and the other receives both posteriolateral flagella. This is why each daughter inherits a different parent flagella set. Except for the caudal ones, all parent flagella transform into different flagella types in the progeny. The flagella undergo extensive mitotic reorganization, mediated by the reorientation, migration, and segregation of their paired basal bodies, whereby the respective flagella change positions. The parent anterior flagellum transforms into a daughter caudal flagellum in each progeny. Transformation is achieved through exchanging the position of the flagella basal bodies. The basal bodies reorient and migrate left-right towards opposite cell sides; the left basal body moves toward the right side, while the right basal body moves toward the left side of the cell. This basal body migration results in the gradual pulling of the intracytoplasmic portions of the pertinent axonemes inside the cell, leading to a disjunction of anterior crossing of the axonemes and, finally, to the exchange of the flagella exit positions with respect to interphase. The transformed flagellum joins a segregated parent caudal flagellum to form a daughter caudal flagella pair. The parent caudal flagella do not undergo mitotic transformation. In prophase, their basal bodies reorient and separate from each other by left–right lateral movement, according to the axis of mitotic spindles. Each caudal basal body docks near one pole of each of the two mitotic spindles. According to the interphase arrangement of basal body pairs and their segregation during mitosis, the parent caudal flagellum always forms the left caudal flagellum in the progeny (viewing the attached cell from above). A pair of the parent posteriolateral or ventral flagella transforms into a pair of daughter anterior flagella in each progeny. At early mitosis, the parent basal bodies move in pairs corresponding to the interphase basal body arrangement. A prerequisite for further transformation is the segregation of the paired basal bodies that occurs later in mitosis. Independent movement of the posteriolateral and ventral parent basal bodies enables the formation of the anterior crossing that is characteristic for anterior axonemes. In the left half of the dividing cell, the basal bodies of

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A

B

C

Fig. 10.2 Reorganization and transformation of parent flagella. The dividing cells are viewed from dorsal side. Upper row (panel A): Sequence of scanning electron micrographs demonstrates migration of parent anterior flagellar pair (rafl = right anterior flagellum, lafl = left anterior flagellum). The arrowhead (also in row B) points to the exit site of the right parent flagellum, which gradually moves from the right (interphase) to the left cell side. Middle row (panel B): Flagellar reorganization and transformation as visible by immunofluorescence using antibody to polyglycylated tubulin, which selectively labels the parent flagella. Temporal sequence of flagella relocation is indicated by arrow. Lower row (panel C): Color-coded flagellar axonemes (red = anterior, green = posteriolateral, blue = ventral, white = caudal) as in panel B show gradual transformation of the parent flagella up to their final distribution between progeny. In progeny, the transformed parent flagella form anterior and caudal flagellar pairs. Scale bars: 5 μm

the parent posteriolateral flagella cross each other to form the origin of the anterior crossing of the daughter anterior axonemes; in the right half, the reorientation of the basal bodies of the parent ventral flagella also forms the crossing. The mechanisms involved in the rearrangement of Giardia basal bodies/flagella are virtually unknown. Coordination between the migration of basal bodies and prophasic nuclei, facilitated by centrin-dependent attachment of the basal bodies to the nuclear envelope, as suggested recently (Sagolla et al., 2006), points to a role for the nuclei in the movement of basal bodies. However, in mitotic cells that lack mitotic spindles due to the inhibition of spindle assembly, the undivided nuclei are mispositioned, whereas basal bodies segregate correctly (Nohynkova et al., 2000). This indicates that, as in Chlamydomonas (Feldman et al., 2007), mispositioning of the nuclei has little

impact on basal body segregation and suggests that the positioning of migrating basal bodies does not depend on interactions with the nuclear envelope. Instead, interactions with the cell surface via axoneme microtubules may be used to move Giardia basal bodies. In many eukaryotic cells (Vaugham, 2011), microtubules emanating from the centrosomes and interacting with the cell cortex are responsible for centriole/basal body movement. In Giardia, this idea particularly corresponds to the movement of the basal bodies of the anterior flagella. Although the exits of the posteriolateral, ventral, and caudal flagella do not significantly change position during basal body movement, those of the anterior flagella move along the cell periphery from one side of the cell to the other, in accordance with the migration of the associated basal bodies deep within the cell (Nohynkova et al., 2006). The interaction between a specialized region of the

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plasma membrane, where a flagellum exits, and a basal body via cytoplasmic axoneme microtubules seems to be crucial for basal body movement and suggests that force-producing molecules might be located at or near the cell surface.

10.6.3 De Novo Assembly of Daughter Flagella Daughter ventral (V) and posteriolateral (PL) flagella pairs are always formed de novo from basal bodies newly assembled during mitosis. Each parent basal body induces the formation of a new one. Due to the configuration of the Giardia basal body pairs, each basal body pair in the progeny and, consequently the interphase cell consists of one old (parent) and one new basal body. Duplication of Giardia basal bodies seems to follow the conservative process of centriole/basal body duplication seen in other cells (Loncarek and Khodjakov, 2009). First, each new basal body arises in proximity to a parent basal body, indicating that it is formed by the duplication of an existing structure (Nohynkova, unpublished data). Second, although pairs of Giardia basal bodies are not orthogonally arranged in interphase, the new basal body is likely to be formed at right angles with the parent organelle following stringent orientation, which is characteristic of centriole duplication (Tsou and Stearns, 2006; Azimzadeh and Bornens, 2007). This canonical mechanism strictly limits the number of newly assembled basal bodies to only one per parent basal body and cell cycle via an, as yet, not fully understood restriction process (Loncarek and Khodjakov, 2009). Similar control apparently also exists in Giardia. However, there are several aspects that are specific to Giardia basal body propagation. First, duplication occurs during division, as there are no barren or probasal bodies in interphase trophozoites (Brugerolle, 1991). This implies that, in concert with centrioles, Giardia basal body duplication is cell cycle-related but coordinated with mitosis, rather than DNA replication (S-phase of cell cycle), as is the general rule for the propagation of centrioles (Tsou and Stearns, 2006). Second, basal body assembly is extremely fast; the whole process, from prophase to metaphase, is finished within seconds, unlike the procentriole

S.C. Dawson et al. elongation, which begins in S phase and continues until the procentriole reaches the length of its parent centriole during the G2/M phase of cell cycle (Tumova et al., 2007b). Therefore, mechanisms, which control the length of the basal body, must operate during Giardia mitosis. Third, a new basal body immediately nucleates an axoneme, whereas a centriole first undergoes maturation over 1.5 cell cycles, as only a mature centriole, that leaves the cell center and docks at the cell surface can produce a cilium (Dawe et al., 2007). What triggers basal body duplication in Giardia, is unknown. By analogy with centrioles, where disengagement (a stage when a mature centriole and procentriole separate due to the loosening of their tight association and orthogonal orientation) is a prerequisite for duplication (Tsou and Stearns, 2006), disorientation of the paired parent basal bodies, as observed at the beginning of mitosis (Nohynkova et al., 2006), might license each of the basal bodies for duplication. No molecular data regarding basal body duplication, or the coupling of the process with the cell cycle are available in Giardia. Of the several kinases and other proteins shown to be essential for centriole duplication (Loncarek and Khodjakov, 2009), gamma tubulin is almost certainly required for the nucleation of new basal bodies. From about metaphase onward, gamma tubulin is present in the basal body regions of newly formed flagella; it persists during the subsequent interphase and disappears when the cell enters mitosis. The disappearance of gamma tubulin staining early in prophase and its reappearance in later mitotic phases suggests a dynamic association between gamma tubulin and the basal bodies (Nohynkova et al., 2000). Active Aurora kinase localized in the basal body regions between prophase and late anaphase (Davids et al., 2008) may contribute to duplication as well as centrin, co-localizing with reorganizing parent basal bodies (Sagolla et al., 2006). Serine in carboxy-terminal RRTSLY sequence of Giardia centrin is likely to be phosphorylated during mitosis. Short, nascent axonemes are visible within the cytoplasm from about anaphase onward (Cerva and Nohynkova, 1992). As Giardia progresses through the initial division phase, the axonemes elongate until they reach the plasma membrane. The mode of formation of the intracytoplasmic axonemes is unknown but is likely to be independent of the intrafla-

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gellar transport system (IFT) that is essential for the assembly and maintenance of membrane-bound flagella and cilia (Pazour and Rosenbaum, 2002). The new, naked Giardia axonemes grow within the cytoplasm; a system specialized for transport between cell membrane and axoneme microtubules seems to be unnecessary there. In contrast, assembly of the external, membrane-bound portions of axonemes, i.e., of new Giardia flagella, could be IFT-dependent. Although this has not been demonstrated in mitotic cells, localization of components of the IFT, kinesin-2 motors, and IFT raft complex components, and the shortening of all but anterior flagella in interphase cells using dominant-negative constructs for kinesin-2 indicate that the IFT machinery operates in Giardia (Hoeng et al., 2008). Indeed, nearly all IFT components can be found in the Giardia genome (see Chapter 18). The new axonemes are directed toward the cell midline from both halves of the dividing cell. After reaching the cell periphery, they emerge as short daughter flagella visible in later division phases (Tumova et al., 2007b). How the exit site in the plasma membrane is selected and the distal tip of the growing axoneme is guided to the exit site and directed to form either a ventral or posteriolateral flagellum remains unknown. Microtubular capture by some cortical factors and the participation of motor proteins are likely. Besides the kinesin-13 depolymerase, which is localized to the distal tip of each growing axoneme (Dawson et al., 2007b), no such factor has been identified yet.

more cell cycle old. This flagellum represents the final stage in the development of a Giardia flagellum corresponding to a mature flagellum. Over the generations, it never changes its position and function. Each newly assembled flagellum requires three successive cell cycles to complete its development, i.e. to become the left caudal flagellum (Nohynkova et al., 2006). This suggests that, as in other cells (Moestrup, 2000), in a single interphase cell, the different types of Giardia flagella represent the different stages of flagellar development. Specific structures, e.g., marginal plates, dense, and paraflagellar rods, associated with the specific flagella types can be interpreted as transient characteristics of a role played by a flagellum during a given cell cycle; this idea is supported by the observation that they mostly disappear during mitotic reorganization.

10.6.4 Maturation of Flagella As a consequence of parent flagella transformation and de novo flagella assembly, the flagella in each Giardia progeny and, therefore, in each interphase cell, have different chronologic ages (Fig. 10.3). Ventral and posteriolateral flagella are the youngest because they are newly formed every mitosis. Anterior flagella are one cell cycle old; they come from the transformation of either a ventral or posteriolateral flagella pair. The right caudal flagellum is two cell cycle old; it is formed through the transformation of a previous anterior flagellum. The left caudal flagellum, the oldest flagellum within the cell, is three or

10.6.5 Developmental Asymmetry of Microtubular Roots of Caudal Flagella The caudal pair composed of unequally aged flagella (the oldest and second oldest) is another consequence of the flagellar maturation process. This generational asymmetry, which is exclusive to the caudal basal bodies/flagella, seems to be essential for the maintenance of the left-right cell asymmetry that is phenotypically manifested by a ventral disc. This single-copy component of the Giardia cytoskeleton is derived from a microtubular basal body root. Of the two root fibers carried by each caudal basal body in diplomonads, the anterior root is asymmetrically developed in Giardia (Brugerolle 1991). The left one, which is nucleated near a base of the basal body of the mature flagellum, is developed extensively to form the disc, whereas the right one, linked to the basal body of the younger caudal flagellum, is rudimentary. This root seems to represent a disc precursor for the subsequent mitosis, when the right caudal basal body will become mature. The difference between the left and right root development indicates asymmetry in a functional competence between the caudal basal bodies and the surrounding area. In this respect, the Giardia caudal basal bodies correspond to a centriolar pair and centrosome in animal cells. In Giardia, however, the centrosome(s) has/have not yet been defined, although several proteins, associ-

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Fig. 10.3 Heterogeneity of flagellar apparatus and flagellar maturation. A–C: Discrimination between origins of flagella by immunostaining. Daughter anterior afl and caudal (afl) flagella (B) come from transformation of parent flagella (A) (A, B: anti-polyglycylated tubulin labeling); daughter ventral (vfl) and posteriolateral (pfl) flagellar pairs are formed de novo (C: anti-acetylated alpha tubulin labeling). Diagram (panel D) demonstrates a model of transformation of a newly formed flagellum (red) (M-phase 0) during three consecutive cell cycles (M-phase 1 to 3) to become a mature flagellum (M-phase 3). Note age heterogeneity of flagella in interphase cell (M-phase 3) (red = the oldest, left caudal flagellum, green = two-cell cycle-old right caudal flagellum, blue = onecell cycle-old anterior flagella, yellow = the youngest, newly formed ventral and posteriolateral flagella)

ated with both scaffolding, e.g., centrin and gamma tubulin, and signaling, namely Aurora kinase, protein kinase B and protein phosphatase 2A, localize to basal body regions (Nohynkova et al., 2000; Lauwaet et al., 2007; Davids et al., 2008). According to a recent protein analysis, Giardia may represent one of those eukaryotes possessing basal bodies without an association with the centrosomes (Hodges et al., 2010). Binucleated diplomonads are generally considered to be doubled cells equipped with a duplicated set of the same, developmentally separated basal body/flagella units. As demonstrated recently, during division of Giardia, the flagellar apparatus is reconstituted as a single unit, exchanging components between mas-

tigonts. Thus, in contrast to the current understanding, a Giardia cell can be characterized as a developmentally linked double tetrakont (Nohynkova et al., 2006).

10.7 Ventral Disc Whereas persistent parent basal bodies represent templates for new basal bodies formed in a conservative fashion, new daughter discs are assembled independently of the parent structure, which disintegrates simultaneously with their formation. As a modified microtubular root, the new disc is always formed de novo.

Chap. 10 Cell Cycle Regulation and Cell Division in Giardia

10.7.1 Parent Ventral Disc Disassembly A dividing Giardia is attached from the beginning of the division up to the assembly of new daughter discs (Nohynkova et al., 2000; Tumova et al., 2007b) although some studies assumed that only swimming trophozoites divide (Ghosh et al., 2001; Benchimol, 2004b). During this attachment phase, the parent disc cytoskeleton undergoes dramatic structural changes, which finally lead to disc disassembly and the detachment of the dividing cell necessary for further progress of cytokinesis (Fig. 10.4). The disappearance of the parent disc prior to cell splitting has been reported repeatedly (Lavier 1939; Soloviev 1963; Cerva and Nohynkova 1992; Kulda and Nohynkova, 1995; Ghosh et al., 2001), but details regarding disc disas-

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sembly have been provided only recently (Tumova et al., 2007b). Shortly after the onset of division, the proximal (minus) ends of the disc microtubules are released from the basal body complex. This step is apparently necessary for the reorganization of the parent flagella and nuclear division. Immunofluorescence microscopy showed that the free disc cytoskeleton retains its interphase position and shape, which then gradually deforms by opening and folding the originally overlapping spiral microtubular array. Changes in the array shape are underlined by ultrastructural changes manifested by the progressive shortening and eventual loss of dorsal microribbons. Consequently, when mitosis is completed, only barren disc microtubules are located beneath the plasma membrane at the ven-

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Fig. 10.4 Disassembly of parent disc. Detachment of the disc cytoskeleton from basal bodies (arrows) is indicated by aster in A. B, C: Alterations of the microtubular cytoskeleton of the disc are demonstrated by immunostaining (anti-acetylated tubulin). Arrowheads point to opening of overlap of the ends of the disc cytoskeleton. Panel D: Note phosphorylated Aurora kinase (green) located in deforming cytoskeleton of parent disc during anaphase and telophase (blue = DAPI stained nuclei). Shortening of dorsal microribbons (dr) and their absence (arrow) during disassembly of the disc cytoskeleton as demonstrated by TEM (E). Note normal length of the dorsal microribbons (dr) in interphase cell (F)

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tral cell side and disappear when the dividing cell detaches. Release of the disc microtubules from basal bodies, gradual disassembly of the disc skeleton, and reorganization of the parent flagella preclude attachment by negative pressure as well as any other model of attachment based on the functioning of the disc cytoskeleton and ventral flagella (Chapter 18). Hence, the mechanism enabling cell adherence is even less clear in dividing Giardia than it is in non-dividing cells. Significant mitotic expansion of the ventrolateral flange, as demonstrated by SEM (Tumova et al., 2007b), indicates that it could be involved in the attachment. The ventrolateral flange is a cytoplasmic lip that surrounds the disc and extends up to the point of emergence of the caudal flagella in G. intestinalis. The adhesive capacity of the flange has been demonstrated experimentally (Erlandsen et al., 2004). It remains an open question whether actin localized by immunofluorescence staining at the periphery of the disc and cell body in interphase (Feely et al., 1982) could participate in the mechanism of attachment of a dividing cell if classical actin-binding and microfilament-associated proteins are lacking in Giardia (Morrison et al., 2007). At mitosis, even localization data for actin are not available. The disc cytoskeleton is stable and highly elaborate and is formed primarily of tubulins and beta giardin (Crossley et al., 1986). Several other proteins, e.g., annexin XIX (Bauer et al., 1999), alpha-17 giardin (Weiland et al., 2005), and SALP-1 (Palm et al., 2005), are localized to the disc in epitope-tagging experiments, suggesting that the protein composition of the disc is far more complex. Hence, different molecules are likely to participate in the stepwise disassembly of this complicated structure but none of them has been identified yet. Katanin, a microtubule-severing protein with ATPase activity that is present in the Giardia genome (Morrison et al., 2007), is among the candidates. In Chlamydomonas, katanin acts to prevent flagellar basal bodies from resorbing flagella during mitosis (Rasi et al., 2009). In Giardia, neither localization nor expression data for katanin are known during the cell cycle. Immunolocalization of activated Aurora kinase along the opened disc cytoskeleton (Davids et al., 2008) prior to its disappearance points to the importance of phosphorylation in the regulated disassembly of the disc. It has been shown in other cells that, through protein hyperphosphorylation,

S.C. Dawson et al. some skeletal filaments are converted to a soluble form. Degradation of as yet unknown cross-linking proteins that form numerous parallel horizontal bridges between neighboring microribbons appears to be crucial for the deformation of the Giardia disc. Similar deformation was observed in isolated disc skeletons after extraction of the cross-bridges with Triton X-100 (Holberton, 1981) and in encysting cells prior to disc fragmentation (Midlej and Benchimol, 2009). One possible scenario, based on cytologic observations, could be that removing the cross-bridges accelerates the decomposition of microribbons, leading to the destabilization and rapid depolymerization of the disc microtubules.

10.7.2 De Novo Assembly of Daughter Ventral Discs Although the ventral adhesive disc is essential for Giardia survival and pathogenicity, until recently, the formation of this organelle during the division process was unclear (Elmendorf et al., 2003). There were discrepancies between the models based on light and electron microscopy studies. Scarce models implied that the existing parent disc persists and induces the assembly of a daughter disc (Solari et al., 2003). Recent findings, based on a combination of observations of live cells, immunostaining and electron microscopy clearly showed that the two daughter discs are formed de novo on the anterior dorsal side of the attached parent cell, with their ventral sides exposed on the parent cell surface (Tumova et al., 2007b). The parent disc cytoskeleton, although not a template, might act as a donor of building material. Short microtubules arising from segregated basal body pairs appear after reorientation of the parent anterior flagella, starting at about anaphase. On either sides of the dividing cell, the short microtubules emanate from a region (MTOC) close to a base at the proximal end of a basal body of a separated parent caudal flagellum. These microtubules elongate to form a disc platform beneath the plasma membrane. The new discs have the two-fold rotational symmetry to each other: each new disc is turning counterclockwise. Assembly terminates after the detachment of the dividing cell, i.e., during the second phase of Giardia division (Tumova et al., 2007b).

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Fig. 10.5 Assembly of daughter ventral discs. Transmission (A, C) and scanning (B) electron microscopy shows development of two daughter discs (arrowheads) on dorsal side of the attached parent cell. In A, arrow points to remnants of a parent ventral disc cytoskeleton. Parent ventrolateral flange (vlf) persists and forms finger-like protrusions (B). Very short dorsal microribbons are attached to microtubules of newly developed daughter disc cytoskeleton (C and inset). (bb = basal body, ax = intracytoplasmic axoneme of reorganizing parent flagella, n = nucleus). D, E: Rotational symmetry of arrangement of microtubular cytoskeletons of newly formed daughter discs when observed the dividing parent cell from above. Both daughter discs expose their ventral sides; arrowhead points to side of overlapping of the daughter disc cytoskeleton (D). Disassembling parent disc cytoskeleton is visible in the same cell (F: arrow) (red = anti-tubulin, blue = DAPI)

The order in which the new disc cytoskeleton is assembled is opposite to that leading to the disintegration of the parent disc (Fig. 10.5). A microtubule sheet is formed first, followed by the gradual assembly of microribbons. A short microribbon with trilaminar ultrastructure is adjacent to the cytoplasmic side of each microtubule in telophase and elongates to reach normal interphase length during the free-swimming division phase when the fine structure of the disc is being completed with the assembly of a lateral crest (a projecting rim surrounding the disc and com-

posed of unknown fibrous material) and the ventrolateral flange. To form the disc cytoskeleton, the fast-growing microtubules emanating from the MTOC must gradually curve to form a spiral platform. Although, in general, fast-growing microtubules are less stiff than slow-growing ones (Janson and Dogterom, 2004), the mechanisms underlying the synchronized bending of newly growing disc microtubules and the control of their length remain unknown. Short filaments (side arms) of unknown composition are visible between

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disc microtubules and the plasma membrane by transmission electron microscopy (Holberton and Ward, 1981) indicate that, as with pellicle microtubules in plant cells and some protists e.g. trypanosomes, interactions between disc microtubules, and the plasma membrane may participate in driving the newly assembling disc microtubules. Evidence for a contribution of cross-bridges to the shaping of the disc cytoskeleton comes from detergent studies (Holberton and Ward, 1981). The biochemical background of the assembly of basic components of daughter discs is also unknown. A rapidly dividing cell might prefer protein recycling rather than synthesis. Solubilized proteins from the old disc might be used to build up components of the new discs. In vitro, soluble giardins reassemble from the solution as filaments, tending to aggregate laterally into ribbon-like structures with the same periodicity as native microribbons (Crossley and Holberton, 1983, 1985). It remains unknown whether this also happens in vivo. The same question concerns tubulins from the parent disc microtubules. Higher concentrations of soluble tubulins arising from the disassembly of the old disc could accelerate the rapid elongation of new microtubules. All five tubulin-specific chaperones that are important for microtubule assembly are present in the Giardia genome (Morrison et al., 2007). However, no data regarding the kinetic behavior of proteins from disc microtubules, microribbons or other structural components during division are available. Another unresolved question concerns the relationship between the median body and newly forming discs. It has repeatedly been suggested that short microtubules from the median body could represent either a pool or seeds for nucleating microtubules required for the rapid assembly of daughter discs. However, only indirect evidence pointing to a biogenetic relationship between these two structures exists. The gradual disappearance of the median body during the extremely short initial phase of mitosis (Sagolla et al., 2006) apparently results from the disassembly of microtubules and coincides with the gradual, de novo assembly of daughter discs. In contrast to the majority of microtubular structures, which depolymerize only from one side because the other side is blocked by docking to the MTOC, there is no known organizing center in the median body so they can

S.C. Dawson et al. quickly disassemble from both ends. The presence of the median body in all species of the genus Giardia that is in those diplomonads possessing adhesive disc, and its absence in all diplomonad genera without the disc, also suggests that these structures are related. Providing further support for this hypothesis, microtubules from the median body have the ability to grow microtubules at both, the minus and plus ends, as demonstrated in nucleation experiments in vitro (Holberton and Ward, 1981) and in mutants defective for kinesin-13 microtubule depolymerizing motor (Hoeng et al., 2008), where median body microtubules are significantly elongated (see Chapter 18).

10.8 Cytokinesis Cytokinesis is the final event of cell cycle that divides one cell into two daughter cells (Fig. 10.6). Although details concerning how the process proceeds differ between eukaryotes (e.g., the position of the division site is determined by the mitotic spindle in animals, the bud-neck in budding yeast and by positioning of the nucleus in fission yeast), the plane of cytokinesis is always perpendicular to the axis of the segregated chromosomes. In animals and yeast, partitioning of mother cell into two daughters is achieved via constriction by the actomyosin ring (Barr and Gruneberg, 2007). In Giardia, the plane of cytokinesis has been a point of controversy for decades. Incongruous models have been proposed (Kabnick and Peattie, 1990; Ghosh et al., 2001; Solari et al., 2003), either based on single observations (Solari et al., 2003; Benchimol, 2004a) or derived theoretically from other observations, e.g., single nucleus labeling of episomal DNA by FISH in interphase cells (Yu et al., 2002). Most of these models could not explain the duplication of other cell systems, namely the nuclei and flagellar apparatus. As indicated by the pioneering work of Soloviev (1963) who also first described the movement of dividing nuclei and their left–right division, and recently confirmed by others (Kulda and Nohynkova, 1995; Sagolla et al., 2006; Tumova et al., 2007b), Giardia undergoes cell division along the sagittal plane. The frontal planes of the daughter cells are perpendicular to the frontal plane of the parent. A sequence of events

Chap. 10 Cell Cycle Regulation and Cell Division in Giardia

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Fig. 10.6 Cytokinesis. A division site (arrowhead) between newly formed daughter discs as shown by scanning electron microscopy (A) and in a live cell (B). C–E: Live dividing cells shortly before detachment (C) and free-swimming (D, E). Free-swimming progeny connected with a short cytoplasmic bridge (arrow in E). F–H: Elongation of the narrow bridge (arrow) between caudal ends of adherent daughter cells; in H, arrowhead points to a site of final abscission

showing that Giardia divides via the unidirectional ingression of a cleavage furrow, followed by adhesion-dependent abscission, has been described in parallel with the morphogenesis of daughter ventral discs (Tumova et al., 2007b). The underlying molecular mechanisms behind the three-phase division process are unknown. However, an actomyosin contractile ring, as observed in animal cells and yeast, is obviously not present in Giardia. The division site is selected in the initial phase of the Giardia division. This site is marked by a shallow surface depression placed centrally on the anterior dorsal side of the attached parent cell in about anaphase when two parallel mitotic spindles lie orthogonal to the longitudinal cell axis. Furrowing is achieved through the assembly of plasma membrane in between the microtubular cytoskeletons of the daughter discs. The plane of cytokinesis is thus perpendicular to the axis of the mitotic spindles. What triggers cytokinesis, how the site is determined and the furrow formed is unknown. Positioning of the site indicates that either the spindles, as in animal cells, or separated basal bodies/ flagella, as suggested by, e.g., Trypanosoma brucei (Hammarton et al., 2007), might be involved in the site/furrow specification. To ensure that each progeny receives a single copy of the genome, it is important that the initiation of cytokinesis and furrow formation does not occur until the chromosomes have been segregated. Coordination is achieved by

coupling the initiation of cytokinesis with the inactivation of mitotic cyclin-dependent kinases. In budding yeast, which, like Giardia, undergo a variant of closed mitosis, mitosis and cytokinesis are coordinated via the mitotic exit network (MEN) (Yeong et al., 2002). Although Giardia has well conserved homologues of several MEN proteins, suggesting similar control (Morrison et al., 2007), it appears to lack the activator of a key GTPase in this signaling pathway, as no homologs for Bfa1p and Bud2p, proteins which form the GTPase activating complex in budding yeast, were identified in the Giardia genome (Morrison et al., 2007). It is, therefore, an open question whether MEN proteins in Giardia can be used to accomplish the same function as in yeast. During the free-swimming phase of Giardia division, the cleavage furrow progresses in the anterior posterior direction, between the caudal axonemes of daughter cells, splitting the detached parent cell along the longitudinal body axis. For this phase, membrane biosynthesis is crucial. Inhibition of the synthesis of sphingolipids, essential components of eukaryotic membranes, blocks the progression of furrowing (Sonda et al., 2008). Mechanistically, the two daughter cells separate ventral-to-ventral (“face-to-face”) with two-fold rotational symmetry (biradial, similar to the ying-jang symbol), which is most obvious from the counterclockwise winding of the ventral discs in both

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daughters (Tumova et al., 2007b). As in trypanosomes (Hammarton et al., 2007), unidirectional remodeling of the cell shape does not correspond to the constriction of a contractile ring, as occurs in animals and yeast. Indeed, myosin II, one of the key components of the actomyosin contractile ring was not found in Giardia (Elmendorf et al., 2003; Morrison et al., 2007), and this species is likely to also lack homologs of the Rho family of small GTPases (RhoA, Cdc42) (Pellegrin and Mellor, 2005) that control actomyosin ring assembly and furrow ingression in other eukaryotes. A contribution of actin to Giardia cytokinesis is currently unclear. The Giardia genome contains a single actin gene but homologs of proteins mediating actin sequestration and polymerization (profilins and formins); actin crosslinking and microfilament specific motor proteins are not encoded (Morrison et al., 2007). Ultrastructural alterations observed in cytokinesis after treatment with the actin filamentdestabilizing agent cytochalasins D (Correa and Benchimol, 2006), may be secondary (indirect), as normal growth characteristics change only 48 hours after exposure to cytochalasins (Castillo-Romero et al., 2009), which implies that Giardia undergoes at least four normal division cycles prior to any observed growth changes. Increased numbers of cells are blocked in the second stage of cytokinesis after the inhibition of Aurora kinase (Davids et al., 2008), indicating that Aurora kinase could participate in the control of ingression rather than selecting the division site, as is usual in other eukaryotes. The most unusual event in Giardia cytokinesis occurs when, after swimming, progeny that are connected tail-to-tail adhere to the substratum via their newly assembled adhesive discs and crawl apart (moving away from one another), while still connected by a narrow intercellular cytoplasmic bridge (Fig. 10.6). The bridge gradually elongates and finally breaks (cracks) in the middle (Tumova et al., 2007b). This phase of Giardia division partially resembles the adhesion-dependent, myosinII-independent cytokinesis of Dictyostelium amoebae (Uyeda and Nagasaki, 2004). Physical forces generated from the opposite movement of the daughter cells may contribute to abscission. How the locomotion is driven and whether there are any

S.C. Dawson et al. specific severing proteins at the site of abscission remain to be identified.

10.9 Asymmetry and Aging in Giardia Division Cytokinesis in Giardia is symmetrical and the two daughters copy the parent cell. However, they are not identical. Although morphologically indistinguishable, they differ in the age of basal bodies/flagella (Nohynkova et al., 2006), and in the positioning of inherited nuclei (Ghosh et al., 2001; Sagolla et al., 2006; Tumova et al., 2007b). From this point of view, the division of Giardia is polarized; only one daughter cell inherits the oldest, i.e., mature basal body/flagellum. As parent basal bodies persist during division and the mature basal body does not transform (see above), over several generations one cell will inevitably inherit an increasingly old mature basal body/flagellum. It is currently unknown whether asymmetrical inheritance of the basal bodies is a mechanism of asymmetrical replicative aging, similar to that seen in bacteria and yeast (Henderson and Gottschling, 2008; Macara and Mili, 2008), and whether Giardia has a limited lifespan.

10.10 Conclusions It is evident that, although much progress has been made in recent years toward an understanding of the division of cells, nuclei, and the cytoskeletal apparatus of Giardia, much remains to be learned about the molecular background of these processes and their control mechanisms.

References Adam RD (2000) The Giardia lamblia genome. Int J Parasitol 30(4): 475–484 Akhmanova A and Hoogenraad CC (2005) Microtubule plusend-tracking proteins: mechanisms and functions. Curr Opin Cell Biol 17(1): 47–54 Amin-Hanjani S and Wadsworth P (1991) Inhibition of spindle elongation by taxol. Cell Motil Cytoskeleton 20(2): 136– 144 Asakawa K, Toya M, et al. (2005) Mal3, the fission yeast EB1 homologue, cooperates with Bub1 spindle checkpoint to prevent monopolar attachment. EMBO Rep 6(12): 1194– 1200

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The Giardia Mitosomes Jan Tachezy and Pavel Doležal

Abstract Mitosomes represent the simplest forms of mitochondria. The long independent evolution of the mitosomes bearing unicellular eukaryotes like Giardia intestinalis transformed mitochondria into tiny double membrane bound vesicles, which have lost organellar genome, respiratory chain as well as the capacity to generate ATP. While publication of G. intestinalis genome sequence has brought great source of data for deciphering mitosomal functions, biochemical approaches have struggled with difficulties in organelle isolation techniques due to its miniature size and low abundance. Thus to date the sole metabolic pathway harbored in the G. intestinalis mitosomes is the formation of FeS clusters. This chapter reviews recent successes in the research of these fascinating organelles and proposes perspective research directions.

11.1 Introduction Mitosomes are minimized forms of mitochondria that have completely lost their mitochondrial genome and the majority of mitochondrial pathways, including respiration, the citric acid cycle, and ATP synthesis. They have been found in parasitic protists living either in oxygen-poor environments, such as Giardia intestinalis (Tovar et al., 2003) and Entameba histolytica (Mai et al., 1999; Tovar et al., 1999), or as intracellular parasites, including microsporidia (Williams et al., 2002) and Cryptosporidium parvum (Riordan et al., 1999). It is likely that mitosomes are also present in some anerobic free-living protists (Hampl and Simpson, 2008). The absence of classical mitochondria, together with early phylogenetic analyses based on SSU rRNA genes, led to the hypothesis that Giardia intestinalis

H. D. Luján et al. (eds.), Giardia © Springer-Verlag/Wien 2011

11 and other so-called “amitochondrial” protists represent deep-branching eukaryotes that diverged before the acquisition of mitochondria (Cavalier-Smith, 1987; Adam, 2001; Best et al., 2004). Known as the Archezoa hypothesis, this attractive concept began to look less likely when genes regarded as mitochondrial in origin, including those coding for chaperonin 60 (CPN60) (Clark and Roger, 1995; Bui et al., 1996); Horner et al., 1996; Roger et al., 1998), mitochondrial-type heat shock protein 70 (mitHSP70) (Bui et al., 1996; Germot et al., 1996, 1997; Morrison et al., 2001; Arisue et al., 2002), pyridine-nucleotide transhydrogenase (Clark and Roger, 1995), cysteine desulfurase (GiiscS) (Tachezy et al., 2001) and other protein members of the FeS cluster assembly machinery (Katinka et al., 2001), were identified in the genomes of all Archezoan groups. It was inferred that these genes were originally part of the genome of endosymbiotic proteobacteria, which gave rise to mitochondria, and that during the endosymbiont to mitochondrion transformation, they were transferred into the nuclear genome of the host cell. The absence of mitochondria in “amitochondriates” was thus explained by the secondary loss of these organelles due to adaptation of certain protists to an anerobic environment or to intracellular parasitism. However, later studies that focused on the subcellular localization of the mitochondrial gene products revealed that mitochondria were not completely lost but that they are present in reduced forms named mitosomes to which the mitochondrial proteins localized. Evidence for the mitochondrial origin of giardial mitosomes comes from (i) the presence of a double membrane surrounding the mitosome, (ii) the similarity of targeting, translocation, and processing of mitosomal proteins compared to mitochondrial ones, and (iii) the presence of mitochondrial-type iron–sulfur cluster assembly machinery (ISC) (Tovar et al., 2003; Dolezal

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et al., 2005). Today, no truly amitochondrial eukaryote, either primary or secondary, is known. Mitosomes evolved independently in various lineages of protists. There are no doubts about the mitochondrial origin of mitosomes in species that are embedded within a group of organisms with regular mitochondria. For example, microsporidians, which possibly all harbor mitosomes, belong to the group of fungi, the members of which usually possess fully equipped mitochondria (Reinders et al., 2006). However, the origin of mitosomes found in G. intestinalis

is still debated. Analysis of the complete G. intestinalis genome sequence did not reveal any new information about mitosomes (Morrison et al., 2007; Franzen et al., 2009), and mitosomal marker proteins known to reside within these organelles were suggested to have been acquired by lateral gene transfer (Morrison et al., 2007). This review is devoted to summarizing recent knowledge and opinions about G. intestinalis mitosomes, their biogenesis and their function, with the aim of contributing to the understanding of these still enigmatic organelles.

A

B

C

Fig. 11.1 Mitosomes of Giardia intestinalis as seen by immunofluorescent labeling of IscU, with two trophozoite nuclei stained with DAPI (B), Nomarski differential contrast (A). Full arrows on the electron micrograph point to central mitosomes distributed between axonemes of caudal flagella (C). The electron micrograph was kindly provided by Pavla Tmová, Charles University in Prague, Czech Republic

Chap. 11 The Giardia Mitosomes

11.2 Morphology and Cellular Distribution Mitosomes of Giardia intestinalis are tiny ovoid organelles less than 0.2 μm in size (an average of 184 × 140 nm). The number of mitosomes ranges from 25 to 100 per cell (Tovar et al., 2003; Dolezal et al., 2005; Regoes et al., 2005). There are two types of mitosomes distributed within the cell. The central mitosomes form a rod-like structure, which is invariably present in all cells between the two nuclei (Fig. 11.1). Electron microscopy revealed that the rod-like structure consists of several tightly packed mitosomes that are organized between axonemes of the caudal flagella. The rest of the mitosomes (peripheral mitosomes) are scattered within the cytosol of Giardia, often in lateral and posterior regions of the cells. Central mitosomes were suggested to be important for mitosome segregation to the daughter progeny. Regoes et al. (2005) observed double rod-like structures in the early stages of mitosis, suggesting that the central mitosomes divide together with basal bodies of the flagella. Later studies on G. intestinalis mitosis revealed that the basal bodies replicate at a later stage of mitosis, which is preceded by parental flagella migration and reorientation (Nohynkova et al., 2006). Only parental caudal flagella do not change their function or position in the daughter progeny. Consequently, each daughter cell possesses one parental caudal flagellum, whereas the second caudal flagellum develops during cell division from the parent anterolateral flagellum. Our investigation of mitosome distribution during G. intestinalis mitosis revealed that the central mitosomes separate from the rod-like structure during mitosis. The central mitosomes split, with some migrating left and some right, in association with the basal bodies of caudal flagella, and mostly likely segregated to the daughter progeny together with the caudal flagella.

11.3 Protein Targeting, Translocation, and Maturation During the evolution of mitochondria, a large part of the protomitochondrial genome was transferred into the nucleus of the host cell (Gray et al., 1999). This evolutionary step was possible only because of the in-

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novation of mechanisms for retargeting nuclear-encoded proteins into these organelles (Dolezal et al., 2006). The solution to this task included the acquisition of N-terminal presequences or internal signals on mitochondrial proteins for protein targeting and the introduction of mitochondrial membrane translocases. Although the primary structure of targeting presequences is not conserved, they are characterized by the ability to form a positively charged amphipathic α-helix, which is first recognized and then translocated by the outer membrane translocase (TOM). The positive residues of the presequence are then recognized by the inner membrane translocase, TIM23 (Donzeau et al., 2000; Meier et al., 2005; Neupert and Herrmann, 2007), and the membrane potential across the inner mitochondrial membrane forces the presequence to pass through the channel of TIM23 by electrophoresis (Martin et al., 1991). After translocation, the presequences may interfere with the function and/ or stability of the mature protein (Mukhopadhyay et al., 2007), so a mitochondrial processing peptidase (MPP) is recruited to recognize and cleave off the presequences before the proteins mature within the organelles. These mechanisms mediate the import of hundreds of proteins required for various mitochondrial functions. A similar system was also found in hydrogenosomes of Trichomonas vaginalis, specific forms of mitochondria producing ATP and molecular hydrogen under anerobiosis.

11.3.1 Protein Targeting The mode of protein targeting to G. intestinalis mitosomes is similar to that operating in mitochondria and hydrogenosomes. However, the mitosomal system seems to be considerably simplified, reflecting limited mitosomal functions and, consequently, the limited number of proteins that have to be delivered to the mitosomes. Although it is difficult to estimate the size of the complete mitosomal proteome, so far only about 20 proteins have been shown to be mitosomally targeted (Jedelský et al., 2010). In a mechanism similar to that in mitochondria, the targeting of mitosomal proteins depends on N-terminal presequences, which are processed upon entering the organelles, or on internal targeting signals (Dolezal et al., 2005; Regoes et al., 2005). Mitosomal presequences are also able to

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deliver proteins to hydrogenosomes as well as mitochondria, which strongly supports a common mode of protein targeting and translocation into these organelles (Dolezal et al., 2005; Regoes et al., 2005). However, only a few mitosomal presequences have been identified and studied experimentally. Three presequences found at the N-termini of ferredoxin, IscU, and IscA, differ significantly from typical mitochondrial presequences. They are rather short, consisting of 10–18 amino acids, with only a single positively charged residue (arginine) at the cleavage site motif [(ARV)R(F/L)(L/I)T] recognized by the giardial processing peptidase. In mitochondrial presequences, there are several additional positively charged residues (arginines and/or lysines) at N-terminally distal positions from the processing site, which increase the overall positive charge of presequences. In contrast, these mitosomal presequences do not contain any distal arginine or lysine, and consequently, the positive charge of mitosomal presequences is considerably lower. These properties have been suggested to reflect lower mitosomal membrane potential and modifications in the mechanisms of protein translocation across the mitosomal membrane as well as unique properties of the processing peptidase (see below). Interestingly, a fourth N-terminal presequence was recently identified in mitosomal glutaredoxin. The conserved part of the protein is preceded by a 77-amino acid extension, which is required for protein translocation and that is absent in the mature protein (Rada et al., 2009). However, it is not clear how the glutaredoxin preprotein is processed, as neither the giardial processing peptidase nor yeast MPP is able to cleave off the presequence in vitro. The N-terminal cleavable presequence-dependent mechanism of protein targeting is typical for the delivery of mitochondrial matrix proteins, whereas non-cleavable inner signals mediate the targeting of hydrophobic membrane proteins (Chacinska et al., 2009). Interestingly, several matrix proteins that are delivered into giardial mitosomes also lack a cleavable presequence. For example, mitochondrial cysteine desulfurases (IscS) invariably possess N-terminal presequences, whereas giardial IscS lacks such an extension. Interestingly, when giardial IscS is expressed in T. vaginalis, it is targeted to the hydrogenosomes, indicating the presence of an inner targeting signal that is recognized by hydrogenosomal translocases.

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Unlike GiiscS, the hydrogenosomal IscS possesses an 8-amino acid targeting presequence. However, when this presequence is removed, hydrogenosomal IscS is also delivered to hydrogenosomes, as is giardial IscS. Similarly, when giardial IscU is expressed in Giardia without a presequence, some of the proteins are still delivered to mitosomes, although the delivery is less efficient (Dolezal et al., 2005). Cleavable presequenceindependent transport of IscU was also observed in Saccharomyces cerevisiae (Gerber et al., 2004). Therefore, the inner targeting presequences present in some matrix proteins are sufficient for protein delivery into the organelle, although N-terminal presequences increase the efficiency of the transport. In addition to IscS presequences seem to be lost in other mitosomal proteins, such as Hsp70, and Cpn60. However, translocation of other proteins, such as mitosomal ferredoxin, that do not possess an inner targeting signal is fully dependent on cleavable presequences (Dolezal et al., 2005; Regoes et al., 2005).

11.3.2 Mitosomal Processing Peptidase The MPP is a heterodimeric zinc metallopeptidase that most likely evolved from a monomeric α-proteobacterial protease related to the Rickettsia prowazekii-processing peptidase by gene duplication and subsequent subunit specialization (Kitada et al., 2007). A catalytic β-MPP subunit binds a zinc cation via amino acid residues of the conserved motif HXXEHX76E of the M16 family of metallopeptidases (Gakh et al., 2002). A regulatory α-MPP subunit is characterized by the presence of a flexible glycinerich loop that is essential for substrate recognition (Nagao et al., 2000; Nishino et al., 2007). The two subunits form a negatively charged cavity that accommodates and immobilizes the presequence during processing by electrostatic interaction. Hence, the processing activity of MPP is a result of the cooperative action of both subunits; neither the β nor the α subunit is active alone (Arretz et al., 1994; Saavedra-Alanis et al., 1994; Gakh et al., 2002). The processing peptidase found in Giardia mitosomes (GPP) appears to be unique among eukaryotes because it functions efficiently as a monomer corresponding to the catalytic β-MPP subunit and thus lacks a functional equivalent of the α-MPP subunit

Chap. 11 The Giardia Mitosomes

(Smid et al., 2008). Kinetic parameters of monomeric GPP (Vmax = 0.27 mM/min; Km = 8.4 mM) are comparable to those published for heterodimeric MPPs. Comparisons of GPP with yeast MPP and hydrogenosomal heterodimeric processing peptidase (HPP) using mitosomal, mitochondrial, and hydrogenosomal substrates showed that GPP is able to process only mitosomal presequences. The substrate specificity of GPP can be explained by its unique structure. In heterodimeric MPPs, the glycine-rich loop of the α subunit recognizes the substrate and moves it to the catalytic site of the β subunit, where the proximal arginine residue of the substrate cleavage motif interacts with the catalytic site, whereas the distal positive residues of the mitochondrial presequence bind to negatively charged residues within the polar cavity formed by the α/β MPP subunits (Shimokata et al., 1998) and stabilize the substrate-MPP complex. In monomeric GPP, the negatively charged region of its active site interacts directly with the cleavage motif of the substrate. The rest of the predicted GPP cavity is positively charged, unlike the β subunit of MPP. The difference in the charge distribution of the GPP cavity is due to the absence of distal positively charged residues in the mitosomal presequences, and this, together with the absence of an α MPP subunit, may explain the inability of GPP to cleave mitochondrial-type presequences. The simplicity of GPP is consistent with the highly reduced proteome of mitosomes: whereas MPP has to recognize and process hundreds of mitochondrial proteins required for the function of complex mitochondrial metabolic pathways, GPP is likely required for the processing of only a few mitosomal proteins (Smid et al., 2008). The unique single subunit-type structure of GPP may be considered to be an ancestral feature, with GPP evolving directly from an ancestral single subunit α protebacterial peptidase. An alternative explanation involving lateral gene transfer for the origin of GPP has been suggested (Morrison et al., 2007). However, phylogenetic analysis revealed that GPP clusters together with mitochondrial and hydrogenosomal β subunits, which suggests that the single-subunit structure of GPP results from reductive evolution including the loss of an α subunit of MPP. This analysis did not support either the ancestral or the lateral gene transfer hypothesis as the origin of the simple GPP structure (Smid et al., 2008).

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11.3.3 Protein Import Machinery The interior of eukaryotic cells is sectioned by lipid membranes into cellular compartments that carry out specialized cellular functions. Whereas the lipid bilayer prevents proteins and other biomolecules from freely diffusing into and out of the cell, specific molecular machinery mediates the translocation of newly synthesized or nascent proteins in the form of unfolded polypeptides across the bilayer into their proper compartments. The role of the translocon is complemented by the presence of a molecular address (e.g., signal peptide, mitochondrial targeting sequence) on the transported protein, which is specifically recognized by a membrane-associated receptor and that subsequently triggers the initial step of the translocation event. The translocation requires energy stored in the form of membrane potential or nucleotide triphosphates, which are hydrolyzed by an associated motor molecule (Wickner and Schekman, 2005). Upon translocation, the unfolded polypeptides undergo assisted folding by molecular chaperones. If a protein contains an N-terminal address sequence, specific organellar peptidases remove the peptide prior to the folding step. Mitosomes and mitochondria stay aside the endomembrane system, which receives most of its proteins in a co-translational mode through the action of Sec61 translocase. During the course of evolution, massive gene losses occurred in the mitochondrial genomes. Some genes were irreversibly abandoned, and some were transferred to the host cell nucleus, with the result that the number of protein-coding genes in the mitochondrial DNA shrunk to 3–67 (Gray et al., 1999). Current mitochondria accept 99% of their proteins post-translationally after the nuclear-encoded proteins are translated on free cytosolic ribosomes. Mitosomes walked step further and lost their entire organellar genome (Chacinska et al., 2009). The two mitochondrial membranes define four sub-compartments, each accommodating a specific set of residential proteins. To deliver proteins into (i) the outer and (ii) inner mitochondrial membranes, (iii) the intermembrane space, and (iv) the mitochondrial matrix, eukaryotes have created several distinct protein import pathways. The protein complexes responsible for this translocation mostly represent ancient eukaryotic innovations; however, some of the core components have homologs operating in bacterial membranes,

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suggesting that these were “borrowed” from bacteria (Alcock et al., 2010). All mitochondrial proteins pass through the Tom40 import channel in the outer mitochondrial membrane. This protein has a β-barrel structure serving as an entry channel for unfolded polypeptides. Several other components are built around Tom40 and constitute the TOM complex. The complex operates as a molecular machine recognizing soluble substrates with either N-terminal targeting presequences or internal targeting signals (Chacinska et al., 2009). Whereas the first interaction is mediated by the soluble domain of the Tom20 receptor, subsequent interactions require the Tom70 receptor, which docks chaperones such as Hsp70 and Hsp90 loaded with hydrophobic substrates. The cooperative action of Tom20 and Tom70 ensures the binding of more complicated substrates (Yamamoto et al., 2009). Proteins are then transferred to Tom22, and from here they are inserted into the Tom40 channel. So-called small Tom proteins play a role in the assembly of the TOM complex or assist in the transport process (Gentle et al., 2007; Petrakis et al., 2009). In the intermembrane space, proteins are sorted by four independent pathways that separately guide proteins to a particular sub-compartment. Proteins of the outer membrane are assembled by the sorting and assembly machinery (SAM) complex, which acts as a specialized molecular machine for β-barrel proteins (Paschen et al., 2003; Gentle et al., 2004). Sam50 is the central component of the SAM complex, and several partner proteins assist Sam50 in binding and releasing substrates. β-barrel proteins, as well as membrane proteins of the inner mitochondrial membrane, must be chaperoned by a complex of small Tims, which bind to hydrophobic patches on the polypeptide and prevent protein aggregation (Petrakis et al., 2009). Heteromeric assemblies of small Tims also help in the translocation of other classes of membrane proteins, such as mitochondrial carriers, that are destined for the inner mitochondrial membrane. Instead of SAM complex, the carrier protein with the chaperones docks at TIM22 complex in the inner membrane and, upon the release of the chaperones, is inserted into the membrane (Wagner et al., 2008). Tim22 serves as the core channel-forming component of the complex and requires only membrane potential as an energy source. Two complexes, TIM23 and

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PAM (presequence translocase associated motor), cooperate during the transport of proteins across the inner mitochondrial membrane. Whereas the TIM23 complex constitutes the membrane channel, receiving proteins from the TOM complex (Mokranjac and Neupert, 2010), PAM functions as an ATP-dependent machine providing energy for the movement of translocated proteins through the channel. Individual proteins of the TIM23 complex recognize the substrates at the exit of the TOM complex, form the membrane channel and mediate association with the PAM complex (Chacinska et al., 2005). The role of the PAM complex is to recruit and modulate the activity of the motor molecule Hsp70, which directly binds to the translocated polypeptide. Proteins entering the mitochondrial matrix undergo proteolytic cleavage to remove the targeting presequence, a process mediated by MPP. The Cpn60-Cpn10 system then assists the folding of the polypeptide into its native conformation (Chacinska et al., 2009). The availability of the genome sequence of G. intestinalis has dramatically spurred molecular research of its cellular functions. However, whereas bioinformatic analyses revealed that homologous proteins mediate various essential processes, not a single component of the mitochondrial/mitosomal translocation machinery was identified in the genome (Morrison et al., 2007). This could either mean that (i) G. intestinalis has lost or replaced these components to form a unique mitosomal machinery, (ii) G. intestinalis never had these proteins in its membranes due to ancient evolutionary independence, or (iii) sequences of these components have diverged to such an extent that they are beyond the sensitivity of our bioinformatic algorithms. Current data indicate that the explanation may involve all three hypotheses (Fig. 11.2). Mitosomes appear to contain only a single β-barrel protein in their outer membrane. Using a hidden Markov model designed to uncover proteins of the porin_3 family, a protein with all the hallmarks of Tom40 was identified. The protein is predicted to have a β-barrel structure built of 16 β-sheets and contains a C-terminal β-signal to target β-barrels to the SAM complex. In a cluster analysis, it showed affinity for other Tom40 molecules. Moreover, in G. intestinalis it was found to localize to the mitosomal membranes in a complex of unknown composition (Dagley et al., 2009). Overall sequence divergence

Chap. 11 The Giardia Mitosomes

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Fig. 11. 2 Schematic representation of protein import pathway in mitosomes of G. intestinalis. Components present in mitosomes are shown in color. Components that are known to participate in the protein import into mitochondria of animals and fungi are shown in gray color

precluded the use of heterologous functional studies, although the protein is recognized and imported into mitochondria of S. cerevisiae. The presence of a β-barrel protein bearing a β-signal would indicate the existence of a functional SAM complex that can take up a protein and assemble it in the outer membrane. Surprisingly, G. intestinalis is thus far the only eukaryotic organism lacking a homolog of Sam50; other mitosome-bearing protists possess them (Dolezal et al., 2010). From an evolutionary perspective, the absence of Sam50 in the mitosomal membrane most likely represents a secondary trait. The relationship between Sam50 and bacterial Omp85 (BamA) is considered one of the strongest pieces of evidence for the bacterial origin of mitochondria (Gentle et al., 2004). Recently, intermembrane space was shown to harbor a disulfide relay system consisting of two proteins Mia40 and Erv1, which together serve toward the import of substrate proteins carrying characteristic patterns of cysteine residues (Mesecke et al., 2005). Oxidoreductase Mia40 directly binds these proteins via disulfide bonds while sulfhydryl oxidase Erv1 reoxidizes

Mia40 and shuffles electrons to cytochrome C. In case of G. intestinalis, mitosomes do not contain either the proteins with consensus cysteine motif or the components of redox chain (Mesecke et al., 2005). Remarkably, no Tim proteins have been found in G. intestinalis, and though it is possible that these proteins are as yet unidentified due to extreme sequence divergence, it is plausible that G. intestinalis has a different inner membrane translocase(s). However, the import of mitosomal proteins relies on the same motor complex as homolog of Pam18, a crucial component of PAM complex, has been found in mitosomes (Dolezal et al., 2005). The protein contains an N-terminal transmembrane domain as well as a Cterminal J-domain, which stimulates the activity of mitosomal Hsp70 (Regoes et al., 2005). In addition, our recent data suggest that G. intestinalis mitosomes contain a Pam16 homolog (Jedelský et al., 2010). Pam16 has a regulatory role in the PAM complex. Pam16 is N-terminally anchored in the inner membrane in a manner similar to Pam18, but it differs from this protein in that its J-domain lacks three residues bearing the chaperone-stimulating activity.

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Altogether, these data suggest that a functional motor complex consisting of Hsp70, Pam18 and Pam16 is present at the inner mitosomal membrane assisting the translocase to pull the substrate protein into the mitosomal matrix. Whether the motor complex assists a different translocase remains to be verified experimentally. It is important to mention that TIM translocases require the presence of a membrane potential, which has not been detected by any means in G. intestinalis mitosomes. Hence, the loss of Tim proteins may have followed the loss of respiratory chain components that generate the proton gradient across the inner mitochondrial membrane and that have also been shown to be in physical contact with TIM translocases (van der Laan et al., 2006; Saddar et al. 2008).

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Upon translocation, the N-terminal targeting sequence is removed by a processing peptidase (see Sect. 11.3.2 above) and the mature polypeptide then undergoes assisted folding. Hsp70 not only serves as the importing motor molecule but also assists in the subsequent folding step (Liu et al., 2001). The stability of interactions between Hsp70 and the unfolded protein follows the ATP–ADP cycle of the chaperone. The speed of this cycle is regulated by co-chaperones that stimulate either the ATPase activity of Hsp70 (Hsp40) or the release of ADP from the ADP-Hsp70 complex (Mge1). In addition to Hsp70 (Regoes et al., 2005), G. intestinalis mitosomes contain a homolog of Mge1 (GrpE) (Jedelský et al., 2010). Although several putative type I J-domain proteins are present in G. intestinalis genome,

Fig. 11. 3 Iron–sulfur cluster assembly machinery in mitosomes of G. intestinalis. Components that were identified in mitosomes are shown in color. Components known to participate in this process in mitochondria of Saccharomyces cerevisiae, that were not found in mitosomes, are shaded gray for comparison

Chap. 11 The Giardia Mitosomes

no mitosomal Hsp40 homologs have yet been identified (Walsh et al., 2004). For more difficult substrates, the Cpn60 and Cpn10 chaperonins oligomerize into an Anfinsen cage and mediate proper protein folding. Cpn60 is present in G. intestinalis mitosomes despite the lack of an obvious N-terminal targeting presequence (Regoes et al., 2005). We have recently identified a homolog of Cpn10 in mitosomes (Jedelský et al., 2010), and in this respect, mitosomes seem to have retained most of the mitochondrial protein-folding machinery.

11.4 FeS Cluster Assembly Machinery The formation of FeS clusters is an essential mitochondrial function mediated by the FeS cluster (ISC) assembly machinery, which facilitates maturation of FeS proteins. The process proceeds in two consecutive steps: the formation of transient FeS clusters on molecular scaffold proteins, and the transfer of the transient FeS clusters to target apoproteins. Importantly, the ISC machinery is required not only for the maturation of mitochondrial apoproteins but also for the formation of FeS centers in other cell compartments (Lill et al., 1999; Lill, 2009). Giardia genome data mining together with proteomic investigations of mitosomes (Jedelský et al., 2010) revealed that mitosomes possess all the key components of the ISC machinery (Fig. 11.3). The core components are cysteine desulfurase (IscS), which releases sulfur from cysteine, and IscU, which provides a molecular scaffold for transient FeS cluster formation. Mitosomes also contains Nfu, which may serve as an alternative molecular scaffold. Components with a proposed role in the transfer of transient FeS clusters to target apoproteins include the chaperone, Hsp70, with co-chaperones, HscB (Dna-J protein, Jac1 in S. cerevisiae) and Mge1 (bacterial GrpE). Mitosomes also contain monothiol glutaredoxin 5, which has a conserved CGFS motif (Grx5). Although the exact role of Grx5 is not clear, its ability to form a homodimeric structure that coordinates a [2Fe2S] cluster suggests that Grx5 may also serve as an alternative FeS scaffold (Rada et al., 2009). Interestingly, the [2Fe2S] cluster bridging two Grx monomers is anchored by four cysteinyl residues. Two cysteinyl residues are provided by the

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cysteines of the CGFS motif of each monomer, whereas the other two cysteine residues are provided by the tripeptide glutathione (GSH). Although GSH was believed to be absent in Giardia, our searches in the Giardia genome revealed the presence of genes coding for two key enzymes required for GSH synthesis: glutamate-cysteine ligase and GSH synthase (Rada et al., 2009). Thus, GSH is likely present in Giardia at low concentrations that were not detected in the previous biochemical study (Brown et al., 1993). IscA is another mitosomal protein that is likely involved in FeS cluster formation, although its role is unclear. In general, there are two main families of IscA-like proteins (Vinella et al., 2009). The ATC-I family includes bacterial ErpA, which was shown to bind FeS clusters and to transfer them to apo-IspG and IspH proteins, which catalyze isopentenyl diphosphate biosynthesis in E. coli (Loiseau et al., 2007), and its eukaryotic mitochondrial ortholog, IscA2 (Isa2). The ATC-II family includes bacterial IscA, a member the of iscSUA gene cluster with a general role in FeS cluster formation (Zheng et al., 1998), eukaryotic IscA1 (Isa1), which resides in mitochondria, and SufA, a member of the SUF system, which is an alternative FeS cluster assembly machinery in bacteria and plastids (Vinella et al., 2009). Both IscA1 and IscA2 are present in virtually all mitochondria. Their proposed roles are to act as scaffold proteins for transient FeS clusters (Pelzer et al., 2000; Krebs et al., 2001; Song et al., 2009) and/or to serve as iron donors (Ding et al., 2004). Whether their roles are specific or redundant is still under debate (Jensen and Culotta, 2000; Pelzer et al., 2000). Studies of IscA, ErpA, and SufA mutant E. coli strains revealed that these proteins are redundant under anerobic conditions. Under aerobic conditions, however, both IscA and ErpA appear to be essential (Vinella et al., 2009). Interestingly, the Giardia mitosome, as well as Trichomonas hydrogenosomes, contains only IscA-2 type proteins, whereas IscA-1 type proteins are absent. We can speculate that the presence of only a single type of IscA proteins in mitosomes and hydrogenosomes could be associated with the growth of Giardia and Trichomonas in anerobic environments. Another possible explanation is that IscA-1 was lost together with majority of mitochondrial FeS proteins, including aconitase, for which IscA-1 was required and that

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IscA-2 was retained either for the maturation of a specific set of mitosomal and hydrogenosomal FeS protein(s) or as an iron transporter (Ding et al., 2004). A possible role of giardial IscA in mitosomal iron transport is also supported by an absence of frataxin. Frataxin is an iron-binding protein that donates iron to the IscS/IscU complex or regulates FeS cluster formation (Stehling et al., 2004; Bencze et al., 2006; Foury et al., 2007). It is invariably present in all eukaryotes containing ISC-type FeS cluster assembly machinery, including organisms with mitosomes, such as E. cuniculi (Goldberg et al., 2008) and C. parvum (Abrahamsen et al., 2004). A partial frataxin sequence has also been found in the diplomonad Spironucleus vortens, which is a close relative of Giardia (our unpublished data). However, our exhaustive bioinformatic search for frataxin in the genomes of the three G. intestinalis strains currently available at GiardiaDB failed to identify any frataxin ortholog. Thus, it would be interesting to test whether the unique absence of frataxin in Giardia is compensated for by giardial IscA-1 to mediate iron transport, and whether frataxin can fulfil this function in other mitosomes that do not contain IscA proteins. The presence of core components of the FeS cluster assembly machinery in the proteome of Giardia mitosomes corroborate the previous finding that a mitosome-enriched fraction catalyzed the formation of FeS clusters on apoferredoxin (Tovar et al., 2003). Nevertheless, the mitosomal machinery seems to be less complex than that of mitochondria. For example, mitosomes do not contain Isd11, which forms a functional heterodimer with cysteine desulfurase (Adam et al., 2006; Wiedemann et al., 2006). Interaction of Isd11 with eukaryotic IscS and its importance for FeS cluster assembly was demonstrated in yeast (Wiedemann et al., 2006) and human cells (Shi et al., 2009). The formation of a functional IscS/Isd11 complex was also shown in Trachipleisthophora hominis, an organism with mitosomes (Goldberg et al., 2008). However, Isd11 is not always an essential component for the function of IscS, as its absence was noticed, in addition to Giardia, also in C. parvum and it is generally absent in bacteria (Richards and van der Giezen, 2006). Ind1 is another component that has not been identified in mitosomes to date. In mitochondria, Ind1 is required for the maturation of the FeS protein of

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multi-subunit respiratory complex I (Bych et al., 2008; Sheftel et al., 2009). Orthologs of Ind1 are also present in hydrogenosomes of T. vaginalis. These organelles contain a highly reduced form of complex I consisting of only two catalytic subunits, Tvh-47 and Tvh-22, which require FeS clusters for their activity (Hrdy et al., 2004). The absence of Ind1 likely reflects the absence of a specific substrate, as no traces of complex I are present in Giardia and other mitosomes. Mitochondrial protein Iba57 forms a complex with IscA type scaffold proteins (Isa1p, Isa2p), which play a specific role in the assembly of [4Fe4S] clusters and in the functional activation of mitochondrial radical-SAM FeS proteins (Gelling et al., 2008). As in the case of Ind1, the absence of Iba57 in mitosomes likely reflects the absence of the corresponding substrate. The question of what is the substrate requiring FeS cluster formation in Giardia mitosomes is an intriguing one. In fact, no FeS protein has been identified in these organelles so far, except for FeS proteins that are members of ISC machinery itself, such as IscU and [2Fe2S] ferredoxin. Thus, the mitosomal machinery is likely required for the biogenesis of FeS proteins outside of mitosomes. In S. cerevisiae, ISC machinery is required for the maturation of cytosolic FeS proteins. Three components have been implicated in the export of a thus far unknown mitochondrial compound that is donated to the cytosolic FeS assembly machinery. These components include inner mitochondrial membrane ABC half-transporter Atm1 (Kispal et al. 1997), intermembrane-space sulfhydryl oxidase Erv1 (Lange et al., 2001), and GSH (Sipos et al., 2002). However, neither Atm1 nor Erv1 ortholog was identified in Giardia mitosomes. Thus, the importance of mitosomal ISC machinery for the formation of cytosolic FeS clusters remains to be clarified.

11.5 Energy Metabolism and Membrane Potential There is no evidence suggesting that Giardia mitosomes possess any pathway leading to ATP synthesis, although it is likely that ATP is required for at least two mitosomal functions associated with the ATPdependent activity of mtHsp70 and CPN60: import

Chap. 11 The Giardia Mitosomes

and maturation of nuclear-encoded mitosomal proteins, and transfer of FeS clusters to apoproteins. However, no components of the tricarboxylic acid cycle, fatty acid β-oxidation, the respiratory electron transport chain or the ATP synthase complex have been identified in the Giardia genome (Morrison et al., 2007; Franzen et al., 2009). In T. vaginalis hydrogenosomes, oxidative phosphorylation is also absent. However, hydrogenosomes possess a specific set of enzymes, including pyruvate:ferredoxin oxidoreductase and hydrogenase, that enables ATP synthesis by substrate-level phosphorylation. In Giardia, these proteins are not present in the mitosome-enriched fraction (Jedelský et al., 2010), and their activities are associated with the cytosol or the trophozoite membrane fraction (Lloyd et al., 2002a; Townson et al., 1996). Therefore, mitosomal energy requirements could be met only by import of ATP from the cytosol to the organelle. Indeed, a unique ADP/ATP carrier characterized in E. histolytica mitosomes has been suggested to supply ATP for energyrequiring processes within these organelles (Chan et al., 2005). Interestingly, this ADP/ATP transporter does not rely on a membrane potential. Uniquely among eukaryotes, the mitochondrial ADP/ATP transporter of the microsporidian Encephalitozoon cuniculi was replaced by an unrelated type of nucleotide transporter used by plastids and some intracellular bacteria, which may provide ATP to mitosomes of this parasite (Tsaousis et al., 2008). However, no candidate for ADP/ATP transport was identified by recent exhaustive bioinformatic and proteomic studies in G. intestinalis. Thus, elucidation of how ATP is delivered to the G. intestinalis mitosomes is one of the key challenges for future investigations. Another intriguing area of research is the identification of the source of reducing equivalents, which are required for the formation of FeS clusters. In mitochondria, electrons required by ISC machinery are provided by a short electron chain consisting of the [2Fe2S] ferredoxin and ferredoxin: NADP+ reductase (FNR) (Mühlenhoff et al., 2002). The presence of this chain was predicted in the mitosomes of C. parvum and E. cuniculi. However, [2Fe2S] ferredoxin, but not FNR, was found in giardial mitosomes (Dolezal et al., 2005). In our search for putative mitosomal oxidoreductases, we identified a diflavo-protein named GiOR. The protein consists of a flavodoxin-like FMN-

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binding domain connected to a FAD-binding pocket and NADP(H)-binding site. Recombinant GiOR utilizes NADPH to donate electrons to various artificial substrates, such as dichlorophenolindophenol and methylviologene (our unpublished data). The identity of the natural electron acceptor for GiOR remains to be determined. Nevertheless, the presence of NADPH-dependent GiOR in mitosomes suggests the involvement of pyridine nucleotides in mitosomal electron transport. Translocation of proteins across the inner mitochondrial membrane depends on membrane potential, which is generated mainly by the cytochrome-dependent respiratory chain. However, this pathway is absent in mitosomes, and any alternative system, such as one involving pyridine-nucleotide transhydrogenase, has not yet been identified. Membrane potential-sensitive dyes such as Rhodamine 123, which are used to locate mitochondria, can label some vesicular structures and the perinuclear region in G. intestinalis (Lloyd et al., 2002b). However, these structures are visibly distinct from mitosomes in size and number as well as cellular distribution, and their nature is unclear. It is likely that the membrane potential of G. intestinalis mitosomes is too low to be detected by the usual mitochondrial dye trackers. Moreover, membrane potential might not be essential for mitosomal protein translocation. As discussed above (see Sect. 11.3.1), mitochondrial targeting presequences are positively charged, so the membrane potential across the inner mitochondrial membrane forces the presequence to pass through the channel of a TIM complex by electrophoresis (Martin et al., 1991). However, mitosomal presequences do not possess positive residues except the arginine of the proximal cleavage motif, and the overall charges of the presequences are considerably lower than those of mitochondrial presequences. Therefore, the role of electrophoresis in mitosomal protein translocation seems to be limited or negligible.

11.6 Interaction with Other Cellular Structures Mitochondria are highly dynamic organelles that can quickly rebuild their architecture as well as establish contacts with other organelles to exchange material or

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to forward a signal to other corners of the cell. Most important among these interactions are (i) mitochondrial fusion/fission cycle, (ii) interactions with the endoplasmic reticulum and (iii) contacts with the cell cytoskeleton (Detmer and Chan, 2007). The relevance of these best-studied interactions to G. intestinalis mitosomes will be further discussed. Animal and fungal mitochondria undergo constant transformations of their shape; thus, they appear as reticular networks rather than distinct rounded vesicles. The expansion of the mitochondrial membranes, occurring especially in the animal cells, mirrors the increasing metabolic capacity as well as the participation of mitochondria in the signaling pathways. Mitofusins and Opa1 mediate specific fusion events between the outer and inner mitochondrial membranes, respectively, allowing for the spatial exchange of material within mitochondrial compartments (Hoppins and Nunnari, 2009). In addition, dynaminbased division machinery continuously divides the tubular network into smaller vesicles (Shibata et al., 2009). G. intestinalis does not contain homologs of any of fusion/fission-participating proteins, and nor does it use the FtsZ-based division machinery employed by bacteria and by mitochondria of several protist groups. According to immunofluorescent labeling of fixed cells, mitosomes are usually present in stable numbers per cell, and there are no data to suggest that variations in mitosome counts occur under different metabolic/stress conditions. Live imaging approaches will be necessary to assess these aspects of mitosomal dynamics. Mitochondria come into close contact with the endoplasmic reticulum to form a physical basis for Ca2+-dependent intercommunication and to exchange lipids and lipid precursors. This interorganellar tethering is mediated by several different proteins, including one isoform of mitofusin (de Brito and Scorrano, 2008), none of which seem to be present in the G. intestinalis genome. However, a recent report by Elias et al. (2008) indicated that Sec20, one of 17 SNARE proteins identified in the G. intestinalis genome, might have at least partial mitosomal distribution. Sec20 is a typical “tail-anchored” protein, with a soluble SNARE domain exposed to the cytosol and a C-terminal transmembrane domain anchoring the protein in the mem-

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brane. Such a topology enables SNARE proteins to form specific trans-SNARE bundles, thus bringing opposing membranes close together and initiating membrane fusions. The function of Sec20 is to mediate transport of vesicles between the endoplasmic reticulum and the Golgi apparatus (Sweet and Pelham, 1992). The mitosomal distribution of Sec20 could represent a unique strategy of linking of mitosomal membranes to the endomembrane system. However, more functional studies are needed before such an interaction can be confirmed. We have recently identified another mitosomal tail-anchored protein (Jedelský et al., 2010), the function of which is related to several diverse cellular processes, such as lipid transfer in vesicular fusion. The protein is called VAMP-associated protein (VAP) and was originally identified as a SNARE-interacting molecule in the neuronal synapse (Skehel et al., 1995), then later shown to be capable of various independent protein–protein interactions on the endoplasmic reticulum membranes. Identification of its molecular partners is essential to characterize VAP function and its involvement in mitosomal metabolism and biogenesis.

11.7 Perspectives Mitosomes of G. intestinalis are believed to represent highly reduced mitochondria. An intriguing question concerns the nature of the mitochondrial homolog from which these organelles evolved. G. intestinalis is a member of the excavate group. Previously, these organisms were considered among basal eukaryotes based on small-subunit ribosomal RNA phylogeny and the lack of certain typical cellular structures, such as mitochondria (Cavalier-Smith, 1987; Adam, 2001; Best et al., 2004). However, later phylogenetic reconstructions using improved bioinformatic tools revealed a lack of resolution in the deepest part of the eukaryotic tree and did not support the basal position of Giardia (Embley and Martin, 2006). More recently, excavates have been considered to belong to the basal groups of eukaryotes based on a specific mechanism of cytochrome c and c1 biogenesis (Cavalier-Smith 2010). Accordingly, the root of eukaryotes was placed between Excavata and Euglenozoa, a group of protists that includes trypanosomatids (Pusnik et al., 2009;

Chap. 11 The Giardia Mitosomes

Cavalier-Smith, 2010). Evidence for an early origin of trypanosomatids include the lack of the Tom40 complex, the presence of a simple and single inner membrane translocase, and short targeting presequences required for the delivery of proteins into organelles (Cavalier-Smith, 2010). It has been suggested that a single β-barrel voltage-dependent anion channel (VDAC) found in trypanosomatids evolved from proteobacterial porin precursors and that Tom40 evolved later by gene duplication and specialization of this porin (Pusnik et al., 2009; Cavalier-Smith, 2010). If these assumptions are valid, the mitochondrial homolog from which the Giardia mitosome evolved may have been less complex than mitosomes of other protists such as microsporidia. Thus, the observed simplicity of mitosomal pathways, particularly the protein import machinery, may reflect retention, rather than reduction, of ancestral features (CavalierSmith, 2010). However, comparison of import machineries between G. intestinalis and the closely related protist T. vaginalis did not support this hypothesis. Our current bioinformatic searches in the T. vaginalis genome and analysis of the hydrogenosome proteome revealed that hydrogenosomes have rather complex protein import machinery, including all three types of β-barrel proteins (Tom40, Sam50, and VDAC), Tim17 homologs and small Tims present in the intermembrane space. Therefore, it is more likely that the simplicity of mitosomal import machinery as well as its metabolic pathways reflects specific adaptations of G. intestinalis to its parasitic life style in an oxygen-poor environment than the simple nature of the mitochondrial ancestor. In conclusion, FeS cluster assembly is a key, if not the only, biosynthetic function of Giardia mitosomes. However, a number of questions concerning this function remain unanswered: (i) What is the mechanism of ATP import to the organelle? (ii) What is the source of reducing equivalents, and what electron transport system is used? (iii) Which compound(s) is responsible for the formation of FeS clusters in extramitochondrial cellular compartments, and how is this compound delivered to a cytosolic machinery? To answer these intriguing questions, many more functional studies are required. Given that functional studies currently suffer from a lack of amenable methods for reverse genetics in G. intestinalis, more focus in this area is required to answer these questions.

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Acknowledgements The research described in this chapter was supported by Czech Ministry of Education (LC 07032, MSM0021620858).

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Signaling Pathways in Giardia lamblia Tineke Lauwaet and Frances D. Gillin

Abstract Giardia trophozoites colonize the ever-changing small intestinal environment and must constantly react to external mucosal signals in order to “decide” whether to multiply and cause disease or to differentiate into cysts. Similarly, upon ingestion, cysts react to stimuli from the new host in order to excyst. Giardia is an excellent model to study signaling because its life cycle can be completed in vitro and genome analyses revealed a limited but broad selection of signaling proteins. Encystation is an entry into dormancy, while excystation is a rapid cellular awakening. Although the stimuli for encystation and excystation are known, understanding of the transduction of these important signals is incomplete. The localization of the various signaling proteins to universal or Giardiaspecific structures and their redistribution in response to environmental signals will provide insights into their functions in the Giardia cell cycle and differentiation. However, research on signaling proteins and pathways in Giardia is hampered by the lack of specific antibodies, substrates, and inhibitors. The most striking finding is the very large number of Nek kinases in the Giardia genome. The Neks are promising targets for further studies and their function and regulation will likely disclose more insights into the regulation of Giardia motility, cell, and life cycle. Here, we summarize current published information on Giardia signaling in growth, encystation, and excystation.

12.1 Introduction The protozoan parasite Giardia lamblia exists in two life cycle stages: the motile trophozoite and the dormant infectious cyst. Trophozoites attach to

H. D. Luján et al. (eds.), Giardia © Springer-Verlag/Wien 2011

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small intestinal enterocytes and can cause symptoms. Trophozoites have to proliferate and respond to their constantly changing environment in order to establish and maintain infection. As the trophozoites reach the lower part of the small intestine, they differentiate into cysts that are excreted in the feces and can survive outside the human body. Upon infection of another host, the cyst, which cannot attach, must quickly sense and adapt to its new environment by excysting in the small intestine, before it is expelled. Thus, the ability to quickly respond to diverse environments and the decision to grow or differentiate are crucial for Giardia’s survival and infectivity. In eukaryotes, important cellular functions are regulated by signal transduction and specific functions of universal signaling proteins are determined by their localization to specific subcellular structures or compartments. During the Giardia cell and life cycle, the cytoskeleton undergoes major changes and inhibition of several cytoskeletal signaling proteins results in decreased encystation and excystation, demonstrating that signaling is essential in the Giardia cell cycle and differentiation. In this review, we summarize current knowledge on Giardia signaling in growth and differentiation.

12.2 Giardia Phosphatases and Kinases Many crucial cellular decisions and activities are regulated by phosphorylation of proteins by specific protein kinases. Elucidating the diversity of protein kinases and their targets is basic to understanding the biological potential of an organism. Giardia genome annotation revealed a kinome with 276 putative protein kinases (Morrison et al., 2007). The kinases that have been studied to date are listed in Table 12.1. Giardia has only serine/threonine kinases and no histi-

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Table 12.1 The Giardia protein and lipid kinases studied to date Kinase

Uniprot Accession No.

Giardia gene ID

Reference

Aurora kinase

A8BBM9

GL50803_5358

Davids et al. (2008)

PKAc

A8BIF7

GL50803_11214

Abel et al. (2001); Gibson et al. (2006)

PKAr

A8B7Q5

GL50803_9117

Gibson et al. (2006)

PKB

A8BFC8

GL50803_11364

Kim et al. (2005)

ERK1

A8BU24

GL50803_17563

Ellis et al. (2003)

ERK2

A8BS66

GL50803_22850

Ellis et al. (2003)

PI3K1

A8B5J3

GL50803_14855

Cox et al. (2006)

PI3K2

A8B8M9

GL50803_17406

Cox et al. (2006)

PKCE-like

Q1H8W8

GL50803_86444

Bazan-Tejeda et al. (2007)

dine or classical tyrosine kinases, although tyrosine phosphorylation has been well documented (Parsons et al., 1993; Morrison et al., 2007). The giardial kinome is described as ‘the most compact known in any eukaryote’. However, the most striking finding is that the Nek or “never in mitosis associated (NIMA)-related kinases” are highly over-represented in the giardial kinome with ~180 members. The Giardia genome contains kinases in the groups of: Nek, cyclin dependent kinases (CMGC), calmodulin dependent protein kinases, sterile kinases (STE), PKA/G/C containing (AGC), casein kinase 1 (CK1) and tyrosine kinase-like (TKL). The numbers of kinases per family are lower than in human but similar to yeast, except for the Nek family (Morrison et al., 2007). In general, ciliated and flagellated cells seem to have more Nek in their genome than non-ciliated cells (Parker et al., 2007). For example, the parasitic ameba E. histolytica has a comparable size kinome (307 putative kinases) but only 1 NIMA kinase (Anamika and Srinivasan, 2007) while the flagelated T. brucei, T. cruzi, and L. major have 156–179 kinases including 20–22 Neks (Parsons et al., 2005). In mammalian cells, the family of Nek kinases consists of 11 members. Nek2, Nek6, Nek7 and Nek9 have roles in mitotic progression, cilium formation, and the DNA damage response (O’Regan et al., 2007), while the other Neks are expected to have functions in axonemal microtubule regulation (O’Regan et al., 2007). In ciliated algae such as Chlamydomonas and Tetrahymena, Neks localize to the cilia and basal bodies and have roles in flagellar disassembly and length

control (Mahjoub et al., 2002; Bradley and Quarmby, 2005; Wloga et al., 2006), while in human ciliated cells, Nek mutations lead to ciliary diseases (Smith et al., 2006). Their over-representation in the Giardia genome suggests an important role for these kinases, but no functions have been described to date. Parker et al. (2007), speculated that the high number of Giardia Neks might be necessary to regulate the complex process of flagellar maturation and segregation during Giardia cytokinesis (Nohynkova et al., 2006). However, 137 of the 180 Neks are predicted to be catalytically inactive (Morrison et al., 2007). Most of them have ankyrin and/or coiled–coiled domains which could function in mediating protein–protein interactions and localization (Li et al., 2006). Signaling requires that cellular responses be rapid and often transient. Thus, activity of protein phosphatases is crucial to balance and limit the effects of kinase activities. Giardia phosphatases have been recently reviewed by Andreeva and Kutuzov (2008) and Kutuzov and Andreeva (2008) (Table 12.2). Similar to other protozoa (Naula et al., 2005), Giardia has no receptor-type protein tyrosine phosphatase (PTP) (Andreeva and Kutuzov, 2008). However, Giardia has ectophosphatases that are inhibited by free P-tyrosine and by inhibitors of acid and tyrosine phosphatase (Amazonas et al., 2009). Giardia has one protein tyrosine phosphatase (PTP) and one PTP-like (PTPL) protein, which is structurally related to PTP but has no catalytic activity. Giardia has one low molecular weight tyrosine phosphatase (LMW-PTP), which in humans inhibits

Chap. 12 Signaling Pathways in G. lamblia

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Table 12.2 Giardia protein phosphatases classified according to Andreeva and Kutuzov (2008) and Kutuzov and Andreeva (2008) Uniprot accession No.

Giardia gene ID

PP1

A8B4W2

GL50803_14568

PP2A

A8BED3

GL50803_5010

PP4/6

A8BYQ2

GL50803_2053

PP5-like

A8BLH6

GL50803_2198

PP5-like

A8B5G7

GL50803_13524

PP5-like

A8B3F5

GL50803_6441

PP5-like

A8BSI3

GL50803_7588

PP5-like

A8BSU3

GL50803_101904

PP5-like

A8BQ55

GL50803_31309

PP5-like

A8BJH3

GL50803_16570

PP5-like

A8BSX2

GL50803_14545

Serine/threonine phosphatase

A8BD58

GL50803_10711

Serine/threonine phosphatase

A8BLT6

GL50803_14311

Serine/threonine phosphatase

A8B747

GL50803_15214

Serine-threonine phosphatases Phosphoprotein phosphatases (PPP)

Mg–Mn-dependent protein phosphatases (PPM or PP2C-related) PP2C, putative

A8B8V6

GL50803_11740

Phosphatase

A8BGW7

GL50803_14404

PP2C-like protein

A8BBQ9

GL50803_9293

PP2C, putative

A8B5X1

GL50803_14650

PP2C

A8B8N7

GL50803_32312

PP2C, putative

A8BVA7

GL50803_4288

Protein tyrosine phosphatases Non-receptor PTP

A8B908

GL50803_25035

PTP-like (inactive)

A8BKZ7

GL50803_7512

Dual specificity phosphatases (DSPs) Atypical DSP (YVH1)

A8BPG1

GL50803_4357

Atypical DSP (YVH1)

A8BS30

GL50803_15112

DSP12

A8B9F0

GL50803_36315

CDC14

A8BAI9

GL50803_9270

PTEN

A8B7C9

GL50803_16728

Myotubularin

A8BYI5

GL50803_8210

Myotubularin

A8BR06

GL50803_13875

Myotubularin (inactive)

A8BTI9

GL50803_112811

CDC25/Acr2

A8B3D6

GL50803_4369

LMW-PTP

A8BYX6

GL50803_14456

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cell growth and counteracts growth factor responses. Apart from the tyrosine-specific phosphatases, Giardia has several dual specificity phosphatases including two mitogen-activated protein kinase phosphatases (MKPs) and one homolog of Yeast gene VH1 (YVH1) related phosphatase. The PfYVH1 protein in Plasmodium dephosphorylates Tyr and Ser residues and is suggested to be involved in rRNA processing and cell-cycle progression (Kumar et al., 2004). Other dual specificity phosphatases (DSPs) are cell division cycle phosphatases Cdc14 and Cdc25, which play crucial roles in cell division. Phosphatase and tensin homolog (PTEN), a phosphatidylinositol-3,4,5trisphosphate 3-phosphatase is implicated in a variety of human diseases (Ooms et al., 2009). Giardia PTEN is homologous to fungal and Cryptosporidium PTEN (Andreeva and Kutuzov, 2008). Giardia has two homologs of myotubularins which are DSPs that dephosphorylate phosphatidylinositol 3-phosphate or phosphatidylinositol-(3.5)-biphosphate. Although no Giardia homolog of protein phosphatase 5 (PP5) was found, one Giardia phosphatase has a PP5 characteristic N-terminal TPR domain (Andreeva and Kutuzov, 2008). However, this TPR domain was not similar to the one from PP5 and seemed to be related to the TPR domain of plant TPR proteins suggesting that Giardia PP5 is not related to the other eukaryotic PP5s but originated independently (Andreeva and Kutuzov, 2008). Giardia has clear homologs of the serine/threonine phosphatases PP1 and PP2A and distant homologs of PP4/PP6 (Kutuzov and Andreeva, 2008). So far, only gPP2Ac has been functionally studied (see below) (Lauwaet et al., 2007b) and future studies are needed to elucidate the functions of the other phosphatases.

12.3 Signaling in the Cell Cycle During Giardia cell division, trophozoites undergo semi-open mitosis with the formation of two spindles, one for each nucleus (Sagolla et al., 2006). During encystation, trophozoites undergo an extra round of karyogamy, without cytokinesis, and during excystation, the excyzoite quickly divides two times to form four trophozoites, emphasizing the complexity and importance of cell-cycle regulation (Adam, 2001). However, the signaling underlying Giardia cell di-

T. Lauwaet and F. D. Gillin

vision is not well understood. In higher eukaryotes, chromosomal segregation during the cell cycle is tightly controlled by four major families of mitotic serine/threonine kinases: aurora, polo, Nek, and cyclin dependent (Cdk) kinases (Malumbres and Barbacid, 2007). The importance of serine-threonine kinases in Giardia cell division was demonstrated by adding the serine-threonine kinase inhibitor staurosporin to excysting cells. Staurosporin did not inhibit excystation but prevented the differentiation of excyzoites into trophozoites (Alvarado and Wasserman, 2009). Polo kinases (Plks) are involved in mitotic entry and progression (Malumbres and Barbacid, 2007). In Giardia one Plk and six putative cdk homologs have been identified (Morrison et al., 2007) including 3 CDC2-like G1 CDKs, and a CDK5 homolog, but their localization and functions have not been reported. The Cdk family contains critical cell-cycle regulating kinases that are composed of a catalytic subunit (Cdk) and a regulatory subunit cyclin. Overexpression of certain cdk leads to cell-cycle misregulation and carcinogenesis in mammals (Malumbres and Barbacid, 2007). Cdk homologs are reported in Giardia, however, phylogenetic studies could not identify “clear orthologs of transcription-related Cdk” (Best et al., 2004; Guo and Stiller, 2004; Morrison et al., 2007). So far, Giardia aurora kinase (AurK) is the only cell-cycle kinase that has been functionally analyzed and shown to be crucial for cell division (Davids et al., 2008). In mammalian cells, there are three AurKs (AurKs A, B and C) that have distinct localization patterns (Carmena et al., 2009). Giardia AurK is most homologous to mammalian AurK A, which localizes to centrosome and spindle poles in mitotic cells. Inhibition of mammalian AurK A leads to aberrant centrosomal separation (Carmena et al., 2009). In interphase cells, Giardia AurK localizes to the nuclei. During mitosis, AurK is activated by phosphorylation on a Thr residue in the active site loop and relocates to the basal bodies in prophase, to the spindle microtubules in metaphase and anaphase and to the parental attachment disk in cytokinesis, in addition to localization to the paraflagellar dense rods and the median body. Treatment of trophozoites with pharmacologic AurK inhibitors arrested cells in cytokinesis and resulted in inhibition of growth, and increased microtubular nucleation (Davids et al., 2008). In other pro-

Chap. 12 Signaling Pathways in G. lamblia

tozoan parasites, cell-cycle kinases have important roles and have been proposed as drug targets (Naula et al., 2005; Grant, 2008). For example, T. brucei aurora kinase (TbAUK1) is involved in nuclear division and cytokinesis and is essential for infection of the mammalian host (Jetton et al., 2009). Inhibition of TbAUK1 with the AK B specific inhibitor hesperadin resulted in an altered cell morphology and inhibition of cell-cycle progression (Jetton et al., 2009). T. brucei NIMA related kinase C (TBNRKC) is a homolog of Nek2 and localizes to the basal bodies. Overexpression and knockdown studies showed TBNRKC is involved in controlling basal body separation and cell division of procyclic cells (Pradel et al., 2006). The P. falciparum genome has four Nek (Ward et al., 2004) of which Nek 2 and Nek 4 are predominantly expressed in gametocytes and are essential for the completion of meiosis (Reininger et al., 2009). These data, together with the abundance of Nek in the Giardia genome, suggest that the Giardia cell cycle might include useful drug targets as long as they are sufficiently divergent from the host proteins. The recent cell-cycle synchronization studies might aid in understanding the signaling underlying the Giardia cell cycle (Poxleitner et al., 2008; Reiner et al., 2008).

12.4 Signaling in the Life Cycle Giardia is one of the few protozoans that can be induced to differentiate in vitro by mimicking the physiological characteristics of its in vivo environments. The differentiation processes of trophozoites into cysts (encystation) and of cysts into trophozoites (excystation) are clearly distinct and their regulation is described separately below.

12.4.1 Encystation Encystation occurs in response to changes in the trophozoites’ environment. Several studies reported that increased bile concentrations and depletion of cholesterol or interfering with its binding to the Giardia cholesterol Ck surface receptor leads to encystation (Gillin et al., 1988; Kaul et al., 2001; Arguello-Garcia et al., 2009).

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Various groups have focused on the role of conserved signaling proteins that are key in eukaryotic cell growth and differentiation and have demonstrated a crucial role for these proteins in Giardia encystation. Signaling in Giardia encystation has recently been reviewed (Lauwaet et al., 2007a; Lauwaet and Gillin, 2008; Arguello-Garcia et al., 2009; Carranza and Lujan, 2010). 14-3-3 proteins are a family of phosphoserine/ threonine binding proteins that play a role in cell cycle and cell differentiation processes and are highly conserved in eukaryotes. Giardia has one 14-3-3 protein (g14-3-3) which is phosphorylated on Thr214 and polyglycylated on Glu246. g14-3-3 is phosphorylated in vegetative and encysting trophozoites and is involved in binding to phosphorylated peptide targets, while polyglycylation is stage dependent and regulates cellular localization (Lalle et al., 2010). Mutant non-polyglycylated protein localizes to the nuclei and cytoplasm whereas the polyglycylated form only localizes to the cytoplasm (Lalle et al., 2006). In encysting cells, 14-3-3 protein localizes to the nuclei and the cytoplasm (Lalle et al., 2006). Future studies are necessary to identify the kinase that phosphorylates g14-3-3, determine the g14-3-3 protein targets, and elucidate its function in encystation. The mitogen activated protein kinase (MAPK) pathway is usually activated by mitogens and extracellular signals such as growth factors and regulates cell proliferation and survival (Junttila et al., 2008). Giardia has homologs of two members of the MAPK family: extracellular signal regulated kinases 1 and 2 (Ellis et al., 2003). ERK1 and ERK2 are serine/threonine kinases that are activated by phosphorylation on the Thr and Tyr in their catalytic loops. Phosphorylation and thus activity of both ERK1 and ERK2 was lower during encystation (Ellis et al., 2003). ERK1 localizes to the basal bodies, median body, disk, ventral groove, and caudal flagella. During encystation, ERK1 localization to the median body and caudal flagella is lost and it accumulates in the ventral groove. In non-encysting trophozoites, ERK2 localizes to the nuclei, anterior and caudal flagella, and the plasma membrane. During encystation, ERK2 localization to the nuclei and flagella is lost, and it becomes more cytoplasmic (Ellis et al., 2003). MEK and MEKK kinases which are upstream activators of ERK are present in the Giardia genome (Arguello-Garcia et al., 2009).

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T. Lauwaet and F. D. Gillin

aPFR bb pPFR cPFR

PP2Ac

PKAc

Fig. 12.1 Immunolocalization of protein phosphatase 2A and protein kinase A catalytic subunits in vegetative trophozoites (Abel et al., 2001; Lauwaet et al., 2007b). bb, basal bodies; aPFR/pPFR/cPFR, anterior, posterior-lateral, caudal paraflagellar dense rods

Protein kinase A (PKA) is a cAMP-dependent serine/threonine kinase that is central to numerous signaling cascades (Sim and Scott, 1999). PKAc is kept inactive in a holoenzyme form composed of 2 catalytic and 2 regulatory subunits. It is activated and released upon cAMP binding to the regulatory subunit. In many organisms, localization and specificity are dictated by regulatory and targeting subunits. In Giardia both the regulatory and catalytic subunits of PKA localize to the basal bodies and to the paraflagellar dense rods (PFRs) that run along the intracellular portions of the anterior and caudal flagella (Abel et al., 2001; Gibson et al., 2006) (Fig. 12.1). During encystation, its localization to the PFR disappears and its activity is increased. High-density vegetative cultures have decreased endogenous cAMP levels and PKA localizes only to the basal bodies. However, refeeding high-density cell cultures with fresh medium restored cAMP levels and localization of PKAc (Abel et al., 2001). This suggests that PKA localization and function may be responsive to external stimuli, such as growth factors or nutrients in the media. Additional studies are needed to define roles of ERK and PKA in encystation. Protein phosphatase 2A (PP2A) is a serine/threonine phosphatase whose function is regulated by methylation and phosphorylation of the catalytic subunit and the holoenzyme composition (Xu et al., 2006; Cho and Xu, 2007). The catalytic domain of Giardia PP2A (gPP2Ac) is crucial for both encystation and excystation. Similar to PKAc, PKAr, ERK1, and ERK2, the localization of gPP2Ac changes during en-

cystation. In vegetative cells, gPP2Ac localizes to the basal bodies and anterior, caudal and posterior PFR (Lauwaet et al., 2007b) (Fig. 12.1). In encysting cells, gPP2Ac localization to the anterior PFR is lost, while their localization to the basal bodies and other PFR is maintained. Encysting cells expressing gPP2Ac antisense had fewer encystation secretory vesicles (ESVs), reduced CWP-1 protein levels, and fewer cysts compared to the wild-type cells. Since gPP2Ac and PKAc localize to the same structures and disappear from the anterior PFR during encystation, it is likely that both participate in the same signaling pathway and regulate the functions of basal bodies and PFR (Abel et al., 2001; Lauwaet et al., 2007b). Each pair of flagella is thought to have a distinct role in motility and attachment and all are interiorized during encystation. The anterior flagella are proposed to play a role in “steering” swimming trophozoites. Localization of signaling proteins to flagella and/or PFR may regulate their functions. Therefore, the disappearance of PKAc and gPP2Ac from the anterior PFR during encystation may reflect decreased flagellar activity during this entry into dormancy. PKA is known to activate PP2A and regulate the phosphorylation of important signaling proteins (Ahn et al., 2007). Protein kinase B (PKB/Akt) is a growth-factorregulated serine/threonine kinase involved in the regulation of eukaryotic cell survival and cell-cycle progression (Yang et al., 2004). gPKB activity has been demonstrated and the gPKB transcripts increase after 17-h encystation, suggesting that this kinase may be involved in the regulation of encystation (Kim et al., 2005). Sequence and phylogenetic analyses have identified two Giardia phosphatidylinositol-3P-kinases, GiPI3K1 and GiPI3K2, that are classified as members of class I and class III PI3K, respectively. Both GiPI3K1 and GiPI3K2 likely phosphorylate phosphatidyl inositols (PtdIns) and their intermediates in the plasma membrane and are associated with intracellular membranes (Cox et al., 2006). Transcripts of both GiPI3K1 and GiPI3K2 are upregulated in encystation and growth was inhibited by the reversible PI3K inhibitor LY294002 (Cox et al., 2006). Homologs of other PI kinases and phosphatases that are likely to act on PtdIns and their possible downstream signaling proteins such as PKA, PKB, g14-3-3, ERK1, and ERK2 have been identified in the Giardia genome

Chap. 12 Signaling Pathways in G. lamblia

(Morrison et al., 2007). However, the interaction of PI kinases and these Giardia signaling proteins needs to be confirmed in in vitro phosphorylation studies. A kinase annotated as a PKA, PKG, and PKC (AGC) kinase subfamily member was reported to localize to the cytoplasm and to the caudal side of trophozoites by means of heterologous antibodies against mammalian PKCbetaII (Bazan-Tejeda et al., 2007). Within minutes after induction of encystation, this protein translocates from the cytoplasm to the membrane, suggesting it is activated. However, within 30 min, it localized back in the cytoplasm. The protein levels were very low in vegetative cells but increased in encystation (Bazan-Tejeda et al., 2007). Despite the progress that has been made on elucidating the role of individual signaling proteins in Giardia encystation, the complete signaling pathway remains unknown. The authors of a recent review (Arguello-Garcia et al., 2009) suggested for the first time an encystation signaling pathway that is partially based on the current Giardia experimental signaling data described above, and partially on in silico data from human homologs of Giardia signaling proteins. Future experimental studies are necessary to validate the speculative parts of this pathway.

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cysts from human feces can also be highly variable (Boucher and Gillin, 1990). So far only PP2A, calcium/calmodulin and PKAmediated signaling pathways have been shown to be essential for excystation (Abel et al., 2001; Reiner et al., 2003; Lauwaet et al., 2007b). Inhibitors of PP2A and PKA blocked excystation when added to cysts in the in vitro stage that mimics their passage through the stomach. In addition, excystation of gPP2Ac antisense cysts was decreased compared to controls and the PP2A inhibitor okadaic acid inhibited resumption of motility when added to the later stage of excystation (Lauwaet et al., 2007b). The highly conserved calcium binding protein calmodulin localizes to the basal body area in Giardia trophozoites and inhibitor studies showed that intracellular calcium levels and calmodulin are important in the later stages of excystation (Reiner et al., 2003). Taken together these data suggest that initiation of excystation is regulated by PP2A and PKA, while PP2A and Ca2+/calmodulin signaling help control emergence and resumption of motility. Since these three signaling proteins all localize constitutively to the basal bodies, it is likely that future studies will reveal other signaling proteins and pathways that are important in Giardia cell differentiation and use basal bodies are a docking site.

12.4.2 Excystation Excystation needs to be tightly regulated to prevent opening of the cyst in the stomach where the parasite would be killed by the low pH of the gastric juices. However, the low pH of the stomach is necessary for successful excystation as it triggers the excystation process (Bingham and Meyer, 1979; Boucher and Gillin, 1990). The molecular mechanism by which signals are perceived across the impervious cyst wall is yet unknown. In contrast to encystion, excystation is a very fast process that is completed within 2 h, suggesting that signaling is very important. Although the in vitro excystation method has been described two decades ago, it remains a challenging technique that is highly sensitive to inter-laboratory variation, explaining why only few quantitative excystation studies have been published. The efficiency of excystation of human Giardia cysts formed in vitro depends on the biological quality or maturity of the cysts. Excystation of

12.5 Conclusion Mucosal pathogens such as Giardia reside in highly changing environments and must constantly react and adjust to signals from the external milieu in order to survive and cause disease. Giardia is a good model to study signaling because its life cycle can be completed in vitro and the recent genome analyses revealed a limited but broad selection of signaling proteins. The localization of the various signaling proteins to universal or Giardia-specific structures and their relocalization in response to environmental signals is likely to shed light on the function of these proteins and these structures in the Giardia cell cycle and differentiation. However, research on signaling proteins and pathways in Giardia is hampered by the lack of specific antibodies, substrates, and inhibitors. The large number of Nek kinases in the Giardia genome make promising targets for future research

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and their function and regulation will likely disclose more insights in Giardia motility, cell and life cycle regulation.

Acknowledgements We thank G. Manning (Salk Institute, La Jolla, CA) for critical reading of the manuscript.

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Transcription and Recombination in Giardia Rodney D. Adam

Abstract Our understanding of the transcription and recombination of Giardia has evolved substantially with recent experimental, genomic and population genetic data. Earlier assumptions that Giardia is an asexual organism have been called into serious question by the finding of the meiotic genes in the genome, the documentation that genetic material can be transferred from one nucleus to another during the process of encystation, and the evidence from population genetic data supporting the occurrence of meiotic recombination within Genotype A2. The different genotypes vary significantly in the way they handle introduction of plasmid DNA and their levels of allelic heterozygosity, indicating differences in their processes of DNA recombination. Transcription in Giardia is typical of that of other eukaryotes with its use of a 5c cap, polyadenylated tail, and its (rare) use of introns. However, it follows the minimalist approach that Giardia follows in many areas of metabolism, with an apparently abbreviated set of transcription factors and promoters that vary significantly from those of other eukaryotes and may also be simplified in their structure and function. The answers to how Giardia handles transcription and recombination with its minimalist approach may also shed light on the more complex systems used by other organisms.

13.1 Transcription 13.1.1 Overview A number of unique questions regarding gene expression are raised by Giardia’s possession of two nuclei that are diploid or approximately diploid. Early stud-

H. D. Luján et al. (eds.), Giardia © Springer-Verlag/Wien 2011

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ies using in situ hybridization of radioactive rDNA sequence demonstrated that both nuclei contain approximately equal numbers of rDNA genes (Kabnick and Peattie, 1990). Transcription from both nuclei has been documented by detecting the uptake of tritiated uridine in equal quantities in both nuclei. Thus, both nuclei are transcriptionally active at similar levels, raising the additional complication of how gene expression is controlled simultaneously in the two nuclei.

13.1.2 General Transcription Factors For eukaryotic promoters with a TATA box, RNA polymerase II (RNAPII) transcription is initiated with the binding of TATA-binding protein (TBP) to the TATA box followed by the recruitment of additional transcription factors and RNA polymerase (For promoters without a TATA box, an additional protein binds first, followed by attachment of TBP.). TBP along with TBP-associated factors (TAFs) forms TFIID. This binding of TFIID is facilitated by TFIIA. Subsequently, TFIIB is recruited to the transcriptional complex. Although none of these proteins have been directly studied from Giardia, a TBP candidate was identified in a genomic survey of the Giardia genome looking specifically for transcription-related genes (Best et al., 2004). The Giardia TBP is highly diverged from that of other eukaryotes, and in fact, is farther from the eukaryote crown than the archael TBPs. Neither TFIIA nor TFIIB were identified in the survey, perhaps related to the remarkable divergence of the Giardia TBP from that of other eukaryotes. TBP is the most highly conserved of the transcription initiation factors, so the inability to identify these other factors in an organism with a highly divergent TBP is not surprising.

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13.1.3 RNA Polymerases

Table 13.2 RNAPII general transcription factors

RNAPII polymerizes the transcripts encoded by most protein-coding genes. The yeast RNAPII holoenzyme is formed of 12 subunits, called Rpbs, some of which are common to the RNA polymerases I and/or III. Rpb1, 2, 3, 4, 7, 9, and 11 are unique to RNAPII, while the others are also found in the other polymerases (Table 13.1). All except Rbp4 and 9 have been identified in the Giardia genome (Seshadri et al., 2003). Of note, neither Rbp4 nor Rbp9 are required in yeast for viability. Interestingly, yeast Rpb9 mutants impaired in their ability to control transcriptional start site. Two forms of Rpb5 were found (Seshadri et al., 2003), which are about as divergent from each other as from the yeast Rpb5. This raises that question of whether the different Giardia polymerases use a different form of Rpb5, but that possibility has not been experimentally tested. In most eukaryotes, RNAPII is highly susceptible to the mushroom toxin, alpha-amanitin, with inhibition of transcription at concentrations of 1 ug/ml. In contrast, RNAPIII transcription is inhibited by 10 ug/ml, while RNAPI transcription is resistant to 1 mg/ul. However, RNAPII transcription in Giardia is highly resistant to amanitin, even at concentra-

Table 13.1 RNA polymerase subunits Subunit #

RNAPI

RNAPII

RNAPIII

1

a

b

c

2

a

b

c

3

a

b

a

4

Missing from Giardia, yeast has one

5

Giardia has two; yeast has one

6

Shared by all three polymerases

7

a

8

Shared by all three polymerases

9

a

10

Shared by all three polymerases

b

Missing in Giardia

11

a

12

Shared by all three polymerases

b

c

c

a

Types a, b, and c are found in RNAPI, II, and III, respectively.

TFIIA

Not found

TFIID

Giardia TBP is highly divergent; TAFs have not been identified

TFIIB

Not found

TFIIE

Not found

TFIIH

P90, P80, Cdk7, P44, and P34 found

TFIIJ

Not commented on

tions of 1 mg/ml (Seshadri et al., 2003). The activity of amanitin against RNAPII transcription occurs as a result of binding to the amanitin bridge domain of RPB1. Co-crystallization studies have suggested that hydrogen bonds between yeast Rpb1 and amanitin have been suggested involve a leucine at residue 722 and a serine at 769 which are highly conserved in eukaryotes but substituted in Giardia. Giardia Rpb1 has substitutions at an additional four residues that have been associated with amanitin resistance in other organisms. RNA polymerase I (RNAPI) is responsible for transcribing the ribosomal RNAs and apart from the identification of several RPBs specifically associated with RNAPI (Table 13.2), no studies of RNAPI have been reported. In view of the high level of amanitin resistance of Giardia RNAPII, any studies attempting to distinguish between RNAPI and RNAPII transcription will be more challenging. The one RNAPI transcription factor identified in Giardia is RRN3, a conserved transcription that binds to RNAPI during transcription. RNA Polymerase III (RNAPIII) is responsible for transcribing tRNAs as well as several other small RNA molecules. Rpc1 is the Rpb1 paralog that is found in RNAPIII, and the Giardia Rpc1 has substitutions associated with amanitin resistance at only one of the six sites that are substituted in Rpb1 and is the same as the yeast and human Rpc1 at all six of these positions. That fits with the finding that Giardia RNAPIII transcription was 85% inhibited by 50 ug/ml of amanitin, which is similar to the degree of inhibition found in other eukaryotes. BRF has also been identified in Giardia (Best et al., 2004). BRF, along with TBP, is a component of TFIIIB.

Chap. 13 Transcription and Recombination in Giardia

13.1.4 Specific Transcription Factors and Regulated Transcription Specific transcription factors have been studied primarily in the setting of the encystation process. These include MyB, which is induced during encystation and upregulates cwp1-3 (Sun et al., 2002; Huang et al., 2008). Two GARP family transcription factors regulate many genes, including cwp1 and constitutive regulation of ran 24 (Sun et al., 2006). ARID binds to specific AT-rich Inr sequences and works in transactivation of cwp1 25 (Wang et al., 2007). WRKY transcription factors also regulate cwp1 and cwp2 (Zhang and Wang, 2005; Pan et al., 2009). These are discussed in more detail in Chapter 23.

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as the Inr element. However, it is notable that “sterile transcripts” are abundant in Giardia (Elmendorf et al., 2001a). These antisense transcripts are polyadenylated and are not yet known to have a function. Therefore, it is possible that these transcripts result from rather loose control of transcription because of promoters that are not highly precise in their nature. However, a subsequent analysis using SAGE data from the genome project accompanied by the use of 5c RACE has suggested that antisense transcripts originate frequently from gene promoter sites. There was no evidence that the antisense transcripts were the result of local chromatin unwinding. These data are most consistent with the hypothesis that the general AT-rich nature of these promoters renders them rather promiscuous.

13.1.5 Promoters 13.1.6 Modification of mRNAs RNAPII transcription is initiated when the appropriate protein complexes bind to the TATA box, which is at about –30 with respect to the transcription initiation, and/or the Inr, which is at the transcription start site, followed by the recruitment of RNA polymerase and additional transcription factors. The consensus sequence of the TATA box is “TATA A/T A A/T”. In addition, many eukaryotic promoters have an upstream promoter element, called the CCAAT box, which is located at about –70. Frequently, additional upstream regions have roles in enhancing or repressing transcription. As we shall see, Giardia promoters vary significantly from the eukaryotic consensus. The first systematic investigation of Giardia promoters was a genomic evaluation of the 5c untranslated regions of multiple cytoskeletal genes (Holberton and Marshall, 1995). Their analysis and prior results obtained with cytoskeletal and other genes indicated that the transcripts have very short 5c UTRs, typically equal to or less than 14 nt (reviewed in Adam (2000)). The transcription start site is surrounded by an A-rich region, and in addition, there is an AT-rich region at approximately –30 which may be the Giardia equivalent of the CAAT box. Subsequently, several promoters have been analyzed, including ran (Sun and Tai, 1999), GDH (Yee et al., 2000), and alpha-2-tubulin (Elmendorf et al., 2001b). The total promoter length is up to 60 bp and is AT-rich at the transcription initiation site, suggesting that this AT-rich region may act

After and during eukaryotic transcription, several modifications to the transcript are performed by the cellular machinery, including placement of a methylguanosine cap on the 5c end, intron splicing, and polyadenylation. Early reports suggested that Giardia transcripts were not capped (Yu et al., 1998). However, subsequent reports have suggested the presence of a cap by showing that Giardia transcripts are blocked at their 5c ends and that the genome encodes the necessary enzymes (Hausmann et al., 2005). Three enzymatic reactions are required, (1) Hydrolysis of the triphosphate to a diphosphate by RNA triphosphatase, (2) Capping of the 5c end with GMP by RNA guanylyltransferase, and (3) methylation of the cap by RNA methyltransferase (Hausmann et al., 2005). They demonstrated that the RNA triphosphatase required a divalent metal cation, which places it in a category with the fungal and protozoan triphosphates, and distinct from plants and metazoans. They also showed a requirement for a 7cmethyl-guanosine cap for efficient translation of mRNA in Giardia. With most eukaryotes, the ribosome scans the transcript beginning from the 5c cap for an AUG from which to initiate translation. When the transcript is >20 nt in length, the ribosome frequently advances to the second inframe AUG. In fact, optimal efficiency is obtained with an 80 nt UTR. Since most Giardia transcripts are <20 nt in length, the optimal length of the UTR was

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studied in Giardia. The investigators found that the optimal length was 9 nt or less and that extending the 5c UTR up to 21 nt significantly reduced the translational efficiency (Li and Wang, 2004). Higher eukaryotes utilize eIF4B, eIF4H, and eIF4G in ribosome to translocate the transcript to the correct for initiation of translation, and orthologous genes for these proteins are absent from the Giardia genome. The absence of these genes coupled with the efficiency translation of the short transcripts suggests that Giardia may initiate translation without the requirement of a ribosome scanning mechanism. Introns are strikingly uncommon in Giardia with a total of three identified by comparing the genome with expressed sequence tag sequences (Morrison et al., 2007). The experimentally verified introns include a 35 nt intron in the mitosomal ferredoxin protein gene (Nixon et al., 2002), a 109 nt intron in the ribosomal protein Rp17a (Russell et al., 2005) and a 200 nt intron in an unnamed ORF (Russell et al., 2005). Eukaryotic intron splicing occurs within the confines of a spliceosome which is composed of five uridinerich small nuclear RNAs (U1, U2, U4, U5, and U6) in addition to many proteins. Candidate orthologs for all five of the genes encoding these snRNAs have been identified in the Giardia genome (Chen et al., 2008a) as well as the genes for many spliceosomal proteins. These findings certainly raise the question of why Giardia would maintain this machinery for these few genes. The polyadenylated tails are relatively short and sometimes begin only a few nucleotides after the stop codon. A conserved seven nucleotide sequence (AGTPuAAPy) precedes the polyA tail by about 10 nucleotides and is assumed to be the polyadenylation signal (Adam, 2001). In addition, there is a vsp-specific sequence (ACTPyAGPuT) immediately preceding the polyadenylation signal, which sometimes includes the stop codon (Svard et al., 1998; Ey et al., 1999). Only six of 23 yeast polyadenylation pathway genes were found in the Giardia genome (Morrison et al., 2007). Nonsense or frameshift mutations in eukaryotes result in accelerated degradation of RNAPII transcripts mediated by the nonsense-mediated mRNA decay (NMD) pathway, resulting in transcript levels for these abnormal transcripts that are 1–20% those of functional transcripts. In Giardia, the levels of

R. D. Adam

nonsense transcripts are about half that of intact transcripts. Up-frameshift 1 (UPF1) is a highly conserved component of the NMD pathway. The ortholog is present in Giardia and its overexpression reduces the levels of nonsense transcripts, suggesting that at least part of the NMD pathway is found in Giardia (Chen et al., 2008c). In addition, UPF1 overexpression reduces the transcript levels of the cyst wall protein genes, cwp1, cwp2, and cwp3, and impairs in vitro encystation (Chen et al., 2008b).

13.2 Genome Structure The haploid genome of Giardia is approximately 12 Mb in size and is divided among five chromosomes that range in size from about 1 to 3.8 Mb each, depending on the isolate and chromosome homolog (Adam, 1988, 5761/id). The chromosomes terminate with the TAGGG telomeric repeat (Adam et al., 1991). Higher level structure with organization into chromatin is maintained by histones which diverge from most eukaryotic histones at the C-termini (Yee et al., 2007). However, the linker histone, H1, was not detected in the genome, so it is possible that linking of adjacent nucleosomes in Giardia differs from that in other eukaryotes. The chromosomes were initially characterized by PFGE accompanied hybridization with a set of chromosome-specific probes (Adam et al., 1988). However, the total DNA content was estimated at eight or more times haploid genome size, suggesting a minimum ploidy of eight. This discrepancy was ultimately resolved when flow cytometry evaluation of trophozoites in different phases of DNA replication as well as Giardia cysts were compared with Escherichia coli as a standard (Bernander et al., 2001). The authors demonstrated that trophozoites spend most of their time in the G2 phase of replication Therefore, trophozoites are tetraploid in G1, and since the nuclei are similar in appearance and have similar DNA quantities, the reasonable assumption is that each nucleus is diploid. However, since the possibility remained that the nuclei had different complements of chromosomes, in situ hybridization with probes from each of the five chromosomes was used to confirm that each nucleus contained a full set of chromosomes (Yu et al., 2002). In most cases, the probes hybridized to the two nuclei

Chap. 13 Transcription and Recombination in Giardia

with equal intensity, supporting the proposal that the nuclei are each diploid. The initial PFGE evaluation demonstrated substantial size variation among chromosome homologs, especially for chromosome 1 (Adam et al., 1988). Subsequent studies demonstrated that the size variation reflected differences in the type and quantity of repetitive DNA in the subtelomeric regions. Approximately 30% of the size difference of the chromosome 1 homologs could be explained by differences in the number of subtelomeric rDNA molecules, while the remainder of difference was probably due to other repetitive DNA (Adam et al., 1991; Adam, 1992). In fact, the rDNA molecules from different genetically related isolates. The CAT, P1 (Portland1), and ISR isolates that were part of one study are all Genotype A1; yet varied substantially in locations of the rDNA repeats. Likewise, different clones of the ISR isolate demonstrated substantial differences from each other. In contrast to the variability at the chromosome ends, the central regions of the Genotype A1 isolates are remarkably constant as evidenced by identical restriction digestion patterns with infrequently digesting enzymes such as NotI, and by the lack of allelic heterozygosity in the sequence of the WB genome (Morrison et al., 2007). Of note, the genome libraries used for the genome project and the assembly approach tended to exclude the subtelomeric regions, so these regions are not part of the assembly. Taken together, these studies give us picture in which the internal regions of the chromosomes are remarkably homogeneous and stable while the subtelomeric regions demonstrate substantial variability even within closely related clones and isolates. However, it is worth noting that even within the more central regions of chromosomes there may be allelic differences in repeat copy numbers of tandem-repeat-containing vsp genes. VspC5 has a 105 bp tandem repeat for which the copy number in different alleles may vary from 17 to 26, while the copy number of the 195 bp repeat of vspA6 varies from 8 to about 22 (Yang and Adam, 1994; Yang et al., 1994). In addition to the rDNA repeats in the subtelomeric regions, there are also several other repeat regions, including three different retroposons found at the ends of several chromosomes (Arkhipova and Morrison, 2001; Prabhu et al., 2007). These retroposons all belong to the LINE (long interspersed nuclear element) family

215

and include one presumed pseudogene (GilD) and two with intact reading frames (GilM and GilT). Their potential roles in recombination in Giardia have not been studied. Vsp genes in a linear array were found at one end of chromosome 5 (Prabhu et al., 2007), but it appears unlikely that the vsp genes contribute substantially to the variation at the chromosome ends.

13.3 DNA Replication and Recombination In concert with many other functions of Giardia, the DNA replication apparatus appears to be fairly minimal. Only two origin recognition complex proteins (Orc4 and Orc1/Cdc6) were identified in the genome (Morrison et al., 2007), and there were no regulatory initiation proteins. There were three B-type DNA polymerases. Trophozoites divide by binary fission, which is generally assumed to be canonical mitotic replication. Sexual reproduction has not been documented and earlier studies using isoenzyme patterns of multiple enzymes involved in metabolism suggested that replication was primarily clonal in nature. However, this assumption failed to explain the low level of allelic heterozygosity (even the higher level of heterozygosity seen in the GS isolate). When diploid or polyploid organisms reproduce asexually over an extended period of time, mutations accumulate asymmetrically in the chromosome homologs, since mitotic crossing over is not very efficient in homogenizing the chromosomes. In species of bdelloid rotifers that have replicated asexually for millions of years, the two chromosome homologs have diverged so far that they would no longer be capable of pairing (Welch and Meselson, 2000). In Giardia, there is the additional requirement of homogenizing the DNA in the two nuclei. Although the answers to these interesting questions are not yet complete, there are interesting recent data that give partial explanations. Most of the enzymes that are involved in DNA replication have the ability to function in mitosis and/ or meiosis because of their roles in processes that are common to both mitosis and meiosis. However, a few genes are more specific to meiosis in a wide variety of eukaryotes. All of the genes that are known to be required for meiosis in other eukaryotes have also

216

R. D. Adam

been found in the Giardia genome, including Dmc1, Spo11, Mnd1, Hop1, and Hop2 (Ramesh et al., 2005). These genes all have full open reading frames, and transcription has been documented for some of them (Melo et al., 2008). Thus, occult sexual reproduction was suggested by this finding. Subsequently, population genetic analysis of a series of field isolates from Peru has suggested meiotic sexual reproduction in Genotype A2 isolates. In this study, JH, a Genotype A2 laboratory isolate (Nash and Keister, 1985; Nash et al., 1985) and five fecal isolates from a highly endemic region of Peru were sequenced at four loci from three chromosomes. Three of these loci showed sequence differences, which overall were at a level near 1%. Two of the isolates were from siblings and were identical, but the

other four demonstrated substantial differences from each other and from JH. The first significant finding was that the five distinct isolates (JH and four field isolates) demonstrated phylogenetic relationships that differed depending on which locus was evaluated. This finding is expected if there is sexual reproduction, but not if reproduction is mostly or completely clonal. Another prediction of sexual reproduction is meiotic crossing over, which tends to occur more frequently in organisms with smaller genome sizes. For the 9.5 kb of contiguous sequence from the chromosome 3 locus, there were two potential crossing over sites (Fig. 13.1). These findings strongly suggest that the Genotype A2 isolates in this setting are undergoing sexual replication. However, there are a several important caveats

B. Chromosome 5 Hypothetical protein 17200 431590

cAMP-dependent protein kinase A catalytic subunit 1648-2727

Sun/nucleolar family protein 2780-5008

Leucine-rich repeat protein 5011-7374

Inositol hexakisphosphate kinase 7393-8322

Translation initiation factor 83199563

Fig. 13.1 Chromosome 5 open reading frames and SNPs across 9.5 kb for six genotype A2 G. lamblia isolates. ORFs are depicted in vertical striped boxes: top boxes are forward, bottom boxes are reverse. ORF names are written above the box and if the ORF is a hypothetical protein, its corresponding GiardiaDB ORF number is provided. JH is the reference Genotype A2 isolate, while 55, 246, 303/305, and 335 are Genotype A2 field isolates. Isolates 303/305 are from siblings and are identical. Hash marks along each horizontal line represent SNPs in the field isolates, compared to JH. Open arrows with an asterisk depict an insertion/deletion (indel) fig. 2 from Cooper et al. (2007)

Chap. 13 Transcription and Recombination in Giardia

to note. First, it is possible for an organism to undergo parasexual reproduction in which two organisms combine and reassort their chromosomes but without true meiosis (Birky Jr, 2010). This occurs in Candida albicans, which has the meiotic gene repertoire, but in which true meiotic reproduction has never been documented (Forche et al., 2008). Second, recombination between Genotypes A and B isolates was not observed in this setting (unpublished data). However, I would note here that two recent manuscripts have suggested that recombination occurs between Genotypes A and B (Teodorovic et al., 2007; Lasek-Nesselquist et al., 2009). Thirdly, recombination has not yet been observed within Genotype A1 isolates such as WB and may be difficult to evaluate because of the lack of heterogeneity among these isolates. Further genomic analyses will be required to determine the extent of recombination within and between genotypes and may help address whether these different genotypes truly represent separate species. Since sexual replication has never been seen in vegetatively growing trophozoites, it is of interest to speculate whether sex might occur during encystation or excystation. To this end, it is of interest that during the process of encystation, plasmid DNA can be exchanged between the nuclei (Poxleitner et al., 2008). If this DNA transfer also involves entire chromosomes, this would explain the homogenization of the two nuclei, and if chromosomes were occasionally lost in the process and required redoubling, it might also explain the low level of allelic heterozygosity. It would be especially interesting to know if there is a difference between WB and GS in this process, since WB has nearly undetectable allelic heterozygosity, while it is about 0.5% in GS. If it is important to note, however, that this process of selfing would not explain the evidence of recombination between different organisms, unless a similar process can involve two separate organisms. The difference between WB and GS is also demonstrated in how they handle integration of stably integrated plasmids. For WB, episomes introduced as circular plasmids remain as episomes and do not integrate into the genome. However, if these episomes are linearized and contain sequences at each end of the linearized sequence that allow homologous integration, these linearized pieces of DNA will integrate precisely (Singer et al., 1998). However, the circular plasmid that

217

replicates episomally in WB will not replicate in GS. Instead, it will sometimes integrate randomly into the genome in a way that does not appear to be homologous in nature. The reason for the differences between these two isolates has not been explained.

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218 Forche A, Alby K, Schaefer D, Johnson AD, Berman J, and Bennett RJ (2008) The parasexual cycle in Candida albicans provides an alternative pathway to meiosis for the formation of recombinant strains. PLoS Biol 6: e110 Hausmann S, Altura MA, Witmer M, Singer SM, Elmendorf HG, and Shuman S (2005) Yeast-like mRNA capping apparatus in Giardia lamblia. J Biol Chem 280: 12077–12086 Holberton DV and Marshall J (1995) Analysis of consensus sequence patterns in Giardia cytoskeleton gene promoters. Nucleic Acids Res 23: 2945–2953 Huang YC, Su LH, Lee GA, Chiu PW, Cho CC, Wu JY, and Sun CH (2008) Regulation of cyst wall protein promoters by Myb2 in Giardia lamblia. J Biol Chem 283: 31021–31029 Kabnick KS and Peattie DA (1990) In situ analyses reveal that the two nuclei of Giardia lamblia are equivalent. J Cell Sci 95(Pt 3): 353–360 Lasek-Nesselquist E, Welch DM, Thompson RC, Steuart RF, and Sogin ML (2009) Genetic exchange within and between assemblages of Giardia duodenalis. J Eukaryot Microbiol 56: 504–518 Li L and Wang CC (2004) Capped mRNA with a single nucleotide leader is optimally translated in a primitive eukaryote, Giardia lamblia. J Biol Chem 279: 14656–14664 Melo SP, Gomez V, Castellanos IC, Alvarado ME, Hernandez PC, Gallego A, and Wasserman M (2008) Transcription of meiotic-like-pathway genes in Giardia intestinalis. Mem Inst Oswaldo Cruz 103: 347–350 Morrison HG, McArthur AG, Gillin FD, Aley SB, Adam RD, Olsen GJ, Best AA, Cande WZ, Chen F, Cipriano MJ, Davids BJ, Dawson SC, Elmendorf HG, Hehl AB, Holder ME, Huse SM, Kim UU, Lasek-Nesselquist E, Manning G, Nigam A, Nixon JE, Palm D, Passamaneck NE, Prabhu A, Reich CI, Reiner DS, Samuelson J, Svard SG, and Sogin ML (2007) Genomic minimalism in the early diverging intestinal parasite Giardia lamblia. Science 317: 1921–1926 Nash TE and Keister DB (1985) Differences in excretory-secretory products and surface antigens among 19 isolates of Giardia. J Infect Dis 152: 1166–1171 Nash TE, McCutchan T, Keister D, Dame JB, Conrad JD, and Gillin FD (1985) Restriction-endonuclease analysis of DNA from 15 Giardia isolates obtained from humans and animals. J Infect Dis 152: 64–73 Nixon JE, Wang A, Morrison HG, McArthur AG, Sogin ML, Loftus BJ, and Samuelson J (2002) A spliceosomal intron in Giardia lamblia. Proc Natl Acad Sci USA 99: 3701–3705 Pan YJ, Cho CC, Kao YY, and Sun CH (2009) A novel WRKYlike protein involved in transcriptional activation of cyst wall protein genes in Giardia lamblia. J Biol Chem 284: 17975–17988 Poxleitner MK, Carpenter ML, Mancuso JJ, Wang CJ, Dawson SC, and Cande WZ (2008) Evidence for karyogamy and exchange of genetic material in the binucleate intestinal parasite Giardia intestinalis. Science 319: 1530–1533 Prabhu A, Morrison HG, Martinez CR III, and Adam RD (2007) Characterisation of the subtelomeric regions of Giardia lamblia genome isolate WBC6. Int J Parasitol 37: 503–513 Ramesh MA, Malik SB, and Logsdon JM Jr (2005) A phylogenomic inventory of meiotic genes; evidence for sex in Giardia and an early eukaryotic origin of meiosis. Curr Biol 15: 185–191

R. D. Adam Russell AG, Shutt TE, Watkins RF, and Gray MW (2005) An ancient spliceosomal intron in the ribosomal protein L7a gene (Rpl7a) of Giardia lamblia. BMC Evol Biol 5: 45 Seshadri V, McArthur AG, Sogin ML, and Adam RD (2003) Giardia lamblia RNA polymerase II: amanitin-resistant transcription. J Biol Chem 278: 27804–27810 Singer SM, Yee J, and Nash TE (1998) Episomal and integrated maintenance of foreign DNA in Giardia lamblia. Mol Biochem Parasitol 92: 59–69 Sun CH, Palm D, McArthur AG, Svard SG, and Gillin FD (2002) A novel Myb-related protein involved in transcriptional activation of encystation genes in Giardia lamblia. Mol Microbiol 46: 971–984 Sun CH, Su LH, and Gillin FD (2006) Novel plant-GARPlike transcription factors in Giardia lamblia. Mol Biochem Parasitol 146: 45–57 Sun CH and Tai JH (1999) Identification and characterization of a ran gene promoter in the protozoan pathogen Giardia lamblia. J Biol Chem 274: 19699–19706 Svard SG, Meng TC, Hetsko ML, McCaffery JM, and Gillin FD (1998) Differentiation-associated surface antigen variation in the ancient eukaryote Giardia lamblia. Mol Microbiol 30: 979–989 Teodorovic S, Braverman JM, and Elmendorf HG (2007) Unusually low levels of genetic variation among Giardia lamblia isolates. Eukaryot Cell 6: 1421–1430 Wang CH, Su LH, and Sun CH (2007) A novel ARID/Brightlike protein involved in transcriptional activation of cyst wall protein 1 gene in Giardia lamblia. J Biol Chem 282: 8905–8914 Welch DM and Meselson M (2000) Evidence for the evolution of bdelloid rotifers without sexual reproduction or genetic exchange. Science 288: 1211–1215 Yang Y and Adam RD (1994) Allele-specific expression of a variant-specific surface protein (VSP) of Giardia lamblia. Nucleic Acids Res 22: 2102–2108 Yang YM, Ortega Y, Sterling C, and Adam RD (1994) Giardia lamblia trophozoites contain multiple alleles of a variantspecific surface protein gene with 105-base pair tandem repeats. Mol Biochem Parasitol 68: 267–276 Yee J, Mowatt MR, Dennis PP, and Nash TE (2000) Transcriptional analysis of the glutamate dehydrogenase gene in the primitive eukaryote, Giardia lamblia. Identification of a primordial gene promoter. J Biol Chem 275: 11432–11439 Yee J, Tang A, Lau WL, Ritter H, Delport D, Page M, Adam RD, Muller M, and Wu G (2007) Core histone genes of Giardia intestinalis: genomic organization, promoter structure, and expression. BMC Mol Biol 8: 26 Yu DC, Wang AL, Botka CW, and Wang CC (1998) Protein synthesis in Giardia lamblia may involve interaction between a downstream box (DB) in mRNA and an anti-DB in the 16S-like ribosomal RNA. Mol Biochem Parasitol 96: 151–165 Yu LZ, Birky CW Jr, and Adam RD (2002) The two nuclei of Giardia each have complete copies of the genome and are partitioned equationally at cytokinesis. Euk Cell 1: 191–199 Zhang Y and Wang L (2005) The WRKY transcription factor superfamily: its origin in eukaryotes and expansion in plants. BMC Evol Biol 5: 1

Intracellular Protein Trafficking Adrian B. Hehl

Abstract The secretory transport capacity of Giardia is perfectly adapted to its changing environment and is able to deploy essential protective surface coats as well as molecules, which act on host epithelia. The lumen-dwelling trophozoites take up nutrients by bulk endocytosis through peripheral vesicles or by receptor-mediated transport. Despite its versatility and fidelity, the giardial trafficking machinery appears to be the product of a general secondary reduction process that led to minimization of all components identified so far. Giardia is emerging as a model for the investigation of synthesis, transport, and assembly of highly effective biopolymers, a hallmark of all perorally transmitted protozoan and metazoan parasites. The cell biology of this simplified and highly derived organism allows unique insights into the function of minimal systems, which can be studied in an uncluttered cellular environment.

14.1 Introduction As an intestinal lumen-dwelling parasite, which can transform into an environmentally resistant cyst form, Giardia interfaces with its environment via secreted proteins and carbohydrates. The most obvious result of secretory activity is the prominent cyst wall, which is deposited on the surface of encysting cells and whose description dates back to Grassi (1879). The discovery of the variant-specific surface protein coat and antigenic variation pointed to additional major protein trafficking pathways to the plasma membrane of trophozoites. In addition to classical exocytosis of membrane proteins, there is also evidence for nonconventional secretion of metabolic enzymes and fac-

H. D. Luján et al. (eds.), Giardia © Springer-Verlag/Wien 2011

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tors that modulate the response of the gut epithelium to infection. Endocytic uptake and transport of nutrients from the complex environments in both the natural habitat and the cell culture are not well understood. In contrast to other Diplomonads, the Giardiinae lack a cytostome organelle with which the bulk of fluid phase transport is handled. Instead, specialized organelles termed peripheral vesicles (PVs) which are arrayed just below the plasma membrane appear to provide an all-in-one solution to nutrient uptake, digestion, and retrograde transport of building blocks to the interior of the cell (Lanfredi-Rangel et al., 1998). Only recently, a few publications have systematically and rigorously addressed the basic issues of endocytic trafficking in order to reveal the principles of retrograde transport and the role of these peculiar organelles. During the past two decades, several research groups have investigated intracellular protein trafficking in more detail. This work was kicked off by a first publication on transport of cyst wall material in encystation-specific vesicles in differentiating trophozoites (Reiner et al., 1990). It became clear that despite an intracellular organization with strongly reduced complexity, Giardia trophozoites have an efficient membrane trafficking system capable of directing numerous cargo proteins to their correct destination along distinct transport pathways. This capability for constitutive and stage-regulated secretion contrasts with the apparent simplicity of the machinery involved and its molecular underpinnings: although many conserved elements have been identified, genomic analysis revealed a significant and consistent reduction of key components on every level. Most striking is the absence of certain compartments or even entire organelles such as a Golgi apparatus with biochemically defined cisternae and an endo-

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somal system that intersects with it, as well as key protein components such as vesicle-tethering factors. Combined with the induction of a prominent secretory pathway in encysting cells, this makes Giardia a unique cell biological model to investigate minimal solutions for membrane transport.

14.2 Organelles and Machineries of the Membrane Transport System The definition of the giardial membrane-bounded organelle system is based on both morphological appearance (Lindmark, 1988; Reiner et al., 1989; McCaffery and Gillin, 1994; Lanfredi-Rangel et al., 1998; Benchimol, 2004) and direct or indirect detection of marker proteins (Reiner et al., 1989; Marti et al., 2003b; Tovar et al., 2003; Touz et al., 2004; Hernandez et al., 2007; Elias et al., 2008). The latter has been problematic because correct localization of markers with heterologous antibodies is possible only if proteins are conserved to a very high degree. Normally this requires generation of specific antibodies against the giardial homologs (Marti et al., 2003b; Elias et al., 2008). Although the fine structures remain to be defined, the combined data reveal highly simplified organizational principles on the one hand and highly specialized features on the other. On a morphological and molecular level only three clearly identifiable organelle systems have been delineated in trophozoites: an extensive endoplasmatic reticulum (ER) which is continuous with the nuclear envelopes as in all other eukaryotes, the peripheral vesicles, and mitochondrial remnant organelles (mitosomes). In differentiating trophozoites an additional set of large organelles, the encystation-specific vesicles (ESVs), is generated and disappears again upon secretion of the cyst wall material. Trafficking pathways to and from these organelle systems have been identified and in some cases characterized in more detail. According to the current state of information, the ER (together with ESVs in differentiating cells) comprises the entire secretory system in Giardia. Similarly, PVs are the only identifiable organelles in the endocytic pathway. This is in line with the low number of conserved members of key protein families involved in membrane transport.

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14.2.1 Endoplasmatic Reticulum and Golgi Apparatus The giardial ER has an intricate bilaterally symmetrical structure and extends from the nuclear envelopes through the entire cell body. The very existence of this archetypical eukaryotic organelle in Giardia had been doubted (Feely et al., 1990; Meyer, 1994) although electron microscopy data indicated the presence of ER membranes (Reiner et al., 1990; McCaffery and Gillin, 1994). Definitive identification of the organelle was reported by two groups in the same year (Lujan et al., 1996a; Soltys et al., 1996). In both papers, antibodies raised against the conserved giardial Hsp70/BiP homolog (Gupta et al., 1994) were used to localize this marker by fluorescence and/or immunoelectron microscopy. Membrane bound and soluble protein disulfide isomerases (PDIs) have been used as markers in subsequent studies revealing the same distribution pattern (Knodler et al., 1999; Stefanic et al., 2006; Abodeely et al., 2009). Live cell confocal microscopy with ER-Tracker™ and EM tomography confirmed the general tubular nature and subcellular distribution of the ER organelle (Abodeely et al., 2009). ER membranes are found throughout the cytoplasm but do not permeate the space occupied by PVs. However, apparent contact points and continuities between the two organelle systems have been detected and are the basis for a recent endocytic transport hypothesis (Abodeely et al., 2009). Glycosylation and ER quality control: All available data indicate that Giardia possesses a conventional ER with respect to secretory trafficking, although some elements of the post-translational modification machinery are missing completely. Co-translational import and folding of secreted proteins are supported by a conserved machinery for translocation (Svard et al., 1999), chaperones, and the five members of the PDI family of proteins (Knodler et al., 1999; Morrison et al., 2007). The latter are unusual in that they have a single thioredoxin domain instead of the normal two or three in PDIs of other eukaryotes. This domain structure appears to be a primary basic feature and not the product of secondary reduction (Knodler et al., 1999). Giardial PDIs most likely play a major role in assisting the folding of the cysteine-rich VSPs and the high cysteine membrane proteins (HCMPs) (Davids et al., 2006a).

Chap. 14 Intracellular Protein Trafficking

While the factors for protein folding in the ER are conserved in large parts, the entire calnexin-calreticulin machinery for quality control of N-glycosylated secreted proteins is missing (Samuelson et al., 2005; Banerjee et al., 2007, 2008). This is in line with the absence of conventional GlcNAc2Man9Glc3 glycans linked to Asn residues on secreted proteins which are exported from the ER (Samuelson et al., 2005). Although complex branched glycans were identified initially (Morelle et al., 2005), rigorous genomic and biochemical analysis revealed that Giardia is missing nucleotide sugar transporters (Banerjee et al., 2008) and has only a single (Alg7) of the usual twelve glycosyltransferases required for the synthesis the dolichol-PP-GlcNAc2Man9Glc3 precursor in the ER (Samuelson et al., 2005). Thus, Asn-linked glycosylation in the giardial ER is limited to the addition of GlcNAc1–2 to proteins. Nevertheless, a recent analysis demonstrated a sizable N-glycome in Giardia with distinct aspects of stage regulation (Ratner et al., 2008). Golgi: the earliest electron microscopic examination of cysts and trophozoites revealed a conspicuous absence of organelles, i.e. ER, Golgi, and mitochondria (Sheffield and Bjorvat, 1977). Later claims that a Golgi organelle existed (Gillin et al., 1991; Lujan et al., 1995a; Lanfredi-Rangel et al., 1999; Dacks et al., 2003) remain unsubstantiated, although occasionally parallel membrane arrays can be observed in some trophozoites. As a classical Golgi we define a series of biochemically distinguishable, dynamic, steadystate compartments in which most or all secreted proteins are delayed for post-translational maturation before being sorted and transported to their final destinations by vesicular transport. Whether these cisternae are arranged as an easily detectable ordered stack with a defined cis (receiving) and trans (exporting) face is irrelevant since there are numerous examples of perfectly functional, delocalized Golgi systems. Since no known Giardia organelle meets these criteria, and no classical markers for the Golgi such as GM130, galactosyl transferases, or the trans-Golgi network marker Rab6 can be identified, it is likely that a bona fide Golgi was lost during evolution. Current molecular phylogenetic data support the idea that the last common eukaryotic ancestor already had a complex membrane trafficking system with a steadystate Golgi apparatus (Dacks and Field, 2007). This is

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not to say that there are no post ER trafficking compartments in Giardia. ESVs of encysting trophozoites have many hallmarks of Golgi cisternae but also clear differences (see below). Components of the normally Golgi-associated coatomer (COPI) coat complex were detected on ESV membranes (Stefanic et al., 2006), but these antibodies against a giardial subunit also label punctuate structures in trophozoites (Marti et al., 2003b). Indirect evidence for the existence of post ER trafficking compartments comes also from identification of a putative KDEL-receptor (Erd2) homolog (GiardiaDB GL50803_4502) in the Giardia genome. This protein is likely involved in retrieval of ER resident proteins such as the chaperone Hsp70/ BiP from distal compartments (Stefanic et al., 2006). Molecular machineries: Accumulating evidence indicates secondary loss of major compartments and functions associated with membrane transport in Giardia. However, Giardia still possesses a core machinery with the three classical coat complexes (COPII, coatomer [COPI], and clathrin) and two adaptor protein complexes. COPI and adaptor protein complexes are normally associated with the Golgi apparatus. All but one subunit of the heteroheptameric COPI complex can be identified in the Giardia genome. In addition, small GTPases of the Arf and Arf-like family which are involved in COPI recruitment to membranes (Murtagh et al., 1992; Marti et al., 2003) were detected. At least one of the two giardial Arf1 homologs is sufficiently conserved to rescue a lethal yeast Arf1/2 double mutant (Lee et al., 1992). Only two complete hetero-tetrameric adaptor protein (AP) complexes corresponding to an AP1 and an AP2/3 equivalent have been identified. The giardial AP1 is involved in secretory transport (Touz et al., 2004), whereas the second complex localizes to PVs and appears to be involved in endocytic processes (Rivero et al., 2010). Currently, Giardia is the only known eukaryote with only two AP complexes. Since APs evolved early from a proto F-COP-AP complex by coordinated gene duplication of their subunits (Schledzewski et al., 1999; Marti et al., 2003b), the reason for the presence of only two complexes in Giardia (secondary loss or divergence before a second duplication event) remains to be determined. Interestingly, a giardial clathrin heavy chain was readily identified in the genome and the protein was localized to PVs and mature ESVs (Marti

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et al., 2003b; Gaechter et al., 2008). However, a light chain homolog appears to be missing. Together with the lack of evidence for clathrin-coated transport intermediates and membrane buds, this could point to an alternative role for this coat protein subunit. Rab GTPases, SNAREs (soluble N-ethylmalemide-sensitive factor attachment protein receptors), and their effectors belong to universally conserved protein families whose members control membrane fusion events and compartment identities. Because these factors interact specifically with membranes of organelles their diversity is a good indicator for the level of compartment complexity in a cell. Organization of the giardial trafficking pathways and compartment structure appears to require only seven Rab proteins (Marti et al., 2003b) in contrast to the large Rab complements of other basal protozoa such as Trichomonas vaginalis (65 Rabs) (Lal et al., 2005) and Trypanosoma brucei (16) (Ackers et al., 2005), or the anaerobic Entamoeba histolytica (>90 Rabs) (Saito-Nakano et al., 2005). The Rab proteins consist mostly of a GTPase domain and are functionally defined by about 30 amino acids at their C-terminus, which includes prenylation sites. For this reason, the sequence constraints are high, and it is unlikely that additional giardial Rabs, which are diverged beyond recognition, will be identified in the future. In Giardia three predicted exocytic (Rab1, 2a/b) (Langford et al., 2002; Marti et al., 2003b) and one predicted (recycling) Rab11 homolog are well conserved, and cluster robustly with orthologs from other eukaryotes in phylogenetic analyses. The three remaining Rab family members (F, D, and 32) cannot be assigned to a specific Rab subgroup (Marti et al., 2003b; Morrison et al., 2007). Consistent with experimental data indicating a simple compartment structure with few trafficking pathways (Hehl and Marti, 2004), only a small number of factors which mediate vesicle docking and membrane fusion were identified. Members of the SNARE family of proteins are highly divergent and therefore more difficult to identify with confidence. Using BLAST search algorithms only seven SNARE proteins (Dacks and Doolittle, 2002, 2004; Marti et al., 2003b; Morrison et al., 2007) were initially identified in the completed genome. Three are putative v-SNARES, and four are syntaxin (t-SNARE) homologs anchored in target compartment membranes.

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Application of more sensitive methods using Hidden Markov Model (HMM) profiles revealed 15 (Kloepper et al., 2007) and 17 (Elias et al., 2008) predicted SNARE family members, respectively, that were classified according to the QabcR rule (Bock et al., 2001). Subcellular localization data of epitope-tagged variants generated by fluorescence microscopy (Elias et al., 2008) supported the idea that Giardia lacks a bona fide Golgi, and provides an excellent starting point for a detailed analysis of SNARE function. Because no Golgi compartment stacks were identified in Giardia it is not surprising that golgins and other Golgi matrix proteins, which act as organizers of this organelle, cannot be found in the genome database. Large coiled-coil tethering factors of the golgin family or generally conserved tethering complexes, i.e. GARP (Golgi-associated retrograde protein), COG (conserved oligomeric Golgi), and exocyst mediate initial long-range capture of transport vesicles before SNARE-driven membrane fusion, and appear to be absent as well. Exceptions are the TRAPPI (transport protein particle) and the HOPS (homotypic fusion and vacuolar protein sorting) complexes. Four of the usual seven subunits of the former, and three of the six subunits of the latter can be identified with high confidence in the Giardia genome database (Koumandou et al., 2007). Overall, despite the conservation of several key factors and complexes, the genomic analysis of the machineries mediating intracellular transport shows consistent reduction of complexity.

14.2.2 Peripheral Vesicles and Endocytic Transport The peripheral vesicles (PVs) constitute a very conspicuous organelle system in transmission EM micrographs of trophozoites. The electron lucent PVs appear approximately oval shaped and typically ~150 nm long. The organelles are clustered in a narrow zone just below the plasma membrane of the entire dorsal side as well as in the small region at the center of the ventral disk. Although easily detectable, their diverse functions are not well understood. In the absence of a cytostome, the PVs are the only known endocytic organelles (Tai et al., 1993) capable of accumulating fluid phase

Chap. 14 Intracellular Protein Trafficking

and membrane-bound molecules. However, PVs also seem to have lysosomal properties (Feely and Dyer, 1987; Lindmark, 1988; McCaffery and Gillin, 1994), which means they acidify and mature to digestive organelles. In addition to hydrolases, PVs contain also cathepsins (Ward et al., 1997; Thirion et al., 2003). The detection of CWPs in the lumen of PVs also suggests a role in regulated secretion of cyst wall protein (Reiner et al., 1990). The dynamics of uptake of fluid phase and membrane cargo was not investigated until recently. Two studies presented data suggesting that PVs periodically open to the environment either via a channel or by fusion with the plasma membrane (PM) and take up soluble material (Gaechter et al., 2008; Abodeely et al., 2009) before closing again. This reversible, transient fusion with the PM effectively flushes the PV, releasing its contents to the environment and replacing this volume with extracellular medium. Uptake of fluid phase markers by this type of environmental sampling can be visualized directly. Combined with confocal live-cell imaging it was possible to determine the dynamics of exchange between the PV system and the environment, and to visualize and measure endocytic transport. PVs in living and in chemically fixed cells can also be visualized using Lysotracker™ (Gaechter et al., 2008; Rivero et al., 2010). More importantly, by using fluorescence recovery after photobleaching (FRAP) it could be shown that there is no lateral exchange of fluid phase markers between PV organelles (Gaechter et al., 2008), while some markers (e.g. casein) were transported rapidly further toward the cell interior, i.e. the ER or the associated tubulo-vesicular network (TVN) (Abodeely et al., 2009). The in vitro data suggest that uptake of soluble material from the environment into PVs is not very discriminatory, but further retrograde transport is selective, allowing only certain, as yet not defined, substances to cross over into the proximal ER. Fluorescently labeled dextrans, which are frequently used as markers for fluid phase endocytosis are not transported beyond PVs, whereas casein is rapidly shuttled to the ER (Abodeely et al., 2009). A recent study shows that import of LDL, which is not present in the gut lumen but abundant in cell culture medium, is mediated via PVs and dependent on the predicted adaptor protein 2/3 complex (designated as AP2 by the authors) (Rivero et al., 2010).

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14.3 Secretory Transport During Growth and Differentiation Secretory transport plays an essential role in Giardia: every life cycle stage with the exception of the resting cyst in the environment is known to secrete specific soluble and membrane-bound proteins (Fig. 14.1). Accumulation of cyst wall material in, and export from ESVs (the specialized organelles of encysting parasites) is the most conspicuous manifestation of secretory transport in this parasite. Yet, maintaining the integrity of the protective VSP coat alone is likely to require a major proportion of secretory resources. Export of both cyst wall material and VSPs requires distinct secretory pathways, which do not intersect beyond the ER and are subject to stage regulation. Because both transport routes are active for many hours in encysting trophozoites, differentiating parasites have to increase their synthetic as well as their organizational capacity considerably. This manifests in the de novo establishment of an additional secretory pathway dedicated to the export of the cyst wall material.

14.3.1 Proliferating Trophozoites Excreted-secreted products of trophozoites are most likely essential pathogenicity determinants. The major membrane proteins are the transmembrane-anchored VSPs of which normally only one variant is expressed at a time (Adam et al., 1988). VSP exodomains are also released by cleavage in the conserved C-terminal domain (Papanastasiou et al., 1996) and become soluble antigens. This results in a constant turnover and export of VSPs at the surface independent of antigenic variation. Thus, synthesis and export of VSPs and also other cysteine-rich, non-variable proteins (Davids et al., 2006a) targeted to the plasma membrane constitute likely a major part of the secretory activity of trophozoites. In addition to the approximately 200 VSPs, more than 500 proteins with a signal sequence are currently predicted in the Giardia Genome Database. Although most of those will not be secreted, it is likely that a considerable number are exported to the surface or released to the environment by conventional membrane transport. Because Giardia lacks Golgi cisternae and delay of secreted proteins in post ER compartments during export has

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A. B. Hehl

Fig. 14.1 Model depicting the major exocytic and endocytic trafficking pathways and the organelles of the membrane transport system. Arrows indicate postulated and confirmed trafficking routes for various cargo proteins described in the text. It is important to note that no trafficking pathway has been elucidated completely, yet. N nucleus; ER endoplasmatic reticulum; PV peripheral vesicle; ESV encystation-specific vesicle; fl, fluid fraction of the cyst wall material; co, condensed fraction of the cyst wall material. Organelle-associated coat protein complexes are indicated in italics. Confirmed cargo sorting steps (boxed s) and partitioning of cyst wall material (boxed p) are indicated

not been observed (Marti et al., 2003a), these components accumulate only at their final destination after leaving the ER. Current data support direct trafficking of VSPs and likely other constitutively secreted soluble and membrane proteins from the ER to the PM (Marti et al., 2003a; Touz et al., 2003; Hehl and Marti, 2004). Interestingly, export of VSPs was sensitive to brefeldin A (Lujan et al., 1995a), a fungal metabolite which inhibits Arf1-dependent recruitment of COPI to Golgi membranes and results in fragmentation of the Golgi organelle in higher eukaryotes. This suggests that VSP transport occurred via a COPI-positive organelle, although this has not been identified. The membrane-anchored VSPs have a short conserved cytoplasmic tail, CRGKA, which is posttranslationally modified by palmitoylation (Papanastasiou et al., 1997; Touz et al., 2005) and citrullination of the arginine residue (Touz et al., 2008). Only few trafficking signals have been characterized in trophozoites: C-terminal sequences that direct VSPs to the PM, and an encystation-specific protease, ESCP (which is also expressed in tropho-

zoites albeit at a lower level (Touz et al., 2002b)), to PVs, respectively. The C-terminal domain of VSPs, which includes the cytoplasmic tail, the hydrophobic transmembrane sequence, and a short stretch of the exodomain, is well conserved. It made sense that targeting signal(s) directing export from the ER and trafficking to the PM would be localized in this invariable region. Indeed, the VSP C-terminal domain is necessary and sufficient for correct secretion of a heterologous Toxoplasma gondii SAG1 surface antigen exodomain (Kasper et al., 1984; Tomavo et al., 1992) that was used as a reporter (Marti et al., 2002). Removal of the CRGKA cytoplasmic tail alone or the entire C-terminal domain resulted in accumulation of this reporter in the ER (Marti et al., 2003a) suggesting that this sequence has an essential function in VSP targeting. When the VSP exodomain is left intact and only CRGKA is removed, the protein still traffics to the PM (Touz et al., 2003), but the possibility that the reporter is co-exported by interaction with endogenous secreted VSP in these transgenic cells has not been tested.

Chap. 14 Intracellular Protein Trafficking

ESCP is synthesized as a membrane-anchored pro-protein and targeted to lysosomes in trophozoites (Touz et al., 2002b, 2003, 2004) by a conserved YXX)-type targeting signal in its short cytoplasmic tail. Targeting of chimeric reporters with a ESCP-derived TM and cytoplasmic tail and a H7 VSP exodomain to PVs is dependent on this YRPI motif (Touz et al., 2003). This transport is dependent on the activity of adaptor protein complex 1 (Touz et al., 2004). A Golgi-like sorting function has been invoked for ESCP secretion, but data showing significant amounts of tagged ESCP on the cell surface (Touz et al., 2003) point to YXX)-mediated retrieval of ESCP from the PM as an alternative explanation. The establishment of axenic culture systems (Keister, 1983) was a prerequisite for the systematic identification of soluble secretory products. In vitro (Katelaris et al., 1994; Roxstrom-Lindquist et al., 2005; Muller et al., 2007) and rodent models (Davids et al., 2006b; Li et al., 2007) for the study of the effects of secreted substances on the host are also well established. Research on secretion in proliferating trophozoites focused on identifying virulence factors, products which cause pathology in the host intestine or which interfere with immunological reactions to giardial infection. While no evidence for the production of secreted toxins was found (Smith et al., 1982), a number of secreted proteins have been identified which potentially interact with host cells on different levels. The best documented effects of Giardia co-cultivation with epithelial cells are a reduction of epithelial barrier function (Teoh et al., 2000) and induction of apoptosis (Chin et al., 2002; Panaro et al., 2007). Secreted proteases were detected in the supernatants of cultured trophozoites (Jimenez et al., 2000; Rodriguez-Fuentes et al., 2006; de Carvalho et al., 2008) although their targets remain unknown. Secreted products are also implicated in the observed degradation of epithelial barrier function in the gut and induction of enterocyte apoptosis (Chin et al., 2002; Troeger et al., 2007) but none have been identified yet. Other secretory products affected the uptake of glucose and phenylalanine in the intestine of mice (Samra et al., 1988). Taken together there is solid support for the idea that Giardia interferes with epithelial function, presumably via soluble or membrane-bound secreted proteins, but these factors and their precise interaction(s) with host cells await characterization.

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In addition to factors which directly interfere with the integrity of the gut epithelium, secreted (glyco) proteins that modulate certain aspects of the intestinal immune response appear to play a role in trophozoite survival. Secreted immunogenic glycoproteins were identified recently, but their roles remain to be determined (Jimenez et al., 2007). More importantly, substantial amounts of soluble VSP exodomains are released after cleavage in the conserved domain at the base of the transmembrane anchor (Papanastasiou et al., 1996). The effect of secreted VSPs, however, is unclear. Although the majority of proteins released by trophozoites are likely exported through the secretory pathway, there are also robust data for nonconventional protein export. Recently it was shown that contact with Giardia elicits distinct changes in chemokine expression in cultured intestinal epithelial cells (Roxstrom-Lindquist et al., 2005). Secreted metabolic enzymes (Ringqvist et al., 2008) were found to alter the microenvironment. An example is the proposed depletion of arginine at the host parasite interface resulting in suppression of NO production (Eckmann et al., 2000). The pathway through which the giardial metabolic enzymes arginine deiminase, ornithine carbamoyl transferase, and enolase are secreted upon contact with epithelial cells in vitro is unknown (Ringqvist et al., 2008). These exported effectors and immunogens (Palm et al., 2003) are cytoplasmic proteins, contain no N-terminal signal peptide, and are therefore not detected in vesicular transport pathways.

14.3.2 Encysting Trophozoites The most striking secretion process in the giardial life cycle occurs during stage differentiation of trophozoites. Secretion of the cyst wall material (CWM) is the only known regulated export pathway. It manifests in neogenesis of ESVs followed by deposition of this extracellular matrix on the surface of the differentiated cell (Fig. 14.1). Protocols for the induction of differentiation in vitro (Gillin et al., 1987) were essential for the investigation of the encystation process. Several protocols are currently used in laboratories. The two-step method (bile deprivation, followed by supplementation with porcine bile and an increase of the pH to 7.85) (Boucher and Gillin, 1990) is the

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most frequently used. Alternative methods such as cholesterol deprivation (Lujan et al., 1996b) or incubation with high concentrations of bile extract in the medium are also used for specific applications which require a high proportion of viable cysts. Encysting trophozoites can be followed from the induction to the formation of the cyst wall. The twostep and the cholesterol deprivation methods are the most useful for the investigation of protein trafficking and cyst formation. Although the time from induction to cyst formation varies considerably between laboratories, this process takes at least 14–20 h. Synthesis and co-translational import of the structural proteins of the cyst wall CWP1–3 (Lujan et al., 1995b; Mowatt et al., 1995) into the ER take place during the first ~7 h post induction. The three members of the CWP family are soluble proteins with a distinct structure comprising a central domain with several leucine-rich repeats (Lujan et al., 1995b). The CWM biopolymer has a surprisingly low complexity considering how effectively it acts as a biological barrier on the surface of the cyst. The three CWPs are clearly paralogous and constitute about 40% of this extracellular matrix; the rest is made up of a simple E1–3 GalNAc homopolymer (Jarroll et al., 1989; Gerwig et al., 2002). A non-VSP type 1 integral membrane protein termed HCNCp is a member of a large group of cysteine-rich factors which are potentially secreted stage specifically (Davids et al., 2006a). Indeed, while an epitope-tagged variant of the HCNCp investigated in this study localized to the plasmalemma in trophozoites, it was detected in ESVs during encystation and was partially secreted to the cyst wall or the PM – cyst wall interface (Davids et al., 2006a). While trafficking of the major proteins of the CW is clearly through ESVs, it is completely unclear when and where in the exocytic pathway the poly-GalNAc sugar component is synthesized and how it is finally incorporated into the cyst wall structure. Synthesis of the galactosamine from glucose and its polymerization is mediated by pathways whose components are upregulated transcriptionally and allosterically after induction of encystation (Macechko et al., 1992; Lujan et al., 1995a; Das and Gillin, 1996; Van Keulen et al., 1998; Bulik et al., 2000). Synthesis of CWP1–3 mRNAs peaks at ~7 h p.i., and the newly produced protein is seen to accumulate from about 2 h p.i. in emerging ESVs (Konrad et al., 2010). CWP export from the ER to ESVs is completed after 8–10 h

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p.i. (Hehl et al., 2000) in parasites encysting in vitro. The fact that ESVs only contain CWPs and no constitutively secreted proteins strongly suggests that the former are sorted away from the latter during ER export (Reiner et al., 1990; Marti et al., 2003a). The first half of the encystation process is therefore dedicated to synthesis and accumulation of the CWM in this newly established secretory pathway. The sorting of the CWPs to ESVs in the absence of a constitutive Golgi apparatus has hallmarks of cis Golgi compartment neogenesis. The membrane carriers, if any, for trafficking of the CWM from the ER to ESVs are currently unknown, but the process appears to be dependent on COPII coat formation and the small GTPase Rab1 (Stefanic et al., 2009). Segregation of the CWPs to ESVs appears to be determined by dominant signals on CWPs and occurs at the level of ER export (Marti et al., 2003a). Rather than defined targeting sequences, entire domains of CWPs, specifically the LLRs, are essential for sorting into ESVs (Hehl et al., 2000; Marti et al., 2003a; Sun et al., 2003). Thus, like Golgi cisternae, ESVs delay secretory cargo, presumably for maturation, before regulated secretion. However, unlike a Golgi, ESVs contain only one type of cargo, the CWM. Interestingly, transient association of COPI components with ESVs (Marti et al., 2003b), their sensitivity to brefeldin A (Lujan et al., 1995a; Marti et al., 2003a), and dependence of ESV genesis and maturation on giardial Sar1 and Arf1 GTPases, respectively (Stefanic et al., 2009), indicate that these organelles might be stage-regulated Golgi-like cisternae. The exact nature of ESVs and their function in regulated secretion in the context of reductive evolution are currently under investigation in several laboratories. After completion of ESV formation, the CWPs are not immediately secreted. The CWM is further delayed in ESVs for several hours, presumably to allow for post-translational maturation before being secreted in fluid form to cover the entire cell surface where it eventually polymerizes. CWP2 has a 121 residue C-terminal extension rich in basic amino acids (Lujan et al., 1995b). Proteolytic processing of this domain, which has no homolog in the other two CWPs, is currently the only proteolytic modification described for CWPs. In addition to processing, the enzymatic formation of disulfide (Hehl et al., 2000) and isopeptide (Davids et al., 2004) bonds between CWPs appears

Chap. 14 Intracellular Protein Trafficking

to play a major role in the export process. Although the evidence clearly implicates specific cleavage of pro-CWP2 by a cysteine protease, there is a controversy as to which enzyme is responsible (Touz et al., 2002b; DuBois et al., 2008). Previous investigations indicated that the entire C-terminal extension of ~13 kDa (Gottig et al., 2006) might be removed from CWP2. However, the small C-terminal portion of the native or the transgenic CWP2 has never been visualized directly (Sun et al., 2003). Although processing of CWP2 was found to occur before secretion of the CWM, it was not correlated with expression kinetics or maturation and morphology of ESVs. More recently, analysis of a CWP variant with epitope tags at both termini showed that cleavage of CWP2 occurred during the maturation stage of ESVs, before secretion of the CWM (Konrad et al., 2010). Western analysis suggested removal of a short fragment of ~5 kDa between 8 h and 10 h post induction in vitro. The Cterminal domain of CWP2 was implicated in sorting of all three CWPs from the ER to ESVs (Gottig et al., 2006). The authors invoked an export mechanism based on sequestration to and protrusion from specialized subdomains of the ER, rather than conventional export. The relation between the ESVs and the ER remains unclear. On the one hand there is a clear morphological separation between the two organelle systems as well as exclusion of cargo and resident factors such as PDIs and Hsp70/BiP from ESVs. The latter, however, appears to be cycling through ESVs and may be retrieved by a KDEL receptor protein (Stefanic et al., 2006). On the other hand, the current data are not clear about how ESVs arise and whether nascent organelles at least could constitute highly specialized ER subdomains (Touz et al., 2002a; Marti et al., 2003a; Gottig et al., 2006). In particular, the spatial proximity of the two organelles and the vigorous exchange of the CWM between established ESVs raises the question whether they ever become truly independent before the secretory process begins (Stefanic et al., 2009). The use of dual-tagged CWP2 and CWP::GFP chimeras to analyze the processes during the maturation phase of ESVs also revealed a hitherto unnoticed sorting and partitioning of the CWM (Konrad et al., 2010). In particular, co-localization of CWP1 with CWP3 or the C-terminus of CWP2 showed that the CWM is partitioned into two biophysically distinct

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fractions in maturing ESVs. CWP3 forms a condensed core structure together with the small CWP2 C-terminus after processing of pro-CWP2. How this condensation process is triggered and what is required exactly are unknown, but an unrelated, earlier study showed that expression of giardial CWP1 and CWP2 in human embryonic kidney-293 cells led to the formation of granules and secretion to the culture medium (Abdul-Wahid and Faubert, 2004). This suggests that condensation is an inherent property of CWPs. In particular, the basic C-terminal extension of CWP2 has been implicated in granule formation during encystation in this study. Conversely, in encysting Giardia CWP1 and the mature N-terminal CWP2 remain in a fluid state until secretion and distribution on the surface of the forming cyst (Konrad et al., 2010). Thus, each fraction contains a mature CWP2 product. Live cell analysis using GFP-tagged variants showed that these components are highly motile within an ESV organelle network. These studies demonstrate that the immobile ESVs are laterally connected via dynamic membrane tubular channels (Stefanic et al., 2009; Konrad et al., 2010). After partitioning of the fluid and condensed fractions of the CWM the former is sorted away into compartments, which localize close to the cell periphery. The upshot is that this fluid material is rapidly and quantitatively secreted within a few minutes. This first layer of the cyst wall is laid down at the same time as morphological transformation of the differentiating cell takes place. Polymerization is rapid and results in the formation of a structurally resistant matrix. The condensed CWM remains completely in internal compartments and is secreted slowly over the course of several hours. Although processing of CWP2 is not required for condensed core formation and sequential secretion of CWM, it is necessary for correct partitioning of CWP2. It was demonstrated that unprocessed CWP2 becomes sequestered in the condensed core and is exported only during the second secretion. This resulted in the formation of morphologically normal cysts, which were not water resistant, however (Konrad et al., 2010). Taken together, regulated secretion and cyst wall formation appear to be more complex than previously thought and require several hours from the time the first layer is established until the cyst becomes water resistant.

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14.3.3 Excysting Parasites When passing through the low pH environment of the stomach Giardia cysts are triggered to initiate excystation. Secreted proteases released from the endosome-lysosome peripheral vesicle system play a brief but essential role in the liberation of the excyzoite (Bernander et al., 2001) from the cyst wall. As a major secreted factor essential for the excystation process, a cathepsin B type protease was identified (Ward et al., 1997). This enzyme, designated CP2, was identified by affinity purification using a biotinylated variant of the specific inhibitor E-64. The inhibitor was also used to determine the subcellular localization of CP2 in PVs or their equivalents in cysts. Secretion of the enzyme into the space between the plasma membrane and the cyst wall is triggered during excystation. CWPs are phosphorylated during export (Slavin et al., 2002). This post-translational modification needs to be reversed for excystation: inhibition of acid phosphatase activity during the first phase of in vitro excystation almost completely abolished the process (Slavin et al., 2002).

14.4 Summary The secretory transport capacity of Giardia is perfectly adapted to its changing environment and is able to deploy essential protective surface coats and molecules, which act on host epithelia. Nevertheless, the giardial trafficking machinery appears to be the product of a general secondary reduction process that led to minimization of all components identified so far. Despite the low complexity of the organelles and machineries involved, these diverse protective surface antigens as well as secreted proteins are delivered with great fidelity. Of special interest are VSPs of trophozoites and the extracellular matrix polymer of cysts, which confer environmental resistance in the respective life cycle stages. Recent and ongoing work in several laboratories is revealing the basic principles behind the transport events responsible for the assembly of these key biological barriers. However, significant work to elucidate the details of these mechanisms, in particular in the light of their astonishingly low complexity, still needs to be done.

A. B. Hehl

For example, it is becoming clear that Giardia could serve as a model for the investigation of synthesis, transport, and assembly of simple but highly effective biopolymers, a key feature of all perorally transmitted protozoan and metazoan parasites. Another example is the PV system: these organelles appear to be at the crossroads of endocytic and exocytic transport. Although the data are still scarce, PVs are emerging as the major sorting station for the transport of soluble as well as membrane-bound factors in and out of the cell. As such, they could provide a protected space for both final modification and/or activation of secreted molecules on the one hand; on the other hand, PVs can act as an intracellular containment system. This would allow controlled separation of nutrients from potentially harmful substances taken up in bulk by constant sampling of the fluid extracellular environment. As is also the case for ESVs, a better understanding of the biological role of trafficking pathways to and from PVs requires elucidation of the “organelle cycle”, i.e. the processes that govern organelle genesis, maintenance, and maturation.

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Post-transcriptional Gene Silencing and Translation in Giardia Pablo R. Gargantini, César G. Prucca and Hugo D. Luján

Abstract The control of gene expression in Giardia lamblia includes several mechanisms already described in higher eukaryotes, but with some interesting features for this early-branching organism. Here we describe two gene expression control systems in Giardia, posttranscriptional gene silencing (PTGS) and translation, and the close interaction between them. For the first mechanism, all the components were identified as being active in this cell, their sequences were analyzed and their localization was identified. Even more important was the implication of this mechanism in the process of antigenic variation in this parasite, which reflects the involvement of the RNAi pathway in variant-specific surface protein (VSP) regulation and switching. Regarding the translational system, the principal characteristics of this parasite are the lack of ribosome scanning mechanism and a prokaryotic resemblance in the small ribosomal subunit recruitment process. Even though the presence of some, but not all, eukaryotic initiation factors could represent a simplified “cap-dependent” process, there is also the possibility that microRNAs could be involved in translation regulation. In general, we can assume that this intestinal parasite has either simplified the gene expression control machinery due to their parasitic life style or, on the other hand, we are privileged witnesses of how the evolutionary process takes place.

15.1 Introduction Gene expression in any organism can be modulated at any stage from transcription to post-translational modifications (White and Sharrocks, 2010). In general, gene expression includes the regulation at chromatin domains, transcriptional events, post-tran-

H. D. Luján et al. (eds.), Giardia © Springer-Verlag/Wien 2011

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scriptional modifications, RNA transport, translation, and mRNA degradation. In eukaryotes, after the DNA is transcribed and the mRNA is generated, translation of mRNA into proteins is regulated by modulating capping, splicing, and addition of a poly (A) tail, nuclear export rate, and sequestration of the RNA transcript in some particular regions of the cell. There is also a regulation during protein synthesis that allows a rapid and reversible control of gene expression at this initial stage. This complex process involves initiation factors and their interactions with ribosomal initiation complexes and is regulated by mechanisms that include a scanning-dependent modulation from these initiation factors and also through RNA-binding proteins and microRNAs affecting individual mRNAs (Jackson et al., 2010). Although transcription in Giardia lamblia has some characteristics of prokaryotic organisms, it is distinctly eukaryotic as the transcript is produced within both nuclei of the parasite, is polyadenylated, and transported to the cytoplasm for translation (see Adam, this volume). Additionally, one of the most important RNA-processing complexes in eukaryotes, the spliceosome, is present in the last common ancestor to extant eukaryotes since four mRNA introns were identified in the genome of this early-branching protist (Morrison et al., 2007). Giardia introns are variable in length (32–220 bp) with conserved 5′ and 3′ motifs and have similar sizes to introns found in the corresponding genes in different isolates (Franzén et al., 2009). Thus, some form of splicing mechanisms may have evolved very early during eukaryotic evolution, or lost in this organism due to its parasitic life style. On the other hand, the Giardia genome presents short intergenic regions and short untranslated regions (UTRs) that are more characteristic of prokaryotes. The Giardia genome is ~11.7 Mb in size, distributed on five chromosomes. It is compact in structure and

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content, and has simplified machinery for DNA replication, transcription, RNA processing, and most metabolic pathways (Morrison et al., 2007). Here we describe two gene expression control stages in Giardia, post-transcriptional gene silencing (PTGS) and translation, and the close interaction between them.

15.2 The RNAi Pathway in Giardia In the last years, a large amount of information regarding the mechanism of regulation of gene expression indicates that virtually all animals and plants utilize small RNA molecules to fine tune the control of protein expression (Cerutti and Casas-Mollano, 2006). From the simplest to the most complex living form, organisms must be able to confront the changes that take place in the environment in which they grow and reproduce. To confront these problems, these organisms have developed the capacity to regulate the expression of specific genes, activating some ones and silencing others under specific stimulus. Considered one of the most outstanding discoveries in recent years, the RNA interference (RNAi) process has become one of the most useful tools used in gene expression studies, leading to the understanding of specific functions in organisms where genetic approaches do not work. Early experiments performed in plants revealed that when additional copies of a flower color gene were introduced into plant cells, the new generated flowers were colorless (Napoli et al., 1990). This phenomenon was called “co-suppression”. Similar features were then observed in the fungus Neurospora crassa and the process, in this case, was named “quelling” (Cogoni et al., 1996). Two years later, a similar mechanism was described in Caenorhabditis elegans and was called RNAi (Fire et al., 1998). Since then, a growing amount of evidence has indicated that double-stranded RNA (dsRNA) plays a very important role in the regulation of several types of gene silencings, leading to the inhibition of translation, destruction of the messenger RNA (mRNA) as well as chromatin remodeling and DNA elimination (Hannon, 2002). Like other organisms, Giardia presents an RNAi machinery, although highly simple. Recently, several

P. R. Gargantini et al.

researchers revealed exciting insights about the importance of small RNAs in this parasite (Ullu et al., 2004, 2005; Prucca et al., 2008; Saraiya and Wang, 2008; Kolev and Ullu, 2009).

15.2.1 The Mechanism of RNAi: General Features RNAi is a genetic mechanism for control of gene expression that involves specific small RNA molecules. These small RNAs are able to trigger the silencing of specific genes based on nucleotide homology between their sequences (Yang and Nowotny, 2009). Basically, long dsRNA molecules are diced into small RNAs of restricted length, which are able to recognize specific mRNAs, to trigger their destruction or inhibition of translation. Although the final purpose of these small molecules is the inhibition of expression of the protein product encoded by mRNAs, the way in which these molecules are generated presents differences. When long stem-loop structures are transcribed in the nucleus, the RNase III enzyme Drosha recognizes and dices them into smaller molecules called pre-micro RNAs (pre-miRNAs). These 70 nucleotides premiRNAs are then transferred to the cytoplasm by means of Exportin 5. When outside of the nucleus, these molecules are recognized by a second enzyme of the RNase III endonuclease family called Dicer, which processes these pre-miRNAs into mature miRNAs (Filipowicz et al., 2005; Tang, 2005). Small interfering RNAs (siRNAs) are generated by a different pathway. When long dsRNAs are recognized by Dicer in the cytoplasm, they are cleaved into small 21- to 26-nucleotide-long molecules. After the generation of any kind of small RNA molecules takes place (miRNAs and/or siRNAs), they should be loaded into an Argonaute (Ago) containing protein complex called RISC (RNA-induced silencing complex) to guide the specific down-regulation of cognates sequences by means of either translation inhibition or destruction of mRNAs. Upon loading of the small RNA molecule into this protein complex, one of the strands is destroyed (the passenger strand) and the remnant strand (guide strand) serves as guide for specific recognition of the target sequence (Tomari and Zamore, 2005).

Chap. 15 Post-transcriptional Gene Silencing and Translation in Giardia

Since miRNAs present not fully complementary identity with mRNAs, they are able to inhibit gene expression by blocking the translation of several mRNAs, in which these sequences found homology. This feature is a classical difference between siRNAs and miRNAs production (Carthew and Sontheimer, 2009). The first class of small RNAs targets genes that present fully complementary sequences (from which they were generated) and generally it leads to the destruction of mRNAs. However, miRNAs can regulate expression of related genes. These genes present partial complementary sequences (usually at the 3′ untranslated region, 3′-UTR) and the degree of silencing depends on the number of miRNAs that are bound to that region. Furthermore, small RNA molecules can also form duplexes with DNA sequences leading to the silencing of specific genes at the transcriptional level, producing epigenetic changes, or in some cases, DNA elimination (Matzke and Birchler, 2005).

15.2.2 The RNAi Machinery 15.2.2.1 Dicer When dsRNAs are in the cytoplasm, they are recognized by a multidomain protein member of the RNase III family known as Dicer (MacRae et al., 2006). This enzyme is present in a large group of organisms that have a fully active RNAi pathway. Based on protein characterization of human and Drosophila melanogaster Dicer enzymes, the information indicates that these endonucleases are multimeric proteins that present an RNA helicase domain, a PAZ domain, two RNase III catalytic domains, a dsRNA binding domain and a domain with an unknown function (DUF283) (Bernstein et al., 2001; Meister and Tuschl,

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2004). Although these features are conserved among several eukaryotes, more important differences between the Dicer-like enzymes are in the dsRNA binding domain and in the PAZ domain (Cerutti and CasasMollano, 2006). In the canonical RNAi pathway, small RNA molecules generated by Dicer activity present a 21- to 26-nt-long sequence with a phosphate at the 5′-end and a 3′ two-nucleotide overhang (MacRae et al., 2006). These two conserved features of the small RNAs are critical to continue the pathway and to be loaded into the RISC complex (Nowotny and Yang, 2009). Giardia Dicer was initially identified by our group in 2002 and called “Giardia Bidentate RNase III” because the lack of domains present in Dicer enzymes from higher eukaryotes. In contrast to many higher eukaryotes (Cerutti and Casas-Mollano, 2006), only one member of the Dicer family is present in Giardia. In an outstanding report by Doudna’s laboratory, these authors presented the complete structural analysis of the full length Giardia Dicer and measure its activity in vitro (MacRae et al., 2006). This protein (Giardia genome ORF accession number: GL: 50803_103887) has approximately 82 kDa and possesses two RNase III domains and one PAZ domain. Interestingly, Giardia Dicer lacks the DExD/H RNA helicase domain as well as the double-stranded RNA binding motif present in other Dicer homologs. While screening the G. lamblia genome database to find sequences coding for ORFs with significant similarity to DEAD and DExH-box proteins, 31 putative RNA helicases were found, including 21 DEAD- and 10 DExH-box proteins (divided in 6 DEAH- and 4 Ski2p-box proteins), suggesting that they could be functional RNA helicase orthologues (Gargantini and Lujan, unpublished data) (Fig. 15.1).

Homo sapiens Dcr1 (1922 aa) DEX Dc

HELIc

D UF283

PA Z domain

RN Ase III (a-b)

DSRM

Giardia lamblia Dcr (754 aa) PAZ domain

RNAse III (a-b)

Fig. 15.1 Comparison between human and Giardia domain architectures of Dicer-like proteins. The conserved domains and their diagrams are shown to scale

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Based on activity assays using a recombinant Giardia Dicer, this enzyme was capable to generate small RNAs in vitro (25- to 27-nt long) from a dsRNA molecule. It was also shown that Giardia Dicer activity depends on the presence of magnesium, and this enzyme fully supports RNAi in Schizosaccharomyces pombe (MacRae et al., 2006). Moreover, a recent report by Prucca et al. (2008) showed that Dicer is expressed during the entire parasite life cycle and, similar to some higher eukaryotes, Giardia Dicer localized to the cytoplasm. In vitro evaluation of the enzyme activity shows that its activity is dependent of ATP and that this enzyme is able to recognize and process virtually any dsRNA, leading to the generation of small RNAs of 25–27 nt in length (Prucca et al., 2008). The molecular structural data indicate that the 65 Å distance between the PAZ and the RNase III domain explains the length of the 25-nt long products generated in the Dicer activity assay. Moreover, a two-metal ion mechanism of dsRNA cleavage was proposed (MacRae et al., 2006). Under this scenario, the two adjacent RNase III domains present two metal ions in their active site and this interaction was implicated in the hydrolysis of each strand of the dsRNA molecule. The superposition of Giardia Dicer and human Argonaute 1 indicates the presence of a pocket that binds the 3′ two-nucleotide overhand present in the small RNAs generated by the RNAi pathway (MacRae et al., 2006). The molecular models of the Dicer enzyme shows that this protein present a “L” shape, and compared with the molecular structure of the human Dicer (in which the RNA helicase domain is present), Giardia Dicer could be modeled with the RNase III domain in the top or the base of the L form (Sashital and Doudna, 2010).

15.2.2.2 Argonaute Proteins The Argonaute (Ago) proteins are present in several organisms, from animals and plants to fungi and protists, even in Archaea (Ma et al., 2005). Ago proteins are involved in the RNAi mechanism and other related pathways; however, it has also been proposed that this protein family is involved in development, stem cell maintenance, and cancer (Carmell et al., 2002). Although the number of Argonaute genes varies from one organism to another, it seems like these proteins

P. R. Gargantini et al.

do not present overlapping functions and have specialized activities. Again, Giardia does have a unique Ago gene in its genome. At the molecular level, the proteins that belong to this family share two domains: the PIWI and the PAZ domains. This family can be classified into three well-defined groups based on amino acid sequence differences: the Argonaute (Ago), the Piwi, and the Worms Argonaute (WAGO) groups. Ago proteins localize in the cytoplasm and in the nucleus, as well as in discrete foci, associated with the P-bodies in which the maintenance and storage of mRNA molecules take place (Hock and Meister, 2008). The RNA molecules that interact with Ago proteins are generated from dsRNA molecules by Dicer enzymatic activity. Argonaute proteins that belong to the PIWI group are expressed mostly in germ cell lines and are involved in the control of transposable elements. PIWI-associated RNAs are generated from a cascade of different dsRNA molecules, in which the small RNA that results from the activity of one PIWI protein becomes the substrate for the activity of another group member (Thomson and Lin, 2009). The last group, named WAGO, was described based on the structure of proteins present in worms, and the RNAs associated with them result from the activity of Dicer on long dsRNA molecules (Pratt and MacRae, 2009). Most of the information regarding the molecular structure of Argonaute proteins was obtained from crystallization of prokaryotic Argonaute associated with DNA molecules. These proteins present two lobes, a structural form that allows the binding of the nucleic acid from both sides. The PAZ domain, that is present in the amino terminal portion of the protein, interacts with the 3′-end of the small DNA molecule. The carboxy terminal portion of Argonaute presents the MID and Piwi domains. The 5′-terminal portion of DNA interacts with the MID domain and with the Piwi domain via coordination of a divalent cation forming a pocket. The Piwi domain also adopts an RNase-H like fold that allows the hydrolysis of the DNA molecule (Hock and Meister, 2008; Pratt and MacRae, 2009; Nowotny and Yang, 2009). Analysis of Argonaute proteins from different species shows a high degree of conservation among them. Moreover, the presence of the characteristic dual domain structure (amino terminal PAZ domain followed

Chap. 15 Post-transcriptional Gene Silencing and Translation in Giardia Homo sapiens PIWIL1 (861 aa) PAZ domain

Divergent PAZ domain

237

15.2.2.3 RNA-dependent RNA-polymerase (RdRP)

Piwi domain

Piwi domain

Giardia lamblia Piwi(754 aa)

Fig. 15.2 Comparison between human and Giardia domain architectures of Argonaute–Piwi proteins. The conserved domains and their diagrams are shown to scale

by a carboxyl terminal PIWI domain) is also well conserved (Cerutti et al., 2006) (Fig. 15.2). When the only Argonaute protein present in Giardia (Giardia genome ORF accession number: GL: 50803_2902) is included in the above-mentioned analysis, the results indicate that Giardia Ago is a 100 kDa protein that presents a well-defined PIWI domain and a highly divergent PAZ domain (Prucca et al., 2008). In a recent report by Saraiya and Wang (2008), a functional analysis of the Giardia Argonaute was performed. This protein seems to be essential for the parasite survival, since the down regulation of his expression leads to a cell growth inhibition. Additional assays using a recombinant Argonaute indicate that this protein is able to bind the 7-methyl guanosine cap of mRNAs, as well as small RNA molecules (Saraiya and Wang, 2008). In addition, another recent report also analyzed the expression of this enzyme indicating that this gene is expressed during the entire cell cycle of the organism and localizes to the cytoplasm of the cell (Prucca et al., 2008). Similar to that of Saraiya et al., Prucca et al. report indicates that the down regulation of the Giardia Argonaute expression leads to the inhibition of cell growth. This can be awarded to the fact that these enzymes are involved in the maintenance of the genome stability controlling the activity of transposons (Ullu et al., 2005).

RNA recognition motif

RdRP domain

RNA-dependent RNA-polymerases are enzymes with the capacity to synthesize RNA molecules based on a RNA template. RdRPs are not widely distributed among eukaryotes. Some organisms present fully active RNAi pathways but their genomes do not present any gene encoding a polymerase of this kind (Siomi and Siomi, 2009). It is possible that this enzyme is not necessary for the function of the RNAi pathway because the dsRNA could be generated by different pathways. However, when the silencing process must spread to regions outside of the initially targeted gene or involve the generation of trans-acting siRNAs, only the organisms that present an active RdRP will be able to accomplish these processes (Cerutti et al., 2006). Biochemical studies indicate that RdRPs are involved in the process of RNA antisense strand generation using aberrant sense RNAs, leading to the generation of long dsRNA molecules (Cogoni and Macino, 1999; Fagard et al., 2000; Prucca et al., 2008) (Fig. 15.3). The Giardia genome encodes only one RdRP (Giardia genome ORF accession number: GL: 50803_ 102515) that is different from the Giardia virus encoded RdRP (White and Wang, 1990). This gene encodes a basic protein with a molecular weight of 155 kDa. This polymerase of RNA shares high homology with RdRPs present in other eukaryotes. Analysis of his expression indicates that this enzyme, similar to Dicer and Argonaute, is expressed during the entire life cycle of the parasite. HA-tagged versions of this enzyme indicate a sub-cellular localization outside the nuclei, surrounding both structures, most likely associated with the ribosomes of the rough endoplasmic reticulum. Additional assays indicate that this enzyme is able to generate antisense RNA

RdRP domain

Arabidopsis thaliana RDR6 (1196 aa)

Giardia lamblia RdRP (2001 aa)

Fig. 15.3 Comparison between Arabidopsis and Giardia domain architectures of RdRP proteins. The conserved domains and their diagrams are shown to scale

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strands when more than one homolog RNA is present. This activity is independent of the presence of small RNAs functioning as primers (Prucca et al., 2008).

15.2.3 Small RNA Molecules in Giardia and Their Putative Biological Functions In the first report in which small RNA from Giardia was identified, Ullu et al. (2005) constructed and sequenced a library of RNAs from 20- to 30-nt-long and using BLAST identified 403 sequences. After an extensive analysis of the identified sequences, most of them correspond to structural RNAs (including rRNA, tRNAs, and snoRNAs); otherwise, a small percent of the identified small RNA match open reading frames, intergenic regions, and putative ORFs. Within the small RNAs that match ORFs, 8% of them present homology with the retroposon GilT, an element that is known to inhabit the Giardia genome. The presences of small RNAs for this element were confirmed by Northern blot using a sense probe to hybridize enriched small RNAs molecules. Small RNAs were founded in that fraction, indicating that these small RNAs may be involved in the control of this mobile element (Ullu et al., 2005). In the same year, another report indicated the presence of snoRNAs in Giardia. In this report, Yang et al. (2005) identified 20 snoRNAs from a cDNA library generated from a size fraction of 50- to 250-nt RNAs. The snoRNAs are a group of non-coding RNAs involved in several aspects of rRNA maturation (Kiss, 2002). After the analysis of the obtained clones, 16 box C/D and 4 box H/ACA small RNAs were identified (Yang et al., 2005). The authors indicate that the genes of small RNAs found in this study match intergenic regions of Giardia genome and their transcription is promoter independent. The promoter region of the genes from where the small RNAs are transcribed presents A-T rich elements in their upstream sequences, a feature already observed in other protein-coding genes. This observation leads to the hypothesis that the generation of this small RNAs involves RNA polymerase II (Yang et al., 2005). Some years later, the previously identified snoRNAs were suggested to be miRNAs precursors. In a detailed study (Saraiya and Wang, 2008), the authors identified, after isolating, cloning, and sequencing,

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some of the already known snoRNAs and observed that one of the small RNAs, known as miR2, presents the features of a product of Dicer activity. This small RNA is generated by Dicer activity on an previously identify snoRNA named GlsR17 (Yang et al., 2005). This miRNA shows recognition sites in the 3′ untranslated region of hundreds of Giardia genes, including 22 variant-specific surface proteins (VSPs). Additional experiments using a reporter gene fused to repetitions of the target sequence show that the expression is down regulated but the level of mRNAs is still unmodified, being indicative of an inhibition of translation, a classical effect of the action of miRNAs (Siomi and Siomi, 2009). The report also shows that the cellular localization of the precursor GlsR17 is in only one of the two nuclei of Giardia, in contrast to the mature miR2, which localizes predominantly in the cytoplasm. This observation leads to the question of how the GlsR17 is transported to the cytoplasm to be processed by Dicer (cytoplasm localization, Prucca et al., 2008) since neither Drosha nor Exportin 5 homologs are found in Giardia. It was postulated that snoRNAs are potential precursors of miRNA. This observation leads to the possibility for a new role in the regulation of VSP expression (Saraiya and Wang, 2008). Similar biological importance of small RNA was proposed by our group in a recent report. The results from a complete detailed study indicated that a system comprising Dicer, Argonaute, and RdRP is involved in the regulation of VSP expression (Prucca et al., 2008). In that work, we identified simultaneous transcription of several VSP genes, isolated and sequenced antisense sequences for the VSPs’ mRNAs, characterized the RNAi enzymes involved in the pathway and identified small RNAs which present homology with VSPs RNAs. In addition, when a reduction of the expression of two of these enzymes took place (Dicer and RdRP), the mechanism of antigenic variation was disrupted leading to the generation of trophozoites that present simultaneous expression of several VSPs in their surface (Fig. 15.4 A, B). Taken together, this evidence supports the involvement of the RNAi pathway in VSP regulation (Prucca et al., 2008). Another recent report identified in silico 10 miRNAs candidates in the Giardia genome suggesting that both small interfering RNAs and miRNAs are involved in different steps of control of VSP expression in Giardia (Chen et al., 2009).

Chap. 15 Post-transcriptional Gene Silencing and Translation in Giardia

Dicer-AS

RdRP-AS

A

B

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Fig. 15.4 Direct immunofluorescence assay on Dicer-AS (A) or RdRP-AS-transfected (B) Giardia trophozoites with FITC-conjugated monoclonal antibody 9B10 (VSP9B10) and TRITC-conjugated monoclonal antibody 5C1 (VSP1267). When Giardia Dicer or RdRP expression was knocked down, trophozoites expressing VSP9B10 on the surface also expressed VSP1267 (merged image, A and B)

The regulation of gene expression by means of small RNA molecules has become one of the most studied mechanisms in the recent years. The involvement of these tiny molecules in the silencing of several genes indicates that this is a very primitive mechanism. Giardia is considered an early-branching eukaryote (Adam, 2001), and in the last years a growing amount of evidence indicates that this parasite posses a fully active RNAi system (composed of Dicer, Argonaute and RNA-dependent RNA-polymerase). Moreover, this mechanism has been associated with the maintenance of genomic integrity, metabolism of the rRNA, and recently it is has been implicated in the regulation of antigenic variation. It is clear that the RNAi field has become an exciting new world for the “Giardiologists”, and a lot of research will be necessary to understand how this mechanism works and the implication on the gene regulation in this organism.

15.3 The Translational Machinery The regulation of protein synthesis occurs principally at the initial stage rather than during polypeptide elongation or termination, allowing a rapid, reversible and spatial control of gene expression (Jackson et al., 2010). The mechanisms that regulate translation evolved independently of the translation apparatus, emerging at different times during the evolution. Initiation mechanisms developed independently in both bacterial and archaeal–eukaryal lineages (Kirpides and Woese, 1998; Benelli and Londei, 2009). The recruitment of small ribosomal subunit to the mRNA is the principal step in all organisms for the beginning of translation. While in prokaryotes this is

mediated through the direct binding of the ribosomal subunit to a sequence called Shine-Dalgarno (SD) located near the start codon, in eukaryotes as their mRNAs lack this SD sequences, the translation initiation depends upon the recognition of the 5′-cap structure of the mRNA by a series of Initiation Factors (eIFs) that finally recruit the 40S ribosomal subunit (Hernández, 2009). As a deep-branched eukaryote, G. lamblia has a number of prokaryotic characteristics, such as short 5′-UTRs and the general absence of introns, but also of eukaryotic nature with its nuclear transcript production, polyadenylation, and RNA transport to the cytoplasm (Adam, 2000).

15.3.1 Is This Short Enough? The analyses of some G. lamblia transcripts have revealed that they have short 5′-UTRs (from 0 to 14 nucleotides) in contrast to the ~90 nucleotides present in RNAs from mammals (Kozak, 1987) and ~52 nucleotides in yeast (Cigan and Donahue, 1987). One exception is the glucosamine-6-phosphate isomerase B gene, having one of its two transcripts a 146-nt 5′UTR (Knodler et al., 1999). Similarly, short 5′-UTRs have also been identified among the mRNAs from other anaerobic protozoa such as Entamoeba histolytica (Bruchhaus et al., 1993) and Trichomonas vaginalis (Davis-Hayman et al., 2000). These short 5′-UTRs could reflect the presence of unique protein synthetic machinery in these closely related primitive eukaryotes. It suggests that Giardia promoters might be near the beginning of the ORF, as confirmed for the GDH promoter where the 44-bp upstream gene sequence provided maximal transcriptional activity in a trans-

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fection assay (Singer et al., 1998) or by deletion and mutational analyses of the Ran promoter, which possesses a region (from −51 to −2) having the maximal promoter activity. In this case, further upstream regions do not contribute to promoter function (Sun and Tai, 1999). It was also found that the 5′-UTR in Giardia mRNA can be reduced to a single nucleotide without affecting its translation efficiency or causing any leakiness, while if this 5′-UTR is longer than 9 nucleotides the efficiency decrease in contrast to what happens in mammalian in vitro system where if the 5′-UTR is shorter than 20 nucleotides the translation efficiency is reduced and presents leakiness in the ribosome scanning process (Kozak, 1991). This suggests that ribosome scanning involved in translation initiation among the more advanced eukaryotes may not exist in Giardia (Li and Wang, 2004), which is supported by the absence of eIF4B, eIF4H, and eIF4G homologs in Giardia that are involved in ribosome scanning (Dever, 2002; Prevot et al., 2003). This unique mechanism of translation initiation probably falls between that of a prokaryote and a eukaryote with a potentially close link with that in the Archaea. The transcripts also have relatively short 3′-UTRs of 10–30 nt (Adam, 2000) with short poly(A) tails typically beginning approximately 10–30 nucleotides beyond the stop codon; the polyadenylation signal proposed is the sequence AGTPuAAPy (Peattie et al., 1989; Adam, 1991).

15.3.2 A Prokaryotic Resemblance There are different mechanisms of translation initiation between prokaryotes and eukaryotes, in the first ones the base pairings between the 16S RNA and the mRNA direct the small ribosomal subunit recruitment, while in eukaryotes it is based on protein–protein and protein–RNA interactions (Sachs et al., 1997). The Shine-Dalgarno sequence of prokaryotic mRNAs (a 6-nt purine-rich sequence in front of the coding region, complementary to the 3′-end of 16S rRNA) (Gold, 1988) helps to bind and position the ribosome at the start site for protein synthesis, being absent from eukaryotic mRNAs. Even an anti-SDlike sequence is present at the 3′-end of 16S-like rRNA in G. lamblia (Sogin et al., 1989), the Giardia mRNAs with short 5′-UTRs do not usually contain

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the SD sequence (Adam, 1991). Another translation initiation signal in the mRNA of Escherichia coli that promotes efficient protein synthesis even in the absence of SD (Sprengart et al., 1996) is a Downstream Box (DB) that consists of 13 nt located downstream of the initiation codon and complementary to a region in the 16S rRNA (Sprengart et al., 1990). There is an identifiable DB homolog within each of the ORFs of Giardia mRNAs. This DB homolog could be involved in recruiting the small ribosomal subunit through base pairing with the anti-DB in the 16S-like rRNA, bringing the initiation codon near the decoding region in the small ribosomal subunit (Yu et al., 1998). Other important components in the translation machinery are the aminoacyl-tRNA synthetases (AARSs). They ensure the fidelity of protein synthesis by correctly acylating a tRNA species with its cognate amino acid (Ibba and Söll, 2000), having an exquisite specificity for their substrates (amino acid and tRNA). Even it is accepted that each cell contains one AARS for each canonical amino acid, some Archaea such as Methanococcus jannaschii, Methanococcus thermoautotrophicum, and Methanococcus maripaludis have a ProRS that is able to catalyze the formation of cysteinyl-tRNA (Cys-tRNA) besides the prolyl-tRNA (Stathopoulos et al., 2000). In Giardia there are two enzymes, ProCysRS and CysRS, for Cys-tRNA formation. Giardia ProCysRS, like the archaeal enzymes, requires tRNA for cysteine activation, whereas proline activation proceeds in the absence of tRNA (Bunjun et al., 2000). Cysteine plays an important role in G. lamblia (as part of the cysteine-rich proteins expressed in the trophozoites surface or maintaining the lowoxygen environment by cysteine and proteins containing cysteine) so the improved capacity to form Cys-tRNA may be needed under certain circumstances during its life cycle.

15.3.3 Bridging the Gap It has been proposed that because of the loss of the Shine-Dalgarno motif in mRNAs, others specific components of the translation machinery were incorporated tending to ensure a more efficient recruitment of the 40S ribosomal subunit by the mRNA (Hernández, 2009).

Chap. 15 Post-transcriptional Gene Silencing and Translation in Giardia

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Table 15.1 Eukaryotic translation initiation factors H. sapiens

Subunit

kDa

Function

Giardia homolog (assemblage A, isolate WB)

kDa

eIF1

12.7

Stimulates binding of eIF2–GTP–Met-tRNAi to 40S subunits; and prevents premature eIF5-induced hydrolysis of eIF2-bound GTP and Pi release. Participate in the initiation codon selection and ribosomal scanning.

GL50803_1657

12.1

eIF1A

16.4

Participate in the initiation codon selection and ribosomal scanning cooperating with eIF1. Stimulates binding of eIF2–GTP–Met-tRNAi to 40S subunits.

GL50803_8708

18.1

eIF2

α β γ

36.1 38.3 51.1

Catalyzes the first regulated step of protein synthesis initiation, promoting the binding of the initiator tRNA to 40S ribosomal subunits.

GL50803_13943 GL50803_91398 GL50803_2970

38.1 37.1 52.3

eIF2B

α β γ δ ε

33.7 38.9 31.5 50.2 80.3

Is a GTP exchange factor essential for protein synthesis. Activates its eIF2 substrate by exchanging eIF2-bound GDP for GTP.

GL50803_91911 GL50803_7652 GL50803_16129 GL50803_16598 GL50803_39587

36.7 42.2 78.2 55.3 145.6

eIF3

A (θ) B (η) C D (ζ) E F G (δ) H (γ) I (β) J (α) K L M

Prevent the large ribosomal subunit from binding the small subunit before it is ready to commence elongation. Promotes attachment of 43S complexes to mRNA and subsequent scanning.

– GL50803_15495 – – – GL50803_7896 – GL50803_16823 GL50803_13661 – – – –

– 98.5 – – – 31.3 – 37.3 38.4 – – – – 43.2

166.6 92.5 105.9 63.9 52.2 37.5 35.6 39.9 36.5 28.9 25.0 66.7 42.5

eIF4A

46.1

DEAD-box ATPase and ATP-dependent RNA helicase.

GL50803_10255

eIF4B

69.16

Enhances the helicase activity of eIF4A. Is an RNA-binding protein.

–

eIF4E eIF4E2

25.1 28.37

Involved in directing ribosomes to the cap structure of mRNAs.

GL50803_17261 GL50803_7990

Enhances the helicase activity of elF4A. Binds eIF4A, eIF4E, eIF3, PABP, SLIP1 and mRNA.

–

–

–

eIF4G-1

175.4

– 25.1 19.88

eIF4H

27.39

Enhances the helicase activity of eIF4A. Is an RNA-binding protein.

–

eIF5

49.2

Induces hydrolysis of eIF2-bound GTP on recognition of the initiation codon.

GL50803_14614

16.6

eIF5A-1

20.1

Stimulates ribosomal peptidyl-transferase and may be involved in nucleocytoplasmic mRNA transport.

GL50803_15538

38.2

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P. R. Gargantini et al.

Fig. 15.5 Alignment of Giardia homologs eIF4E1 and eIF4E2

In modern eukaryotes, most mRNAs are translated by a so-called ‘cap-dependent’ mechanism, a sophisticated process that requires at least 12 eukaryotic initiation factors, some of which are multimeric (Kozak, 1987). Genes encoding homologues of eIF1; eIF1A; eIF2 α, β, γ; eIF2B α, β, γ, δ, ε; eIF3 B, F, H, I; eIF4A, eIF4E, eIF5, and eIF5A were identified in the Giardia genome, suggesting that capped mRNA may be present and translated in this organism. However, as mentioned above, homologs of other initiation factors such as eIF4B, eIF4G, and eIF4H are apparently missing. So the protein synthetic machinery should be more simplified in Giardia and despite being able to assemble all the existing components even without the scanning process (Table 15.1). The posttranscriptional modification at the 5′-end of most eukaryotic mRNAs, small nuclear RNAs (snRNAs), and small nucleolar RNAs (snoRNAs) is the addition of a 7-methylguanosine (m7G) cap (Furuichi and Shatkin, 2000; Shuman, 2001). In the case of snRNAs and snoRNAs, their cap is then further methylated at its N2 position in the cytoplasm or nucleus to yield an N2,N2,7-methylguanosine (m2,2,7G) cap (Ro-Choi, 1999). The m7GpppN-cap structure is required for the recruitment of mRNA by the translational machinery, whereas the m2,2,7GpppNcap plays crucial roles in gene expression, such as mRNA splicing, methylation, pseudouridylation, rRNA processing and ribosome assembly (Ro-Choi, 1999). The eukaryotic initiation factor 4E (eIF4E) recognizes the m7GpppN-cap of mRNAs; this factor is also a protein that modulates the overall rate of translation and the mRNA selectivity of the translation apparatus (Furuichi and Shatkin, 2000; Gingras et al., 1999). There are two eIF4E homologs in G. lamblia (designated Giardia eIF4E1 and eIF4E2) that are not similar to each other at all except for some

of the conserved residues in the 170-amino-acid core and are highly divergent from all the other eIF4Es. The functional importance of eIF4E is illustrated by the lethality of eIF4E gene disruption in Saccharomyces cerevisiae (Altmann et al., 1989) but neither eIF4E1 nor eIF4E2 from Giardia was capable of rescuing the growth of yeast lacking endogenous eIF4E (Li and Wang, 2005) (Fig. 15.5). eIF4E2, but not eIF4E1, could be the cap-binding protein in the translation initiation complex of Giardia and the m7GpppN-cap is most likely the cap of Giardia mRNAs and functions in translation initiation. It is not unlikely that a simple conjugation between the 5′-cap and the initiation codon may constitute the essential and sufficient structural basis in the mRNA to initiate translation in Giardia (Li and Wang, 2005). We can summarize that the mechanisms of regulating initiation fall into two broad categories: those that impact on the eIFs (or ribosomes), and therefore affect virtually all scanning-dependent initiation events; and those that impact on the mRNA itself, either through sequence-specific RNA-binding proteins or through microRNAs (miRNAs), and are therefore potentially selective for certain mRNAs. Since Giardia lacks some of the components described for the scanning of mRNAs before translation, the identification of a miRNA mechanism could fulfill the need for a translation regulation process.

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P. R. Gargantini et al. Siomi H and Siomi CM (2009) On the road to reading the RNA-interference code. Nature 457: 396–404 Sogin ML, Gunderson JH, Elwood J, Alonso HJ, and Peattie DA (1989) Phylogenetic meaning of the kingdom concept: an unusual ribosomal RNA from Giardia lamblia. Science 243: 75–77 Sprengart ML, Fatscher HP, and Fuchs E (1990) The initiation of translation in E. coli: apparent base pairing between the 16S ribosomal RNA and downstream sequences of the mRNA. Nucleic Acids Res 18: 1719–1723 Sprengart ML, Fuchs E, and Porter AG (1996) The downstream box: an efficient and independent translation initiation signal in Escherichia coli. EMBO J 15: 665–674 Stathopoulos C, Li T, Longman R, Vothknecht UC, Becker H, Ibba M, and Söll D (2000) One polypeptide with two aminoacyl-tRNA synthetase activities. Science 287: 479–482 Sun CH and Tai JH (1999) Identification and characterization of a ran gene promoter in the protozoan pathogen Giardia lamblia. J Biol Chem 274: 19699–19706 Tang G (2005) siRNA and miRNA: an insight into RISCs. Trends Biochem Sci 30: 106–114 Thomson T and Lin H (2009) The biogenesis and function PIWI proteins and piRNAs: progress and prospect. Annu Rev Cell Dev Biol 25: 355–376 Tomari Y and Zamore PD (2005) Perspective: machines for RNAi. Genes Dev 19: 517–529 Ullu E, Tschudi C, and Chakraborty T (2004) RNA interference in protozoan parasites. Cell Microbiol 6: 509–519 Ullu E, Lujan HD, and Tschudi C (2005) Small sense and antisense RNAs derived from a telomeric retroposon family in Giardia intestinalis. Eukaryot Cell 4: 1155–1157 White RJ and Sharrocks AD (2010) Coordinated control of the gene expression machinery. Trends Genet 26(5): 214–220 White TC and Wang CC (1990) RNA dependent RNA polymerase activity associated with the double-stranded RNA virus of Giardia lamblia. Nucleic Acids Res 18: 553–559 Yang CY, Zhou H, Luo J, and Qu LH (2005) Identification of 20 snoRNA-like RNAs from the primitive eukaryote, Giardia lamblia. Biochem Biophys Res Commun 328: 1224–1231 Yu DC, Wang AL, Botka CW, and Wang CC (1998) Protein synthesis in Giardia lamblia may involve interaction between a downstream box (DB) in mRNA and an anti-DB in the 16S-like ribosomal RNA. Mol Biochem Parasitol 96(1–2): 151–165

Antigenic Variation in Giardia Theodore E. Nash

Abstract Giardia lamblia trophozoites undergo surface antigenic variation where one member of a family of related proteins, variant specific surface proteins (VSPs), is expressed but periodically replaced by another. It is the only intestinal dwelling organism to do this. Switching occurs spontaneously in culture in vitro in the absence of defined environmental triggers. The two major Giardia groups infecting humans differ in rates of VSP switching, which vary from once every 6.5 to 12–13 generations, nature of the vsp repertoires and occurrence of switching during encystation/excystation. VSPs are cysteine-rich with many CXXC motifs whose positions are indicative of the VSP subgroup. The cysteines are non-reactive most likely because of disulfide bond formation, which creates conformational epitopes. The most characteristic feature of VSPs is an absolutely conserved carboxyl terminal CRGKA tail, which is post-translationally modified by palmitoylation of the cysteine and modification of the arginine to citrulline. The function of these modifications is unclear but both affect antibody-mediated cytotoxicity. A well-conserved membrane-spanning region is immediately proximal to the tail. VSPs vary dramatically in size and a subset has tandem repeating amino acid units near the amino terminus that can differ in size, number, sequence, and antigenicity. Some are known to be immunodominant. Unique surface Zn finger and GGCY motifs of unclear function are present in a majority. VSPs are secreted into the medium by an undefined proteolytic process that cleaves the VSP close to the membrane-spanning region; the fate of the residual palmitate-containing fragment has not been studied. With the exception of closely related VSPs, they are antigenically distinct and possess individual features that impart differenti-

H. D. Luján et al. (eds.), Giardia © Springer-Verlag/Wien 2011

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ating physical chemical characteristics. Similar to the role of surface proteins of other organisms that undergo antigenic variation, VSPs are essential for the development of immunity, immune selection, and immune evasion as well as biological selection. The latter operates in humans and experimental animals in the absence of an adaptive immune system or before adaptive immune responses develop. Only certain VSPs are compatible in the intestine of specific host and organisms that express these survive and grow. Additionally, populated VSPs are also unrecognized by the host’s immune system. These constraints likely limit the number of VSPs that can be expressed in an infected individual. VSPs are most diverse at the amino terminus, the portion of the VSPs that interfaces most directly with the intestine and the immune responses of the host. Antibodies directed to the amino terminus are cytotoxic in vitro and lead to immune selection and replacement with VSPs unrecognized by the host in vivo. Experimental animals immunized to all VSPs are resistant to challenge infection. The WB isolate has a repertoire of about 270 vsp genes, only one of which is present on the surface of an organism. How switching occurs is unclear. Unlike the process in African trypanosomes, the process is epigenetic since there is no gene movement and vsps are not overly represented at the telomeres. There is good evidence that RNAi-based post-transcriptional mechanisms are involved in the control of VSP expression but how switching occurs and only one VSP is expressed remain unclear.

16.1 Introduction Giardia lamblia undergoes surface antigenic variation where one member of a family of antigenically

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16.2 Switching Characteristics

Fig. 16.1 Immunoelectronmicroscopy using a Mab to specific VSP that demonstrates the VSP densely covers the entire parasite including the flagella (Pimenta et al., 1991)

distinct but structurally similar variant-specific surface protein, VSP, is at times replaced by another (Adam et al., 1988, 1992; Nash et al., 1988; Bruderer et al., 1993; Nash, 2003). VSPs densely coat the entire surface and flagella of trophozoites (Nash and Keister, 1985; Pimenta et al., 1991; Svard et al., 1998) (Fig. 16.1). The presence of antigenic variation was suspected after repeated surface labeling studies of the passaged Giardia cultures and clones showed unexplainable changes of radiolabeled proteins over time. Antigenic variation was definitively proven after the development of VSP-specific monoclonal antibodies (Mab) that resulted in complement-independent cytotoxicity to trophozoites expressing the VSP specific to the Mab (Nash and Aggarwal, 1986; Nash et al., 1988). After exposure of Giardia clones expressing mostly one variant surface antigen to VSP-specific cytotoxic monoclonal antibodies, only a few trophozoites survived and subsequently grew and repopulated the culture. The original VSP expressed was replaced by other VSPs that differed in size and antigenicity from the originally expressed VSP. Repetition of the process using clones derived from the above surviving trophozoites yet again resulted in the selection of another set of trophozoites expressing other VSPs that were antigenically unique. Subsequent studies defined the characteristics of antigenic variation in vitro, presence and course in infections in vivo in animal models and humans, nature of the VSP family of proteins, genomic organization, partial demonstration of controlling mechanisms, and biological relevance.

In Giardia lamblia VSP switching occurs spontaneously in vitro in the absence of obvious stimuli or anti VSP antibodies (Nash et al., 1988). During growth only one VSP is detected on the surface of each trophozoite and only its transcript is detected in Northern blots (Adam et al., 1988; Mowatt et al., 1991). Antigenic variation occurs in all Giardia lamblia isolates and likely occurs in other Giardia species; Giardia muris possesses vsp genes and its surface reacts varyingly to monoclonal antibodies (Mabs) directed to its surface similar to other VSP reacting Mabs (Ropolo et al., 2005). Switching differs in some aspects between the two major types of Giardia, initially separated into Groups 1, 2, and 3 (Nash et al., 1985) but more commonly referred to as Assemblages A (Groups 1 and 2) and B (Group 3) (Monis et al., 1996). Rates of switching also differ slightly but significantly even in the same isolate. A switch event occurs about once in 12–13 generations in isolate WB (WB) (Smith et al., 1982), the prototypic Assemblage A isolate and significantly faster at about once every 6.5 generations for the Assemblage B prototypic isolate GS (GS) (Nash and Keister, 1985; Nash et al., 1985, 1990a). In culture, which assumes little or no bias in expression, VSP switching occurred about once every 12 generations for two WB VSPs and significantly slower at once every 13 generations for a third WB VSP. Over time, the comparatively small difference in switching rates among VSPs could have biological significance and lead to a decreased representation of the slower switching WB VSP in culture or non-selective host. A slower switching VSP could conceivably result in a staggered or late occurrence of a new VSP, possibly prolonging infection. Two VSPs can be found on a single trophozoite when switching occurs during growth in vitro (Nash et al., 2001). The original VSP is lost with a half-life of about 17 h and is replaced by another VSP. In the WB isolate switching also occurs during encystation/excystation, which was felt to be a way to diversity VSP expression and lead to reinfection within the same or similar hosts (Svard et al., 1998; Carranza et al., 2002). However, GS trophozoites expressing VSPH7 did not switch suggesting that the two major Giardia groups differ not only in switching rates but also in ability to switch while undergoing

Chap. 16 Antigenic Variation in Giardia

encystation/excystation. In WB expressing WB 1267 one report documented that VSP9B10 was additionally expressed during encystation in vitro (Carranza et al., 2002). It is not clear how universal dual VSP express occurs during encystation; this was not mentioned during encystation when VSP TSA417 was expressed during encystation (Svard et al., 1998). Von Allmen et al. (2004) not only confirmed switching associated with encystation/excystation but also observed that inoculation with cysts that did not express VSPH7 resulted in expression of VSPH7 after infection of naïve mice. In contrast, when naive mice were inoculated with non-VSPH7 expressing trophozoites, VSPH7 was not expressed. The authors suggested that cysts had been “reset” and express VSPH7, which did not occur after infection with trophozoites.

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presence, composition, number, size of tandem repeats, which are located close to the signal peptide.

16.4 Genomic Organization Unlike a number of other organisms that undergo antigenic variation, WB vsps are not concentrated near the telomeres and do not undergo gene movement or change of sequence when expressed. VSPs are found in all 5 chromosomes but are most common in chromosome 4(81) followed by chromosome 5(65). About half of the vsps occurs as tandem arrays but a small minority are also found in tail-to-tail or headto-head pairs.

16.3 Basic Description of VSPs

16.5 Spatial Organization, Antigenicity, and Motifs

VSPs are a unique family of an estimated 270 genes in WB (Adam et al., 2010) characterized by the presence of numerous CXXC motifs (Adam et al., 1988), a conserved almost carboxyl terminal membrane anchor of about 38 amino acids (aa) (Gillin et al., 1990; Mowatt et al., 1991) and an absolutely conserved terminal CRGKA cytotoxic tail (Gillin et al., 1990; Mowatt et al., 1991; Davids et al., 2006), which distinguishes VSPs from other CXXC motif containing proteins (Davids et al., 2006). Although almost all VSPs have significant similarity to each other, they are in fact diverse and may differ greatly in size (Adam et al., 2010), presence, number and position of motifs, presence and number of tandem repeats (located close to the amino terminus) and antigenicity (Nash, 2003; Adam et al., 2010). The sequences of the membrane-spanning anchor region are different enough so that most but not all of the time, differences in their sequence can be used to identify individually expressed VSPs (Bienz et al., 2001a; Adam et al., 2010). Analyses of the relatively few VSPs shown to be present on the trophozoite’s surface, indicated most possessed unique Zn finger and GGCY motifs (Nash, 2003). However, review of entire WB vsp gene repertoire revealed these motifs were variably present in putative vsps (Adam et al., 2010). WB vsps differ in size from 222 to 6777 bases as well as the presence, number, and location of motifs, and the

From carboxyl to amino terminus, the amino acid sequence, and structure of VSPs change from well conserved to less conserved (Nash, 2003). The carboxyl terminus ends in an absolutely conserved CRGKA sequence followed by a very similar but not perfectly conserved 38 amino acid membrane-spanning region (Mowatt et al., 1991; Gillin et al., 1990). The positions of the CXXC become less preserved from the carboxyl to the amino terminus as well (Nash, 2003). The N-terminal region, which is most distal from the parasite surface membrane is the most divergent part of the VSP and usually antigenically distinct (Nash et al., 1990b), consistent with the biological purpose of antigenic variation. It is this region that interfaces with the host, reacts with cytotoxic Mabs (Mowatt et al., 1994; Stager et al., 1997; Nash et al., 2001), and is the initial target of antibodies produced to VSPs in infected animals (Müller et al., 1996; Stager et al., 1997, 1998), and experimental human infections. Some VSPs possess tandem repeats (Adam et al., 2010; Chen et al., 1996; Yang and Adam, 1995a). The biological significance of tandem repeats is not known but may consist of 2–30 repeating units and may be immunodominant. One Mab to epitopes formed in a tandem repeat region is cytotoxic to Giardia expressing this repeat at extremely high titers (Nash and Aggarwal, 1986). Their strategic location near the amino terminus suggests an important but

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unclear role. Antibodies to the conserved membranespanning region and tail have not been detected following natural or experimental human (Nash et al., 1990b) or animal infections (Stager et al., 1997; Bienz et al., 2001a). Although VSPs for the most part are antigenically distinct certain VSPs almost certainly share antigenic determinants to varying degrees. These include VSPs with differing number of identical tandem repeats and vsps with significant, identical, or very similar regions in common such as the H7 family of vsps in isolate GS (Nash et al., 1995; Nash and Kulakova, unpublished results). In addition, there is some conservation of sequence around and including an extended GGCY motif in the carboxyl regions of some VSPs that has been shown to invoke antibody responses (Müller et al., 1996; Stager et al., 1997; Bienz et al, 2001b). The Zn finger motif, CxxCHxxCxxC is unique to Giardia and is made up of a combination of LIM and RING Zn fingers, mammalian Zn finger binding motifs (Nash, 1992; Nash and Mowatt, 1993; Zhang et al., 1993). Surrounding proximal and distal cysteine spacing has some conservation as well. No other organism has a Zn finger motif on its surface. It

is likely that the closely related motif, CxxCxxxCxxC that substitutes other amino acids in the histidine position such as glutamate, also functions as a Zn finger as well. Although the role of the Giardia Zn finger is not known, because of its surface location, it more plausibly acts to attach and bind to other proteins, possibly even other VSPs, rather than function as DNA-binding transcription factors. VSPs possessing Zn fingers bind Zn in vitro in standard Zn finger binding assays and have the usual binding affinities for other cations (Nash and Mowatt, 1993). Zn was found in a native VSP by one group (Lujan et al., 1995) but not confirmed by another laboratory (Papanastasiou et al., 1997a). Large amounts of VSP are released into the culture medium and by analogy into the lumen of the intestines (Nash et al., 1993; Papanastasiou et al., 1996). VSPs are commonly protease resistant (Papanastasiou et al., 1997a; Nash et al., 1991) and likely functional in the intestine. Therefore, there is the potential for VSPs and/or Zn motifs to bind Zn or other cations following release into the lumen/mucosa of the intestines. The function of the GGCY motif is unknown. Although carbohydrate-binding sites are present in VSPs, no carbohydrate was found in

Location of CXXC, GGCY, and Zn finger motifs in VSPs CXXC GGCY VSPH7 Zn Finger VSP1267 VSP1269 VSP9B10 VSPA6 VSP4A1 VSP65 VSPJSA-417 VSPA5 VSP136 0

200

400

600

BP from carboxyl terminus

Fig. 16.2 Diagrammatic figure showing the spacing of CXXC, GGCY, and Zinc finger motifs in a small subset of VSPs initially described. Recent data show GGCY and Zinc finger motifs and not always found in VSPs (Nash, 2003)

Chap. 16 Antigenic Variation in Giardia

analyses of a purified native GS VSPH7. However, another group detected carbohydrate in GS VSPH7 and another VSP (Papanastasiou et al., 1997; Hiltpold et al., 2000). VSPs fall into related groups more recently analyzed through sequence analyses (Yang and Adam, 1995a, b; Nash et al., 1995; Ey et al., 1992, 1996; Adam et al., 2010). Structural relatedness of VSPs can be inferred through analysis of the location and spatial arrangement of the numerous CXXC motifs; presence, sequence, and number of tandem repeats; and the presence, number, and position of the GGCY and Zn finger motifs. In the few VSPs analyzed in this manner, the relatedness of VSPs became more evident (Nash, 2003) (Fig. 16.2). This type of analyses may yield additional information compared to sequence alone. The three dimensional structure of VSPs has not been solved and would be particularly difficult because of the numerous cysteine residues that are nonreactive because they are most likely bound to each other. This also explains the relatively frequent presence of conformational epitopes. Reduction of VSPs destroys many of the epitopes recognized by Mabs or sera from infected hosts. Antibodies made to CRGKA do not react with native VSPs but recognize reduced VSPs suggesting (Lujan et al., 1995) the epitope is sequestered, modified, or bound. At the present time there is no direct evidence indicating VSPs are covalently bound to each other. VSPs are post-translationally modified. Carbohydrates were detected by one group (Papanastasiou et al., 1997b; Hiltpold et al., 2000). However, the cysteine of the cystosolic five amino acid tail is palmitoylated (Papanastasiou et al., 1997; Hiltpold et al., 2000; Touz et al., 2005) and the arginine in the tail modified to citrulline (Touz et al., 2008). Both these modifications alter the cytotoxic effects of monoclonal antibodies. The biological purpose of these modifications is unclear. VSPs are rapidly excreted into the culture medium in vitro as a truncated form (Papanastasiou et al., 1996; Svard et al., 1998; Touz et al., 2005). Current evidence suggests that they are cleaved just proximal to membranous anchor region, releasing the soluble portion without the palmitate into the surrounding medium (Papanastasiou et al., 1996). The fate of the membranous portion has not been determined.

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16.6 Differences Among VSPs As noted earlier there are two major types of Giardia that infect humans; these were initially called Groups 1 and 2, and 3 and later renamed and more popularly known as genotypes or assemblages A or B. These types of Giardia differ in a number of fundamental ways and are most appropriately considered separate species (Nash et al., 1985; Nash, 1992; Franzen et al., 2009; Adam R, Nash TE, and Porcello SF, In preparation). A remarkable difference is absolute or relative restriction of VSPs to their own assemblage. Although a relatively small number of vsps have been studied, assemblages A and B as well as some subgroups appear to have a different repertoire and limited antigenic cross reactivity of vsps genes (Nash et al., 1990b; Nash, 1992). VSPs of WB fall into three related groups, which segregate from the VSPs of the GS isolate (Assemblage B) (Bienz et al., 2001a; Adam et al., 2010). Although VSPs share common features and characteristics, these can vary such as presence and nature of tandem repeats and motifs, location and number of the common and unique motifs, variation of CXXC motifs that result in differences in sequence, structure and antigenicity. These variable features in turn give rise to unique physical chemical attributes such as varying levels of VSP resistance to high concentrations of trypsin or chymotrypsin (Nash et al., 1991) that allow trophozoites to survive in the small intestines. Similar to other parasites that undergo antigenic variation such as Plasmodium falciparum malaria whose variant surface proteins adhere to specific endothelial receptors, it is likely that VSPs also possess different, but not well-defined biological characteristics as well (see below). Both growth inhibiting or cytotoxic immune responses by the host and unique biological characteristics of VSPs contribute different but intersecting roles to establish and maintain infections and contribute to the pathophysiology of disease.

16.7 Immune Responses to VSP and Immune Selection Almost all the information about immunity to Giardia originates from animal model infections. Most of the studies used G. muris infections in mice and

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G. lamblia infections in neonatal mice, adult mice, or gerbils. Almost all studies of VSP immunity employ G. lamblia since it is the only Giardia that undergoes antigenic variation and can also be cultured in vitro enabling control and analysis of VSP expression. Most studies employed clones or cultures of the GS isolate, the prototypic assemblage B isolate but also some used the WB isolate, the prototypic assemblage A organism. Short-term (21 day) experimental infections in humans using similar if not identical cultures/ clones of isolate of GS used in animals, allow some degree of comparison to animal model infections. Because animal infections differ substantially compared to the limited information known about human infections, their relevance to human infections is unclear. Prior to the description of antigenic variation there was little indication that antigenic variation occurred in human or animal infections. At best, the known propensity of some Giardia infections to become chronic was perhaps suggestive, but even so, most human infections self-cured as shown by the classic human experiments of Rendtroff (1954) Additionally, the course of Giardia animal model infections were no more indicative. The expected course of infection, based on the African trypanosome paradigm, is to observe successive waves of a single predominate surface antigen type brought about by immune elimination or growth inhibition of the earlier expressed varying antigen. When immune responses develop to the expressed antigenic type, it is eliminated and replaced by another predominant variant surface protein antigenically distinct from previously experienced VSPs and unrecognized by the immune system of the host. None of the model Giardia infections are ideal to study antigenic variation and none with the possible exception of the neonatal mouse model hinted at changing numbers of single VSP expressing parasites (Stager et al., 1998). Antigenic variation has only been studied in one series of experimental human infections up to day 21 (Nash et al., 1990c). The initial VSP expressed changed and diversified after day 14, when humoral responses to VSPs were first noted. By day 21 no intestinal parasites expressing the original expressed VSP were detected in aspirates of the small intestine. Most Giardia isolates can infect neonatal mice but become resistant to all but the GS isolate infections after weaning. There are a number of variables

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that complicate the interpretation of neonatal mice infections including maturation of the neonatal mice’s immune state, concomitant maternal cross infection, infection and ingestion by sucking mice of biologically active anti-VSPH7 or possibly other anti-VSP antibodies. Adult mouse infection with the GS isolate is unique and possibly not representative because it is the only isolate of those from North America that consistently infects adult mice (Byrd et al., 1994). It also requires an inoculum consisting of a large number of trophozoites that produces an easily detectable, reproducible infection that lasts about 21 days. Gerbils accept most Giardia isolates (Belosevic et al., 1983) and undergoes relatively long-term infections depending on the infecting isolate (Aggarwal and Nash, 1987), develop signs of infection and develop immunity. The major disadvantage is that most gerbils are not pathogen free, reagents not available or optimal and genetics knockouts non-existent. Adult mice infected with GS VSPH7 develop a peak infection just before humoral responses are detected, usually around 7–14 days and apparent self-cure around day 21 (Byrd et al., 1994). Neonatal mice switch after day 7 post infection and self cure by day 22 (Gottstein and Nash, 1991; Müller and Gottstein 1998). Interestingly, in “self-cured” adult mice trophozoites cannot be detected by usual means, but small numbers can be detected in most mice only by culture of the intestinal contents (Byrd et al., 1994). Gerbils as evidenced by recrudescence of “self-cured” animals after administration of corticosteroids also develop subliminal infection (Lewis et al., 1987). Together these results indicate the development of barely detectable chronic infections in these two models and it is plausible that antigenic variation plays a role in maintenance of chronic infection in these models. This is supported by experiments by Bienz et al. (2001a) and Stager et al.(1998) and other papers from the same laboratory (Müller and Gottstein, 1998) that showed considered VSP diversity in mice after antibodies appeared to the initial VSPH7 and even on day 42 when the trophozoites are present in low numbers. Although the pattern of infection is reminiscent of changes or waves of different VSP-expressing organisms over time, the presence of low or barely detectable trophozoites suggests other factors may be controlling infections as well. Whether subliminal infections are common in humans has not been shown but low-level infections,

Chap. 16 Antigenic Variation in Giardia

undetectable by stool examination and even antigen assays (Nash et al., 1987) are well documented in human giardiasis. Humoral immune responses are the most important adaptive immune response controlling antigenic variation in humans and animal model infections. Mabs or polyclonal antibodies to VSPs produced by immunization or through natural infection cause complement-independent cytotoxicity and/or growth inhibition depending on the concentration and type of antibody (Nash and Aggarwal 1986; Aggarwal and Nash, 1987; Nash et al., 1988; Stagger et al., 1997, 1998). It is therefore not surprising that switching in vivo is commonly associated with the detection of antibodies to the expressed VSP. Humans infected with GS expressing VSPH7 start to express other VSPs on day 14 when antibodies are first detected, continue to diversity over the next week and are completely replaced with non-VSPH7 expressing trophozoites by day 21 (Nash et al., 1988). Similar findings occur in neonatal mice (Gottstein and Nash 1991; Stagger et al., 1998), adult mice (Singer et al., 2001), and gerbils (Aggarwal and Nash, 1987) after infection with the same Giardia. Interestingly, in the later study, cross-reactive antibodies to either VSPs or other common surface antigens occur between isolate WB and GS. More recent experiments suggest crossprotecting humoral responses are due to common epitopes between isolates WB and GS (Rivero et al., 2010). A purported mechanism appears to be membrane loss and damage (Hemphill et al., 1996). However, because mutations in the CRGKA tail prevent post-translational modifications and result in loss of antibody-mediated cytotoxicity, another possibility is that cytotoxicity is mediated by transduction activity mediated through the only absolutely conserved motif in VSPs (Touz et al., 2005, 2008). Neonatal nude mice and adult TCRE knockout mice that lack T cell responses, develop high numbers of parasites in the small intestine are unable to undergo self-cure (Gottstein and Nash, 1991; Singer and Nash, 2000). Nevertheless they are able to make antibodies to VSPs and undergo antigenic variation. In neonatal nude mice, ability to self-cure is reestablished after transfer of immune Peyer’s Patch cells (Gottstein et al., 1993). As anticipated, SCID mice have even greater number of trophozoites in the intestines, are unable to undergo self-cure, cannot produce

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antibodies and fail to undergo VSP switching when inoculated with VSPH7 expressing trophozoites (Gottstein et al., 1993; Singer et al., 2001). The results of experiments assessing the requirement of IgA and/or other antibodies for control of infection appear contradictory. One group using the GS VSPH7 infection model in neonatal mice showed that IgA and B cell-deficient mice fail to undergo VSP switching normally but eventually change surface VSP expression late in the course of infection (Stager and Müller, 1997). They also do not resolve the infection. Langford et al. (2000) using G. muris and GS infection in adult mice found that B cells and specifically IgA were essential for control of infection. In contrast Singer and Nash (2000) using the same GS VSPH7 expressing isolate or G. muris infection showed that cure was T cell dependent and not B cell dependent in the adult mice. So, in the neonatal mouse model and the adult mouse model described by Langford et al., humoral antibodies assumed to be anti-VSP responses, were mostly required for control of infection while in the adult mouse described by Singer and Nash (2000), antibodies were not required. Although there are a number of methodological differences in the models, in general the differences in results are not easily reconciled. The timing and response to common epitopes may be important in control of infection (Aggarwal and Nash, 1987). The earliest responses in human and animals are to specific epitopes located to the amino terminal region (Nash et al., 1988; Stager et al., 1997). These responses result in cytotoxic and inhibitory activities that allow repopulation of trophozoites expressing VSP and epitopes unrecognized by the host. Certain VSPs are highly related and it is likely that immune responses to one may be inhibitory to other members. Responses to more internal VSP epitopes, which have some degree of homology among subsets of VSPs also occur (Müller et al., 1996; Bienz et al., 2001b) and result in some growth inhibition to the homologous isolates. Responses to the absolutely conserved tail have not been found in human or animal infections (Müller et al., 1996; Nash TE, unpublished data). Since there is good evidence that the tail resides internally, if humoral responses were produced, they would not be expected to have cytotoxic activity. T cell responses to VSPs have been detected to VSP/H7 infections once in neonatal mice (Gottstein

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et al., 1990) and another time in humans (Gottstein et al., 1991). These are limited studies; to date there is no direct evidence implicating these responses in the control of antigenic variation but may provide T cell help for antibody production. Recent novel experiments by Rivero et al. (2010) more directly implicate immune responses to VSPs are important for protection. Gerbils infected with WB isolate trophozoites manipulated to express many VSPs simultaneously by RNAi inhibition of either Dicer or RNA-dependent RNA polymerase were protected from challenge. Similar protection was obtained following immunization with purified VSPs obtained from the above-mentioned pan VSP expressing trophozoites. Protection was observed from Giardia expressing many VSPs, Giardia expressing specific VSPs, and cysts obtained from infected humans, and partially from GS infections. These results indicate VSP immunity is protective after the host is exposed to many if not all VSPs of WB and there is some cross protection to heterologous assemblage B Giardia. Humoral responses were felt to be protective since gerbils developed strong humoral responses; however, the immune mechanisms involved in protection in this model were not vigorously studied. Similar results could possibly result with infections with Giardia trophozoites held in culture for extended periods of time. These cultures typically express many different VSPs, perhaps most or all of their VSP repertoire. Gerbils infections with either isolate WB or GS show a high degree of protection to homologous isolate challenge and a decreased but still significant level of protection to heterologous challenge (Aggarwal and Nash, 1987). The nature of the cross protection was not known at the time but the above experiment implicate cross-reacting VSPs. Some degree of cross protection to GS was found after gerbils were immunized with purified WB VSPs (Rivero et al., 2010) suggesting that cross protection in gerbils is due to common VSP epitopes.

16.8 Biological Selection The importance of biological selection is well described and accepted in a number of organisms that undergo surface antigenic variation. Perhaps the best

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example is the var proteins of Plasmodia falciparum malaria that mediate binding of schizonts to endothelial cells and induce strong humoral immune and growth inhibitory responses. In Giardia biological selection is an important determinant of VSP selection both in vitro and in vivo and appears to act in concert with immune selection. Although commonly overlooked perhaps because the biological activity and purpose of VSPs are not well defined, biological selection in giardiasis clearly occurs in animal and human infections. The first hint that individual VSPs were physically different was suggested after trophozoites were exposed to high concentrations of trypsin and chymotrypsin in vitro (Nash et al., 1991). Some clones expressing specific VSPs were killed after exposure to high concentrations of trypsin or chymoptrysin, while others were resistant. In susceptible clones almost the entire culture was killed except for a few viable organisms that expressed other VSPs, which were resistant to these proteases and consequently were able to regrow and repopulate the culture. The phenomenon is reminiscent what is observed after the addition of cytotoxic Mabs to a culture expressing a recognized VSP. Biological selection also occurs in vivo and can be seen in two settings. First, changes in expression of VSPs are noted before adaptive immune responses appear and secondly, in animals such as in SCID mice who lack an adaptive immune system. Human volunteers were experimentally inoculated with isolate GS initially expressing many different VSPs but with VSPH7 measured at 0.11–1.2 % and VSP3F6 at 1.0–2.6%. After 1–2 weeks VSPH7 expression increased to between 40.5% and 91.3% in 5/6 experimentally infected human volunteers while a single person expressed a relatively unchanged 2.8% VSPH7. In contrast expression of VSP3F6 became almost non-detectable with only 1/6 volunteers expressing any VSP3F6 at 0.2% (Nash et al., 1988). Therefore, before humoral antibodies could be produced, expression of VSPH7 was “favored” and increased in contrast to expression of VSP3F6. A similar preference for VSPH7 occurs in neonatal mice inoculated with populations expressing between 85 and 95% VSPH7. Expression of VSPH7 increases to 100%, a level never achieved during cultivation in vitro (Gottstein and Nash, 1991). Infections in adult SCID more directly demonstrate biological selection.

Chap. 16 Antigenic Variation in Giardia

Changes in VSP expression cannot be attributed to adaptive immune responses since they are absent in these mice. A panel of Giardia clones derived from GS, each expressing a different VSP, was individually used to infect SCID mice (Singer et al., 2001). The expression of each VSP was analyzed using Mabs specific to each clone over the next 16 days. While VSPH7 and 3 other VSPs were preferred, highly expressed and maintained in SCID mice, 2 VSPs were not and replaced by other VSPs. In contrast, gerbils made immunoincompetent by irradiation and infected with the same GS clones, showed a different pattern of VSP expression compared to SCID mice. Therefore, it appears that a portion of all VSPs are preferred or maintained in specific hosts or under specific host conditions. Of the “acceptable” VSPs only those previously unrecognized by the adaptive immune system are able to grow and populate the host intestine. If this is true, VSPs that are expressed in a particular host will be reduced and limited compared to its repertoire because only a subset of VSPs will be immunologically naïve to the host and at the same time compatible with the intestinal environment of a specific host.

16.9 VSP Secretion VSPs are constitutively expressed and secreted during growth in culture. They have a signal peptide directing them to the ER and then transported to the surface by an unknown route, which is inhibited by Brefeldin A. Electron microscopy studies visualizes a dense coat of surface VSP (Pimenta et al., 1991; Svard et al., 1998). Most surface labeled VSPs are released into the culture medium by an unknown mechanism (Nash et al., 1983; Papanastasiou et al., 1996) but most likely VSPs are cleaved then secreted as a soluble peptide leaving the hydrophobic carboxyl portion in the membrane (Papanastasiou et al., 1996; Touz et al., 2005). Since VSPs have been detected in peripheral vesicles (Svard et al., 1998) and are internalized during encystation, it is possible that VSPs are internalized, cleaved in the peripheral vesicles and then secreted. Conservation of the secretory mechanisms between Giardia and mammals is suggested because VSPs with their own signal peptides are transported correctly to the surface of COS cells (Nash TE, unpublished data). However, release from

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the surface was not detected and VSPs could not be surface radiolabeled (Nash TE, unpublished data), unlike VSPs on the surface of trophozoites (Nash et al., 1983). Marti et al. (2003) analyzed the requirement for VSP surface secretion using a number chimera protein vectors consisting of an inducible cyst wall promoter, a heterologous Toxoplasmosis surface protein and various lengths of carboxyl terminal VSP. He convincingly showed that the terminal conserved CRGKA was required for secretion to the surface. Touz et al. (2005) as part of other studies employed a different VSP construct that consisted of the strong D tubulin constitutive promoter and the entire VSPH7 gene excluding the terminal CRGKA. VSPH7 was correctly placed on the surface that suggested the tail was not necessary for routing. The later studies have been criticized as being incomplete and suggested use of the almost full length VSP lacking the tail permitted dimerization of mutant VSPs with a full length endogenously secreted VSPs possessing an intact CRGKA allowing the mutant VSP to be carried to the surface. There are obvious differences between the two systems including use and type of promoter, strength of the promoters, presence of almost full length VSP in the later system compared to use of a heterologous chimera protein in the former. Although dimerization is a possible hypothesis, there is no direct proof this occurs and further experiments are needed to understand the disparate results. Mab antibodies and polyclonal antibodies to VSPs are rapidly cytotoxic to trophozoites by a complement-independent mechanism (Nash and Aggarwal, 1986). Trophozoites exposed to Mab were subsequently unable to multiple after transfer to antibody free culture medium. This may simply be due to mechanical entanglement of the flagella that inhibits movement and adherence, or it may be due to perturbation and destruction of the membrane as suggested by one study (Hemphill et al., 1996). The absolute conservation of the CRGKA tail and the rapidity of the process are also consistent with participation of other mechanisms such as signal transduction mediated by the conserved tail. Post-translational modifications of the CRGKA tail occur normally and seem to be essential for antibody-mediated cytotoxicity. VSPs are palmitoylated by an acyl palmitoyl transferase at the conserved cysteine in the tail that changes the membrane localization of VSPs (Touz et al., 2005).

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Mutation of the cysteine to alanine prevents palmitoylation, obviates cytotoxicity and inhibits immune elimination of the organisms expressing the mutated VSP (Touz et al., 2005). In addition to palmitolylation, the tail is also modified by citrullination of the conserved arginine by a unique arginine deiminase. Similarly, prevention of citrullination by replacement of the arginine in the tail also decreases cytotoxicity (Touz et al., 2005). These modifications suggest that in addition to its suggested role in transport, posttranslational modifications are essential for immune elimination. Exactly how this occurs is unclear. The biological consequence of cytotoxic antibodies is the immediate elimination of expressed VSPs recognized by the host, and regrowth and replacement by expressed VSPs that are not recognized by the host.

16.10 Control of Antigenic Variation The mechanisms involved in the control of antigenic variation must elucidate the cardinal cellular and molecular features and findings characteristic of antigenic variation. Namely, in culture each trophozoite expresses one VSP at a time (except at the time of switching) and its transcript is predominate. Switching not only occurs normally in culture in the absence of additional obvious external stimuli but also occurs during encystation/excystation in the certain isolates. In addition there is no obvious vsp gene movement or associated gene rearrangements. The process must explain how identical vsps with virtually identical coding, 3c and 5c untranslated regions are each controlled uniquely so that only one of the two identical vsps is expressed. And, how both transfected constitutively expressed vsps and homologous normally present vsps can be expressed at the same time. Since both nuclei are similar and appear equally active and functional, a mechanism almost certainly exists to coordinate and control of vsp expression between both nuclei. Heterologous or tagged homologous VSPs that are commonly expressed from episomes or less commonly from DNA inserted into the genome offer clues in the control of antigenic variation. In the most common scenario VSPH7 (a GS VSP not present in WB) is expressed in isolate WB (Elmendorf et al., 2001). When the homologous 5c upstream sequence is used

T. E. Nash

in the construct, VSP expression is detected initially but the translation start sites are almost all abnormal. Furthermore, over time the transfected VSP is no longer expressed and only rarely detected on the surface of the trophozoite. On the other hand, if the upstream sequence is changed to a constitutive promoter, commonly D tubulin, the vsp is constitutively expressed and found on the surface of the parasite (Elmendorf et al., 2001; Kulakova et al., 2006). Analysis of these transfectants showed that 2 VSPs are expressed on the trophozoite surface (Kulakova et al., 2006; Prucca et al., 2008), the VSPs normally expressed by the organism and the constitutively expressed vsp. Presumably antigenic variation continues. There are two implications. First, the upstream region of vsps is required and appears to control transcription since its removal and replacement with an active promoter leads to transcription. Since the upstream regions of some vsps are virtually identical and if this region is critical and important in the control of expression, it is unclear how specific control of vsp expression could be achieved. Secondly, two VSPs can both be expressed in a single trophozoite. Therefore, the findings suggest that transcription of one vsp gene does not lead to total inhibition of all others (see below). The only difference from the natural state is the 5c coding region of the transfected vsp is replaced with the D tubulin promoter. VSPs integrated into its own genome with its homologous surrounding non-coding DNA are expressed intermittently as usual but constitutively expressed when the upstream region is replaced with the D tubulin promoter (Nash, unpublished result). Also, a well-conserved 15 nt extended polyadenylation signal (5c-ACTYAGRTAGTRAAY-3c) follows the coding region of vsps that could play a role in post-transcription control. The recent publications of Prucca et al. (Prucca et al., 2008) as well as other studies (Saraiya and Wang, 2008) implicate RNAi in post-transcriptional regulation of vsp transcripts. A more detailed review is found elsewhere in this book. RNA-dependent RNA polymerase, DICER and Argonaute, all essential members of RNAi machinery are present and function in Giardia. The scenario suggested and supported by the authors’ experiments is that all vsps are initially transcribed and every vsp except one is then degraded. The contention is that RdRP synthesizes double-stranded vsp RNA from every vsp mRNA

Chap. 16 Antigenic Variation in Giardia

except one, perhaps the vsp mRNA with the highest transcript concentration. DICER then processes these into siRNA, which combines with Argonaute degrading all but one intact single stranded mRNA vsp that is expressed. This scenario is supported by experiments demonstrating transcription of many vsps by run on assays, presence of siRNA to nonexpressed vsps, and expression of many VSPs on a single trophozoites following knockdown of DICER and to a lesser extent following knockdown with RdRP. This mechanism is consistent with an epigenetic character of antigenic variation and could explain how an organism with two transcriptionally active nuclei can coordinate expression. However, the details are fuzzy. For instance, how does transcription of all vsps occur and how is it controlled? If selection is concentration dependent, what controls vsp transcript abundance? How switching occurs is still not known and the concentration-based trigger for RNAi is at best suggestive. The mechanisms cannot explain how only one of two identical vsps with the same coding, 3c and 5c regions are expressed and controlled differentially (Yang et al., 1994; Kulakova et al., 2006). A positional or random effect would need to be operational. The authors also compared the level of expression of the usual or homologous vsps in trophozoites transfected with another or heterologous vsp or a non-vsp gene. There was a decrease in expression of the original vsp in the heterologous vsp transfected organism compared to non-vsp transfectant. The predicted result would be little or no expression of one of them. Our understanding of the various aspects of antigenic variation in Giardia has made significant strides since its initial description by us about 23 years ago. However, there is much to learn. We do not know the natural course of infection in humans or its role of VSPs and antigenic variation in disease pathogenesis. Responses to VSPs may be modulated and result in tolerance to disease that is commonly seen in heavily endemic populations. There is little doubt that VSPs are important in the development of immunity and control of infection at least in animal models, but they almost certainly have biological function. Antigenic variation occurs in individual organisms but for the most part is studied as populations. Study of single organisms would likely yield a clearer idea of contributing mechanisms and its consequence.

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References Adam RD, Aggarwal A, Lal AA, Delacruz VF, Mccutchan T, and Nash TE (1988) Antigenic variation of a cysteine-rich protein in Giardia-lamblia. J Exp Med 167: 109–118 Adam RD, Yang YM, and Nash TE (1992) The cysteine-rich protein gene family of Giardia lamblia: loss of the CRP170 gene in an antigenic variant. Mol Cell Biol 12: 1194–1201 Adam RD, Nigam A, Seshadri V, Martens CA, Farneth GA, Morrison HG, Nash TE, Porcella SF, and Patel R (2010) The Giardia lamblia vsp gene repertoire: characteristics, genomic organization, and evolution. BMC Genomics: in press Aggarwal A and Nash TE (1987) Comparison of two antigenically distinct Giardia lamblia isolates in gerbils. Am J Trop Med Hyg 36: 325–332 Belosevic M, Faubert GM, Maclean JD, Law C, and Croll NA (1983) Giardia-lamblia infections in Mongolian gerbils – an animal-model. J Infect Dis 147: 222–226 Bienz M, Siles-Lucas M, Wittwer P, and Müller N (2001a) vsp gene expression by Giardia lamblia clone GS/M-83-H7 during antigenic variation in vivo and in vitro. Infect Immun 69: 5278–5285 Bienz M, Wittwer P, Zimmermann V, and Müller N (2001b) Molecular characterisation of a predominant antigenic region of Giardia lamblia variant surface protein H7. Int J Parasitol 31: 827–832 Bruderer T, Papanastasiou P, Castro R, and Kohler P (1993) Variant cysteine-rich surface-proteins of Giardia isolates from human and animal sources. Infect Immun 61: 2937– 2944 Byrd LG, Conrad JT, and Nash TE (1994) Giardia-lamblia infections in adult mice. Infect Immun 62: 3583–3585 Carranza PG, Feltes G, Ropolo A, Quintana SM, Touz MC, and Lujan HD (2002) Simultaneous expression of different variant-specific surface proteins in single Giardia lamblia trophozoites during encystation. Infect Immun 70: 5265– 5268 Chen N, Upcroft JA, and Upcroft P (1996) A new cysteine-rich protein-encoding gene family in Giardia duodenalis. Gene 169: 33–38 Davids BJ, Reiner DS, Birkeland SR, et al. (2006) A new family of giardial cysteine-rich non-VSP protein genes and a novel cyst protein. Plos One 1: e44 Elmendorf HG, Singer SM, Pierce J, Cowan J, and Nash TE (2001) Initiator and upstream elements in the alpha 2-tubulin promoter of Giardia lamblia. Mol Biochem Parasitol 113: 157–169 Ey PL, Khanna K, Andrews RH, Manning PA, and Mayrhofer G (1992) Distinct genetic groups of Giardia-intestinalis distinguished by restriction fragment length polymorphisms. J Gen Microbiol 138: 2629–2637 Ey PL, Bruderer T, Wehrli C, and Kohler P (1996) Comparison of genetic groups determined by molecular and immunological analyses of Giardia isolated from animals and humans in Switzerland and Australia. Parasitol Res 82: 52–60 Franzen O, Jerlstrom-Hultqvist J, Castro E, et al. (2009) Draft Genome sequencing of Giardia intestinalis assemblage B isolate GS: is human giardiasis caused by two different species? PLoS Pathog 5: e1000560

256 Gillin FD, Hagblom P, Harwood J, et al. (1990) Isolation and expression of the gene for a major surface protein of Giardia lamblia. Proc Natl Acad Sci USA 87: 4463–4467 Gottstein B and Nash TE (1991) Antigenic variation in Giardia-lamblia – infection of congenitally athymic nude and scid mice. Parasite Immunol 13: 649–659 Gottstein B, Harriman GR, Conrad JT, and Nash TE (1990) Antigenic variation in Giardia-lamblia – cellular and humoral immune-response in a mouse model. Parasite Immunol 12: 659–673 Gottstein B, Stocks NI, Shearer GM, and Nash TE (1991) Human cellular immune-response to Giardia-lamblia. Infection 19: 421–426 Gottstein B, Deplazes P, and Tanner I (1993) In vitro synthesized immunoglobulin A from nu/+ and reconstituted nu/ nu mice against a dominant surface antigen of Giardia lamblia. Parasitol Res 79: 644–648 Hemphill A, Stager S, Gottstein B, and Müller N (1996) Electron microscopical investigation of surface alterations on Giardia lamblia trophozoites after exposure to a cytotoxic monoclonal antibody. Parasitol Res 82: 206–210 Hiltpold A, Frey M, Hulsmeier A, and Kohler P (2000) Glycosylation and palmitoylation are common modifications of Giardia variant surface proteins. Mol Biochem Parasitol 109: 61–65 Kulakova L, Singer SM, Conrad J, and Nash TE (2006) Epigenetic mechanisms are involved in the control of Giardia lamblia antigenic variation. Mol Microbiol 61: 1533–1542 Langford TD, Housley MP, Boes M (2000) Central importance of immunoglobulin A in host defense against Gardia spp. Infect Immun 70(1): 11–18 Lewis PD, Belosevic M, Faubert GM, Curthoys L, and Maclean JD (1987) Cortisone-induced recrudescence of Giardia-lamblia infections in gerbils. Am J Trop Med Hyg 36: 33–40 Lujan HD, Mowatt MR, Wu JJ, et al. (1995) Purification of a variant-specific surface protein of Giardia-lamblia and characterization of its metal-binding properties. J Biol Chem 270: 13807–13813 Marti M, Regos A, Li Y, et al. (2003) An ancestral secretory apparatus in the protozoan parasite Giardia intestinalis. J Biol Chem 278: 24837–24848 Monis PT, Mayrhofer G, Andrews RH, Homan WL, Limper L, and Ey PL (1996) Molecular genetic analysis of Giardia intestinalis isolates at the glutamate dehydrogenase locus. Parasitology 112 (Pt 1): 1–12 Mowatt MR, Aggarwal A, and Nash TE (1991) Carboxy-terminal sequence conservation among variant-specific surfaceproteins of Giardia-lamblia. Mol Biochem Parasitol 49: 215–228 Mowatt MR, Nguyen BYT, Conrad JT, Adam RD, and Nash TE (1994) Size heterogeneity among antigenically related Giardia-lamblia variant-specific surface-proteins is due to differences in tandem repeat copy number. Infect Immun 62: 1213–1218 Müller N and Gottstein B (1998) Antigenic variation and the murine immune response to Giardia lamblia. Int J Parasitol 28: 1829–1839

T. E. Nash Müller N, Stager S, and Gottstein B (1996) Serological analysis of antigenic heterogeneity of Giardia lamblia variant surface proteins. Infect Immun 64: 1385–1390 Nash T (1992) Surface-antigen variability and variation in Giardia-lamblia. Parasitol Today 8: 229–234 Nash TE (2003) Surface antigenic variation in Giardia lamblia. In: Antigenc variation (A. Craig and A. Schert, eds). London, Academic Press, pp 357–374 Nash TE and Aggarwal A (1986) Cytotoxicity of monoclonalantibodies to a subset of Giardia isolates. J Immunol 136: 2628–2632 Nash TE and Keister DB (1985) Differences in excretory-secretory products and surface-antigens among 19 isolates of Giardia. J Infect Dis 152: 1166–1171 Nash TE and Mowatt MR (1993) Variant-specific surfaceproteins of Giardia-lamblia are zinc-binding proteins. Proc Natl Acad Sci USA 90: 5489–5493 Nash TE, Aggarwal A, Adam RD, Conrad JT, and Merritt JW (1988) Antigenic variation in Giardia-lamblia. J Immunol 141: 636–641 Nash TE, Gillin FD, and Smith PD (1983) Excretory-secretory products of Giardia-lamblia. J Immunol 131: 2004–2010 Nash TE, Mccutchan T, Keister D, Dame JB, Conrad JD, and Gillin FD (1985) restriction-endonuclease analysis of DNA from 15 Giardia isolates obtained from humans and animals. J Infect Dis 152: 64–73 Nash TE, Herrington DA, and Levine MM (1987) Usefulness of an Enzyme-linked-immunosorbent-assay for detection of Giardia antigen in feces. J Clin Microbiol 25: 1169–1171 Nash TE, Conrad JT, and Merritt JW (1990b) Variant specific epitopes of Giardia-lamblia. Mol Biochem Parasitol 42: 125–132 Nash TE, Banks SM, Alling DW, Merritt JW, and Conrad JT (1990a) Frequency of variant antigens in Giardia-lamblia. Exp Parasitol 71: 415–421 Nash TE, Herrington DA, Levine MM, Conrad JT, and Merritt JW (1990c) Antigenic variation of Giardia-lamblia in experimental human infections. J Immunol 144: 4362–4369 Nash TE, Merritt JW, and Conrad JT (1991) Isolate and epitope variability in susceptibility of Giardia-lamblia to intestinal proteases. Infect Immun 59: 1334–1340 Nash TE, Conrad JT, and Mowatt MR (1995) Giardia-lamblia – identification and characterization of a variant-specific surface protein gene family. J Eukaryot Microbiol 42: 604–609 Nash TE, Lujan HT, Mowatt MR, and Conrad JT (2001) Variant-specific surface protein switching in Giardia lamblia. Infect Immun 69: 1922–1923 Papanastasiou P, Hiltpold A, Bommeli C, and Kohler P (1996) The release of the variant surface protein of Giardia to its soluble isoform is mediated by the selective cleavage of the conserved carboxy-terminal domain. Biochemistry 35: 10143–10148 Papanastasiou P, Bruderer T, Li Y, Bommeli C, and Kohler P (1997) Primary structure and biochemical properties of a variant-specific surface protein of Giardia. Mol Biochem Parasitol 86: 13–27 Papanastasiou P, McConville MJ, Ralton J, and Kohler P (1997b) The variant-specific surface protein of Giardia, VSP4A1, is a glycosylated and palmitoylated protein. Biochem J 322: 49–56

Chap. 16 Antigenic Variation in Giardia Pimenta PFP, Dasilva PP, and Nash T (1991) Variant surfaceantigens of Giardia-lamblia are associated with the presence of a thick cell coat – thin-section and label fracture immunocytochemistry survey. Infect Immun 59: 3989–3996 Prucca CG, Slavin I, Quiroga R, et al. (2008) Antigenic variation in Giardia lamblia is regulated by RNA interference. Nature 456: 750–754 Rendtorff RC (1954) The experimental transmission of human intestinal protozoan parasites. II Giardia lamblia cysts given in capsules. Am J Hyg 59: 209–220 Rivero FD, Saura A, Prucca CG, Carranza PG, Torri A, and Lujan HD (2010) Disruption of antigenic variation is crucial for effective parasite vaccine. Nat Med 16: 551–557, 551p following 557 Ropolo AS, Saura A, Carranza PG, and Lujan HD (2005) Identification of variant-specific surface proteins in Giardia muris trophozoites. Infect Immun 73: 5208–5211 Saraiya AA and Wang CC (2008) snoRNA, a novel precursor of microRNA in Giardia lamblia. PLoS Pathog 4: e1000224 Singer SM and Nash TE (2000) T-cell-dependent control of acute Giardia lamblia infections in mice. Infect Immun 68: 170–175 Singer SM, Elmendorf HG, Conrad JT, and Nash TE (2001) Biological selection of variant-specific surface proteins in Giardia lamblia. J Infect Dis 183: 119–124 Smith PD, Gillin FD, Spira WM, and Nash TE (1982) Chronic giardiasis: studies on drug sensitivity, toxin production, and host immune response. Gastroenterology 83: 797–803 Stager S and Müller N (1997) Giardia lamblia infections in B-cell-deficient transgenic mice. Infect Immun 65: 3944– 3946 Stager S, Felleisen R, Gottstein B, and Müller N (1997b) Giardia lamblia variant surface protein H7 stimulates a heterogeneous repertoire of antibodies displaying differential cytological effects on the parasite. Mol Biochem Parasitol 85: 113–124 Stager S, Gottstein B, and Müller N (1997a) Systemic and local antibody response in mice induced by a recombinant pep-

257 tide fragment from Giardia lamblia variant surface protein (VSP) H7 produced by a Salmonella typhimurium vaccine strain. Int J Parasitol 27: 965–971 Stager S, Gottstein B, Sager H, Jungi TW, and Müller N (1998) Influence of antibodies in mother’s milk on antigenic variation of Giardia lamblia in the murine mother-offspring model of infection. Infect Immun 66: 1287–1292 Svard SG, Meng TC, Hetsko ML, McCaffery JM, and Gillin FD (1998) Differentiation-associated surface antigen variation in the ancient eukaryote Giardia lamblia. Mol Microbiol 30: 979–989 Touz MC, Conrad JT, and Nash TE (2005) A novel palmitoyl acyl transferase controls surface protein palmitoylation and cytotoxicity in Giardia lamblia. Mol Microbiol 58: 999– 1011 Touz MC, Ropolo AS, Rivero MR, et al. (2008) Arginine deiminase has multiple regulatory roles in the biology of Giardia lamblia. J Cell Sci 121: 2930–2938 von Allmen N, Bienz M, Hemphill A, and Müller N (2004) Experimental infections of neonatal mice with cysts of Giardia lamblia clone GS/M-83-H7 are associated with an antigenic reset of the parasite. Infect Immun 72: 4763–4771 Yang YM and Adam RD (1995a) Analysis of a repeat-containing family of Giardia lamblia variant-specific surface protein genes: diversity through gene duplication and divergence. J Eukaryot Microbiol 42: 439–444 Yang YM and Adam RD (1995b) A group of Giardia lamblia variant-specific surface protein (VSP) genes with nearly identical 5´ regions. Mol Biochem Parasitol 75: 69–74 Yang YM, Ortega Y, Sterling C, and Adam RD (1994) Giardia lamblia trophozoites contain multiple alleles of a variantspecific surface protein gene with 105-base pair tandem repeats. Mol Biochem Parasitol 68: 267–276 Zhang YY, Aley SB, Stanley SL, and Gillin FD (1993) Cysteine-dependent zinc-binding by membrane-proteins of Giardia-lamblia. Infect Immun 61: 520–524

Section IV Pathology, Treatment and Diagnostics of Giardia and the Host Immune Response

Interaction of Giardia with Host Cells Guadalupe Ortega-Pierres, Maria Luisa Bazán-Tejeda, Rocio Fonseca-Liñán, Rosa María Bermúdez-Cruz and Raúl Argüello-García

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Abstract

17.1 Introduction

In the context of host-Giardia interaction, adhesion of parasites to epithelial cells is considered the initial event that allows establishment of the parasite in the host. During this contact trophozoites also interact with microenvironmental factors to indirectly promote pathophysiologic alterations in the infected host contributing to some extent to the variable clinical outcome of giardiasis. Giardia contains a unique key element, the adhesive disk which is involved in adhesion of Giardia to host cells. This structure together with other specialized parasite elements that include the ventrolateral flange, the flagella, and several parasite molecules located at the intracellular (contractile proteins), epicellular (i.e. cell surface molecules) levels or secreted participate in the colonization of the very hostile environment at the small intestine. Several observations have indicated that trophozoites secrete various molecules that upon interaction with epithelial cells may be responsible for the pathologic changes seen at the enterocyte during the course of giardiasis. Also due to this interaction, alterations related to the transport of a variety of solutes such as chloride and sodium ions as well as high sized proteins have been documented. Moreover changes occur in the arrangement of α-actinin, myosin light chain, F-actin related to cytoskeleton dynamics and of proteins that participate in the intercellular junctions such as claudins and ZO-1 which in turn may lead to the activation of apoptotic signaling. All these manifestations may be related to the parasite strain and are also influenced by the immune, clinical, and nutritional status of the host leading to the onset of giardiasis.

During the life cycle of Giardia, there are complex host-parasite interactions involving successive “crosstalks” between the alternate infective (cyst) and pathogenic (trophozoite) parasite stages and the gastric and intestinal milieus. Owing to these interactions the parasite responds to host stimuli (i.e. low pH, cholesterol, and bile components) and carries out stage conversions (ex- and encystation) with intermediate entities (excyzoite and encyzoite, respectively) resulting in the establishment of the parasite inside and dissemination outside the infected host (Bingham and Meyer, 1979; Gillin et al., 1988; Argüello-García et al., 2009). From a biological, clinical, and epidemiologic perspective, the interaction of trophozoites with small-intestinal epithelia is a key process as it allows parasite colonization of the intestinal epithelium and the onset of the variable symptoms and signs associated with giardial infections. When trophozoites interact with intestinal epithelial cells these parasites may activate pathogenic mechanisms encompassing: (a) adhesion of the parasite to the microvillus border of epithelial cells, (b) interaction with microenvironmental factors to favor parasite metabolic requirements for growth and differentiation at the expense of disequilibrium in the host at cellular and systemic levels, and (c) production and secretion of molecules able to interact with host epithelial cells or that activate innate and specific immune responses. The combined effects of these three levels of interaction contribute importantly to the variable outcome of disease (symptomatic, asymptomatic, acute, chronic, self-limiting, etc.). In this chapter, we analyze both the multifactorial nature and possible role of each of these processes in the pathogenesis of giardiasis.

H. D. Luján et al. (eds.), Giardia © Springer-Verlag/Wien 2011

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Regarding trophozoite adhesion to target cells, the presence of a species-specific ventral disk confers Giardia a great advantage for colonizing the very hostile environment of the small intestinal tract (Fig. 17.1). This highly specialized disk is functionally present while Giardia remains within the infected host and this structure is disassembled in early encystation and in the dormant cyst stage and quite fast reassembled into a functional disk 20 min after excystation induction (Palm et al., 2005). It has been suggested that the disk-less, transitory excyzoite might be “adhered” to epithelial cells by expressing on its surface proteins as α1-giardin that are able to bind glycosaminoglycans (e.g. heparan sulphate) widely expressed on the apical surface of epithelial cells (Weiland et al., 2003). The ventral disk is by far the most important structure mediating the most intimate and direct interaction of trophozoites with epithelial cells, extracellular matrix components (e.g. collagen; Knaippe, 1990) and even

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a variety of inert surfaces (glass, plastic, nylon, etc.). In an evolutionary sense, the ventral disk is very likely the main morphologic marker of evolution and parasitism in Giardia as compared with its phylogenetically closest relatives, the members of the Diplomonad family (from Hexamitinae to Enteromonads). In this group, the morphologic evolutionary trend comprised the progressive reduction of the alimentary groove (cytostome) and the appearance of the adhesive disk. This latter structure allows Giardia to survive in the host and become a member of this family that has most successfully adapted to parasitism (Keeling and Brugerolle, 2006). Among the six species within Giardia genus and the assemblages (A–H) within the species G. duodenalis, there is not compelling evidence for innate differences in adhesion ability of isolates whereby trophozoites from distinct species/genotypes infect different host repertoires. For instance, it has been reported that parasites axenized from symptomatic or asymptomatic children (all from genotype A) had distinct abilities to colonize the intestinal lumen, as assessed in rodent models (Astiazarán-García et al., 2000). However, in another study a Giardia isolate that was lethal in birds and genetically similar to Giardia human isolates produced a chronic infection in mice with a course resembling that of the failure-to-thrive syndrome (Upcroft et al., 1997). Therefore, trophozoite adhesion is interplaying with other parasitic (distinct virulence and pathogenicity of strains) and host (age, nutritional, and immunologic status) factors to determine the establishment and outcome of infection. This complex scenario deserves a dissecting analysis of the interaction of trophozoites with epithelial cells and gut lumen factors and the effects of parasite-released factors on the host.

17.2 Trophozite Adhesion: What is In, What is Out

Fig. 17.1 Ventral view of a Giardia duodenalis trophozoite in which the adhesive disk (AD), the lateral crest (LC), the ventrolateral flange (VLF), and the pairs of: anterior (AF), ventral (VF) and caudal (CF) flagella are shown. IEC6 cells displaying microvilli are underneath the trophozoite. Scale bar: 1 μm

The adhesion of trophozoites to gut epithelia may be considered as the closest Giardia-host interaction as it implies physical contact between cell surfaces of trophozoites and enterocytes. The biologic implication of this process is twofold: while parasite adhesion provides an important step in the pathogenic mechanism, it also serves Giardia as a “defense” pro-

Chap. 17 Interaction of Giardia with Host Cells

cess. Concerning the latter case, parasites attached just beneath the mucus layer and lying down on the microvillus border are able to evade innate immune responses of the host, which include soluble mediators released by enterocytes (chemokines, nitric oxide, and cryptidins) (Eckmann, 2003; Roxstrom-Lindquist et al., 2006), cytotoxic factors in intestinal fluid including digestive enzymes and bile salts, and eventually diet elements as lactoferrin, un/saturated fatty acids, lectins, and natural fibers (Rohrer et al., 1986; Leitch et al., 1989; Grant et al., 2001; Rayan et al., 2005; Ochoa and Cleary, 2009). Even in the presence of an adaptive immune response, where intestinal hypermotility is recognized as a contributing process in tro-

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phozoite clearance in humans and rodent models (Andersen et al., 2006), the adhesive force provided mainly by the ventral disk is an essential factor determining parasite persistence or elimination from the gut. Regarding the mechanism of giardial adhesion, several hypotheses have been proposed on the basis of various studies which are summarized in Table 17.1 These include the generation of hydrodynamic forces (Holberton, 1974); a cytoskeletal regulation of contractile activity (Feely and Erlandsen, 1982; Erlandsen and Feely, 1984; Narcisi et al., 1994), lectin- and surface molecule-mediated adhesion (Farthing et al., 1986; Inge et al., 1988; Katelaris et al., 1995) and the

Table 17.1 Strategies modeling adhesion of Giardia trophozoites to different substrata and agents blocking this process Agent

Possible target/mechanism

Experimental model

Reference

Concentrated/diluted TYI-S33 medium

Cell membrane osmoregulation

Glass, human epithelial cell line (C2BBc-1)

Hansen and Fletcher (2008)

Mucins

Electrostatic repulsion

Polystyrene

Roskens and Erlandsen (2002)

Antibodies

δ-giardin

Glass

Jenkins et al. (2009)

200-kDa surface molecule

Dog and human epithelial cell lines (MDCK, IEC6) and Mongolian gerbils

Hernández-Sánchez et al. (2008)

VSP9B10A-like

Dog epithelial cell line (MDCK)

Bermúdez-Cruz et al. (2004)

Monosaccharides (GluNAc, GalNAc, Glucose, Galactose, Fucose, Mannose-6phosphate) or lectins (Concanavalin A)

Lectins

Human epithelial cell lines (Caco-2, Int-407)

Pegado and de Souza (1994), Magne et al. (1991), Sousa et al. (2001)

D-mannosyl-containing sugars and glycoproteins

Surface mannose-binding lectin

Isolated rat intestinal epithelial cells

Inge et al. (1988)

Colonic mucus

Lectins?

Glass

Zenian and Gillin (1985)

Formononetin

Nucleoside hydrolase?

Glass, human epithelial cell line (Caco-2) and murine small intestinal explants

Lauwaet et al. (2010)

E-64, TPCK

Cysteine proteases

Human epithelial cell line (IEC6)

Rodríguez-Fuentes et al. (2006)

Iodoacetic acid

Energy metabolism (glycolysis)

Polystyrene

Feely and Erlandsen (1982)

Cytochalasin-B

Actin

Polystyrene

Feely and Erlandsen (1982)

Microtubule-acting drugs (Albendazole, mebendazole, thiabendazole, nocodazole and colchicine)

Tubulin

Human epithelial cell lines (Caco-2, Int-407)

Magne et al. (1991), Katelaris et al. (1994), Sousa et al. (2001)

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participation of contacts by the ventrolateral flange/ border (Friend, 1966; Knaippe, 1990; Erlandsen et al., 2004). The suction-based mechanism of attachment has been recognized of primary importance in studies concerning the contributions of physical parameters as viscosity of the medium, where this suction force (in the order of tenths of a microdyne) was calculated to be higher than detaching forces (Jones et al., 1983), and the similar adhesion ability of trophozoites to positively charged, hydrophobic or inert surfaces (Hansen et al., 2006). In a general context, trophozoites may attach to a variety of biologic and inert surfaces. However, the onset and maintenance of this contact is largely due to the concerted activity of molecules, macromolecular systems, and morphologic structures located at the intra- and epicellular levels as are cytoskeleton components, cell surface molecules, ventral disk, and ventrolateral flange (Fig. 17.1). These factors depict the multifactorial nature of giardial adhesion and moreover some elements may have a dual function like cytoskeletal molecules (e.g. α1- and δ-giardins) which have been shown to be exposed also at the trophozite plasma membrane becoming immunogenic as well (Weiland et al., 2003; Jenkins et al., 2009). In the following sections the features and roles of cy-

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toskeletal cell surface-associated and secreted factors in giardial adhesion are further discussed.

17.3 Ancestral Structural Proteins in a Very Evolved Adhesion Apparatus In the adhesion process of Giardia trophozoites, an outstanding characteristic is the contractile activity of the ventral disk, which may be envisaged as a structural marker of Giardia evolution and parasitism (Keeling and Brugerolle, 2006). This activity is the result of the connections among a concentric spiral microtubule network partially cross-bridged by giardins from center to periphery of disk, where the contractile proteins actin, alpha-actinin, myosin, tropomyosin, and vinculin have been detected (Feely et al., 1982). The rim of the adhesive disk is known as the lateral crest (Feely et al., 1982; Narcisi et al., 1994), a noticeably flexible region characterized by the absence of intramembranous particles and cholesterol complexes on its plasma membrane (Erlandsen and Chase, 1974; Chávez and Martinez-Palomo, 1995). The lateral crest is the region of the disk that ultimately projects strongly downwards, establishes physical contact with epithelial cells (Fig. 17.2), and

Fig. 17.2 Micrograph of MDCK epithelial cell monolayer displaying microvilli after interaction with G. duodenalis trophozoites. Dashed traces indicate the horse shoe shape areas where parasites have made contact with the epithelial cell surface. Kindly provided by Bibiana Chavez-Munguía (Department of Infectomics and Molecular Pathogenesis, Cinvestav-IPN, Mexico). Scale bar: 10 μm

Chap. 17 Interaction of Giardia with Host Cells

leaves characteristic deep horseshoe-like imprints on the surface of the gut mucosa (Erlandsen and Feely, 1984). This unique structure is formed by contractile proteins that are phylogenetically ancestral, the early divergent origin of Giardia is supported by tubulin phylogenetic analyses (Edlind et al., 1996; Keeling and Doolittle, 1996) while giardins and the related protein SALP-1 have likewise distinct ancestral histories (Palm et al., 2005). Tubulin and giardins are the major proteins in ventral disk. Initially, giardins were characterized as a pair of 30 kDa proteins (Crossley and Holberton, 1983) but now they have been classified in four groups. Alpha giardin (Morgan and Fernández, 1995, 1997), beta giardin, which is a striated fibre (SF)-assemblin homolog (Weber et al., 1993), gamma giardin (Nohria et al., 1992), and delta giardin (Elmendorf et al., 2001; Jenkins et al.,; 2009). The alpha giardins are a prominent family of 21 members: alpha-1 and -2 giardin (Peattie et al., 1989; Alonso and Peattie, 1992), alpha-3 (Weiland et al., 2003), alpha-7.1, -7.2 and -7.3 giardin (Palm et al., 2003) until 19 giardin (Weiland et al., 2005). Interestingly these genes share an ancestry with human annexins (Morgan and Fernández, 1995; Szkodowska et al., 2002). Several of the alpha giardins are among the most highly expressed proteins in Giardia. For example, alpha-1 and -11 giardins show high levels of transcription of mRNA and the alpha 1 giardin is one of the major immunodominant proteins during acute giardiasis (Weiland et al., 2005; Palm et al., 2003). Additionally, the alpha 2 giardin has been reported as an assemblage A specific protein (Steuart et al., 2008). In general, the different alpha-giardins may have specific sub-domains that confer additionally biochemical characteristics allowing them to display other functions according to its cellular localization. Further studies on the biologic roles of the different alphagiardins are required to fully understand their role in the interaction of Giardia with host cells. Beta-giardin and SALP-1 are present exclusively in the genus Giardia albeit they display weak homology to SF-assemblin (SFA), a microtubule-associated protein that constitutes a fundamental element of ancestral cytoskeletal structures of several single-celled organisms such as flagellated algae and apicomplexan parasites. Indeed, SF assembling is the major struc-

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tural protein of the system I fibers that forms part of the basal apparatus in the flagellate green algae (Lechtreck and Melkonian, 1991). Beta-giardin and SFA share some identity and both present a similar coiled-coil structure of alpha-type fibrous proteins (km-e-f class) that is exhibited by other fibrous proteins such as tropomyosin and myosin (Holberton et al., 1988). In spite of being localized to adhesive disk as well, the gamma-giardin protein sequence that is encoded by a single gene does not show homology to SFA (Nohria et al., 1992) albeit it was shown to interact to end-binding 1 (EB1), a protein closely associated with the microtubule assembling (plus-endtracking) machinery that couples ribosome biogenesis and mitosis (Kang et al., 2010). The latest giardin identified was δ-giardin that was characterized by a screening of G. duodenalis cDNA and genomic libraries with specific antibodies (Elmendorf et al., 2001). The δ-giardin transcript encodes for a 31 kDa protein that is localized at the microribbons of the ventral disk (Jenkins et al., 2009). These several lines of evidence suggest that giardins constitute key elements contributing to the unique form and organization of the ventral disk. Also, these characteristics may suggest that system I fibers and ventral disk may have arisen from molecules able to form a special segmented coiled-coil which can assemble into ancestral but very specialized cytoskeletal structures. Such molecules might have been subjected to a strong evolutionary drift in sequence but not in length and architecture (Weber et al., 1993).

17.4 Molecular Factors Involved in Giardia Adhesion to Host Cells The adhesion of Giardia to epithelial cells is a multistep and complex process involving several adhesion molecules, signaling events, and proteolytic activities together with the parasite structures mentioned above. To establish a successful infection the parasite has to interact initially with the cell surface of the enterocytes and as a result of this interaction several parasite molecules are secreted. The participation of specific trophozoite components in Giardia adhesion ability has been inferred by modeling parasite interaction with target cells/substrates and determining inhibi-

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tion/blockade of adhesion. Table 17.1 includes strategies that have been used to target Giardia adhesion to glass and plastic surfaces and a variety of epithelial cell lines ranging from dog kidney (MDCK) to human intestinal line cells (Caco2 and its sub-line C2BBc-1, IEC6, Int-407) and in some cases isolated rat cells and murine explants. Initial studies carried out with axenic cultures adhered to glass and plastic surfaces, showed that trophozoite adhesion required thiol-reducing agents (cysteine), serum components (Cohn III fraction), and optimal values of temperature (35.5–37oC), pH (6.85–7.0) and ionic strength (200–300 mOsmol/kg) (Gillin and Reiner, 1982). Thereafter a variety of adhesion-blocking agents have been identified. These include physicochemical procedures that affect homeostatic processes, namely tonic shock that deregulate cell volume recovery (Hansen and Fletcher, 2008) and mucosal barriers such as poly-anionic, heavily glycosylated proteins (mucins) that by electrostatic repulsion may impede the contact between trophozoite and substrate (Roskens and Erlandsen, 2002). In this context, it is interesting to note that human small intestinal mucus but not the colonic one promotes Giardia adhesion (Zenian and Gillin, 1985) though some mucins had a detrimental effect on adhesion (Roskens and Erlandsen, 2002). Other blocking agents are antibodies directed to cell surface components of trophozoites (e.g. rabbit antiserum, monoclonal antibodies, and secretory IgA from human milk; Inge et al., 1988; Samra et al., 1991) or to specific surface molecules such as a 200-kDa protein, the variable surface protein VSP9B10A-like and the cytoskeletal, ventral disk-localized protein δ-giardin (Bermúdez-Cruz et al., 2004; Hernández-Sánchez et al., 2008; Jenkins et al., 2009). This approach could certainly be very useful for prophylactic purposes once the immunodominancy and representative traits in Giardia isolates are addressed for these molecules. Heavily glycosylated components on trophozoite surface (i.e. lectin-like proteins and glycoproteins) have been also implicated in trophozoite adhesion since this process was also inhibited with a variety of monosaccharides or some lectins as Concanavalin A (see Table 17.1). Among Giardia lectins, the most studied is Taglin (Trypsin-activated Giardia lectin; Lev et al., 1986), a 30-kDa protein which has been suggested as a possible agent contributing to the microvillus short-

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ening associated with giardiasis (Farthing et al., 1986) that was further identified using taglin-specific monoclonal antibodies as α1- and α2-giardin (Wieland et al., 2003). However it is still unclear whether these giardin-type lectins are targeted by all or some of the adhesion-blocking monosaccharides and lectins aforementioned. Intracellular enzyme activities and macromolecular complexes have been experimentally shown to be critical for trophozoite adhesion as well. Indeed trophozoites treated with the O-methylated isoflavone formononetin lost rapidly the adhesion ability to biologic and inert substrates by a yet undefined mechanism. This is because it was recently suggested that formononetin impaired flagellar motility (Lauwaet et al., 2010) which may participate in the suction force generated underneath the adhesive disk (Holberton, 1974) while Sterk et al. (2007) showed that a giardial purine-metabolizing enzyme (nucleoside hydrolase) was bound and inactivated by isoflavones, and the expression levels of this enzyme are decreased in formononetin-resistant Giardia. In other studies parasite adhesion has been adversely affected by treating trophozoites with compounds directed to enzymes with cysteine residues critical for its activity. These have included the irreversible protease inhibitors L-trans-epoxysuccinylleu-4-guanidinobutylamide (E64) and tosyl-L-phenylalanine chloromethyl ketone (TPCK) (Rodríguez-Fuentes et al., 2006) and iodoacetic acid, and alkylating agent that very likely affects glycolytic enzymes involved in giardial energy metabolism (Feely and Erlandsen, 1982). These observations are in good agreement with the enhancing effect of cysteine for trophozoite adhesion (Gillin and Reiner, 1982). On the other hand, the unique cytoskeleton of Giardia that forms several cellular structures including ventral disk, flagella, median body, and funis (Elmendorf et al., 2003) is a key macromolecular element involved in its adhesion. In several studies, cytoskeleton components have been also targeted by using various benzimidazoles (albendazole, mebendazole, thiabendazole, and nocodazole) and the plant metabolite colchicine, all these binding β-tubulin and significantly decreasing parasite adhesion (Magne et al., 1991; Katelaris et al., 1995; Sousa et al., 2001). Likewise actin-disrupting agents as the mycotoxin cytochalasin B are effective inhibitors of giardial adhesion (Feely and Erlandsen, 1982). In fact, the role

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of cytoskeletal proteins in trophozoite adhesion has been proposed to be in close relation to Giardia virulence (Elmendorff et al., 2003). In the case of cell surface molecules, its role in the adhesion process is likely important in terms of selectivity for small intestinal epithelia because in co-cultures of Giardia and epithelial cell lines, despite of the fact that most trophozoites are adhered by its ventral surface, parasites adhered in various other orientations have been observed as well (Chávez et al., 1986; Inge et al., 1988; Magne et al., 1991; McCabe et al., 1991; Sousa et al., 2001). Furthermore, the adhesive activity of the ventrolateral flange that surrounds the ventral disk and establishes localized contacts with substratum surface reinforces the role of lectins and surface proteins in parasite adhesion (Erlandsen et al., 2004). Several other studies have proposed the participation of other secreted components from Giardia in the interaction with epithelial cells. This has been addressed by identifying immunoreactive proteins in Giardia using infected humans sera (patients with acute giardiasis), among these are variable surface proteins (VSP), alfa-giardins, arginine deiminase (ADI), ornithine carbamoyl transferase (OCT), fructose-1,6-bis phosphate aldolase, and novel proteins such as enolase, SALP-1, GTA-1, GTA-2, UPL-1 (Palm et al., 2003; Ringqvist et al., 2008). ADI (Knodler et al., 1998) and OCT are members of the arginine dihydrolase pathway, found on the surface of many bacteria including Streptococci and Lactobacilli (Vrancken et al., 2009) and protozoa such as Trichomonas vaginalis (Yarlett et al., 1996), and Hexamita inflata (Biagini et al., 2003). ADI deiminates arginine, producing citrulline which is subsequently converted into ornithine and carbamoyl phosphate by OCT. Then carbamate kinase (CK) (Minotto et al., 1999) uses carbamoyl phosphate to phosporylate ADP to generate one molecule of ATP (Galkin et al., 2004); however, the enzyme was not found in the giardia secretome (Ringqvist et al., 2008), suggesting a specific role for ADI and OCT. It is known that in Giardia, arginine is an important substrate for energy production (Edwards et al., 1992), and that it is metabolized to ornithine. In this way by reducing the available arginine concentration (Eckmann et al., 2000), concomitantly the ability of intestinal epithelial cells to generate nitric oxide (NO)

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from arginine (Cendan et al., 1995) is prevented. Interestingly, ADI expression which is already high, is induced upon trophozoite-host cell interaction in vitro though no intracellular arginine has been detected (Knodler et al., 1994), consistent with this a reduced cytokine-induced NO production was observed while recombinant giardial ADI was added to intestinal cells. Further, it has been reported that arginine deprivation may induce apoptosis (Potoka et al., 2003). Recent findings indicate that Giardial ADI may have multiple regulatory roles (Touz et al., 2008), it converts arginine to citrulline in proteins. In this study the identified target was the cytoplasmatic CRGKAtail of variant-specifc surface proteins (VSPs). Also, as expected it was observed that this modification altered the VSP switching. Further, ADI was also subjected to sumoylation, suggesting that its activity may be regulated. Another metabolic enzyme identified in the supernatant fraction of the host-parasite co-cultures is enolase (Ringqvist et al., 2008). Enolase is a metabolic enzyme that has been widely found on the surface, cytoplasm, nucleus or secreted in many prokaryotic and eukaryotic cells (Pancholi, 2001; Sun., 2006). In general, enolases found on surface are related to tissue invasion by pathogens (Liu and Shih 2007) as they bind plasminogen (e.g. plasminogen binding receptor), upon cleavage by activators it is converted to plasmin which can activate colagenase and degrade several matrix proteins. Thus, by providing plasminogen bound to enolase on the surface or secreted in the cellular environment, cells may be equipped with a wide source of proteolytic enzymes to ease their penetration in the extracellular matrix, however its role in parasites remains to be elucidated. As expected from a moon lightning protein, it has been recently described that Entamoeba invadens enolase has a role in encystation as a specific anti-enolase antibody inhibited encystation (Segovia-Gamboa et al., 2010), while in Entamoeba histolytica enolase was found to bind Dnmt2, a DNA and tRNA methyltransferase located in the nucleus, thereby inhibiting its function (Tovy et al., 2010). Further investigation needs to be carried out on Giardia enolase addressing all these novel activities to gain insight on its possible role in the pathogenicity of giardiasis. Other proteins that are secreted in the growth medium by trophozoites are the Giardia VSP (Nash

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et al., 1983; Palm et al., 2003). These proteins are targets for the humoral immune response and they are switched often; they are responsible for the antigenic variation widely documented in experimental human and animal infections (Nash, 2002). The VSP switch has also been seen in response to selection by antibodies or physiologic stress. Although only one gene is expressed at one time, the giardial VSP repertoire has been estimated as 150–200 genes per haploid genome (Nash, 2002). Regarding their expression, it has been shown that VSPH7 is epigenetically regulated (Kulakova et al., 2006), while recent data implicate the RNA interference pathway in antigenic variation of VSP9B10 (Prucca et al., 2008). Nevertheless, the role of these VSP in the pathogenicity of giardiasis remains to be further investigated. A 58 kDa, acidic (pI 4.75) glycoprotein with lectin activity secreted from the P-1 strain into the culture medium (Kaur et al., 2001; Shant et al., 2002, 2005) has been observed. This is an immunodominant and immunoprotective protein with enterotoxic activity in ligated-loop experiments. Furthermore, it binds enterocytes and increases the intracellular Ca2+ concentration, making it a potential disease-causing factor (Shant et al., 2005). A partial amino-acid sequence (ADFVPQVST) has been identified; however, it does not correlate to any open reading-frame in the Giardia WB genome (Morrison et al., 2007). According to molecular weight, pI, and glycosylation pattern this protein may be a VSP protein specific to P-1. Proteases are other secreted proteins that may play an important role in the host-parasite relationship. They are generally related to tissue and cellular invasion, morphogenesis during life cycle, host protein degradation, pathogenicity, virulence, and stimulation and evasion of immune response (Klemba and Goldberg, 2002). Giardia trophozoites contain multiple proteases located in the intracellular – in the recently described ER-like TVN (tubulovesicular network) (Abodeely et al., 2009) and extracellular milieu that play a role in parasite metabolism and physiologic processes (Hare et al., 1989; Parenti, 1989; Williams and Coombs, 1995; Coradi and Guimaraes, 2006). Recent studies on proteases from Giardia trophozoites have indicated that these proteins are involved in immunologic and pathophysiologic processes (Jimenez et al., 2000, 2004; Kaur et al., 2001; Shant

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et al., 2002). Indeed these proteins can be secreted during in vitro culture of trophozoites (Nash and Keister, 1985; Jimenez et al., 2000; Shant et al., 2002; DuBois et al., 2006; Rodriguez-Fuentes, et al., 2006; David et al., 2007; Batista de Carvalho et al., 2008) and interestingly our studies have provided evidence that these molecules are a determinant factor in the adhesion of trophozoites to epithelial cells. Their role in this process may be very important since these proteins may act as virulence factors. Our findings revealed that secreted proteolytic activity of trophozoite proteases was enhanced when parasites were co-cultured with IEC6 epithelial cells (Rodríguez-Fuentes et al., 2006). Furthermore, this activity was strongly inhibited by pre-incubation of live trophozoites with the cystein and serine protease inhibitors E-64 and TPCK with a consequent inhibition of adhesion (Rodríguez-Fuentes et al., 2006) as observed in other protozoa such as Trichomonas vaginalis (Sommer et al., 2005). Although the mechanisms by which proteases participate during Giardia infection are poorly understood, it may be possible that proteases secreted from Giardia trophozoites may activate receptors in the host epithelial cells that together with signaling receptors could induce apoptosis in enterocytes and contribute to epithelial barrier dysfunction by increasing epithelial permeability due to tight junctional ZO-1 disruption in a caspase-3-dependent manner (Chin et al., 2002, 2003). Also a down regulation in claudin-1 and epithelial apoptosis has been observed in human duodenal biopsy specimens (Troeger et al., 2007). Alternatively, this apoptosis referred as anoikis may be induced in response to a disruption of the epithelial cell-matrix (Valentijn et al., 2004). It is clear that some Giardia proteins are directly involved in the damage of the intestinal epithelium; however, the exact mechanisms that derive in the onset of diarrhea need to be further elucidated.

17.5 Consequences of Giardia-Host Cell Interactions Several lines of evidence from experimental infection models help to exemplify that trophozoite-driven mechanisms may have alone or combined severe effects on host components leading to alterations seen in clinical giardiasis. On the one hand, damage of the

Chap. 17 Interaction of Giardia with Host Cells

intestinal epithelium by trophozoite adhesion has been proposed as a major pathogenic mechanism (Inge et al., 1988; Buret et al., 2002; Muller and von Allmen, 2005); however, adhered parasites and parasite excretory/secretory products alter both microvillus architecture and cytoskeletal proteins of enterocytes resulting in altered epithelial barrier function and induction of diarrhea in the host (Buret et al., 2002). In particular, the diffuse loss of epithelial microvillus length contributes to impaired activity of brush border enzymes such as disaccharidases (Farthing, 1997; Mohammed and Faubert, 1995) and lactase promoting lactose intolerance. Thus the diarrhea may be responsible for the hypersecretion of Cl, malabsorption of glucose, water and Na+ as well as the reduction in luminal trypsin, chemotrypsin, and lipase concentrations observed in clinical giardiasis (Farthing, 1997). On the other hand, Giardia trophozoites take up avidly conjugated bile salts and fatty acids, a process that likely interferes with micellar solubilization of fats (Halliday et al., 1988; Farthing, 1996) leading indeed to steatorrhea. In relation to possible toxin-like molecules produced by trophozoites, it has been observed that a 58-kDa excretory/secretory product is cytotoxic for mouse intestinal cells and epithelial cell lines (Shant et al., 2002). Nevertheless, the actions of toxins released by parasites are not the unique factors to trigger pathogenesis. In this sense, the immune response might have undesirable effects as a role for T-cell dependent responses has been proposed to participate in pathogenesis, promoting microvillus shortening at the gut epithelia (Scott et al., 2000) as well as immune reaction involving mast cells, dendritic cells, IgA, and NO (Roxtrom et al., 2006; Andersen et al., 2006; Li et al., 2007). Further, the effect of the pathogen on microvilli has been linked to CD8+ T cells, but the effector mechanisms remain unknown (Scott et al., 2004). Although early studies have shown that only weak inflammatory response and little microvilli shortening (Oberhuber et al., 1997) were observed in 3.2% of Giardia infected humans, an experimental infection in humans demonstrated that 5/10 individuals developed symptoms while only 2/10 displayed intestinal border abnormalities (Nash et al., 1987). Thus, there is no direct correlation between tissue alteration and symptoms.

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For proper function, the intestinal barrier which is composed by enterocyte apical plasma membrane (transcellular barrier) and the tight junctions (paracellular barrier) (Farquhar and Palade, 1963), needs to be intact. Although this has been shown to be strain dependent, Giardia duodenalis trophozoites may induce apoptosis in enterocytes in a caspase-3 dependent fashion as observed in SCBN (non transformed human duodenal epithelial cell line) (Chin et al., 2002) where nuclear condensation and fragmentation as well as epithelium injury were observed. Also, microarray analysis data from human CaCo2 cells in contact with G.duodenalis have revealed that the parasite-host interactions upregulate genes implicated in the apoptotic cascade and the formation of reactive oxygen species (Roxstrom-Lindquist et al., 2005). This was confirmed in 13 biopsies from human patients with chronic giardiasis where increased epithelial apoptosis was detected, additionally epithelial resistance and claudin 1 expression were also reduced (Troeger et al, 2007). Consistent with this, if not all, many giardial infections cause rearrangements of Factin and α-actinin (Teoh et al., 2000), tight junction protein ZO-1 disruption (Chin et al., 2002) with a resulting increased intestinal permeability. Other reports studied the interaction between human intestinal epithelial cell line HCT-8 and G. duodenalis strain WB trophozoites (Panaro et al., 2007) where DNA fragmentation analysis, detection of active caspase 8, 9 (extrinsic and intrinsic pathways), and consequently caspase-3 as well as degradation of its substrate PARP (Protease-Activated Receptor Protein) demonstrated that indeed this parasite induces apoptosis. Consistent with this, a down regulation and up regulation of Bcl-2 and Bax, respectively were also observed. Though this remains to be elucidated, these findings together with the disruption of ZO-1 and/or down regulation of claudin 1 during interaction of parasite with its host cells (Panaro et al., 2007) may suggests that a particular apoptotic process such as anoikis may take place (Valentijn et al., 2004). Interestingly, all these effects may be counteracted by SGLT-1 (sodium-dependent glucose co-transporter) (Yu et al., 2008), since SGLT-1 transfected Caco2 cells caspase-3-dependent apoptosis due to Giardia sonicates is inhibited in the presence of high level glucose (25 mM), suggesting that an increased sugar uptake has a protective effect against apoptosis. Induction

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of apoptosis in host cells seems to be a more general phenomenon since other protozoa such as Trichomonas foetus (Singh et al., 2004), Acanthamoeba castellanii (Zheng et al., 2004), Trichomonas vaginalis (Kummer et al., 2008) have also been proven to induce apoptosis in the cells that they infect and this may facilitate infection as has been demonstrated for Entamoeba histolytica (Becker et al., 2010).

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Acknowledgments We are grateful to Arturo Pérez-Taylor for technical assistance in the preparation of this manuscript. The work was partially supported by a grant from ECOS-ANUIES M06-S03 and from Conacyt No. 128426.

References 17.6 Conclusions The mechanism of Giardia attachment to epithelial cells is a multi-factorial process in which parasite structures as well as molecules either on the trophozoite surface or secreted participate with consequences for the outcome of the infection caused by this parasite in the host. Although several studies have revealed the role of these structures and of some molecules in this phenomenon, there are still several aspects that need further analysis. These include a detailed characterization of Giardia virulence factors together with their ligands and effects in the host’s cells. Also the regulation of gene expression for these virulence factors is of much interest since this will allow understanding how this parasite deals with such a hostile environment at the small intestine and still causes disease. Further of great importance is the identification of such virulence factors in the various Giardia assemblages thus a more accurate definition of virulence can be defined in this parasite. In this context comparative genomics can provide a platform to investigate key areas of pathogenesis through future investigations. In addition, studies are needed to define connections between adhesion and cytoskeleton components in both parasite and host epithelial cells, involvement of signaling systems in this interaction, and possible processing of adhesion molecules as well as its regulation by host factors. Because high-throughput methods are becoming increasingly standard and affordable, an explosion of new data is likely to occur. Finally it should be noticed that the new insights in adhesion molecules could have enormous implications for the development of therapeutic agents against Giardia. Surface and secreted parasite components such as proteases that are expressed upon interaction with host cells are candidates since some of them have indispensable functions in Giardia and very likely do not show antigenic variation.

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272 Inge PM, Edson CM, and Farthing MJ (1988) Attachment of Giardia lamblia to rat intestinal epithelial cells. Gut 29: 795–801 Jenkins MC, O’Brien CN, Murphy C, Schwarz R, Miska K, Rosenthal B, and Trout JM (2009) Antibodies to the ventral disc protein δ-giardin prevent in vitro binding of Giardia lamblia trophozoites. J Parasitol 95: 895–899 Jimenez JC, Uzcanga G, Zambrano A, Di Prisco MC, and Lynch NR (2000) Identification and partial characterization of excretory/secretory products with proteolytic activity in Giardia intestinalis. J Parasitol 86: 859–862 Jimenez JC, Fontaine J, Grzych JM, Dei-Cas E, and Capron M (2004) Systemic and mucosal responses to oral administration of excretory and secretory antigens from Giardia intestinalis. Clin Diagn Lab Immunol 11: 152–160 Jones RD, Lemanski CL, and Jones TJ (1983) Theory of attachment in Giardia. Biophys J 44: 185–190 Kang K, Kim J, Yong TS, and Park SJ (2010) Identification of end-binding 1 (EB1) interacting proteins in Giardia lamblia. Parasitol Res 106: 723–728 Katelaris PH, Naeem A, and Farthing MJ (1994) Activity of metronidazole, azithromycin and three benzimidazoles on Giardia lamblia growth and attachment to a human intestinal cell line. Aliment Pharmacol Ther 2: 187–192 Katelaris PH, Naeem A, and Farthing MJ (1995) Attachment of Giardia lamblia trophozoites to a cultured human intestinal cell line. Gut 37: 512–518 Kaur H, Ghosh S, Samra H, Vinayak VK, and Ganguly NK (2001) Identification and characterization of an excretorysecretory product from Giardia lamblia. Parasitology 123: 347–356 Keeling PJ and Brugerolle G (2006) Evidence from SSU rRNA phylogeny that Octomitus is a sister lineage to Giardia. Protist 157: 205–212 Keeling PJ and Doolittle WF (1996) Alpha-tubulin from earlydiverging eukaryotic lineages and the evolution of the tubulin family. Mol Biol Evol 13: 1297–1305 Klemba M and Goldberg DE (2002) Biological roles of proteases in parasitic protozoa. Annu Rev Biochem 71: 275–305 Knaippe F (1990) Giardia lamblia attachment to biological and inert substrates. Microsc Electron Biol Cel 14: 35–43 Knodler LA, Edwards MR, and Schofield PJ (1994) The intracellular amino acid pools of Giardia intestinalis, Trichomonas vaginalis, and Crithidia luciliae. Exp Parasitol 79(2): 117–125 Kummer S, Hayes GR, Gilbert RO, Beach DH, Lucas JJ, and Singh BN (2008) Induction of human host cell apoptosis by Trichomonas vaginalis cysteine proteases is modulated by parasite exposure to iron. Microb Pathog 44: 197–203 Kulakova L, Singer SM, Conrad J, and Nash TE (2006) Epigenetic mechanisms are involved in the control of Giardia lamblia antigenic variation. Mol Microbiol 61: 1533–1542 Lauwaet T, Andersen Y, Van de Ven L, Eckmann L, and Gillin FD (2010) Rapid detachment of Giardia lamblia trophozoites as a mechanism of antimicrobial action of the isoflavone formononetin. J Antimicrob Chemother 65: 531–534 Lechtreck KF and Melkonian M (1991) Striated microtubuleassociated fibers: identification of assemblin, a novel 34-kD

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Chap. 17 Interaction of Giardia with Host Cells Nohria A, Alonso RA, and Peattie DA (1992) Identification and characterization of gamma-giardin and the gamma-giardin gene from Giardia lamblia. Mol Biochem Parasitol 56: 27–37 Ochoa TJ and Cleary TG (2009) Effect of lactoferrin on enteric pathogens. Biochimie 91: 30–34 Oberhuber G, Kastner N, and Stolte M (1997) Giardiasis: a histologic analysis of 567 cases. Scand J Gastroenterol 32: 48–51 Palm JE, Weiland ME, Griffiths WJ, Ljungström I, and Svärd SG (2003) Identification of immunoreactive proteins during acute human giardiasis. J Infect Dis 187: 1849–1859 Palm D, Weiland M, McArthur AG, Winiecka-Krusnell J, Cipriano MJ, Birkeland SR, Pacocha SE, Davids B, Gillin F, Linder E, and Svard S (2005) Developmental changes in the adhesive disk during Giardia differentiation. Mol Biochem Parasitol 141: 199–207 Panaro M, Cianciulli A, Mitolo V, Mitolo CI, Acquafredda A, Brandonisio O, and Cavallo P (2007) Caspase-dependent apoptosis of the HCT-8 epithelial cell line induced by the parasite Giardia intestinalis. FEMS Immunol Med Microbiol 51: 302–309 Pancholi V (2001) Multifunctional alpha-enolase: its role in diseases. Cell Mol. Life Sci 58: 902–920 Parenti DM (1989) Characterization of a thiol proteinase in Giardia lamblia. J Infect Dis 160: 1076–1080 Peattie DA, Alonso RA, Hein A, and Caulfield JP (1989) Ultrastructural localization of giardins to the edges of disk microribbons of Giardia lamblia and the nucleotide and deduced protein sequence of alpha giardin. J Cell Biol 109: 2323–2335 Pegado MG and de Souza W (1994) Role of surface components in the process of interaction of Giardia duodenalis with epithelial cells in vitro. Parasitol Res 80: 320–326 Potoka DA, Upperman JS, Zhang XR, Kaplan JR, Corey SJ, Grishin A, Zamora R, and Ford HR (2003) Peroxynitrite inhibits enterocyte proliferation and modulates Src kinase activity in vitro. Am J Physiol (Gastrointest Liver Physiol) 285: G861–G869 Prucca CG, Slavin I, Quiroga R, Elías EV, Rivero FD, Saura A, Carranza PG, Luján HD (2008) Antigenic variation in Giardia lamblia is regulated by RNA interference. Nature 456: 750–754 Rayan P, Stenzel D, and McDonnell PA (2005) The effects of saturated fatty acids on Giardia duodenalis trophozoites in vitro. Parasitol Res 97: 191–200 Ringqvist E, Palm JE, Skarin H, Hehl AB, Weiland M, Davids BJ, Reiner DS, Griffiths WJ, Eckmann L, Gillin FD, and Svard SG (2008) Release of metabolic enzymes by Giardia in response to interaction with intestinal epithelial cells. Mol Biochem Parasitol 159: 85–91 Rodríguez-Fuentes GB, Cedillo-Rivera R, Fonseca-Liñán R, Argüello-García R, Munoz O, Ortega-Pierres G, and YepezMulia L (2006) Giardia duodenalis: analysis of secreted proteases upon trophozoite-epithelial cell interaction in vitro. Mem Inst Oswaldo Cruz 101: 693–696 Rohrer L, Winterhalter KH, Eckert J, and Kohler P (1986) Killing of Giardia lamblia by human milk is mediated by unsaturated fatty acids. Antimicrob Agents Chemother 30: 254–257

273 Roskens H and Erlandsen SL (2002) Inhibition of in vitro attachment of Giardia trophozoites by mucin. J Parasitol 88: 869–873 Roxstrom-Lindquist K, Palm D, Reiner D, Ringqvist E, and Svard SG (2006) Giardia immunity – an update. Trends Parasitol 22: 26–31 Samra HK, Ganguly NK, and Mahajan RC (1991) Human milk containing specific secretory IgA inhibits binding of Giardia lamblia to nylon and glass surfaces. J Diarrhoeal Dis Res 9: 100–103 Scott KG, Logan MR, Klammer GM, Teoh DA, and Buret AG (2000) Jejunal brush border microvillous alterations in Giardia muris-infected mice: role of T lymphocytes and interleukin-6. Infect Immun 68: 3412–3418 Scott KG, Yu LC, and Buret AG (2004) Role of CD8+ and CD4+ T lymphocytes in jejunal mucosal injury during murine giardiasis. Infect Immun 72: 3536–3542 Segovia-Gamboa NC, Chávez-Munguía B, Medina-Flores Y, Cázares-Raga FE, Hernández-Ramírez VI, Martínez-Palomo A, Talamás-Rohana P (2010) Entamoeba invadens, encystation process and enolase. Exp Parasitol 125: 63–69 Shant J, Bhattacharyya S, Ghosh S, Ganguly NK, and Majumdar S (2002) A potentially important excretory-secretory product of Giardia lamblia. Exp Parasitol 102: 178–186 Shant J, Ghosh S, Bhattacharyya S, Ganguly NK, and Majumdar S (2005) Mode of action of a potentially important excretory – secretory product from Giardia lamblia in mice enterocytes. Parasitology 131: 57–69 Singh BN, Lucas JJ, Hayes GR, Kumar I, Beach DH, Frajblat M, Gilbert RO, Sommer U, and Costello CE (2004) Tritrichomonas foetus induces apoptotic cell death in bovine vaginal epithelial cells. Infect Immun 72: 4151–4158 Sommer U, Costello C, Hayes G, Beach D, Gilbert R, Lucas J, and Singh B (2005) Identification of Trichomonas vaginalis cysteine proteases that induce apoptosis in human vaginal epithelial cells. J Biol Chem 280: 23853–23860 Sousa MC, Goncalves CA, Bairos VA, and Poiares-Da-Silva J (2001) Adherence of Giardia lamblia trophozoites to Int407 human intestinal cells. Clin Diagn Lab Immunol 8: 258–265 Sterk M, Müller J, Hemphill A, and Müller N (2007) Characterization of a Giardia lamblia WB C6 clone resistant to the isoflavone formononetin. Microbiology 153 4150–4158 Steuart RF, O’Handley R, Lipscombe RJ Lock RA, and Thompson RC (2008) Alpha 2 giardin is an assemblage A-specific protein of human infective Giardia duodenalis. Parasitology 135: 1621–1627 Sun H (2006) The interaction between pathogens and the host coagulation system. Physiology 21: 281–288 Szkodowska A, Müller MC, Linke C, and Scholze H (2002) Annexin XXI (ANX21) of Giardia lamblia has sequence motifs uniquely shared by giardial annexins and is specifically localized in the flagella. J Biol Chem 277: 25703–25706 Teoh DA, Kamieniecki D, Pang G, and Buret AG (2000) Giardia lamblia rearranges F-actin and α-actinin in human colonic and duodenal monolayers and reduces transepithelial electrical resistance. J Parasitol 86: 800–806 Troeger H, Epple H, Schneider T, Wahnschaffe U, Ullrich R, Burchard G, Jelinek T, Zeitz M, Fromm M, and Schulzke J (2007) Effect of chronic Giardia lamblia infection on epi-

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Primary Microtubule Structures in Giardia Scott C. Dawson

Abstract Giardia intestinalis is a common parasitic protist with a complex microtubule cytoskeleton critical for cellular function and transitioning between the cyst and trophozoite life cycle stages. The giardial microtubule cytoskeleton is comprised of highly dynamic and stable structures including the eight flagella, the ventral disc, the median body, and the funis. Novel microtubule structures like the ventral disc are essential for the parasite’s attachment to the intestinal villi to avoid peristalsis. Fundamental areas of giardial cytoskeletal biology remain to be explored and knowledge of the molecular mechanisms of cytoskeletal functioning is needed to better understand Giardia’s unique biology and pathogenesis. The completed Giardia genome combined with new molecular genetic tools and live imaging will aid in the characterization and analysis of cytoskeletal dynamics throughout the giardial life cycle.

18.1 Introduction Giardia’s complex microtubule (MT) cytoskeleton is of critical importance throughout both of its life cycle stages – the environmentally resistant cyst stage and the swimming intestinal trophozoite stage (Adam, 2001; Elmendorf et al., 2003). Cysts are ingested from contaminated food or water and excyst in the small intestine of the animal host. Flagellar motility may play a mechanical role in the initial opening of the cyst, in addition to contractile or other MT-mediated forces (Feely, 1986; Buchel et al., 1987). A quadrinucleate, slightly rounded trophozoite then exits the cyst and subsequently elongates, flattens, and undergoes cytokinesis. Trophozoites are thought to partially complete cytokinesis prior to excystation, H. D. Luján et al. (eds.), Giardia © Springer-Verlag/Wien 2011

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although it is not clear at what point in the excystation process this occurs, nor whether meiosis is involved (Poxleitner et al., 2008). Flagellated trophozoites attach to the intestinal microvilli using a specialized microtubule structure, the ventral disc. The assembly of fully functional ventral discs following excystation allows the trophozoite to readily attach to surfaces. Trophozoites colonize the small intestine, undergoing cell division approximately once every 6–8 hours. Prior to cytokinesis, new dual mitotic spindles segregate chromosomes and new microtubule structures (ventral disc, axonemes, etc.) are assembled. Trophozoites eventually differentiate to become cysts and are released to infect new hosts (Roxstrom-Lindquist et al., 2006). During encystation, cytoskeletal movements, combined with the assembly of the cyst wall, remodel trophozoites from a flattened teardrop shape to the more ovoid shape characteristic of the cyst (Midlej and Benchimol, 2009). The flagella are internalized during cyst formation, yet do not completely resorb; they continue to beat inside the newly formed cyst (Midlej and Benchimol, 2009). The MT spiral of the ventral disc gradually opens and becomes horseshoe-shaped before the disc is fragmented and partially disassembled by unknown mechanisms. The median body, a MT-based structure of unknown function present in interphase trophozoites, is not observed in the cyst stage. Cytoskeletal disassembly and assembly are clearly required for encystation and excystation, but the details of cytoskeletal dynamics during these important transitions in the giardial life cycle have not been described at the molecular level. Despite its small size, Giardia has a complex three-dimensional ultrastructure with novel cytoskeletal elements of unknown function and composition (Fig. 18.1). In addition to the eight motile flagella, there are several unique microtubule-based structures

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– the median body, ventral disc, and funis (Dawson 2010; and described in detail below). During mitosis, two mitotic spindles form around the nuclear envelope and kinetochore microtubules segregate chromosomes (described in more detail in another Chapter). Other MT structures may be present during less well-

characterized stages such as excystation. Giardia’s MT cytoskeleton is an important determinant of cell shape, cell polarization, and intracellular trafficking (Elmendorf et al., 2003) and is essential for key aspects of its life cycle including motility, attachment, intracellular transport, cell division, and encystation/

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Fig. 18.1 Stable and dynamic elements of the microtubule cytoskeleton. A Giardia trophozoite, visualized using DIC, is shown in (A). The characteristic teardrop shape is visible along with the ventral disc (vd), the bare area (ba), and the four flagellar pairs (afl = anterior, pfl = posteriolateral, vfl = ventral, cfl = caudal). (B) Schematic representation of the primary elements of the trophozoite MT cytoskeleton. (C) Immunostained MT cytoskeleton (red, using the TAT1 antibody) showing the primary MT arrays of the trophozoite ventral disc (vd), four pair of flagella (afl = anterior, pfl = posteriorlateral, vfl = ventral, cfl = caudal), the median body (mb), and the two nuclei (in DAPI, blue). Panels D and E show the dynamic nature of the median body and flagella using cytoskeletal drugs that sequester tubulin subunits and inhibit polymerization (10 μm nocodazole, D), and that stabilize microtubule depolymerization (20 μm Taxol, E). Note the shortened flagella and increased cytoplasmic staining in D, and the elongated median body and flagella in E. Scale bar = 2 μm

Chap. 18 Primary Microtubule Structures in Giardia

excystation. Beyond its clinical relevance, study of the giardial cytoskeleton also informs basic cell biology, molecular biology and cellular evolution (Elmendorf et al., 2003). This chapter focuses on the structure and dynamic movements of the giardial microtubule cytoskeleton. While the actin cytoskeleton undoubtedly plays essential roles in giardial biology, polymerized actin filaments have not been identified cytologically and are only inferred from immunostaining using heterologous antibodies or from the binding of TRITCphalloidin (Castillo-Romero et al., 2009). Future work should include more detailed molecular analyses of the structure, function, and proteins associated with the actin cytoskeleton in Giardia.

18.2 Molecular Components of the Cytoskeleton Many cytoskeletal proteins are deeply conserved in eukaryotic evolution. The giardial genome possesses cytoskeletal proteins (see Table 18.1) that are structural (e.g., α-, β-, γ-, δ-, ε-tubulin, and canonical actin), proteins that regulate cytoskeletal movements (e.g., 24 kinesins and 14 dyneins), protein regulating microtubule dynamics (e.g., XMAP215, katanin, and EB1), and proteins that modify tubulin (e.g., tubulin tyrosine ligases). While actin is present in the giardial genome, proteins that modulate actin dynamics appear to be missing (Morrison et al., 2007); thus the giardial actin cytoskeleton should receive more research attention. Two actin related proteins (ARPs) have been identified in the giardial genome, although they appear to be more related to nuclear, or chromatin-remodeling ARPs, rather than ARPs that regulate actin polymerization dynamics. The primary cytoskeletal elements in Giardia are the eight flagella, the ventral disc, and the median body (Fig. 18.1). Many flagellar proteins have been identified in the giardial genome (Table 18.1). It has been proposed that upwards of 500 proteins comprise the eukaryotic flagellum (Luck, 1984; Dutcher, 1995; Ostrowski et al., 2002; Pazour et al., 2005); however, some flagellar components appear to be lineage-specific. The giardial genome contains over one hundred, microtubule-associated, flagellar, and basal body proteins (see Table 18.1). Flagellar structural components

277

include the protofilament ribbons (Rib43a and Rib72), the central pair (PF16, PF20, and hydin), the radial spokes (rsp3 and rsp9), and nexin links (PF2). Also present are basal body associated proteins (e.g., centrin, δ-tubulin, and ε-tubulin) and components of the BBSome. Many proteins associated with flagellar assembly are also conserved in the giardial genome. Some flagellar-associated proteins such as the annexins (e.g., α-giardins) appear specific to Giardia, although it is unclear whether they contribute to flagellar function or structure (Vahrmann et al., 2008). Molecular components of other structures, such as the ventral disc and median body, are not well characterized. Few proteins associated with the ventral disc have been identified. Biochemical strategies such as 2D protein electrophoresis and immunoprecipitation have yielded a handful of disc associated proteins (Feely, 1990), but to which disc structural element (microribbons, crossbridges, etc.) each of these proteins localizes is unknown. Three separate gene families of “giardins” localize generally to the ventral disc: 1) annexins, including at least three α-giardins (Peattie, 1990; Bauer et al., 1999; Weiland et al., 2003, 2005); 2) striated fiber (SF)-assemblins, including β-giardin, δ-giardin, and the divergent SALP-1 (Palm et al., 2005); and 3) γ-giardin, a novel protein (Nohria et al., 1992). Only one protein – the median body protein – has been reported to have specific localization to the median body (Marshall and Holberton, 1993). Annexins are membrane-scaffold proteins that bind in a Ca+2-regulated manner to the periphery of membranes, particularly to those containing negatively charged, acidic phospholipids. In Giardia, annexins (α-giardins) comprise a large gene family of 22 homologs associated with the cytoskeleton. Weiland et al. (2005) localized 13 of the 22 annexins using epitope-tagging and acetone fixation. Of the 22 annexins, four localize to the disc (α2-giardin, α3-giardin, α5-giardin, and α17-giardin), and at least six localize to flagella (α9- and α10-giardins specifically to ventral flagella). Further, α14-giardin has recently been confirmed to associate with axonemal MTs (Vahrmann et al., 2008). Other annexins were not localized and several annexins (α4-giardin, α7.1-giardin, α8-giardin, and α11-giardin) were purportedly lethal – meaning the investigators were unable to create stable transformants using native promoters. The

278

S. C. Dawson

Table 18.1 Giardial homologs of cytoskeletal proteins. The presence of putative homologs of microtubule-associated, flagellar, BBSome, and basal body associated genes indicate that over 80 such proteins are present in the Giardia WBC6 (Assemblage A) genome (Morrison et al., 2007). Homologs were identified by best mutual BLAST hits (e = 5 × 10–2) and compared to a handcurated list of Chlamydomonas flagellar proteins that were discovered by biochemical, genetic, and bioinformatic methods (Pazour et al., 2005). Protein families are indicated by PFAM or Interpro designations. Shaded rows indicate giardial homologs of putative flagellar or basal body proteins that have only been identified in proteomic (Ostrowski et al., 2002; Li et al., 2004; Kilburn et al., 2007; Keller and Marshall, 2008) or bioinformatic studies (FABP) and await experimental confirmation. Cytoskeletal proteins known in other organisms, such as some IFT proteins that are not readily identifiable in the genome, are not presented GiardiaDB

Gene name

Family

PFAM

Molecular function

References

GL50803_103676

alpha-tubulin 1

tubulin

PF00091

MT metabolism

Morrison et al. (2007)

GL50803_112079

alpha-tubulin 2

tubulin

PF00091

MT metabolism

Morrison et al. (2007)

GL50803_101291

beta-tubulin 1

tubulin

PF00091

MT metabolism

Morrison et al. (2007)

GL50803_136021

beta-tubulin 2

tubulin

PF00091

MT metabolism

Morrison et al. (2007)

GL50803_136020

beta-tubulin 3

tubulin

PF00091

MT metabolism

Morrison et al. (2007)

Microtubules (MTs)

Tubulin modification (7) GL50803_95661

tubulin tyrosine ligase

tubulin tyrosine ligase PF03133

Morrison et al. (2007)

GL50803_14498

tubulin tyrosine ligase

tubulin tyrosine ligase PF03133

Morrison et al. (2007)

GL50803_8592

tubulin tyrosine ligase

tubulin tyrosine ligase PF03133

Morrison et al. (2007)

GL50803_10382

tubulin tyrosine ligase

tubulin tyrosine ligase PF03133

Morrison et al. (2007)

GL50803_9272

tubulin tyrosine ligase

tubulin tyrosine ligase PF03133

Morrison et al. (2007)

GL50803_8456

tubulin tyrosine ligase

tubulin tyrosine ligase PF03133

Morrison et al. (2007)

GL50803_10801

tubulin tyrosine ligase

tubulin tyrosine ligase PF03133

Morrison et al. (2007)

MT dynamics/regulators (10) GL50803_14373

dynamin

dynamin

PF00350

Morrison et al. (2007)

GL50803_14048

EB1

EB1

PF03271

Dawson et al. 2007b)

GL50803_96399

xmap215

XMAP215 family

none

Morrison et al. (2007)

GL50803_91480

stu2

Stu2 family

none

Morrison et al. (2007)

GL50803_11953

katanin (p80)

WD domain, G-beta repeat

PF00400

Morrison et al. (2007)

GL50803_15368

katanin (p60)

AAA ATPase family

PF00004

Morrison et al. (2007)

GL50803_16535

tubulin-specific chaperone E

cap_gly MT binding domain

PF01302

Morrison et al. (2007) (Continued)

Chap. 18 Primary Microtubule Structures in Giardia

279

Table 18.1 (Continued) GiardiaDB

Gene name

Family

PFAM

Molecular function

References

GL50803_5374

tubulin-specific chaperone B

cap_gly MT binding domain

PF01302

Morrison et al. (2007)

GL50803_16893

tip elongation aberrant protein 1

kelch2 motif

PF0646

Morrison et al. (2007)

GL50803_15054

kelch-repeat containing protein

kelch2 motif

PF0646

Morrison et al. (2007)

Gamma Turc/Tusc complex (3) GL50803_17429

gcp-2

Spc97_Spc98

PF04130

MT metabolism

Morrison et al. (2007)

GL50803_12057

gcp-3

Spc97_Spc98

PF04130

MT metabolism

Morrison et al. (2007)

GL50803_114218

gamma-tubulin

tubulin

PF00091

MT metabolism

Morrison et al. (2007)

Basal-body associated (11) GL50803_104685

caltractin

caltractin

PF00036

Signalling

Meng et al. (1996)

GL50803_6744

centrin

centrin

PF00036

Signalling

Belhadri et al. (1995)

GL50803_5462

delta-tubulin

tubulin

PF00091

MT metabolism

Morrison et al. (2007)

GL50803_6336

epsilon-tubulin

tubulin

PF00091

MT metabolism

Morrison et al. (2007)

GL50803_5167

VFL3

centriole positioning

none

GL50803_15956

FAP52

basal body proteome BUG14

SSF50978

GL50803_15455

PACRG1

basal body proteome as BUG21; PACRG parkin co-regulated gene. E04F6.2 like protein

PF10274

GL50803_13372

FAP45

BUG28

SSF50978

GL50803_32375

POC18

basal body proteome

none

Protein interaction

Keller et al. (2005)

GL50803_33762

POC1

basal body proteome WD repeat protein

SSF50978

Protein interaction

Keller et al. (2005)

GL50803_14135

NDK7

(BUG5) in basal body proteome as BUG5

Merchant et al. (2007) Protein interaction

Keller et al. (2005) Keller et al. (2005)

Keller et al. (2005)

BBSome (5) GL50803_8738

BBS1

none

Morrison et al. (2007)

GL50803_23934

BBS2

none

Morrison et al. (2007)

GL50803_10529

BBS4

TPR_1 tetratricopeptide repeat

PF01515

Protein interaction

Morrison et al. (2007)

GL50803_8146

BBS5

DUF1448 domain of unknown function

PF07289

Protein interaction

Morrison et al. (2007) (Continued)

280

S. C. Dawson

Table 18.1 (Continued) GiardiaDB

Gene name

Family

PFAM

Molecular function

References

GL50803_8508

BBS8

TPR_1 tetratricopeptide repeat

PF01515

Protein interaction

Morrison et al. (2007)

Axoneme structure (30) axonemal dyneins (IDAs) GL50803_100906

IAD-1alpha

IAD-1alpha dynein heavy chain (DHC) family

PF03028

Flagellar structure

Wickstead et al. (2007)

GL50803_94440

IAD-1beta

IAD-1beta dynein heavy chain (DHC) family

PF03028

Flagellar structure

Wickstead et al. (2007)

GL50803_40496

IAD-4

IAD-4 dynein heavy chain (DHC) family

PF03028

Flagellar structure

Wickstead et al. (2007)

GL50803_37985

IAD-4

IAD-4 dynein heavy chain (DHC) family, partial

PF03028

Flagellar structure

Wickstead et al. (2007)

GL50803_111950

IAD-5

IAD-5 dynein heavy chain (DHC) family

PF03028

Flagellar structure

Wickstead et al. (2007)

Axonemal dyneins (ODAs) GL50803_17265

OAD-alpha

OAD-alpha dynein heavy chain (DHC) family

PF03028

Flagellar structure

Wickstead et al. (2007)

GL50803_17243

OAD-beta

OAD-beta dynein heavy chain (DHC) family

PF03028

Flagellar structure

Wickstead et al. (2007)

GL50803_16450

rsp3

radial spoke protein 3

PF06098

Flagellar structure

Morrison et al. (2007)

GL50803_17278

rsp9

radial spoke protein 9; A subunit in the radial spoke head; (pf17)

none

Flagellar structure

Morrison et al. (2007)

GL50803_114462

axonemal p66 (RSP6)

outer dynein armnone docking complex subunit 2 (ODA-DC 2)

Flagellar structure

Morrison et al. (2007)

PF2

component of dynein regulatory complex (DRC) of flagellar axoneme; trypanin

Flagellar structure

Morrison et al. (2007)

PF20

central pair WD-repeat PF00400 protein of the central pair; associates with the intermicrotubule bridge.

Flagellar structure

Morrison et al. (2007)

Radial spokes

Dynein regulatory complex GL50803_16540

PD936484

Central pair GL50803_16500

(Continued)

Chap. 18 Primary Microtubule Structures in Giardia

281

Table 18.1 (Continued) GiardiaDB

Gene name

Family

GL50803_16202

PF16

GL50803_137712

GL50803_14568

PFAM

Molecular function

References

central pair associated PF00514 protein

Flagellar structure

Morrison et al. (2007)

HY3 (FAP74)

(HYD3) similar to mouse hydrocephaly protein hydin HY3

SSF52540

Flagellar structure

Morrison et al. (2007)

PP1

Ser/Thr protein phosphatase PP1alpha 2 catalytic subunit

SSF56300

Signalling

Morrison et al. (2007)

GL50803_11867

RIB43a

associated with protofilament ribbons of flagellar microtubules

PF05914

Flagellar structure

Morrison et al. (2007)

GL50803_41512

RIB72

novel component of the ribbon compartment of flagellar MTs

PS51336

Flagellar structure

Morrison et al. (2007)

GL50803_16263

DIP13

deflagellation inducible protein; Sjogren’s syndrome nuclear autoantigen 1

PD968187

MT Metabolism

Morrison et al. (2007)

GL50803_102248

MBO2

move backward only mutant defective in ciliary waveform.

PD936484

Protein interaction

Morrison et al. (2007)

GL50803_14048

EB1

EB1

PF03271

MT metabolism

Dawson et al. (2007b)

GL50803_5333

calmodulin

EF-hand domain

SSF47473

Signalling

Morrison et al. (2007)

GL50803_7439

PP2A

Ser/Thr phosphatase SSF48371 2A, 65 kDa reg sub A; ARM repeat domain

Signalling

Morrison et al. (2007)

GL50803_14004

long-flagella protein LF4

protein kinase domain PF00069 (MAP kinase)

Flagellar regulation

Morrison et al. (2007)

GL50803_137716

GASP-180

ankyrin repeat family

PF00023

Protein interaction

Elmendorf et al. (2005)

GL50803_5649

alpha10-giardin

annexin

PF00191

Protein interaction

Weiland et al. (2005)

GL50803_15097

alpha14-giardin

annexin

PF00191

Protein interaction

Weiland et al. (2005)

GL50803_15101

alpha17-giardin

annexin

PF00191

Protein interaction

Weiland et al. (2005)

GL50803_7796

alpha2-giardin

annexin

PF00191

Protein interaction

Weiland et al. (2005)

GL50803_7797

alpha5-giardin

annexin

PF00191

Protein interaction

Weiland et al. (2005)

Axoneme-associated

(Continued)

282

S. C. Dawson

Table 18.1 (Continued) GiardiaDB

Gene name

Family

PFAM

Molecular function

References

GL50803_103437

alpha9-giardin

annexin

PF00191

Protein interaction

Weiland et al. (2005)

Axoneme-associated (40 putative) GL50803_12046

SPEF1

SSF47576

Signalling

Merchant et al. (2007)

GL50803_24423

SSA17

SSF51045

Protein interaction

Merchant et al. (2007)

GL50803_13809

TEX9

(FBB15)

SSF46966

Protein interaction

Merchant et al. (2007)

GL50803_4538

DPY30

(FBB12) chromatin modifying protein complex member; C. elegans defective in male sensory behavior

PF05186

Protein interaction

Merchant et al. (2007)

GL50803_15834

CTO59

(FBB5) similar to C21orf59 (CTO59)

GL50803_2523

FAP100

GL50803_87817

FAP118

GL50803_8865

FAP122

GL50803_17567

FAP134

GL50803_6245

Merchant et al. (2007) none

tubby superfamily protein

Merchant et al. (2007)

SSF50978

Protein interaction

Merchant et al. (2007)

PD968187

Signalling

Merchant et al. (2007)

SSF52075

Protein interaction

Merchant et al. (2007)

FAP155

SSF52047

Protein interaction

Merchant et al. (2007)

GL50803_22806

FAP184

SSF46579

Protein modification

Merchant et al. (2007)

GL50803_17116

FAP198

cyt-b5 -like domain in SSF55856 the N terminus

Metabolism

Merchant et al. (2007)

GL50803_15364

FAP251

SSF50978

Protein interaction

Merchant et al. (2007)

GL50803_6455

FAP263

none

GL50803_16768

FAP43

SSF50978

GL50803_21048

FAP47

none

GL50803_102438

FAP44

GL50803_37452

FAP50

GL50803_11321

FAP57

GL50803_95653

FAP59

protein phosphatase PP1 regulatory subunit SDS22

WD40-repeat like

SSF50978

Merchant et al. (2007) Protein interaction

Merchant et al. (2007) Protein interaction

none WD40-repeat like

SSF50978

Merchant et al. (2007)

Merchant et al. (2007) Merchant et al. (2007)

Protein interaction

Merchant et al. (2007) Li et al. (2004) (Continued)

Chap. 18 Primary Microtubule Structures in Giardia

283

Table 18.1 (Continued) GiardiaDB

Gene name

Family

PFAM

Molecular function

GL50803_5186

FAP61

GL50803_16709

FAP66

GL50803_10924

FAP73

none

Merchant et al. (2007)

GL50803_9283

FAP74

none

Merchant et al. (2007)

none TPR repeat domain

SSF48452

References Merchant et al. (2007)

Protein interaction Merchant et al. (2007)

GL50803_10879

FAP253

GL50803_8423

FBB11

FBB4 none

Merchant et al. (2007)

GL50803_8942

FBB5

none

Merchant et al. (2007)

GL50803_102438

FBB17

PF00246

GL50803_21110

FBB9

none

GL50803_5787

MOT12

SSF52540

Protein metabolism

Merchant et al. (2007)

GL50803_24043

MOT16

SSF47576

Signalling

Merchant et al. (2007)

GL50803_23330

MOT17

SSF52540

Signalling

Merchant et al. (2007)

GL50803_11638

MOT18

SSF81301

RNA metabolism Merchant et al. (2007)

GL50803_7792

MOT37

SSF52075

Protein interaction

Merchant et al. (2007)

GL50803_4897

MOT39

SSF57850

Protein turnover

Merchant et al. (2007)

GL50803_15868

MOT4

SSF57850

Protein turnover

Merchant et al. (2007)

GL50803_134441

MOT52

microtubule regulation and metabolism

none

Flagellar structure

Merchant et al. (2007)

GL50803_16996

NKRN1

(FAP106)

none

Signalling

Merchant et al. (2007)

GL50803_27147

UNC119

signal transduction protein

SSF81296

Trafficking

Merchant et al. (2007)

CH domain

topoisomerase I-related protein

Merchant et al. (2007)

Protein metabolism

Li et al. (2004) Merchant et al. (2007)

Intraflagellar transport, IFT (18) IFT complex A (2) GL50803_17251

IFT140

intraflagellar transport none protein IFT140

Flagellar transport

Hoeng et al. (2008)

GL50803_16547

IFT122

intraflagellar transport none protein IFT122

Flagellar transport

Briggs et al. (2004)

GL50803_17105

IFT172

intraflagellar transport none protein IFT172

Flagellar transport

Briggs et al. (2004)

GL50803_7664

IFT46

intraflagellar transport none protein IFT46; FAP32

Flagellar transport

Briggs et al. (2004)

IFT complex B (8)

(Continued)

284

S. C. Dawson

Table 18.1 (Continued) GiardiaDB

Gene name

Family

GL50803_112963

IFT52

GL50803_14713

PFAM

Molecular function

References

intraflagellar transport none protein IFT52; osm-6.

Flagellar transport

Briggs et al. (2004)

IFT57

intraflagellar transport none protein IFT57

Flagellar transport

Briggs et al. (2004)

GL50803_9750

IFT74/72

intraflagellar transport none protein IFT74/72

Flagellar transport

Briggs et al. (2004)

GL50803_17223

IFT80

intraflagellar transport PF00400 protein IFT80; WD domain, G-beta repeat

Flagellar transport

Briggs et al. (2004)

GL50803_15428

IFT81

intraflagellar transport none protein IFT81

Flagellar transport

Hoeng et al. (2008)

GL50803_16660

IFT88

intraflagellar transport none protein IFT88

Flagellar transport

Briggs et al. (2004)

IFT complex B associated (4) GL50803_87202

DYF-1

PR protein; TPR5 (FAP259)

SSF48452

Protein interaction

Merchant et al. (2007)

GL50803_16707

DYF-3

D. rerio cystic kidney disease gene qilin; FAP22; Clusterin associated protein

PF10234

Protein interaction

Merchant et al. (2007)

GL50803_9098

DYF-11

MT associated TRAF3 interacting protein (FAP116)

PF10243

Trafficking

Merchant et al. (2007)

GL50803_16375

DYF-13

(FBB2) required for ciliogenesis in C. elegans

SSF48452

Protein interaction

Merchant et al. (2007)

GL50803_114885

KAP

kinesin-associated protein; non-motor subunit of kinesin-II complex

PF05804

Flagellar transport

Morrison et al. (2007)

GL50803_16456

GiKIN2a

kinesin-2

PF00225

Flagellar transport

Hoeng et al. (2008)

GL50803_17333

GiKIN2b

kinesin-2

PF00225

Flagellar transport

Hoeng et al. (2008)

GL50803_93736

cytoDHC-1b

cytoDHC1b, putative IFT cytoplasmic dynein 1b family

PF03028

Flagellar transport

Wickstead et al. (2007)

SF-assemblin

PF06705

Baker et al. (1988)

none

Nohria et al. (1992)

PF06705

Elmendorf et al. (2001)

IFT motors (4)

Ventral disc-associated (8) GL50803_4812

beta-giardin

GL50803_17230

gamma-giardin

GL50803_86676

delta-giardin

SF-assemblin

(Continued)

Chap. 18 Primary Microtubule Structures in Giardia

285

Table 18.1 (Continued) GiardiaDB

Gene name

Family

PFAM

Molecular function

References

GL50803_4410

SALP-1

SF-assemblin

PF06705

Palm et al. (2003)

GL50803_7796

alpha2-giardin

annexin

PF00191

Weiland et al. (2005)

GL50803_11683

alpha3-giardin

annexin

PF00191

Weiland et al. (2005)

GL50803_7797

alpha5-giardin

annexin

PF00191

Weiland et al. (2005)

GL50803_15101

alpha17-giardin

annexin

PF00191

Weiland et al. (2005)

Median body associated (2) GL50803_16343

median body protein

MBP

none

Holberton et al. (1995)

GL50803_14048

EB1

EB1

PF03271

Dawson et al. (2007b)

Spindle, kinetochore-associated (3) GL50803_15248

bub2

TBC domain

PF00566

Morrison et al. (2007)

GL50803_100955

mad2

mitotic checkpoint protein

PF00557

Morrison et al. (2007)

GL50803_14048

EB1

EB1

PF03271

Dawson et al. (2007b)

MT motor proteins Other flagellar kinesins (22) GL50803_13825

GiKIN1

kinesin-1

PF00225

Wickstead et al. (2006)

GL50803_6262

GiKIN3a

kinesin-3

PF00225

Wickstead et al. (2006)

GL50803_102101

GiKIN3b

kinesin-3

PF00225

Wickstead et al. (2006)

GL50803_112846

GiKIN3c

kinesin-3

PF00225

Wickstead et al. (2006)

GL50803_16650

GiKIN4

kinesin-4

PF00225

Wickstead et al. (2006)

GL50803_16425

GiKIN5

kinesin-5

PF00225

Wickstead et al. (2006)

GL50803_102455

GiKIN6a

kinesin-6

PF00225

Wickstead et al. (2006)

GL50803_15134

GiKIN6b

kinesin-6

PF00225

Wickstead et al. (2006)

GL50803_15962

GiKIN7

kinesin-7

PF00225

Wickstead et al. (2006)

GL50803_4371

GiKIN8

kinesin-8

PF00225

Wickstead et al. (2006)

GL50803_10137

GiKIN9a

kinesin-9

PF00225

Wickstead et al. (2006)

GL50803_6404

GiKIN9b

kinesin-9

PF00225

Wickstead et al. (2006)

GL50803_16945

GiKIN13

kinesin-13

PF00225

Dawson et al. (2007b)

GL50803_8886

GiKIN14a

kinesin-14

PF00225

Wickstead et al. (2006)

GL50803_13797

GiKIN14b

kinesin-14

PF00225

Wickstead et al. (2006) (Continued)

286

S. C. Dawson

Table 18.1 (Continued) GiardiaDB

Gene name

Family

PFAM

Molecular function

References

GL50803_7874

GiKIN16a

kinesin-16

PF00225

Wickstead et al. (2006)

GL50803_16161

GiKIN16b

kinesin-16

PF00225

Wickstead et al. (2006)

GL50803_16224

GiKIN20

orphan

PF00225

Wickstead et al. (2006)

GL50803_17264

GiKIN21

orphan

PF00225

Wickstead et al. (2006)

GL50803_14070

GiKIN22

orphan

PF00225

Wickstead et al. (2006)

GL50803_112729

GiKIN23

orphan

PF00225

Wickstead et al. (2006)

GL50803_11442

GiKIN24

orphan

PF00225

Wickstead et al. (2006)

GL50803_11177

KLC

kinesin light chain

Morrison et al. (2007)

Other Dynein Heavy Chains (7) GL50803_17478

cytoDHC

cytoDHC cytoplasmic f dynein heavy chain family

MT metabolism

Wickstead et al. (2007)

GL50803_103059

dynein heavy chain (DHC) family

PF03028

MT metabolism

Wickstead et al. (2007)

GL50803_8172

dynein heavy chain (DHC) family, partial

PF03028

MT metabolism

Wickstead et al. (2007)

GL50803_16804

dynein heavy chain (DHC) family

PF03028

MT metabolism

Wickstead et al. (2007)

GL50803_101138

dynein heavy chain (DHC) family

PF03028

MT metabolism

Wickstead et al. (2007)

GL50803_10538

dynein heavy chain (DHC) family

PF03028

MT metabolism

Wickstead et al. (2007)

GL50803_29256

axonemal dynein heavy chain, partial

PF03028

MT metabolism

Wickstead et al. (2007)

Dynein light chains (10) GL50803_4236

DYNLT1 (Tctex1/ LC9)

Tctex-1 family

PF03645

MT metabolism

Wickstead et al. (2007)

GL50803_4463

LC1

dynein light chain (DLC) family

PF01221

MT metabolism

Wickstead et al. (2007)

GL50803_7578

LC5?

dynein light chain (DLC) family

PF01221

MT metabolism

Wickstead et al. (2007)

GL50803_9848

LC8

dynein light chain (DLC) family

PF03645

MT metabolism

Wickstead et al. (2007)

GL50803_13575

DYNLT2 (Tctex2/ LC19)

Tctex-1 family

PF01221

MT metabolism

Wickstead et al. (2007)

GL50803_14270

roadblock/LC7

roadblock-related dynein light chain

PD03259

MT metabolism

Wickstead et al. (2007)

GL50803_27308

LC4

dynein light chain (DLC) family

PF01221

MT metabolism

Wickstead et al. (2007) (Continued)

Chap. 18 Primary Microtubule Structures in Giardia

287

Table 18.1 (Continued) GiardiaDB

Gene name

Family

PFAM

Molecular function

References

GL50803_15606

Tctex-I

Tctex-1 family

PF03645

MT metabolism

Wickstead et al. (2007)

GL50803_17371

DYNLT1 (Tctex1/ LC9)

Tctex-1 family

PF03645

MT metabolism

Wickstead et al. (2007)

GL50803_15124

roadblock/LC7

roadblock/LC7 domain family

PD03259

MT metabolism

Wickstead et al. (2007)

axonemal dynein light PF10211 chain family, p28

MT metabolism

Wickstead et al. (2007)

Dynein light intermediate chain (1) GL50803_13273

axonemal DLIC

Dynein intermediate chain (3) GL50803_6939

IC70

dynein intermediate chain (DIC) family

PF05783

MT metabolism

Wickstead et al. (2007)

GL50803_10254

IC138

dynein intermediate chain (DIC) family

PF05783

MT metabolism

Wickstead et al. (2007)

GL50803_33218

IC78

dynein intermediate chain (DIC) family, Flagellar outer dynein arm intermediate chain, ODA-IC1

PF05783

MT metabolism

Wickstead et al. (2007)

localization of another four annexins was not investigated (α12-giardin, α13-giardin, α18-giardin, and α19-giardin). The data on annexin localization to specific structures in the disc is somewhat contradictory, however (Elmendorf, Dawson et al. 2003) . In early studies, Peattie et. al. used 2D electrophoresis to identify annexins and produce polyclonal antibodies that recognized microribbons. This antibody preparation was likely contaminated with co-migrating β-giardin (Elmendorf et al., 2003), thus the published microribbon localization of either β-giardin or the α-giardins is suspect (Elmendorf et al., 2003). A probable mechanism for generating conformational changes in the disc leading to attachment could be via disc-associated annexins (α3-giardin, α5-giardin, and α17-giardin) directly linking the rigid structure of the disc to the more flexible membrane, and modulating this linkage in response to intracellular calcium fluxes. Annexins thus could represent excellent candidates for rational drug design in Giardia if their functions and mutant phenotypes are better characterized. β-giardin is an SF-assemblin homolog that assembles into 2.5 nm filaments in vitro, and co-purifies

with giardial MTs (Holberton, 1981; Crossley and Holberton, 1985). In Chlamydomonas SF-assemblin forms 2.5 nm non-motile contractile filaments at the base of the flagella (Weber et al., 1993). SF-assemblin knockdowns cause flagellar assembly defects (Lechtreck and Grunow 1999). It is possible that βgiardin, δ-giardin, and SALP-1 form the structural basis of the microribbons upon which other associated proteins assemble (Feely, 1990). Other proteins comprising the ventral disc, such as γ-giardin, have no known homologs in other organisms. The functional role and precise localization of γ-giardin in the disc remains unknown. On the basis of complexity of the disc structure, it is highly likely that many discassociated proteins remain to be discovered.

18.2.1 The Role of the Ventral Disc in Giardial Attachment One of the more striking features of the trophozoite is the presence of the large and complex ventral disc (VD) structure (Fig. 18.2). This highly organized

288

S. C. Dawson

A

B

mr

ba

afl

vd vlf

lc

cb fr

vfl

1 μm

200 nm

C

Fig. 18.2 Structural elements of the ventral disc. (A) Scanning electron micrograph of a detergent-extracted cytoskeleton, showing the MT spiral array of the ventral disc with the microribbons (vd = ventral disc, ba = bare area, lc = lateral crest, vlf = ventrolateral flange, fn = funis, vfl = ventral flagella. afl = anterior flagella). (B) High resolution SEM image of a detergent-extracted ventral disc highlighting the microribbons (mr) and crossbridges (cb) that connect microribbons of the spiral. (C) Schematic representation of the ventral disc showing the MTs, microribbons and crossbridges. SEM and TEM images courtesy of Joel Mancuso, UC Berkeley

right-handed spiral MT structure is essential to virulence as it promotes giardial attachment to the intestinal microvilli (Elmendorf et al., 2003). Attachment is a critical aspect of the giardial life cycle and virulence, as it allows parasites to colonize the intestine and resist peristalsis. The ventral disc is composed of three main structural elements: 1) a concave right-handed spiral MT array; 2) trilaminar microribbons attached perpendicularly to the length of the MT spiral and extending into the cytoplasm; and 3) crossbridges that horizontally link the microribbons (Holberton, 1973a, 1981; Holberton and Ward, 1981; Feely et al., 1982; Crossley and Holberton 1983, 1985). The microtu-

bules spiral about one and one quarter turns. A “bare area”, central to the region where the disc spiral MTs overlap, contains numerous membrane-bound vacuoles (Elmendorf et al., 2003). Microribbons, associated with the MT spiral, are ~25 nm thick, and extend 150–400 nm into the cytoplasm (Holberton, 1973a, 1981). The trilaminar microribbons are believed to be comprised of two sheets of globular subunits sandwiched around a fibrous inner core (Holberton, 1981). The spacing between the microtubules and microribbons of the disc spiral is uniform (~250-300 nm), and they are linked by numerous crossbridges. “Sidearms”, or electron dense structures of unknown com-

Chap. 18 Primary Microtubule Structures in Giardia

position (Holberton, 1973a), appear to link the microtubule spiral to the plasma membrane. The MT-associated structures (microribbons, crossbridges, and sidearms) (Feely, 1990) may act to mediate disc conformational changes enabling attachment. Many proteins comprising these primary disc structures remain to be determined. To date no known proteins associated with microtubule dynamics or movements (e.g., kinesins or dyneins), or more generic microtubule-associated proteins (MAPs) have been shown to associate with the ventral disc. Finally, a fibrillar structure of unknown composition – the lateral crest – surrounds the ventral disc and may have contractile functions (Kulda and Noynkova, 1995). The primary structure of the ventral disc is microtubule-based, but actin has been reported to localize to the lateral crest and periphery of the disc. Complicating this view of ventral disc structure, various heterologous actin antibodies have produced contradictory localizations – possibly due to the divergence of the giardial actin gene (Elmendorf et al., 2003; Morrison et al., 2007). The proposed function of the lateral crest as a modulator of attachment dynamics is intriguing, although microfilaments have not been identified cytologically in either the disc or lateral crest (Chavez and Martinez-Palomo, 1995). Microbial attachment to surfaces is widespread and diverse, permitting microbes to colonize surfaces and, likely, physiologically alter the local environment of attachment. Attachment mechanisms include focal adhesions and podosomes in amoebae and fibroblasts (Décavé et al., 2002; Evans and Matsudaira, 2006; Pellegrin and Mellor, 2007), and bacterial holdfasts (Tsang et al., 2006). To proliferate and colonize the host small intestine, Giardia must remain attached to the intestinal villi. The parasites attach to biological substrates (in vivo attachment), as well as to inert laboratory substrates like plastic or glass (in vitro attachment). Giardial trophozoites use the ventral disc to orient ventral side “down” to either biological or inert substrates via an undefined mechanism (Holberton, 1973b, 1974) that might involve suction (Hansen et al., 2006). Disc mediated attachment in Giardia likely involves disc conformational changes and structural dynamics that generate a negative pressure beneath the disc (Hansen et al., 2006). Thus, in contrast to attachment mechanisms commonly used by other microbes, ventral disc-mediated attachment

289

appears to be without precedent in the microbial world. The mechanism of giardial attachment to in vivo or in vitro substrates remains somewhat controversial. As new disc-associated proteins are identified, molecular genetic analysis of both structural and regulatory disc-associated proteins, combined with visualization of live ventral disc dynamics, will be pivotal in assessing the mechanism of giardial attachment. However, proposed models of giardial attachment to surfaces can be broadly summarized in several categories that include: ligand-independent interactions (electrostatic or van der Waals forces) (Hansen et al., 2006), ligand-dependent interactions (Nash et al., 1983; Inge et al., 1988; Magne et al., 1991; Ortega-Barria et al., 1994; Sousa et al., 2001), clutching mechanisms (Holberton, 1973a, 1973b; Feely and Erlandsen, 1981; Inge et al., 1988; Elmendorf et al., 2003), or suction-mediated mechanisms (Holberton, 1973a, b, 1974; Feely and Erlandsen, 1981; Elmendorf et al., 2003; Hansen et al., 2006; Hansen and Fletcher, 2008) (reviewed in Elmendorf et al., 2003). Each of the proposed models is not necessarily mutually exclusive. Multiple mechanisms may contribute to various aspects of in vivo attachment. Nonetheless, disc-mediated suction is likely sufficient for in vitro attachment (Hansen et al., 2006; Hansen and Fletcher, 2008). Cytological descriptions, rather than molecular genetic functional analyses, underlie most models of giardial attachment (Holberton, 1973a, 1974; Benchimol, 2004; Piva and Benchimol, 2004; Mariante et al., 2005). Light or electron microscopy has been used to observe attached trophozoites in various experimental situations. Based on such observations, two mechanisms involving dynamic movements of the disc or flagella have been proposed to produce suction, and thus generate a negative pressure differential under the disc. Perhaps one of the more compelling proposed mechanisms of attachment is that conformational changes of the disc could be sufficient to produce suction-based attachment by generating suction in vitro, or alternatively, “clutching” in vivo (Elmendorf et al., 2003). The disc is reported to undergo a conformational change upon attachment to become less concave (Sousa, 2001), thus the principal structural components of the disc (MTs, microribbons, crossbridges and/or motor pro-

290

teins) could generate and maintain attachment forces. To date, however, no disc-associated protein has been shown to be necessary or sufficient to modulate disc conformational changes. Microtubules are highly conserved linear polymers comprised of heterodimers of α- and β-tubulin. In general, microtubule-based cellular structures are rapidly assembled, disassembled, and remodeled into distinct arrays such as the ventral disc, funis, axonemes, median body, or mitotic spindles. Individual MT polymers exhibit intrinsic dynamic instability where MTs exist either in growth (polymerization) phases or and shrinkage (depolymerization) phases. MT dynamics are also actively modulated by various MT regulators including those that regulate assembly, disassembly, or rates of catastrophe. Various drugs affect the dynamic nature of MTs. Thus MT-disrupting and MT-stabilizing drugs are valuable tools to probe the assembly dynamics of microtubules in Giardia. As the disc is comprised primarily of a large (over 5 μm in length) microtubule spiral, intrinsic or regulated MT dynamics may contribute to disc conformational dynamics responsible for attachment. Anti-microtubule drugs affect the dynamics of MTs (although generally not stable, polymerized MTs) by both sequestering tubulin monomer pools and inhibiting polymerization (nocodazole, colchicine, and oryzalin) or by stabilizing growing MTs (Taxol) (Long, 1994; Pellegrini and Budman, 2005; Bhattacharyya et al., 2008). Cytoskeletal drugs would negatively impact attachment if intrinsic or regulated MT dynamics were involved in generating disc conformational dynamics. Multiple studies have investigated the role of microtubule dynamics in attachment using anti-microtubule drugs with somewhat conflicting conclusions (Feely and Erlandsen, 1982; Mariante et al., 2005). In general, however, it appears that MTs of the eight flagella, the median body, and the mitotic spindles are dynamic as they are sensitive to these drugs. The ventral disc MTs are unaffected (Sagolla et al., 2006) in interphase. One would expect drugs that affect MT polymerization/depolymerization to affect all cytoskeletal structures during their assembly. Thus drugs that limit MT polymerization, such as albendazole, have minimal effects on attachment in interphase (short incubation periods), and effects such as severe deformation of the disc are only observed after long incubation periods involving multiple

S. C. Dawson rounds of cell division (Chavez et al., 1992; Oxberry et al., 1994). The lack of intrinsic or active MT dynamics does not imply that the disc structure itself is not dynamic; rather it implies that the MTs of the disc do not undergo rapid polymerization/depolymerization in interphase.

18.2.2 The Structure and Putative Function of the Median Body The median body forms the crooked giardial “smile” (Fig. 18.1), and is another MT array of unknown function (Piva and Benchimol, 2004; Elmendorf et al. 2003). This novel structure is present on the dorsal side of trophozoites roughly perpendicular to the caudal axonemes, posterior to the ventral disc. The median body is generally described as a “haystack” of semi-organized or bundled MTs. Median body MTs appear to be dynamic during interphase as they are sensitive to both MT stabilizing and MT depolymerizing drugs (Sagolla et al., 2006; Dawson et al., 2007). Median body MT dynamics have recently been shown to be regulated by the depolymerizing kinesin motor protein, kinesin-13 (Dawson et al., 2007). This is in contrast to other recent works in which antibodies targeting various tubulin modifications localized to the median body, possibly indicating the presence of more stable microtubules. It is possible that the median body possesses a mixture of dynamic and more stable microtubules, and that median body MTs – stable or dynamic – may still have a yet undiscovered critical function in the giardial life cycle. Several clues to median body function derive from analyses of median body structure and shape throughout the life cycle. The shape and presence of the median body varies during the cell cycle; it disappears altogether following mitosis, prior to disc division (Sagolla et al., 2006). Remaining attached to the villi during cell division would prevent Giardia from being swept through the digestive tract by peristalsis. Thus the median body may serve as a reservoir of tubulin subunits for duplicating MT structures, such as the daughter ventral discs, prior to cytokinesis (Brugerolle, 1975; Feely, 1990). This would permit the rapid assembly of the ventral disc so that trophozoites could quickly reattach to the intestinal villi. In support of this hypothesis, Brugerolle identified small

Chap. 18 Primary Microtubule Structures in Giardia

A

291

C

B

afl

afl afl vd vfl

afl

vfl

pfl

mb vfl pfl

vfl

pfl pfl

cfl

cfl

cfl cfl

D

E afl

fn

vfl fn

cfl fn

cfl pfl cfl

Flagellar motility

F

Dorsolateral tail flexion

G afl afl cfl pfl

vfl

afl

afl pfl vfl

Directional movement

Rotational (tumbling) movement

Fig. 18.3 Flagellar structure and flagellar-based movements. Scanning electron micrograph of the trophozoite ventral side (A) (scale bar = 5 μm) highlighting the four flagellar pairs (afl = anterior, pfl = posteriorlateral, vfl = ventral, cfl = caudal); image courtesy of Joel Mancuso, UC Berkeley. In panels B and C, the long cytoplasmic regions and the membrane-bound portions of all eight axonemes are visible (B = anti-tubulin-immunostaining, red; C = anti-tubulin immunostaining, red; anti-alpha14-annexin labels the membrane-bound axonemes (Szkodowska et al., 2002) in green; blue = DAPI; mb = median body). Scale bars = 5 μm. Panels D–G diagram various flagellar movements generated by each flagellar pair. In attached cells (D), flagellar motility is primarily evident in the ventral or anterior flagella. In panel E, “dorsolateral tail flexion”, or the lateral and/or dorsal flexing of the posterior end of the cell is attributed to the funis or caudal complex that may modulate caudal flagellar beating. Left-right directional movement has been attributed to the anterior flagella (F), and forward or downward movement to the anterior and ventral flagella. Rotational or tumbling movement (G) has been ascribed to anterior and/or posteriolateral flagellar beating. Ventral flagellar beating during tumbling is also apparent and may contribute to tumbling or positioning

292

“appendages” similar to the disc microribbons on median body MTs (Brugerolle, 1975). In addition, Crossley et al. (1986) showed β-giardin also localized to the median body of some cells. An alternative function of the median body has also been proposed, implicating this structure in detachment. To date, the function of the median body remains enigmatic; few studies have investigated the “reservoir” hypothesis or this alternative “detachment” hypothesis (Piva and Benchimol, 2004).

18.2.3 Flagellar Structure and Motility Giardia belongs to a phylogenetic group of protists termed diplomonads, whose defining characteristics are eight flagella and two nuclei (Feely 1990). Flagellar motility (Fig. 18.3) is required for Giardia to find suitable sites for attachment and subsequent colonization of the intestinal villi (Campanati et al., 2002). Giardia’s eight flagella are organized into four bilaterally symmetrical and functionally unique pairs: the anterior, the caudal, the posteriolateral, and the ventral (Fig. 18.3). In general, eukaryotic flagella extend from a basal body or centriole and are surrounded by a specialized flagellar membrane after they project from the cell surface. In Giardia, the eight flagellar basal bodies nucleate the axonemes and are positioned mainly between the two nuclei in the cell interior. The anterior basal bodies are located toward the anterior ends of the two nuclei. The axonemes are distinguished by the presence of long, cytoplasmic regions. Each axoneme also has a membrane-bound portion, and the ratio of the length of the cytoplasmic region to the length of the membrane-bound portion varies between each flagellar pair. The transition zones of all axonemes are restricted to regions proximal to the basal bodies, rather than to the entire cytoplasmic axoneme (Hoeng et al., 2008). The caudal axonemes run longitudinally along the anterior-posterior axis of the cell, exiting, and extending ~4 μm at the extreme posterior tip. The anterior axonemes cross over the ventral disc spiral before exiting and extending about ~8 μm at the anterior regions on the right and left sides of the trophozoite. The ventral axonemes exit and extend about 12 μm at the ventral side, posterior to the ventral disc in the “ventrolateral flange” region. Finally, the posteriolateral axonemes

S. C. Dawson are positioned at the lower third of the cell, angling outward and extending about 8 μm from the cell body. Electron-dense “collars” are found at the regions where each flagellum exits the cell body (Hoeng et al., 2008). This axonemal organization also distinguishes giardial flagella from those of other diplomonads (Adam, 2001). Giardia’s flagella produce complex and coordinated movements that are involved in motility, cell division, and possibly excystation or attachment (Fig. 18.3). The ventral flagella beat synchronously, while the anterior and posteriolateral flagellar pairs beat asynchronously. The anterior and ventral flagellar pairs are primarily required for forward and downward directional movements (Campanati et al., 2002). Ventral flagellar beating is also proposed to mediate suction-based attachment via the “hydrodynamic model” (Holberton, 1974). Rotational or tumbling movements of the trophozoites are associated with the beating of the anterior and/or posteriolateral flagella. Despite having a motile axonemal structure, the caudal flagella flex, rather than beat. This lack of beating of the caudal flagella has been attributed to the presence of a MT sheet, the “caudal complex” surrounding the cytoplasmic regions of the caudal axonemes. The funis is essentially a flared out region of at the midpoint of the cell body and derives from the MTs of the caudal complex. The “tail” region of trophozoites bends dorsally as well as laterally. These flexing movements have been termed “dorsal tail flexion” (Carvalho and Monteiro-Leal, 2004) and may derive from the flexing of the caudal complex, caudal axonemes, or funis (Fig. 18.3). Dorsal tail flexion has been associated with detachment (Owen, 1980), although this flexing also occurs when trophozoites are not attached. The microtubule sheets are thought to slide over each another and over the caudal axonemes during flexion, and dynein is proposed to mediate this sliding (Campanati et al., 2002). The role of flagellar beating in attachment also remains open to debate. Early attachment models invoked ventral flagellar beating as a means to generate a hydrodynamic force (Holberton, 1974) to create suction. These models are based on early reports that the ventral flagella beat when cells are attached, and stop beating when cells detach. Whether these observations reflect causality (ventral flagellar beating

Chap. 18 Primary Microtubule Structures in Giardia

causes attachment) or correlation (ventral flagella beat at the same time cells attach) remains to be determined. In spite of this logical discrepancy, several theoretical or observational arguments propose that the hydrodynamic currents generated by the ventral flagella (Holberton 1974; Ghosh et al., 2001) cause attachment. Even if it is not required to generate hydrodynamic currents, flagellar motility is doubtless important for positioning the cell parallel to the substrate and for movement toward suitable areas for attachment and colonization. Thus mechanistic studies should consider the relative contributions of both disc conformational dynamics and flagellar motility to giardial attachment. The canonical eukaryotic motile flagellum (Manton 1952) consists of a ring of nine doublet microtubules surrounding a central pair of singlet microtubules (“9 + 2”). This structure is conserved among diverse eukaryotic microbes (Porter and Sale, 2000; Smith and Yang, 2004; Nicastro et al., 2006). The microtubule structure of the axoneme has associated inner- and outer-arm dynein ATPases, nexin links between the outer doublet microtubules (Heuser et al., 2009), and radial spokes projecting from the A tubule of each outer doublet microtubules toward the singlet central pair microtubules. In spite of the extensive cytoplasmic regions, giardial axonemes have a conserved structure akin to more commonly studied flagella in experimental systems such as Chlamydomonas. Giardial axonemes possess the motile conserved 9 + 2 microtubule structure in both the cytoplasmic and membrane bound regions. Each of the eight giardial axonemes has radial spokes, dynein arms, and the outer doublets, and the central microtubule pair (Clark and Holberton, 1988; Elmendorf et al., 2003; Carvalho and Monteiro-Leal, 2004). Not all of the four flagellar pairs have characteristic flagellar waveforms, however (e.g., the causal axonemes tend to bend rather than beat). Giardia maintains a unique identity for each flagellar pair, and to date, several different pairs of axonemes have specific proteins that localize exclusively to either the cytoplasmic or membrane-bound regions. Such proteins associating with specific axonemal pairs include GASP-180, a member of a novel family of coiledcoil proteins (Elmendorf et al., 2005), and several α-giardins (Szkodowska et al., 2002; Weiland et al., 2005).

293

18.2.4 Structure and Putative Function of Axoneme-Associated Elements Novel axoneme-associated structures are associated with each flagellar pair in trophozoites (Friend, 1966) – their presence also serves as way to define the identity of each flagellar pair. Such axoneme-associated structures include: 1) the “marginal plate” associated with the anterior axonemes (Friend 1966), 2) the fin-like structures that extend from the ventral axonemes (Kulda and Nohynkova, 1995), 3) the electron dense material that is associated with the posteriolateral axonemes, and 4) the microtubules of the “caudal complex” or “funis” that surround and extend from the caudal axonemes. None of the giardial axoneme-associated structures are homologous to the paraflagellar rod (PFR), an extraaxonemal structure physically attached to the trypanosome axoneme (Blaineau et al., 2007). Giardial extra-axonemal structures may confer on each flagellar pair a unique structural identity and, likely, a unique functional role in motility or even attachment (Campanati et al., 2002). The axoneme-associated structures are both compositionally and functionally understudied (Elmendorf et al., 2003). The “marginal plate” and “striated fiber” structures associated with the anterior axonemes are located slightly dorsal to the anterior regions of the disc spiral arrays (Kulda, 1995). The “funis” (also described as the “caudal complex”) associates with the caudal axonemes, and has been the subject of several ultrastructural studies (Benchimol et al., 2004). The funis is comprised of MT sheets apparently nucleated from bands of linked MTs in the nuclear region of the caudal basal bodies (Benchimol et al., 2004). These MT sheets fan out laterally at the emergence of the ventral axonemes (Benchimol et al., 2004). Microtubule ends of the funis appear to be anchored in the cytoplasmic regions of the posteriolateral axonemes. Filamentous links to the underlying plasma membrane have also been reported (Benchimol et al., 2004). The funis has no known function, yet has been suggested to either have a structural role in maintaining the giardial cell shape or a potential role in the dorsolateral flexion of the posterior “tail” region during detachment (Benchimol et al., 2004). “Dorsolateral tail flexion” has thus been attributed to the funis (Ghosh et al., 2001), the caudal complex (Carvalho and Monteiro-Leal, 2004) or the caudal flagella (Campanati et al., 2002).

294

18.3 Flagellar Assembly and Interphase Flagellar Length Maintenance Because the growing membrane-bound axoneme excludes ribosomes, complex and coordinated targeting and transport of components synthesized in the cytoplasm is required to transport flagellar building blocks to distal elongating tip. Thus axonemes are assembled by extension and elongation at the distal tip rather than at the basal body. As originally described (Kozminski et al., 1993), intraflagellar transport (IFT) ensures the delivery of axonemal building blocks from the cell body to the distal flagellar tips through the continuous and bidirectional movement of large proteinaceous particles or “rafts” (reviewed recently in Rosenbaum and Witman, 2002; Scholey 2003). Recent identification of links between proper flagellar function and human ciliary diseases such as polycystic kidney disease (Pazour et al., 2000; Haycraft et al., 2001; Lin et al., 2003) and Bardet-Biedl syndrome (Li et al., 2004; Snell et al., 2004) underscore the importance of understanding flagellar assembly and length dynamics (Sloboda, 2002). Despite the fact that Giardia is a parasitic protist with eight flagella and thus may have certain derived features (Knight, 2004), IFT-based flagellar assembly (Briggs et al., 2004) appears to be as highly conserved as the structure of the giardial axoneme (Hoeng et al., 2008). The kinesin-2 heterotrimeric complex is comprised of two kinesin-2 homologs and the kinesin-associated protein (KAP) (Wedaman et al., 1996), and powers the anterograde movement of IFT proteinaceous rafts along the outer doublet of axonemes. The retrograde movement of IFT rafts toward the cell body is mediated by cytoplasmic dynein 1b (Signor et al., 1999). Homologous components of both the retrograde and anterograde IFT complexes (A and B), IFT complex B associated components (DYF-1, DYF-3, DYF-11, and DYF-13) and the kinesin-II heterotrimeric complex and IFT dynein are present in the giardial genome (see Table 18.1). The primary anterograde IFT motor in Giardia is the kinesin-2 heterotrimeric complex (Briggs et al., 2004; Morrison et al., 2007); Giardia does not contain homologs of the homodimeric OSM-3 complex found in both metazoans and ciliates. In Giardia, both IFT complex A and B components localize to the cytoplasmic and membrane-bound regions of axonemes. Kinesin-2::GFP fusions (GiKI-

S. C. Dawson N2a and GiKIN2b) and components of the IFT complex A (IFT140) and complex B (IFT81) raft localize along the length of axonemes and form foci at the eight distal flagellar tips and the flagellar pore regions. Thus, IFT particles likely dock on cytoplasmic portions of axonemes, and are observed to accumulate at the beginning of the membrane bound regions and at the distal flagellar tips. These areas likely represent the start and endpoints of the giardial IFT pathway (Hoeng et al., 2008). The lengths of flagella are also dynamic, and stable flagellar length is a balance between flagellar assembly and flagellar disassembly. Thus the equilibrium lengths of the eight giardial flagella are also maintained during interphase. Flagellar assembly and length maintenance rely upon intraflagellar transport (IFT) to provide building blocks to the distal flagellar tip (Kozminski et al., 1995). Cytoplasmic regions of giardial axonemes are not segregated from the site of protein synthesis. Is IFT is required for assembly of the cytoplasmic portions of axonemes? Previous work has demonstrated that cytoplasmic regions of axonemes, particularly the non-motile caudal pair, have a conserved flagellar ultrastructure, possessing the outer double MTs, canonical radial spokes, axonemal dynein arms, and the central microtubule pair (Clark and Holberton, 1988; Elmendorf et al., 2003; Carvalho and Monteiro-Leal, 2004). Thus the transition zone of all eight axonemes is restricted to small regions proximal to the basal bodies (as in Chlamydomonas), rather than to the entire cytoplasmic region. IFT raft proteins and the heterotrimeric kinesin-2 motor complex are in the Giardia genome (see Table 18.1) and have been shown to have conserved functions in assembly of the membrane-bound regions of flagella (Hoeng et al., 2008). In support, cytoplasmic axoneme length is unaffected by morpholinos that interfere with kinesin-2 expression or by the overexpression of a dominant negative kinesin-2 (Hoeng et al., 2008; Carpenter and Cande, 2009). Cytoplasmic regions of giardial axonemes do not require kinesin-2 for their assembly, suggesting that cytoplasmic axonemes are assembled by an IFT-independent mechanism. In summary, an IFT-independent mechanism may be responsible for cytoplasmic giardial axonemal assembly, whereas IFT-mediated assembly is required for the external membrane-bound regions. IFT particles and motors might assemble on cytoplasmic re-

Chap. 18 Primary Microtubule Structures in Giardia

gions of axonemes rather than at basal bodies in Giardia, and IFT-mediated axoneme assembly appears to be required only for membrane-bound regions of axonemes. Both IFT-mediated and non-IFT mediated assemblies of axonemes have been shown to occur simultaneously in the same cell in other organisms (Han et al., 2003; Briggs et al., 2004). The mechanism and temporal sequence by which the extra axonemal-associated structures (e.g., marginal plate, caudal complex or funis) are assembled during cell division remain unclear (Hoeng et al., 2008). As mentioned above, equilibrium flagellar length is a balance between rates of flagellar assembly and disassembly. In terms of axoneme disassembly, a depolymerizing kinesin – kinesin-13 – has been shown to be involved in flagellar length regulation in Giardia. Overexpression of a dominant negative kinesin13 resulted in long flagella, indicating a role of kinesin-13 in flagellar disassembly (Dawson et al., 2007b). In support, Giardia’s flagellar length is also sensitive to both MT stabilizing and destabilizing drugs (Dawson et al., 2007b). Treatment with the MT stabilizing drug Taxol results in all flagella extending over three times the average interphase length. Interestingly, the anterior flagellar length is less affected by microtubule drugs than the other flagellar pairs (Dawson et al., 2007b). Thus, it is possible that the different axonemes, such as the anterior axonemes, assemble at different rates. Thus, hierarchical levels of regulation act to maintain a stable flagellar length in Giardia, and include both intrinsic microtubule dynamics, and active assembly (by IFT) and active axonemal MT disassembly by kinesin-13.

18.4 Duplication and Division of Cytoskeletal Structures Giardia cell division and duplication of the disc and flagella are discussed in another chapter. In short, initial studies of giardial cell division indicated a lack of mitotic spindles, and novel, unprecedented mechanisms of chromosome segregation or nuclear division were proposed (Solari et al., 2003; Benchimol, 2004). However, two extranuclear spindles have recently been reported in Giardia (Sagolla et al., 2006). Chromosomes are segregated along the left–right (L–R) axis, and cytokinesis occurs along the longitudinal

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axis, perpendicular to the spindle (Sagolla et al., 2006). The eight flagella and basal bodies are not resorbed prior to cell division, and are instead involved in the organization and positioning of the two spindles. The giardial spindle MTs radiate from one of the flagellar basal bodies near each spindle pole, forming a sheath around the nuclear envelope. Each spindle pole is associated with at least one axoneme. The nuclear envelope remains, forming a barrier between cytoplasmic microtubule arrays and chromatin; there is no evidence of mixing of the chromatin between nuclei (Sagolla et al., 2006). Daughter cells inherit one copy of each parent nucleus. Presumptive kinetochore microtubules penetrate at the spindle poles through large polar openings in the nuclear membrane (Sagolla et al., 2006). Likely more than one microtubule is attached per kinetochore in Giardia. The internal (presumably kinetochore) microtubules extend only a few microns into the nucleus near the chromatin in late stage (anaphase B) nuclei.

18.5 The Cytoskeleton and Encystation/ Excystation In colonizing the small intestine, Giardia first excysts, then trophozoites find suitable site for attachment, attach to the intestinal villi, divide, and eventually encyst before passing into the environment. Each of these important stages in the giardial life cycle is facilitated by dynamics of the microtubule cytoskeleton. After ingestion, cysts transform into the swimming trophozoite stage and colonize the small intestine. Morphological aspects of excystation have been investigated using scanning electron microscopy (Feely 1986; Buchel et al., 1987). The stages of excystation are less well characterized with respect to cytoskeletal movements or dynamics, however, and such dynamics obviously play an important role in excystation. Flagellar motility has been suggested to play a mechanical role in the initial opening of the cyst; however, other contractile or MT-mediated forces have not been excluded (Feely, 1986; Buchel et al., 1987). A rounded trophozoite exits the cyst prior to progressive elongation and cytokinesis. The subsequent assembly of fully functional flagella and ventral discs allows the trophozoite to swim and attach to surfaces. Encysta-

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tion and excystation thus require flagellar disassembly and assembly; however, the details or stages of flagellar assembly/disassembly dynamics during these important transitions in the giardial life cycle have not been described at the cytological or molecular level. Prior to excystation, the trophozoite may undergo meiosis and nuclear fusion (Poxleitner et al., 2008). Cytoskeletal movements, in conjunction with the assembly of the cyst wall, transform trophozoites into the environmentally resistant cyst form. The shape of the trophozoite morphs from a flattened teardrop shape to a rounded ovoid shape as the cyst wall is assembled and cytoskeletal rearrangements occur during encystation (Midlej and Benchimol, 2009). The ventral disc structure transforms from a closed spiral disc to a horseshoe-shaped structure, and is subsequently fragmented and partially disassembled in the cyst. Flagella are internalized during cyst formation, yet do not completely resorb. Notably, internalized flagella are known to continue to beat inside the newly formed cyst (Midlej and Benchimol, 2009). With respect to both encystation and excystation, the mechanisms underlying cytoskeletal assembly, disassembly, and cytoskeletal movements remain virtually undescribed.

18.6 Perspectives Fundamental areas of giardial cytoskeletal biology remain to be explored. These include obtaining high resolution images of microtubule and actin-based cytoskeletal structures, identifying their protein composition, and assessing the role of microtubule-associated proteins in dynamic cytoskeletal movements throughout the giardial life cycle. Further investigations of cytoskeletal mechanisms should include: 1) giardial attachment via the ventral disc; 2) the function of the enigmatic median body; 3) biogenesis of the disc; and 4) morphogenesis during encystation/excystation. As most efforts to study the Giardia cytoskeleton have been cytological, future work should emphasize understanding details of Giardia’s elaborate MTbased structures and elucidating the molecular mechanisms of dynamic cytoskeletal movements. The mechanisms of some Giardia-specific microtubule dynamics – such as attachment, cell division, and encystation/excystation – are essentially uncharacterized at the molecular level. Previous technical

S. C. Dawson limitations have made studying giardial protein function at this level difficult (Davis-Hayman and Nash, 2002, Elmendorf et al., 2003), but recently developed molecular tools permit detailed analysis of cytoskeletal mechanisms (Carpenter and Cande, 2009). Reverse genetic tools to generate dominant negative mutants (Dawson et al., 2007b; Gaechter et al., 2008), or antisense (Touz et al., 2005), and morpholinobased knockdowns (Carpenter and Cande, 2009) combined with a completed genome, permit the identification and characterization of novel structural components and the mechanisms underlying Giardia’s cytoskeletal dynamics. Further, the initial cytological and ultrastructural analyses of the cytoskeleton need to be updated and revisited at higher resolution using current state-of-the-art fixation techniques and microscopy. Current methodologies and the availability of a complete genome will enable a more complete understanding of the complex cytoskeleton in Giardia.

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298 Kilburn CL, Pearson CG, et al. (2007) New Tetrahymena basal body protein components identify basal body domain structure. J Cell Biol 178(6): 905–912 Knight J (2004) Giardia: not so special, after all? Nature 429(6989): 236–237 Kozminski KG, Johnson KA, et al. (1993) A motility in the eukaryotic flagellum unrelated to flagellar beating.” Proc Natl Acad Sci U S A 90(12): 5519–5523 Kozminski KG, Beech PL, et al. (1995) The Chlamydomonas kinesin-like protein FLA10 is involved in motility associated with the flagellar membrane. J Cell Biol 131(6 Pt 1): 1517–1527 Kulda J and Nohynkova E (1995) Giardia in humans and animals. In: Parasitic Protozoa, vol. 10 (J.P. Kreier, ed.). Academic Press, Inc., San Diego, pp 225–423 Lechtreck KF and Grunow A (1999) Evidence for a direct role of nascent basal bodies during spindle pole initiation in the green alga Spermatozopsis similis. Protist 150(2): 163– 181 Li JB, Gerdes JM, et al. (2004) Comparative genomics identifies a flagellar and basal body proteome that includes the BBS5 human disease gene. Cell 117(4): 541–552 Lin F, Hiesberger T, et al. (2003) Kidney-specific inactivation of the KIF3A subunit of kinesin-II inhibits renal ciliogenesis and produces polycystic kidney disease.” Proc Natl Acad Sci U S A 100(9): 5286–5291 Long HJ (1994) Paclitaxel (Taxol): a novel anticancer chemotherapeutic drug. Mayo Clin Proc 69(4): 341–345 Luck DJ (1984) Genetic and biochemical dissection of the eucaryotic flagellum. J Cell Biol 98(3): 789–794 Magne D, Favennec L, et al. (1991) Role of cytoskeleton and surface lectins in Giardia duodenalis attachment to Caco2 cells. Parasitol Res 77(8): 659–662 Manton, I. a. C., B. (1952) An electron microscope study of teh spermatozoid of Sphagnum. J Exp Bot 3: 265–275 Mariante RM, Vancini RG, et al. (2005) Giardia lamblia: evaluation of the in vitro effects of nocodazole and colchicine on trophozoites. Exp Parasitol 110(1): 62–72 Marshall J and Holberton DV (1993) Sequence and structure of a new coiled coil protein from a microtubule bundle in Giardia. J Mol Biol 231(2): 521–530 Meng T-C, Hetsko ML and Gillin FD (1996) Inhibition of Giardia lamblia excystation by antibodies against cyst walls and by wheat germ agglutinin. Infect Immun 64: 2151– 2157 Merchant SS, Prochnik SE, Vallon O, Harris EH, Karpowicz SJ, et al. (2007) The Chlamydomonas genome reveals the evolution of key animal and plant functions. Science 318: 245–250 Midlej V and Benchimol M (2009) Giardia lamblia behavior during encystment: how morphological changes in shape occur. Parasitol Int 58(1): 72–80 Morrison HG, McArthur AG, et al. (2007) Genomic minimalism in the early diverging intestinal parasite Giardia lamblia. Science 317(5846): 1921–1926 Nash TE, Gillin FD, et al. (1983) Excretory-secretory products of Giardia lamblia. J Immunol 131(4): 2004–2010 Nicastro D, Schwartz C, et al. (2006) The molecular architecture of axonemes revealed by cryoelectron tomography. Science 313(5789): 944–948

S. C. Dawson Nohria A, Alonso RA, et al. (1992) Identification and characterization of gamma-giardin and the gamma-giardin gene from Giardia lamblia. Mol Biochem Parasitol 56(1): 27– 37 Ortega-Barria E, Ward HD, et al. (1994) Growth inhibition of the intestinal parasite Giardia lamblia by a dietary lectin is associated with arrest of the cell cycle. J Clin Invest 94(6): 2283–2288 Ostrowski LE, Blackburn K, et al. (2002) A proteomic analysis of human cilia: identification of novel components. Mol Cell Proteomics 1(6): 451–465 Owen RL (1980) The ultrastructural basis of Giardia function. Trans R Soc Trop Med Hyg 74(4): 429–433 Oxberry ME, Thompson RC, et al. (1994) Evaluation of the effects of albendazole and metronidazole on the ultrastructure of Giardia duodenalis, Trichomonas vaginalis and Spironucleus muris using transmission electron microscopy. Int J Parasitol 24(5): 695–703 Palm D, Weiland M, et al. (2005) Developmental changes in the adhesive disk during Giardia differentiation. Mol Biochem Parasitol 141(2): 199–207 Pazour GJ, Dickert BL, et al. (2000) Chlamydomonas IFT88 and its mouse homologue, polycystic kidney disease gene tg737, are required for assembly of cilia and flagella. J Cell Biol 151(3): 709–718 Pazour GJ, Agrin N, et al. (2005) Proteomic analysis of a eukaryotic cilium. J Cell Biol 170(1): 103–113 Peattie DA (1990) The giardins of Giardia lamblia: genes and proteins with promise. Parasitol Today 6(2): 52–56 Pellegrin S and Mellor H (2007) Actin stress fibres. J Cell Sci 120(Pt 20): 3491–3499 Pellegrini F and Budman DR (2005) Review: tubulin function, action of antitubulin drugs, and new drug development. Cancer Invest 23(3): 264–273 Piva B and Benchimol M (2004) The median body of Giardia lamblia: an ultrastructural study. Biol Cell 96(9): 735–746 Porter ME and Sale WS (2000) The 9 + 2 axoneme anchors multiple inner arm dyneins and a network of kinases and phosphatases that control motility. J Cell Biol 151(5): F37– F42 Poxleitner MK, Carpenter ML, et al. (2008) Evidence for karyogamy and exchange of genetic material in the binucleate intestinal parasite Giardia intestinalis. Science 319(5869): 1530–1533 Rosenbaum JL and Witman GB (2002) Intraflagellar transport. Nat Rev Mol Cell Biol 3(11): 813–825 Roxstrom-Lindquist K, Palm D, et al. (2006) Giardia immunity – an update. Trends Parasitol 22(1): 26–31 Sagolla MS, Dawson SC, et al. (2006) Three-dimensional analysis of mitosis and cytokinesis in the binucleate parasite Giardia intestinalis. J Cell Sci 119(Pt 23): 4889–4900 Scholey JM (2003) Intraflagellar transport. Annu Rev Cell Dev Biol 19: 423–443 Signor D, Wedaman KP, et al. (1999) Role of a class DHC1b dynein in retrograde transport of IFT motors and IFT raft particles along cilia, but not dendrites, in chemosensory neurons of living Caenorhabditis elegans. J Cell Biol 147(3): 519–530 Sloboda RD (2002) A healthy understanding of intraflagellar transport. Cell Motil Cytoskeleton 52(1): 1–8

Chap. 18 Primary Microtubule Structures in Giardia Smith EF and Yang P (2004) The radial spokes and central apparatus: mechano-chemical transducers that regulate flagellar motility. Cell Motil Cytoskeleton 57(1): 8–17 Snell WJ, Pan J, et al. (2004) Cilia and flagella revealed: from flagellar assembly in Chlamydomonas to human obesity disorders. Cell 117(6): 693–697 Solari AJ, Rahn MI, et al. (2003) A unique mechanism of nuclear division in Giardia lamblia involves components of the ventral disk and the nuclear envelope. Biocell 27(3): 329–346 Sousa MC, Concalves CA, Bairos VA and Poiares-Da-Silva J (2001) Adherence of Giardia lamblia trophozoites to Int407 human intestinal cells. Clin Diagn Lab Immunol 8: 258–265 Szkodowska A, Muller MC, et al. (2002) Annexin XXI (ANX21) of Giardia lamblia has sequence motifs uniquely sdhared by giardial annexins and is specifically localized in the flagella. J Biol Chem 277(28): 25703–25706 Touz MC, Conrad JT, et al. (2005) A novel palmitoyl acyl transferase controls surface protein palmitoylation and cytotoxicity in Giardia lamblia. Mol Microbiol 58(4): 999–1011 Tsang PH and Li G, et al. (2006) Adhesion of single bacterial cells in the micronewton range. Proc Natl Acad Sci U S A 103(15): 5764–5768

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Pathophysiological Processes and Clinical Manifestations of Giardiasis Andre G. Buret and James Cotton

Abstract Giardiasis represents one of the most common parasitic diseases of humans, food-producing animals and pets, and it occurs throughout the World. Infected hosts may present with a broad spectrum of symptoms ranging from asymptomatic carriage to acute or chronic diarrhea. Some hosts may develop post-infectious chronic disorders in the gastrointestinal tract as well as at extraintestinal sites. For unknown reasons, Giardia infection does not cause any sign of overt inflammation in the gut. Of the 7 distinct genetic Giardia duodenalis “assemblages” described to this day, Assemblages A and B can infect human hosts. Both assemblage A and assemblage B isolates are capable of causing symptomatic diarrheal disease, and the same assemblage has been reported to cause differing lengths of symptomatic infection. Much remains to be learned about the role of parasite genotype in pathogenesis. The immune or humoral status of the host greatly contributes to the variable manifestations of giardiasis, as both humoral- and cellular-mediated immune responses are involved in parasite clearance as well as in pathophysiology. Infection with Giardia is able to trigger chronic gastrointestinal disorders such as irritable bowel syndrome, and may cause disease at extraintestinal sites, via mechanisms that have yet to be identified. The pathophysiological mechanisms that occur during G. duodenalis infection are not completely understood. Research findings to date indicate that shortly after colonization of the small intestinal lumen, Giardia trophozoites heighten the rates of enterocyte apoptosis, decrease intestinal barrier function, and these changes lead to diffuse shortening of small intestinal brush border microvilli, maldigestion, and

H. D. Luján et al. (eds.), Giardia © Springer-Verlag/Wien 2011

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malabsorption, via the activation of CD8+ T-lymphocytes. Infection also increases intestinal chloride secretion, and accelerates small intestinal transit. The combination of these events contributes to the diarrheal disease seen in symptomatic infections. Several parasitic factors, although still incompletely characterized, have been directly implicated in the pathophysiology of giardiasis. Further identification of these factors is sorely needed as it may also help explain symptom variability. Clearly, the pathogenesis of giardiasis is multifactorial. The identification of host and microbial factors responsible for symptoms in giardiasis offers promising research avenues to better understand a variety of enteric infections and chronic gastrointestinal disorders.

19.1 Introduction Every year, millions of people world-wide are infected by Giardia duodenalis (syn. G. lamblia, G. intestinalis), which was recently added to the WHO’s “Neglected Disease Initiative” (Savioli et al., 2006). In food-producing animals and in pets, the infection can reduce weight gain, and may become a concern for zoonotic transmission (Ortega and Adam, 1997; Savioli et al., 2006). Infection occurs following ingestion of infectious cysts either directly via the fecaloral route, or indirectly via contaminated food or water. Excysted trophozoites colonize the lumen of the small intestine, without invading host tissues or entering the bloodstream (Ortega and Adam, 1997). The mechanisms whereby this parasite causes intestinal disease remain incompletely understood. The pathophysiological processes and symptoms produced by Giardia infections are multifactorial, and mediated by parasitic as well as host immune factors. There is

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also increasing evidence to suggest that the pathophysiology of giardiasis can lead to the development of chronic gastrointestinal and extraintestinal disorders. This chapter discusses the various clinical manifestations of giardiasis, and provides a state-of-the-art review of how Giardia-mediated pathophysiological processes may contribute to disease, during the infection as well as in post-infectious manifestations.

19.2 Clinical Manifestations of Giardiasis The clinical manifestations of G. duodenalis infection are highly variable. Infected hosts may present with a spectrum of symptoms ranging from asymptomatic carriage to acute or chronic symptomatic infection that manifests as diarrheal disease. Some hosts may develop more complex symptoms post-infectiously that will be discussed in the following paragraphs. However, in the majority of cases, the gastrointestinal symptoms associated with giardiasis include diarrhea, bloating, abdominal pain, nausea, vomiting, as well as anorexia and failure to thrive. Most commonly, symptoms start 7–10 days after infection (range 3–25 days post-infection). While chronically infected hosts may continue to have diarrhea, they may also become constipated, and symptoms may rarely be associated with signs of “microscopic duodenal inflammation” (Hanevik et al., 2007; Morken et al., 2008). In most cases, Giardia infection does not cause any sign of overt inflammatory cell infiltration. For reasons that remain obscure, some infected hosts may remain asymptomatic, while they still can pass infectious cysts capable of infecting susceptible hosts (IshHorowicz et al., 1989). Weight loss or reduced weight gain has been associated with giardiasis. Experimental infections in rodents indicate that at least part of this effect may be due to reduced food intake during the infection (Araujo et al., 2008). The detrimental effects of giardiasis on body weight have also been reported in humans, food-producing animals, and pets (Buret et al., 1990; Olson et al., 1996, 1997; Aloisio et al., 2006; Kohli et al., 2008; Hoar et al., 2009; Geurden et al., 2010). In addition to the zoonotic potential of the infection (Buret et al., 1990; Hoar et al., 2009; Geurden et al., 2010) discussed elsewhere in this book, the ef-

A. G. Buret and J. Cotton

fects of Giardia on body weight remain a serious concern to the food-producing animal industry. Controlled experimental infection studies in lambs have clearly established a cause-to-effect relationship between Giardia infection and weight loss (Olson et al., 1995; Aloisio et al., 2006). G. duodenalis trophozoites can develop drug resistance to standard anti-parasitic treatments (Upcroft et al., 1996; Muller et al., 2007; Sterk et al., 2007), and refractory giardiasis due to parasite drug resistance has been reported in both healthy and immunocompromised individuals (Abboud et al., 2001; Aronson et al., 2001; Hanevik et al., 2007; Morch et al., 2008). (For a complete review of G. duodenalis drug resistance see Chapter 22.) To date, G. duodenalis has been reported to exist in 7 distinct genetic “assemblages”. Assemblages A and B can infect human hosts (Robertson et al., 2010). Both assemblage A and assemblage B isolates are capable of causing symptomatic diarrheal disease (reviewed in Robertson et al., 2010), and the same assemblage has been reported to cause differing lengths of symptomatic infection in healthy individuals (Hanevik et al., 2007). Apparently therefore, the parasite genotype alone cannot fully explain why infected individuals experience such a broad range of symptoms or lengths of infection. Various reports from Europe, Africa, South America, Asia, and Australia have attempted to correlate parasite genotype with symptomatology. Some have suggested that Assemblage B may cause more severe disease, but there are also conflicting studies reporting otherwise (Homan and Mank, 2001; Read et al., 2002; Aydin et al., 2004; Haque et al., 2005; Gelanew et al., 2007; Kohli et al., 2008; Pelayo et al., 2008; Sahagun et al., 2008; Ajjampur et al., 2009; Robertson et al., 2010). This controversy may reflect the fact that both microbial and host factors contribute to disease (see below), making this type of study difficult to internally control for. More research is needed to dissect the role of parasite genotype in pathogenesis. Research has investigated whether another factor that may contribute to the variable clinical manifestations of giardiasis may be the size of the infectious dose. Studies in mice infected with G. muris demonstrated that the length of the latent period before cyst excretion occurs is directly related to the number of infectious cysts ingested, but the size of the infectious

Chap. 19 Pathophysiological and Clinical Manifestations of Giardiasis

dose did not affect the number of cysts excreted in the host feces (Belosevic and Faubert, 1983). Using another animal model, Mongolian gerbils infected with human G. duodenalis isolates, pathophysiological alterations in the intestinal mucosa occurred independently of the number of trophozoites administered (Araujo et al., 2008). The early study by Rentdorff demonstrated that ingestion of 10 cysts sufficed to cause giardiasis in human volunteers (Rendtorff, 1954). More recent human experimental infections did not investigate symptom variability in association with infectious dose (Nash et al., 1987, 1990). Therefore, whether and how infectious dose may contribute to symptom variability still remains unclear. The immune or humoral status of the host greatly contributes to the variable manifestations of giardiasis, as both humoral- and cellular-mediated immune responses are involved in parasite clearance as well as in pathophysiology, as discussed further in the chapter (Eckmann, 2003). (For a complete review on the immunology of G. duodenalis infection see Chapter 21.) Giardiasis tends to be self-limiting in hosts with competent immune systems. In contrast, immunodeficient hosts are more likely to suffer from chronic diarrheal disease. Chronic giardiasis in humans with compromised immune systems, due to common variable immunodeficiency (CVID) or acquired immune deficiency syndrome (AIDS), can result in chronic dehydration and anorexia (Carcamo et al., 2005; Onbasi et al., 2005; Oksenhendler et al., 2008). However, refractory giardiasis has been reported in healthy patients without immune deficiencies, again, implicating that other factors are involved in the development of chronic diarrheal disease (Eckmann, 2003; Hanevik et al., 2007). Experimental infections in healthy humans with competent immune systems suggest that individuals previously infected with G. duodenalis are less likely to become re-infected, or develop symptomatic giardiasis following re-infection, which indicates that symptomatic giardiasis is more likely to develop in naïve individuals (Nash et al., 1987, 1990). In areas where endemic giardiasis is common, resident populations can develop symptomatic giardiasis when infected with a new strain of G. duodenalis, and will most likely not become infected or will remain asymptomatic following infection with the endemic strain (Istre et al., 1984). Therefore, further research

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is required to determine how various hosts may develop acquired immunity and resistance to symptoms in giardiasis. Children between two and five years old that have recently been weaned from their mother’s breast milk are highly susceptible to infection with Giardia. As infants, they are protected from infection by their mothers’ breast-milk, via a combination of anti-microbial substances such as immunoglobulin A (IgA) and other antibodies, or products like epidermal growth factor, which may all combine to confer antiinfective properties to maternal milk (Lunn et al., 1999; Buret et al., 2002; Tellez et al., 2003). Should young children susceptible to infection contract giardiasis, it can result in nutrient malabsorption and malnutrition potentially causing long-term, detrimental side-effects including failure to thrive and even impaired cognitive development (Sullivan et al., 1991; Newman et al., 2001; Berkman et al., 2002; AlMekhlafi et al., 2005; Silva et al., 2009; Botero-Garces et al., 2009; Goto et al., 2009). Nutrient malabsorption and failure to thrive as a result of G. duodenalis infection has also been reported in newborn calves and rodent models, indicating that these pathophysiological effects are not restricted to humans (Olson et al., 1997; Astiazaran-Garcia et al., 2000).

19.3 Chronic Gastrointestinal Disorders Associated with Giardiasis There is increasing evidence to suggest that infection with Giardia leads to the development of chronic disorders in the gastrointestinal tract as well as at extraintestinal sites. Extraintestinal manifestations that have been recorded in association with giardiasis are listed in Table 19.1, and include several circulating, skin, joint and muscular, or ocular disorders, and/or micronutrient deficiencies. These disorders appear to resolve following patient clearance of Giardia trophozoites, implicating parasitic colonization as the causative agent of disease. Although similar extraintestinal manifestations have been reported in individuals afflicted by chronic gastrointestinal inflammatory disorders or infected with other enteric pathogens (Ardizzone et al., 2008; Girschick et al., 2008; Timani and Mutasim, 2008), the pathophysiological mechanisms responsible for the post-infectious development of extraintestinal disorders remain unknown.

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Table 19.1 As discussed in the text, giardiasis may lead to post-infectious chronic disorders of the intestine including Irritable Bowel syndrome. In addition, this table lists the various extraintestinal clinical manifestations that have been associated with G. duodenalis infection Type of manifestation

Reference

Circulating Eosinophilia

(Farthing et al., 1983; Dos Santos and Vituri Cde, 1996; Canonne et al., 2000)

Hypokalemia

(Cervello et al.,1993; Addiss and Lengerich, 1994; Genovese et al., 1996)

Increased circulating IgE (Farthing et al., 1983; Di Prisco et al., 1993, 1998; Perez et al., 1994; Pietrzak et al., 2005) Joint and muscular Arthalgia

(Meza-Ortiz, 2001)

Arthritis

(Goobar, 1977)

Hypokalemic induced myopathy

(Cervello et al.,1993; Genovese et al., 1996)

Reactive arthritis

(Woo and Panayi 1984; Layton et al., 1998; Letts et al., 1998; Tupchong et al., 1999; Carlson and Finger, 2004)

Micronutrient deficiencies Iron

(Ertan et al., 2002; Demirci et al., 2003; Culha and Sangun, 2007; Abou-Shady et al., 2010)

Zinc

(Karakas et al., 2001; Ertan et al., 2002; Demirci et al., 2003; Abou-Shady et al., 2010)

Vitamin A

(Quihui-Cota et al., 2008)

Vitamin B12

(Cordingley and Crawford, 1986; Springer and Key, 1997)

Ocular Non-progressive retinal lesions

(Pettoello Mantovani et al., 1990; Corsi et al., 1998)

Retinal arteritis

(Knox and King, 1982)

Uveitis

(Carroll et al., 1961; Knox and King, 1982; Anderson and Griffith, 1985)

Skin Erythema nodosum

(Giordano et al., 1985; Harries and Taylor, 1986; Lammintausta et al., 2001)

Granuloma annulare

(Pietrzak et al., 2005)

Pruritus

(Goobar, 1977; Hamrick and Moore 1983; Clyne and Eliopoulos, 1989; Spaulding, 1990; SanchezCarpintero and Vazquez-Doval, 1998; Giacometti et al., 2003)

Urticaria

(Weisman, 1979; Farthing, 1983; Clyne and Eliopoulos, 1989; Martinez Rodriguez et al., 1992; Giacometti et al., 2003)

Wells’ syndrome

(Canonne et al., 2000)

Clinical studies have shown that individuals infected with G. duodenalis may have elevated serum levels of IgE antibodies against food antigens, and are at heightened risk of developing food allergy (Hamrick and Moore, 1983; Di Prisco et al., 1993, 1998; Giacometti et al., 2003). It has been proposed that increased intestinal permeability during G. duodenalis infection results in the increased uptake of food antigens, which in turn leads to the production of IgE an-

tibodies to these antigens. Upon secondary exposure to antigens, sensitized mast cells are activated and induce host enteropathy. This observation is further supported by studies demonstrating that G. duodenalis infection results in delayed local and sub-cutaneous mast cell hyperplasia (Hardin et al., 1997). The latter may contribute to the occurrence of urticaria and other skin disorders associated with giardiasis (Hamrick and Moore 1983; Di Prisco et al., 1993;

Chap. 19 Pathophysiological and Clinical Manifestations of Giardiasis

et al., 1998; Giacometti et al., 2003). However, whether extraintestinal manifestations such as urticaria and pruritis occur as a result of G. duodenalis-mediated increases in small intestinal permeability has yet to be conclusively demonstrated. Similarly, the causal link between translocation of food antigens resulting from increased intestinal permeability and the resulting mast cell hyperplasia remains to be established in giardiasis. Experimental evidence has shown that intestinal hypersensitivity develops in genetically pre-disposed individuals following stress-induced increases in small intestinal permeability (Buret, 2006; Yang et al., 2006). In addition, other reports have shown that intestinal hypersensitivity can develop in the absence of genetic susceptibility, suggesting that genetic predisposition may not be required for individuals infected with G. duodenalis to develop similar disorders, which has yet to be verified (O’Connell, 2003; Heyman, 2005). Therefore, further investigation is required in order to determine if the pathophysiological events that cause G. duodenalis-induced intestinal hypersensitivity are dependent upon increased small intestinal permeability and, also, if these events occur with higher prevalence in individuals with genetic predisposition. Irritable bowel syndrome (IBS) is a chronic disorder of the gastrointestinal tract that is characterized by recurrent abdominal pain and altered bowel habits; it is believed to be caused by biological and psychosocial factors that occur in the absence of a descriptive cause of disease (Longstreth et al., 2006). Research findings indicate that acute enteric infection may result in post-infectious IBS (PI-IBS) (Thornley et al., 2001; Marshall et al., 2006). There is increasing evidence to suggest that G. duodenalis infection can also result in the development of PI-IBS, via mechanisms that remain obscure (Hanevik et al., 2007; Morken et al., 2009b). In affected individuals, it appears that the infection may represent a triggering factor for the development of IBS, and the presence of the parasite is not required for symptoms to persist (D’Anchino et al., 2002). The development of PI-IBS in individuals previously infected with G. duodenalis appears to be largely biological in origin and not dependent on psychosocial factors (Morken et al., 2009b). Yet, the etiology of G. duodenalis PI-IBS remains largely unknown and further studies examining bacterial overgrowth as a

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possible contributing trigger of post-infectious IBS following G. duodenalis infection have produced conflicting results. Lactulose breath tests are negative for individuals that have developed PI-IBS as a result of G. duodenalis infection (Morken et al., 2008). Whereas, a follow-up study established that patients instilled with a live fecal culture from a healthy individual had their IBS symptoms temporarily improved, while similar patients given antibiotics showed no signs of improvement (Morken et al., 2009a). Therefore, disturbances to the microbiota, if present, are not solely responsible for triggering PI-IBS in patients previously infected with G. duodenalis. Therefore, the etiology of chronic intestinal disorders such as food allergy and irritable bowel syndrome following G. duodenalis infection remains poorly understood. Further investigation is required in order to determine the pathophysiological mechanism responsible for the development these disorders post-infectiously. Campylobacter jejuni is another common enteropathogen responsible for PI-IBS (Marshall et al., 2006). This infection facilitates the systemic dissemination of enteric bacteria, including those of the normal flora (Lamb-Rosteski et al., 2008; Kalischuk et al., 2009). As increased intestinal permeability following infection with Campylobacter jejuni may also contribute to the production of symptoms in patients with inflammatory bowel disease (IBD) (Lamb-Rosteski et al., 2008; Kalischuk and Buret, 2009), future research needs to assess if G. duodenalis may facilitate the development of IBD symptoms via similar mechanisms.

19.4 Pathophysiological Processes Causing Symptoms in Giardiasis The pathophysiological mechanisms that occur during G. duodenalis infection are not completely understood. However, research has identified several host pathophysiological responses that are believed to contribute to disease. Shortly after colonization of the small intestinal lumen, G. duodenalis trophozoites heighten rates of enterocyte apoptosis, decrease intestinal barrier function, and these changes lead to diffuse shortening of small intestinal brush border microvilli. Infection also increases intestinal chloride secretion, and accelerates small intestinal transit. The

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combination of these events is responsible for the diarrheal disease during symptomatic infections. The following section describes the known pathophysiological mechanisms of giardiasis, and critically discusses how they may contribute to the symptoms associated with giardiasis.

19.4.1 Giardia Promotes Excessive Enterocyte Apoptosis Apoptosis, also called programmed cell death, is characterized by biochemical and morphological events that include cell rounding, membrane blebbing, cytoskeletal disassembly, chromatin condensation, and DNA fragmentation (reviewed in Kroemer et al., 2009). In apoptosis, at least part of these events can be ascribed to the activity of cysteinyl aspartate proteases (caspases) that are divided into two subgroups: initiator caspases (caspase-2, -8, -9 and -10) and executioner caspases (caspase-3, -6 and -7) (reviewed in Luthi and Martin, 2007). In resting cells, caspases are kept in an inactive precursor form, and they are proteolytically cleaved only following the induction of apoptosis, via the activation of the extrinsic or intrinsic apoptotic-signalling cascade reviewed in Li and Yuan (2008). Both pathways culminate in the activation of executioner caspases, but each uses different initiator caspases to proteolytically process and activate executioner caspases. In the extrinsic cascade, caspase-8 or -10 is activated following binding of a death receptor (Fas) to its respective ligand, while the intrinsic cascade results in the activation of caspase-9 following an increase in the permeability of the outer mitochondrial membrane (OMM) (Li and Yuan, 2008). In the gastrointestinal tract, the induction of enterocyte apoptosis is a highly regulated process that contributes to the homeostatic turn-over of epithelial cells (Hall et al., 1994). During homeostatic cell renewal, enterocyte apoptosis does not alter epithelial permeability (Madara and Madara et al., 1987; Pappenheimer, 1987). However, dysregulation of enterocyte apoptosis has been associated with various forms of gastrointestinal disease, and occurs during infection with G. duodenalis (Jones and Gores, 1997; Ramachandran et al., 2000; Troeger et al., 2007). Indeed, colonization of the small intestine by G. duode-

A. G. Buret and J. Cotton

nalis trophozoites quickly results in heightened rates of enterocyte apoptosis (Chin et al., 2002; Troeger et al., 2007; Panaro et al., 2007). Microarray analyses of enterocytes exposed to parasitic products observed a significant activation of genes involved in the apoptotic cascade (Roxstrom-Lindquist et al., 2005). Data available to date indicate that heightened rates of enterocyte apoptosis during giardiasis occur via activation of caspase-3 and caspase-9, increased expression of pro-apoptotic Bax and reduced expression of antiapoptotic Bcl-2, and are associated with cleavage of poly(ADP-ribose) polymerase (PARP) (Chin et al., 2002; Panaro et al., 2007). In resting cells, Bcl-2 antagonizes the action of pro-apoptotic proteins involved in the intrinsic apoptotic-signaling pathway, such as Bax, by maintaining the potential of the outer mitochondrial membrane (OMM) (reviewed by Adachi et al., 1997; Burlacu, 2003; Youle and Strasser, 2008). Therefore, the decrease in Bcl-2 and concomitant increase in Bax trigger the intrinsic apoptoticsignaling pathway, and results in the release of other pro-apoptotic molecules, including cytochrome c and apoptosis inducing factor, ultimately activating caspase-9 and caspase -3 (Youle and Strasser, 2008). Caspase-3 cleaves the DNA-repair protein PARP, which prevents PARP from repairing DNA, hence allowing apoptosis to proceed (Kaufmann et al., 1993; Nicholson et al., 1995). G. duodenalis increases expression of caspase-8 to trigger the extrinsic apoptotic signaling cascade (Panaro et al., 2007). As activation of Proteinase-activated receptors on the apical surface of enterocytes is capable of triggering similar events, the possible role played by parasitic proteases in this pathophysiological effect warrants further investigation (Chin et al., 2003; Flynn and Buret, 2004). These observations however do not necessarily imply that parasitic products are able to directly activate caspase-8. Indeed, the induction of caspase-8 may also occur via activation of caspase-3, which may cleave caspase-6, another caspase-8 activator, which further amplifies the cascade (Slee et al., 1999). The products responsible for heightened enterocyte apoptosis in giardiasis are yet to be identified, and the molecular cascade leading to this phenomenon is incompletely understood. However, from the information available to date, it appears that the elevated enterocyte apoptosis during Giardia infection may represent a significant player in the pathophysiology of giardiasis, and

Chap. 19 Pathophysiological and Clinical Manifestations of Giardiasis

that the effect may be mediated by both parasitic and host factors.

19.4.2 Giardia Disrupts Intestinal Barrier Function The intestinal epithelium functions as a selective barrier that separates the underlying host tissues from noxious luminal contents including bacteria and food antigens. Enterocytes maintain this barrier by forming apical junctional and adherent complexes that are formed through the association of various transmembrane and cytosolic plaque proteins, and several cytosolic regulatory proteins. Decreased intestinal barrier function has been found in several forms of gastrointestinal disease, including enteric infection and various chronic gastrointestinal disorders (O’Hara and Buret, 2008; Turner, 2009). The maintenance of epithelial barrier integrity is delicately regulated. Drug- or immune-induced enterocyte apoptosis, or treatment with bacterial lipopolysacchardide (LPS), may increase intestinal permeability (Katz et al., 1989; Sun et al., 1998; Abreu et al., 2000; Gitter et al., 2000; Yu et al., 2005). Decreased intestinal barrier function can be caused by the breakdown of proteins associated with enterocyte tight junctions, such as cytoskeletal F-actin and the cytoplasmic plaque protein zonula occludens-1 (ZO-1), and various tight junction proteins including various claudins (Fanning et al., 1998; Wittchen et al., 1999). Increases in intestinal permeability may be associated with flocculation of F-actin, or trafficking of zonula occludens-1 (ZO-1) from the cell membrane to the cytosol (Ma et al., 1995, 1999). Conversely, increased apical glucose uptake via the sodium-coupled-glucose transporter-1 (SGLT-1) transporter protects intestinal epithelia from LPS-induced enterocyte apoptosis and the subsequent increase in transepithelial permeability (Yu et al., 2005, 2006). While this endogenous rescue mechanism may also operate against Giardia antigens, the observations discussed above clearly indicate that this self-protective phenomenon is overridden during giardiasis in vivo, via mechanisms that remain obscure (Ma et al., 1995, 1999). Infection with Giardia increases intestinal permeability, and results in alterations within host enterocytes whereby F-actin, ZO-1, and α-actinin, a

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component of the actomyosin ring responsible for regulating paracellular flow across the intestinal epithelium, are relocated from the cell periphery to the cytosol (Teoh et al., 2000; Scott et al., 2002). Interestingly, ZO-1 relocation induced by Giardia can also be prevented if the intestinal epithelium is pre-treated with epidermal growth factor, further suggesting a possible role for this milk peptide in anti-Giardia protection (Buret et al., 2002). F-actin and ZO-1 relocation from the periphery to the cytosol, during G. duodenalis infection, requires the activation of caspase-3, which implies a cause-to-effect relationship between Giardia-induced apoptosis and the loss of intestinal barrier integrity (Chin et al., 2002). Caspase-3 may cause the relocation of F-actin and ZO-1 via its effect on myosin light chain kinase (MLCK) and Rho kinase (ROCK), two proteins that regulate intestinal tight junctional complexes (Mills et al., 1998; Coleman et al., 2001; Sebbagh et al., 2001). Indeed, cleavage of both MLCK and ROCK by caspases-3 results in their constitutive activation (Mills et al., 1998; Coleman et al., 2001; Sebbagh et al., 2001). The Giardia-mediated disruption of F-actin and ZO-1 is, at least partially, dependent on activation of MLCK, but it is unknown if activation of caspase-3 is required for the MLCK-mediated disruption of tight junctions that occurs during infection (Scott et al., 2002). Studies in mice have observed that elevated intestinal permeability in vivo occurs during the peak of trophozoite infection, and returns to normal after parasite clearance (Scott et al., 2002). This may in part explain why extraintestinal manifestations in giardiasis (Table 19.1) appear to resolve following patient clearance of Giardia trophozoites. Consistent with this observation, increased macromolecular uptake across the intestinal wall occurs at the height of Giardia infection, but not following trophozoite clearance (Hardin et al., 1997). Studies in human subjects have demonstrated that chronic giardiasis significantly increases intestinal permeability (Troeger et al., 2007). Whether or not G. duodenalis trophozoites stimulate the ROCK-mediated disruption of intestinal epithelial tight junctional proteins, and whether this process is also dependent upon caspase-3, remains to be determined. The activation of either ROCK or MLCK by G. duodenalis trophozoites or their parasitic products, either through caspase-3 dependent or independent mechanisms, could potentially explain

308

why patients with chronic giardiasis have reduced expression levels of claudin-1 (Troeger et al., 2007), another tight junctional protein contributing to intestinal barrier function (Inai et al., 1999). In summary, by inducing enterocyte apoptosis and activating MLCK, Giardia infection relocates and/or degrades various proteins associated with enterocyte tight junctions, and induces cytoskeletal contractility, which in turn increases gut permeability. Further investigation is required in order to elucidate all the mechanisms that mediate disruption of epithelial tight junctional structure and function during infection.

19.4.3 Giardia Induces a Diffuse Shortening of Brush Border Microvilli and Causes Electrolyte Transport Abnormalities In an attempt to better understand the mechanisms responsible for diarrhea in giardiasis, studies using experimental infections observed that Giardia causes a diffuse shortening of microvilli along the small intestinal epithelium (Scott et al., 2000, 2004). Consistent with the hypothesis that during giardiasis immunopathophysiology may be secondary to the disruption of intestinal barrier, the Giardia-induced shortening of brush border microvilli is mediated by activated CD8+ T lymphocytes (Scott et al., 2000, 2004). Brush border microvilli surface area is not reduced in infected hosts devoid of mature lymphocyte populations, and the injury is independent of CD4+ lymphocytes (Scott et al., 2000, 2004). Early human studies have produced conflicting results on the effects of giardiasis on mucosal absorptive surface area (Oberhuber et al., 1997). However, using mucosal morphometry combined with impedance spectroscopy in humans, it has now been clearly established that duodenal mucosal surface area is decreased in patients with chronic giardiasis (Troeger et al., 2007). This loss of epithelial absorptive area is independent of villous atrophy, which may or may not occur during giardiasis (Scott et al., 2000, 2004). These effects appear to be strain dependent (Cevallos et al., 1995; Oberhuber et al., 1997; Troeger et al., 2007; Behera et al., 2008; Koot et al., 2009). The diffuse shortening of brush border microvilli decreases the total absorptive surface area, and reduces the expression of brush border enzymes. In-

A. G. Buret and J. Cotton

deed, giardiasis causes deficiencies of a broad range of digestive enzymes (Table 19.2). These deficiencies are responsible for maldigestion. In addition, the loss of epithelial microvilli, where nutrient-coupled electrolyte transporters reside, directly causes malabsorption of glucose and electrolytes, which in turn reduces water absorption (Buret et al., 1990, 1991, 1992; Nain et al., 1991; Cevallos et al., 1995; Scott et al., 2000, 2004). Undigested carbohydrates are processed by the colonic microbiota into short chain fatty acids, and impaired nutrient absorption produces an osmotic gradient within the lumen of the small intestine that contributes to intestinal distension, rapid peristalsis, and malabsorptive diarrhea (Robayo-Torres et al., 2006). Consistent with these observations, clinical studies have shown that patients infected with G. duodenalis may have elevated fecal levels of short chain fatty acids, indicative of carbohydrate malabsorption (Morken et al., 2009a). One report indicates that lactase activity can take weeks to normalize following infection with Giardia, which may contribute to diarrheal disease even after trophozoite clearance (Faubert, 2000). In addition to causing diarrheal disease via small intestinal malabsorption, other studies have demonstrated that G. duodenalis infection results in increased intestinal transit rates that appear to be dependent upon mast cell degranulation and adaptive immune responses (Li et al., 2006, 2007). Giardia also increases chloride secretion (Gorowara et al., 1992; Cevallos et al., 1995; Troeger et al., 2007), which leads to secretion of water, hence further contributing to the production of diarrhea (Baldi et al., 2009). In inflammatory bowel disease, heightened rates of enterocyte apoptosis allow for water and ions to cross the intestinal epithelial barrier, resulting in leak-flux diarrhea (Schulzke et al., 2006). Whether this pathophysiological mechanism is also implicated in giardiasis is unknown. In summary, infection with Giardia causes diarrheal disease via a combination of factors that include microvillus shortening, maldigestion and malabsorption, ion hypersecretion, and increased rates of intestinal transit (Fig. 19.1). As microvillus shortening, and hence maldigestion and malabsorption, are dependent upon active host lymphocytes, intestinal injury and malfunction in giardiasis cannot directly result from trophozoite attachment and/or parasite virulence factors.

Chap. 19 Pathophysiological and Clinical Manifestations of Giardiasis

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H2O

H2O

Fig. 19.1 The pathophysiology of Giardia duodenalis-mediated diarrheal disease is multifactorial. (1) After exycstation, Giardia trophozoites colonize the lumen of the small intestine and induce heightened rates of enterocyte apoptosis via the activation of caspase-3 and caspase-9. (2) Following the induction of apoptosis, zonula occludens 1 (ZO-1), F-actin, and α-actinin are relocated from apical junctional complexes to the cell interior via activated caspase-3 and myosin light chain kinase (MLCK). (3) The G. lamblia-mediated breakdown of apical junctional complexes causes an increase in intestinal permeability that facilitates the translocation of various luminal antigens, including microbial factors and food antigens, into the sub-epithelial compartment that promotes the recruitment of host lymphocytes. (4) CD8+ lymphocyte populations induce the diffuse shortening of brush border microvilli resulting in brush border enzyme deficiencies, and small intestinal malabsorption. Trophozoites also induce the secretion of chloride ions. Together, the accumulation of undigested carbohydrates and secretion of ions generates an osmotic gradient within the small intestinal lumen that results in the loss of water, intestinal distension, and rapid peristalsis that ultimately causes malabsorptive and hypersecretory diarrheal disease. Small intestinal malabsorption is believed to be the major cause of diarrhea during Giardia infection, while chloride hypersecretion further contributes to symptoms of diarrheal disease

19.5 Role of Parasitic Factors in the Pathogenesis of Giardiasis A number of parasitic factors have been implicated in the pathophysiology of giardiasis (Table 19.3). Factors produced by Giardia trophozoites have been shown to induce specific immune responses, contribute to immune evasion and, potentially, the establishment of chronic infection. Oral administration of yet unidentified excretory/secretory Giardia products to mice may result in a dominant Th2-type response and, rarely, eosinophil infiltration (Jimenez et al., 2004). However, it should be noted that the latter inflamma-

tory response is not observed in natural infection with this parasite. Variant-specific surface proteins (VSPs) expressed on Giardia trophozoites are involved in antigenic variation and evasion of host humoral defences, such that Giardia trophozoites with a different surface VSP appear as the host begins to produce antibodies against the current immunodominant VSP (Byrd et al., 1994; Singer et al., 2001; Prucca and Lujan, 2009). It has been proposed that VSPs may confer zoonotic infectivity to Giardia, but this hypothesis has yet to be tested in controlled experimental conditions (Singer et al., 2001). Arginine deiminase produced by Giardia contributes to parasite immune

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Table 19.2 Enzyme deficiencies induced by Giardia and/or giardiasis Enzyme

Study model

Type of Giardia challenge

Reference

Maltase

Humans, Mice, Gerbils, cattle, cells in vitro

Live parasites, soluble extracts

(Duncombe et al., 1978; Gillon et al., 1982; Welsh et al., 1984; Gillon and Ferguson, 1984; Anand et al., 1985; Faubert, 1988; Belosevic et al., 1989; Buret et al., 1990, 1991, 1992; Favennec et al., 1991; Daniels and Belosevic, 1992, 1995; Chenu et al., 1992; Mohammed and Faubert, 1995a,b; O’Handley et al., 2001)

Isomaltase

Gerbils, Mice

Live parasites

(Belosevic et al., 1989; Nain et al., 1991)

Sucrase

Humans, Mice, Gerbils

Live parasites, soluble extracts

(Duncombe et al., 1978; Gillon et al., 1982; Anand et al., 1982, 1985; Welsh et al., 1984; Gillon and Ferguson, 1984; Faubert, 1988; Belosevic et al., 1989; Buret et al., 1990,1991; Daniels and Belosevic, 1992; Chenu et al., 1992; Mohammed and Faubert, 1995a,b)

Lactase

Humans, Mice, Gerbils, Rats

Live parasites, soluble extracts

(Duncombe et al., 1978; Gillon et al., 1982; Anand et al., 1982, 1985; Welsh et al., 1984; Gillon and Ferguson, 1984; Faubert, 1988; Ish-Horowicz et al., 1989; Belosevic et al., 1989; Daniels and Belosevic, 1992, 1995; Chenu et al., 1992; Cevallos et al., 1995; Mohammed and Faubert, 1995; Singh et al., 2000; O’Handley et al., 2001)

Trehalase

Mice, Gerbils

Live parasites, soluble extracts

(Gillon and Ferguson, 1984; Belosevic et al., 1989; Daniels and Belosevic, 1992, 1995; Mohammed and Faubert, 1995; Mohammed and Faubert, 1995)

Glucoamylase

Mice

Live parasites

(Nain et al., 1991)

Saccharase

Gerbils

Live parasites

(Faubert, 1988)

Alkaline phosphatase

Humans, Mice, Gerbils, Rats, cells in vitro

Live parasites

(Anand et al., 1982; Welsh et al., 1984; Faubert, 1988; Favennec et al., 1991; Mahmood et al., 2005)

Trypsin

Human patients, cells in vitro

Live parasites, soluble extracts

(Chawla et al., 1975; Seow et al., 1993)

Chymotrypsin

Humans

Live parasites, soluble extracts

(Chawla et al., 1975; Carroccio et al., 1997)

evasion; first, it suppresses enterocyte NO production which was shown to help kill Giardia trophozoites (Eckmann et al., 2000; Ringqvist et al., 2008), and second, it enhances trophozoite antigenic variation by modifying VSPs (Touz et al., 2008). Giardia trophozoites also produce thiol proteinases that cleave human IgA (Parenti, 1989). Other still unknown parasitic factors are also known to modulate host innate immune responses in dendritic cells (Kamda and Singer, 2009). Several parasitic factors, albeit still incompletely characterized, have been directly implicated in the pathophysiology of giardiasis. For example, a 58 kilodalton (kDa) “enterotoxin” produced by Giardia trophozoites activates signal transduction path-

ways in enterocytes resulting in excessive ion secretion and intestinal fluid accumulation (Gorowara et al., 1992, 1994; Kaur et al., 2001; Shant et al., 2002, 2004, 2005). Unidentified Giardia products also induce the disruption of enterocyte tight junctional proteins via activation of caspase-3 and MLCK (Teoh et al., 2000; Buret et al., 2002; Chin et al., 2002; Scott et al., 2002). Soluble extracts from Giardia trophozoites, ranging from 32 to 200 kDa in size, induce disaccharidase deficiencies (Mohammed and Faubert, 1995a). Finally, trophozoite attachment to enterocytes is mediated by several parasitic factors, including δ-giardinan (Jenkins et al., 2009), surface lectins (Inge et al., 1988; Pegado and de Souza, 1994; Katelaris et al., 1995; Sousa et al.,

Chap. 19 Pathophysiological and Clinical Manifestations of Giardiasis

311

Table 19.3 List of Giardia parasitic factors that may contribute to disease pathogenesis, and their mechanisms of action Effect of parasitic factor

Mechanism of action

Factor

Reference

Unknown cysteine protease

(Rodriguez-Fuentes et al., 2006)

Establishment of infection 1. Enterocyte attachment

VSPs

(Bermudez-Cruz et al., 2004)

Surface lectins

(Inge et al., 1988; Pegado and de Souza, 1994; Katelaris et al.,1995; Sousa et al., 2001)

δ-giardian

(Inge et al., 1988; Pegado and de Souza, 1994; Katelaris et al., 1995; Sousa et al., 2001; Bermudez-Cruz et al., 2004; Rodriguez-Fuentes et al., 2006; Jenkins et al., 2009)

2. Zoonotic infectivity (Singer et al., 2001; Prucca and Lujan, 2009)

VSPs

(Singer et al., 2001)

1. Antigenic switching

Arginine deiminase

(Touz et al., 2008)

Variant-specific Surface Proteins

(Nash et al., 1990; Byrd et al.,1994; Prucca and Lujan, 2009)

2. Cleavage of human IgA

Thiol proteinase

(Parenti, 1989)

3. Modulation of dendritic cell responses

Unknown

(Kamda and Singer, 2009)

4. Reduce enterocyte NO expression

Arginine deiminase

(Eckmann et al., 2000; Ringqvist et al., 2008)

1. Eosinophil infiltration

Unknown

(Jimenez et al., 2004)

2. Th2 immune response

Unknown

(Jimenez et al., 2004)

Immune evasion

Induction of specific immune responses

Pathophysiological responses in enterocytes 1. Disruption of tight junction proteins

(Teoh et al., 2000; Chin et al., 2002; Scott et al., 2002; Buret et al., 2002)

a. Activation of MLCK

Unknown

(Scott et al., 2002)

b. Induction of apoptosis

Unknown

(Chin et al., 2002; Yu et al., 2008)

2. Ion hypersecretion

(Gorowara et al., 1992, 1994; Jimenez et al., 2004)

a. Activation of signal transduction pathways

58 kDa enterotoxin

(Gorowara et al., 1992, 1994; Shant et al., 2004, 2005)

3. Luminal fluid accumulation

58 kDa enterotoxin

(Kaur et al., 2001; Shant et al., 2002)

4. Disaccharidase deficiencies

Unknown protein(s) (ranged 32 to 200 kDa)

(Mohammed and Faubert, 1995b)

2001), unknown Giardia cysteine proteases (Rodriguez-Fuentes et al., 2006), and VSPs (Bermudez-Cruz et al., 2004).

Further identification of Giardia parasitic factors implicated in pathogenesis may also help explain symptom variability. Indeed, recent observations indi-

312

cate that α-2 giardin may be a protein specific to assemblage A isolates (Steuart et al., 2008). However, its exact role remains unclear in the context of infection, and whether this factor may be involved in pathogenesis is yet to be assessed. Furthermore, the induction of enterocyte apoptosis may be strain and stimulus specific. Indeed, some G. duodenalis isolates fail to induce enterocyte apoptosis in intestinal epithelial cells, where parasitic products from other strains do (Chin et al., 2002). The identification of parasitic factors responsible for symptomatic giardiasis represents a key direction toward our understanding of this disease, and possibly new effective control measures.

19.6 Role of Host Factors in Pathogenesis As illustrated above, host lymphocytes activated during the infection appear to be responsible for the loss of epithelial surface and subsequent maldigestion and malabsorption seen in giardiasis. The possible implication of other host factors in disease pathophysiology warrants further research. The Giardia deiminase-induced depletion of epithelial arginine prevents enterocytes from synthesizing nitric oxide, a compound known to inhibit trophozoite growth and encystation (Eckmann et al., 2000). Arginine depletion has been shown to induce enterocyte apoptosis in other models (Philip et al., 2003; Potoka et al., 2003). However, a direct role for host arginine depletion in the context Giardia infection remains to be established. The anti-apoptotic SGLT-1-mediated enterocyte rescue appears to be overcome during the infection (Yu et al., 2008). This host defence mechanism requires an intact microtubular network, and is characterized by increased apical expression and Vmax of the SGLT-1 transporter (Yu et al., 2008). Whether host factors activated by infection in vivo may directly contribute to the inhibition of this endogenous protective mechanism is yet to be established. The identification of host or microbial factors responsible for symptoms in giardiasis offers promising research avenues in our attempts at controlling disease in a variety of enteric infections and chronic gastrointestinal disorders (Moss et al., 1996; Kim et al., 1998; McCole et al., 2000; Ciccocioppo et al., 2001; Di Sabatino et al., 2003).

A. G. Buret and J. Cotton

19.7 Conclusion In conclusion, the pathophysiological processes and variable clinical manifestations of Giardia infection remain incompletely understood. Based on the findings available to date, the causes of symptom production in giardiasis are clearly multifactorial, and implicate parasite as well as host factors that have yet to be fully identified. Similarly, the reasons why certain hosts develop chronic disease while others remain asymptomatic cannot simply be explained on the grounds of varying degrees of protective immunity. Figure 19.1 offers an overview of our current understanding of the pathophysiological processes in giardiasis. How some of these events may be modulated in the host to yield different clinical outcomes requires further investigation. The induction of enterocyte apoptosis appears to be one of the initial triggers for disease pathogenesis. As this effect is strain-dependent, a better understanding of how different Giardia genotypes may induce various degrees of epithelial injury offers a promising ground for future research. Finally, the infection is able to trigger chronic gastrointestinal disorders such as irritable bowel syndrome, and may cause disease at extraintestinal sites, via mechanisms that have yet to be identified. As some of these processes have been observed in a number of other disorders of the gastrointestinal tract, a better understanding of their biological basis may offer a rational basis to manage enteric disease far beyond the scope of giardiasis.

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A. G. Buret and J. Cotton Oberhuber G, Kastner N, and Stolte M (1997) Giardiasis: a histologic analysis of 567 cases. Scand J Gastroenterol 32(1): 48–51 Oksenhendler E, et al. (2008) Infections in 252 patients with common variable immunodeficiency. Clin Infect Dis 46(10): 1547–1554 Olson ME, et al. (1995) Effects of giardiasis on production in a domestic ruminant (lamb) model. Am J Vet Res 56(11): 1470–1474 Olson ME, et al. (1997) Giardia and Cryptosporidium in dairy calves in British Columbia. Can Vet J 38(11): 703–706. Olson ME, Morck DW, and Ceri H (1996) The efficacy of a Giardia lamblia vaccine in kittens. Can J Vet Res 60(4): 249–256 Onbasi K, et al. (2005) Common variable immunodeficiency (CVID) presenting with malabsorption due to giardiasis. Turk J Gastroenterol 16(2): 111–113 Ortega YR and Adam RD (1997) Giardia: overview and update. Clin Infect Dis 25(3): 545–549; quiz 550 Panaro MA, et al. (2007) Caspase-dependent apoptosis of the HCT-8 epithelial cell line induced by the parasite Giardia intestinalis. FEMS Immunol Med Microbiol 51(2): 302– 309 Parenti DM (1989) Characterization of a thiol proteinase in Giardia lamblia. J Infect Dis 160(6): 1076–1080 Pegado MG and de Souza W (1994) Role of surface components in the process of interaction of Giardia duodenalis with epithelial cells in vitro. Parasitol Res 80(4): 320–326 Pelayo L, et al. (2008) Giardia infections in Cuban children: the genotypes circulating in a rural population. Ann Trop Med Parasitol 102(7): 585–595 Perez O, et al. (1994) Evaluation of the immune response in symptomatic and asymptomatic human giardiasis. Arch Med Res 25(2): 171–177 Pettoello Mantovani M, et al. (1990) Intestinal giardiasis associated with ophthalmologic changes. J Pediatr Gastroenterol Nutr 11(2): 196–200 Philip R, Campbell E, and Wheatley DN (2003) Arginine deprivation, growth inhibition and tumour cell death: 2. Enzymatic degradation of arginine in normal and malignant cell cultures. Br J Cancer 88(4): 613–623 Pietrzak A, et al. (2005) Cutaneous manifestation of giardiasis – case report. Ann Agric Environ Med 12(2): 299–303 Potoka DA, et al. (2003) Peroxynitrite inhibits enterocyte proliferation and modulates Src kinase activity in vitro. Am J Physiol Gastrointest Liver Physiol 285(5): G861–G869 Prucca CG and Lujan HD (2009) Antigenic variation in Giardia lamblia. Cell Microbiol 11(12): 1706–1715 Quihui-Cota L, et al. (2008) Impact of Giardia intestinalis on vitamin a status in schoolchildren from northwest Mexico. Int J Vitam Nutr Res 78(2): 51–56 Ramachandran A, Madesh M, and Balasubramanian KA (2000) Apoptosis in the intestinal epithelium: its relevance in normal and pathophysiological conditions. J Gastroenterol Hepatol 15(2): 109–120 Read C, et al. (2002) Correlation between genotype of Giardia duodenalis and diarrhoea. Int J Parasitol 32(2): 229–231 Rendtorff RC (1954) The experimental transmission of human intestinal protozoan parasites. II. Giardia lamblia cysts given in capsules. Am J Hyg 59(2): 209–220

Chap. 19 Pathophysiological and Clinical Manifestations of Giardiasis Ringqvist E, et al. (2008) Release of metabolic enzymes by Giardia in response to interaction with intestinal epithelial cells. Mol Biochem Parasitol 159(2): 85–91 Robayo-Torres CC, Quezada-Calvillo R, and Nichols BL (2006) Disaccharide digestion: clinical and molecular aspects. Clin Gastroenterol Hepatol 4(3): 276–287 Robertson LJ, et al. (2010) Giardiasis – why do the symptoms sometimes never stop? Trends Parasitol 26(2): 75–82 Rodriguez-Fuentes GB, et al. (2006) Giardia duodenalis: analysis of secreted proteases upon trophozoite-epithelial cell interaction in vitro. Mem Inst Oswaldo Cruz 101(6): 693–696 Roxstrom-Lindquist K, et al. (2005) Giardia lamblia-induced changes in gene expression in differentiated Caco-2 human intestinal epithelial cells. Infect Immun 73(12): 8204–8208 Sahagun J, et al. (2008) Correlation between the presence of symptoms and the Giardia duodenalis genotype. Eur J Clin Microbiol Infect Dis 27(1): 81–83 Sanchez-Carpintero I and Vazquez-Doval FJ (1998) Cutaneous lesions in giardiasis. Report of two cases. Br J Dermatol 139(1): 152–153 Savioli L, Smith H, and Thompson A (2006) Giardia and Cryptosporidium join the ‘Neglected Diseases Initiative’. Trends Parasitol 22(5): 203–208 Schulzke JD, et al. (2006) Disrupted barrier function through epithelial cell apoptosis. Ann NY Acad Sci 1072: 288–299 Scott KG, et al. (2000) Jejunal brush border microvillous alterations in Giardia muris-infected mice: role of T lymphocytes and interleukin-6. Infect Immun 68(6): 3412–3418 Scott KG, et al. (2002) Intestinal infection with Giardia spp. reduces epithelial barrier function in a myosin light chain kinase-dependent fashion. Gastroenterology 123(4): 1179– 1190 Scott KG, Yu LC, and Buret AG (2004) Role of CD8+ and CD4+ T lymphocytes in jejunal mucosal injury during murine giardiasis. Infect Immun 72(6): 3536–3542 Sebbagh M, et al. (2001) Caspase-3-mediated cleavage of ROCK I induces MLC phosphorylation and apoptotic membrane blebbing. Nat Cell Biol 3(4): 346–352 Seow F, Katelaris P, and Ngu M (1993) The effect of Giardia lamblia trophozoites on trypsin, chymotrypsin and amylase in vitro. Parasitology 106(Pt 3): 233–238 Shant J, et al. (2002) A potentially important excretory-secretory product of Giardia lamblia. Exp Parasitol 102(3–4): 178–186 Shant J, et al. (2004) The alteration in signal transduction parameters induced by the excretory-secretory product from Giardia lamblia. Parasitology 129(Pt 4): 421–430 Shant J, et al. (2005) Mode of action of a potentially important excretory – secretory product from Giardia lamblia in mice enterocytes. Parasitology 131(Pt 1): 57–69 Silva RR, et al. (2009) Association between nutritional status, environmental and socio-economic factors and Giardia lamblia infections among children aged 6-71 months in Brazil. Trans R Soc Trop Med Hyg 103(5): 512–519 Singer SM, et al. (2001) Biological selection of variant-specific surface proteins in Giardia lamblia. J Infect Dis 183(1): 119–124 Singh KD, et al. (2000) Effect of Giardia lamblia on duodenal disaccharidase levels in humans. Trop Gastroenterol 21(4): 174–176

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318 Yang PC, et al. (2006) Chronic psychological stress in rats induces intestinal sensitization to luminal antigens. Am J Pathol 168(1): 104–114; quiz 363 Youle RJ and Strasser A (2008) The BCL-2 protein family: opposing activities that mediate cell death. Nat Rev Mol Cell Biol 9(1): 47–59 Yu LC, et al. (2005) SGLT-1-mediated glucose uptake protects intestinal epithelial cells against LPS-induced apoptosis and barrier defects: a novel cellular rescue mechanism? FASEB J 19(13): 1822–1835

A. G. Buret and J. Cotton Yu LC, Turner JR, and Buret AG (2006) LPS/CD14 activation triggers SGLT-1-mediated glucose uptake and cell rescue in intestinal epithelial cells via early apoptotic signals upstream of caspase-3. Exp Cell Res 312(17): 3276–3286 Yu LC, et al. (2008) SGLT-1-mediated glucose uptake protects human intestinal epithelial cells against Giardia duodenalisinduced apoptosis. Int J Parasitol 38(8–9): 923–934

Immunology of Giardiasis Steven M. Singer

Abstract Most infections with Giardia are controlled by the host within a few weeks, suggesting the ability of the immune system to control the infection. Giardia induces a robust production of IgA in human and animal infections and it has been thought for many years that antibody was the primary mechanism by which the immune system exerts this control. Many studies have indicated that numerous other immune activities are also activated by Giardia infections and that these may also be important mechanisms for controlling the infection. Recent studies have begun to elucidate how the immune response to Giardia is regulated, both how the innate response to the parasite is activated and possible ways in which the parasite may have evolved to manipulate the host response. This chapter will examine what is known about the induction of innate and adaptive responses to Giardia and how they contribute to the control of infection.

20.1 Infections in Humans Perhaps the most important question to address when trying to understand the immunology of giardiasis is whether infections naturally lead to the development of immunity. The ability of most individuals to control the Giardia infections within a few weeks suggests that there is an effective immune response. Studies of repeat infections in humans lend further support to the ability of the immune system to develop effective memory responses. Immune memory has been demonstrated in outbreaks whereby local residents, who are more likely to have had previous exposure to the parasite, exhibit significantly reduced rates of symptomatic disease compared to visitors. A 1965 outbreak in Aspen, Colorado yielded an 11.3% rate of diarrheal

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disease caused by Giardia among visiting skiers compared to only 5% in local residents (Gleason, 1970). A 1981 study of Giardia in the neighboring community of Aspen Highlands showed that long-term residents suffered less in an outbreak than individuals who had recently moved to the area (Istre et al., 1984). IsaacRenton and colleagues (1994) found clear evidence for prior infection leading to protection against symptomatic Giardia infection in a series of outbreaks in British Columbia. The mechanism of protection was not examined in these studies, and it is possible that prior infection may provide resistance to symptomatic disease rather than re-infection, similar to acquired immunity in malaria. A recent analysis of a cohort of children in Brazil with Giardia infection found a clear decrease in lactoferrin in stool, a marker of inflammation, during second and third Giardia infections (Kohli et al., 2008). This suggests that acquired immunity may indeed promote reduced development of symptomatic disease rather than sterile immunity. The development of chronic giardiasis in patients suffering from hypogammaglobulinemia has implicated antibodies as a major mechanism for controlling this infection. In particular, patients with either common variable immunodeficiency (CVID) or Xlinked agammaglobulinemia (XLA) are at increased risk of symptomatic giardiasis (Webster, 1980; Perlmutter et al., 1985). Interestingly, CVID is characterized by both B-cell and T-cell deficiency, as well as agammaglobulinemia (Herbst et al., 1994). Similarly, XLA can affect cell types other than B cells, making it difficult to conclude that the lack of antibodies alone is responsible for the inability of these patients to control Giardia infections. Indeed, chronic giardiasis is not a typical infection in patients with selective IgA deficiency, suggesting that there are other important aspects of CVID and XLA which need to be considered.

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Numerous studies have demonstrated IgA and IgG responses to Giardia in humans (Nash et al., 1987; Ljungström and Castor, 1992), and a wide variety of antigens have been proven to stimulate immune responses. Perhaps the best-characterized antigens of Giardia are the variant-specific surface proteins (VSPs, see Chap. 16). As their name implies, the parasite is able to change the pattern of VSP antigen expression during the course of a single infection. Thus, even though these proteins are often targeted by host antibodies, they are problematic for development of vaccines and diagnostics. Many investigators have also sought to identify non-variant antigens targeted by the immune response. Other antigens recognized by human antibodies include surface proteins (82 kDa and 65 kDa polypeptides), the cytoskeletal giardins, heat shock proteins, lectins, and tubulin in the disk and flagella (Einfeld and Stibbs, 1984; Moss et al., 1990; Crossley and Holberton, 1983, 1985; Char et al., 1992; Farthing et al., 1986). A recent study used sera from an outbreak in Sweden to identify commonly recognized protein antigens and identified sixteen additional targets (Palm et al., 2003). Much work remains to determine if any of these antigens might be useful for diagnostics or vaccines. Studies in patients with T cell deficiencies indicate that limited T cell responses do not increase susceptibility to Giardia infection. HIV-positive individuals with giardiasis have marginally lower anti-Giardia immunoglobulin levels along with severe CD4+ deficiencies, but are still able to resolve infections despite these immunological deficiencies (Janoff et al., 1988). These data suggest that Giardia itself is not opportunistic in individuals with HIV; infection rates of as low as 6.2% have been reported in HIV-positive individuals (Barrett et al., 2008; Viriyavejakul et al., 2009). Similarly, immunodeficient patients with thymic dysplasia (DiGeorge syndrome) also do not commonly suffer severe diarrheal manifestations from giardiasis (Webster, 1980). While CD4 T cells are clearly important for producing strong IgA responses, low numbers may be sufficient for control of giardiasis in humans. Several recent studies have tried to elucidate the role of cytokines made by T cells in human giardiasis. In one study, peripheral blood lymphocytes from naïve individuals produced interferon (IFN)-J in response to trophozoites (Ebert, 1999). It was unclear if these individuals had been previously exposed to the parasite,

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and IFN-J could have been produced by NK cells, rather than T cells in this context. Analysis of serum cytokine levels in infected humans does not indicate a strong bias toward either a Th1 or a Th2 response. Sera from infected adults in Poland contained elevated levels of both Th1 and Th2 cytokines (IL-5, IL-6, and IFN-J; Matowicka-Karna et al., 2009). A similar study in patients from Turkey found elevated levels of IL-2, but not IL-4 or IL-10 in infected patients (Bayraktar et al., 2005a). An analysis in infected children found elevated TNF-D and sIL-2R levels, but not IL-1E, IL-6, or IL-8 in uncomplicated giardiasis (Bayraktar et al., 2005b). Children with allergic complications, primarily urticaria, had higher levels of IgE and all cytokines analyzed. These cytokines could all be produced by activated mast cells, rather than T cells, however. A recent study in Mexico correlated high serum levels of MCP-1, IFN-J, IL-4, and IL-5 with increased duration of G. duodenalis infection, while high levels of IL-8 were associated with shorter durations (Long et al., 2010). Considered together these data indicate that humans are able to produce a broad array of cytokines in response to infection with Giardia. Similar to studies with antibodies, however, it is unclear which responses actually contribute to parasite control.

20.2 Infections in Animals Studies in mice allow for direct testing of the importance of particular aspects of the immune response. There exists a wide variety of strains with spontaneous and targeted mutations in multiple components of the immune system as well as antibodies for in vivo blockade and in vitro analyses. Several different animal models have been used, including infections in neonatal and adult mice with G. duodenalis, infections in adult mice with G. muris and infections in gerbils with G. duodenalis. These different models have occasionally produced discordant results, but in a few cases, identifying the reasons for these discrepancies has highlighted important facets of this disease.

20.3 The Antibody Response Because humans with immunoglobulin deficiencies develop chronic giardiasis and because infection gen-

Chap. 20 Immunology of Giardiasis

erates strong anti-parasite IgA responses, many investigators have examined the role of antibodies in controlling infections in animals. Snider and colleagues treated mice with anti-IgM antibodies in order to deplete naïve B cells and prevent antibody responses. They found that infection with G. muris was prolonged in these mice, consistent with the model that antibodies were necessary for control of this infection (Snider et al., 1985). X-linked immunodeficient (XID) mice carrying a mutation in the same gene that is altered in human XLA (btk), also develop prolonged infections with G. muris (Snider et al., 1988), again consistent with an important role for antibodies in giardiasis. However, XID mice acquired resistance to secondary infections as well as their wild-type controls (Skea and Underdown, 1991), suggesting that control of primary and secondary infections could involve different immune mechanisms. Moreover, XID mice actually produced even more anti-parasite IgA than wild-type controls (Snider et al., 1988), suggesting that this mutation has a more complicated pheno-

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type and that a lack of antibody production might not be the key defect in terms of Giardia infection. Infections of adult mice with G. duodenalis first indicated that B cells were not necessary for control of infections. Mice which lack B cells due to deletion of the P exons of the Ig heavy chain (PMT mice) controlled G. duodenalis infections as quickly as wild-type controls (Singer and Nash, 2000a). This experiment does not indicate that antibodies cannot control the infection, only that other mechanisms exist in the mouse which can control G. duodenalis infections in a similar time frame. μMT mice can actually produce limited amounts of IgA in some cases (Macpherson et al., 2001), but failed to make anti-Giardia IgA when infected with G. duodenalis (Fig. 20.1). Interestingly, Langford et al. (2002) reported that PMT mice exhibited a significant delay in clearance of G. muris infections, suggesting that there are differences in the interactions between these two parasite species and mice. One possible difference is that mice make different types of immune responses

Fig. 20.1 Anti-parasite IgA in wild-type and PMT mice. Mice were infected for 14 days and intestinal fluid was collected by flushing a segment of jejunum with PBS. In vitro cultures of GS/M(H7) were adhered to glass slides, fixed with acetone, and incubated with 1:20 dilutions of the intestinal fluid. Anti-IgA conjugated with FITC was used to visualize antibody responses and propidium iodide (PI) was used to visualize the parasite nuclei. Not every parasite is recognized by IgA in wild-type mice due to antigenic variation

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to the two parasite species. Alternately, non-antibody effector mechanisms which can eradicate G. duodenalis might be unable to eradicate G. muris. IgA is believed to be the most relevant antibody isotype for Giardia infections since it is the major isotype in the intestinal lumen. IgA is transported across epithelial layers by the poly-Ig receptor (pIgR) into the intestinal lumen and other secretions. IgA deficient mice have a profound defect in their ability to control both primary and secondary infections with G. muris (Langford et al., 2002). This is consistent with the idea that the antibody response, and IgA in particular, are essential in acquired immunity in the G. muris mouse model. Direct confirmation of differences in the role of IgA in controlling G. muris and G. duodenalis was obtained using mice lacking the poly-Ig receptor (pIgR). These mice fail to transport IgA into the intestinal lumen and developed chronic infections with G. muris, but not with G. duodenalis (Davids et al., 2006). This implies that G. duodenalis may be more sensitive than G. muris to non-antibody effector mechanisms in mice, or perhaps that G. muris is merely more sensitive to antibody-mediated cytotoxicity. Identification of non-antibody effectors which can eliminate G. duodenalis could be an exciting approach to treatment or vaccination of the human disease. Antibodies can be cytotoxic to Giardia in vitro. Serum from G. muris infected mice has been shown to agglutinate trophozoites and induce lysis with complement (Belosevic and Faubert, 1987). Complement-independent cytotoxicity of monoclonal antibodies targeting Giardia has also been described (Nash and Aggarwal, 1986). Furthermore, when sera containing high levels of anti-Giardia IgA were systemically administered to mice, they provided transient protection for the host against G. muris (Davids et al., 2006). Antibodies may also provide protection against G. duodenalis in mice. During our study of IL-6 deficient mice, we noted a significant delay in parasite elimination (Zhou et al., 2003). Examination of anti-parasite IgA responses in these mice and in wild-type controls indicated that all mice produced anti-parasite IgA, but that early during the infection that IgA reacted with only a subset of the parasites. Presumably this reflected antibodies reacting with VSPs expressed by a subset of parasites used for the ex vivo portion of the study. Interestingly, IgA anti-

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bodies reacting with all parasites examined could be detected 60 days post-infection, at the same time that the infection was eliminated from the mice (Zhou et al., 2003). These results suggest that antigenic variation by G. duodenalis (see Chap. 16) prevents antibodies from effectively controlling infections until such time as the antibodies recognize either invariant epitopes or all potential variant antigens. Several approaches have been used to identify nonvariant antigens conserved between Giardia strains. Since polyIgR deficient mice accumulated IgA in the serum rather than transport it into the lumen and because these antibodies could passively transfer protection, these sera were used to identify immunodominant antigens (Davids et al., 2006). Several 25–50 kDa antigens conserved between the GS(M) and WB/C6 strains were identified, but most of the 22 to >200 kDa antigens belonged exclusively to WB/ C6. A total of 12 proteins were identified by mass spectrometry: OCT, ADI, D-enolase, D-1, D-2, D-7.1, and D-11-giardins, as well as E-giardin, D2-tubulin, Giardia trophozoite antigen-2, uridine phosphorylase-like protein 1, and fructose-1,-6-bisphosphate aldolase. Antibodies to several of these antigens were also detected in the breast milk of infected humans, suggesting similar immune responses across the host species (Téllez et al., 2005). Other non-variant antigens include a 57 kDa antigen (Einfeld and Stibbs, 1984), a 49 kDa glycosylphosphatidylinositol (GPI) anchored protein (Das et al., 1994), and an 8 kDa fatty acid binding protein (Hasan et al., 2002).

20.4 The Cellular Immune Response Data from both G. muris and G. duodenalis infections in mice consistently demonstrate an essential role for CDC4+ T cells in controlling infections. Mice lacking a thymus, treated with antibodies against CD4, or with a deletion in the T cell receptor E chain locus all experience prolonged infection with both Giardia species (Stevens et al., 1978; Heyworth, 1989; Singer and Nash, 2000a). Moreover, adoptive transfer of CD4 T cells into T cell deficient recipients restores resistance to infection in both models (Scott et al., 2000; Singer, unpublished). It is possible that the role of CD4 cells is merely to provide help for B cell production of IgA, but depletion of CD4 cells prolonged G. duodenalis

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infections in B-cell deficient mice, suggesting an antibody independent role for T-cells (Singer and Nash, 2000a). In contrast to the role for CD4 T cells, rapid control of G. duodenalis infections in E2-microglobulin knockout mice which are class I MHC deficient and lack CD8 T cells ruled out a role for CD8+ T cells in protective immunity (Singer and Nash, 2000a). Finally, while DE-TCR deficient mice were unable to control G. duodenalis infections, mice with a deletion in the T cell receptor G locus cleared infections similarly to wild type (Singer and Nash, 2000a). In addition to a role in protective immunity, recent data have indicated a role for T cells in disease pathology. Scott et al. (2000) reported that microvillous atrophy and reduced levels of intestinal dissacharidases could be observed following infection of wild-type mice with G. muris. Infection of T-cell-deficient nude mice failed to produce either of these changes, despite having a greater parasite load. Interestingly, the brush border changes could be induced in naïve nude mice by adoptive transfer of CD8 T cells from infected wildtype mice, but not by CD4 cells. This suggests that the immune response responsible for inducing symptoms is distinct from that responsible for parasite control.

20.5 Cytokines in Giardiasis In many infectious diseases, CD4 T cell responses become polarized into distinct subsets characterized by production of specific cytokines, i.e., IFN-J in Th1 responses, IL-4 in Th2 responses, and IL-17 in Th17 responses. Data on the cytokines produced by T cells during Giardia infections is minimal. Venkatesan et al. (1996) stimulated mesenteric lymph node T cells from G. muris infected BALB/c and B10.D2 mice with the mitogen ConA. They observed elevated production of IL-5 in BALB/c mice and elevated production of both IL-5 and IFN-J in the B10.D2 strain. Treatment of B10.D2, but not BALB/c, mice with anti-IFN-J led to enhanced cyst output while anti-IL-4 treatment had no effect in either strain. These data suggest that IFN-J responses contribute to parasite control. Studies, in the G. duodenalis model found no significant difference between trophozoite counts in C57BL/6 mice with and without a targeted deletion of the IFN-J gene, although there was a small trend toward prolonged infection in these mice (Singer and

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Nash, 2000a). In contrast, mice lacking IL-4, IL-4RD, or STAT-6 (a key signaling molecule for IL-4 and IL13 responses) on a BALB/c background were actually difficult to colonize with Giardia. We have previously interpreted the results as indicating no definitive role for either Th1 or Th2 responses in parasite control, but additional studies are needed before excluding that IFN-J may have a protective effect. Data from G. duodenalis infections in mice lacking IL-6 or TNF-D have been much clearer. Elimination of either cytokine led to significant increases in the number of trophozoites in the small intestine following infection (Bienz et al., 2003; Zhou et al., 2003, 2007). In both cases, the increase in parasite numbers was transient, indicating that the immune system can compensate for their absence. In the case of IL-6 deficiency, as noted above, the pattern of IgA responses was consistent with the idea that the eventual clearance of the parasite was due to the production of IgA which could recognize either invariant parasite antigens or all of the different VSPs expressed by the parasite. The enhanced parasite loads seen early in the infection correlated to higher levels of IL-4 mRNA (Bienz et al., 2003) and lower levels of NOS2 mRNA in the intestine (Li et al., 2006). This suggests that one role of IL-6 during the response to Giardia is to shape the development of the adaptive response. It could also be involved in regulating the types of effector mechanisms which are induced following infection (see below). Compared to IL-6 deficient mice, TNF-D-deficient mice and wild-type mice treated with anti-TNF-D had a less pronounced defect in parasite control (Zhou et al., 2007). These mice eliminated the infection after 28 days whereas IL-6 deficient mice required 60 days, and wild-type mice required only 14. Similar to the IL-6 knockouts, TNF-D-deficient mice produced normal levels of anti-parasite IgA. These data are all consistent with the idea that antibody-independent mechanisms exist in mice which can rapidly control G. duodenalis infections, while antibodies have an important role later in the infection.

20.6 Innate Immunity The development and polarization of an immune response is greatly influenced by the early events of the

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infection. In giardiasis, the parasite initially contacts epithelial cells lining the intestinal tract and later dendritic cells in the sub-mucosa are exposed to parasite antigens which they can process and present to T cells to initiate the adaptive response. Release of chemokines by the intestinal epithelium may function to recruit both innate and adaptive immune cells upon infection with Giardia. Two studies have examined the response of intestinal epithelial cell lines to Giardia. Using semi-quantitative RT-PCR, Jung et al. (1995) found that live parasites failed to induce transcription of IL-8, MCP-1 (CCL2), TNF-D, GMCSF, IL-1D, IL-1E, IL-10, or TGF-E1. In contrast, Roxström-Lindquist and colleagues (2005) used a microarray to examine transcription and identified increased expression of CCL2, CCL20, and CXCL1-3 from polarized Caco2 cells in response to Giardia. Secretion of CCL20 was further confirmed using ELISAs. These chemokines are responsible for the recruitment of many types of immune cells, including neutrophils, dendritic cells, T cells, and macrophages. Roles for these chemokines in giardiasis have not been examined directly, although (as noted above) high serum levels of MCP-1 (CCL2) have recently been correlated with more extended bouts of Giardia

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infection (Long, 2010). CCL2 was the only cytokine examined in both studies and the results were discordant. This might reflect differences in the detection methods employed or differences in the differentiation status of the Caco2 cells used for stimulation. While Jung et al. stimulated cells upon reaching confluency, Roxström-Lindquist and colleagues allowed the cells to differentiate for 3 weeks prior to being tested. It should be noted that while some infections with Giardia are accompanied by inflammation in the small intestine, this is not the case in the majority of human infections and does not appear to correlate with the severity of symptoms (Oberhuber and Stole, 1990). In the adult mouse model of G. duodenalis infection, little overt inflammation is noted (Fig. 20.2), although we have not examined chemokine production in these mice. Dendritic cells (DCs) are able to recognize numerous types of pathogens through a family of Toll-like Receptors (TLRs). TLR signaling typically leads to DC maturation, secretion of pro-inflammatory cytokines and an enhanced ability to activate naïve T cells. G. duodenalis has the capacity to inhibit pro-inflammatory cytokine production by DCs in response to several different TLR agonists, including

Fig. 20.2 Lack of inflammation in G. duodenalis infected mice. Segments of jejunum were fixed in formaldehyde, embedded in paraffin, sectioned and stained with hematoxylin and eosin. No obvious differences are seen between uninfected mice (left) and mice infected for 7 days (right) with the GS/M(H7) strain

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Fig. 20.3 MBL binding to G. duodenalis trophozoites. Parasites were allowed to attach to slides, fixed with acetone and incubated with serum from MBL deficient mice alone A or supplemented with 5 Pg/ml rMBL B–D. 1 mM GlcNAc C or D-methyl-galactopyrannoside D were included as competitors. After washing, samples were treated with anti-mouse MBL followed by a FITC conjugated goat anti-mouse secondary antibody

LPS (Kamda and Singer, 2009). There was also an effect on DC maturation. Exposure of bone marrow derived DCs to Giardia extracts led to slightly elevated expression of the costimulatory molecules CD80 and CD86, and CD40 levels similar to that induced by LPS. In contrast, parasite extract was a poor inducer of cytokines, inducing no IL-10 or IL-12 and only small amounts of IL-6 and TNF-D. While LPS alone strongly induced the secretion of IL-6, IL-12, IL-10, and TNF-D, co-incubation of DCs with LPS and parasite extracts led to reduced levels of IL-6, IL-12, and TNF-D. Interestingly, IL-10 levels were increased by co-incubation of DCs with LPS and parasite extract and treatment with anti-IL10R antibody partially restored production of IL-12. Furthermore, inhibition of IL-12 secretion was even more significantly reversed by treatment with wortmannin, an inhibitor of phosphoinositide-3 kinase (PI-3K). These data suggest that Giardia may activate PI-3K in dendritic cells, resulting in reduced production of IL-12 that might direct development of Th1 cells. The ability of Giardia antigens to actively direct development of Th2 responses was demonstrated by Jimenez and colleagues (2009). Immunization of mice using an excretory/secretory fraction of parasite antigens led to specific IL-4 responses to these parasites. Interestingly, development of this IL-4 response required proteolytic activity as it was inhibited by heat denaturation and addition of the protease inhibitor E-64. In addition to being recognized by cellular components of the innate immune system, there is evidence

that Giardia can be recognized by soluble factors. Samuelson and colleagues (2005) have recently demonstrated the presence of the sugar N-acetyl-glucosamine (GlcNAc) on cytosolic and surface proteins of Giardia trophozoites. This sugar is a ligand for the mannose binding lectin (MBL) and it has recently been shown that MBL is able to bind to strain WB trophozoites in vitro and that lysis by normal human serum can be inhibited by addition of GlcNAc to inhibit binding (Evans-Osses et al., 2010). Figure 20.3 shows that MBL binding to trophozoites of strain GS can also be inhibited by soluble GlcNAc. The importance of lysis by complement in vivo has not yet been shown and given the parasite’s location in the gut lumen, may not be a major component of parasite eradication.

20.7 Anti-parasite Effector Mechanisms 20.7.1 Defensins While antibodies can by cytotoxic for Giardia trophozoites, numerous studies indicate that antibody-independent mechanisms exist which can also participate in immunity to this infection. Indeed, the studies cited above suggest that control of G. duodenalis in the mouse model is due first to these antibody-independent mechanisms and later to the production of IgA. Human milk and duodenal fluids have been shown to be lethal for Giardia trophozoites, suggesting that that they have anti-Giardia activities

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that could help prevent establishment of infection or possibly contribute to the elimination of infection (Reiner et al., 1986). In 1994, Aley et al. examined the ability of a variety of antimicrobial peptides to kill Giardia trophozoites. This group found that the mouse intestinal D-defensins, cryptdins 2 and 3, were toxic for trophozoites, but the related peptides, cryptdins 1 and 6 were not. Humans express two D-defensins HD5 and HD6 in the intestinal tract and three more in neutrophil granules (HNPs 1–3). This study tested HNP-1 and HNP-2, and found that while HNP-2 was effective, much higher doses of HNP-1 were required to kill trophozoites. These data indicate that specific D-defensins are clearly able to kill trophozoites of G. duodenalis, but that there is a level of specificity involved which remains unclear. The role of cryptdins was examined in the G. muris mouse model in a review by Eckmann (2003) using mice lacking the protease matrilyin (MMP-7). Matrilysin cleaves the pro-peptides from all D-defensin precursors in mice and, therefore, mice lacking this enzyme have no detectable D-defensin activity. G. muris levels in the small intestine of MMP-7 deficient mice were actually lower in MMP-7 deficient mice than in wild-type mice at one-week post infection, and were not statistically different at 3 and 7 week time points (Eckmann, 2003). This indicates that the cryptdins do not help eliminate G. muris in mice. It was suggested that the defensins may function indirectly to promote G. muris infection by altering the commensal flora of the small intestine, which is known to effect the ability of G. duodenalis to colonize mice (see Sect. 20.8). Interestingly, the data presented showed that while wild-type mice had a decrease in parasite load of ~50-fold between weeks 1 and 3, the parasite loads in the MMP-7 deficient mice remained relatively constant. Additional studies of the role of defensins in animals and the ability of human intestinal defensins to kill Giardia are clearly needed.

(SNP) and 3-Morpholinosydnonimine hydrochloride (SIN-1) can inhibit trophozoite growth, kill parasites and inhibit differentiation from trophozoite to cyst and vice versa (Fernandes and Assreuy, 1997; Eckmann et al., 2000). Interestingly, the ability of intestinal epithelial cells in culture to produce NO was inhibited by live Giardia. Consumption of arginine by the parasite reduced its availability for conversion to NO by NOS2 (Eckmann et al., 2000). Whether arginine would be limiting in an in vivo situation is unclear. The importance of NO in the mouse model was studied using NOS2 (inducible nitric oxide synthase) deficient mice, the drug NG-nitro-L-arginine methyl ester (L-NAME) which inhibits all 3 isoforms of NOS and N-iminoethyl-L-lysine (L-NIL) which is specific for NOS2 (Li et al., 2006). Mice lacking NOS2 and mice treated with L-NIL both eliminated G. duodenalis parasites with kinetics identical to that seen in untreated wild-type mice. However, L-NAME treatment consistently resulted in prolonged infections, even when used to treat NOS-deficient mice (Li et al., 2006). This suggested that either NOS1 (neuronal NOS) or NOS3 (endothelial NOS) plays a role in the elimination of the parasite. Experiments with 7-nitroindazole (7-NI) and NOS1 confirmed that the neuronal isoform is required to rapidly eliminate the parasite (Andersen et al., 2006; Li et al., 2006). Indeed, Giardia infection leads to enhanced expression of NOS activity in the enteric nerves which control intestinal motility (Fig. 20.4) and rates of intestinal transit are markedly elevated in mice following infection with either G. duodenalis or G. muris (Li et al., 2006; Andersen et al., 2006). Interestingly, the increased transit was seen in wild-type mice but not in SCID mice, indicating that adaptive immune responses are required for the change in motility (Andersen et al., 2006; Li et al., 2006). Enhanced transit rates could be blocked using L-NAME, indicating that NO production was also involved.

20.7.2 Nitric Oxide

20.7.3 Mast Cells

Nitric oxide (NO) is an important effector mechanism for the control of several intracellular pathogens. In vitro experiments have indicated that NO donors such as glutathione-S-nitric oxide (GSNO), S-Nitrosoacetyl-penicillamine (SNAP), sodium nitroprusside

NO is known to be an inhibitory neurotransmitter and is able to relax smooth muscle. Increases in intestinal transit, however, require coordinated contraction and relaxation of these muscles in order to generate increased propulsion. Increases in the contractile

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A

B

C

D

Fig. 20.4 nNOS activity in enteric nerves of infected mice. The myenteric plexus was exposed, fixed and stained for NADPH Diaphorase activity, a marker of nNOS activity in these nerves. A and B are samples of jejunum. C and D are sample of colon. A and C are naive mice while B and D are mice infected for 7 days with G. duodenalis. Staining and imaging by Terez Shea-Donohue, University of Maryland Medical School

force of intestinal smooth muscle have also been observed in mice and gerbils following infection with G. duodenalis (Deselliers et al., 1997; Li et al., 2007). The neurotransmitter cholecystokinin (CCK) was recently shown to be important for mediating enhanced muscle contractility following Giardia infection (Li et al., 2007). Strips of longitudinal muscle from the intestines of Giardia infected mice exposed to CCK exhibited significantly greater increases in contractile force compared to muscle preparations from uninfected mice. In contrast, several other neurotransmitters including histamine, 5-hydroxytrptamine, and a PAR-1 agonist induced similar changes in muscle from infected and uninfected mice. The response to CCK was blocked by the addition of ketotifen, a compound which inhibits degranulation of mast cells, and by pre-treatment of tissues with compound 48/80, a mast cell agonist intended to deplete mast cells of their granule contents before treatment with CCK. Together these data suggest that CCK responsive mast cells in

the intestine are activated following Giardia infection, leading to muscular contraction. Coordination with an increase in muscle relaxation generated by NO leads to an increased rate of transit of intestinal contents and a more rapid elimination of the parasite. Interestingly, elevated CCK levels were reported in a small number of patients with giardiasis (Leslie et al., 2003). The primary role of CCK in the gastrointestinal tract is to signal the presence of fat in the lumen, leading to contraction of the gall bladder and release of bile into the lumen to aid in fat solubilization and absorption. Giardia scavenges lipids and consumes host bile, suggesting that the induction of CCK release by the parasite should be advantageous for the parasite. Erlich and colleagues first demonstrated a failure of mice with defects in mast cell function to control infections with G. muris (Erlich et al., 1983). Mast cells were similarly shown to be important in controlling G. duodenalis using mice with mutations in the gene for c-kit as well as antibodies to c-kit which pre-

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vent mast cell development and activation (Li et al., 2004). Furthermore, infections in wild-type mice lead to a massive infiltration of mast cells and the release of granule contents, as indicated by high serum levels of both histamine and mouse mast cell protease-1. The effects on motility are therefore two-fold, infection leads to an increase in the number of mast cells in the intestinal tract as well as an increase in CCK. It remains to be shown whether CCK can activate these mast cells directly or whether CCK acts on another cell type which functions as an intermediary. The importance of mast cells in human giardiasis has also not been directly addressed. However, several studies have correlated elevated levels of serum IgE with symptomatic giardiasis, particularly in patients exhibiting signs of urticaria or allergy (Di Prisco et al., 1988; Giacometti et al., 2003).

20.8 Gut Ecology The mammalian intestinal tract is anything but a sterile environment. In order to establish a successful infection, Giardia must elude not just host defenses, but also competition from other microbes, both commensals and other pathogens. The importance of the interaction between Giardia and commensal organisms was noted during some of the early studies to establish an adult mouse model of infection with G. duodenalis. The first studies focused on differences among Giardia isolates and discovered that Assemblage B strains such as GS could colonize mice, while assemblage A strains like WB could not (Byrd et al., 1994). Even using the GS strain, however, we experienced variability between experiments which we later were able to correlate to the supplier of the mice (Singer and Nash, 2000b). While attempts to infect mice obtained from the Jackson Laboratories were routinely successful, attempts with mice purchased from Taconic Farms almost always resulted in failure to colonize the mice. Housing mice from Taconic with mice from Jackson in the same cage resulted in transfer of resistance to infection within a few weeks. In contrast, treating the Taconic mice with a cocktail of antibiotics made them susceptible to infection, indicating that the presence of bacteria was responsible (Singer and Nash, 2000b). This effect was seen even in mice lacking adaptive immune systems, indicating that cross-protective an-

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tibodies could not be involved. More recently, in vitro studies have suggested that bacterial secretions might be toxic for the parasites. Supernatants from certain Lactobaccilli could kill Giardia, suggesting that probiotic approaches might be useful in treatment or prevention of giardiasis (Perez et al., 2001). Indeed, treatment of gerbils with L. johnsonii prior to and during G. duodenalis infection led to a marked decline in the level and duration of cyst shedding (Humen, 2005) and oral L. casei reduced trophozoite burdens and cyst shedding in mice (Shukla et al., 2008). Another recent study examined the effect of co-infection with an intestinal helminth on Giardia infection (von Allmen et al., 2006). Mice infected with Trichinella spiralis, a strong inducer of Th2 and mucosal mast cell responses, followed by Giardia exhibited much more pronounced Giardia infections. Thus, although mast cells are key components of the protective response to Giardia, other changes induced by this helminth contributed to success of the protozoan. Millions of Giardia infections occur each year in regions of the world endemic for intestinal helminthes, as well as enteric bacteria and protozoa. Studies of Giardia infection in isolation are difficult in humans, but may also be inadequate for a true understanding of the immune response to this infection.

20.9 Summary G. duodenalis is able to activate multiple components of the innate and adaptive immune systems. IgA and mast cells are known to be major contributors to control of this infection, while complement, antimicrobial peptides and nitric oxide may also exert a protective effect. The interplay between the innate and adaptive response and the role of concurrent infection with both commensal and pathogenic microbes requires much more study.

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329 Einfeld DA and Stibbs HH (1984) Identification and characterization of a major surface antigen of Giardia lamblia. Infect Immun 46: 377–383 Erlich JH, Anders RF, Roberts-Thomson IC, Schrader JW, and Mitchell GF (1983) An examination of differences in serum antibody specificities and hypersensitivity reactions as contributing factors to chronic infection with the intestinal protozoan parasite, Giardia muris, in mice. Aust J Exp Biol Med Sci 61: 599–615 Evans-Osses I, Ansa-Addo EA, Inal JM, and Ramirez MI (2010) Involvement of lectin pathway activation in the complement killing of Giardia intestinalis. Biochem Biophys Res Commun 395: 382–386 Farthing MJ, Pereira ME, and Keusch GT (1986) Description and characterization of a surface lectin from Giardia lamblia. Infect Immun 51: 661–667 Fernandes PD and Assreuy J (1997) Role of nitric oxide and superoxide in Giardia lamblia killing. Braz J Med Biol Res 30: 93–99 Giacometti A, Cirioni O, Antonicelli L, D’Amato G, Silvestri C, Del Prete MS, and Scalise G (2003) Prevalence of intestinal parasites among individuals with allergic skin diseases. J Parasitol 89: 490–492 Gleason NN, Horwitz MS, Newton LH, and Moore GT (1970) A stool survey for enteric organisms in Aspen, Colorado. Am J Trop Med Hyg 3: 480–484 Hasan SM, Maachee M, Córdova OM, Diaz de la Guardia R, Martins M, and Osuna A (2002) Human secretory immune response to fatty acid-binding protein fraction from Giardia lamblia. Infect Immun 4: 2226–2229 Herbst EW, Armbruster M, Rump JA, Buscher HP, and Peter HH (1994) Intestinal B cell defects in common variable immunodeficiency. Clin Exp Immunol 95: 215–221 Heyworth MF (1989) Intestinal IgA responses to Giardia muris in mice depleted of helper T lymphocytes and in immunocompetent mice. J Parasitol 75: 246–251 Humen MA, De Antoni GL, Benyacoub J, Costas ME, Cardozo MI, Kozubsky L, Saudan KY, Boenzli-Bruand A, Blum S, Schiffrin EJ, and Perez PF (2005) Lactobacillus johnsonii La1 Antagonizes Giardia intestinalis in vivo. Infect Immun 73: 1265–1269 Isaac-Renton JL, Lewis LF, Ong CS, and Nulsen MF (1994) A second community outbreak of waterborne giardiasis in Canada and serological investigation of patients. Trans R Soc Trop Med Hyg 88: 395–399 Istre GR, Dunlop TS, Gaspard GB, and Hopkins RS (1984) Waterborne giardiasis at a mountain resort: evidence for acquired immunity. Am J Public Health 6: 602–604 Janoff EN, Smith PD, and Blaser MJ (1988) Acute antibody responses to Giardia lamblia are depressed in patients with AIDS. J Infect Dis 157: 798–804 Jimenez JC, Fontaine J, Grzych JM, Capron M, and Dei-Cas E (2009) Antibody and cytokine responses in BALB/c mice immunized with the excreted/secreted proteins of Giardia intestinalis: the role of cysteine proteases. Ann Trop Med Parasitol 103: 693–703 Jung HC, Eckmann L, Yang SK, Panja A, Fierer J, MorzyckaWroblewska E, and Kagnoff MF (1995) A distinct array of proinflammatory cytokines is expressed in human colon

330 epithelial cells in response to bacterial invasion. J Clin Invest 95: 55–65 Kamda JD and Singer SM (2009) Phosphoinositide 3-kinasedependent inhibition of dendritic cell interleukin-12 production by Giardia lamblia. Infect Immun 77: 685–693 Kohli A, Bushen OY, Pinkerton RC, Houpt E, Newman RD, Sears CL, Lima AAM, and Guerrant RL (2008) Giardia duodenalis assemblage, clinical presentation and markers of intestinal inflammation in Brazilian children. Trans R Soc Trop Med Hyg 102: 718–725 Langford TD, Housley MP, Boes M, Chen J, Kagnoff MF, Gillin FD, and Eckmann L (2002) Central importance of immunoglobulin A in host defense against Giardia spp. Infect Immun 70: 11–18 Leslie FC, Thompson DG, McLaughlin JT, Varro A, Dockray GJ, and Mandal BK (2003) Plasma cholecystokinin concentrations are elevated in acute upper gastrointestinal infections. QJM 96: 870–871 Li E, Zhou P, Petrin Z, and Singer SM (2004) Mast cell-dependent control of Giardia lamblia infections in mice. Infect Immun 11: 6642–6649 Li E, Zhou P, and Singer SM (2006) Neuronal nitric oxide synthase is necessary for elimination of Giardia lamblia infections in mice. J Immunol 176: 516–521 Li E, Zhao A, Shea-Donohue T, and Singer SM (2007) Mast cell-mediated changes in smooth muscle contractility during mouse giardiasis. Infect Immun 75: 4514–4518 Ljungström I and Castor B (1992) Immune response to Giardia lamblia in a water-borne outbreak of giardiasis in Sweden. J Med Microbiol 36: 347–532 Long KZ, Rosado JL, Santos JI, Haas M, Al Mamun A, DuPont HL, Nanthakumar NN, and Estrada-Garcia T (2010) Associations between mucosal innate and adaptive immune responses and resolution of diarrheal pathogen infections. Infect Immun 78: 1221–1228 Macpherson AJ, Lamarre A, McCoy K, Harriman GR, Odermatt B, Dougan G, Hengartner H, and Zinkernagel RM (2001) IgA production without mu or delta chain expression in developing B cells. Nat Immunol 2: 625–631 Matowicka-Karna J, Dymicka-Piekarska V, and Kemona H (2009) IFN-gamma, IL-5, IL-6 and IgE in patients infected with Giardia intestinalis. Folia Histochem Cytobiol 47: 93–97 Moss DM, Mathews HM, Visvesvara GS, Dickerson JW, and Walker EM (1990) Antigenic variation of Giardia lamblia in the feces of Mongolian gerbils. J Clin Microbiol 28: 254–257 Nash TE and Aggarwal A (1986) Cytotoxicity of monoclonal antibodies to a subset of Giardia isolates. J Immunol 7: 2628–2632 Nash TE, Herrington DA, Losonsky GA, and Levine MM (1987) Experimental human infections with Giardia lamblia. J Infect Dis 156: 974–984 Oberhuber G and Stolte M (1990) Giardiasis: analysis of histological changes in biopsy specimens of 80 patients. J Clin Pathol 43: 641–643 Palm JE, Weiland ME, Griffiths WJ, Ljungström I, and Svärd SG (2003) Identification of immunoreactive proteins during acute human giardiasis. J Infect Dis 12: 1849–1859

S. M. Singer Perez PF, Minnaard J, Rouvet M, Knabenhans C, Brassart D, De Antoni GL, and Schiffrin EJ (2001) Inhibition of Giardia intestinalis by extracellular factors from lactobacilli: an in vitro study. App Env Micro 67: 5037–5042 Perlmutter DH, Leichtner AM, Goldman H, and Winter HS (1985) Chronic diarrhea associated with hypogammaglobulinemia and enteropathy in infants and children. Dig Dis Sci 12: 1149–1155 Reiner DS, Wang CS, and Gillin FD (1986) Human milk kills Giardia lamblia by generating toxic lipolytic products. J Infect Dis 154: 825–832 Roxström-Lindquist K, Ringqvist E, Palm D, and Svärd S (2005) Giardia lamblia-induced changes in gene expression in differentiated Caco-2 human intestinal epithelial cells. Infect Immun 73: 8204–8208 Samuelson J, Banerjee S, Magnelli P, Cui J, Kelleher DJ, Gilmore R, and Robbins PW (2005) The diversity of dolichol-linked precursors to Asn-linked glycans likely results from secondary loss of sets of glycosyltransferases. Proc Natl Acad Sci USA 102: 1548–1553 Scott KG, Logan MR, Klammer GM, Teoh DA, and Buret AG (2000) Jejunal brush border microvillous alterations in Giardia muris-infected mice: role of T lymphocytes and interleukin-6. Infect Immun 68: 3412–3418 Shukla G, Pushpa Devi P, and Sehgal R (2008) Effect of Lactobacillus casei as a probiotic on modulation of Giardiasis. Dig Dis Sci 53: 2671–2679 Singer SM and Nash TE (2000a) T-cell-dependent control of acute Giardia lamblia infections in mice. Infect Immun 68: 170–175 Singer SM and Nash TE (2000b) The role of normal flora in Giardia lamblia infections in mice. J Infect Dis 181: 1510– 1512 Skea DL and Underdown BJ (1991) Acquired resistance to Giardia muris in X-linked immunodeficient mice. Infect Immun 59: 1733–1738 Snider DP, Gordon J, McDermott MR, and Underdown BJ (1985) Chronic Giardia muris infection in anti-IgM-treated mice. I. Analysis of immunoglobulin and parasite-specific antibody in normal and immunoglobulin-deficient animals. J Immunol 134: 4153–4162 Snider DP, Skea DL, and Underdown BJ (1988) Chronic giardiasis in B-cell-deficient mice expressing the xid gene. Infect Immun 56: 2838–2842 Stevens DP, Frank DM, and Mahmoud AA (1978) Thymus dependency of host resistance to Giardia muris infection: studies in nude mice. J Immunol 120: 680–682 Téllez A, Palm D, Weiland M, Alemán J, Winiecka-Krusnell J, Linder E, and Svärd S (2005) Secretory antibodies against Giardia intestinalis in lactating Nicaraguan women. Parasite Immunol 27: 163–169 Venkatesan P, Finch RG, and Wakelin D (1996) Comparison of antibody and cytokine responses to primary Giardia muris infection in H-2 congenic strains of mice. Infect Immun 64: 4525–4533 Viriyavejakul P, Nintasen R, Punsawad C, Chaisri U, Punpoowong B, and Riganti M (2009) High prevalence of Microsporidium infection in HIV-infected patients. Southeast Asian J Trop Med Public Health 40: 223–228

Chap. 20 Immunology of Giardiasis von Allmen N, Christen S, Forster U, Gottstein B, Welle M, and Muller N (2006) Acute trichinellosis increases susceptibility to Giardia lamblia infection in the mouse model. Parasitology 133: 139–149 Webster AD (1980) Giardiasis and immunodeficiency diseases. Trans R Soc Trop Med Hyg 74: 440–443 Zhou P, Li E, Zhu N, Robertson J, Nash T, and Singer SM (2003) Role of interleukin-6 in the control of acute and

331 chronic Giardia lamblia infections in mice. Infect Immun 71: 1566–1568 Zhou P, Li E, Shea-Donohue T, and Singer SM (2007) Tumour necrosis factor alpha contributes to protection against Giardia lamblia infection in mice. Parasite Immunol 29: 367–374

Vaccination Against Giardia Peter Lee, Aws Abdul-Wahid and Gaétan Faubert

Abstract There are many reasons to justify the development of an anti-Giardia vaccine. Herein, we review the challenges encountered for developing a suitable vaccine, including the rationale for a vaccine and the importance of giardiasis as a public health issue in developing and developed countries. We also present a review of the candidate vaccine antigens derived from the trophozoite and cyst stages of the parasite, with arguments for the merits of developing vaccines aimed at either reducing the pathology of the disease, as well as blocking the transmission of the parasite to susceptible hosts. Finally, we conclude the chapter by relating our experiences in devising transmissionblocking vaccines using giardial cyst wall protein-2 as antigen as well as the different strategies we have employed for its delivery to gut-associated lymphoid tissues, with the intention of stimulating a protective local immune response.

21.1 Introduction Giardia is an extracellular protozoan dwelling in the lumen of the intestine of its host; therefore mucosal antibodies provide the most important adaptive mechanism of host defense. The literature is rich with information describing the host’s immune response to Giardia, implicating the importance of the role played by both B and TH cells (Roberts-Thompson and Mitchell, 1979; Snider et al., 1988; Faubert, 2000; Faubert et al., 2002; Roxström-Lindquist et al., 2006). The control of Giardia spp. requires a localized mucosal immune response that comprises a balanced Ag-specific CD4+ TH response (IFN-J and IL-4) as well as parasite-specific local mucosal IgA and IgG (Faubert, 2000; Roxström-Lindquist et al., 2006; Abdul-Wahid and Faubert, 2008). H. D. Luján et al. (eds.), Giardia © Springer-Verlag/Wien 2011

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Although, mucosal antibodies are stimulated during a natural infection, only a percentage of the infection-stimulated polyclonal antibodies are likely to confer protection. The majority of the mucosal antibodies are most likely directed against non-protective epitopes, thereby signaling the presence of the parasite instead of playing a role in its elimination. Therefore, the preparation of a protective vaccine formulation requires a characterization of the epitope(s) capable of stimulating the production of protective antibodies. This is supported by the work of Roberts-Thompson and Mitchell (1979) who were the first to develop a vaccine against Giardia by systemically injecting mice with a Giardia muris trophozoite crude extract emulsified in Freund’s complete adjuvant in peritoneal cavities and hind foot pads. The vaccine failed to protect susceptible C3H/He mice from challenge with G. muris cysts. We now know Giardia crude trophozoite lysates, used as vaccine antigens, are poor immunogens (Djamiatun and Faubert, 1998). In addition, the selection of the systemic route for vaccinating C3H/He mice might have also been inappropriate, since it promotes the production of circulating instead of local intestinal antibodies. Another aspect to consider in the development of a vaccine is the duration of protection. If a vaccine generates short-term protection, it fails to induce the formation of memory cells, leaving the host unprotected against future encounters with the parasite. The effectiveness of memory cells relies, in part, on the incubation period of the pathogen. In the case of Giardia, which has a long incubation period (7–15 days), maintaining detectable protective antibodies at the time of infection is not necessary, since this long incubation period gives the memory B cells time to respond by producing high levels of local antibodies. Therefore, a vaccine for giardiasis should be designed primarily to induce high levels of memory B cells.

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Although there are a multitude of reasons to justify the development of a vaccine against Giardia, the three most important ones may be the following: First, Giardia has been recognized as a re-emerging opportunistic pathogen (Pond, 2007) affecting highrisk groups such as young children, the elderly, and those who are immuno-compromised. Second, although Giardia is ubiquitous worldwide, giardiasis is a severe debilitating infection affecting thousands annually especially in developing countries due to its higher prevalence in those areas. Third, Giardia has zoonotic potential and its main mode of transmission is via non-treated surface water, which is used for drinking, washing food, and personal hygiene especially in developing countries whereby access to clean drinking water is not guaranteed.

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ing-vaccine (TBV) is most appropriate in regions whereby a reservoir source can be identified. In fact, the aim of a Giardia TBV is to reduce the burden of infective cysts released into the environment, to the point where transmission can no longer be sustained in an endemic area (Kaslow, 2002). First incepted in the early 1980s, TBVs represent a novel approach to controlling parasitic organisms, since they directly interfere with transmission and can be more beneficial and cost effective than prophylactic or therapeutic vaccines, accomplishing both tasks simultaneously (Kaslow, 2002). Thus far, parasite TBVs have been investigated for schistosomiasis, malaria, leishmaniasis cryptosporidiosis and recently giardiasis (Abdul-Wahid and Faubert, 2007).

21.2 Targetting Transmission versus Pathology

21.2.1 Factors to Consider Before Developing Anti-Giardia Vaccine for Developing Countries

Parasites have complex life cycles, hence the preparation of vaccines against protozoa or helminthes is challenging. Giardia has a rather simple direct life cycle and yet, it offers at least two strategies for vaccine development. The vaccine can specifically aim at the trophozoite or cyst stage of the life cycle. The vegetative stage is responsible for the acute phase of the infection, the pathology to the villi and for the diarrhea. The cyst form is responsible for its survival out of the host and its subsequent transmission to susceptible hosts. The obvious question to be posed for Giardia vaccine design is which stage to target? Designing a vaccine geared against pathology is most appropriate for regions where the infection is endemic and may be considered more as immunotherapeutic, since it reduces the severity of symptoms, hence ameliorating a quicker recovery of the host. For example, in developed countries, child care workers, children attending daycare centers, school aged children, international travelers, hikers, campers, and swimmers which are at risk would stand to benefit from this type of vaccine (Faubert et al., 2002). On the other hand, in developing countries, this type of vaccine can serve as a prophylactic, in addition to acting as an immunotherapeutic treatment against giardiasis symptoms in endemic areas. Alternatively, a transmission-block-

In developing countries, particularly in Asia, Africa, and Latin America, about 200 million people have symptomatic giardiasis with some 500,000 new cases reported each year (Dib et al., 2008). Infections result from ingesting cysts present in contaminated water used for washing fresh food or for recreational purposes. With the rapid growth rate of urbanization in Africa and Asia and global warming, the level of safe water supply is diminishing, making these locations vulnerable to higher risk of water-related diseases. Hence, access to clean drinking water is a crucial concern for the governments of developing countries (Pond, 2007). In a study focusing on giardiasis prevalence in South Asia, South East Asia, and the Far East, Dib et al. (2008) remarked higher levels in urban than in rural areas, more infections among poor communities, and slightly higher prevalence in males than in females with age range of 2- to 5-yearold children, and among university students, and oldaged people. Since the main cause of Giardia transmission can be attributed to inherent economical and other deficiencies within the social infrastructure of developing countries, vaccination against giardiasis proves extremely challenging due to its endemicity among the population. For example, children are particularly affected since their immune systems may not be fully

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developed or may be compromised due to nutritional deficiencies or other infections. As a result, administering an immunotherapeutic vaccine formulation may be ineffective since the vaccine is unable to stimulate a compromised immune system. A TBV strategy aimed at domestic and livestock animals, which live in close proximity of humans may be useful since Giardia can be zoonotic (Faubert, 2000). However, faced with the choice of either developing an immunotherapeutic or TBV strategy, an immunotherapeutic vaccine would be of more immediate benefit than a TBV, provided that the immune system of the target population is not compromised. We believe that committing funds to build infrastructures for providing and maintaining clean drinking water should be top priority to reduce giardiasis incidence in developing countries instead of developing a vaccine. Prevention through continuous health education would also contribute to the overall solution by increasing awareness about food and water contamination, the avoidance of swimming in unclean water, and proper hygiene (Dib et al., 2008).

21.2.2 Factors to Consider Before Developing Anti-Giardia Vaccine for Developed Countries In developed countries, giardiasis presents a different picture. The majority of the population is not immuno-compromised with incidence being prominent mainly in children, especially in daycare centers, and among travelers returning from endemic regions. Approximately 100 cases are reported annually in Japan, whereas it is over 9000 in Canada and at least 20,000 in the USA (Pond, 2007). This increasing incidence in recent years is due to the deteriorating conditions of surface water and/or the infrastructure to provide clean drinking water. At least 325 water-borne outbreaks of parasitic protozoan diseases have been reported in the literature, with Giardia duodenalis and Cryptosporidium parvum accounting for the majority of outbreaks (O’Handley and Olson, 2006). Infected livestock has long been incriminated as a source for the water-borne transmission of giardiasis. In fact, Giardia prevalence studies in beef and dairy cattle are reporting rates varying from 15 to 60% (Buret et al., 1990; Ralston et al., 2003). More-

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over, calves are more likely to shed Giardia cysts than adults, resulting in an increased concentration of Giardia cysts being detected in agricultural runoff during peak calving time (O’Handley and Olson, 2006). In general, giardiasis in cattle has been linked to confinement practices on dairy farms and feedlot confinement on beef farms (Ruest et al., 1998; Ralston et al., 2003). Management of fecal waste from cattle living in confinement is important since water run-off can reach surface water or even contaminate groundwater. One outbreak of human water-borne giardiasis in the USA, implicating pasture run-off has been reported (Weniger et al., 1983). Since fecal waste is used as fertilizer, there are major concerns with spreading manure onto agricultural land used for the production of crops and livestock grazing because of the potential for contaminated fecal material reaching surface water (Barwick et al., 2003). It is clear that Giardia, originating from livestock animals, presents one of the greatest problems to the human population in general, but especially in developed countries due to increased livestock operations. Therefore, a TBV designed for vaccinating livestock is an interesting solution to stop the spreading of Giardia cysts into the environment.

21.3 Candidate Antigens for a Vaccine Against Giardia Pathology Most studies focusing on using Giardia antigens as vaccine candidates have been done using the WB isolate (ATCC access number 30957), originating from a duodenal aspirate of a 30-year-old human male obtained in 1979 in Bethesda, Maryland. This isolate and others can be easily grown axenically in TYI-S-33 culture medium supplemented with cysteine. The availability of culture media and the mouse model of the disease with its natural parasite, G. muris, have largely contributed to our knowledge of Giardia antigens. A variety of antigens usually derived from crude extracts of trophozoites from the WB isolate or other isolates grown in vitro have been used by different laboratories to study acquired immunity. However, the Giardia WB isolate commonly used by most laboratories, may differ due to the conditions used in each laboratory for cultivating the trophozoites. Differences even between trophozoites obtained from

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symptomatic and asymptomatic patients have been reported (Moss et al., 1991). Hence, it is difficult to compare the results on acquired immunity or vaccines prepared in different laboratories. The antigens identified in Giardia trophozoites having potential as target antigens can be classified into five different groups according to their physiologic and somatic properties.

21.3.1 The Heat Shock Proteins (HSPs) Heat shock proteins are chaperones expressed by living cells in order to help them survive extrinsic and intrinsic stresses. Giardia trophozoites live in the intestine, which is a stressful habitat, and several HSPs secreted by Giardia trophozoites have been identified (Gupta et al., 1994). Serum antibody response to Giardia HSP-57 has been studied in children with persistent diarrhea due to giardiasis. Interestingly, HSP-57 antibodies revealed that only the level of IgM increases while the levels of IgA and IgG were similar to non-infected children. Contrary to the children clearing the infection, the switch from an IgM to IgG or IgA response did not occur in patients with chronic giardiasis (Gupta et al., 1994). This failure may explain, in part, the occurrence of chronic giardiasis since IgA antibodies are playing a major role in the clearance of the infection from the small intestine (Faubert, 2000; Roxström-Lindquist et al., 2006). This study indicates the existence of trophozoite proteins having a negative effect on the immune system. To our knowledge, there are no reports in the literature using HSPs as target antigens for vaccination in giardiasis.

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21.3.3 The Giardins Giardins represent a family of proteins found only in Giardia spp., and consist of a group of ~30-kDa proteins found in microribbons, attached to microtubules in the disk cytoskeleton of the trophozoite or in association with other organelles (Holberton et al., 1988). The interest in giardins as vaccine antigens stems from the fact that these somatic proteins are not present in the cytoskeletons of host cells and because they are highly conserved among different giardial isolates. In addition, giardins appear to be highly immunogenic (Roxström-Lindquist et al., 2006). Several laboratories have raised antibodies against the giardins as a tool to study their structures but there is only one study on the destructive effects of anti-giardin-antibodies on trophozoites. Pre-treatment of G. duodenalis trophozoites with anti-recombinant G-giardin sera affected the morphology of the trophozoites and inhibited trophozoite binding to the surface of culture slides (Jenkins et al., 2009). The authors concluded that these results may lead to the development of immunotherapeutic agents against giardiasis.

21.3.4 The Tubulins Tubulins are cytoskeleton proteins found in the flagella, ventral disk, funis, and median body of Giardia (Holberton et al., 1988). They represent a primary target for the immune system since they are found in many organelles in the trophozoites. However, there is less interest in using them as a prime target antigen for vaccine since they are also found in the cytoskeletons of the host cell.

21.3.2 The Lectins

21.3.5 Variant Surface Proteins (VSPs)

These glycoproteins are abundant on the plasma membrane of trophozoites. Therefore, they are important molecules to consider, since they are among the first foreign proteins detected by the local immune system. Activation of G. duodenalis lectins by proteases from the human duodenum has been reported (Lev et al., 1986). Unfortunately, the role played by the lectins with regards to the immune response to Giardia remains to be elucidated.

Antigenic variation is considered a major mechanism allowing parasite evasion from the host’s immune system, leading to chronic infections despite continuous immune pressure (Deitsch et al., 2009). Antigenic variation in Giardia involves VSPs, which are cysteine-rich integral membrane proteins that posses a variable extracellular N-terminal region and a conserved C-terminal. VSPs form a thick coat covering the entire surface of the trophozoite; and are among

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the first foreign molecules recognized by the host’s immune system. However, only one of about 200 VSP genes encoded by the Giardia genome is expressed on the surface of individual Giardia cells at any given time (Prucca and Lujan, 2009), but switching expression to an antigenically distinct VSP occurs even in culture (Prucca et al., 2008). A functional role of antibodies in the selection of phenotypic variants during the course of infection has been proposed because a humoral immune response in Giardia-infected hosts coincides with the elimination of the original VSP (Gottstein and Nash, 1991). The fact that antigenic variation is not observed in athymic mice and the initial surface variant antigens remained unchanged after encystation, indicate that the phenomenon of antigenic variation, in giardiasis, is also driven by the immune system of the host. For a more complete review on trophozoite antigens, used in studies to protect laboratory animals (see Faubert, 2000).

21.4 Vaccines Designed to Reduce Pathology 21.4.1 Introduction The first vaccine aimed at reducing pathology can be attributed to Vinayak et al. (1992) who orally immunized inbred mice with a G. duodenalis 56 kDa surface antigen. Their vaccine not only inhibited the establishment of trophozoites by blocking intestinal colonization, but also prevented their multiplication and expedited their elimination from the gut thereby reducing pathology. Subsequently, Olson and colleagues generated a Giardia vaccine geared to reduce pathology in companion animals such as cats and dogs (Olson et al., 1996). The vaccine formulation was primarily based on a crude G. duodenalis cell extract, and was reported to provide protection in kittens by reducing or eliminating intestinal trophozoites and fecal cyst excretion. Fort Doge Animal Health (Division of Wyeth/Pfizer) subsequently licensed the vaccine formulation and commercialized it after being granted a license by the USDA, releasing it as the first Giardia vaccine to prevent disease and reduce cyst shedding. The vaccine is currently marketed as GiardiaVax• for dogs and Felo-O-Vax GiardiaVax• for cats. Unfortunately, there is no information available in the

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literature on how this commercial vaccine stimulates the immune system of the recipient.

21.4.2 Hurdles in Constructing a Vaccine Using Giardia Trophozoite Proteins Since trophozoites reside in the intestinal lumen and do not usually invade tissues, the stimulation of the immune system is limited to the intestinal mucosal sites. In vitro experiments have shown a poor stimulation of Th1 and Th2 cytokines (Djamiatun and Faubert, 1998; Roxström-Lindquist et al., 2006). When extracts of G. muris trophozoites were used to stimulate the production of cytokines by Peyer’s patch and spleen cells from infected mice, IL-4, IL-5, and IFN-J were not detected (Djamiatun and Faubert, 1998). Likewise, the gene expression of several cytokines in vitro by human intestinal epithelial cells, five hours after G. duodenalis infection, did not reveal the stimulation of cytokines (Roxström-Lindquist et al., 2006). These results suggest that trophozoites are poor immunogens or are capable of down-regulating the immune system. In addition, the phenomenon of antigenic variation (Prucca and Lujan, 2009), which has been reported in animal models and humans (Gottstein and Nash, 1991), is also a major hurdle in the preparation of a vaccine to prevent or even limit the pathology associated with giardiasis. However, Prucca et al. (2008) recently proposed that the control of surface antigen expression in Giardia involves a mechanism similar to RNA-interference (RNAi). They reported a reduction in the expression of components of the RNAi pathway (RNA-dependent RNA polymerase and Dicer enzymes knocked-down) leading to a change from single to multiple VSP expressions in individual Giardia trophozoites (Prucca et al., 2008). The authors, concluded that the host should be able to prevent infection by simultaneously developing specific immune responses to all variable surface molecules instead of just one (Prucca and Lujan, 2009). Therefore, deregulating antigenic variation could be useful in generating vaccines preventing the infection and pathology since Giardia’s mechanism of evasion of the host’s immune reactions may depend on switching expression among antigenically distinct VSPs. Recently, Lujan et al. (Rivero et al., 2010) proposed an alternative vaccine strategy involving the use of

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VSPs to prevent infection and reduce pathology. Prophylactic oral vaccination with either trophozoites simultaneously expressing many VSPs or a purified VSP cocktail mix consisting of VSPs from transgenic cells was able to confer strong protection in gerbils against a challenge infection with G. duodenalis. Antigenic variation is of importance for Giardia to evade the host immune response. Infections with trophozoites expressing a unique VSP on their surface were highly resistant to challenge by trophozoites expressing the original VSP. They were not protected with trophozoites expressing an antigenically different VSP. For more information on antigenic variation (see Prucca and Lujan, 2009; Gottstein and Nash, 1991).

21.4.3 Immune Responses Required for Reducing the Pathology Secretory antibodies of the IgA and IgM isotypes are attractive candidates for immune defense against Giardia, because they are secreted in large quantities into the intestinal lumen. The mechanisms by which IgA exerts its anti-giardial functions are not well understood, but are likely to involve immune expulsion by immobilizing or detaching the trophozoites from the intestinal epithelium or acting on the mucus layer rather than killing the trophozoites (Langford et al., 2002). The use of an attenuated Salmonella typhimurium vaccine constructed to express and to deliver a portion of G. duodenalis trophozoite variant surface protein 7 (VSPH7) to the intestinal mucosa of mice (Stäger et al., 1997) provided some insight on the role played by specific antibodies. Oral administration of the VSPH7-expressing recombinant Salmonella stimulated the production of Ag-specific serum IgG and intestinal IgA antibodies. The anti-VSPH7 antibody concentration stimulated by vaccination was similar to that induced following infections in mice with G. duodenalis clone GS/M-83-H7. Isotype analysis of the vaccine-elicited serum antiVSPH7 IgG antibodies demonstrated the presence of IgG1- and/or IgG2b antibody production. No significant IgG2a anti-VSPH7 antibody production was detected in infected or in vaccinated animals. Taken together, these results indicate that the presentation of VSPH7 in the context of a recombinant S. typh-

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imurium may stimulate an Ag-specific TH2-type of an immune response. Antibodies against giardins may also play a role in inhibiting or preventing trophozoite attachment to the intestinal villi. Jenkins et al. (2009) demonstrated that binding of G-giardin specific antibodies to the ventral disc affects the ability of trophozoites to bind or to remain attached to inanimate surfaces, such as glass. In addition, G. duodenalis trophozoites treated with anti-giardin sera were noticeably less mobile than trophozoites treated with control sera. This phenomenon is not without precedent. It has been previously reported that antibodies to whole G. duodenalis blocked in vitro binding of trophozoites to enterocytes and glass culture surfaces (Samra et al., 1991). The mechanisms involved to prevent attachment of G. duodenalis trophozoites to glass surfaces remain unknown. We can speculate that antibodies specific to the cytoskeleton interfere with trophozoite binding or hinders the flexibility of the ventral disk, thereby preventing parasite attachment to gut epithelial cells.

21.4.4 Success or Failure of Vaccine in Reducing the Pathology Thus far, studies testing the efficacy of GiardiaVax• have indicated a failure of the vaccine to ameliorate the symptoms or reduce the pathology of giardiasis in animals. Stein et al. (2003) assessed the efficacy of Giardia vaccination as a treatment for giardiasis in 16 experimentally infected cats using GiardiaVax•. Following establishment of the infection and signs of giardiasis, eight cats in the treatment group received vaccine injected subcutaneously on weeks four, six, and 10 post infection. By week 28, five of eight vaccinated cats and seven of eight control cats had patent Giardia infections. In addition, the vaccine did not completely eliminate the parasite from experimentally infected cats within the study period. Since clinical signs were minimal in both groups of cats, it could not be determined whether vaccination lessened severity of clinical disease. It must be acknowledged that the results may have been influenced by the large inoculation dose. Anderson et al. (2004) assessed the effectiveness of GiardiaVax• with 20 asymptomatic antigen

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positive dogs at a research facility, whereby 10 were vaccinated and 10 received a placebo. Feces were then monitored monthly for the presence of Giardia antigens and cysts for up to six months. At weeks 4, 8, 12, and 16 following vaccination, there were more Giardia-positive dogs in the vaccinated group as compared with the controls. At week 20, an equal number of dogs were Giardia positive, and at week 24, fewer dogs were positive in the vaccinated group than in the control group. Since there was no significant difference between both groups, they concluded that the vaccine was not an effective treatment for asymptomatic canine giardiasis. Uehlinger et al. (2007) have tested GiardiaVax• in dairy cows and calves, although it is intended and marketed for dogs. Six two-week old calves were vaccinated subcutaneously with the vaccine and six control calves received a subcutaneous injection of sterile phosphate-buffered-saline mixed with adjuvant. Injections were repeated after 28 days, and on day 39. Calves were challenged orally with 1 u 105 purified G. duodenalis cysts from a naturally infected calf. The circulating IgG antibody stimulated by GiardiaVax• vaccine was not successful in preventing the pathology and reducing cyst shedding in calves. Vaccinated calves tended to excrete more G. duodenalis cysts in their feces than non-vaccinated calves. Moreover, intestinal trophozoite load did not differ between vaccinated and non-vaccinated calves.

21.5 Transmission-blocking Vaccines Against Giardia Using Cyst Wall Protein 2 The development of a TBV against Giardia has been facilitated by the development of a protocol allowing the development of the Giardia cell in vitro, thereby allowing the study of the cellular basis of encystation as well as expression of encystation-specific antigens (McCaffery et al., 1994). The cyst wall contains two major groups of proteins (Meng et al., 1996). Group I consists of several broad-band proteins, within the molecular weight range of 26–46 kDa. They are expressed as early as three hours post-encystation in vitro, and are primarily identified using specific antibodies. These proteins localize in the interior of the encysting-specific vesicles (ESVs) and are also found on the surface of the cyst wall. Group II proteins are mostly glycoproteins whose molecular weights range from 66 to 140 kDa, and can be identified by antibodies and wheat germ agglutinin. Unlike group I, group II proteins localize exclusively to the surface of the cyst wall. Therefore, group I proteins may serve as attractive antigen candidates for TBV since they are determinants of differentiation and are expressed throughout the process of encystation. The identification and cloning of group I proteins have been the subject of intensive research efforts, and to date, two main classes of encystation-specific proteins have

Table 21.1 Encystation-specific surface-bound group I proteins Protein family

Genbank accessions No.

Discernible characteristics

Localization

Reference

– N-terminal signal peptide – CXXC motif – Zn binding fingers – C-terminal CRGKA motif

– Plasma membrane of encysting trophozoites and excyzoites

Carranza et al. (2002)

– N-terminal signal peptide – >20 CXXC/CXC motifs – GGCY motifs – C-terminal CRRSKAV

– Nucleus of trophozoites – ESVs in encysting cells – Plasma membranes of excyzoites

Davids et al. (2006)

– N-terminal signal peptide – 4–5 LRR motifs – C-terminal Cysteine-rich regions – >16 Cysteine residues

– ESVs in encysting cells – Cyst wall

Lujan et al. (1995) Lujan et al. (1995) Sun et al. (2003)

Variable surface proteins (VSP) VSP9B10B

AF293416

High Cysteine Membrane Proteins (HCMp) HCNCp

DQ1444994

Cyst wall proteins (CWP) CWP1 CWP2 CWP3

U09330 U28965 AY061927

340

P. Lee et al.

Table 21.2 Encystation-specific enzymes present amongst group I proteins Enzyme

Genbank accessions No.

Stage-specific expression

Role in encystation

Localization

Reference

Bip

AAA19123

– Upregulated during encystation

– Chaperone

ER/ESV

Lujan et al. (1996)

PDI

AAF20171

– Slight upregulation during encystation

– ER folding enzyme that ensures proper disulfide bond formation – Transglutaminase activity that is essential for cyst wall protein crosslinking at the surface

ER/ESV Plasma membrane

Knodler et al. (1999)

ESCP

AF293408

– Encystation-specific

– Cathepsin-C-like cysteine protease – Catalyzes the removal of C-terminal tail moiety from Pro-CWP2

PV/ESV

Touz et al. (2002b)

GSP

AAK97081

– Encystation-specific

– Ca2+ binding protein – Helps maintain soluble environment in ESV

ESV

Touz et al. (2002a)

CW

Not determined

– Encystation-specific

– N-acetylgalactosaminyl transferase enzyme that converts UDPGalNAc to N-acetylgalactosamine polysaccharides

ESV/cyst wall

Karr et al. (2004)

PP2A

XP767901

– Constitutively expressed

– Serine/threonine protein phosphatase – Involved in regulating giardial differentiation

Basal bodies Centrosomes Ventral disk

Lauwaet et al. (2007)

been identified: surface-bound proteins and enzymes (Tables 21.1 and 21.2). Surface-bound proteins can be subdivided into three families: (i) the CWPs (Lujan et al., 1995; Sun et al., 2003), (ii) the high cysteine membrane proteins (Davids et al., 2006), and (iii) the encystation-specific VSPs (Carranza et al., 2002). In addition to surface-bound proteins, there are a number of enzymes that are intimately associated with encystation including: heavy-chain binding proteins (Lujan et al., 1996), protein disulfide isomerases (Knodler et al., 1999) encystation-specific cysteine proteases (Touz et al., 2002b), granule-specific calcium binding proteins (Touz et al., 2002a), the protein phosphatase 2A (Lauwaet et al., 2007), and cyst wall synthases (Karr and Jarroll, 2004).

21.5.1 Biochemical Composition of the Cyst Wall Gas chromatography and mass spectrometry studies have demonstrated that purified, filamentous cyst walls of mature G. duodenalis and G. muris cysts are composed of approximately 57% proteins, and 43%

carbohydrates by dry weight, of which galactosamine is the major constituent (Jarroll et al., 2001). Currently, three CWP-encoding genes have been cloned and sequenced (Fig. 21.1). However, only two have been functionally and biochemically characterized (Lujan et al., 1995; Sun et al., 2003). cwp1 and cwp2 (Genbank accession no. U09330 and U28965, respectively) have molecular weights of 26 and 39 kDa, respectively, and are synthesized as early as one hour post-encystation, forming a stable complex within five minutes after their synthesis while co-localizing to ESVs. Both proteins contain five tandem copies of leucine-rich repeats (LRR), N-terminal secretion signals, and several predicted N-glycosylation sites (Lujan et al., 1995, 1997). Hehl et al. (2000) confirmed that the N-terminal domain plays a role in directing nascent CWPs to the secretory pathway, and the LRR domain is essential in incorporating CWPs into the cyst wall. In addition, cwp1 and cwp2 share a high degree of sequence identity, yet cwp2 is distinguishable from cwp1 by the presence of a strongly basic carboxyl-terminal tail (Fig. 21.1). Recent studies have shown that the C-terminal tail moiety serves the dual role of directing nascent or full length Pro-CWP2

Chap. 21 Vaccination Against Giardia

341

CWP1

(Pro)-CWP2

CWP3

Fig. 21.1 Structure and features of Giardia CWP1, 2, and 3. All three CWPs share similar features and domains. However, CWP2 differs from the other two by the presence of a 13 kDa C-terminal region expressed by the nascent Pro-CWP2. This moiety is removed by encystation-specific cysteine protease (ESCP) to yield a 26 kDa mature protein that is similar to CWP1 and CWP3

to the secretory pathway, as well as the recruitment of ESVs for shuttling CWPs to the cell surface (AbdulWahid and Faubert, 2004; Gottig et al., 2006). Using CWP2 monoclonal antibodies 8C5.C11 and 7D2, it was shown that Pro-CWP2 undergoes proteolysis, presumably in peripheral vesicles, by an encystationspecific cysteine protease. This protease removes the 13 kDa tail moiety and results in the appearance of a mature 26 kDa protein (CWP2) in the mature cyst wall (McCaffery et al., 1994; Meng et al., 1996; Touz et al., 2002b). Despite the high degree of homology between CWP1 and CWP2, their encoding genes are found on chromosomes III and IV, respectively (Fig. 21.1).

21.5.2 Local Immune Response to CWPs Pro-CWP2 can be considered as the most important target protein (Fig. 21.2). Its expression is required for the formation of ESVs and the initiation of the intracellular trafficking of nascent cyst wall material (Gottig et al., 2006). Immuno-biochemical studies revealed that Pro-CWP2 forms a stable complex with

Fig. 21.2 Confocal micrograph illustrating the specific expression of CWP2 by encysting Giardia trophozoites and cysts, but not trophozoites. G. duodenalis trophozoites were induced into encystation for 24 hrs, and then fixed and incubated with mAb 8C5.C11. CWP2-bound antibodies were detected with Cy-3coupled secondary rabbit anti-mouse pAb. Nuclei were stained with DAPI and cells were mounted and visualized using a BioRad Radiance confocal microscope

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Table 21.3 Percentage distribution of T and B lymphocyte subsets (/106 cells) in the PP of G. muris infected BALB/c mice

Non-infected

Mean number cells u 106) isolated from PPs (u

T-lymphocyte subsets CD4+ (%)

CD8+ (%)

B-lymphocytes (%)

6.2 r1.1

11.2 r1.3

3.8 r0.8

55.8 r2.3

5.8 r2.3 6.0 r0.6

16.6 r0.8* 18.3 r1.0*,**

2.9 r0.2* 4.3 r0.3**

67.5 r0.7* 67.8 r1.8*

G. muris infected Acute phase Elimination phase *

Denotes significance when comparing against non-immunized mice (Holm-Sidak one-way ANOVA, p < 0.001). Denotes significance when comparing pairwise against Acute stage (Holm-Sidak one-way ANOVA, p < 0.001). Modified from Abdul-Wahid and Faubert (2008).

**

other CWPs as soon as five minutes following its synthesis (Lujan et al., 1995). We believe cwp2 is of significant biological importance to Giardia spp. since the protein’s sequence seems to be conserved in this parasite irrespective of the assemblage or the species (Unpublished observations). Before using Pro-CWP2 as a candidate antigen for TBV, it is important to determine how the immune system responds to ProCWP2 and CWP2 in infected mice. We reported (Abdul-Wahid and Faubert, 2008) that an increase in the percentage of local TH-cells occurs in mice infected with G. muris (Table 21.3). Therefore, we tested whether these cells, collected from PP and MLN, recognized cyst-specific antigens by analyzing the production of cyst Ag-specific IL-4 and IFN-J secretion in response to stimulation with either rPro-CWP2 or purified Giardia cyst wall (PCW) proteins (Fig. 21.3A). Regardless of the phase of infection or the antigen used for in vitro stimulation, similar numbers of Ag-specific IFN-J and IL-4 TH secreting cells were counted. These observations confirmed that a balanced TH1 and TH2 cytokine response is produced in response to CWPs, and especially with Pro-CWP2. In addition, a significant level of local IgA and IgG antibodies against PCW and Pro-CWP2 antigens was detected in intestinal lavage samples with no significant levels of local IgM, indicating that a committed local antibody response was raised against PCW and Pro-CWP2 during a primary infection (Fig. 21.3B). We determined the effect of Pro-CWP2 antibodies on cyst formation in vitro. Encysting cultures were incubated with filter sterilized intestinal lavage samples obtained from Giardia-infected (day 40) or control mice, for 24 h. Following incubation, the number of

cysts was determined by flow cytometry (Fig. 21.4). An average of 2000 cysts was detected after 24 hours post-encystation. The addition of intestinal lavage fluid from G. muris-infected mice significantly reduced the number of cysts to 1000, which represents an ~50% reduction as compared to control encysting cultures. Based on these observations, we hypothesized that a component of the host’s immune response to a primary infection is directed against cyst antigens, which is playing a role in acquired immunity against a secondary infection.

21.5.3 Use of rPro-CWP2 as an Oral Vaccine The first vaccination attempt of a TBV for giardiasis was done by directly administering recombinant Pro-CWP2 (rPro-CWP2) protein into mice, by oral gavage. Although the administered protein was a recombinant form expressed in E. coli, it was nonetheless immuno-reactive when tested with mAb 8C5.C11. rPro-CWP2 was capable of stimulating an immune response comparable to that obtained with an extract of encysting cells admixed with cholera toxinB (CtB) (Larocque et al., 2003). This immunization protocol stimulated the production of local IgA antibodies as well as serum IgG1 and IgG2a antibodies. In addition, mRNA for TH1 and TH2 cytokines was detected in spleen- and PP-derived leukocytes taken from immunized animals. The vaccine stimulated anti-CWP2 intestinal IgA antibodies and reduced the number of cysts in feces by ~70%. These results led us to investigate other avenues for presentation of the TBV to gut-associated lymphoid tissues.

Chap. 21 Vaccination Against Giardia

40

A

343

B

, Peyer s patches

700

IgA

30

ug/mg Total lgA

Δ SFU/2 × 105 cells

600

20

500 400 300 200

10

100 0

0 PCW

rCWP2

PCW

rCWP2

PCW

IFN-γ

IL-4 40

1800

MLN

IgG 1500

30

ng lgG/ml

Δ SFU/2 × 105 cells

rCWP2

20

1200 900 600

10 300 0

0 PCW

rCWP2

PCW

rCWP2

PCW

rCWP2

IFN-γ

IL-4 Uninfected

Acute phase

Elimination phase

Fig. 21.3 Immune response against CWPs. A Enumeration of Giardia-specific IL-4 and IFN-J cytokine producing local lymphocytes by ELISPOT. PP (top panel) and MLN (bottom panel) derived lymphocytes were collected from uninfected or G. muris infected BALB/c mice at the acute and elimination phases of infection and stimulated in vitro with PCW or rPro-CWP2. The numbers of Ag-specific IL-4 and IFN-J secreting cells were measured. Results from Giardia-infected mice were significantly different when compared to the results obtained from uninfected animals. B Quantification of Ag-specific local IgA (top panel) and IgG (bottom panel) antibodies by ELISA. Intestinal lavage samples were collected from uninfected or G. muris infected BALB/c mice at the acute and elimination phases of infection and tested for the presence of antibodies specific to PCW or rPro-CWP2. Asterisk designates significance when compared to uninfected, while double asterisk designates significance when compared pairwise to acute phase (p < 0.05; Holm-Sidak One-way ANOVA). Modified from Abdul-Wahid and Faubert (2010)

21.5.4 Lactic Acid Bacteria (LAB) as a Live TBV Delivery Vehicle We investigated the possibility of having CWP2 expressed in situ and delivered to gut-associated lymphoid tissues by virtue of non-pathogenic LAB (Wells and Mercenier, 2008). This approach has several ad-

vantages, namely the possibility of having the Ag expressed by the delivery vehicle, bypassing the difficulties associated with the expression and purification of sufficient quantities of rPro-CWP2 and the need of co-administering a mucosal adjuvant. Lactococcus lactis and Streptococcus gordonii are LAB currently studied for use as live antigen delivery vehicles to mu-

344

P. Lee et al.

Number of cysts formed in vitro

5000

4000

3000

2000

1000

0 Control

Uninfected

Giardia-infected

+ Intestinal flushes

Fig. 21.4 Effect of intestinal antibodies on cyst production in vitro. Encysting Giardia trophozoites were incubated with or without filter sterilized intestinal lavage samples for 24 h. The number of generated cysts was assessed by flow cytometry. The data presented represent the results of three independent experiments. Asterisk designates significance when compared to control, while double asterisk designates significance when compared to uninfected; p < 0.05 Holm-Sidak One-way ANOVA

cosal sites. Since both vehicles differ in capability to persist within the small intestine and in their mode of antigen delivery we determined which one would be superior as a delivery system (Mercenier et al., 2000; Medina and Guzman, 2001; Kimoto et al., 2003; Lee and Faubert, 2006a). Oral immunization of BALB/c mice with either recombinant L. lactis or S. gordonii expressing CWP2 anchored onto the cell surface significantly reduced cyst release following heterologous challenge infections with G. muris (Lee and Faubert, 2006b; Lee et al., 2009). The efficacy of L. lactis and S. gordonii, as vehicles for the delivery of CWP2 to the intestinal mucosal site is presented in Table 21.4. Mice immunized with engineered S. gordonii shed ~40% less cysts than their L. lactis immunized counterparts (Fig. 21.5). On the other hand, L. lactis-immunized mice also experienced a reduction in total number of shed cysts (~70%) while the duration of cyst output was unaltered. Both LAB constructs produced a significant up-regulation

Table 21.4 Summary of differences between vaccine constructs Vaccine constructs Lactococcus lactis

Streptococcus gordonii

Expression type

Inducible

Constitutive

cwp2 DNA maintenance

Episomal

Chromosomal

cwp2 copy No.

~30

1

Ag presentation

Cell surface anchored

Cell surface anchored

Gut colonization

No (transient, ~48 h)

Yes (persistent, ~28 days)

No. of administrations

6

4

Booster period (days)

13

28

Cell types stimulated

CD4+T-cells, CD19+B-cells

CD4+T-cells, CD19+B-cells

Ab response

Secretory IgA

Secretory IgA and IgG

Cytokines

IL-4 and IFN-J

IL-4 and IFN-J

Polarity

Balanced

IFN-J predominant

T-helper profile

TH1/TH2

TH1

Cyst output (cysts/g feces)

1.3 r 0.3 u 105

0.4 r0.1 u 105

Cyst output duration (days)

20/20

12/20

Time reduction (%)

0

40

Cumulative cyst shedding reduction (%)

71

90

Characteristics

Immunology

Vaccine efficacy

Modified from Lee et al. (2009).

Chap. 21 Vaccination Against Giardia

A

Cyst output (×105) per gram feces

Cyst output (×105) per gram feces

18.0

345

16.0 14.0 12.0 10.0 8.0 6.0 4.0 2.0

6

B

5 4 3 2 1 0

0.0 6

8

10

12

14

16

18

20

Non L. lactis L. lactis L. lactis S. gordonii S. gordonii S. gordonii M6 CWP2 CWP2 M6 immunized

Days post challenge

Fig. 21.5 A Cyst release pattern over a 15-day period. Feces were collected daily from all mice individually and cyst counts were performed and determined as mean number rSEM u 105 cysts/g fecal material. B Mean number (rSEM) of cysts/gram feces during a 15-day period. The total number of cysts counted for each group was divided by the total amount of feces (g) collected. Asterisk indicates significance (p = 0.05) when compared to the non-immunized group, whereas double asterisk indicates significance when compared pairwise to L. lactis-CWP2 (p < 0.001; Holm-Sidak One-way ANOVA). Data presented are representatives of three independent experiments. Modified from Lee et al. (2009)

Table 21.5 Percentage distribution of CD4+/CD8+/CD19+ cells (/106 cells) in PP and MLN of immunized mice Mouse groups

% CD4+

% CD8+

%CD19+

PP (%)

MLN (%)

PP (%)

MLN (%)

PP (%)

Non-immunized

23.3 r1.9

55.0 r2.6

6.6r1.5

16.6 r1.0

57.6 r2.0

L. lactis

20.2 r1.6

50.9 r3.8

6.0r1.7

15.7 r3.8

L. lactis-M6

27.7 r2.1

57.1 r3.8

6.1r1.4

14.8 r1.0

L. lactis-CWP2

30.0 r2.4*

60.0 r3.9*

6.6r1.0

16.0r0.4

S. gordonii

23.4 r1.9

52.0 r1.6

7.5r0.7

S. gordonii-M6

24.4 r2.0

54.0 r1.6

S. gordonii-CWP2

35.4 r1.8

61.5 r3.1

*,**

*

MLN (%) 20.8r1.9

*

30.9r0.2*

*

71.0r0.8

31.3r0.6*

73.8r1.4*,***

32.2r0.2*

69.6r1.1

17.2 r0.4

*

62.2r1.6

26.4r1.6*

9.4r2.3

15.3 r0.5

63.8r0.9*

25.9r0.6*

10.2 r3.0

14.5 r0.6

*

30.1r0.5*

67.0r2.2

*

Denotes significance when comparing against non-immunized mice (Holm-Sidak one-way ANOVA, p < 0.001). Denotes significance when comparing pairwise against L. lactis-CWP2 immunized mice (Holm-Sidak one-way ANOVA, p < 0.001). *** Denotes significance when comparing pairwise against S. gordonii-CWP2 immunized mice (Holm-Sidak one-way ANOVA, p < 0.001). Modified from Lee et al. (2009). **

of TH- and B-cells in the PP and MLN compartments. However, administration of CWP2-expressing S. gordonii resulted in a larger change in mucosal TH- and B-cell populations (Table 21.5). The humoral response stimulated by the engineered S. gordonii resulted in the production of twice as much CWP2-specific IgA antibody when compared to the delivery by L. lactis (Fig. 21.6). Only the S. gordonii vehicle was capable of stimulating CWP2-specific IgG mucosal antibodies. Lastly, L. lactis stimulated a balanced CWP2-specific IFN-J and IL-4 responses, whereas administration of recombinant S. gordonii construct resulted only in a

prominent CWP2-specific IFN-J response in the MLN and PP compartments (Fig. 21.6). We concluded that S. gordonii was the superior live vaccine delivery vehicle because of its abilities to: (i) continuously stimulate the intestinal immune system due to its longer persistence in the gut; (ii) stimulate a higher percentage of T- and B-lymphocytes locally; (iii) induce a higher concentration of CWP2specific IgA; (iv) induce significantly higher levels of CWP2-specific intestinal IgG; and (v) stimulate the production of higher levels of CWP2-specific IFN-J. Interestingly, oral administration of the engineered

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P. Lee et al.

Humoral response intestinal IgA

Cellullar response , Peyer s patches

B 3.0

500

400

Cytokine SFU ratios in PP cells

CWP2 specific IgA (μg/mg Total IgA)

A

*

300

*

200

100

0

1.5 1.0 0.5

1.0 1.5 2.0 2.5 3.0 S. gordonii

CWP2

Mesenteric lymph nodes Cytokine SFU ratios in MLN cells

CWP2 specific IgG (μg/mg Total IgG)

L. lactis CWP2

3.0

8

*

6

4

2

0 L. lactis M6

TH2

0.5

Non immunized

Intestinal IgG

Non L. lactis immunized

T H1

0.0

Non L. lactis L. lactis L. lactis S. gordonii S. gordonii S. gordonii M6 M6 immunized CWP2 CWP2

10

*

2.5 2.0

*

2.5 2.0 1.5 1.0 0.5

TH1

0.0

T H2

0.5 1.0 1.5 2.0 2.5 3.0 Non immunized

L. lactis S. gordonii S. gordonii S. gordonii CWP2 M6 CWP2

L. lactis CWP2

S. gordonii

CWP2

Fig. 21.6 A CWP2-specific antibody ELISA. Results are expressed as the ratio of the amount of CWP2-specific antibodies (Pg) to the amount of total antibody (mg) in the sample. Asterisk indicates significance p < 0.001 (Holm-Sidak One-way ANOVA). Data presented are representatives of three independent experiments. B Post-immunization TH subset profiles in PP and MLN. Results are expressed as either IFN-J/IL-4 SFU or IL-4/IFN-J SFU ratios. Non-immunized animals served as a baseline control demonstrating what normal TH subset profile is exhibited in PP and MLN. A ratio of 2 or more indicates TH subset polarization and is significant (p < 0.001, Student’s t-test). Asterisk indicates polarization. Data presented are representatives of two independent experiments. Modified from Lee et al. (2009)

Table 21.6 Percentage distribution of systemic and mucosal T and B lymphocyte subsets (/106 cells) in DNA immunized mice Mouse groups

%CD4+ T-cells

%CD19+ B-Cells

MLN (%)

SPLN (%) PP (%)

Non16.0r0.6 immunized

50.2r0.6

27.0r0.7

3.3r0.4 15.7r0.4

13.0r0.4

66.5r2.3 28.0r1.3

48.5r2.4

15.2r0.7

48.8r1.2

25.9r0.9

3.3r0.2 15.3r0.3

11.1r0.3

66.0r3.3 28.7r1.1

51.1r1.0

Pro-CWP2 19.8r2.5*,** 55.2r2.5*,** 29.8r0.9

3.5r0.2 14.8r0.4

12.6r0.2

72.8r0.8 32.0r1.4*,** 52.3r1.0

pCDNA3

*

PP (%)

% CD8+ T-Cells

MLN (%) SPLN (%) PP (%)

MLN (%)

SPLN (%)

Denotes significance when comparing against non-immunized mice (Holm-Sidak one-way ANOVA, p < 0.001). Denotes significance when comparing pairwise against pCDNA3 immunized mice (Holm-Sidak one-way ANOVA, p < 0.001). Modified from Abdul-Wahid and Faubert (2007). **

Chap. 21 Vaccination Against Giardia

CWP2-specific intestinal (IgA)

B 1250

300

1000

200

750

100

500 300

0

0

C

CWP2-specific serum (IgG)

D

5

Subclass of CWP2-specific serum IgG 2.7

Log10 of end-point titre

IgG (ng /ml)

CWP2-specific intestinal (IgG)

400

IgG (ng/ml)

μg/mg Total IgA

A

347

4 3 2 1

1.8

0.9

0.0

0

Total IgG CWP2

pCDNA3

IgG1

IgG2a

Unimmunized

Fig. 21.7 Quantification of the DNA vaccine stimulated CWP2-specific local and systemic antibodies. Serum and Intestinal lavage samples were collected from DNA immunized and control mice one week following the last immunization and tested for the presence of CWP2-specific antibodies by ELISA. Panel A shows the concentration of CWP2-specific intestinal IgA antibodies. Panels B and C show the concentration of CWP2-specific IgG in the intestines and serum, respectively. Panel D shows the determination of the subclass of CWP2-specific IgG in the serum. Asterisk indicates significance at when compared to the unimmunized group, whereas double asterisk indicates significance when compared pairwise to pCDNA (p < 0.05; Holm-Sidak One Way ANOVA). Data presented are representative of three independent experiments. Modified from Abdul-Wahid and Faubert (2007) Table 21.7 Summary of outcomes of the different CWP2-based Giardia TBV trials Strategy Oral delivery of rProCWP2 + CtB Live delivery of CWP2 by S. gordonii

Live delivery of CWP2 by L. lactis

S. typhimuriummediated bactofection of a Pro-CWP2-encoding DNA vaccine

Immunization schedule – 4 administrations every 7 days by oral gavage – 4 administrations on days 1, 2, 29 and 30 by oral gavage – 6 administrations on days 1, 2, 3, 15, 16 and 17 by oral gavage – 3 administrations on days 1, 14 and 28 by oral gavage

[CWP2] delivered 500 μg rPro-CWP2

~2.83 μg delivered by 109 CFUs per administration ~11.3 Pg delivered by 109 CFUs per administration 10 ng of cwp2pCDNA3 delivered by 109 CFUs

Stimulated immune responses – Balanced TH1/TH2 immune response – CWP2-specific local IgA – Predominant TH1 immune response – CWP2-specific local IgA and IgG – Balanced TH1/TH2 immune response – CWP2-specific local IgA only – Predominant TH1 immune response – CWP2-specific local IgA and IgG

Summary of outcomes – 70% reduction of cumulative cyst shedding – 90% reduction in cumulative cyst shedding and 40% reduction in shedding time – 70% reduction in cumulative cyst shedding with no effect on shedding time – 70% reduction in cumulative cyst shedding with 25% reduction of cyst shedding time

P. Lee et al.

S. gordonii was able to induce an intestinal immune response similar to that obtained after a primary infection which is the ultimate quality sought from a vaccine (Venkatesan et al., 1996; Djamiatun and Faubert, 1998; Abdul-Wahid and Faubert, 2008).

21.5.5 Efficacy of the TBV Using a DNA Vaccine Strategy

CWP2 pCDNA3

Stimulation index

250

A Unimmunized pCDNA3 CWP2

200 150 100 50 0

4 3 2 1

Cysts output (× 104 per gram feces)

4

Induction of immune responses by DNA vaccines at the level of the intestinal mucosa can be achieved either by: (i) mucosal administration of plasmids formulated with cationic lipids; (ii) oral administration of microencapsulated DNA vaccines; or (iii) oral administration of a bactofection vehicle or using attenuated bacteria like S. typhimurium (Weiss, 2003). The Salmonella-delivered mucosal DNA vaccine is based on the bacteria’s ability to cross the epithelial lining of the gut by invading intestinal M-cells and infecting underlying antigen-presenting cells (APCs) like macrophages and dendritic cells. Once internalized by APCs, the attenuated bacteria are effectively killed

5

Cysts (× 104 per gram feces)

348

750

6

8

14 16 12 10 Days-post-infection

18

20

B

625 500 375

** * 250 125 0 Unimmunized

pCDNA3

CWP2

Fig. 21.9 Efficacy of the Pro-CWP2-encoding DNA vaccine in reducing cyst shedding. DNA immunized and control mice (n = 12 mice per group) were challenged with 105 G. muris cysts a week following the last immunization. The number of G. muris cysts released in fecal pellets was monitored for a period of 20 days. Panel A shows the effect of DNA immunization on the pattern of cyst shedding. Panel B shows the reduction of the total number of cysts released over the entire study period by infected mice following DNA immunization. Asterisk indicates significance when compared to unimmunized control group, whereas double asterisk indicates significance when compared pairwise to the pCDNA3 immunized group (p < 0.05; HolmSidak One-way ANOVA). Modified from Abdul-Wahid et al. (2007)

0 PP

MLN

SPLN

Fig. 21.8 Lymphoproliferation of cells in response to stimulation with CWP2. Cells were isolated from the PPs, MLNs and spleens of DNA immunized or control mice (n = 12 mice per group) and cultured in the presence of r-Pro-CWP2 for 72 hours. Results of the lymphoproliferation are represented as a stimulation index (cpm of Ag stimulated cells/cpm of unstimulated cell). A stimulation index greater than 1.5 is considered significant. The dotted line refers to the lymphoproliferation response observed using cells from non-immunized mice. Asterisk indicates significance at when compared to the pCDNA3 unimmunized group, whereas double asterisk indicates significance when compared pairwise to either CWP2 immunized MLN or pleen derived cells (p < 0.05; Holm-Sidak One Way ANOVA). Modified from Abdul-Wahid and Faubert (2007)

in phago-lysosomes, which results in the release of their plasmid load; ultimately leading to host cell transfection. Once the DNA vaccine-encoded antigen is expressed, the transfected intestinal APCs drain to the MLN where they can present it via the MHC class I and class II pathways to lymphocytes leading to the induction of Ag-specific mucosal as well as systemic immune responses (Weiss, 2003; Darji et al., 2000). We used S. typhimurium (STM1 strain) as a bactofection vehicle for rPro-CWP2 (Abdul-Wahid and Faubert, 2007). Oral administration of 109 CFUs of the rPro-CWP2 DNA vaccine-baring STM1 resulted

Chap. 21 Vaccination Against Giardia

in an increase in the number of TH- and B-cells than their L. lactis immunized counterparts in the PP, MLN, and spleen of immunized animals (Table 21.6). Analysis of the immune responses revealed the production of CWP2-specific intestinal IgA and IgG antibodies (Fig. 21.7), comparable to that observed when CWP2 was delivered by recombinant L. lactis. The oral DNA vaccine led to the development of a predominant CWP2-specific IFN-J production at the level of PPs (Fig. 21.8), which we attributed to the local presentation of Pro-CWP2 in the context of the Salmonella delivery vehicle inducing the production of IFN-J by host leukocytes to eradicate the infecting bacteria (Eckmann and Kagnoff, 2001). The analysis of the DNA vaccine’s efficacy following a challenge infection revealed a reduction in the duration of cyst shedding and a reduction in total of cyst shedding (Fig. 21.9). A summary of different CWP2-based Giardia TBV strategies is presented in Table 21.7.

References Abdul-Wahid A and Faubert GM (2004) Similarity in cyst wall protein (CWP) trafficking between encysting Giardia duodenalis trophozoites and CWP-expressing human embryonic kidney-293 cells. Biochem Biophys Res Commun 324: 1069–1080 Abdul-Wahid A and Faubert GM (2007) Mucosal delivery of a transmission-blocking DNA vaccine encoding Giardia lamblia CWP2 by Salmonella typhimurium bactofection vehicle. Vaccine 25: 8372–8383 Abdul-Wahid A and Faubert GM (2008) Characterization of the local immune response to cyst antigens during the acute and elimination phases of primary murine giardiasis. Int J Parasitol 38: 691–703 Anderson KA, Brooks AS, Morrison AL, Reid-Smith RJ, Martin SW, Benn DM, and Peregrine AS (2004) Impact of Giardia vaccination on asymptomatic Giardia infections in dogs at a research facility. Can Vet J 45: 924–930 Barwick RS, Mohammed HO, White ME, and Bryant RB (2003) Factors associated with the likelihood of Giardia spp. and Cryptosporidium spp. in soil from dairy farms. J Dairy Sci 86: 784–791 Buret A, denHollander N, Wallis PM, Befus D, and Olson ME (1990) Zoonotic potential of giardiasis in domestic ruminants. J Infect Dis 162: 231–237 Carranza PG, Feltes G, Ropolo A, Quintana SM, Touz MC, and Lujan HD (2002) Simultaneous expression of different variant-specific surface proteins in single Giardia lamblia trophozoites during encystation. Infect Immun 70: 5265–5268 Darji A, Zur Lage S, Garbe AI, Chakraborty T, and Weiss S (2000) Oral delivery of DNA vaccines using attenuated Salmonella typhimurium as carrier. FEMS Immunol Med Microbiol 27: 341–349

349 Davids BJ, Reiner DS, Birkeland SR, Preheim SP, Cipriano MJ, McArthur AG, and Gillin FD (2006) A new family of giardial cysteine-rich non-VSP protein genes and a novel cyst protein. PLoS ONE 1: e44 Deitsch KW, Lukehart SA, and Stringer JR (2009) Common strategies for antigenic variation by bacterial, fungal and protozoan pathogens. Nat Rev Microbiol 7: 493–503 Dib HH, Lu SO, and Wen SF (2008) Prevalence of Giardia lamblia with or without diarrhea in South East, South East Asia and the Far East. Parasitol Res 103: 239–251 Djamiatun K and Faubert GM (1998) Exogenous cytokines released by spleen and Peyer’s patch cells removed from mice infected with Giardia muris. Parasite Immunol 20: 27–36 Eckmann L and Kagnoff MF (2001) Cytokines in host defense against Salmonella. Microbes Infect 3: 1191–2000 Faubert G (2000) Immune response to Giardia duodenalis. Clin Microbiol Rev 13: 35–54 Faubert GM, Lee P, and Wahid AA (2002) Giardia duodenalis. In: Infections of the gastrointestinal tract (M.J. Blaser, P.D. Smith, J.I. Ravdin, H.B. Greenberg, and R.L. Guerrant, eds.), Second Edition. Philadelphia, Baltimore, New York, London, Buenos Aires, Hong Kong, Sydney, Tokyo, Lippincott, Williams, and Wilkins, pp 979–1006 Gottig N, Elias EV, Quiroga R, Nores MJ, Solari AJ, Touz MC, and Lujan HD (2006) Active and passive mechanisms drive secretory granule biogenesis during differentiation of the intestinal parasite Giardia lamblia. J Biol Chem 281: 18156–18166 Gottstein B and Nash TE (1991) Antigenic variation in Giardia lamblia: infection of congenitically athymic nude and scid mice. Parasite Immunol 13: 649–659 Gupta RS, Aitken K, Falah M, and Singh B (1994) Cloning of Giardia lamblia heat shock protein HSP70 homolog: implications regarding origin of eukaryotic cells and of endoplasmic reticulum. Proc Natl Acad Sci USA 91: 2895–2899 Hehl AB, Marti M, and Kohler P (2000) Stage-specific expression and targeting of cyst wall protein-green fluorescent protein chimeras in Giardia. Mol Biol Cell 11: 1789–1800 Holberton D, Baker DA, and Marshall J (1988) Segmented alpha-helical coiled-cell structure of the protein giardin from the Giardia cytoskeleton. J Mol Biol 204: 789–795 Jarroll EL, Macechko PT, Steimle PA, Bulik D, Karr CD, van Keulen H, Paget TA, Gerwig G, Kamerling J, Vliegenthart J, and Erlandsen S (2001) Regulation of carbohydrate metabolism during Giardia encystment. J Eukaryot Microbiol 48: 22–26 Jenkins MC, O’Brien CN, Murphy C, Schwartz R, Miska K, Rosenthal B, and Trout JM (2009) Antibodies to the ventral disc protein G-giardin prevent in vitro binding of Giardia lamblia trophozoites. J Parasitol 95: 895–899 Karr CD and Jarroll EL (2004) Cyst wall synthase: N-acetylgalactosaminyltransferase activity is induced to form the novel N-acetylgalactosamine polysaccharide in the Giardia cyst wall. Microbiology 150: 1237–12343 Kaslow DC (2002) Transmission-blocking vaccines. Chem Immunol 80: 287–307 Kimoto H, Nomura M, Kobayashi M, Mizumachi K, and Okamoto T (2003) Survival of lactococci during passage through mouse digestive tract. Can J Microbiol 49: 707– 711

350 Knodler LA, Noiva R, Mehta K, McCaffery JM, Aley SB, Svard SG, Nystul TG, Reiner DS, Silberman JD, and Gillin FD (1999) Novel protein-disulfide isomerases from the early-diverging protist Giardia lamblia. J Biol Chem 274: 29805–29811 Langford TD, Housley MP, Boes M, Chen J, Kagnoff MF, Gillin FD, and Eckmann L (2002) Central importance of immunoglobulin A in host defense against Giardia spp. Infect Immun 70: 11–18 Larocque R, Nakagaki K, Lee P, Abdul-Wahid A, and Faubert GM (2003) Oral immunization of BALB/c mice with Giardia duodenalis recombinant cyst wall protein inhibits shedding of cysts. Infect Immun 71: 5662–5669 Lauwaet T, Davids BJ, Torres-Escobar A, Birkeland SR, Cipriano MJ, Preheim SP, Palm D, Svard SG, McArthur AG, and Gillin FD (2007) Protein phosphatase 2A plays a crucial role in Giardia lamblia differentiation. Mol Biochem Parasitol 152: 80–89 Lee P and Faubert GM (2006a) Expression of the Giardia lamblia cyst wall protein 2 in Lactococcus lactis. Microbiology 152: 1981–1990 Lee P and Faubert GM (2006b) Oral immunization of BALB/c mice by intragastric delivery of Streptococcus gordonii-expressing Giardia cyst wall protein 2 decreases cyst shedding in challenged mice. FEMS Microbiol Lett 265: 225–236 Lee P, Abdul-Wahid A, and Faubert GM (2009) Comparison of the local immune response against Giardia lamblia cyst wall protein 2 induced by recombinant Lactococcus lactis and Streptococcus gordonii. Microbes Infect 11: 20–28 Lev B, Ward H, Keusch T, and Pereira ME (1986) Lectin activation in Giardia lamblia by host protease: a novel hostparasite interaction. Science 232: 71–73 Lujan HD, Mowatt MR, Conrad JT, Bowers B, and Nash TE (1995) Identification of a novel Giardia lamblia cyst wall protein with leucine-rich repeats. Implications for secretory granule formation and protein assembly into the cyst wall. J Biol Chem 270: 29307–29313 Lujan HD, Mowatt MR, Conrad JT, and Nash TE (1996) Increased expression of the molecular chaperone BiP/GRP78 during the differentiation of a primitive eukaryote. Biol Cell 86: 11–18 Lujan HD, Mowatt MR, and Nash TE (1997) Mechanisms of Giardia lamblia differentiation into cysts. Microbiol Mol Biol Rev 61: 94–304 McCaffery JM, Faubert GM, and Gillin FD (1994) Giardia lamblia: Traffic of a trophozoite variant surface protein and a major cyst wall epitope during growth, encystation, and antigenic switching. Exp Parasitol 79: 236–239 Medina E and Guzman CA (2001) Use of live bacterial vaccine vectors for antigen delivery: potential and limitations. Vaccine 19: 1573–1580 Meng TC, Hetsko ML, and Gillin FD (1996) Inhibition of Giardia lamblia excystation by antibodies against cyst walls and by wheat germ agglutinin. Infect Immun 64: 2151–2157 Mercenier A, Muller-Alouf H, and Grangette C (2000) Lactic acid bacteria as live vaccines. Curr Issues Mol Biol 2: 17–25 Moss DM, Mathews HM, Visvesvara GS, Dickerson JW, and Walker EW (1991) Purification and characterization of Gi-

P. Lee et al. ardia lamblia antigens in the feces of Mongolian gerbils. J Clin Micobiol 29: 21–26 O’Handley RM and Olson ME (2006) Giardiasis and cryptosporidiosis in ruminants. Vet Clin North Am Food Anim Pract 22: 623–643 Olson ME, Morck DW, and Ceri H (1996) The efficacy of a Giardia lamblia vaccine in kittens. Can J Vet Res 60: 249– 256 Pond K (2007) World Health Organization (WHO).Water recreation and disease. In: Plausibility of associated infections: Acute effects, sequelae and mortality. Chap. 5 Protozoa and Trematodes. Available at http://www.who.int/water_ sanitation_health/bathing/recreadischap5.pdf. Prucca CG and Lujan HJ (2009) Antigenic variation in Giardia lamblia. Cell Microbiol 11: 1706–1715 Prucca CG, Slavin I, Quiroga R, Elias EV, Rivero FD, Sauna A, Carranza PG, and Lujan HD (2008) Antigenic variation in Giardia lamblia is regulated by RNA interference. Nature 456: 750–754 Ralston BJ, McAllister TA, and Olson ME (2003) Prevalence and infection pattern of naturally acquired giardiasis and cryptosporidiosis in range beef calves and their dams. Vet Parasitol 114: 113–122 Rivero FD, Saura A, Prucca CG, Carranza PG, Torri A, and Lujan HD (2010) Disruption of antigenic variation is crucial for effective parasite vaccine. Nat Med 16: 551–557 Roberts-Thompson IC and Mitchell GF (1979) Protection of mice against Giardia muris infection. Infect Immun 24: 971–973 Roxström-Lindquist K, Palm D, Reiner D, Ringqvist E, and Svärd SG (2006) Giardia immunity – an update. Trends Parasitol 22: 26–31 Ruest N, Faubert GM, and Couture Y (1998) Prevalence and geographical distribution of Giardia spp. and Cryptosporidium spp. in dairy farms in Quebec. Can Vet J 39: 697– 700 Samra HK, Ganguly NK, and Mahajan RC (1991) Human milk containing specific secretory IgA inhibits binding of Giardia lamblia to nylon and glass surfaces. J Diarrhoeal Dis Res 9: 100–103 Snider DP, Skea D, and Underdown BJ (1988) Chronic giardiasis in B-cell-deficient mice expressing the xid gene. Infect Immun 56: 2838–2842 Stäger S, Gottstein B, and Müller N (1997) Systemic and local antibody response in mice induced by a recombinant peptide fragment from Giardia lamblia variant surface protein (VSP) H7 produced by a Salmonella typhimurium vaccine strain. Int J Parasitol 8: 965–971 Stein JE, Radecki SV, and Lappin MR (2003) Efficacy of Giardia vaccination in the treatment of giardiasis in cats. J Am Vet Med Assoc 11: 548–551 Sun CH, McCaffery JM, Reiner DS, and Gillin FD (2003) Mining the Giardia lamblia genome for new cyst wall proteins. J Biol Chem 278: 21701–21708 Touz MC, Gottig N, Nash TE, and Lujan HD (2002a) Identification and characterization of a novel secretory granule calcium-binding protein from the early branching eukaryote Giardia lamblia. J Biol Chem 277: 50557– 50563

Chap. 21 Vaccination Against Giardia Touz MC, Nores MJ, Slavin I, Carmona C, Conrad JT, Mowatt MR, Nash TE, Coronel CE, and Lujan HD (2002b) The activity of a developmentally regulated cysteine proteinase is required for cyst wall formation in the primitive eukaryote Giardia lamblia. J Biol Chem 277: 8474–8481 Uehlinger FD, O’Handley RM, Greenwood SJ, Guselle NJ, Gabor LJ, Van Velsen CM, Steuart RF, Barkema HW (2007) Efficacy of vaccination in preventing giardiasis in calves. Vet Parasitol 146: 182–188 Venkatesan P, Finch RG, and Wakelin D (1996) Comparison of antibody and cytokine responses to primary Giardia muris infection in H-2 congenic strains of mice. Infect Immun 64: 4525–4533 Vinayak VK, Kum K, Khanna R, and Khuller M (1992) Systemic-oral immunization with 56 kDa molecule of Giardia

351 lamblia affords protection in experimental mice. Vaccine 10: 21–27 Weiss S (2003) Transfer of eukaryotic expression plasmids to mammalian hosts by attenuated Salmonella spp. Int J Med Microbiol 293: 95–106 Wells JM and Mercenier A (2008) Mucosal delivery of therapeutic and prophylactic molecules using lactic acid bacteria. Nat Rev Microbiol 6: 349–362 Weniger BG, Blaser MJ, Gedrose J, Lippy EC, and Juranek DD (1983) An outbreak of waterborne giardiasis associated with heavy water runoff due to warm weather and volcanic ash fall. Am J Public Health 73: 868–872

Diagnosis of Human Giardiasis Huw V. Smith† and Theo G. Mank

Abstract Although progress has been made in the non-morphological diagnosis of Giardiasis, examination (usually of feces) by microscopy remains the cornerstone in the diagnosis of intestinal parasitic infections. Routine examination of stool specimens is the best known and most frequently performed laboratory procedure worldwide in which almost all intestinal parasites can be detected at the same time. In Europe, standard methods in the laboratory diagnostics of enteric parasitoses, including Giardia, comprise the direct microscopic examination of the wet mount, and the examination of material from a stool concentrate. There is however an increasing trend towards routine use of fixatives, permanent staining, and multiple sampling techniques as these methods have been proven to enhance the diagnostic yield considerably. Advances in the biomedical sciences however have led to the non-morphological laboratory diagnosis of Giardiasis. Immunoassays to detect copro-antigens are gradually being introduced into routine diagnostics, particularly in larger laboratories processing large numbers of samples. PCR-based methods not only have exquisite sensitivity and specificity but also enables Giardia assemblage identification, but only few diagnostic laboratories are well placed to undertake these methods. These new parasite specific techniques may aid diagnosis and should be considered for adoption into the routine diagnostic algorithm. Once they have been evaluated fully and compared with accepted methods in individual laboratories or as interlaboratory quality assurance investigations they can be regarded as an adjunct to the conventional

H. D. Luján et al. (eds.), Giardia © Springer-Verlag/Wien 2011

22

detection and identification techniques used for laboratory diagnosis.

22.1 Introduction The flagellated protozoan parasite G. duodenalis (syn: G. lamblia) is a common cause of diarrhoea in humans and is probably the most commonly detected intestinal parasite in human stools, worldwide. G. duodenalis infections occur both in developing regions, where sanitation is poor or non-existent and where drinking untreated surface water is common, and in developed regions with strong market economies where effective sanitation and public health measures are practiced and drinking water is treated and piped to dwellings. Accordingly, the prevalence of giardiasis in developing regions is ~20% (4–43%) compared to ~5% (3–7%) in developed regions (Roxström-Lindquist et al., 2006). Exact incidence and prevalence data depend on the population examined. Globally, G. duodenalis causes an estimated 2.8 × 108 cases per year (Lane and Lloyd, 2002), whereas in Asia, Africa and Latin America, about 200 million people have symptomatic giardiasis with ~500,000 new cases reported annually (WHO, 1996). Between 100,000 and 2.5 million Giardia infections occur annually in the United States (Nasmuth, 1996). In Scotland, giardiasis was made a laboratory reportable disease in 1999, and in 2008–2009, 4.2 cases per 100,000 individuals were reported. Most cases (>90%) report chronic diarrhoea, and a significant proportion appear to be imported. In the Netherlands, the prevalence of infection varies between 2 and 14%, being high in patients who consult their general

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practitioner with persistent diarrhoea and low (2%) in asymptomatic subjects (Mank et al., 1998; de Wit et al., 2001).

22.1.1 Early Studies and Their Impact on Diagnosis Although Giardia has become increasingly recognised in the past 50 years, it was first observed in 1681 by Antony van Leeuwenhoek, the pioneering microscopist from Delft, Netherlands, in his watery stools. Ford (2005) states that a letter written in his native early modern Dutch and dated November 4, 1681 was translated into English for the Royal Society and read to the Fellows at their meeting of November 9, 1681. In that letter, van Leeuwenhoek described what he observed in his stool sample using his single lens microscope. “I have sometimes also seen tiny creatures moving very prettily; some of them a bit bigger, others a bit less, than a blood-globule but all of one and the same make. Their bodies were somewhat longer than broad, and their belly, which was flattish, furnished with sundry little paws, wherewith they made such a stir in the clear medium and among the globules, that you might even fancy you saw a woodlouse running up against a wall; and albeit they made a quick motion with their paws, yet for all that they made but slow progress.” (Ford, 2005). Dobell (1920) concluded from van Leeuwenhoek’s description that these were Giardia trophozoites (Ford, 2005), but there was no evidence that van Leeuwenhoek could

have observed trophozoites using a ‘primitive’ microscope until 2004. Ford (2005) determined that van Leeuwenhoek could have seen Giardia trophozoites through his simple, single lens microscope by observing satisfactory motile and air dried G. duodenalis trophozoites through a similar single lens microscope with a magnification of 295× (Plate 1a). More than 350 years since van Leeuwenhoek’s discovery, microscopy still remains the mainstay of Giardia diagnosis today. Centuries past since its discovery, during which time Giardia received minimal attention and it was only in the 1960s that this protozoan parasite was formally recognised as a pathogen causing human diarrhoea by the medical community (Feachem et al., 1983). Within five years of this recognition, the first waterborne outbreak of giardiasis was documented in 1965, and in 1987, Nash and colleagues fulfilled Koch’s postulates (rules for proving that an organism causes disease) for experimental human infections. Thus, its commonness in humans, its ability to cause outbreaks of human disease and the fulfilment of Koch’s postulates have made Giardia a well recognised and important human pathogen.

22.2 Giardia Diagnosis 22.2.1 Giardia and Human Giardiasis G. duodenalis parasitises the small intestine of man and other vertebrates and is the species responsible for hu-

Excystation

Encystation

Trophozoite Fig. 22.1 Giardia duodenalis life cycle

Cyst

Chap. 22 Diagnosis of Human Giardiasis

man disease. Transmission can occur via any route by which material contaminated with viable cysts excreted by infected hosts can reach the mouth of a susceptible host. It’s life cycle is direct, requiring no intermediate host, and the parasite exists in two distinct morphological forms, namely, the reproductive trophozoite which parasitises the enterocytes of the upper small intestine, and the environmentally resistant cyst, voided in the faeces, which is the transmissive and infective stage (Fig. 22.1). Cysts are resistant to many external environments and can survive for prolonged periods of time in moist micro-environments.

22.2.2Symptoms and Basis for Laboratory Investigations Giardia infection can present different clinical pictures ranging from acute to chronic disease and asymptomatic carriage. In immunocompetent individuals the disease is self-limiting, the acute phase being normally short-lived and followed by the chronic phase, which is the common disease presentation. Typically, the symptoms accompanying infection include diarrhoea, flatulence, upper intestinal cramps, abdominal distension, nausea, weight loss and malabsorption (Wolfe, 1979). The most prominent symptom is protracted diarrhoea which can be mild, with the passage of semi-solid stools, or intense and debilitating when the passage of watery, voluminous stools which are sometimes frothy, greasy, offensive and float on water (Plate 1b). In chronic disease (estimated to occur in 30–50% of symptomatic cases), malaise, weight loss and other features of malabsorption may become prominent. By this time, stools are usually pale or yellow, frequent and of small volume. Occasionally episodes of constipation intervene with nausea and diarrhoea precipitated by the ingestion of food. Malabsorption in vitamins A and B12 and D-xylose can occur. Disaccharidase deficiencies (most commonly lactase) are frequently detected in chronic cases. The asymptomatic cyst passing stage has prevalence rates varying from 13 to 76% however, its duration is not known (Lopez et al., 1980; Wolfe, 1984). The factors determining the variability in clinical outcome in giardiasis are poorly understood. Host factors, such as immune status, nutritional status and age,

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as well as differences in virulence and pathogenicity of G. duodenalis isolates are recognised as important determinants for the severity of infection (Thompson et al., 1993; Buret, 1994; Homan and Mank, 2001). The incubation period is usually 1–2 weeks. On average, the prepatent period (time from infection to the initial detection of parasites in stools) is 9.1 days (Rendtorff, 1979). As the prepatent period can exceed the incubation period, initially symptoms can occur in the absence of cysts being excreted in faeces. Cyst excretion can approach 107 g–1 faeces (Danciger and Lopez, 1975). In severe infections up to 14 billion parasites can occur in a diarrhoeal stool whereas in a moderate infection up to 300 million parasites can occur (Lin, 1985). Symptoms usually disappear following successful treatment, but infection can lead to chronic sequelae which can occur sporadically for years. Childhood infection is associated with nutritional shortfalls, stunting and wasting, poorer cognitive function and failure to thrive, independent of diarrhoea, while inflammatory bowel disease can be a consequence in adulthood (Savioli et al., 2006; Ortega-Pierres et al., 2009). In young children, “failure to thrive” is frequently due to giardiasis, and all infants being investigated for causes of malabsorption should have a diagnosis of giardiasis excluded. Stools are often loose or watery in the acute stage and intermittently in the chronic stage, and may contain only trophozoites. Immediate wet film examination of a freshly-passed (hot) stool specimen can identify intact motile organisms, but unless recently voided stools are kept warm, trophozoite motility will not be observed and stools should be processed accordingly to detect immotile trophozoites (Sects. 22.4, 22.5, 22.11), cysts (Sects. 22.6, 22.11), Giardia antigen (Sect. 22.12) and/or Giardia DNA using polymerase chain reaction (PCR) based assays (Sect. 22.16). In the last 30 years, giardiasis has received more recognition as a common intestinal pathogen in the clinical diagnostic laboratory. Widespread documentation of numerous endemic and epidemic outbreaks of giardiasis, its documented foodborne and waterborne transmission (Smith and Paget, 2007), especially through community water supplies (Karanis et al., 2006), has focused the attention of public health professionals on its commonness and increased the

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demand for diagnostic laboratories to identify it (Lopez, et al., 1980; Wolfe, 1984; Girdwood, 1989; Casemore, 1990). Some risk factors associated with Giardia infection are presented in Table 22.1. In the diagnostic laboratory, special consideration should be given to Giardia in the aetiology of diarrhoeal disease in developed regions of the world for the following groups: • Individuals with prolonged duration (>14 days) of diarrhoeal complaints. • Immunocompetent neonates and pre-school children attending nurseries, etc. • Travellers. • Immunocompromised individuals with reduced protection against the development of clinical disease

(e.g. concurrent infections, congenital immunological defects (IgA deficiency), immunosuppressive therapy for transplantation surgery and neoplasia, and renal dialysis) (Cook, 1990).

22.2.3Giardia Species and Assemblages The recent application of molecular techniques to subdivide G. duodenalis has revealed high levels of genetic diversity within this species. Currently, G. duodenalis consists of six recognised variants or assemblages (assemblages A, B, C, D, E and F), each having a varying degree of host specificity (Cacciò and Ryan, 2008; see Chapter 2). Only G. duodenalis

Table 22.1 Risk factors associated with G. duodenalis infection Risk factor

Association

Postulated reasons

Age

Children are more likely to be infected than adults and excrete cysts in larger numbers

1. Behaviour: greater risk of exposure 2. Immunology: lack of immunity 3. Diagnosis: infections in adults may more frequently be asymptomatic and thus less likely to be diagnosed

Gender

Some studies indicate males are more likely to be infected than females

1. Behaviour: greater risk of exposure 2. False result due to biased sampling

Nutritional status

Malnourished individuals more likely to be infected than well-nourished individuals

1. Hypochlorhydria 2. Reduced intestinal immune functions 3. Low enzyme activity 4. Poor intestinal motility

Breast-feeding

Non-breast-fed infants more likely to be infected than breast-fed infants

1. Contamination: greater risk of exposure to cysts in bottled milk 2. Immunology: acquisition of antibodies in breast milk 3. Cysticidal properties of breast milk

Diet

Consumption of leafy vegetables may be a risk factor

1. Often consumed raw 2. May be washed in contaminated water

Urban/rural

Some studies suggest city dwellers more likely to be infected than those in rural areas

1. High population density and overcrowding 2. Poverty and poor sanitation

Seasonality

Incidence may increase during cooler and wetter seasons

Cysts more likely to survive under cool, damp conditions

Socio-economic status

Associated with lower socio-economic status

1. Poverty and poor sanitation 2. Over-crowding 3. Inadequate water supplies 4. Lack of health education 5. Use of night soil as fertiliser

Socio-economic status

Associated with higher socio-economic status

1. Under-reporting in low socio-economic groups as reduced access to health-care 2. Access to international travel

Chap. 22 Diagnosis of Human Giardiasis

assemblages A and B cause human disease (Cacciò et al., 2005; Cacciò and Ryan, 2008) but at present, it is not completely clear what the relationship between clinical symptomatology and infection with these assemblages is because of contrasting results linking assemblage A to mild, intermittent diarrhoea and assemblage B to severe, acute/persistent diarrhoea and vice versa (Homan and Mank, 2001; Read et al., 2002; Sahagún et al., 2008). Clearly, this is an important area for further investigation.

22.3 Brightfield, Phase Contrast (PC) and Differential Interference Contrast (DIC) Microscopy Morphometry (the accurate measurement of size and shape of suspended organisms) and morphology (the accurate identification of organelles in suspended organisms) are fundamental diagnostic criteria in microscopy. At low (10×) magnification much internal detail of unstained trophozoites and cysts is not readily visible by bright field microscopy. PC and DIC are contrast-enhancing optical techniques that enhance the contrast of transparent specimens such as trophozoites and cysts through a phase shift of light, causing a reduction in brightness, based on refractive indices. Using the 20× or 40× (dry) objective, Nomarski differential interference contrast (DIC) microscopy provides an especially sharp image of trophozoite and cyst perimeter for measurement and a clearer image of trophozoite and cyst content.

22.3.1Micrometry In diagnostic parasitology, definitive diagnosis of intact life cycle forms often necessitates the measurement of the size and shape (morphometrics) of the organism in question, in order to ensure that its morphometrics fall within the accepted range of these standard parameters for the species in question. The light microscope can be used for crude measurement of relatively large objects (>1 mm) by measuring the object using the Vernier scales commonly fitted to mechanical stages. The measurement of microscopic objects (<1 mm) is achieved by using a stage micrometer in conjunction with an eye-piece microm-

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eter. Objects are measured in Systeme International (S.I.) units, and the standard unit of measurement for bright field microscopy is the micron (μ = 0.001 mm). The stage micrometer consists of a 76 × 26 mm glass slide which has a millimetre scale graduated in microns, permanently mounted on it. The eye-piece micrometer is a disc of transparent glass or plastic bearing a graduated scale which is placed in one of the eye-pieces of a binocular microscope. The scale is usually 1 cm in length and is sub-divided into millimetre intervals. When the microscope is focused on the object to be measured, both the scale on the eye-piece micrometer and the image of the object are seen simultaneously in focus. The standard scale on the stage micrometer is usually 1 or 2 ml. When making measurements, the appropriate objective lens dependent on the magnification required’ is chosen, and the number of divisions corresponding to the length or breadth of the image of the object is read on the scale of the eye-piece micrometer. The observed measurement is translated into real length (which corresponds to the number of eye-piece micrometer divisions representing the chosen parameter to be measured) by substituting the stage micrometer for the object and determining the number of divisions on the eye-piece micrometer corresponding to a definite number of divisions of the millimetre scale on the stage micrometer, under the same magnification. Remember that your calculation, in real length, of the value of the division on the eye-piece micrometer scale will only be valid for the magnification of the objective chosen. You will have to recalculate the value of a division on the eye-piece micrometer for each objective of differing magnification on the microscope. Because morphometrics is a fundamental component of diagnostic parasitology, repetitive measurements of similar objects present in a single sample or of various objects of varying sizes in sequential samples, necessitating the use of a variety of magnifications, will have to be performed. By determining the micrometer value of the eye-piece scale for each objective used, constant interchange of objects and stage micrometer can be overcome enabling rapid calculation of morphometrics, in millimetres, or fractions thereof, to be performed using any objective lens.

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22.3.2Trophozoite Morphometry and Morphology G. duodenalis trophozoites contain two nuclei (binucleate) located in the anterior of the organism and the shape resembles that of a pear cut in half along its long axis (pyriform). Trophozoites measure 12–18 μm

a n

vd

m a

c p

v

Key: a = anterio-lateral flagellum, c = caudal flagellum, m = median bodies, n = nucleus, p = posterio-lateral flagellum, v = ventral flagellum.

b

in length, from their broad (anterior) end to their narrow (caudal or posterior) end, 6–9 μm in width, and 2–4 μm in thickness. Each trophozoite has a dorsal and ventral surface, the dorsal surface being convex (Plate 1c). A ventro-lateral flange separates the dorsal from the ventral surface. On the anterior half of the flattened ventral surface is located a distinctive concave (ventral) disc with a raised ridge at its anterior end (Plate 1c). This is the attachment organelle, enabling trophozoite attachment to host enterocytes. In the fluid-filled lumen of the small intestine maintaining niche is an important survival strategy and impressions of the ventral disc can be seen on the microvillous surfaces of enterocytes in scanning electron micrographs. Trophozoites possess four pairs of flagella arranged in bilateral symmetry. G. duodenalis trophozoites possess anterio-lateral, ventro-lateral, posterio-lateral and caudal pairs of flagella with the caudal pair being ventral. The axonemes of the four pairs of symmetrical flagella arise from basal granules located at the anterior pole of the two nuclei. The axonemes of the three posterior facing pairs of flagella (ventro-lateral, posterio-lateral and caudal) run between the two nuclei (Fig. 22.2a). Trophozoites attach and detach from the microvillous surface of enterocytes. Motile trophozoites exhibit forward movement during which they tend to rotate around their longitudinal axes displaying both a tumbling movement resembling that of a falling leaf and an up and down movement referred to as “skipping”. Two “claw-hammer” shaped median bodies, composed of microtubules, lie transversely in the mid-

n Table 22.2 Characteristic morphological features of G. duodenalis trophozoites by Nomarski DIC microscopy

c a

Key: a = axostyle (flagellar axoneme), c = crescentic fragments of the ventral disc, n = nucleus. Fig. 22.2 Giardia duodenalis; line drawing of trophozoite (a) and cyst (b) identifying significant morphological features.

Binucleate, resembling a pear cut in half longitudinally with a convex dorsal surface, measuring 12–18 μm in length, from their broad (anterior) end to their narrow (caudal) end and 6–8 μm wide, and 2–4 μm thick. Four pairs of flagella arranged in bilateral symmetry, all of which are directed caudally. Distinctive concave disc with a raised ridge at its anterior end located in the anterior half of the ventral surface. Two “claw-hammer” shaped median bodies lying transversely in the mid-portion of the organism.

Chap. 22 Diagnosis of Human Giardiasis

portion of the organism. The function of the median bodies remains unknown. Trophozoites multiply by binary fission and following nuclear division, two daughter trophozoites with the same complement of morphological features and organelles are produced. The characteristic morphometry and morphology of suspended G. duodenalis trophozoites as seen by DIC microscopy are presented in Table 22.2.

22.3.3Cyst Morphometry and Morphology Exposure to bile salts and alkaline pH as trophozoites pass down the small intestine induces them to encyst, rounding up and forming the immature binucleate cyst. During encystation, flagella are lost but axonemes are retained and following encystation, ovoid to ellipsoid cysts with dimensions of 8–14 μm long and 6–10 μm wide occur in the lower small intestine and large intestine and are voided in faeces. The cyst contains fewer identifiable organelles than the trophozoite. Apart from the nuclei, flagellar axonemes can often be seen running diagonally along the long axis of the cyst as can the crescentic fragments of the ventral disc (Plate 1d and e; Fig. 22.2b). Whilst encysted, the nuclei of the single trophozoite undergo division to produce a mature cyst containing four nuclei located at one pole. The characteristic morphometry and morphology of recently voided, suspended G. duodenalis cysts as seen by Nomarski DIC microscopy are presented in Table 22.3. Not all of the above features will be seen in a single preparation therefore, both unstained and stained preparations may be necessary. Most, but not all these features can be seen in either unstained

Table 22.3 Characteristic morphological features of G. duodenalis cysts by Nomarski DIC microscopy Ellipsoid to oval, smooth walled, colourless and refractile. 8–14 × 6–10 μm (length × width). Mature cysts contain four nuclei displaced to one pole of the organism. Flagellar axonemes (axostyle) lying diagonally across the long axis of the cyst. Two “crescentic bodies” (fragments of the ventral disc) lying transversely in the mid-portion of the organism.

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direct (unconcentrated) or concentrated preparations. Nuclei are not as visible in saline suspensions as they are in formalin-fixed material by bright-field microscopy. Lugol’s iodine is a useful temporary stain for direct films and concentrates as it highlights cyst nuclei by staining them yellow to brown (Sect. 22.6.1.1.1).

22.4 Rationale for Laboratory Diagnosis of Infection Typically, the stage of the Giardia life-cycle present in faeces is the environmentally robust cyst which does not multiply outside the body of the host. In instances of florid diarrhoea, trophozoites will also be voided, as intestinal transit time is reduced. Classically, stool analysis is the primary approach to disease diagnosis, having the advantage that it is not an invasive technique. Wet (temporary) mounts are examined. Wet mounts can be faecal suspensions or concentrates and may be unstained (suspended in saline or formalin-ether/formalin-ethyl acetate) or stained (iodine, which is primarily a stain for cysts, or other temporary or permanent stains). As Giardia cannot be expanded in in vitro culture readily from faeces, the maximum numbers of parasites voided in the stool will be the maximum number detectable. The microscopic identification of G. duodenalis cysts relies upon the morphometry and morphology of suspended organisms (see Sect. 22.3), although the use of various staining methods enhances or reveals internal structures (e.g. organelles) and/ or surface topography. Examine the stool macroscopically and record whether it is formed, soft, unformed, or liquid and whether it contains evidence of blood or mucus. Stools with evidence of blood or mucus may contain other protozoan parasites and should be examined with the minimum of delay by direct microscopy to ensure that these stages are still intact and motile. Formed stools, without any evidence of blood or mucus, are normally examined following concentration within 24 h of voiding, and are stored refrigerated until then. When long storage or transit times, which can result in the deterioration of trophozoite and/or cyst morphology, are anticipated the use of a preservative should be considered.

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Concentration techniques are unsatisfactory for trophozoites, and the direct examination of emulsified stools or the examination of fixed samples with or without the examination of permanently stained smears is necessary for their demonstration. Permanent conventional stains aid the identification of Giardia and serve as a permanent record to document the presence of the parasite. If unfixed stool suspensions cannot be examined in the daily routine, or if sample transportation is protracted, fixatives, which preserve trophozoite and cyst morphology, should be considered. Formalin (40% formaldehyde in water), buffered formalin (6.10 g Na2HPO4 and 0.15 g NaH2PO4 are mixed thoroughly and the powder is stored in an airtight container. Add 0.8 g of mixture to 1 litre of 10% (or 5%) formalin) and mercuric chloride (Schaudinn’s fluid; polyvinyl alcohol (PVA)) are traditional fixatives for parasites, but they have limitations. Formalin retains cyst morphology far better than trophozoite morphology and is not recommended for trophozoite preservation and there are clear health and safety precautions with the use and disposal of mercury-based fixatives. Prolonged formalin fixation also reduces PCR sensitivity. Effective alternative fixatives exist and include sodium acetate-acetic acid-formalin (SAF; 1.5 g sodium acetate, 2.0 ml glacial acetic acid; 4.0 ml formaldehyde (37–40% solution), 92 ml distilled water), merthiolate-iodine-formaldehyde (MIF) stain and fixative, as well as commercial preparations. An aliquot of stool can be preserved in SAF, MIF or PVA fixatives for microscopical examination and/or conventional staining and examination later. PVA, SAF and MIF provide good preservation of trophozoite and cyst morphology (Plate 1f). Garcia and Bruckner (1997) consider that SAF produces ‘softer’ morphology than mercuric chloride fixation, and that SAF followed by iron haematoxylin staining produces a better permanent morphological preparation than SAF followed by Trichrome stain. Where possible, fixation in SAF is recommended. SAF fixation of stools increases the detection rate of intestinal protozoa compared with unfixed stools (Mank et al., 1995). Examination of SAF-fixed stools (microscopy of both concentrated sediment and per-

H.V. Smith and T.G. Mank manent stained direct smear) revealed intestinal protozoa in 60.3% of SAF-fixed samples compared with 36% of unfixed samples. Badparva et al. (2009) found that SAF (followed by haematoxylin staining) outperformed MIF (followed by Trichrome staining) and SAF, PVA and MIF followed by carbol-fuchsin staining, but none was as good as formalin followed by haematoxylin staining for highlighting G. duodenalis cyst morphology and maximising cyst detection. Pietrzak-Johnston et al. (2000) found that EcoFix (Meridian Diagnostics, Inc., Cinncinnati, OH, USA) was an acceptable commercial alternative to formalin for wet preparations. Specimens preserved in SAF can be stored for several months for use in a Proficiency Testing scheme, and the concordance rates between initial and resubmitted reports of the same specimen are about 80% for pathogenic protozoa (G. duodenalis, Entamoeba histolytica/dispar and Dientamoeba fragilis) (Libman et al., 2008). However, Utzinger et al. (2010) found only moderate agreement between European clinical diagnostic parasitology reference centres for diagnosing pathogenic intestinal protozoa using ether-concentration of SAF preserved faecal samples, and advocated continued external quality assessment to further enhance both accuracy and uniformity in parasite diagnosis. The absence of cysts in a single stool sample cannot exclude infection; therefore at least three stool specimens should be examined by a competent microscopist prior to suggesting other diagnostic procedures. The erratic nature of cyst excretion, the requirement for experienced staff for microscopic identification and the relatively low detection rate (Sect. 22.13) have precipitated the development of alternative methods for the diagnosis of giardiasis. When trophozoites or cysts, Giardia antigen or DNA cannot be found following extensive examination of repeat stool samples, and giardiasis is suspected clinically, more invasive procedures, such as the examination of duodenal or jejunal fluid and/or tissue biopsy may be indicated (Sects. 22.9, 15). The fact that laboratory identification consists of multiple analyses using different matrices identifies the difficulties encountered in Giardia diagnosis.

Chap. 22 Diagnosis of Human Giardiasis

22.5 Examination for Trophozoites and Cysts in Un-Concentrated (Direct) Stool Samples Analysing recently voided, or freshly preserved, unconcentrated stools for trophozoites and/or cysts by direct microscopy is the most straightforward laboratory investigation. The tip of a clean wooden applicator stick is used to remove a small sample (about 2 mg) of faeces which is then emulsified thoroughly in 150 mM NaCl pre-warmed to 37ºC in a test tube. For liquid stools ~50 μl of stool is mixed with ~25 μl pre-warmed saline. Mucus strands present in liquid stools can be mixed with saline on a microscope slide. A smear of the correct transparency is made on a clean microscope slide. An acceptable thickness can be achieved when either the hands of your watch or the print on this page can just be read when viewed through the preparation. If the smear is too thin or thick, trophozoites and cysts will be missed. A coverslip is placed onto the faecal suspension and the slide is examined (Sect. 22.8) using the 10× objective. If a trophozoite- or cyst-like object is seen, examine it under the 40× (dry) to determine whether the characteristics of the object match with those in Tables 22.1 and/or 22.2. Examine the remainder of the preparation to determine the numbers of trophozoites/cysts present. Trophozoite and/or cyst abundance can be recorded as scanty (1+), moderate (2+) or numerous (3+). Lugol’s iodine can be added to increase the contrast of cyst organelles, as it stains Giardia cyst nuclei yellow or brown. Lugol’s iodine preparations should be viewed within 15 min of preparation, otherwise overstaining of cyst inclusions will occur. If objects of the correct size and shape are seen, but no diagnostic inclusions can be recognised, another emulsion should be examined. Cyst excretion can be intermittent, some infected humans may be low cyst excretors (Danciger and Lopez, 1975) and symptoms can occur before parasites are voided in stool. Repeated examinations on a weekly basis may be necessary until the infection becomes patent (Jokipii and Jokipii, 1977). For these reasons, timing of stool collection is important. Where parasites are numerous, direct smear examination in saline or Lugol’s iodine may be sufficient for diagno-

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sis. Generally, formol-ether concentration of cysts is the preferred option (Sect. 22.6.1.1) but flotation using zinc sulphate can occasionally demonstrate cysts when other techniques have failed (Sect. 22.6.2).

22.6 Concentration of Cysts From Faeces 22.6.1Biophysical Methods Methods for the preferential concentration of life cycle stages in faecal suspensions were devised to maximise the likelihood of detecting parasites in stools. The objective is to separate any parasites present from the faecal bulk in the specimen, including faecal particulates and other interferents. The efficiency of these methods is dependent on the density on the parasite(s) sought and the robustness of the life cycle stage in question. Cysts (and helminth ova and larvae) can withstand concentration procedures but trophozoites cannot, hence the direct examination of wet films or permanently stained smears of stools is recommended for trophozoites (Sect. 22.4, 22.5, 22.11). This rationale provides the clinical laboratory diagnostician the maximum opportunity of detecting and identifying the maximum numbers of diagnostic stages in the life cycle. Two methods, sedimentation and flotation, with centrifugation, are effective for cysts, as they withstand concentration procedures. Centrifugation enhanced recoveries. Concentration methods can be monophasic or biphasic. In monophasic concentration, the biophysical properties of only one solution are employed as opposed to biphasic concentration where the biophysical properties of two, normally immiscible, solutions are employed. Monophasic sedimentation is seldom used in diagnostic laboratories as it is more time consuming, but examples of monophasic flotation with centrifugation include brine, zinc sulphate and sucrose flotation. Formol-ether (ethyl acetate) concentration is the best example of biphasic sedimentation with centrifugation. Biphasic flotation is not used for cyst concentration in the clinical laboratory. The advantage of using biphasic immiscible solutions is that different stool components will have

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greater affinities with one or other liquids and will settle in them preferentially. This preferential settlement allows us to separate the different components by analysing each immiscible liquid.

Faecal debris and fat Diethyl ether or ethyl acetate

22.6.1.1 Formol-Ether (Ethyl Acetate) Concentration Cysts settle more rapidly when a stool suspension is centrifuged, but faecal particulates also settle, masking cysts in the film examined. Larger faecal particulates can be removed before centrifuging by filtering the emulsified stool through a sieve whose aperture size is large enough for parasites to pass through, but retains the larger particulates. As this process is more efficient than sedimentation by gravity a smaller faecal sample (500 mg–1 g: the size of a pea) is sufficient for examination. Although centrifugation concentrates particulates more quickly, faecal debris which can obscure parasites remains. The efficiency of detection is increased by adding 10% formalin for fixation and preservation of parasites, and ether (or ethyl acetate) to remove fats and oils. Formalin and ether are also microbiocidal and provide user protection. After centrifugation, a fatty plug, which may adhere to the inner walls of the tube, can be seen at the interface of the two liquids (Fig. 22.3). The ether layer, the fatty plug and the formalin below it are discarded and the whole pellet is resuspended and retained for examination. There are many modifications to this procedure, and the following protocol, based on the method of Allen and Ridley (1970; Sect. 22.6.1.1.1) is typical of the method used in diagnostic laboratories. Less distortion of protozoan cysts occurs with this method than with zinc sulphate flotation. This method is rapid, effective, achieving a concentration of 15–50-fold dependent upon the parasite sought, and provides a good concentrate of protozoan cysts and helminth eggs which are diagnostically satisfactory. 22.6.1.1.1 Formol-Ether (Ethyl Acetate) Concentration 22.6.1.1.1.1 Materials 15-ml conical glass centrifuge tubes, disposable wooden applicator sticks, sieve (425 μm aperture size 38 mm diametera), 50-ml Pyrex beakerb centrifuge with 15-ml swing-out buckets, glass microscope

Formalin

Sediment containing parasite ova, larvae and cysts

Fig. 22.3 Formol-ether (ethyl acetate) concentration. Diagram of the four layers seen in a conical centrifuge tube after centrifugation.

slides (76 × 26 mm), coverslips (22 × 32 mm), diamond marker, bright-field microscope with ×10 and ×40 objective lenses. 10% formalin (10% of 40% formaldehyde in water), diethyl ether (or ethyl acetate), Lugol’s iodine (iodine crystals 5.0 g, KI 10 g, distilled water 100 ml. Dissolve KI in distilled water, then add iodine crystals). Store in a dark glass bottle out of direct sunlight where it will remain stable for several weeks. 22.6.1.1.1.2 Method 1. Sample approximately 500 mg–1 g faeces with an applicator stickc and place in a clean centrifuge tube containing 7 ml of 10% formalin. If the stool is liquid, dispense about 750 μl into the centrifuge tube. 2. Break up the sample thoroughly and emulsify with an applicator stick. 3. Filter the resulting suspension through a sieve into a beaker and pour the filtrate back into the same tube.a,d 4. Add 3 ml of diethyl ether (or ethyl acetatee) to the formalinised solution, place a gloved thumb on top of the tube and shake the solution vigorously for 30 sec. Invert the tube a few times during this procedure and release the pressure developed gently by removing your thumb slowly.

Chap. 22 Diagnosis of Human Giardiasis f

5. Centrifuge the tube at 750× g for 60 sec. 6. Loosen the fatty plug with a wooden stick by passing the stick between the inner walls of the tube and the plug. Discard the plug and the fluid both above and below it by inverting the tube, allowing only the last one or two drops to fall back into the tube. Resuspend the pellet by agitation. 7. Pour the whole, or the majority of the resuspended pellet onto a microscope slide, or transfer the resuspended contents onto a microscope slide with a Pasteur pipette, apply a coverslip, and examine for the presence of parasites using the ×10 objective lens.g 8. Identify any definitive morphological features under the ×40 objective.h 9. Assess the numbers of parasites present.i a

425 μm aperture size, 38 mm diameter is equivalent to 36 mesh British Standard (BS 410-86) or 40 mesh American Standard (ASTM E11-81). b The skirt of the sieve should fit neatly into the rim of the beaker. c The sample should include portions from the surface and from within a formed stool. d Debris trapped on the sieve is discarded. Both the sieve and the beaker should be washed thoroughly in running tap water between each of the samples. e Ethyl acetate, although less flammable than diethyl ether is nevertheless flammable, therefore the procedure should be performed in well ventilated areas, ensuring that they contain no naked flames. Avoid prolonged breathing or skin contact. f Centrifugation at speeds higher than 750× g for long periods of time is not advised since some helminth ova may rupture and collapse at higher centrifugal speeds. g Too large a pellet is indicative of one or more of the following: centrifuging above the recommended speed and/or time, insufficient shaking, taking too large a faecal sample. h If protozoan cysts of the correct size and shape can be seen, but no diagnostic inclusions can be recognised, add a drop or two of Lugol’s iodine either to the fluid at the edge of the coverslip, and re-examine the preparation when the iodine has diffused into the fluid under the coverslip (about five min), or to the resuspended pellet from another concentrate prior to applying the coverslip. Lugol’s iodine stains nuclei

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and glycogen masses in cysts yellow to brown. Lugol’s iodine preparations should be viewed within 15 min of preparation, otherwise overstaining of cyst inclusions will occur. i Numbers can be recorded as scanty, moderate or numerous. A commercial device for concentrating helminth ova, larvae and protozoan cysts based on a modification of the formalin-ether method of Ritchie is available. Sold as the Fecal Parasite Concentrator (FPC, Evergreen Scientific, Los Angeles, California 90058, USA)’ it is an enclosed system consisting of two polypropylene tubes, a flat-bottomed tube used for emulsifying the stool, and a conical tube used for centrifugation, with an interconnecting sieve. Both fresh and preserved (10% formalin, SAF, MIF, and PVA) stool specimens can be used.

22.6.2Centrifugal Flotation The specific gravity of protozoan cysts, helminth ova and larvae ranges between 1.05 and 1.15 and the flotation principle exploits a liquid suspending medium which is denser than the parasites to be concentrated. When mixed with flotation fluid, parasites rise to the surface and can be skimmed out of the surface film. For a flotation fluid to be useful in diagnostics, the suspending medium must not only be heavier than the object to be floated but also must not produce shrinkage sufficient to render the object undiagnosable. Originally brine, a concentrated aqueous NaCl solution, which has a specific gravity (sp. gr.) between 1.12 and 1.20 depending on the impurity of the salt used, was employed, but cysts can become badly shrivelled or collapse in brine, if not examined between 5 and 20 min after flotation. For these reasons, zinc sulphate flotation, which cause less plasmolysis, was introduced. The efficiency of cyst recovery using flotation is increased by the addition of a centrifugation step after the sample has been emulsified. Centrifugation sediments larger faecal particulates producing a flotation meniscus enriched for cysts, with fewer contaminating particulates. Zinc sulphate centrifugal flotation provides an effective concentration method for protozoan cysts, helminth ova and larvae from stool, which are undistorted and viable. The most useful concen-

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tration of zinc sulphate for floating the commonly encountered parasites has a specific gravity of 1.180. A specific gravity of 1.20 is recommended for formalinised specimens (Smith, 1992). Faecal samples (500 mg–1 g) are thoroughly mixed in a 15-ml centrifuge tube with 10 ml of distilled water, strained through a sieve (see footnote a in 22.6.1.1.1) to remove large particles. Suspended particles are removed by decanting the supernatant following centrifugation (750× g, 1min). The pellet is resuspended, and centrifuged until the supernatant fluid is clear (usually 2–3 times). The pellet is then resuspended in zinc sulphate flotation fluid (1.180 sp. gr.). Initially 3–4 ml of flotation fluid is added to resuspend the pellet, then sufficient fluid is added to fill the centrifuge tube to within 5 mm of its brim. The tube is centrifuged (750× g, 2 min) and once stationary, the tube is stood vertically in a test tube rack. The preparation can be sampled either (i) after 10 min standing by carefully removing the material floating in the surface film with a Pasteur pipette or by several loopsful of a wire loop, bent at right angles, onto a clean microscope slide or (ii) by carefully pipetting sufficient zinc sulphate solution to form a slight positive meniscus at the brim of the centrifuge tube, and gently placing a coverslip onto the rim of the tube. This should be left for 10 min, then lifted vertically, in one movement, together with its hanging drop and placed onto a clean microscope slide. Cysts (and other parasites) are examined using the 10× objective lens and confirmed using the morphological and morphometric criteria in Tables 22.1 and 22.2 under the 40× objective. Cysts which rise to the surface will begin to sink after about an hour, therefore the sample should be removed from the meniscus after the stated time. Prolonged exposure of cysts to zinc sulphate may cause them to distort, making identification difficult, therefore preparations should be examined as soon as possible.

22.7 Giardia Requests as Part of an Enteropathogenic Parasite Screen Specialised parasitology diagnostic and reference laboratories will be requested to investigate a sample for the presence of a variety of parasitic enteropathogens, including Giardia. In the absence of preserved or unpreserved, recently voided stool samples, where

H.V. Smith and T.G. Mank the vegetative and transmissive stages of enteropathogenic protozoa can be sought, most specialised laboratories will use the formol-ether (ethyl acetate) method for enteropathogenic parasite screens (Section 22.6.1.1). Microscopy on unconcentrated and concentrated samples should be performed to determine the presence of either trophozoites and cysts or small numbers of cysts, respectively. Furthermore, after reading the wet film, the sample can be analysed under the UV filter of a fluorescence microscope for the presence of Cyclospora and Isospora oocysts, as the oocysts of Cyclospora and the oocysts, sporoblasts and sporocysts of Isospora autofluoresce a sky blue colour under UV light. If brightfield microscopy is unhelpful further, specific investigations can include immunofluorescence microscopy for cysts on microscope slides using commercially available kits (Sect. 22.12.1), Giardia stool antigen detection using immunoassays such as the enzyme-linked immunosorbent assay (ELISA) or the immunochromatographic (dipstick) assay (Sects. 22.12.2.1, 12.2.2). Other laboratory diagnostic approaches are identified in Section 9.

22.7.1Triple Faeces Test (TFT) The examination of a single stool sample underestimates G. duodenalis occurrence as fluctuations in trophozoite abundance and intestinal motility result in sporadic trophozoite/cyst excretion. In Europe, fresh, unpreserved stool specimens are generally requested for examination, and patients are asked to submit more than one stool samples because parasites are shed intermittently. A limitation to this approach is that vegetative stages can be missed because of delays in stool processing and/or low compliance with the request to submit multiple stool samples. As microscopy of stools forms the cornerstone of detection in clinical parasitology laboratories, to overcome this limitation, van Gool et al. (2003) recommended the use of the triple faeces test (TFT) which combines (i) multiple sampling on 3 consecutive days, (ii) the use of a fixative (SAF), (iii) a concentration method and (iv) an easy-to-use permanent stain (Chlorazol Black dye) for routine clinical use (Fig. 22.4). van Gool et al. (2003) compared TFT results with those of formol-ether concentration of a single fresh

Chap. 22 Diagnosis of Human Giardiasis

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T.F.T.- Test

Day 1 SAF

Day 2 Unpreserved

Day 3 SAF

Ridley

(oo)cyst ova spores Screening Iodine wet preparation

Negative

Positive: direct identification

No further action

Positive: uncertain

Chlorazol black dye permanent stain

Fig. 22.4 Laboratory handling of the triple faeces test (TFT)

stool specimen (n = 544) and found that 209 and 106 cases, respectively, were diagnosed with infection by one or more parasitic species (P < 0.005). Pathogens were detected by TFT and formol-ether concentration in 94 and 39 cases, respectively, and nonpathogenic species in 288 and 124 cases, respectively (P < 0.05). Compliance with the vial filling instructions in the TFT vial sets occurred in 462 of 544 (85%) of cases and the additional costs for the sampling devices, laboratory reagents and handling of the TFT were acceptable. TFT is an effective method for detecting intestinal parasites in stool samples in routine clinical practice.

12 ml of a 1% solution of phosphotungstic acid hydrate and, finally, 20 ml of glacial acetic acid. Work in a fume cupboard and store Basic solution, which has a working lifetime of 1 year, in a dark bottle at room temperature (RT). (ii) Grind 5 g Chlorazol Black E powder in a pestle and mortar with a small amount of Basic solution for at least 3 min until a smooth paste is obtained. Add more basic solution and grind for 5 min. Allow the solution to settle for a few min. and pour the supernatant into a dark bottle. Add more basic solution to the remaining sediment and repeat grinding until all the powder has dissolved. The solution must be stored at RT and left for 4–6 weeks to ripen. During this time, a dark sediment settles out, leaving the supernatant black and cherry-coloured. Chlorazol Black solution has a working lifetime of 1 year. (iii) Basic solution and Chlorazol Black solution are combined to make Final Chlorazol Black solution (FCB). It is advisable to have two FCB solutions consisting of different ratios of Basic solution and Chlorazol Black to accommodate different staining times (during the working day [2–4 h] where 2 parts of Chlorazol Black solution are added to 1 part of Basic solution; and overnight [4–24 h] where 1 part of Chlorazol Black solution is added to 2 parts of Basic solution). If parasites do not stain well enough (too dark or too light) change either the dilution or the staining period until parasites are stained properly. Always include a positive control. (iv) The albumin working solution is prepared by making a 1:10 dilution of stock albumin (4% albumin w/v in distilled water) in distilled water. The working solution should be prepared daily and stored at RT. The stock solution of albumin should be stored at 4ºC.

22.7.1.1 Preparation of Chlorazol Black Stain The permanent stain, Chlorazol Black is used for demonstrating vegetative stages and cysts of intestinal parasitic protozoa. The stain is composed of four working solutions which must be prepared prior to use, namely (i) Basic solution, (ii) Chlorazol Black solution, (iii) Final Chlorazol Black solution (FCB), and (iv) albumin solution. (i) Basic solution is made by adding the following ingredients in the following order. To 628 ml distilled water, add 160 ml 96% ethanol, 160 ml methanol,

22.7.1.1.1Chlorazol Black Staining of SAF-Preserved Specimens 1. Place 1 drop of albumin working solution (iv) on a labelled slide, spread it with a cotton bud and leave to air dry.a 2. Mix the SAF-preserved sample thoroughly, place a cotton bud in the SAF and spread the SAF material over the slide. From each SAF sample make two slides (one thick and one thin smear).b 3. Allow the slides to dry at RT (10–15 min).

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4. Calculate staining times (iii) and place the slide in the appropriate FCB solution.c,d 5. Dehydrate stained slides by placing in 70% ethanol (1 min), then directly without drying in 96% ethanol (1 min), then immediately into 100% ethanol (5 min).d,e 6. Add mounting solution (Euperal green) to the wet, stained area and cover with a coverslip, then place slides in a 37ºC incubator for 15 h (or overnight). 7. Examine the smears using oil-immersion objectives. Use the 50× objective to screen and examine for at least for 10 min before reporting a negative result. If a trophozoite or cyst is observed, use the 100× objective to study the morphological features in detail.f a

Albumin ‘glues’ the SAF-specimen to the glass slide. b Always prepare two slides: one thin and one thick. After staining and dehydration the smear can sometimes appear thinner than expected. c FCB solution can be used for 4 weeks. d All containers used for staining should be covered to prevent evaporation of reagents. e The dehydration solutions (ethanol) can become black from the Chlorazol Black stain. If this happens refresh the ethanol solutions. f When the smear is stained correctly, the background stains light blue/grey. Protozoa can be recognized as blue/grey structures with dark (blue / black)

H.V. Smith and T.G. Mank nuclei. Cysts tend to stain darker than trophozoites. Helminth ova are difficult to recognise as they become overstained. Iron Haematoxylin Kinyoun is a useful replacement for Chlorazol Black in the TFT test if Cryptosporidium spp. Cyclospora cayetanensis and Isospora belli oocysts are sought. Oocysts stain red and are readily detectable. Additionally, automated staining machines can be used with the Iron Haematoxylin Kinyoun stain (Palmer, 1991; Mank et al., 1995).

22.8 Microscopical Examination of Samples Each sample must be examined in a systematic manner. Observation should commence using the 10× objective ensuring that the entire coverslip or sample is viewed. A suggested scheme is as follows: Commence in the upper left-hand corner of the sample working across the slide from left to right, one field width at a time, until the upper right-hand edge of the sample is reached. Move down one field height and continue working across the slide from right to left, field by field, until the right-hand edge of the sample is reached. Continue in this manner until the end of the sample (lower right-hand corner) is reached. During the observation period the fine focus should be adjusted continuously so that the depth of the sam-

Plate. 22.1 Morphology of Giardia duodenalis trophozoites and cysts in various microscopic preparations. (a) G. duodenalis trophozoites stained with methyl violet seen through a copy of van Leeuwenhoek’s simple, single lens microscope (magnification = 295×). Note the morphology of the trophozoites and compare to the criteria identified in Table 2. (b) Stool from giardiasis case. Note that it is voluminous, frothy, greasy and floats on water. (c) Dorsal and ventral aspects of a G. duodenalis trophozoite. (Nomarski DIC microscopy, magnification = 1000×). On the dorsal aspect, note the pyriform shape, the two anteriorly located nuclei and the flagella directed caudally. On the ventral aspect, note the pyriform shape, the ventral disc occupying the majority of the anterior surface and the flagella arranged in bilateral symmetry and directed caudally. (d) Immature cyst stained with the nuclear fluorogen 4′6-diamidino-2-phenyl indole (DAPI). (Photographed under combined Nomarski DIC and UV microscopy, magnification = 1250×). Note the two nuclei at one pole of the cyst, the flagellar axonemes (axostyle) lying diagonally across the long axis of the cyst and the two “crescentic bodies” (fragments of the ventral disc) lying transversely in the mid-portion of the cyst. DAPI highlights the two nuclei which fluoresce sky blue. (e) G. duodenalis cyst photographed under Nomarski DIC microscopy (magnification = ×1250). Note the nuclei at the upper pole of the cyst, the flagellar axonemes (axostyle) lying diagonally across the long axis of the cyst and the “crescentic body” (fragment of the ventral disc) lying horizontally in the lower mid-portion of the cyst. (f) G. duodenalis trophozoite in a diarrhoeic, air dried, stool stained with Giemsa (magnification 1250×). Note the pyriform shape, the two anteriorly located nuclei each with a central karyosome, the flagella arranged in bilateral symmetry and directed caudally and the two “claw-hammer” shaped median bodies lying transversely in the mid-portion of the trophozoite. (g) G. duodenalis cyst in stool fixed in SAF fixative and stained with Iron Haematoxylin Kinyoun stain (magnification = 1000×). Note the distinct cyst wall, the stained cyst and two distinct nuclei at one pole, each with a central karyosome. (h) G. duodenalis cysts in stool stained with a commercial FITC-labelled ant-G. duodenalis monoclonal antibody (magnification = 500×). Note the bottle green fluorescence around the perimeter of each cyst

Chap. 22 Diagnosis of Human Giardiasis

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ple is also viewed. G. duodenalis cysts are refractile under bright-field microscopy. When an object is located, it is inspected under the high (dry, 40×) objective and either verified or disregarded. Higher magnification (100× oil immersion lens) may be necessary to visualise definitive morphological characteristics. Wet mounts can be sealed with nail varnish or a proprietary sealant to prevent coverslip movement when using an immersion lens.

22.9 Infection in the Absence of Detectable Cysts Cysts will not be detectable in clinical samples from all human giardiasis cases, and the absence of trophozoites and cysts in repeated submissions of samples from symptomatic individuals does not necessarily indicate the absence of infection. Apart from recuperating immunocompetent cases, where trophozoite and cyst abundance can be low, low cyst abundance can occur because of fluctuations in trophozoite abundance and intestinal motility, resulting in sporadic cyst excretion. In these instances, and particularly when clinical suspicion is high, trophozoite and cyst negative stool samples should be subjected to antigen and/or PCR-based detection, as sufficient Giardia antigen or DNA from trophozoites should be present in faeces. For PCR-based methods, nested PCR methods, being more sensitive than direct PCR methods, are likely to have a higher diagnostic index, particularly when single copy gene loci are used. When trophozoites or cysts, Giardia antigen or DNA cannot be found following extensive examination of repeat stool samples, and giardiasis is suspected clinically, more invasive procedures, such as the examination of duodenal or jejunal fluid and/or tissue biopsy may be indicated. Both are invasive procedures, requiring close liaison with other health professionals and should be used with caution. Duodenal or jejunal fluid can be sampled by endoscopy or the Enterotest® (a rubber-lined weighted gelatine capsule containing a nylon string which is swallowed whilst the free end of the string is taped to the side of the mouth. The Enterotest® is left in place for 4–8 h, by which time the string extends to its full length, and its distal half becomes saturated with bile-stained mucus. Once removed, the mucus is scraped off the string,

H.V. Smith and T.G. Mank mixed with warm (37ºC) saline, and trophozoites present in the mucus are detected microscopically as they are still motile. Duodenal or jejunal aspirates can reveal the presence of motile trophozoites that can be cultured in vitro in TYI-S-33 (Keister, 1983) for research purposes, while histopathology of biopsy material can reveal the presence of trophozoites adjacent to villi. The fact that laboratory identification consists of multiple analyses using different matrices identifies the difficulties encountered with Giardia diagnosis. More sensitive detection techniques, which detect G. duodenalis sub-cellular components, should also be considered for adoption into the routine diagnostic algorithm. These techniques, and other research-orientated techniques, are included in this chapter and should prove useful for laboratory and epidemiological investigations and research into parasite biology. Once they have been evaluated fully and compared with accepted methods in individual laboratories or as interlaboratory quality assurance investigations they can be regarded as an adjunct to the conventional detection and identification techniques used for laboratory diagnosis.

22.10Immunomagnetic Separation (IMS) for Giardia Cysts IMS is based on the principle of attaching antibodies to magnetisable beads, mixing the antibody coated beads with the suspended target in question and attracting the antibody coated bead-target complex towards a permanent magnet, which concentrates the target. Giardia IMS kits use monoclonal antibodies (mAbs) reactive with surface exposed epitopes on G. duodenalis cysts which are covalently bound to iron-cored latex beads. Mixing mAb bound beads with G. duodenalis cysts generates a bead-cyst complex, which can be concentrated from the suspension by attraction in a magnetic field generated by a permanent neodymium magnet. Once attracted to the magnet, bead-cyst complexes are concentrated from the suspending fluid which is removed by aspiration. Bead-cyst complexes can be dissociated in an acidic solution, liberating cysts that are both concentrated and purified (Campbell and Smith, 1995; http:// products.invitrogen.com/ivgn/product/73002?CID =Search-Product).

Chap. 22 Diagnosis of Human Giardiasis

IMS is not normally used for routine clinical diagnosis because there are sufficient cysts present in either unconcentrated stools or stools concentrated using biophysical methods (Sect. 22.6). IMS is limited by its time and cost constraints for routine diagnostics, but IMS is an option when investigating specific cases of infection or disease, particularly when the index of clinical or epidemiological suspicion is high. IMS enhances assay sensitivity as it enables the analysis of larger sample volumes and their concentration from 10 ml down to 100 μl. As with Cryptosporidium oocysts (Smith, 2008), IMS kit performance can be influenced by G. duodenalis assemblage and isolate, antibody affinity and isotype, cyst epitope expression density and the physico-chemical environment can affect epitope-paratope interaction. IMS detection of cysts in stools (n = 127) was more sensitive (27.5% by IMS, followed by immunofluorescence staining) than the methods of both Faust et al. and Lutz (15.7%) (Souza et al., 2003). Bifulco and Schaeffer (1993) reported recoveries of 82% for G. duodenalis cysts from water using IMS, while Hu et al. (2004) reported recoveries >89.0% for G. duodenalis cysts suspended in water. Not only does IMS reduce particulates and interferents, its concentration/purification step is superior to the centrifugal sedimentation and flotation methods normally used for cyst concentration.

22.11Permanent Staining – Detection of Giardia Trophozoites and Cysts in Faecal Smears by Giemsa Staining Several combinations of the original polychrome methylene blue and eosin (Romanowsky stain) exist. The principle in each Romanowsky staining method (e.g. Leishman, Giemsa, Field, Wright, J.S.B.) is that the basic methylene blue and oxidised azures combine with the acid eosin in watery solution and stain chromatin reddish-purple and cytoplasm grey-blue (Plate 1f). Air dried, methanol fixed, thin faecal films are stained with an alcoholic solution of freshly prepared Giemsa stain, differentiated in buffered water, air dried and examined microscopically for the presence of parasites. Giemsa stain is usually bought as solution but can be prepared as follows: Giemsa powder 3.8 g;

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methyl alcohol 250 ml; glycerine 250 ml. Place the powder in a mortar and grind thoroughly. Add the glycerine to the mortar a little at a time, grinding and mixing with each addition. Add about half of the methyl alcohol; continue to grind and mix. Pour the contents of the mortar into a hard glass (e.g. Pyrex) stoppered bottle. Pour the remainder of the methyl alcohol into the mortar, mix and grind up the residue of the stain and add the whole contents of the mortar to the bottle. Incubate for 24 h at 37°C giving the bottle an occasional shake. Filter through Whatman’s No. 1 filter paper before dispensing into hard glass bottles. Giemsa stain must be diluted with buffered water (pH 6.8) before use and the working stain should be prepared freshly for each batch of samples.

22.11.1Method 1. Wear gloves. Prepare thin faecal smears and air dry (see Sect. 22.5). 2. Fix in methanol (94%) for 1 min. 3. Dilute Giemsa stain 1 in 25 (4%) with phosphate buffered (pH 6.8) distilled watera. 4. Place slide with film side upwards on a horizontal staining racka and pipette 1.5 ml of the stain carefully to cover the filmb. 5. Stain for 30 min. 6. Differentiate by flooding slide with buffered distilled waterc. 7. Place slide at an angle to drain and dry, do not blot. 8. Examine the stained film using the 10×, 40× and 100× oil immersion lensesd. a

Stock solutions are prepared as follows: Stock solution A: Na2HPO4 9.5 g per litre; stock solution B: KH2PO4.H2O 9.2 g per litre. Add 49.6 ml of stock solution A to 50.4 ml of stock solution B and make up to I litre with 900 ml of distilled water. Determine pH with a pH meter and adjust if necessary. Buffer tablets of the correct pH are available. b For large numbers of slides, a Coplin jar can be used. c Avoid prolonged washing of film after staining or water-soluble methylene blue will be removed, leaving the cytoplasm of parasites unstained. About 20 sec should be sufficient.

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d

before mAb application. If a permanent record is required, a separate sample should be taken and placed in the chemical preservative of choice. If the test result is negative, giardiasis cannot be excluded and further stool samples should be tested.

Flagella and nuclei stain reddish pink, cytoplasm stains blue. Ensure that the morphological and morphometric characteristics identified in Tables 22.2 and 22.3 and Plate 1f are identified.

22.12Immunological Methods 22.12.1Antigen Detection Using Antibodies Labelled with Fluorescent Reporters

22.12.2Antigen Detection Using Antibodies Labelled with Enzyme and Other Reporters 22.12.2.1 Enzyme Immunoassays

Immunofluorescence assays using mAbs were developed to increase the sensitivity of detection. Commercial kits utilise mAbs which recognise surface-exposed cyst epitopes and the fluorescence visualised defines the maximum dimensions of the cyst (Plate 1h). Both direct and indirect immunofluorescence procedures are available: indirect fluorescence is more time-consuming but the use of a second antibody for indirect detection can increase assay sensitivity. Detection antibodies are usually conjugated to fluorescein isothiocyanate (FITC) which fluoresces ‘bottle green’ when viewed under the blue filter block of a fluorescence microscope (excitation 490 nm; emission 510 nm), but can be conjugated to different reporters if subspecies discrimination of cysts is required. For maximum sensitivity, concentration (either using formol-ether [Sect. 22.6.1.1] or IMS [Sect. 22.10]) of the sample prior to immunofluorescence detection is recommended. Although this method is more time-consuming than bright-field microscopy, translucent cysts and cysts with poor morphology can be detected. This is especially useful for environmental samples http://www. epa.gov/microbes/1623de05.pdf. A disadvantage of immunofluorescence using air-dried smears is that cyst contents can become distorted reducing the potential for definitive identification through recognising organelles. Various commercial kits are available whose protocols can differ. Unfixed or 10% formalin fixed specimens are normally suitable; however, the suitability of formalin fixation for different cyst epitopes should be determined from kit inserts and/or evaluated on known positives. Where formalin is found to be unsuitable, the 10% formalin used in the formol-ether concentration method can be substituted with PBS or distilled water if a concentrate is to be analysed. Sample concentrates are air dried onto microscope slides and fixed (e.g. acetone or methanol)

The detection of antigen in faeces can overcome the diagnostic issues associated with erratic cyst excretion, trophozoite and cyst disintegration in vivo, the low detection rate associated with microscopy, and the inability to detect trophozoites in jejunal juice or biopsy, particularly if the assay detects antigens that are common to both trophozoites and cysts. The antigen detection assays described cannot substitute or replace routine requests for microscopy for ova, cysts/ oocysts and parasites (OCP) as only G. duodenalis antigen is detected. The ELISA detects Giardia antigen in faeces, and some diagnostic laboratories prefer ELISA to microscopy. Numerous methods have been described, and target antigens range from the multitude present in aqueous trophozoite extracts to individual Giardia specific molecules such as Giardia stool antigen, present in trophozoites and cysts and which has a relative molecular mass of 65 kDa (GSA 65; Rosoff et al., 1989). The methods described in the literature are antigen capture ELISAs which utilise antibodies to capture Giardia stool antigen on the solid phase and to detect its presence and develop the coloured reaction product. In addition, ELISAs offer much needed quality assurance to diagnosis, simplify mass screening procedures in suspected outbreak settings (e.g. waterborne, Green et al., 1990) and also are clinically helpful for treatment follow-ups.

22.12.2.2 Lateral Flow Immunochromatographic (Dipstick) Assays The speed of antigen-antibody interaction in lateral flow immunochromatography is far more rapid than in ELISA, where interaction between antigen and the immobilised capture antibody occurs through the mo-

Chap. 22 Diagnosis of Human Giardiasis

lecular diffusion of the antigen. In lateral flow immunochromatography, all fluids are drawn through a membrane enclosed in a cassette, by a wicking action rather than by diffusion, which dramatically increases the speed of antigen–antibody interaction (minutes), and provides a result within 15 min. Soluble G. duodenalis antigens in the test sample are drawn up the membrane where they come into contact with, and bind to, immobilised antibodies raised against G. duodenalis antigens. Positive reactions are qualitative and are seen as a coloured band at a specific location on the membrane, normally identified by a line marked on the cassette. Positive and negative controls are included in each assay, and the test is easy to perform as no concentration step is required prior to testing. The format of immunochromatographic assays can vary from commercial kit to commercial kit. As for antigen detection by ELISA, the laboratory diagnostician should always determine whether any contraindications apply to the use of a commercial test and any fixative (e.g. PVA) used. Immunochromatographic assays provide diagnostic laboratories with a convenient and rapid alternative method for performing antigen detection assays for Giardia on stool samples. Immunochromatographic assays can be invaluable in cases of infection in the absence of detectable trophozoites and/or cysts (Sect. 22.9) and can prove useful in laboratories without trained microscopists.

22.13 Sensitivity of Detection in Faeces Diagnosis of giardiasis is dependent upon demonstrating intact parasites (cysts and/or trophozoites) by microscopy, parasite products by immunoassay and/ or parasite DNA in faeces or duodenal/jejunal aspirates and serology. Cyst excretion can be intermittent and some infected persons can be low cyst excretors. Diagnosis based on the detection of cysts in faecal concentrates can be inefficient. The likelihood of detecting cysts, by bright-field microscopy, in a single stool sample from an infected human is between 35 and 50%, whereas between 6 and 10 stool specimens from an infected human need to be examined to achieve a detection rate of 70–90% (Sawitz and Faust, 1942), reflecting the erratic nature of cyst excretion in symptomatic cases.

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The reported sensitivity of ELISA for detecting Giardia antigens in stool samples has been evaluated on numerous occasions (e.g. Faubert, 2000). The sensitivity and specificity of ELISA are comparable (Goldin et al., 1990) to those of microscopic examination for cysts in stool or better. According to Faubert (2000), all studies using the ELISA-GSA 65 antigen report greater sensitivity with ELISA than microscopy on a single specimen, and the sensitivity varies between 95 and 100%, with 100% specificity reported when used with stools from patients infected with various intestinal parasites (Behr et al., 1997; Rosoff and Stibbs, 1986). Rosoff et al. (1989) reported that the ELISA-GSA 65 can detect Giardia infection in at least 30% more cases than microscopy. Antigen detection ELISAs have been used to diagnose infection when standard microscopic techniques have failed. Lateral flow immunochromatographic (dipstick) assay specificity is reported to be high. Some (e.g. h t t p : / / w w w. d p d . c d c . g ov / d p d x / h t m l / f r a m e s / diagnosticprocedures/body_dp_stoolpara_antigens. htm) report equal or better sensitivity than conventional microscopic methods while others (e.g. Weitzel et al., 2006) report reduced sensitivity. Amar et al. (2002) found that 0.5 and 0.05 pg of DNA per reaction mixture were sufficient to amplify the tpi gene fragment from assemblages A and B, equivalent to 50 and 5 copies of the tpi gene, respectively. No correlation was found between reproducibility in triplicate qPCR-RFLP and cyst numbers detected by microscopy, but in eight of 20 positive samples, five or fewer cysts were detected per objective field (Amar et al., 2003). Using three real-time (qPCR) assays (tpi; gdh; orfC4). Almeida et al. (2010) detected DNA from a single trophozoite (4–8 target copies).

22.14 Antibody Detection Symptomatic individuals have elevated circulating antibody responses to trophozoite antigens. IgG antibodies to surface expressed trophozoite antigens occurred in 81% of symptomatic cases and 12% of controls (Smith, 1984). IgM antibody levels fall to control levels between two and three weeks after drug treatment, indicating that IgM antibody might be a useful indicator of current infection (Smith, 1984), but IgG antibodies remain detectable for up to 18 months in most cases

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following drug treatment. Both immunofluorescence and ELISA are comparable for detecting IgA and IgG anti-Giardia antibodies in the sera of individuals with proven infection. ELISA sensitivity varies with the antibody isotype detected, IgM being most sensitive (Faubert, 2000) and intact trophozoites provide greater sensitivity than trophozoite extracts when used as an antigen in the IgG ELISA. The Western blot assay does not detect antibodies in all samples from patients with proven giardiasis, although higher circulating antibody titres occur in sera from symptomatic compared to asymptomatic cases. Western blot assay sensitivity increases when purified Giardia proteins are used as antigens (Faubert, 2000). Currently, the usefulness of serological assays for diagnosing human giardiasis is debatable because a) different geographical isolates have different antigenic identity, b) in chronic disease, parasites cause immunodepression, in the absence of the acute, immuno-responsive stage of the disease and c) antigenic variation down-regulates antibody production (Faubert, 2000). Serology may not reveal differences in serum antibody responses between symptomatic and asymptomatic patients (Faubert, 2000). As yet, a common, nonvariant, immunodominant antigen, useful for serodiagnosis, has not been identified, but Giardia-specific α-giardins could prove useful for the serodiagnosis of acute giardiasis (Palm et al., 2003).

H.V. Smith and T.G. Mank (2007) compared upper endoscopy with fluorescence antibody detection of cysts in 3 stool specimens (Sect. 22.12.1) from each of 31 patients (9, 22, median age 39 years, range 19–63 years) presenting with persistent diarrhoea after returning from the tropics or subtropics. Upper gastrointestinal endoscopy was performed before and after treatment and lower gastrointestinal endoscopy was performed subsequently. Cysts were detected in 16 of 31 patients while histology of duodenal biopsies and microscopy of duodenal fluids revealed parasites in 8 and 3 patients, respectively, indicating that upper endoscopy was less sensitive than cyst detection using fluorescence labelled antibodies for giardiasis diagnosis. Similarly, the study of Chew et al. (2008) in a UK population did not support the approach of routine duodenal biopsy for diagnosing giardiasis. Neither light microscopy nor SEM examination of tissue is now used for routine diagnosis. Biopsy is an invasive, expensive and time-consuming technique and, as not all regions of the intestine are infected, sampling errors can arise. Biopsy has been replaced with alternative techniques. Light or electron microscopy of biopsy tissue is still useful when investigating histopathological and cyto-architectural changes associated with infection.

22.16Molecular Diagnosis – Nucleic Acid Detection Methods 22.15 Biopsy Because of the insensitivity of trophozoite and cyst detection and the variability of cyst excretion, human giardiasis can be diagnosed histologically by detecting trophozoites adjacent to the microvillus region of the intestinal mucosa obtained by biopsy. Special staining procedures offer little advantage over haematoxylin and eosin. In haematoxylin and eosin-stained sections, intact and sectioned trophozoites (seen as crescents) can be seen either apposed to the microvillus region of the intestinal mucosa or in the lumen. In instances, much of the morphology and morphometry described in Sect. 22.3 can be identified. Although upper endoscopy (microscopy of duodenal fluid and biopsy histology) is recognised as a useful tool for Giardia diagnosis, particularly when cysts cannot be detected in faeces, Wahnschaffe et al.

PCR-based methods are more sensitive than conventional and immunological assays for detecting G. duodenalis in faeces, but the sensitivity of published methods can vary. Molecular techniques are often restricted to specialist laboratories but are necessary to determine G. duodenalis assemblages and sub-assemblages. Identifying G. duodenalis assemblages infecting humans (A and B) is necessary as it helps determine the epidemiology of disease and likely transmission routes. As DNA sequence based Giardia surveys have also found assemblages A and B in nonhuman hosts, the zoonotic potential of some assemblage A and B isolates must be borne in mind (Cacciò and Ryan, 2008; Ortega-Pierres et al., 2009). Molecular sub-typing methods are less developed for G. duodenalis than for other protozoan pathogens, but loci on the following genes have been targeted

Chap. 22 Diagnosis of Human Giardiasis

(small subunit ribosomal RNA (ssu-rRNA), β giardin (bg), glutamate dehydrogenase (gdh), elongation factor 1-alpha (ef-1), triose phosphate isomerase (tpi), GLORF-C4 (C4) and the inter-genomic rRNA spacer region (IGS) (Cacció and Ryan, 2008). Cacciò and Ryan (2008) highlight potential typing and sub-typing complications caused by (i) intra-isolate sequence heterogeneity (the presence of mixed templates that influence the identification of subtypes within each assemblage) and (ii) the unreliable assignment of isolates to G. duodenalis assemblages generated by different genetic markers, especially when single genetic markers are used.

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DNA recovery (Smith and Nichols, 2009) as they can result in DNA loss, but there should be an adequate number of trophozoites and/or cysts present in human stool samples to extract sufficient G. duodenalis DNA for PCR-RFLP and sequence analysis. Prior to routine adoption in clinical laboratories, both the variability between methods and the recognised difficulties in amplifying nucleic acids from faecal specimens by PCR must be overcome as faecal samples can contain many PCR inhibitors. In addition to bilirubin and bile salts, complex polysaccharides are also significant inhibitors. Boiling faecal samples in 10% polyvinylpolypyrrilidone (PVP) before extraction can also reduce inhibition (Smith and Nichols, 2009).

22.16.1Extraction of G. duodenalis DNA from Stools Stools, mucoid secretions, tissue biopsies, paraffin embedded sections, etc. can be submitted to clinical laboratories for assemblage and sub-assemblage identity, but the majority of samples will be stools. There is no standardised method for extracting DNA from small numbers of partially purified cysts and the sensitivity of most methods described has not been addressed fully. The method described for Cryptosporidium spp. oocysts (Nichols and Smith, 2004; Nichols et al., 2006a,b; Smith, 2008) should be applicable, as G. duodenalis cysts are not as robust as Cryptosporidium oocysts. G. duodenalis DNA can be extracted either following partial purification of cysts using the flotation, sedimentation or IMS (Almeida et al., 2010) techniques described above, or from cysts in faeces or on microscope slides following zirconia bead extraction (Amar et al., 2002). Although expensive, IMS can be used to concentrate cysts from aqueous extracts of stools for subsequent DNA extraction (Almeida et al., 2010), particularly when cyst abundance is low. Almeida et al. (2010) used IMS purified cysts suspended in water with no further DNA extraction protocol. If concentration by formol-ether sedimentation is the routine laboratory procedure, cyst concentrates suitable for lysis and amplification by PCR can be made by substituting distilled water for the 10% formalin normally used, as for Cryptosporidium oocysts (Nichols et al., 2006a). Commercial DNA purification columns can be used following validation of

22.16.2Primer, Gene Locus Selection, PCR and Mixed Infections Care is necessary when choosing primers and loci. Assemblage A or B is reported more frequently than mixed assemblage infections. A thorough understanding of the usefulness, strengths and weaknesses of PCR-based amplification at each locus chosen is required (Cacciò and Ryan, 2008; Cacciò, 2010; see Chapter 2). Few studies have compared the efficacy of amplification and differentiation of assemblages present. The most robust information regarding assemblage/subtype information has been derived from the study of three genetic loci (ssu-rRNA; tpi; gdh) by PCR-RFLP and/or sequencing amplicons. Loci that have been tested and validated in numerous laboratories are the best options to adopt and are advocated here. A multi-locus approach to characterising G. duodenalis A and B isolates is essential for accuracy and increased confidence in diagnosis and various ssuRNA and other loci are available for species (and subtype) determination (see Chapter 2). Multiplexing, real-time PCR and melting curve analysis offer prospects for multiple species and assemblage detection in automated procedures (Amar et al., 2003; Almeida et al., 2010). The increased sensitivity of real-time PCR guarantees increased speed of detection and qualitative diagnosis while the quantitative nature of the assay will be invaluable in estimating parasite DNA load. ‘Closed-tube’ assay formats reduce the danger of contamination from ‘carry over’. Quantita-

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tive real time PCR and high resolution melt (HRM) analysis should prove useful for the future, once issues surrounding matrix inhibition are overcome. Currently, there are no standardised quantitative real time PCR methods. Detecting mixed infections and determining their clinical importance can be problematic if only a single sample is analysed, particularly if infection and cyst excretion dynamics, clinical signs and symptoms, and health sequelae differ for different infecting assemblages and sub-assemblages. The inability to detect mixed species consistently can compromise our understanding of the epidemiology of human disease and the health sequelae of giardiasis. In endemic areas of disease, mixed infections may be more common than presumed. Mixed infections can be a consequence of sporadic infection, waterborne or foodborne outbreaks of disease (Smith et al., 1995) if more than one infectious assemblage is present in the contaminating vehicle(s), and identifying the correct molecular tools to determine them, through round robin testing, is clearly an important consideration for the future.

22.16.2.1Molecular Diagnosis in Routine Clinical Practice PCR-based diagnostics can be highly sensitive and specific, but their use in routine diagnostic laboratories remains limited due to standardising DNA extraction, the influence of PCR inhibitors and cross contamination (Verweij et al., 2003; ten Hove et al., 2007). Real-time PCR can reduce labour and reagent costs and the risk of cross-contamination, and offers the possibility of detecting multiple targets in a single multiplex reaction. Such a multiplex real-time PCR has been described for the simultaneous detection of E. histolytica, G. duodenalis and C. parvum/hominis, demonstrating high sensitivity and specificity with species specific DNA controls and a range of welldefined stool samples (Verweij et al., 2004). A recent study (ten Hove et al., 2007) demonstrated that a multiplex real-time PCR approach is a feasible diagnostic alternative in the clinical laboratory for detecting G. duodenalis, E. histolytica and C. parvum/hominis infections in patients consulting their General Practitioner because of gastrointestinal symptoms.

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22.16.3 Reporting Results of PCR-RFLP/ Sequencing Examination Negative results should be reported as ‘NO G. duodenalis DNA detected’. Positive results should be reported as ‘G. duodenalis DNA detected’ inserting the assemblage(s) (and sub-assemblages) identified. Identify mixtures. The typing tool(s) used must be identified (locus/loci and method).

22.17Shipping of Cysts and Cyst DNA for Quality Assurance and Round Robin Testing The exchange of cysts and cyst DNA between diagnostic laboratories for quality assurance and round robin testing should be encouraged. Effective, validated protocols for cyst identification, DNA extraction, primer selection and amplification and identification using PCR-based methods can only be determined by round robin testing. Human infectious G. duodenalis cysts fall into Biosafety Level II organisms and their shipping must conform to IATA Dangerous Goods regulations (http://www.iata.org/ps/ publications/9065.htm). Cysts can be shipped airdried onto microscope slides (either stained or unstained) or dried onto filter paper (e.g. Whatman FDA cards, http://www.whatman.com/products/? pageID=7.31.31) and cyst DNA can be extracted in the recipient laboratory. One benefit of using slides containing stained cysts is that they can be enumerated in both donor and recipient laboratories, thus adding an extra level of quality assurance to the system. Cyst DNA can also be applied onto filter paper, dried and then shipped. Different regulations might apply to different countries and laboratories should investigate these prior to shipment.

References Allen AVH and Ridley DS (1970) Further observations on the formol ether concentration technique for faecal parasites. J Clin Pathol 23: 545–546 Almeida A, Pozio E, and Cacciò SM (2010) Genotyping Giardia duodenalis cysts by new real-time PCR assays: detection of mixed infections in human samples. Appl Environ Microbiol, in press. doi:10.1128/AEM.02305-09

Chap. 22 Diagnosis of Human Giardiasis Amar CF, Dear PH, Pedraza-Diaz S, Looker N, Linnane E, and McLauchlin J (2002) Sensitive PCR-restriction fragment length polymorphism assay for detection and genotyping of Giardia duodenalis in human feces. J Clin Microbiol 40: 446–452 Amar CFL, Dear PH, and McLauchlin J (2003) Detection and genotyping by real-time PCR/RFLP analyses of Giardia duodenalis from human faeces. J Med Microbiol 52: 681– 683 Badparva E, Fallahi Sh, Sepahvand A, Pournia Y, Rashnoo ShM (2009) The comparison of the efficacy of various fixatives on diverse staining methods of Giardia lamblia cyst. Pak J Biol Sci 12(17): 1212–1216 Behr MA, Kokoskin E, Gyorkos TW, Cédilotte L, Faubert GM, and MacLean JD (1997) Laboratory diagnosis for Giardia lamblia infection: a comparison of microscopy, coprodiagnosis and serology. Can J Infect Dis 8: 33–38 Bifulco JM and Schaefer FW III (1993) Antibody-magnetite method for selective concentration of Giardia lamblia cysts from water samples. Appl Environ Microbiol 59: 772–776 Buret A (1994) Pathogenesis – how does Giardia cause disease? In: Giardia from molecules to disease (R.C.A. Thompson, J.A. Reynoldson, and A.J. Lymbery, eds.). CAB, Wallingford, pp 128–135 Cacciò SM and Ryan U (2008) Molecular epidemiology of giardiasis. Mol Biochem Parasitol 160: 75–80 Cacciò SM, Thompson RCA, McLauchlin J, and Smith HV (2005) Unravelling Cryptosporidium and Giardia epidemiology. Trends Parasitol 21: 430–437 Cacciò, 2010 (see Chapter 2). Campbell AT and Smith HV (1997) Immunomagnetisable separation of Cryptosporidium parvum oocysts from water samples. Water Sci Technol 35: 397–401 Casemore DP (1990) Foodborne protozoal infection. Lancet 336(8728): 1427–1432 Chew TS, Hopper AD, and Sanders DS (2008) Is there a role for routine duodenal biopsy in diagnosing giardiasis in a European population? Scand J Gastroenterol 43: 1219– 1223 Cook GC (1990) Protozoan infections of the small intestine and colon. Curr Opin Infect Dis 3: 256–262 Danciger M and Lopez M (1975) Numbers of Giardia in the feces of infected children. Am J Trop Med Hyg 24: 237– 242 de Wit MAS, Koopmans MPG, Kortbeek LM, van Leeuwen NJ, Bartelds AIM, and van Duynhoven THP (2001) Gastroenteritis in sentinel general practice, the Netherlands. Emerg Infect Dis 7: 82–91 Dobell C (1920) The discovery of the intestinal protozoa of man. Proc Roy Soc Med, Section of the History of Medicine, XIII: 1–15 Faubert G (2000) Immune response to Giardia duodenalis. Clin Microbiol Rev 13: 35–54 Feachem RG, Bradley DJ, Garelick H, and Mara DD (Eds) (1983) Sanitation and disease: health aspects of excreta and wastewater management. John Wiley & Sons, Chichester, U.K. Ford BJ (2005) The discovery of Giardia. Microscope 53: 147– 153

375 Garcia LS and Bruckner DA (1997) Diagnostic medical parasitology, 3rd edn. American Society for Microbiology Press, 1325 Massachusetts Avenue, N.W. Washington, DC 20005, USA Girdwood RWA (1989) Protozoan’ infections in the immunocompromised patient – the parasites and their diagnosis. J Med Microbiol 30: 3–16 Goldin AJ, Apt W, Aguilera X, Zulantay I, Warhurst DC, and Miles MA (1990) Efficient diagnosis of giardiasis among nursery and primary school children in Santiago, Chile by capture ELISA for the detection of faecal Giardia antigens. Am J Trop Med Hyg 42: 538–545 Green E, Warhurst D, Williams J, Dickens T, and Miles M (1990) Application of a capture enzyme immunoassay in an outbreak of waterborne giardiasis in the United Kingdom. Eur J Clin Microbiol Infect Dis 9: 424–428 Homan WL and Mank TG (2001) Human giardiasis: genotype linked differences in clinical symptomatology. Int J Parasitol 31: 822–826 Hu J, Feng Y, Ong SL, Ng WJ, Song L, Tan X, and Chu X (2004) Improvement of recoveries for the determination of protozoa Cryptosporidium and Giardia in water using method 1623. J Microbiol Methods 58: 321–325 Jokipii AMM and Jokipii L (1977) Prepatency of giardiasis. Lancet 309 (8021): 1095–1097 Karanis, P, Kourenti K, and Smith HV (2006) Waterborne transmission of protozoan parasites: a worldwide review of outbreaks and lessons learnt. J Water Health 5: 1–38 Keister DB (1983) Axenic culture of Giardia lamblia in TYI-S33 medium supplemented with bile. Trans R Soc Trop Med Hyg 77: 487–488 Lane S and Lloyd D (2002) Current trends in research into the waterborne parasite Giardia. Crit Rev Microbiol 28: 123– 147 Libman MD, Gyorkos TW, Kokoskin E, and Maclean JD (2008) Detection of pathogenic protozoa in the diagnostic laboratory: result reproducibility, specimen pooling, and competency assessment. J Clin Microbiol 46: 2200–2205 Lin SD (1985) Giardia lamblia and water supply. J Am Water Works Assoc 77: 40–47 Lopez CE, Dykes AC, Juranek DD, Sinclair SP, Conn JM, Christie RW, Lippy EC, Schultz MG, and Mires MH (1980) Waterborne giardiasis: a community wide outbreak of disease and a high rate of asymptomatic infection. Am J Epidemiol 112: 495–507 Mank TG, Zaat JO, Blotkamp J, and Polderman AM (1995) Comparison of fresh versus sodium acetate acetic acid formalin preserved stool specimens for diagnosis of intestinal protozoal infections. Eur J Clin Microbiol Infect Dis 14: 1076–1081 Mank TG, Zaat JOM, van Eijk JThM, Polderman AM, and Deelder AM (1998) Persistent diarrhoea in a general practice population in the Netherlands, prevalence of protozoal and other intestinal infections. In: Proceedings of the IXth International Congress of Parasitology, Chiba, Japan, pp 803–807 Nash TE, Herrington DA, Losonsky GA, and Levine MM (1987) Experimental human infections with Giardia lamblia. J Infec Dis 156: 974–984 Nasmuth K (1996) A homage to Giardia. Curr Biol 6: 1042

376 Nichols RAB and Smith HV (2004) Optimisation of DNA extraction and molecular detection of Cryptosporidium parvum oocysts in natural mineral water sources. J Food Protec 67: 524–532 Nichols RAB, Campbell BM, and Smith HV (2006b) Molecular fingerprinting of Cryptosporidium species oocysts isolated during water monitoring. Appl Environ Microbiol 72: 5428–5435 Nichols RAB, Moore JE, and Smith HV (2006a) A rapid method for extracting oocyst DNA from Cryptosporidium positive human faeces for outbreak investigations. J Microbiol Methods 65: 512–524 Ortega-Pierres G, Smith HV, Caccio SM, and Thompson RCA (2009) New tools provide further insights into Giardia and Cryptosporidium biology. Trends Parasitol 25: 410–416 Palm JED, Weiland ME-L, Grifths WJ, Ljungström I, and Svärd SG (2003) Identication of immunoreactive proteins during acute human giardiasis. J Infect Dis 187: 1849–1859 Palmer J (1991) Modified Iron Hematoxylin/Kinyoun stain. Clin Microbiol News 13: 39–40 Pietrzak-Johnston SM, Bishop H, Wahlquist S, Moura H, Da Silva ND, Da Silva SP, and Nguyen-Dinh P (2000) Evaluation of commercially available preservatives for laboratory detection of helminths and protozoa in human fecal specimens. J Clin Microbiol 38: 1959–1964 Read C, Walter J, Robertson ID, and Thompson RCA (2002) Correlation between genotype of Giardia duodenalis and diarrhoea. Int J Parasitol 32: 229–231 Rendtorff RC (1979) The experimental transmission of Giardia lamblia among volunteer subjects. In: Waterborne transmission of giardiasis (W. Jakubowski and J.C. Hoff, eds.). U.S. Environmental Protection Agency, Office of Research and Development, Environmental Research Centre, Cincinnati, Ohio 45268, USA, EPA-600/9-79-001, pp 64– 81 Rosoff JD and Stibbs HH (1986) Isolation and identification of a Giardia lamblia-specific stool antigen (GSA-65) useful in coprodiagnosis of giardiasis. J Clin Microbiol 23: 905–910 Rosoff JD, Sanders CA, Seema SS, DeLay PR, Hadley WK, Vincenzi FF, Yajko DM, and O’Hanley PD (1989) Stool diagnosis of giardiasis using a commercially available enzyme immunoassay to detect Giardia-specific antigen 65 (GSA 65). J Clin Microbiol 27: 1997–2002 Roxström-Lindquist K, Palm D, Reiner D, Ringqvist E, and Svärd SG (2006) Giardia immunity – an update. Trends Parasitol 22: 26–31 Sahagún J, Clavel A, Goñi P, Seral C, Llorente MT, Castillo FJ, Capilla S, Arias A, and Gómez-Lus R (2008) Correlation between the presence of symptoms and the Giardia duodenalis genotype. Eur J Clin Microbiol Infect Dis 27: 81–83 Savioli L, Smith HV, and Thompson RCA (2006) Giardia and Cryptosporidium join the ‘Neglected Diseases Initiative’. Trends Parasitol 22: 203–208 Sawitz WG and Faust EC (1942) the probability of detecting intestinal protozoa by successive stool examinations. Am J Trop Med 22: 130–136 Smith PD (1984) Human immune responses to Giardia lamblia. In: Giardia and giardiasis. Biology, pathogenesis and epidemiology (S.L. Erlandsen and E.A. Meyer, eds.). Plenum Press, New York and London, pp 201–218

H.V. Smith and T.G. Mank Smith HV (1992) Intestinal protozoa. In: Medical parasitology: a practical approach (P.M. Hawkey and S.H. Gillespie, eds.). IRL Press, Oxford University Press, Oxford, UK, pp 79–118 Smith HV (2008) Diagnostics. In: Cryptosporidium and cryptosporidiosis, 2nd edn (R. Fayer and L. Xiao, eds.). Taylor & Francis, Boca Raton, Florida, USA, pp 173–208 Smith HV and Nichols RAB (2009) Parasite concentration and nucleic acid extraction methods for clinical, food and environmental samples used for direct molecular applications. In: Lieu D (Ed) Handbook of nucleic acid purification. CRC Press, Taylor & Francis Group, Boca Raton, Florida, USA, pp 317–377 Smith HV and Paget T (2007) Giardia. In: Simjee S (Ed) Foodborne diseases. Humana Press, Totowa, NJ, USA, pp 303– 336 Smith HV, Robertson LJ, and Ongerth JE (1995) Cryptosporidiosis and giardiasis: the impact of waterborne transmission. J Water SRT – Aqua 44(6): 258–274 Souza DSM, Barreiros JT, Papp KM, Steindel M, Simões CMO, and Barardi CRM (2003) Comparison between immunomagnetic separation, coupled with immunofluorescence, and the techniques of Faust et al. and of Lutz for the diagnosis of Giardia lamblia cysts in human feces. Rev Inst Med Trop S Paulo 45(6): 339–342 ten Hove R, Schuurman T, Kooistra M, Moller L, van Lieshout L, and Verweij J (2007) Detection of diarrhoea-causing protozoa in general practice patients in the Netherlands by multiplex real-time PCR. Clin Microbiol Infect 13: 1001– 1007 Thompson RCA, Reynoldson JA, and Lymbery AJ (1993) Giardia from molecules to disease and beyond. Parasitol Today 9: 313–315 Utzinger J, Botero-Kleiven S, Castelli F, Chiodini PL, Edwards H, Köhler N, Gulletta M, Lebbad M, Manser M, Matthys B, N’goran EK, Tannich E, Vounatsou P, and Marti H (2010) Microscopic diagnosis of sodium acetate-acetic acid-formalin-fixed stool samples for helminths and intestinal protozoa: a comparison among European reference laboratories. Clin Microbiol Infect 16: 267–273 van Gool T, Weijts R, Lommerse E, and Mank TG (2003) Triple faeces test: an effective tool for detection of intestinal parasites in routine clinical practice. Eur J Clin Microbiol Infect Dis 22: 284–290 Verweij JJ, Schinkel J, Laeijendecker D, van Rooyen M, van Lieshout L, Polderman AM (2003) Real-time PCR for the detection of Giardia lamblia. Mol Cell Probes 17: 223–225 Verweij JJ, Blangé RA, Templeton K, Schinkel J, Brienen EAT, van Rooyen MAA, van Lieshout L, and Polderman AM (2004) Simultaneous detection of Entamoeba histolytica, Giardia lamblia and Cryptosporidium parvum in fecal samples using multiplex real-time PCR. J Clin Microbiol 42: 1220–1223 Wahnschaffe U, Ignatius R, Loddenkemper C, Liesenfeld O, Muehlen M, Jelinek T, Burchard GD, Weinke T, Harms G, Stein H, Zeitz M, Ullrich R, and Schneider T (2007) Diagnostic value of endoscopy for the diagnosis of giardiasis and other intestinal diseases in patients with persistent diarrhea from tropical or subtropical areas. Scand J Gastroenterol 42: 391–396

Chap. 22 Diagnosis of Human Giardiasis Weitzel T, Dittrich S, Möhl I, Adusu E, and Jelinek T (2006) Evaluation of seven commercial antigen detection tests for Giardia and Cryptosporidium in stool samples. Clin Microbiol Infec 12: 656–659 Wolfe MS (1979) Giardiasis. Pediatr Clin N Am 26(2): 295– 303

377 Wolfe MS (1984) Biology, pathogenensis and epidemiology. In: (S.L. Erlandsen and E.A. Meyer, eds.). Giardia and giardiasis. Plenum Press, New York and London, pp 147–161 World Health Organisation (1996) The world health report, 1996. World Health Organisation, Geneva

Section V Methods for Giardia Research

Methods for Giardia Culture, Cryopreservation, Encystation, and Excystation In Vitro Barbara J. Davids and Frances D. Gillin

Abstract The main objective of this chapter is to present the most widely accepted methods for routine culturing, encysting, and excysting Giardia lamblia in vitro. In addition, we include basic methods to cryopreserve and recover Giardia trophozoites. Encystation and excystation methods described were originally optimized for strain WB clone C6 (ATCC 50803; the first genome strain), and can serve as a foundation for other strains or species of Giardia. Throughout the chapter, we highlight important factors and procedural details that should assist one to improve or maintain high efficiencies of Giardia growth and differentiation. We also include “recipe” cards for all media and solutions used for protocols detailed in the chapter for ease of use in laboratory settings. Although we have attempted to summarize the literature, we only have direct experience with the strain and procedures used in our laboratory and cannot critically evaluate others’ methods.

23.1 Introduction It must be emphasized that Giardia is a very fastidious organism with great biologic variation between isolates. Success in developing culture techniques that permit Giardia growth and differentiation in vitro without contaminating bacteria or fungi (axenic) was realized in the 1970s by E. A. Meyer (1970, 1976) and by L. Diamond (1978), with key contributions by D. Keister (1983). The need for consistent and unlimited numbers of a single isolate of cells for biochemical, molecular, and cellular studies of infectious protozoa was the driving force for the development of media.

H. D. Luján et al. (eds.), Giardia © Springer-Verlag/Wien 2011

23

This would permit comparative studies with less variability than studying random uncharacterized in vivo isolates. The ability to culture isolates paved the way to reproduce the lifecycle in vitro and dissect the giardial differentiation process with the goal to identify and understand pathways, proteins, or biologic factors that may be targeted to control giardiasis or interrupt the lifecycle. Today, a growing number of Giardia isolates are available, as reviewed in this book by Molestina and Lujan. Initial axenic cultures of Giardia were grown by Meyer in a medium that was very cumbersome to make and required human serum and chick embryo extracts (Karapetyan, 1962; Meyer, 1970). Later, Diamond discovered that medium developed to culture Entamoeba and trichomonad species (“TYI-S-33”) (Diamond et al., 1978) could support axenic growth of Giardia trophozoites (Visvesvara, 1980). In 1983, Keister (1983) modified Diamond’s medium to specifically enhance giardial growth and these changes are still used (“complete modified TYI-S-33”; see Sect. 23.2.2, Fig. 23.2). Keister’s modifications of TYI-S-33 were addition of bile, higher concentrations of cysteine, removal of the vitamin-Tween 80 mixture, pH change to 7–7.2, and sterilization by filtration, rather than autoclaving. Modified TYI-S-33 is suitable for most Giardia strains/species and is the most commonly used medium for trophozoite culture. A comprehensive description of culture media is reviewed in Clark and Diamond (2002), including reference to a serum-free medium (Wieder et al., 1983). We do not cover establishment of new giardial cultures from clinical samples, which remains challenging. Once routine culture techniques were established for Giardia, the physiologic factors that induced differentiation in vitro was the next line of investiga-

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tion. Trophozoites primarily colonize in the proximal small intestine below the entry of the common bile duct (Gillin et al., 1987; Adam, 1991; Oberhuber et al., 1997) where they may attach to enterocytes and are often found between the mucus layer and epithelia. Residency in this area is loosely modeled by the neutral-basic, nutrient-rich, bile-containing, reducing (cysteine) TYI-S-33 medium (Keister, 1983). Some trophozoites detach and/or swim around in the small intestine and are unable to re-attach. As these parasites travel with host intestinal bulk flow, conditions in the intestinal lumen change significantly and signal these trophozoites to differentiate into cysts (Adam, 2001; Eichinger, 2001; Arguello-Garcia et al., 2009; Carranza and Lujan, 2010). Cyst wall synthesis and transport is specific to encystation and leads to assembly of an extracellular matrix that will protect the cells in the environment and from acid as they pass through the stomach of a host to initiate a new infection. This process is referred to as encystation and the known signal transduction pathways leading to and development of the cyst wall assembly are detailed in Chapters 12 and 14 of this book. Encystation: The primary criterion for encystation is efficient production of mature, biologically active cysts that are capable of excystation (see below). Expression of cyst wall proteins and encystation secretory vesicles (ESV; see below Sect. 23.2.4) are early events that are necessary, but not sufficient for production of mature cysts. To induce encystation of Giardia in vitro, Gillin et al. (1987) initially evaluated where in the gut the highest abundance of cysts are found to begin to predict possible biologic signals for induction. They used the G. lamblia strain WB C6 in the suckling mouse model of infection. Early in infection, cysts were found in the mid to lower portion of the jejunum while later the majority of cysts were in the large intestine and cecum. Clearly the small intestine contained factor(s), especially bile, which could induce encystation. It has been suggested that bile stimulates encystation by sequestering cholesterol (Lujan et al., 1996, 1997). In the same study (Gillin et al., 1987) it was shown that primary bile salts alone could elicit a modest encystation response greater than secondary bile salts, also suggesting that co-factors calcium and iron may stimulate encystation (primary salts bind both). Later, a higher number and quality of biologically active cysts were obtained by the addition of fatty acids,

B. J. Davids and F. D. Gillin

adjusting medium pH to slightly alkaline (7.8; Gillin et al., 1988), and adding lactic acid hemicalcium salt (“LA”; Gillin et al., 1989). Together, one of the most widely used methods for giardial encystation is based on these studies, which incorporates a growth period in TYI-S-33 without the customary low concentration of bile (“pre-encystation”) followed by encystation in a medium with porcine bile (rather than bovine bile) plus LA at pH 7.8. Details and recipes for this encystation method can be found below (Method 1; Sect. 23.2.4, I). Another common method (Method 2; Sect. 23.2.4, II) for encysting Giardia in vitro does not require a pre-encystation period, but requires excess bovine bile (Sun et al., 2003). A similar method, not evaluated by the authors of this chapter, was reported in 1991 (Kane et al., 1991) using excess bovine bile in the absence of LA. Method 2 is generally favored over Method 1 because it takes less time to produce abundant cysts capable of excysting in vitro (“biologically active cysts”). In general, the efficiency of encystation can be rather variable between laboratories and even over time in a single laboratory due to sources of variation discussed below (see Sect. 23.2.1). Also, other methods have been published for in vitro encystation (i.e. Kane et al., 1991; Reiner et al., 1995; Lujan et al., 1996; Kaul et al., 2001), but we cannot evaluate them critically here or provide adequate methodology guidance because we have not compared them side by side. Potential mechanisms of in vitro encystation are discussed elsewhere in recent publications (Lujan et al., 1997; Arguello-Garcia et al., 2009; Carranza and Lujan, 2010). Excystation: Not surprisingly, the physiologic signals that induce excystation of in vitro derived cysts are significantly different from those for encystation. Excystation of both fecal cysts and cysts prepared in vitro is highly variable and the latter depends on the quality of the encystation. Several methods were published for excystation of G. lamblia fecal cysts before the 1990s (i.e. Bingham and Meyer, 1979; Rice and Schaefer, 1981; Hautus et al., 1988), but these methods were not optimal for excystation of in vitro derived G. lamblia cysts. It was apparent from reports of fecal cyst excystation that exposure of cysts to low pH was necessary to initiate excystation. Later, Boucher and Gillin (1990) optimized the protocol of Rice and Schaefer (1981) to delineate how excystation could be improved for in vitro derived cysts. The resulting method in-

Chap. 23 Methods for Giardia Culture, Cryopreservation, Encystation and Excystation

volves 2 steps; the first is “induction” by low pH which models exposure of cysts to acid in the stomach and the second is “emergence” which is initiated by a protease-rich/slightly alkaline solution modeling exposure of cysts to pancreatic juices in the proximal small intestine. The quality of encystation and age of cysts was also monitored and had a crucial impact on excystation efficiency. Detailed protocols determined by Boucher and Gillin (1990) for completion of the G. lamblia lifecycle in vitro are explained in Sect. 23.2.6.

23.2 Materials Prepare all solutions in double-distilled water (ddH2O).

23.2.1 General Considerations Over the past few decades, several researchers (Diamond et al., 1978; Lujan et al., 1997; Clark and Diamond, 2002) have noted lot-to-lot variations in reagents used to make media for Giardia culture, as well as for completing its lifecycle in vitro. These variations can greatly affect their ability to support growth, kinetics, and efficiency of encystation and thus excystation. It is important to realize that not all water is of the same quality and that many of the ingredients used to prepare the media are “biological” or highly complex. The three major “biological” components that consistently affect growth and the lifecycle of Giardia are casein digest (trypticase), yeast extract, and serum. Trypticase and yeast extract are sometimes combined as “biosate”. Commercial preparations of bile can also have a significant impact on growth and encystation. Also, L-cysteine is highly labile and oxidizes to cystine, which does not support efficient growth or encystation. These variables, as well as strain differences (Reiner et al., 1993), may help explain the laboratory to laboratory differences in the kinetics of growth and encystation, and efficiency of in vitro encystation and excystation. To promote consistency and reproducibility we and others (i.e. Diamond et al., 1978; Clark and Diamond, 2002) recommend that each laboratory tests new lots of reagents for culture over ~10 culture cycles (some lots do not support growth and can be toxic!),

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and monitors efficiency of encystation and excystation. As Clark and Diamond (2002) recommend: “… test the ability of each new lot of reagent to support growth before starting to use it”. Because of the time and effort of testing lots of reagents and serum, some laboratories purchase large amounts of the best media components. Suppliers generally agree to hold the entire lot secure during a testing period. Store casein and yeast extract at room temperature in a dry place and serum at –80°C until use. Be sure to cryopreserve cells as they acclimate to biologic ingredient(s) in case of future contamination (Sect. 23.2.3). A few words of caution: In the following sections, all methods and procedures described are those that have been used in the authors’ laboratory. In some cases, small variations from original publications are substituted or noted to reflect what is currently used in the laboratory. For example, we no longer use antibiotics or antifungals in any growth or differentiation media or solutions as these may conceal contamination. Piperacillin (500 Pg/ml) and amikacin (125 Pg/ ml), and other antibiotic combinations (see review by Clark and Diamond (2002)), may be used if necessary without apparently affecting growth or differentiation (Gault et al., 1985; Gillin et al., 1987). Other methods for growth or differentiation that are published elsewhere are referenced and may be equivalent or better, but we have no direct experience with them. Because Giardia is a very fastidious organism, the media and procedures are complex and strains vary and clones undergo physiologic selection, we cannot guarantee success with ours or others’ methods. Also, the exact components for media, tubes, etc. may not be available worldwide. Therefore, the authors cannot guarantee results even if our procedures are followed verbatim. All volumes for media or solutions can be downor up-scaled according to the needs of the laboratory.

23.2.2 Growth Medium and Cultivation of Giardia Trophozoites In Vitro The primary medium used for axenic cultivation of Giardia is Keister’s (1983) modification of TYI-S-33 (Diamond et al., 1978). Giardia should be subcultured two times a week with varying volumes of inocula and cultured at 37°C. We use screw-cap borosilicate

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B. J. Davids and F. D. Gillin

glass tubes (9 ml) for transfers, but 15 and 50 ml polystyrene/propylene tubes and larger flasks are suitable for upscaling the experiments. Cells used for transfers should be from a healthy culture; not more than 80–90% confluence (no noticeable cell masses at the bottom of the tube) and the morphology of the cells should be teardrop in shape (not roundish). Because Giardia is sensitive to oxygen, all culture tubes and other media tubes or flasks should be nearly full to minimize gas exchange. A detailed protocol to prepare complete modified TYI-S-33 medium is in-

cluded below, as well as convenient “recipe” cards (Figs. 23.1 and 23.2). Brand names are given only for convenience. They indicate what our laboratory currently uses and are not an endorsement. Detailed protocol to make complete modified TYI-S33 medium: (i)

100X Bovine Bile Dissolve 26 g of bovine bile

(ii)

(i.e. bovine and ovine bile; Sigma-Aldrich B8381) into a

O. 2 Filter sterilize with 0.22 μM filter unit and store at 4°C. Fig. 23.1 Recipe card for 100u bovine bile

Prepare a stock solution of 100u bovine bile (Fig. 23.1). Dissolve 26 g of bovine and ovine bile (i.e. Sigma-Aldrich B8381) into a final volume of 500 ml of ddH2O. Filter sterilize with 0.22 PM filter unit (i.e. Millipore Steritop, GP Millipore Express® PLUS Membrane) and store at 4°C. Replace every ~2 months or if the bile precipitates. Purchase heat-inactivated bovine (adult or calf) serum from a supplier or heat-inactivate serum in the laboratory. If you did not purchase heatinactivated serum, you will need to preheat a waterbath to 56°C, submerge and periodically agitate serum bottle for 30 min to inactivate complement. Store defrosted and heat-inac-

C omp le te Mo d ifie d T YI- S33 M edium REAGENTS.......... FINAL VOLUME = 1 L Double distilled water............. 500 ml Casein digest............. 20 g Yeast extract............. 10 g Glucose (dextrose)............. 10 g NaCl............. 2 g KH2PO4............. 0.6 g K2HPO4.3H2O.............1.3 g L-Cysteine HCl............. 2 g L-Ascorbic acid............. 0.2 g Ferric ammonium citrate............. 0.012 g Dissolve all of the above, pH to 7.1, and then QS with ddH2O to 890 ml. Filter sterilize

Bovine serum, heat inactivate............. 100 ml 100X Bovine bile............. 10 ml

Fig. 23.2 Recipe card for complete modified TYI-S-33 medium

Chap. 23 Methods for Giardia Culture, Cryopreservation, Encystation and Excystation

tivated serum at 4°C. Note: We have encountered bottles of serum that have apparently been over-heated and appear darker or have precipitates. We discard these bottles. (iii) To make 1 liter of complete modified TYI-S-33 medium (Fig. 23.2):

and cannot comment on which is the best. Instead, we detail below a method used routinely in our laboratory that consistently works well. This protocol has been in the laboratory for many years and was likely developed from one of Dr. L. Diamond’s original protocols. (i)

1. Combine and dissolve the following reagents: 500 ml ddH2O, 20.0 g casein digest, 10.0 g yeast extract, 10.0 g glucose, 2.0 g NaCl, 0.6 g KH2PO4, 1.3 g K2HPO4 . 3H2O, 2.0 g L-cysteine HCl monohydrate (i.e. Sigma-Aldrich C7880), 0.2 g L-ascorbic acid, and 0.012 g ferric ammonium citrate. Note: if biosate (2:1 casein digest to yeast extract) is preferred, use 30.0 g per 1 liter in place of the casein digest and yeast extract. 2. pH the solution with 5 M NaOH to 7.1, and then add ddH2O quantity sufficient (QS) to 890 ml volume. Filter sterilize with 0.22 PM filter unit (i.e. Millipore Steritop, GP Millipore Express® PLUS Membrane). Do not autoclave any components of modified TYIS-33 medium. 3. After filtration, add 10 ml of sterile 100u bovine bile and 100 ml of heat-inactivated bovine serum.

Prepare Giardia Freezing Medium (FM; Fig. 23.3): 1. Dissolve 3.5 g sucrose into 8.8 ml of DMSO (cell culture grade). Add 62 ml of complete modified TYI-S-33 medium and an additional 20 ml of heat-inactivated bovine serum. Mix well on a magnetic stirrer, then filter sterilize with 0.22 PM filter (i.e. Millipore Millex syringe driven filter unit) (DO NOT autoclave). Use immediately or store at –20°C until use (see #2). 2. Aliquot 5 ml of FM into 15 ml culture tubes and store at –20°C until use. Vortex aliquots following defrosting to resuspend the FM. If you notice that the FM has a white, flaky precipitate that does not easily go back into solution discard it (and remaining aliquots from same sample preparation) and prepare fresh FM.

(ii) Complete modified TYI-S-33 can be stored for several days at 4°C. The presence of a white precipitate/crystals (likely cystine) at the bottom of the bottle is one indication that the medium is no longer useable and should be discarded. Culture medium can be aliquoted into 50 ml culture tubes, parafilmed, and stored at –20°C for future use. Check sterility of media by incubating one or more tubes containing media only at 37°C. Also, streak out samples of media periodically on LB agar plates.

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Protocol for Cryopreservation of Giardia Trophozoites: 1. Culture trophozoites until roughly 80% confluent to late logarithmic phase. Note: if cells can be seen by eye at the bottom

Giardia Freezing Medium Sucrose

3.5 g

DMSO (cell culture grade)

8.8 ml

23.2.3 Cryopreservation of Giardia Trophozoites Grown In Vitro

Mix on stirrer until dissolved, and then add:

TYI-S33 medium

62 ml

There are a number of published methods to cryopreserve Giardia trophozoites from in vitro culture (i.e. Diamond, 1964; Meyer and Chadd, 1967; Warhurst and Wright, 1979; Phillips et al., 1984), but we have not used those exact recipes or methods described

Bovine serum (additional)

20 ml

Fig. 23.3 Recipe card for complete freezing medium

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of tube as a white mass then the cells have already reached stationary phase and these cells will not be of optimal quality for cryopreservation. 2. Ice tubes of cells until they detach from the culture tube. Concentrate cells by centrifugation for 5 min at 600ug and 4°C. 3. Decant spent medium and add 2 ml of room temperature FM per cell pellet (from 15 ml starting culture). Resuspend cells briefly in FM, and then transfer immediately to a cryo tube (i.e. sterile Nunc 1 ml tube, Nalgene Nunc International). All manipulations need to be done under sterile conditions. 4. Place cryo tube into a room temperature Nalgene Cryo 1°C Freezing Container (Cat. # 5100-0001; be sure to fill tank to line with isopropanol as instructed by manufacturer) and put at –80°C for at least 24 h. Move cryo tubes containing Giardia to a liquid nitrogen tank for long-term storage. (iii)

Thawing Giardia from Liquid Nitrogen: 1. Remove a cryo tube with Giardia from the liquid nitrogen tank and immediately agitate in a 37°C water bath just until defrosted (and no longer). 2. Transfer the cells into a 9 ml borosilicate glass tube with ~8 ml of pre-warmed (37°C) complete modified TYI-S-33 medium. Allow cells to attach to surface of the tube for a minimum of 1 h at 37°C. 3. Decant used FM once attachment is observed and refresh tube with 9 ml of prewarmed (37°C) complete modified TYI-S33 medium. 4. Start to subculture cells once they are confluent – usually within 24 h.

23.2.4 Encystation of Giardia In Vitro Many protocols have been used to induce expression of cyst wall proteins and encystation secretory vesicles (ESV) (i.e. Gillin et al., 1987, 1988, 1989; Schupp et al., 1988; Kane et al., 1991; Reiner et al., 1995; Lujan et al., 1996; Kaul et al., 2001; Sun et al.,

2003). However, induction of cysts that resist water lysis and are competent to excyst is more challenging. Here, we review only two methods (Gillin et al., 1989; Sun et al., 2003) used in our laboratory to induce Giardia differentiation with high efficiency. Both methods were optimized with Giardia strain WB clone C6 (ATCC 50803), but may be suitable for other strains or isolates. Method 1 (Boucher and Gillin, 1990; Gillin et al., 1989) includes a pre-encystation period where parasites are grown in the absence of bile products, whereas Method 2 (Sun et al., 2003) does not entail pre-encystation and encystation is stimulated directly with excess bile. We define a good encystation as one that results in a significant number of viable cysts that are biologically active or capable of excysting (Gillin et al., 1989). It is important to recognize that not all cysts are equivalent. In the past, it has been demonstrated that preparations of in vitro produced viable cysts or even fecal cysts (Schupp and Erlandsen, 1987; Schupp et al., 1988; Gillin et al., 1989) surviving hypotonic lysis may vary in their ability to excyst in vitro or are not able to infect rodents even though they may appear cystic. There is also variability in in vitro encystation efficiency within a single laboratory using the same protocol. In some cases, this may result when cysteine is oxidized, water-quality issues arise, trophozoites have been in culture too long, or perhaps encystation was set up with cells in stationary phase of the cell-cycle, or other unknown factors we have not defined. (I) Encystation Method 1 – originally reported in Gillin et al. (1989) and modified by Boucher and Gillin (1990). (i)

Freshly (same-day) prepare 1 liter of pre-encystation medium (bile starvation; Fig. 23.4): 1. Combine and dissolve the following reagents: 500 ml ddH2O, 20.0 g casein digest, 10.0 g yeast extract, 10.0 g glucose, 2.0 g NaCl, 0.6 g KH2PO4, 1.3 g K2HPO4386 . 3H2O, 2.0 g L-cysteine HCl monohydrate (i.e. Sigma-Aldrich C7880), 0.2 g L-ascorbic acid, and 0.012 g ferric ammonium citrate. Note: In the event of using biosate, add 30.0 g per 1 liter in place of the casein digest and yeast extract.

Chap. 23 Methods for Giardia Culture, Cryopreservation, Encystation and Excystation

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Pre-Encystation Medium REAGENTS.......... FINAL VOLUME = 1 L Double distilled water.......... 500 ml Casein digest.......... 20 g Yeast extract.......... 10 g Glucose (dextrose).......... 10 g NaCl.......... 2 g KH2PO4.......... 0.6 g K2HPO4.3H2O..........1.3 g L-Cysteine HCl.......... 2 g L-Ascorbic acid.......... 0.2 g Ferric ammonium citrate.......... 0.012 g Dissolve all of the above, pH to 7.1, and then QS with ddH2O to 900 ml. Bovine serum, heat inactivate.......... 100 ml Fig. 23.4 Recipe card for pre-encystation medium

2. pH the solution to 7.1, and then QS with ddH2O to 900 ml volume. Filter sterilize with 0.22 PM filter unit (i.e. Millipore Steritop, GP Millipore Express® PLUS Membrane). 3. Following filtration, add 100 ml of heat-inactivated bovine serum to complete medium. (ii)

40X Porcine Bile Dissolve 1 g of porcine bile (i.e. Sigma-Aldrich B8631) into a O. 2 Filter sterilize with 0.22 μM filter unit and store at 4°C.

Pre-encystation: Fig. 23.5 Recipe card for 40u porcine bile

1. Ice logarithmic-phase trophozoites until cells detach and count. We use a hemocytometer for counting, but a coulter counter is fine. If you use a coulter counter, be sure to look at cells under a microscope to be sure they are healthy and not clumped. 2. Fill 9-ml screw-cap borosilicate glass tubes with freshly prepared room temperature pre-encystation medium and inoculate tubes at a density of 5000 trophozoites/ml. 3. Culture cells for 3 d at 37°C. Note: Cells will grow more slowly without the low concentration of bovine bile. (iii)

Following 3 d of pre-encystation, prepare fresh encystation medium #1:

A. Prepare stock solution of 40u porcine bile (Fig. 23.5). Dissolve 1.0 g porcine bile (i.e. Sigma-Aldrich B8631) into a final volume of 100 ml of ddH2O. Filter sterilize with a 0.22 PM filter unit (i.e. Millipore Steritop, GP Millipore Express® PLUS Membrane) and store at 4°C. B. Freshly (same day) prepare a 20u stock of L-lactic acid hemicalcium salt (LA; Fig. 23.6) by dissolving 0.55 g of LA into 50 ml of water. Filter sterilize with 0.22 PM filter unit (i.e. Millipore Steriflip-GP filter unit). C. Methods to make 1 liter of complete encystation medium #1 (Fig. 23.7):

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2. pH the solution with 5 M NaOH to 7.8, and then QS with ddH2O to 825 ml volume. Filter sterilize with 0.22 PM filter unit (i.e. Millipore Steritop, GP Millipore Express® PLUS Membrane). 3. Following filtration aseptically add: 25 ml 40u porcine bile, 50 ml 20u LA, and 100 ml of heat-inactivated bovine serum. Place medium in 37°C waterbath or incubator to warm before use.

20X Lactic Acid (Hemicalcium Salt) Dissolve 0.55 g of L-Lactic acid hemicalcium salt (i.e. Sigma-Aldrich L2000) into 50 ml of water. Filter sterilize with 0.22 μM filter unit. Fig. 23.6 Recipe card for 20u lactic acid hemicalcium salt

1. Combine and dissolve the following reagents: 500 ml ddH2O, 20.0 g casein digest, 10.0 g yeast extract, 10.0 g glucose, 2.0 g NaCl, 0.6 g KH2PO4, 1.3 g K2HPO4 . 3H2O, 2.0 g L-cysteine HCl monohydrate (i.e. Sigma-Aldrich C7880), 0.2 g L-ascorbic acid, and 0.012 g ferric ammonium citrate. Note: In the event of using biosate, add 30.0 g per 1 liter in place of the casein digest and yeast extract.

(iv)

Induce encystation: A. Invert tubes 3–5u and discard pre-encysting medium and non-attached trophozoites. Add 9 ml of warmed encystation medium #1 to attached cells and place tubes upright in 37°C incubator. Culture tubes for 42– 48 h. Note: The time for optimal cyst production and recovery may vary with media and strain.

Encystation Medium #1 REAGENTS.......... FINAL VOLUME = 1 L Double distilled water.......... 500 ml Casein digest.......... 20 g Yeast extract.......... 10 g Glucose (dextrose).......... 10 g NaCl.......... 2 g KH2PO4.......... 0.6 g K2HPO4.3H2O..........1.3 g L-Cysteine HCl.......... 2 g L-Ascorbic acid.......... 0.2 g Ferric ammonium citrate.......... 0.012 g Dissolve all of the above, pH to 7.8, and then QS with ddH2O to 825 ml. Filter sterilize with 0.22 μM filter unit, and then add aseptically:

Bovine serum, heat inactivate.......... 100 ml 40X Porcine bile.......... 25 ml 20X Lactic acid.......... 50 ml

Fig. 23.7 Recipe card for encystation medium #1

Chap. 23 Methods for Giardia Culture, Cryopreservation, Encystation and Excystation

A

B

C

389

D

E

Fig. 23.8 Examples of G. lamblia trophozoites, cysts, and an excyzoite. A Non-encysting trophozoite imaged live with DIC optics. B Encysting trophozoite-containing ESV imaged live with DIC optics. C Type 1 cyst. D Type 2 cyst. E Excysting excyzoite, methanol fixed. Bar is 10 PM

B. ESV counts. Encystation secretory vesicles (ESV) are prominent cytoplasmic vesicles that transport cyst wall precursors. They appear as protuberances with Nomarski differential interference contrast (DIC) optics (Reiner et al., 1989). They also appear dark under phase contrast. ESV are generated after trophozoites are stimulated by encystation conditions and the presence of these vesicles can be used to evaluate the kinetics and predict quality of encystation (Stefanic et al., 2009). We suggest that each laboratory initially check several time points to evaluate kinetics of ESV generation, say 4, 12, 21 h. If ESV are not observed or not abundant in the trophozoite population, it is likely that the encystation may have a problem. In this case, we suggest optimizing encystation (see Sect. 23.2.5). Please note that appearance of ESV is an early event in encystation and does not guarantee development of biologically active cysts. A DIC image of a vegetative trophozoite and an encysting trophozoite with ESV can be viewed in Fig. 23.8A and B. To count ESV: 1. Pre-warm (37°C) a microscope slide on a slide warmer. 2. Ice a tube of encysting trophozoites and allow cells to detach (10–15 min). 3. Invert tube 3–5u, remove 1 ml of culture and transfer to 1.5 ml Eppendorf centrifuge tube. Concen-

trate cells by centrifugation for 3–5 min, 1500u g at 4°C. Remove supernatant leaving ~100 Pl of media. 4. Resuspend trophozoites and add cells to the prewarmed slide for 2–5 min, at 37°C. 5. Mount slides with a coverslip and count ESV in at least 50 random cells using a 40u objective equipped with DIC (best) or phase contrast optics. ESV are present throughout the cytoplasm of a trophozoite, so be sure to use the fine-focus to view entire area between the dorsal and ventral plasma membranes. Record each ESV count and calculate the average number of ESV/cell, and average number of cells with ESV. (v)

Following differentiation: harvest, water-treat, and count cysts (Gillin et al., 1989). A. Cool sterile ddH2O (~1 liter) to 4°C. B. Harvesting of cysts and hypotonic lysis of remaining trophozoites, and incomplete cysts: 1. Ice encysting cultures for at least 10 min. Invert 3–5u and transfer medium containing encysting trophozoites and cysts to a 50 ml conical centrifuge tube. 2. Sediment cysts by centrifugation for 5 min, 4°C, 150u g. 3. Resuspend cyst pellet in 50 ml of refrigerated ddH2O and place on ice for 10–20 min. 4. Repeat steps 2–3, at least two more times. Resuspend final washed cyst pellet in ddH2O and note volume. If you plan

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to store the cysts at 4°C for later use, it is best to fill the centrifuge tube to the top with refrigerated ddH2O. 5. Count water-resistant cysts. Remove an aliquot of cyst suspension and dilute 1:1 in trypan blue stain (i.e. Invitrogen 15250-061) and count type 1 and 2 cysts (Gillin et al., 1989) in a hemocytometer with DIC or phase-contrast optics.

(ii)

A. Prepare a 20u stock of lactic acid hemi-calcium (LA; Fig. 23.6) by dissolving 0.55 g of LA into 50 ml of water. Filter sterilize with 0.22 PM filter unit (i.e. Millipore SteriflipGP filter unit). Use only on the same day LA is prepared. B. Methods to make 1 liter of complete encystation medium #2 (Fig. 23.9).

Definitions: Type 1 cysts: Cysts are oval and phase bright, the cell body is evenly distributed within the cyst wall (see Fig. 23.8C). The cyst wall is readily visible. Cytoskeletal structures such as the fragmented disc or flagella resemble fecal cysts and can be visualized inside the cyst. These cysts are trypan-blue negative (white) and are the best quality for excystation.

1. Combine and dissolve the following reagents: 500 ml ddH2O, 20.0 g casein digest, 10.0 g yeast extract, 10.0 g glucose, 2.0 g NaCl, 0.6 g KH2PO4, 1.3 g K2HPO4 . 3H2O, 2.0 g L-cysteine* (i.e. Sigma-Aldrich 168149), 0.2 g L-ascorbic acid, 0.012 g ferric ammonium citrate, and 12.5 g bovine and ovine bile (i.e. Sigma-Aldrich B8381). Note: In the event of using biosate, add 30.0 g per 1 liter in place of the casein digest and yeast extract. 2. pH the solution to 7.8 with 5 M NaOH, and then QS with ddH2O to 850 ml volume. Filter sterilize with 0.22 PM filter unit (i.e. Millipore Steritop, GP Millipore Express® PLUS Membrane). 3. Following filtration, aseptically add 50 ml of 20u LA and 100 ml of heat inactivated bovine serum to complete the medium. Place medium in 37°C waterbath or incubator to warm before use.

Type 2 cysts: The cell body or cytoplasm appears distorted or shrunken away from the cyst wall (see Fig. 23.8D). These cysts are trypan-blue positive (blue). >40% type 1 cysts is usually considered high enough to start an excystation. 6. Store water-resistant cysts in ddH2O at 4°C until excystation. Optimal excystation of cysts was obtained within the first 2 days after water treatment (Boucher and Gillin, 1990). (II) Encystation Method 2 – originally reported in Sun et al. (2003). (i)

Set up trophozoite cultures. 1. Freshly prepare complete modified TYIS-33 medium as described above (Sect. 23.2.2). 2. Ice logarithmic-phase trophozoites until cells detach and count. 3. Fill 15 ml centrifuge tubes with 14 ml complete modified TYI-S-33 medium and inoculate tubes at a density of 5 u 105 trophozoites per ml. 4. Culture cells upright for 18–21 h at 37°C.

Freshly (same-day) prepare 1 liter encystation medium #2.

(iii)

Induce encystation. 1. Replace TYI-S-33 medium with prewarmed (37°C) freshly prepared complete encystation medium #2. Invert tubes 3–5u and pour off growth medium and unattached trophozoites. Add 15 ml of warmed encystation medium #2 and place tubes upright in 37°C incubator. The time of greatest type 1 cyst production (generally 40–48 h, but sometimes

* We use a new, unopened bottle (2.5 g) of L-cysteine for each encystation.

Chap. 23 Methods for Giardia Culture, Cryopreservation, Encystation and Excystation

391

Encystation Medium #2 REAGENTS

FINAL VOLUME = 1 L

Double distilled water.......... 500 ml Casein digest.......... 20 g Yeast extract.......... 10 g Glucose (dextrose).......... 10 g NaCl.......... 2 g KH2PO4.......... 0.6 g K2HPO4.3H2O.......... 1.3 g L-Cysteine*.......... 2 g L-Ascorbic acid.......... 0.2 g Ferric ammonium citrate.......... 0.012 g Bovine and ovine bile.......... 12.5 g *Use a new 2.5 g bottle of L-Cysteine (not HCl monohydrate) for each encystation. Dissolve all of the above, pH to 7.8, and then QS with ddH2O to 850 ml. Filter sterilize with 0.22 μM filter unit, and then add aseptically:

Bovine serum, heat inactivate.......... 100 ml 20X Lactic acid.......... 50 ml

Fig. 23.9 Recipe card for encystation medium #2

earlier) should be determined in preliminary experiments as cysts do not survive long at 37°C (Bingham et al., 1979). 2. Count ESV as recommended above (Sect. 23.2.4. iv, part B of encystation method 1). 3. Following differentiation: harvest, watertreat, and count cysts as described above (Sect. 23.2.4, v, part B of encystation method 1). Store water-resistant cysts in ddH2O at 4°C until excystation, if desired.

23.2.5 Ideas to Optimize Encystation Efficiency, if Needed (1) Inoculate initial cultures at a different concentration of parasites per ml, higher or lower numbers may improve encystation (parasite concentration effect). (2) Try a different type or amount of bile. (3) Substitute L-cysteine HCl for L-cysteine (or vice versa), or

just purchase a fresh bottle of both in case the current stocks in your laboratory have oxidized. (4) Try a different type of culture tube to set up your encystation. (5) Switch to a different lot of biologic reagents (i.e. casein/yeast extract/bovine serum/bile). (6) Defrost a “younger” (fewer culture cycles) tube of Giardia from the liquid nitrogen tank. Note: some subclones of WB (and other isolates) do not encyst well for reasons that are not understood (Reiner et al., 1993).

23.2.6 Excystation of Giardia In Vitro Excystation is the process whereby environmental cues (i.e. H+) pass through the cyst wall to signal the encased parasite to awaken, and with the participation of small intestinal proteases and secretions, eventually results in the release of the excyzoite (newly excysting cell) that can initiate an infection (i.e. Bingham and Meyer, 1979; Bingham et al., 1979). This process must be timed just right as excystation in the stomach would be fatal to the excyzoite. Excyzoites have a

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Excystation Stag e 1 Solution REAGENTS.......... FINAL VOLUME = 25 ml 1X Hanks’ balance salt solution........... 7 ml L-Cysteine*.......... 68 mg L-Glutathione reduced.......... 68 mg Sodium bicarbonate.......... 52 mg Adjust pH to 2.0 with 1N HCl, then QS to 25 ml with ddH2O. *Use a new 2.5 g bottle of L-Cysteine (not HCl monohydrate) for each excystation. This bottle can be from one opened in the same week of use.

Fig. 23.10 Recipe card for excystation Stage 1 solution

variable oval to oblong morphology (see Fig. 23.8E) with an adhesive disc that reassembles within 30 min of excystation (Palm et al., 2005). They have 4 nuclei that contribute nuclear material to four binucleated trophozoites after 2 rounds of division (Bernander et al., 2001). Induction of high efficiency in vitro excystation of in vitro generated cysts depends on the quality of the cysts, as well as the excystation method. We continue to use one method (Boucher and Gillin, 1990). Other published protocols used for excysting fecal cysts can be tried (i.e., Rice and Schaefer, 1981; Schaefer et al., 1984; Feely et al., 1991), but were inefficient in our laboratory. All excystation solutions are only filter sterilized if excyzoites are intended for axenic culture purposes. (i)

(ii)

Freshly prepare complete modified TYI-S-33 as detailed above (Sect. 23.2.2; Fig. 23.2) for the final stage of excystation. Place at 37°C until needed. Prepare excystation Stage 1 solution (25 ml final volume; Fig. 23.10): shortly before beginning excystation, dissolve 68.0 mg L-cysteine (i.e. Sigma-Aldrich 168149), 68.0 mg reduced L-glutathione (i.e. Sigma-Aldrich G6529), and 52.0 mg sodium bicarbonate (i.e. Sigma-Aldrich S6297) in 7 ml 1u Hanks’ balanced salt solution (with sodium bicarbonate, without phenol red, calcium chloride, magnesium chloride, and magnesium sulfate; i.e. Gibco/Invitrogen 14175), then add 15 ml of ddH2O. pH the solu-

(iii)

tion to pH 2 with 1N HCl and QS to 25 ml with ddH2O. Place complete Stage 1 solution in 37°C waterbath to warm. Stage 1 excystation. 1. Place 2 u 106 type 1 cysts in a 1.5 ml Eppendorf tube. Concentrate cysts by centrifugation for 5 min, 4°C, 8000u g. 2. Discard the supernatant and resuspend cysts in 1.5 ml of pre-warmed Stage 1 solution. Vortex briefly to resuspend cysts. 3. Incubate cysts in Stage 1 solution at 37°C for 30 min. 4. Prepare Stage 2 solution approximately 5 min before it is needed (roughly 25 min following induction of Stage 1 excystation).

(iv)

Preparing Stage 2 excystation solution (Fig. 23.11). Dissolve 100.0 mg trypsin Type II-S (from porcine pancreas; Sigma-Aldrich T7409)

Excystation Stag e 2 Solution – 10 ml Trypsin Type II-S.......................100 mg 1X Tyrode’s salt solution, pH 8...10 ml Mix until dissolved.

Fig. 23.11 Recipe card for excystation Stage 2 solution

Chap. 23 Methods for Giardia Culture, Cryopreservation, Encystation and Excystation

(v)

into each 10 ml of Tyrode’s salt solution (with sodium bicarbonate, pH 8; SigmaAldrich T2397). Freshly prepare the volume necessary for each experiment. Each sample of cysts requires 1.5 ml volume of Stage 2 solution. Pre-warm Stage 2 solution in a 37°C waterbath. (Note: To simplify, one can pre-warm the Tyrode’s salt solution (without trypsin) 37°C at the start of excystation and pre-weigh the trypsin in a separate tube, and combine just before use.) Stage 2 excystation and emergence. 1. Briefly vortex cysts directly after incubation in Stage 1 solution. 2. Sediment cysts for 5 min, room temperature, at 8000u g on tabletop centrifuge. 3. Discard supernatant and resuspend cysts in 1.5 ml of pre-warmed Stage 2 excystation solution in the Eppendorf tube. Vortex briefly to resuspend parasites. 4. Incubate tubes at 37°C for 1 h. Vortex gently every 15 min. 5. Briefly vortex samples before sedimenting cysts in tabletop centrifuge at 8000u g at room temperature for 5 min. 6. Discard supernatant and resuspend parasites in 1.5 ml of pre-warmed complete modified TYI-S-33 (made on the day of excystation). 7. Incubate tubes at 37°C for 1 h with periodic agitation. 8. Concentrate excyzoites in tabletop centrifuge at 8000u g at room temperature for 5 min. 9. Aspirate all but ~100 Pl of medium and resuspend before counting excyzoites. 10. Enumerate excyzoites on a hemocytometer. Excyzoites will have motility and this will assist one to differentiate the excyzoites from the non-excysted cysts. See Fig. 23.8E for an image of an excyzoite.

Acknowledgements We thank Laura Johnston (www.helixconcepts.com) for designing and illustrating the recipe cards found throughout the chapter. We also thank David N.

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Lambillotte (IVD Research Inc., Carlsbad, CA) for critically reading the chapter.

References Adam RD (1991) The biology of Giardia spp. Microbiol Rev 55: 706–732 Adam RD (2001) Biology of Giardia lamblia. Clin Microbiol Rev 14: 447–475 Arguello-Garcia R, Bazan-Tejeda ML, and Ortega-Pierres G (2009) Encystation commitment in Giardia duodenalis: a long and winding road. Parasite 16: 247–258 Bernander R, Palm JE, and Svard SG (2001) Genome ploidy in different stages of the Giardia lamblia life cycle. Cell Microbiol 3: 55–62 Bingham AK and Meyer EA (1979) Giardia excystation can be induced in vitro in acidic solutions. Nature 277: 301–302 Bingham AK, Jarroll EL Jr, Meyer EA, and Radulescu S (1979) Giardia sp.: physical factors of excystation in vitro, and excystation vs eosin exclusion as determinants of viability. Exp Parasitol 47: 284–291 Boucher SE and Gillin FD (1990) Excystation of in vitro-derived Giardia lamblia cysts. Infect Immun 58: 3516–3522 Carranza PG and Lujan HD (2010) New insights regarding the biology of Giardia lamblia. Microbes Infect 12: 71–80 Clark CG and Diamond LS (2002) Methods for cultivation of luminal parasitic protists of clinical importance. Clin Microbiol Rev 15: 329–341 Diamond LS (1964) Freeze-preservation of protozoa. Cryobiology 1: 95–102 Diamond LS, Harlow DR, and Cunnick CC (1978) A new medium for the axenic cultivation of Entamoeba histolytica and other Entamoeba. Trans R Soc Trop Med Hyg 72: 431–432 Eichinger D (2001) Encystation in parasitic protozoa. Curr Opin Microbiol 4: 421–426 Feely DE, Gardner MD, and Hardin EL (1991) Excystation of Giardia muris induced by a phosphate-bicarbonate medium: localization of acid phosphatase. J Parasitol 77: 441–448 Gault MJ, Reiner DS, and Gillin FD (1985) Tolerance of axenically cultured Entamoeba histolytica and Giardia lamblia to a variety of antimicrobial agents. Trans R Soc Trop Med Hyg 79: 60–62 Gillin FD, Reiner DS, Gault MJ, et al. (1987) Encystation and expression of cyst antigens by Giardia lamblia in vitro. Science 235: 1040–1043 Gillin FD, Reiner DS, and Boucher SE (1988) Small-intestinal factors promote encystation of Giardia lamblia in vitro. Infect Immun 56: 705–707 Gillin FD, Boucher SE, Rossi SS, and Reiner DS (1989) Giardia lamblia: the roles of bile, lactic acid, and pH in the completion of the life cycle in vitro. Exp Parasitol 69: 164–174 Hautus MA, Kortbeek LM, Vetter JC, and Laarman JJ (1988) In vitro excystation and subsequent axenic growth of Giardia lamblia. Trans R Soc Trop Med Hyg 82: 858–861 Kane AV, Ward HD, Keusch GT, and Pereira ME (1991) In vitro encystation of Giardia lamblia: large-scale production of in vitro cysts and strain and clone differences in encystation efficiency. J Parasitol 77: 974–981

394 Karapetyan A (1962) In vitro cultivation of Giardia duodenalis. J Parasitol 48: 337–340 Kaul D, Rani R, and Sehgal R (2001) Receptor-Ck regulates giardia encystation process. Mol Cell Biochem 225: 167–169 Keister DB (1983) Axenic culture of Giardia lamblia in TYI-S33 medium supplemented with bile. Trans R Soc Trop Med Hyg 77: 487–488 Lujan HD, Mowatt MR, Byrd LG, and Nash TE (1996) Cholesterol starvation induces differentiation of the intestinal parasite Giardia lamblia. Proc Natl Acad Sci USA 93: 7628–7633 Lujan HD, Mowatt MR, and Nash TE (1997) Mechanisms of Giardia lamblia differentiation into cysts. Microbiol Mol Biol Rev 61: 294–304 Meyer EA (1970) Isolation and axenic cultivation of Giardia trophozoites from the rabbit, chinchilla, and cat. Exp Parasitol 27: 179–183 Meyer EA (1976) Giardia lamblia: isolation and axenic cultivation. Exp Parasitol 39: 101–105 Meyer EA and Chadd JA (1967) Preservation of Giardia trophozoites by freezing. J Parasitol 53: 1108–1109 Oberhuber G, Kastner N, and Stolte M (1997) Giardiasis: a histologic analysis of 567 cases. Scand J Gastroenterol 32: 48–51 Palm D, Weiland M, McArthur AG, et al. (2005) Developmental changes in the adhesive disk during Giardia differentiation. Mol Biochem Parasitol 141: 199–207 Phillips RE, Boreham PF, and Shepherd RW (1984) Cryopreservation of viable Giardia intestinalis trophozoites. Trans R Soc Trop Med Hyg 78: 604–606 Reiner DS, Douglas H, and Gillin FD (1989) Identification and localization of cyst-specific antigens of Giardia lamblia. Infect Immun 57: 963–968 Reiner DS, Hetsko ML, Das S, et al. (1993) Giardia lamblia: absence of cyst antigens and reduced secretory vesicle for-

B. J. Davids and F. D. Gillin mation and bile salt uptake in an encystation-deficient subline. Exp Parasitol 77: 461–472 Reiner DS, Hetsko ML, and Gillin FD (1995) A lipoproteincholesterol-albumin serum substitute stimulates Giardia lamblia encystation vesicle formation. J Eukaryot Microbiol 42: 622–627 Rice EW and Schaefer FW 3rd (1981) Improved in vitro excystation procedure for Giardia lamblia cysts. J Clin Microbiol 14: 709–710 Schaefer FW 3rd, Rice EW, and Hoff JC (1984) Factors promoting in vitro excystation of Giardia muris cysts. Trans R Soc Trop Med Hyg 78: 795–800 Schupp DG and Erlandsen SL (1987) Determination of Giardia muris cyst viability by differential interference contrast, phase, or brightfield microscopy. J Parasitol 73: 723–729 Schupp DG, Januschka MM, Sherlock LA, et al. (1988) Production of viable Giardia cysts in vitro: determination by fluorogenic dye staining, excystation, and animal infectivity in the mouse and Mongolian gerbil. Gastroenterology 95: 1–10 Stefanic S, Morf L, Kulangara C, et al. (2009) Neogenesis and maturation of transient Golgi-like cisternae in a simple eukaryote. J Cell Sci 122: 2846–2856 Sun CH, McCaffery JM, Reiner DS, and Gillin FD (2003) Mining the Giardia lamblia genome for new cyst wall proteins. J Biol Chem 278: 21701–21708 Visvesvara GS (1980) Axenic growth of Giardia lamblia in Diamond’s TPS-1 medium. Trans R Soc Trop Med Hyg 74: 213–215 Warhurst DC and Wright SG (1979) Cryopreservation of Giardia intestinalis. Trans R Soc Trop Med Hyg 73: 601 Wieder SC, Keister DB, and Reiner DS (1983) Mass cultivation of Giardia lamblia in a serum-free medium. J Parasitol 69: 1181–1182

Synchronization of Giardia Karin Troell and Staffan Svärd

Abstract Protozoans are distantly related to humans and studies of basic cellular processes in these organisms can reveal how important processes such as the eukaryotic cell cycle evolved. Giardia is not only an important pathogenic protozoan parasite but also a unique biologic model system. It has recently been possible to synchronize Giardia’s cell cycle using compounds that inhibit different steps of the cell cycle such as DNA replication and cytokinesis. Here we discuss these methods and describe detailed protocols for synchronization and the analysis of synchronized cells.

24.1 Introduction Pathogenesis of infectious diseases is often the result of uncontrolled expansion of the infectious microbes, resulting in tissue destruction and inflammation. Protozoan parasites are not an exception to this but surprisingly little is known about the regulation of cell growth and the cell cycle in these organisms (Striepen et al., 2007). Our understanding of cell cycle regulation in these important organisms, including Giardia intestinalis, has been lagging behind, mainly due to the lack of good synchronization protocols (Svärd et al., 2003). Genomic analyses performed during the past four years have shown that G. intestinalis has a typical eukaryotic set-up for cell cycle regulation (Morrison et al., 2007; Franzen et al., 2009). However, the number of regulatory components (e.g. cyclins and cyclin-dependent kinases), is reduced and this makes Giardia an interesting model system to understand the evolution of cell cycle regulation in higher eukaryotes. Here we will summarize what is currently known about synchronization of the medically important human protozoan parasite G. intestinalis.

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Giardia is unusual in containing two, apparently identical, diploid nuclei (Adam, 1991; Bernander, 2001). During vegetative growth, each nucleus cycle between a diploid (2N) and tetraploid (4N) state, resulting in a cellular ploidy of 4N and 8N. During stationary phase, trophozoites are arrested in the G2 phase with a ploidy of 8N (Bernander, 2001). Moreover, it is possible to leave the vegetative cell cycle and enter into a differentiation pathway (encystation), triggered by intestinal stimuli (bile stress or cholesterol starvation). During encystation, a trophozoite undergoes two rounds of DNA replication with a single karyokinesis, to form a quadrinucleate 16N cyst (Bernander, 2001). Although presumed to be asexual, Giardia has low levels of allelic heterozygosity, indicating that the two nuclear genomes may exchange genetic material (Morrison et al., 2007). Recent data demonstrate fusion between nuclei during encystations and this might explain the low levels of allelic heterozygosity (Poxleitner et al., 2008a). When a cyst awakens from dormancy, in response to signals from the host, it undergoes two rounds of cytokinesis, with a single round of nuclear division, to form four binucleate trophozoites, which only then re-enter the cell cycle (Bernander, 2001). Thus, the giardial cell cycle displays unusual features and DNA replication is tightly coupled to vegetative growth and differentiation of the parasite. We have been able to synchronize Giardia cultures by arresting the cells at the G1/S transition point with aphidicolin (Reiner et al., 2008). Aphidicolin is a reversible inhibitor of eukaryotic nuclear DNA replication. It blocks the cell cycle at late G1/early S-phase, and is a specific inhibitor of eukaryotic DNA polymerases A and D (Reiner et al., 2008). Successful synchronization is dependent upon release after arrest in G1/S and we did that by replacing the aphidicolincontaining medium with fresh growth medium. We

arrested trophozoites for 6 h with 5 mg/ml aphidicolin in a stop-release synchronization experiment (Reiner et al., 2008). After release, many cells rapidly (x 0.5 h) entered S phase and the DNA content increased from 4N (G1) to 8N (G2) between 1 and 2 h (Fig. 24.1). By 3 h post-release a 4N subpopulation appeared, indicating that cell division had occurred (Fig. 24.1). The relative size of the 4N peak increased until the 5.5 h time-point and decreased again as the synchronized population re-entered S phase (Fig. 24.1). Flow cytometry analysis was used to estimate the length of time in each different cell cycle stage. Most untreated trophozoites (70–80%) from an exponentially growing population were in G2/M/cytokinesis. Analysis of synchronized cells with a 6 h generation time gave the following estimate of the time the cells were in each cell cycle stage: G1 (0.7 h), S (1.3 h), and G2/M/ cytokinesis (4 h). We could also perform transcriptional analyses of cell-cycle-regulated genes (cyclin B and histones) using the synchronized cells and this showed that these genes are cell-cycle-regulated on the transcriptional level in Giardia (Reiner et al., 2008). The cell cycle can also be synchronized using aphidicolin in combination with nocodazole treatment (Poxleitner et al., 2008b). Nocodazole is an anti-neoplastic agent which has its effect on cells by depolymerizing microtubules (Samson et al., 1979). Cells treated with nocodazole arrest with a G2/Mphase DNA content when analyzed by flow cytometry (Elmendorf et al., 2003; Cooper et al., 2006). It has been suggested that nocodazole has little or no effect against Giardia microtubules since the use of these drugs did not affect the attachment of parasites (Elmendorf et al., 2003). However, in contrast to the results in the attachment study, nocodazole was seen to have a dramatic effect on cells in two other studies (Mariante et al., 2005; Dawson et al., 2007). These two studies show that cells treated with nocodazole became misshapen with abnormal numbers of flagella, irregular dorsal surface, membrane blebs, and disruption of the lateral flange. The drug also affects the microtubule dynamics in dividing cells, resulting in broken spindles and various chromosome segregation defects (Sagolla et al., 2006). In agreement with those latter studies, our unpublished results show that nocodazole seriously affects the Giardia cells. Many of the cells get an altered shape and look irregular, often

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Number of cells (normalized to highest peak)

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DNA content

Fig. 24.1 Flow cytometry analysis of synchronized Giardia trophozoites. The cells were arrested in G1/S with aphidicolin and released from the drug after 6 h (i.e. one generation cycle. The WB strain C6 has a generation time of 6 h). T is time after release from block

Chap. 24 Synchronization of Giardia

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B

A

Number of cells (normalized to highest peak)

Untreated trophozoites

1 h nocodazole

1.5 h nocodazole

8 h nocodazole

24 h nocodazole 4N 8N

16N

32N

DNA content

Fig. 24.2 Treatment of Giardia trophozoites with 100 nM nocodazole. A Fluorescent microscopy analysis of cells stained with ethidium bromide. Note the changes in morphology. B Flow cytometry analysis of nocodazole treated cells showing 32N cells

with a bump in the anterior part of the cell (Fig. 24.2A). Some of the trophozoites have more than 2 nuclei; up to 4 nuclei per cell were seen. Nocodazole blocks cell division but the cells continue their DNA replication, which results in cells with a ploidy of up to 32N (Fig. 24.2B). Thus, nocodazole treatment of Giardia generates severe side-effects. There are also sideeffects from the aphidicolin treatment since the cells continue to grow even if DNA replication is blocked (Hofstetrova et al., 2010). Thus, DNA replication is not coupled to cell growth in Giardia but cell division seems to be blocked when the DNA is not replicated, suggesting a check-point in G2/M that controls DNA replication. However, these side-effects can be reduced by keeping the time (one cell generation or shorter) and concentration (5 ug/ml) of aphidicolin treatment at a minimum (Hofstetrova et al., 2010). The optimal would be to have a drug-free synchronization protocol but aphidicolin-synchronized cells can be used in gene-expression assays, localization assays, differentiation assays, and different cell-biologic assays. A detailed protocol follows below.

24.2 Materials 24.2.1 Cell Culture 1. Diamond’s TYI-S-33 (Tryptone-Yeast extract-IronSerum): 30 g/l BBL Biosate peptone (tryptone and yeast extract mixture) (BD Biosciences 211862), 55.6 mM glucose, 34.2 mM NaCl, 1.14 mM Lascorbic acid, 5.74 mM K2HPO4·3H2O, 4.41 mM KH2PO4, 11.4 mM L-cysteine and 0.038 mM ferric ammonium citrate. The pH is set to 7.0. Sterile filter through a 0.45 Pm filter. Add sterile filtered bovine bile (Sigma B3883) to a final concentration of 0.5 mg/ml and 10% v/v heat-inactivated bovine serum (Gibco 16170-078). 2. 10 ml Nunclon delta flat side tubes (NUNC CS450).

24.2.2 Whole Culture Synchronization 1. Diamond’s TYI-S-33 medium. 2. 50 ml Falcon tubes (NOTE1).

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3. Aphidicolin (Fluka-Sigma Aldrich A10797) is dissolved in dimethyl sulfoxide (DMSO) to a final concentration of 10 mg/ml and is stored in –20°C. 4. Cell scrapers (NUNC 179693). 5. Phosphate buffered saline (PBS): 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.76 mM KH2PO4 (pH is set to 7.4 with HCl).

K. Troell and S. Svärd

2. The cells are sub-cultured twice per week. Before re-inoculation, the cells are kept on ice for 10 min to let the cells detach from the tube surface. The sample is mixed by swirling and 5–10 Pl of confluent trophozoite culture is transferred to a new tube with pre-warmed TYI-S-33 medium.

24.3.2 Measure of Generation Time 24.2.3 Flow Cytometry 24.2.3.1 Fixing Cells 1. Cell fixative: 1% Triton X-100, 40 mM citric acid, 20 mM dibasic sodium phosphate, 0.2 M sucrose, pH is set to 3.0 with HCl. 2. Diluent buffer: 125 mM MgCl2 in PBS. 3. Phosphate buffered saline (PBS): as above.

24.2.3.2 Wash and DNA Labeling 1. Phosphate buffered saline (PBS): as above. 2. Dissolve RNase A in ddH2O to a concentration of 10 mg/ml. 3. Flow buffer: 10 mM Tris (pH 7.5), 10 mM MgCl2. Filter through 0.2 Pm pore size filter. This will remove particles and limit light scatter background. Store at 4°C. 4. Dissolve Ethidium bromide (Sigma-Aldrich E7637) in ddH2O to a concentration of 4 mg/ml. 5. Dissolve Mithramycin A (Sigma-Aldrich M6891) in ddH2O to a concentration of 2.5 mg/ml. 6. DNA staining solution: This solution should be prepared fresh each time. For each sample, use 0.75 Pl Ethidium bromide (4 mg/ml), 6 Pl Mithramycin A (2.5 mg/ml) and 68.25 Pl flow buffer.

This protocol is based on generation time rather than hours. The reason for this is that the generation time for all Giardia isolates differs. For best synchronization result the culture should be arrested as short time as possible but it is still needed that one, or close to one, generation time is allowed to pass with the drug to collect all cells at the same cell cycle stage. Below is described a simple way to determine the generation time of your cells. The knowledge of generation time can also be used together with the data collected with flow cytometry to estimate the length of the different stages during the cell cycle. 1. Fresh cultures with 5 u 104 cells per ml is set up into three separate 10 ml tubes in 37°C TYI-S-33 medium. 2. The generation time of exponentially growing trophozoites is determined by continuous counting of a culture. Every 90 min, for up to 12 h, the cells are detached by chilling the tube on ice for 10 min and then swirled to obtain a homogenized culture. 3. The total cell count is determined in a Bürker chamber. 10 Pl from each tube is administered to the chamber and the averages of counts from the three tubes per time point are used to determine the generation time.

24.3.3 Whole Culture Synchronization

24.3 Methods 24.3.1 Cell Culture 1. Trophozoites from Giardia is axenically maintained in 10 ml flat side tubes in TYI-S-33 medium at 37°C.

This method can be used for collecting either all cells at the G1/S transition point or cells from different cell cycle stages. If the aim is to collect samples from different cell cycle stages, the culture should be released from the drug. The synchronization generally stays excellent for one full cycle and can be kept for up to 2 full cycles.

Chap. 24 Synchronization of Giardia

1. TYI-S-33 enough for all cultures for both aphidicolin treatment and re-feeding to release the cells from the drug is prepared and filtered. The medium is refrigerated. However, appropriate amount should be pre-warmed to 37°C at all steps to avoid cold stress on the cells. 2. Grow cells to ~80% confluence. 3. Chill cells on ice for 10 min and dilute cells 1:10 with fresh medium (NOTE2). The culture should be swirled regularly and kept on ice while being prepared for synchronization. This is important to make sure all cultures are equal from the start. 4. For synchronization with release from the drug, the number of samples plus two DMSO controls is set up. Use one tube or flask per time point when to collect the sample as Giardia trophozoites attach to the surface and need to be kept on ice or scraped to detach. If samples were to be collected from the same culture. they would be heavily influenced by this treatment and the synchronization would be lost. 5. Let the cultures grow for three generation cycles or to approximately 70% confluence before adding the drug. 6. Add aphidicolin, 0.5 Pl/ml culture to each tube (except the controls) and 0.5 Pl/ml culture DMSO to the controls. 7. Let the cultures grow for one, or slightly shorter than one generation cycle before release to allow all cells to reach the synchronization point in the cell cycle. 8. As most cells are attached to the surface, the best way to release the cells from aphidicolin arrest is to pour off the drug-containing medium and add fresh pre-warmed medium. The release is instant, to obtain a sample with all cells arrested in G1 the sample should not be released. Instead, collect cells with aphidicolin containing medium or collect cells one generation time after release. 9. Use a small cell scraper to harvest the cells (NOTE3). If the cells are to be analyzed with flow cytometry as well, make sure to save approximately 7 ml of culture for that analysis (see protocol below). Spin cells at 900 g for 5 min at 4°C and snap-freeze in liquid nitrogen and then move to freezer to preserve the sample for further analysis of RNA, DNA or proteins.

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24.3.4 Flow Cytometry In flow cytometry, cells are individually analyzed. In this case, when cells are stained with DNA-specific dye the strength of fluorescence signal is proportional to the intracellular DNA content in each cell. The method is one of the ways to visualize the success of the cell cycle synchronization as the flowgrams illustrate the number of cells with the different numbers of genome copies (in the case of Giardia 4N for GI cells and 8N for G2 cells). See also Fig. 24.1.

24.3.4.1 Fixing Cells 1. Spin the scraped cell culture at 900 g for 5 min at 4°C. 2. Remove as much of the medium as possible. Some liquid (~50 Pl) needs to be kept to avoid destruction of the often loose pellet. 3. Immediately add 150 Pl of cell fixative. Mix carefully by pipetting the sample twice. Incubate for 5 min in room temperature. 4. Add 350 Pl Diluent Buffer to the sample after the incubation. Mix by pipetting carefully. Do not vortex the sample (NOTE4). 5. The cells could either be kept in 4°C until the day after or continue the protocol immediately.

24.3.5 Wash and DNA Labeling 1. Transfer the sample to a 1.5 ml tube. Spin for 3 min at 900 g. 2. Remove the supernatant carefully to avoid losing cells and add 100 Pl PBS. Mix sample very gently until pellet is dissolved. Spin again for 3 min at 900 g. 3. Remove the supernatant carefully and add 500 Pl PBS and 2.5 Pl RNase A (10 mg/ml). Mix the sample very gently until pellet is dissolved. 4. Incubate samples for 30 min at 37°C. 5. Spin sample for 3 min at 900 g. Remove supernatant and add 75 Pl of DNA staining solution. Mix carefully not to destroy the cells. When the cell pellet is dissolved, add 75 Pl flow buffer and mix carefully. Incubate samples on ice for 20 min and keep covered from light until analysis. 6. Read samples at 405 nm in a flow cytometry analyzer (NOTE5).

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24.4 Notes NOTE1. Depending on the further analysis of the synchronized cells, less or more cells might be needed. However, synchronization of cells in tubes instead of flasks results in more cells per volume and therefore saves aphidicolin. If it is important to easily view the cells during the progression through the cell cycle, flask or flat side tubes should be used. NOTE2. Depending on what the cultures are grown in (10 ml tube, 50 ml tube or flask) the number of cells/ ml needed for 100% confluence differs. This is important to take into consideration if cells or the type of container is changed (tube for flask or vice versa) for synchronization. If, using different containers, there is a great risk of having too many or too few cells in the final culture, which will affect the result of the synchronization. NOTE3. Cells could also be put on ice to be detached from the tube. However, to minimize the time from harvest to fixation use of cell scraper is recommended. NOTE4. The cells are very fragile after the acid cell fixation and it is crucial to be careful when mixing the sample with diluent buffer. If the cells are handled too roughly, they may break and the flow cytometry results will not be accurate. NOTE5. The cells are fixed in acid but the diluent buffer should neutralize the pH to protect the cells and the DNA. However, best results are reached if the flow cytometry analysis is performed within the next two days.

References Adam RD (1991) The biology of Giardia spp. Microbiol Rev 55: 706–732

K. Troell and S. Svärd Bernander R, Palm JE, and Svärd SG (2001) Genome ploidy in different stages of the Giardia lamblia life cycle. Cell Microbiol 3: 55–62 Cooper S, Iyer G, Tarquini M, and Bissett P (2006) Nocodazole does not synchronize cells: implications for cell-cycle control and whole-culture synchronization. Cell Tissue Res 324: 237–242 Dawson SC, et al. (2007) Kinesin-13 regulates flagellar, interphase, and mitotic microtubule dynamics in Giardia intestinalis. Eukaryot Cell 6: 2354–2364 Elmendorf HG, Dawson SC, and McCaffery JM (2003) The cytoskeleton of Giardia lamblia. Int J Parasitol 33: 3–28 Franzen O, et al. (2009) Draft genome sequencing of giardia intestinalis assemblage B isolate GS: is human giardiasis caused by two different species? PLoS Pathog 5: e1000560 Hofstetrova K, et al. (2010) Giardia intestinalis: aphidicolin influence on the trophozoite cell cycle. Exp Parasitol 124: 159–166 Mariante RM, Vancini RG, Melo AL, and Benchimol M (2005) Giardia lamblia: evaluation of the in vitro effects of nocodazole and colchicine on trophozoites. Exp Parasitol 110: 62–72 Morrison HG, et al. (2007) Genomic minimalism in the early diverging intestinal parasite Giardia lamblia. Science 317: 1921–1926 Poxleitner MK, et al. (2008a) Evidence for karyogamy and exchange of genetic material in the binucleate intestinal parasite Giardia intestinalis. Science 319: 1530–1533 Poxleitner MK, Dawson SC, and Cande WZ (2008b) Cell cycle synchrony in Giardia intestinalis cultures achieved by using nocodazole and aphidicolin. Eukaryot Cell 7: 569–574 Reiner DS, et al. (2008) Synchronisation of Giardia lamblia: identification of cell cycle stage-specific genes and a differentiation restriction point. Int J Parasitol 38: 935–944 Sagolla MS, Dawson SC, Mancuso JJ, and Cande WZ (2006) Three-dimensional analysis of mitosis and cytokinesis in the binucleate parasite Giardia intestinalis. J Cell Sci 119: 4889–4900 Samson F, Donoso JA, Heller-Bettinger I, Watson D, and Himes RH (1979) Nocodazole action on tubulin assembly, axonal ultrastructure and fast axoplasmic transport. J Pharmacol Exp Ther 208: 411–417 Striepen B, Jordan CN, Reiff S, and van Dooren GG (2007) Building the perfect parasite: cell division in apicomplexa. PLoS Pathog 3: e78 Svärd SG, Hagblom P, and Palm JED (2003) Giardia lamblia – a model organism for eukaryotic cell differentiation. FEMS Microbiol Lett 218: 3–7

Methods for Giardia Transfection and Gene Expression Janet Yee and Joella Joseph

Abstract The study of gene expression in Giardia intestinalis has revealed several unexpected and intriguing findings in this long-branching eukaryote. Transcription was observed to initiate from AT-rich sequences located extremely close to the start of the coding region of each gene examined. In fact, the AT-rich sequence contains the ATG start codon in the majority of Giardia genes. Moreover, promoter regions are compact and canonical TATA-boxes are either scarce or absent. The Giardia RNA pol II has a reduced number of subunits and its transcription of protein-encoding genes is resistant to high concentrations of D-amanitin. Sterile transcripts or antisense RNA is found corresponding to ~50% of the ORFs in the Giardia genome when steady-state RNA was examined. However, whether this antisense RNA originates in the nucleus as a result of transcription from bidirectional promoters by DNA-dependent RNA polymerases, or copied from sense RNA by a RNA-dependent RNA polymerase in the cytoplasm is still unclear. Nevertheless, it is apparent that epigenetic and post-transcriptional regulations have important roles in the expression of Giardia genes. Several techniques and protocols have been developed and utilized in these gene expression studies. The aim of this section is to describe and discuss the application of some of these techniques.

25.1 Giardia Transfection Transient transfection of Giardia trophozoites by electroporation was first described in 1995 by Yee and Nash for the introduction of plasmid DNA and by Yu et al. for the introduction and expression of in vitro transcribed RNA by a giardiavirus-mediated system. The DNA-based and RNA-based methods were fur-

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ther developed to obtain stable transfection of Giardia trophozoites using puromycin (Singer et al., 1998) and neomycin (Yu et al., 1996b), respectively, as the selectable marker. Since then, Giardia transfection has been used in gene expression, protein transport, and encystation studies described in close to 100 publications. The variables and parameters in designing and performing Giardia transfection experiments are discussed below.

25.1.1 DNA versus RNA Constructs The RNA constructs used in Giardia transfections were based on the giardiavirus (GLV), a doublestranded RNA virus that infects some strains of Giardia (Wang and Wang, 1991). RNA of the firefly luciferase gene flanked by the UTRs of GLV cDNA was generated from linearized plasmid DNA by in vitro transcription from a T7 promoter. Electroporation of this hybrid RNA into WB Giardia already infected with GLV resulted in the synthesis of both the sense and the antisense RNA of luciferase, as well as the expression of luciferase activity, up to 5 days after the transfection. This procedure has been used to elucidate the sequences required for the replication and translation of the giardiavirus RNA in Giardia (Yu et al., 1996a, 1998). However, wider application of the GLV-mediated transfection has been restricted due to the necessity of generating large amounts of in vitro transcripts (50–100 Pg) required per electroporation sample. Furthermore, protein is produced only from the electroporated RNA in Giardia already infected with the giardiavirus as no luciferase activity was detected when GLV-luciferase RNA was electroporated into viral-free Giardia. The initial DNA constructs used in Giardia transfection were based on the P11/LUC plasmid, a

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pGEM-3Zf(–) vector (Promega) containing the firefly luciferase gene inserted in-frame between the 18th and 19th codons of the Giardia glutamate dehydrogenase gene (GDH). The GDH gene and its accompanying 5c (820 bp) and 3c (120 bp) flanking sequences on this plasmid were cloned from Giardia Portland-1 genomic DNA (Yee and Dennis, 1992). The P11/ LUC construct was further modified to produce the 5c'5N plasmid that contains the firefly luciferase gene flanked upstream by a 44 bp HindIII/NcoI fragment containing the minimal promoter of the GDH gene, and flanked downstream by a 131 bp XhoI/KpnI fragment of the 3c non-coding sequence of the GDH gene (Yee et al., 2000). This 5c'5N plasmid, also referred to as pGDH.luc (Elmendorf et al., 2001), has been used as the source for the GDH regulatory sequences found in the wide variety of Giardia transfection vectors that are commonly used today. For example, Sun and Tai (2000) used the GDH promoter to drive the expression of the tet-repressor in developing a tetracycline inducible gene expression transfection vector. Vectors have also been constructed to allow the expression of two different Giardia genes cloned into the same plasmid for transient transfection (Touz et al., 2004). Other available constructs include those that would incorporate an epitope tag of either an AU1 (Elmendorf, 2000), HA, human influenza hemagglutinin (Dolezal, 2005, Kulakova et al., 2006), V5 (Pan et al., 2009), or six histidine residues (Touz et al., 2004; Sun et al., 2005) to the cloned gene for localization of expressed proteins in fixed Giardia cells by immunoflourescent microscopy. Transfection of plasmids encoding GFP-fusion proteins has also been used for protein localization (Hehl et al., 2000) by direct monitoring of the intrinsic fluorescence of GFP. However, the need to expose Giardia, an anaerobe, to oxygen for the detection of GFP is problematic. To address this issue, Regoes and Hehl (2005) adapted the use of the SNAP-tag system (New England Biolabs) to allow the monitoring of proteins expressed from transfected plasmid in living cells. In this system, the gene of interest is expressed as a fusion protein with the SNAP-tag, which is a 20 kDa O6-alkylguanine-DNA alkyltransferase. A synthetic fluorophore linked to benzylguanine, which is a substrate for the SNAP-tag enzyme, is added to the transfected cells and then the fluorophore becomes covalently linked to the fusion protein.

J. Yee and J. Joseph

25.1.2 Transient versus Stable Transfection Transient transfection of a single reporter gene encoding firefly luciferase has been used to analyze the promoter structure of the genes for Ran (Sun and Tai, 1999), GDH (Yee et al., 2000), D2-tubulin (Elmendorf et al., 2001), and a PHD zinc-finger protein (Ong et al., 2002). These analyses were performed by transfecting plasmid constructs containing deletions or mutations of potential gene regulatory sequences that are cloned upstream of the reporter gene, and then measuring the resultant luciferase activity in each sample. The difficulty of using a single luciferase reporter in these analyses is that variations in transfection efficiencies among the samples would also contribute to differences in luciferase activity. This problem can be alleviated by performing at least 3–5 independent transfection experiments with the same set of promoter constructs, and then using the average luciferase activity calculated for each construct for the comparisons. Another option is to use the dual luciferase system (Promega) in which a plasmid vector containing the promoter region to be tested is linked to the firefly luciferase gene, and a control plasmid containing a constitutive promoter linked to the Renilla luciferase gene are co-transfected into Giardia samples. After a recovery period, the firefly luciferase activity detected in each sample is normalized against the Renilla luciferase activity measured in the same sample. This system has been used to identify the regulatory elements in the core histone promoters, where the GDH promoter was used to drive the expression of the Renilla luciferase gene (Yee et al., 2007). Another important factor to consider when using transient transfection for promoter analysis is to ensure that the quantity and quality of the different transfection constructs are equivalent. Plasmid DNA prepared by the double cesium chloride gradient centrifugal method results in the highest luciferase activity per Pg of plasmid DNA transfected, although DNA prepared on Qiagen columns would also give acceptable results albeit with luciferase activities that are 20–50% lower. The DNA concentration for each plasmid construct is usually determined by UVabsorption readings at 260 nm wavelength. However, degraded nucleic acids and other contaminants in some samples can contribute to an overestimation of plasmid DNA concentration. It is therefore vital

Chap. 25 Methods for Giardia Transfection and Gene Expression

that the DNA concentration of all the plasmid constructs is checked by loading equivalent mass of each sample into an agarose gel followed by electrophoresis and ethidium bromide staining. In our laboratory, 250–350 ng of the plasmid DNA, based on the A260 readings, is first linearized with a restriction enzyme before electrophoresis on a 0.7% agarose gel. The fluorescent intensity of the linearized plasmid DNA band in each sample is compared among all samples within the same gel, and the DNA concentration for the samples is then adjusted accordingly. On the basis of the modified DNA concentration, an equivalent amount of the DNA from each sample is again linearized and checked on an agarose gel. A sample protocol for gene promoter analysis with the dual luciferase system in transient transfection of Giardia is outlined below: 1. Grow Giardia trophozoites in screw-capped glass culture tubes at 37°C in TYI-S-33 culture medium supplemented with 1u antibiotic/antimycotic (Invitrogen) until mid-late log phase (~1 u 105–5 u 105 cells/ml). Three 16-ml culture tubes of Giardia cells are required for each DNA construct to be electroporated in triplicate. The cells are harvested by centrifugation of the culture tubes at 1000 u g for 10 min at 4°C. The cell pellets are pooled and resuspended in fresh medium to obtain a final concentration of 3.33 u 107 cells/ml. 2. DNA from all plasmid constructs for testing in Giardia transfections should be prepared by the same method – either by a Qiagen Maxi- or Mega-prep kit or by double cesium chloride gradients. The DNA should be resuspended in a volume of 10 mM Tris pH 8 to obtain a DNA concentration of 4 Pg/Pl or higher as determined by A260 readings, so that the volume of DNA added to each electroporation sample would be minimized. Check the DNA concentrations by digesting a small amount of plasmid DNA from each sample (~250–350 ng) with a restriction enzyme to linearize it, followed by electrophoresis in a 0.7% agarose gel containing ethidium bromide. Examine the gel under UV light and adjust the DNA concentration of each sample according to the relative fluorescent intensity of the linear plasmid DNA band as compared to those from the other samples. The DNA samples should be stored at 4°C to avoid repeated freezing and thawing cycles.

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3. Transfer a 300 Pl aliquot (~107 cells) from the concentrated cell suspension prepared in step 1 into a pre-chilled 0.4 cm gap electroporation cuvette placed on ice. Add 40 Pg of the test plasmid containing the firefly luciferase gene (F-Luc) driven by different upstream sequences and 5 Pg of the control plasmid DNA containing the Renilla luciferase gene (R-Luc) driven by the GDH promoter to the cuvette immediately before electroporation. We routinely add 30–50 Pg of test plasmid DNA for each electroporation sample although Elmendorf et al. (2001) have used as little as 5 Pg of plasmid DNA per sample. 4. Electroporate each cuvette at 350 V, 1000 PF, and 720 : (low-voltage setting on a BTX Electro Cell Manipulator 600). Place the cuvette back on ice for 15 min before transferring the cells to medium in a capped 16-ml glass culture tube. Use a small volume of medium (0.5–1 ml) to rinse the cuvette and add this rinse to the culture tube. Incubate the culture tube at 37°C for a recovery period. We routinely use a recovery period of 6 h, as this was the time point of maximal luciferase activity in a time course experiment (Yee and Nash, 1995) although other laboratories have obtained acceptable levels of luciferase activities after recovery times of 16– 30 h (Sun and Tai, 1999; Elmendorf et al., 2001). 5. Centrifuge the culture tubes at 1000u g for 10 min at 4°C. Resuspend the cell pellet in each tube with 1 ml of phosphate buffered saline (PBS) and then transfer it to a 1.5-ml microfuge tube. Centrifuge the microfuge tube at 1000 u g for 10 min at 4°C and remove the supernatant. Resuspend the cell pellet in each tube with 45 Pl of freshly prepared 1u passive lysis buffer (Promega) and then store at –70°C for at least 1 h. 6. Assay a 20-Pl aliquot of the cell lysate from each sample with 100 Pl of LARII and 100 Pl of Stop&Glo solution from the Dual-Luciferase® Reporter Assay kit (Promega) on the dual luciferase setting of a Turner TD20-20 luminometer (Promega) and record the ratio of F-luc:R-luc. Each DNA construct should be tested in triplicate samples, and each sample should be assayed twice for F-Luc and R-Luc activities. A total of 2–3 independent electroporation experiments should be performed for each set of DNA constructs to be tested.

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Giardia transfections have also been used to study gene expression during encystation, which is the transformation of the trophozoite to the cyst form of this protist. Such experiments require stable transfections of the plasmid constructs due to the time needed to induce encystation in Giardia cultures (Gillin et al., 1987, 1989; Lujan et al., 1996). These studies include the analysis of putative transcription factors and identification of their binding sites in the promoters of encystation-induced genes such as glucosamine-6 -phosphate isomerase, CWP1, and CWP2 (Knodler et al., 1999; Hehl et al., 2000; Sun et al., 2002; DavisHayman et al., 2003; Huang et al., 2008; Pan et al., 2009). As these encystation-inducible promoters appear to contain both negative regulatory elements to repress gene expression in vegetative trophozoites as well as positive regulatory elements to activate gene expression during encystation, it is necessary to assay for the luciferase reporter gene activity in transfected Giardia under both culture conditions. The induction or fold-change in gene expression during encystation can be determined by dividing the luciferase activity obtained in encysting cells by the activity obtained in vegetative trophozoites that are transfected with the same reporter construct (Sun et al., 2002; DavisHayman et al., 2003).

in the cultured Giardia, there were fewer genes affected and less change in the corresponding mRNA and protein levels as compared to neomycin.

25.1.3 Puromycin versus Neomycin for Drug Selection for Stable Transfection

25.2.1 Preparation of Giardia Nuclear Extracts

Puromycin and neomycin are the two drugs used to select for the maintenance of DNA plasmid constructs transfected into Giardia (Singer et al., 1998, Sun et al., 1998). Several laboratories have successfully used either one or both drugs in their transfection studies. However, our own experience showed that incubation of untransfected Giardia cultures with 100–150 Pg/ml neomycin could result in the appearance of trophozoites that are drug resistant after 7 or more days of incubation. In contrast, we have not detected any drug-resistant trophozoites when Giardia cultures are incubated with 25–50 Pg/ml puromycin for the same period (unpublished). Su et al. (2007) showed that maintenance of Giardia cultures under neomycin selection also affected the expression of several endogenous genes. Although puromycin selection also influenced endogenous gene expression

25.2 Gel-shift Assays Gel-shift assays – also known as electrophoretic mobility shift assays (EMSA) and band-shift assays – are used to detect interactions between DNA-binding proteins and DNA, and are based on the observation that the mobility of DNA fragments bound by proteins is reduced in relation to unbound DNA when separated by electrophoresis on a non-denaturing polyacrylamide gel. Gel-shift assays have been used to investigate protein-binding sites in Giardia gene promoters, and conversely to identify the nuclear proteins that bind to the promoters (Sun and Tai, 1999; Yee et al., 2000, 2007; Ong et al., 2002). The source of proteins is typically a crude nuclear extract prepared from Giardia trophozoites (Sun and Tai, 1999, Yee and Dennis, 1994). DNA probes may have either radioactive or non-radioactive labels; the label used impacts how the shifted complexes are visualized. This section will describe the preparation of nuclear extracts and labeled probes, as well as detection of non-radioactive probes.

Nuclear proteins used in gel-shift assays are obtained from Giardia trophozoites in the logarithmic phase of growth. Proteins are extracted using a modification of a method originally described by Andrews and Faller (1991). Briefly, cells are collected by centrifugation, washed in PBS, and lysed with a hypotonic buffer in the presence of a mild detergent. Centrifugation separates the soluble cytoplasmic proteins in the supernatant from the nuclei in the pellet. The pellet is washed to remove residual cytoplasmic proteins. Addition of a high salt buffer to the washed pellet disrupts interactions between proteins and DNA in the nuclei, so that centrifugation of the sample would yield a supernatant containing the extracted soluble nuclear proteins. The following protocol was used to obtain nuclear proteins for gel-shift assays with the glutamate dehydrogenase (GDH) and core histone H4 promoters (Yee and Dennis, 1994; Yee et al., 2000, 2007).

Chap. 25 Methods for Giardia Transfection and Gene Expression

1. Grow Giardia trophozoites in screw-capped glass culture tubes at 37°C in TYI-S-33 medium supplemented with 1u antibiotic/antimycotic (Invitrogen) until mid-late log phase (~1 u 105–5 u 105 cells/ml). We use 20–40 tubes of Giardia culture for each nuclear preparation. Harvest the cells by centrifugation at 1000u g for 10 min at 4°C. Wash the cell pellets with cold PBS and pool all the resuspended cells into a single sterile 15-ml capped tube. Pellet the cells by centrifugation and remove the supernatant. Estimate the packed cell volume (PCV) of the pellet – we usually obtain 50–100 Pl PCV from 20 tubes of culture. 2. Resuspend the cell pellet in 250 Pl of Lysis Buffer A (10 mM HEPES, pH 7.9; 1.5 mM MgCl2, 10 mM KCl) per 100 Pl of PCV. Transfer the resuspended cells to a pre-chilled 1.5-ml microfuge tube and store at –80°C for at least 1 hour before thawing. This freeze and thaw cycle helps in the lysis of the cells. The cells may be stored in the lysis buffer for longer periods at –80°C, but extended storage of a week or more results in reduced yields and reduced DNA binding activity. 3. Remove the sample from storage at –80°C and immediately add 100u Protease Inhibitor Cocktail (BioShop) to 1u, and DTT to a final concentration of 1 mM. Thaw the sample at room temperature and immediately place on ice. 4. Add Nonidet P-40 (NP-40) to obtain a final concentration of 0.2%. Break up any clumps of cells in the lysate with a sterile pestle that can fit inside the microfuge tube. 5. Centrifuge the microfuge tube at 3300u g for 1 min at 4°C. The supernatant, which contains cytoplasmic proteins, should be removed and can be stored as the cytoplasmic extract at –80°C. 6. Wash the remaining cell pellet in 500 Pl of chilled PBS, then centrifuge 3300u g for 1 min at 4°C. Remove the supernatant and store this sample as the cytoplasmic wash at –80°C. 7. Resuspend the nuclear pellet in 250 Pl of Extraction Buffer C (20 mM HEPES, pH 7.9; 20% glycerol, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA). Immediately add Protease Inhibitor Cocktail (BioShop), and DTT to a final concentration of 1 mM. Break up any visible clumps in the sample with a sterile pestle. Incubate the tube on ice for 20 min, with occasional inversion to mix.

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8. Centrifuge the microfuge tube at 13,400u g for 5 min at 4°C. Transfer the supernatant to a new, pre-chilled microfuge tube and save this sample as the nuclear extract. Exchange the high salt buffer of this sample into the protein storage buffer (50 mM Tris-HCl, pH 7.5; 5% glycerol) using a microfiltration unit with a 5000 Da Molecular Weight Cut-off (Millipore). Wash the nuclear extract with 3u its original volume with chilled storage buffer and concentrate to a final volume of 250 Pl. Determine the protein concentration of the nuclear extract by the Bradford assay. Divide the nuclear extract into 50 Pl aliquots in separate microfuge tubes. Flash-freeze the samples with liquid nitrogen and store at –80°C. When stored in this manner, the nuclear extract can be used in gel-shift assays for up to one month with little loss in binding activity and may be used up to two months. As with any proteins sample, repeated freeze-thaw cycles should be avoided. Expected yields of nuclear protein range from 300 to 600 Pg from 20 tubes of Giardia culture (~320 ml of culture). Up to 1400 Pg of nuclear proteins may be obtained from 40 tubes of Giardia culture (~640 ml of culture). When analyzed by SDS-PAGE, the cytoplasmic extract and nuclear extract should have distinct protein-binding patterns (Fig. 25.1) although the cytoplasmic fraction would also contain some nuclear proteins due to premature lysis of a few nuclei, and the nuclear fraction would also contain some residual cytoplasmic proteins that were not removed in the wash step. If a nuclear protein preparation containing minimal contamination of cytoplasmic protein is required, then it is necessary to use a procedure that includes an additional nuclei isolation step as described by Sun and Tai (1999), which is an adaptation of the classical Dignam method (1990). As the two nuclei in a Giardia trophozoite are physically connected to the ventral disk and other cytoskeleton components, it is also possible to adapt a cytoskeleton preparation such as the one described by Crossley and Holberton (1983) to isolate Giardia nuclei (unpublished). In this procedure, the presence of intact nuclei in the cytoskeleton preparation should be examined under the microscope after DAPI staining. The nuclei and cytoskeleton sample can then be resuspended in a high salt buffer (as described in step 7 of the above

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NE

2

3

kDa 250 150 100 80 60

50

40

30

25

20

15

10 1

Fig. 25.1 SDS-PAGE analysis of the protein composition of Giardia cytoplasmic and nuclear extracts. Lane 1 contains 5 Pl of 10–250 kDa Protein Size Ladder (New England Biolabs). Lane 2 contains 5 Pg of Cytoplasmic Extract (CE), and lane 3 contains 5 Pg of Nuclear Extract (NE). Proteins were separated on a 12% Tris-Glycine gel at 150 V and stained with Coomassie Blue

procedure) and the extracted nuclear proteins can be recovered in the supernatant.

25.2.2 Preparation of Probes (for Radioactive and Non-radioactive Detection of Signals) DNA probes used in gel-shift assays represent Giardia gene promoter regions, which are likely to contain

binding sites for nuclear proteins. Probes range in size from 17 bp (Yee and Dennis, 1994; Yee et al., 2000) to 130 bp (Yang et al., 2003), and can be amplified by polymerase chain reaction (PCR) from plasmid constructs containing the promoter region of the gene of interest using specific primers (Yee et al., 2000; Yang et al., 2003). Alternatively, probes may be made as synthetic oligonucleotides. Both radioactive and non-radioactive labels may be used for detection. For radioactive labels, probes may be labeled at either the 5c or 3c ends, or by the incorporation of labeled nucleotides during amplification by PCR (Sun and Tai, 1999; Yee et al., 2000, Yang et al., 2003). Oligonucleotides are labeled at the ~ 5c end with [J32P]ATP by T4 polynucleotide kinase (Sun and Tai, 1999; Yee et al., 2000), while doublestranded probes with 3c overhangs may be filled with [D-32P]dNTP by T4 DNA polymerase (Yang et al., 2003). Excess or unincorporated radiolabeled nucleotides are removed by column chromatography or by ethanol precipitation of the DNA probe. While radioactive probes provide great sensitivity for the detection of shifted complexes and is the most commonly used labeling method, oligonucleotides with non-radioactive labels such as 5c biotin have also been effectively used in gel-shift assays with Giardia nuclear proteins (Yee et al., 2007). We have also used oligonucleotides labeled with digoxygenin (DIG) as probes in gel-shift assays (unpublished). In performing the gel-shift assay, it may be necessary to prepare both single-stranded and doublestranded labeled probes, as proteins in Giardia nuclear extracts have been shown to bind to specific elements in single-stranded oligonucleotides (Yee and Dennis 1994; Sun and Tai, 1999; Yee et al., 2000; Ong et al., 2002). The following is a general protocol for the preparation of double-stranded probes, and can be used for both radioactive and non-radioactive labeled probes. Unlabeled double-stranded competitors may be prepared in a similar manner. 1. Combine the two complementary oligonucleotides in equimolar amounts in annealing buffer (50 M Tris-HCl, pH 7.9; 10 mM MgCl2, 50 mM NaCl, 1 mM DTT) to give the desired final concentration. 2. Heat at 90°C in a water bath for 10 min, then allow to slowly cool to room temperature.

Chap. 25 Methods for Giardia Transfection and Gene Expression

3. Centrifuge the sample briefly to collect condensation and then place on ice for 5 min before use. Single-stranded probes and competitors are also heated at 90°C for 10 min, but are centrifuged and immediately placed on ice to prevent self-complementary annealing.

25.2.3 Preparation of Membrane for Non-radioactive Detection of Gel-shifts Use of radioactive probes permits direct visualization of shifted complexes by autoradiography (Sun and Tai, 1999; Yee et al., 2000; Ong et al., 2002). Non-radioac-

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tive labels cannot be detected directly; thus, visualization of biotin-labeled or DIG-labeled probes requires more extensive preparation. After separation by electrophoresis, bound and unbound DNA probes within the gel are transferred to a membrane by an electric current. The DNA is crosslinked to the membrane by exposure to UV light, then the membrane is washed in buffer containing milk proteins to prevent antibody binding to non-specific sites. The membrane is incubated in buffer containing diluted antibody-enzyme conjugate. After being washed to remove unbound antibody-enzyme conjugate, the membrane is washed in detection buffer. A specific substrate is then added to the membrane, and then the signal produced by the action of the enzyme on the substrate can be detected.

Nuclear protein preps Probe alone

1

2

3

4

5

3him probe

Fig. 25.2 Gel-shift assays with different preparations of Giardia nuclear extract. Each lane contains 2 pmol of 5c biotin-labeled, double-stranded DNA containing three histone motifs in tandem (3him). The sequence of the top strand of the DNA probe is shown beneath the gel with the histone motifs indicated by gray boxes and the g-CAB elements underlined. The first gel lane contains the probe alone; each of the lanes labeled 1–5 also contains 5 Pg of Giardia nuclear extract prepared on separate days. Note: Nuclear protein prep 4 showed markedly reduced binding to the 3him probe compared to the other preparations tested and was discarded

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For clarity, it should be noted that the molecule used for the detection of biotin is a streptavidin-enzyme conjugate. While streptavidin binds with high affinity and specificity to biotin, it is not an antibody. Detection of the DIG label requires an anti-DIG antibody-enzyme conjugate. The following protocol was used by Yee et al (2007) for the detection of biotinlabeled DNA probes: 1. After electrophoresis, transfer the DNA in the gel onto a 0.45 Pm nylon transfer membrane (Magna Probe) using a semi-dry electroblotter (Owl) at 200 V for 30 min. Both the filter paper in contact with plates and the membrane should be soaked in the running buffer used for electrophoresis. 2. Crosslink the transferred DNA to the membrane by placing the membrane facedown on a UVtransilluminator for 4 min. Wash the membrane for 2 min in wash buffer (100 mM maleic acid, pH 7.5; 150 mM NaCl, and 0.3% Tween-20), then incubate in 15 ml of blocking buffer (0.1 M TrisHCl, pH 7.5; 0.15 M NaCl, 0.1% skim milk powder) for 30 min at room temperature or overnight at 4°C. 3. Incubate the membrane for 30 min in 10 ml of 1u blocking buffer with 1 Pl of streptavidin-alkaline phosphatase (Strept-AP; Roche) added to obtain a 1:10,000 dilution of this conjugate. 4. Wash the membrane twice in wash buffer for 15 min each, and then in alkaline detection buffer (0.1 M Tris-HCl, pH 9.5; 0.1 M NaCl) for 5 min. 5. Add 4–6 drops of CDP Star chemiluminescent reagent (Perkin Elmer) to the membrane and evenly distribute over the membrane surface containing streptavidin-alkaline phosphatase conjugate bound to biotin-labeled probes. 6. Pre-incubate the membrane at 37°C for 10 min to enhance the chemiluminescent signal, and place the membrane in the cabinet of the Chemigenius2 Bioimaging System (Syngene) for the detection of biotin-labeled probes. Images are obtained in the absence of light and without filters, and exposure times vary from 15 to 60 min. Provided that binding and electrophoresis conditions are optimized, the addition of 1–2 pmol of biotin-labeled probe in each gel-shift reaction allows for clear visualization of resolved complexes (Fig. 25.2).

J. Yee and J. Joseph

25.3 Identification of Transcription Initiation Sites The core promoter is usually found within 100 bp upstream of the transcription initiation site of most genes since this is the region where the RNA pol II and the basal transcription factors need to assemble. Therefore, the localization of the initiation site for a gene would help in the identification of its promoter. In the vast majority of Giardia genes examined, transcription initiates from an AT-rich sequence, called the initiator, which also contains the ATG translation start codon for the gene (Reviewed by Adam, 2001). The proximity of the transcription initiation site to the translation start site in these Giardia genes results in the short 1–6 nucleotide 5c UTR of the corresponding mRNAs. The techniques that have been used to determine the transcription start sites of Giardia genes will be discussed below.

25.3.1 Primer Extension, S1 Nuclease Protection, and 5c RACE The first characterization of the transcription start sites in Giardia performed by primer extension and S1 nuclease protection assay on the alpha- and beta-tubulin genes (Kirk-Mason et al., 1989) revealed unusually short (6 nucleotides) 5c UTR of these transcripts. Analyses of further Giardia genes by a combination of primer extension, S1 nuclease mapping and RNA sequencing have shown that a short 5c UTR is a common feature of Giardia mRNAs (Mowatt et al., 1991; Alonso and Peattie, 1992; Murtagh et al., 1992; Nohria et al., 1992; Yee and Dennis, 1992; Ey et al., 1993; Sun and Tai, 1999). Most recently, primer extension has also been used to map methylation sites in Giardia 16S rRNA (Yang et al., 2005). Currently, 5c RACE is the technique most commonly used for the identification of transcriptional start sites for Giardia genes (Knodler et al., 1999; Bienz et al., 2001; Elmendorf et al., 2001; Sun et al., 2002; Davis-Hayman et al., 2003). This is probably due to the availability of several commercial kits for 5c RACE and the requirement of radiolabeling and electrophoresis on large-format polyacrylamide gels for the other techniques. Nevertheless, primer extension has the advantage of allowing the identification of

Chap. 25 Methods for Giardia Transfection and Gene Expression

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alternative and less common transcription start sites for a gene in a single experiment. The analysis of 3–5 clones from a 5c RACE experiment is normally performed for a single gene but it is likely that 10 or more clones would need to be analyzed to detect less frequently used transcription start sites.

RNA interference, as well as by transcriptional activation. Consequently, it may be necessary to study both newly synthesized and steady-state RNAs in the analysis of gene expression in Giardia. The following procedure was used to study the transcription of newly synthesized RNAs corresponding to the GDH gene (Yee et al., 2000):

25.3.2 Nuclear Run-on

1. Grow 20–40 tubes of Giardia trophozoites in screw-capped glass culture tubes at 37°C in TYIS-33 medium supplemented with 1u antibiotic/ antimycotic (Invitrogen) until mid-late log phase (1–5 u 105 cells/ml). Harvest cells by centrifugation and wash twice with PBS. 2. Resuspend cell pellet in buffer A (150 mM sucrose, 20 mM KCl, 20 mM HEPES pH 7.2, 3 mM MgCl2, 1 mM DTT, and 1 mg/ml leupeptin) to obtain 3 u 108 cells/ml. Transfer 400 Pl sample containing 1.2 u 108 cells into a microfuge tube and incubate on ice for 5 min. Add lysolecithicin (Sigma) to 500 Pg/ml to permeabilized the cell membrane and incubate on ice for 1 min. 3. Wash the cells twice in buffer A and resuspend into 100 Pl of buffer A at room temperature. Add an equal volume of transcription buffer (2u stock: 20 mM HEPES pH 7.2, 180 mM KCl, 7 mM MgCl2, 50 mM phosphocreatine, 1.2 mg/ml creatine kinase, 4 mM ATP, 2 mM GTP, 2 mM CTP, 100 PCi [D-32P] UTP, 1 mM DTT, 10 Pg/ml leupeptin) and allow transcription to proceed for 10 min at room temperature. This pulse labeling step can also be perform for 1 h at room temperature (Seshadri et al., 2003) or for 30 min at 30°C (Lopez et al., 2003). 4. After transcription, add 600 Pl of TRIZOL-LS reagent (Invitrogen) to each sample and extract RNA according to the manufacturer’s directions. Hybridize the recovered RNA to slot blots containing DNA fragments corresponding to genes of interest. It is essential that the membrane contain an equivalent mass of DNA in each slot. The DNA concentration of each sample should be checked by gel electrophoresis as described in step 2 in the procedure for transient transfection.

While primer extension, S1 nuclease protection, and 5c RACE determine the transcription start sites based on the 5c ends of steady-state mRNAs, the nuclear run-on assay allows the localization of these sites based on newly synthesized RNAs. The use of nuclear run-on assays is essential to detect trans-spliced transcripts, as observed in Leishmania and Trypanosomes, or other post-transcriptional events that would affect the length of the 5c UTR of the mRNAs. For the transcripts of the Giardia GDH gene, nuclear run-on was used (Yee et al., 2000) to confirm the transcription start sites determined previously by primer extension and S1 nuclease protection (Yee and Dennis, 1994). Nuclear run-on has also been used to examine the transcriptional activation of five enzymes involved in cyst wall polysaccharide biosynthesis during encystation (Lopez et al., 2003). Interestingly, Lopez et al noted that the fold-change in the mRNA levels during encystation for the five genes was lower for the nuclear run-on data than those obtained by RNA dot blots. They proposed that an increase in the transcriptional level combined with a decrease in RNA turnover might be responsible for the larger overall increase in the steady-stage mRNA levels observed for these genes during encystation. Seshadri et al. (2003) used nuclear run-on assays to study the alpha-amanitin sensitivity of RNA polymerase II transcription. Intriguingly, they did not detect transcription of D2-tubulin antisense mRNAs in their nuclear run-on assays that Teodorovic et al. (2007) observed by 5c RACE. Recently, Prucca and Lujan (2009) performed nuclear run-on with isolated Giardia nuclei and showed that although several VSP genes are actively transcribed, only mRNA corresponding to one specific VSP gene can be detected among the steady-state RNAs examined by Northern blot analysis. These results suggest that some genes in Giardia are regulated by posttranscriptional processes, such as RNA stability or

We have discussed and provided outlines for several of the commonly used techniques for the study of gene expression in Giardia. Techniques for gene expression knockdowns or knockouts are currently being

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developed. Since Giardia trophozoites are tetraploid, gene replacement by homologous recombination, although possible (Singer et al., 1998), would be technically difficult. However, introduction of hammerhead ribozymes (Dan et al., 2000; Li and Wang, 2006; Chen et al., 2007; Feng et al., 2008) and antisense RNA (Touz et al., 2002, 2005; Huang et al., 2008; Prucca and Lujan, 2009) into Giardia trophozoites has been successful in reducing the expression of several genes. As it appears that an RNA interference pathway exists in Giardia (Prucca and Lujan, 2009), the development of RNAi as a gene knockdown technique should be possible and it would become a major tool for determining gene function in Giardia.

References Adam RD (2001) Biology of Giardia lamblia. Clin Microbiol Rev 14: 447–475 Alonso RA and Peattie DA (1992) Nucleotide sequence of a second alpha giardin gene and molecular analysis of the alpha giardin genes and transcripts in Giardia lamblia. Mol Biochem Parasitol 50: 95–104 Andrews NC and Faller DV (1991) A rapid micropreparation technique for extraction of DNA-binding proteins from limiting numbers of mammalian cells. Nucleic Acids Res 19: 2499 Bienz M, Siles-Lucas M, Wittwer P, and Muller N (2001) VSP gene expression by Giardia lamblia clone GS/M-83-H7 during antigenic variation in vivo and in vitro. Infect Immun 69: 5278–5285 Chen XS, Rozhdestvensky TS, Collins LJ, Schmitz J, and Penny D (2007) Combined experimental and computational approach to identify non-protein-coding RNAs in the deep-branching eukaryote Giardia intestinalis. Nucleic Acids Res 35: 4619–4628 Crossley R and Holberton DV (1983) Characterization of proteins from the cytoskeleton of Giardia lamblia. J Cell Sci 59: 81–103 Dan M, Wang AL, and Wang CC (2000) Inhibition of pyruvate-ferredoxin oxidoreductase gene expression in Giardia lamblia by a virus-mediated hammerhead ribozyme. Mol Microbiol 36: 447–456 Davis-Hayman SR, Hayman JR, and Nash TE (2003) Encystation-specific regulation of the cyst wall protein 2 gene in Giardia lamblia by multiple cis-acting elements. Int J Parasitol 33: 1005–1012 Dignam JD (1990) Preparation of extracts from higher eukaryotes. Methods Enzymol 182: 194–203 Dolezal P, Smid O, Rada P, Zubacova Z, Bursac D, Sutak R, Nebesarova J, Lithgow T, and Tachezy J (2005) Giardia mitosomes and trichomonad hydrogenosomes share a common mode of protein targeting. Proc Natl Acad Sci USA 102: 10924–10929

J. Yee and J. Joseph Elmendorf HG, Singer SM, and Nash TE (2000) Targeting of proteins to the nuclei of Giardia lamblia. Mol Biochem Parasitol 106: 315–319 Elmendorf HG, Singer SM, Pierce J, Cowan J, and Nash TE (2001) Initiator and upstream elements in the alpha2-tubulin promoter of Giardia lamblia. Mol Biochem Parasitol 113: 157–169 Ey PL, Khanna KK, Manning PA, and Mayrhofer G (1993) A gene encoding a 69-kilodalton major surface protein of Giardia intestinalis trophozoites. Mol Biochem Parasitol 58: 247–257 Feng XM, Cao LJ, Adam RD, Zhang XC, and Lu SQ (2008) The catalyzing role of PPDK in Giardia lamblia. Biochem Biophys Res Commun 367: 394–398 Gillin FD, Reiner DS, Gault MJ, Douglas H, Das S, Wunderlich A, and Sauch JF (1987) Encystation and expression of cyst antigens by Giardia lamblia in vitro. Science 235: 1040– 1043 Gillin FD, Boucher SE, Rossi SS, and Reiner DS (1989) Giardia lamblia: the roles of bile, lactic acid, and pH in the completion of the life cycle in vitro. Exp Parasitol 69: 164–174 Hehl AB, Marti M, and Kohler P (2000) Stage-specific expression and targeting of cyst wall protein-green fluorescent protein chimeras in Giardia. Mol Biol Cell 11: 1789–1800 Huang YC, Su LH, Lee GA, Chiu PW, Cho CC, Wu JY, and Sun CH (2008) Regulation of cyst wall protein promoters by Myb2 in Giardia lamblia. J Biol Chem 283: 31021–31029 Kirk-Mason KE, Turner MJ, and Chakraborty PR (1989) Evidence for unusually short tubulin mRNA leaders and characterization of tubulin genes in Giardia lamblia. Mol Biochem Parasitol 36: 87–99 Knodler LA, Svard SG, Silberman JD, Davids BJ, and Gillin FD (1999) Developmental gene regulation in Giardia lamblia: first evidence for an encystation-specific promoter and differential 5c mRNA processing. Mol Microbiol 34: 327–340 Kulakova L, Singer SM, Conrad J, and Nash TE (2006) Epigenetic mechanisms are involved in the control of Giardia lamblia antigenic variation. Mol Microbiol 61: 1533–1542 Li L and Wang CC (2006) A likely molecular basis of the susceptibility of Giardia lamblia towards oxygen. Mol Microbiol 59: 202–211 Lopez AB, Sener K, Jarroll EL, and van Keulen H (2003) Transcription regulation is demonstrated for five key enzymes in Giardia intestinalis cyst wall polysaccharide biosynthesis. Mol Biochem Parasitol 128: 51–57 Lujan HD, Mowatt MR, Byrd LG, and Nash TE (1996) Cholesterol starvation induces differentiation of the intestinal parasite Giardia lamblia. Proc Natl Acad Sci USA 93: 7628–7633 Mowatt MR, Aggarwal A, and Nash TE (1991) Carboxy-terminal sequence conservation among variant-specific surface proteins of Giardia lamblia. Mol Biochem Parasitol 49: 215–227 Murtagh JJ Jr, Mowatt MR, Lee CM, Lee FJ, Mishima K, Nash TE, Moss J, and Vaughan M (1992) Guanine nucleotidebinding proteins in the intestinal parasite Giardia lamblia. Isolation of a gene encoding an approximately 20-kDa ADP-ribosylation factor. J Biol Chem 267: 9654–9662

Chap. 25 Methods for Giardia Transfection and Gene Expression Nohria A, Alonso RA, and Peattie DA (1992) Identification and characterization of gamma-giardin and the gamma-giardin gene from Giardia lamblia. Mol Biochem Parasitol 56: 27–37 Ong SJ, Huang LC, Liu HW, Chang SC, Yang YC, Bessarab I, and Tai JH (2002) Characterization of a bi-directional promoter for divergent transcription of a PHD-zinc finger protein gene and a ran gene in the protozoan pathogen Giardia lamblia. Mol Microbiol 43: 665–676 Pan YJ, Cho CC, Kao YY, and Sun CH (2009) A novel WRKYlike protein involved in transcriptional activation of cyst wall protein genes in Giardia lamblia. J Biol Chem 284: 17975–17988 Prucca CG and Lujan HD (2009) Antigenic variation in Giardia lamblia. Cell Microbiol 11: 1706–1715 Regoes A, Hehl AB (2005) SNAP-tag mediated live cell labeling as an alternative to GFP in anaerobic organisms. Biotechniques 39: 809–810, 812 Seshadri V, McArthur AG, Sogin ML, and Adam RD (2003) Giardia lamblia RNA polymerase II: amanitin-resistant transcription. J Biol Chem 278: 27804–27810 Singer SM, Yee J, and Nash TE (1998) Episomal and integrated maintenance of foreign DNA in Giardia lamblia. Mol Biochem Parasitol 92: 59–69 Su LH, Lee GA, Huang YC, Chen YH, and Sun CH (2007) Neomycin and puromycin affect gene expression in Giardia lamblia stable transfection. Mol Biochem Parasitol 156: 124–135 Sun CH and Tai JH (1999) Identification and characterization of a ran gene promoter in the protozoan pathogen Giardia lamblia. J Biol Chem 274: 19699–19706 Sun CH and Tai JH (2000) Development of a tetracycline controlled gene expression system in the parasitic protozoan Giardia lamblia. Mol Biochem Parasitol 105: 51–60 Sun CH, Chou CF, and Tai JH (1998) Stable DNA transfection of the primitive protozoan pathogen Giardia lamblia. Mol Biochem Parasitol 92: 123–132 Sun CH, Palm D, McArthur AG, Svard SG, and Gillin FD (2002) A novel Myb-related protein involved in transcriptional activation of encystation genes in Giardia lamblia. Mol Microbiol 46: 971–984 Sun CH, Su LH, and Gillin FD (2005) Influence of 5c sequences on expression of the Tet repressor in Giardia lamblia. Mol Biochem Parasitol 142: 1–11 Teodorovic S, Walls CD, and Elmendorf HG (2007) Bidirectional transcription is an inherent feature of Giardia lamblia promoters and contributes to an abundance of sterile antisense transcripts throughout the genome. Nucleic Acids Res 35: 2544–2553 Touz MC, Gottig N, Nash TE, and Lujan HD (2002) Identification and characterization of a novel secretory granule calcium-binding protein from the early branching eukaryote Giardia lamblia. J Biol Chem 277: 50557–50563

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Touz MC, Kulakova L, and Nash TE (2004) Adaptor protein complex 1 mediates the transport of lysosomal proteins from a Golgi-like organelle to peripheral vacuoles in the primitive eukaryote Giardia lamblia. Mol Biol Cell 15: 3053–3060 Touz MC, Conrad JT, and Nash TE (2005) A novel palmitoyl acyl transferase controls surface protein palmitoylation and cytotoxicity in Giardia lamblia. Mol Microbiol 58: 999– 1011 Wang AL and Wang CC (1991) Viruses of parasitic protozoa. Parasitol Today 7: 76–80 Yang H, Chung HJ, Yong T, Lee BH, and Park S (2003) Identification of an encystation-specific transcription factor, Myb protein in Giardia lamblia. Mol Biochem Parasitol 128: 167–174 Yang CY, Zhou H, Luo J, and Qu LH (2005) Identification of 20 snoRNA-like RNAs from the primitive eukaryote, Giardia lamblia. Biochem Biophys Res Commun 328: 1224–1231 Yee J and Dennis PP (1992) Isolation and characterization of a NADP-dependent glutamate dehydrogenase gene from the primitive eucaryote Giardia lamblia. J Biol Chem 267: 7539–7544 Yee J and Dennis PP (1994) The NADP-Dependent glutamate dehydrogenase of Giardia lamblia – a study of function, gene structure and expression. Syst Appl Microbiol 16: 759–767 Yee J and Nash TE (1995) Transient transfection and expression of firefly luciferase in Giardia lamblia. Proc Natl Acad Sci USA 92: 5615–5619 Yee J, Mowatt MR, Dennis PP, and Nash TE (2000) Transcriptional analysis of the glutamate dehydrogenase gene in the primitive eukaryote, Giardia lamblia. Identification of a primordial gene promoter. J Biol Chem 275: 11432–11439 Yee J, Tang A, Lau WL, Ritter H, Delport D, Page M, Adam RD, Muller M, and Wu G (2007) Core histone genes of Giardia intestinalis: genomic organization, promoter structure, and expression. BMC Mol Biol 8: 26–40 Yu DC, Wang AL, Wu CH, and Wang CC (1995) Virus-mediated expression of firefly luciferase in the parasitic protozoan Giardia lamblia. Mol Cell Biol 15: 4867–4872 Yu DC, Wang AL, and Wang CC (1996a) Amplification, expression, and packaging of a foreign gene by giardiavirus in Giardia lamblia. J Virol 70: 8752–8757 Yu DC, Wang AL, and Wang CC (1996b) Stable coexpression of a drug-resistance gene and a heterologous gene in an ancient parasitic protozoan Giardia lamblia. Mol Biochem Parasitol 83: 81–91 Yu DC, Wang AL, Botka CW, and Wang CC (1998) Protein synthesis in Giardia lamblia may involve interaction between a downstream box (DB) in mRNA and an anti-DB in the 16S-like ribosomal RNA. Mol Biochem Parasitol 96: 151–165

Biological Resource Centers for Giardia Research Robert Molestina and Hugo D. Luján

Abstract To succeed as a parasite, Giardia lamblia has evolved a series of complex strategies to evade the host immune system. Importantly, the accessibility of reference strains and reagents is critical to the generation of studies aimed at elucidating the intricate aspects underlying the host-parasite relationship. In this context, the mission of culture collections known as biological resource centers is to provide quality-controlled reference materials to scientists carrying out basic research to aid the development of improved diagnostic tests, vaccines, and therapies.

26.1 Collection of Giardia Strains at the ATCC The accessibility of reference cultures is critical to the generation of peer-reviewed studies. Protozoan strains deposited in culture collections are in fact “biological standards” as they are key components of comparative studies. Thus, such collections, better known as biological resource centers (BRCs), have contributed significantly to the development of scientific research by making reference strains available to the wider scientific community. The Protistology Collection at the American Type Culture Collection (ATCC®) houses the largest service repository of parasitic protozoa in the world. Collecting clinical isolates has been a common practice among parasitologists for decades, with many of them depositing their strains at the ATCC. Giardia researchers are no exception with the first strain (Portland-1) acquired by the Protistology Collection in 1980 (Table 26.1). At present, the Collection holds more than 30 strains of Giardia lamblia from a range of geographical locations. This includes reference

H. D. Luján et al. (eds.), Giardia © Springer-Verlag/Wien 2011

26

strains used in diverse biological studies, most notably, genome-related work. Preservation techniques are critical to the longterm maintenance of cultures at BRCs. These techniques are especially applicable to maintain the earliest possible in vitro passages of strains to ensure genetic and phenotypic stability. Establishing seed and distribution stocks for every strain is a common practice utilized at the ATCC to make certain that organisms distributed to the scientific community are closely similar to the original material provided by the depositor. These practices are performed under a strict quality management system which complies with the requirements of ISO 9001:2000 certified and accredited by the British Standards Institute (BSI).

26.2 The BEI Research Resources Repository The ATCC also currently manages the NIAID Biodefense and Emerging Infections Research Resources Repository (BEI Resources). BEI Resources is focused on acquiring and authenticating several categories of materials for registered members of the research community (http://www.beiresources.org/). These include pathogens newly recognized in the past two decades, re-emerging pathogens, and NIAID Category A, B and C agents. Resources for Giardia researchers housed at BEI include many of the strains listed in Table 1. In addition, BEI Resources is the distribution center for materials generated by the NIH Seattle Structural Genomics Centers for Infectious Diseases (SSGCIDs). The primary mission of the SSGCID is to determine the structure of 75–100 protein targets from NIAID Category A, B, and C agents in addition to emerging and re-emerging infectious disease organisms.

Portland-1

WB

KS

New Orleans-1

UPV:0685:1

UPV:1285:1

30957

50114

50137

50163c

d

30888

WB clone 1267

WB clone A6

JH (NIH:1182)

AB (NIH:0883:1) Human

50582

50583

50584

50585

NF

S2

D3

BR-7

BR-15

EGY (CDC:1088:1)

MR-4

BR-1

203332

203333

203334

PRA-41

PRA-42

PRA-43

PRA-45

PRA-63

WB clone C6

GS clone H7

50581

50803

GS/M

50580

Human

Muskrat

Human

Human

Human

Dog

Lamb

Drinking water

Clone of WB

Human

Clone of WB

Clone of WB

Clone of GS/M

Human

Guinea pig

113:NIH

50558d

Human

UNO/04/87/1

e

Philadelphia, PA, USA

New Orleans, LA, USA

Philadelphia, PA, USA

Afghanistan

Portland, OR, USA

Geographic location

1987

1985

1985

1985

1984

1979

1971

Year of isolation

1983

Brazil

Canada

Ismailia, Egypt

Brazil

Brazil

Calgary, Canada

Calgary, Canada

Botwood, Newfoundland, Canada

N/A

Peru

West Virginia, USA

1990

1986

1988

1990

1990

1986

1986

1991

1983

1983

1982

NIH, Bethesda, MD, USA 1987?

NIH, Bethesda, MD, USA N/A

NIH, Bethesda, MD, USA 1988?

Alaska, USA

NIH, Bethesda, MD, USA 1982

Omaha, NE, USA

Domestic sheep Madison, WI, USA

Domestic cat

Human

Human

Human

Human

Source

50184

50170

Strain

ATCC No.b

Table 26.1 Giardia lamblia strains available at the American Type Culture Collectiona

FD Gillin

LS Diamond

Depositor

1981

1980

Year of deposit

LC Pino and RG Yaeger

1985

GS Visvesvara

WM Wenman

GS Visvesvara

GS Visvesvara

GS Visvesvara

PM Wallis

ME Olson

N/A

FD Gillin

TE Nash

TE Nash

TE Nash

TE Nash

TE Nash

TE Nash

N/A

WD O’Dell

GS Visvesvara

GS Visvesvara

GS Visvesvara

GS Visvesvara

GS Visvesvara

UTI, Inc.

UTI, Inc.

2003

2003

2003

2003

2003

1998

1998

2000 1998

FD Gillin

1996

1996

1996

1996

1996

1996

1995

1988

UTI, Inc.f

TE Nash

TE Nash

TE Nash

TE Nash

TE Nash

TE Nash

LS Diamond

WD O’Dell

CE Kirkpatrick CE Kirkpatrick 1987

CE Kirkpatrick CE Kirkpatrick 1986

LC Pino

CE Kirkpatrick CE Kirkpatrick 1985

FD Gillin

EA Meyer

Isolated by

N/A

(Wenman et al., 1986)

(Abaza et al., 1991)

N/A

N/A

(Wenman et al., 1986)

(Buret et al., 1990)

(Teoh et al., 2000)

(Gillin and Diamond, 1980)

(Nash and Keister, 1985)

(Nash and Keister, 1985)

(Adam et al., 1988; Aggarwal and Nash, 1988; Nash et al., 1988)

(Nash et al., 1988)

(Aggarwal et al., 1989)

(Nash et al., 1985, 1990b)

N/A

(Abaza et al., 1991)

(Kiorpes et al., 1987)

(Mahbubani et al., 1991)

(Campbell et al., 1990)

(Kirkpatrick and Green, 1985)

(Smith et al., 1982)

(Meyer, 1976)

Referencesg

414 R. Molestina and H. D. Luján

D. Hall (DH, NIH:1083-1)

DAN (NIH:0984) Human

AC (NIH:0484:1) Human

BE-1 (IP:0482:1)

BE-2 (IP-0583:1)

PRA-246e

PRA-247e

e

e

PRA-250e

G1M

G2M

NIC (N, NIH:0782)

PRA-252e

PRA-254

Human

Human

Human

Beaver

Beaver

New Orleans, LA, USA

Peru

Peru

Calgary, Canada

Banff National Park, Canada

Dominican Republic

USA

West Virginia, USA

Maryland, USA

USA

N/A

Florida, USA

N/A

N/A

N/A

N/A

N/A

N/A

N/A

N/A

N/A

N/A

N/A

N/A

TE Nash

TE Nash

TE Nash

G Faubert

G Faubert

TE Nash

TE Nash

TE Nash

TE Nash

TE Nash

TE Nash

TE Nash

TE Nash

TE Nash

TE Nash

TE Nash

TE Nash

TE Nash

TE Nash

TE Nash

TE Nash

TE Nash

TE Nash

TE Nash

2007

2007

2007

2007

2007

2007

2007

2007

2007

2007

2007

2007

(Nash et al., 1985)

(Nash et al., 1990a)

(Nash et al., 1990a)

(Nash et al., 1985)

(Nash et al., 1985)

(Nash and Keister, 1985)

(Nash et al., 1990a)

(Nash et al., 1985)

(Nash and Keister, 1985)

(Nash et al., 1990a)

N/A

(Nash et al., 1985)

The table was assembled based on information provided by the depositors in the ATCC deposit forms and personal communications; bThe ATCC trademark and trade name and any and all ATCC catalog numbers are trademarks of the American Type Culture Collection; cdeposited as Giardia cati; ddeposited as Giardia sp.; eAvailable at BEI Resources in addition to the ATCC general collection; fUniversity Technologies International, Calgary, Canada; goldest references citing the strain; N/A, information not available.

a

PRA-251

e

PRA-249

PRA-248

Human

Human

SUG (NIH:0784:1)

PRA-245e

PRA-244

Human

Human

Mario (NIH:1284-1)

Egypt-4

PRA-243e

Human

e

CM (NIH:0883:2)

PRA-242e

Chap. 26 Biological Resource Centers for Giardia Research 415

416

R. Molestina and H. D. Luján

Table 26.2 SSGCID clones harboring Giardia lamblia genes available at BEI Resourcesa BEI No.

SSGCID clone

NCBI accession

Description

NR-16612

GilaA.10478.a.A1

XP_001706262.1

Branched-chain amino acid aminotransferase lateral transfer candidate

NR-16613

GilaA.10520.a.A1

XP_001709175.1

Hydroxyacylglutathione hydrolase

NR-16614

GilaA.10520.b.A1

XP_001708067.1

Hydroxyacylglutathione hydrolase

NR-16615

GilaA.17029.a.A1

XP_001708073.1

Metallo-beta-lactamase superfamily protein

a

Clones available at BEI Resources represent un-induced expression constructs which have been verified by sequencing in both directions from vector primers.

All structures are to be submitted to the Protein Data Bank (PDB), and all materials (clones and protein) generated are to be made publicly available through deposition in BEI Resources. A list of expression clones for different Giardia proteins that are targeted by the SSGCID and available through BEI Resources is shown in Table 26.2. Giardia researchers should consider BEI Resources as a centralized BRC for a variety of biomaterials including antisera, monoclonal antibodies, purified proteins, and plasmid constructs. By centralizing these resources, access to and use of these materials in the scientific community are monitored and quality control of the reagents is assured. Benefits of depositing include secure storage, community access and distribution; all while protecting the intellectual property rights of the depositor.

References Abaza SM, Sullivan JJ, and Visvesvara GS (1991) Isoenzyme profiles of four strains of Giardia lamblia and their infectivity to jirds. Am J Trop Med Hyg 44: 63–68 Adam RD, Aggarwal A, Lal AA, de La Cruz VF, McCutchan T, and Nash TE (1988) Antigenic variation of a cysteine-rich protein in Giardia lamblia. J Exp Med 167: 109–118 Aggarwal A and Nash TE (1988) Antigenic variation of Giardia lamblia in vivo. Infect Immun 56: 1420–1423 Aggarwal A, Merritt JW Jr, and Nash TE (1989) Cysteine-rich variant surface proteins of Giardia lamblia. Mol Biochem Parasitol 32: 39–47 Buret A, denHollander N, Wallis PM, Befus D, and Olson ME (1990) Zoonotic potential of giardiasis in domestic ruminants. J Infect Dis 162: 231–237 Campbell SR, van Keulen H, Erlandsen SL, Senturia JB, and Jarroll EL (1990) Giardia sp.: comparison of electrophoretic karyotypes. Exp Parasitol 71: 470–482

Gillin FD and Diamond LS (1980) Clonal growth of Giardia lamblia trophozoites in a semisolid agarose medium. J Parasitol 66: 350–352 Kiorpes AL, Kirkpatrick CE, and Bowman DD (1987) Isolation of Giardia from a llama and from sheep. Can J Vet Res 51: 277–280 Kirkpatrick CE and Green GAt (1985) Susceptibility of domestic cats to infections with Giardia lamblia cysts and trophozoites from human sources. J Clin Microbiol 21: 678–680 Mahbubani MH, Bej AK, Perlin M, Schaefer FW 3rd, Jakubowski W, and Atlas RM (1991) Detection of Giardia cysts by using the polymerase chain reaction and distinguishing live from dead cysts. Appl Environ Microbiol 57: 3456–3461 Meyer EA (1976) Giardia lamblia: isolation and axenic cultivation. Exp Parasitol 39: 101–105 Nash TE and Keister DB (1985) Differences in excretory-secretory products and surface antigens among 19 isolates of Giardia. J Infect Dis 152: 1166–1171 Nash TE, McCutchan T, Keister D, Dame JB, Conrad JD, and Gillin FD (1985) Restriction-endonuclease analysis of DNA from 15 Giardia isolates obtained from humans and animals. J Infect Dis 152: 64–73 Nash TE, Aggarwal A, Adam RD, Conrad JT, and Merritt JW Jr (1988) Antigenic variation in Giardia lamblia. J Immunol 141: 636–641 Nash TE, Conrad JT, and Merritt JW Jr (1990a) Variant specific epitopes of Giardia lamblia. Mol Biochem Parasitol 42: 125–132 Nash TE, Herrington DA, Levine MM, Conrad JT, and Merritt JW Jr (1990b) Antigenic variation of Giardia lamblia in experimental human infections. J Immunol 144: 4362–4369 Smith PD, Gillin FD, Kaushal NA, and Nash TE (1982) Antigenic analysis of Giardia lamblia from Afghanistan, Puerto Rico, Ecuador, and Oregon. Infect Immun 36: 714–719 Teoh DA, Kamieniecki D, Pang G, and Buret AG (2000) Giardia lamblia rearranges F-actin and alpha-actinin in human colonic and duodenal monolayers and reduces transepithelial electrical resistance. J Parasitol 86: 800–806 Wenman WM, Meuser RU, and Wallis PM (1986) Antigenic analysis of Giardia duodenalis strains isolated in Alberta. Can J Microbiol 32: 926–929

List of Contributors

Aws Abdul-Wahid Department of Cancer Genomics and Proteomics University Health Network – Ontario Cancer Institute Princess Margaret Hospital Room 7–106, 610 University Ave. Toronto, Ontario Canada M5G-2M9 Rodney D. Adam Departments of Medicine and Immunobiology University of Arizona College of Medicine Tucson, AZ, USA Stephen B. Aley Department of Biological Sciences Infectious Diseases and Immunology Program Border Biomedical Research Center University of Texas at El Paso El Paso, Texas, USA Igor C. Almeida Department of Biological Sciences Infectious Diseases and Immunology Program Border Biomedical Research Center University of Texas at El Paso El Paso, Texas, USA Raúl Argüello-García Department of Genetics and Molecular Biology Centro de Investigación y de Estudios Avanzados IPN México, DF México Maria Luisa Bazán-Tejeda Department of Genetics and Molecular Biology Centro de Investigación y de Estudios Avanzados IPN México, DF México

Marlene Benchimol Laboratório de Ultraestrutura Celular Universidade Santa Ursula Rio de Janeiro, Brazil

Rosa María Bermúdez-Cruz Department of Genetics and Molecular Biology Centro de Investigación y de Estudios Avanzados IPN México, DF México

Andre G. Buret James Cotton University of Calgary Calgary (AB) Canada T2N 1N4

Simone M. Cacciò Department of Infectious Parasitic and Immunomediated Diseases Istituto Superiore di Sanità Viale Regina Elena 299 Rome 00161, Italy

Atasi De Chatterjee Department of Biological Sciences Border Biomedical Research Center University of Texas at El Paso El Paso, Texas, USA

Michael Cipriano Section of Microbiology University of California at Davis, Davis, CA 95616, USA

418

Siddhartha Das Department of Biological Sciences Infectious Diseases and Immunology Program Border Biomedical Research Center University of Texas at El Paso El Paso, Texas, USA Barbara J. Davis Department of Pathology University of California at San Diego 214 Dickinson Street San Diego, CA 92103-8416, USA Scott C. Dawson Department of Microbiology One Shields Avenue UC Davis, Davis CA 95616, USA Pavel Doležal Faculty of Science Department of Parasitology Charles University in Prague Prague, Czech Republic Gaétan Faubert Institute of Parasitology Macdonald campus of McGill University 21,111 Lakeshore Boulevard Sainte-Anne-de-Bellevue Québec, Canada H9X 3V9 Rocio Fonseca-Liñán Department of Genetics and Molecular Biology Centro de Investigación y de Estudios Avanzados IPN México, DF México Pablo R. Gargantini Laboratory of Biochemistry and Molecular Biology School of Medicine Catholic University of Córdoba CP X5004ASK, Córdoba Argentina Thomas Geurden Laboratory for Parasitology Faculty of Veterinary Medicine Ghent University, Salisburylaan 133 9820 Merelbeke, Belgium

List of Contributors

Frances D. Gillin University of California at San Diego Department of Pathology 214 Dickinson Street San Diego, CA 92103-8416, USA Adrian B. Hehl Institute of Parasitology University of Zurich, 8057 Zurich Switzerland Edward L. Jarroll Department of Biological Sciences Lehman College, CUNY Bronx, NY, USA Joella Joseph Biology and Chemistry Departments Environmental and Life Sciences Graduate Program Trent University, Peterborough Ontario, Canada K9J 7B8 Harry van Keulen Department of Biological Geological and Environmental Sciences Cleveland State University Cleveland, OH 44115, USA Tineke Lauwaet Department of Pathology, University of California at San Diego 214 Dickinson Street San Diego, CA 92103-8416, USA Peter Lee School of Veterinary Medicine Department of Biochemistry Nippon Veterinary and Life Science University 1-7-1 Kyonancho, Musashino Tokyo, Japan 180-8602 Yvonne Ai Lian Lim Faculty of Medicine Building Department of Parasitology University of Malaya 50603 Kuala Lumpur, Malaysia

List of Contributors

Donald G. Lindmark Department of Biological Geological and Environmental Sciences Cleveland State University Cleveland, OH 44115, USA Hugo D. Luján Laboratory of Biochemistry and Molecular Biology School of Medicine Catholic University of Córdoba, CP X5004ASK Córdoba, Argentina Theo G. Mank Laboratory of Public Health Department of Parasitology Boerhaavelaan 26, 2035 RC Haarlem The Netherlands Robert Molestina Protistology Department American Type Culture Collection Manassas, Virginia, USA Paul T. Monis Australian Water Quality Centre South Australian Water Corporation Adelaide, SA 5000, Australia Hilary G. Morrison Josephine Bay Paul Center Marine Biological Laboratory Woods Hole, MA 02543, USA Ernesto S. Nakayasu Pacific Northwest National Laboratory Richland, WA 99352, USA and Department of Biological Sciences Border Biomedical Research Center University of Texas at El Paso El Paso, Texas, USA Theodore E. Nash Clinical Parasitology Unit and Helminth Immunology Section Laboratory of Parasitic Diseases National Institute of Allergy and Infectious Diseases National Institutes of Health Bethesda, Maryland 20892, USA

419

Eva Nohýnkov 1st Faculty of Medicine Department of Tropical Medicine Charles University 128 00 Prague 2 Czech Republic Merle Olson Bow Valley Research Calgary Alberta, Canada and Department of Biomedical Engineering University of Alberta Edmonton Alberta, Canada Guadalupe Ortega-Pierres Department of Genetics and Molecular Biology Centro de Investigación y de Estudios Avanzados IPN México, DF México Timothy A. Paget Medway School of Pharmacy The Universities of Kent and Greenwich at Medway Chatham Maritime, Kent ME4 4TB César G. Prucca Laboratory of Biochemistry and Molecular Biology School of Medicine Catholic University of Córdoba CP X5004ASK, Córdoba Argentina Phillips W. Robbins Department of Molecular and Cell Biology Boston University Boston, MA 02118-2394, USA Lucy J. Robertson Parasitology Laboratory Department of Food Safety and Infection Biology Norwegian School of Veterinary Science Postbox 8146 Dep., 0033 Oslo Norway

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John Samuelson Department of Molecular and Cell Biology Boston University Boston, MA 02118-2394, USA

Steve M. Singer Department of Biology and Center for Infectious Disease Georgetown University Washington, DC USA Huw V. Smith† Scottish Parasite Diagnostic Laboratory Stobhill Hospital Glasgow G21 3UW Scotland, UK

List of Contributors

Staffan Svärd Department of Cell and Molecular Biology Uppsala University SE-75124 Uppsala, Sweden Jan Tachezy Faculty of Science Department of Parasitology Charles University in Prague Prague, Czech Republic R. C. Andrew Thompson World Health Organization Collaborating Centre for the Molecular Epidemiology of Parasitic Infections School of Veterinary and Biomedical Sciences Murdoch University Murdoch, WA 6150 Australia

Wanderley de Souza Laboratório de Ultraestrutura Celular Hertha Meyer Instituto de Biofísica Carlos Chagas Filho Universidade Federal do Rio de Janeiro Cidade Universitária, Ilha do Fundão Rio de Janeiro 21941-902, Brazil and Instituto Nacional de Metrologia Normalização e Qualidade Industrial Inmetro

Karin Troell National Veterinary Institute 751 89 Uppsala Sweden

Hein Sprong Laboratory for Zoonoses and Environmental Microbiology National Institute for Public Health and Environment (RIVM) Mailbox 63, Antonie van Leeuwenhoeklaan 9 P.O. Box 1, 3720 BA, Bilthoven The Netherlands

Mayte Yichoy Department of Veterinary Pathobiology Texas A & M University Texas 77843, USA and Department of Biological Sciences Border Biomedical Research Center University of Texas at El Paso El Paso, Texas, USA

Janet Yee Biology and Chemistry Departments Environmental and Life Sciences Graduate Program Trent University, Peterborough Ontario, Canada K9J 7B8