Fasciola hepatica: Methods and Protocols (Methods in Molecular Biology, 2137)

Preface
Contents
Contributors
Chapter 1: Maintenance of Life Cycle Stages of Fasciola hepatica  in the Laboratory
	1 Introduction
	2 Materials
		2.1 Maintenance of Lymnaea neotropica Colony in the Laboratory
			2.1.1 Snail Culture
			2.1.2 Oscillatoria Culture
		2.2 Obtaining Eggs from F. hepatica
		2.3 Hatching of Miracidia by Incubation of Eggs
		2.4 Snail Infection
		2.5 Development of F. hepatica Inside the Snail and Metacercariae Obtention
		2.6 In Vitro Excystment of F. hepatica Metacercariae
			2.6.1 Plasticware
			2.6.2 Solutions
			2.6.3 Equipment and Instruments
		2.7 Obtaining F. hepatica Adult Flukes
	3 Methods
		3.1 Maintenance of Lymnaea neotropica Colony in the Laboratory
			3.1.1 Oscillatoria Culture
			3.1.2 Lymnaea neotropica Colony
		3.2 Obtaining Eggs from F. hepatica
		3.3 Hatching of Miracidia by Incubation of Eggs
		3.4 Snail Infection
		3.5 Development of F. hepatica Inside the Snail and Metacercaria Obtention
		3.6 In Vitro Metacercariae Excystment
		3.7 NEJ Culture
		3.8 Obtaining F. hepatica Adult Flukes
	4 Notes
	References
Chapter 2: Microscopical Techniques to Analyze the Hepatic and Peritoneal Changes Caused by Fasciola hepatica Infection
	1 Introduction
	2 Materials
		2.1 Recovery of Peritoneal Fluid
		2.2 Total Peritoneal Cell Count
		2.3 Differential Cell Count Using Immunocytochemistry and Hematoxylin and Eosin Counterstain
		2.4 Pathological Examination
	3 Methods
		3.1 Recovery of Peritoneal Fluid
		3.2 Total Peritoneal Cell Count
		3.3 Differential Cell Count Using Immunocytochemistry and Hematoxylin and Eosin Counterstain
		3.4 Pathological Examination
			3.4.1 Gross Pathology
			3.4.2 Histopathology
	4 Notes
	References
Chapter 3: Isolation of Secreted and Tegumental Surface Proteins from Fasciola hepatica
	1 Introduction
	2 Materials
		2.1 Collection of F. hepatica Excretory-Secretory Proteins In Vitro
		2.2 Filter Device Concentration of Excretory-Secretory Products
		2.3 Acetone Precipitation from Large Volumes
		2.4 Methanol-Chloroform Precipitation from Small Volumes
		2.5 In-Gel Trypsin Digestion
		2.6 Biotinylation of F. hepatica Tegumental Surface Proteins
		2.7 Protein Extraction and Streptavidin Pull-Down of the Biotinylated Proteins
		2.8 Soft Trypsin Digestion of F. hepatica
	3 Methods
		3.1 Collection of F. hepatica Excretory-Secretory Proteins In Vitro
		3.2 Filter Device Concentration of Excretory-Secretory Products
		3.3 Acetone Precipitation from Large Volumes of ES
		3.4 Methanol-Chloroform Precipitation from Small Volumes of ES
		3.5 In-Gel Trypsin Digestion
		3.6 Biotinylation of F. hepatica Tegumental Surface Proteins
		3.7 Protein Extraction and Streptavidin Pull-Down of the Biotinylated Proteins
		3.8 ``Soft´´ Trypsin Digestion of F. hepatica Newly Excysted Juveniles (NEJ)
	4 Notes
	References
Chapter 4: Isolation and Analysis of Fasciola hepatica Extracellular Vesicles
	1 Introduction
	2 Materials
		2.1 Parasite Culture
		2.2 Plasma and Urine Sampling
		2.3 Ultracentrifugation
		2.4 Analyses and Characterization of F. hepatica EVs
		2.5 Apparatus
	3 Methods
		3.1 Parasite Culture
		3.2 EVs Isolation by Differential Centrifugation
		3.3 EVs Isolation from Biological Samples
			3.3.1 Initial Processing of Plasma
			3.3.2 Initial Processing of Urine
		3.4 EVs Isolation by Size Exclusion Chromatography
		3.5 Storage of Functional EVs (Freeze-Drying/Lyophilization in Sucrose 10%)
		3.6 Analyses and Characterization of F. hepatica EVs
			3.6.1 Analysis by Transmission Electron Microscopy (TEM)
			3.6.2 Analysis by Scanning Electron Microscopy (SEM) of F. hepatica Adults
			3.6.3 Immuno-Gold Labeling of Fasciola hepatica EVs
			3.6.4 Fluorescent Labeling of Fasciola hepatica EVs and In Vivo Uptake by Cells in Culture: Confocal Microscopy
			3.6.5 Labeling of EVs Membrane and RNA Components
			3.6.6 Uptake of Labeled F. hepatica EVs
	4 Notes
	References
Chapter 5: Cloning and Heterologous Expression of Protein-Coding Sequences in Escherichia coli
	1 Introduction
	2 Materials
		2.1 Medium and Antibiotics
		2.2 Nucleic Acid Electrophoresis
		2.3 Preparing F. hepatica cDNA
		2.4 Preparing DNA Vector
		2.5 ORF Amplification by PCR
		2.6 Cloning by In Vivo Recombination Using recA+ E. coli Strain
		2.7 Colony PCR to Confirm Recombinant Clones
		2.8 Production of Recombinant Protein Fused to GST
		2.9 Purification of GST-Fused Protein by Glutathione-Sepharose Affinity Chromatography
		2.10 Preparation of KC8 Competent Cells
		2.11 Cloning FhTGR Wild Type and Their Mutant Sequences into TA Cloning Vector and pET28a Expression Vector
		2.12 Expression and Purification of Recombinant FhTGR
	3 Methods
		3.1 Preparing F. hepatica cDNA
		3.2 Preparing DNA Vector
		3.3 ORF Amplification by PCR
		3.4 Cloning by In Vivo Recombination in recA+ E. coli Strain
		3.5 Colony PCR to Confirm Recombinant Clones
		3.6 Production of Recombinant Protein Fused to GST
		3.7 Purification of GST-Fused Protein by Glutathione-Sepharose Affinity Chromatography
		3.8 Preparation of KC8 Competent Cells
		3.9 Cloning FhTGR Wild Type and Their Mutant Sequences into TA Cloning Vector and pET28a Expression Vector
		3.10 Expression and Purification of Recombinant FhTGR
	4 Notes
	References
Chapter 6: Gene Silencing in the Liver Fluke Fasciola hepatica: RNA Interference
	1 Introduction
	2 Materials
		2.1 Parasites
		2.2 Parasite Culture
		2.3 Parasite Viability
		2.4 Generation of Interfering and Reporter RNA Molecules
		2.5 Delivery of Interfering and Reporter RNA Molecules
		2.6 Gene Knockdown Analysis at the mRNA Level
		2.7 Gene Knockdown Analysis at the Protein Level
		2.8 Gene Knockdown Analysis at the Phenotypic Level
	3 Methods
		3.1 In Vitro Excystment, Collection and Culture of NEJs
		3.2 Monitoring the Viability of the Parasites
		3.3 Design and Preparation of siRNA Molecules
		3.4 Design and Preparation of Long dsRNA Molecules (Longer than 250 bp)
		3.5 dsRNA Quantification and Electrophoretic Analysis
		3.6 Precipitation of the dsRNA (Optional)
		3.7 Long dsRNA Labeling
		3.8 Synthesis of Reporter mRNA
		3.9 Purification and Quantification of Mature Capped and Polyadenylated mRNA
		3.10 Delivery of dsRNA by Soaking
		3.11 Delivery of dsRNA by Electrosoaking
		3.12 Monitoring RNA Incorporation in Live Parasites
		3.13 Monitoring RNA Incorporation in Fixed Parasites
		3.14 RNA Isolation for Gene Silencing Analysis at the mRNA Level
		3.15 Reverse Transcription
		3.16 Gene Expression Quantitation by Quantitative RT-PCR (RT-qPCR)
		3.17 mRNA Detection by Whole-Mount In Situ Hybridization (WISH)
		3.18 Analysis of the Gene Silencing at the Protein Level
		3.19 Analysis of the Gene Silencing at the Phenotypic Level
	4 Notes
	References
Chapter 7: Analysis of Gene Expression in Fasciola hepatica Juveniles and Adults by In Situ Hybridization
	1 Introduction
	2 Materials
		2.1 Materials for In Vitro Labeling of RNA Probes with DIG
		2.2 Materials for Probe Quantification by Dot-Blot
		2.3 Materials for Fluorescent ISH on Sections of F. hepatica Adults
		2.4 Materials for WMISH of F. hepatica NEJs
	3 Methods
		3.1 In Vitro Transcription of DIG-Labeled Probes
		3.2 Dot-Blot Quantification of Probes
		3.3 Fluorescent ISH on Cryosections of F. hepatica Adults
		3.4 WMISH of F. hepatica NEJ
	4 Notes
	References
Chapter 8: Evasion of Host Immunity During Fasciola hepatica Infection
	1 Immunity to F. hepatica
	2 Mechanisms of Immune Evasion
	3 Enzymatic Modulators
		3.1 Cathepsins
		3.2 Peroxiredoxin
	4 Nonenzymatic Modulators
		4.1 HDM
		4.2 TLM
	5 Summary
	References
Chapter 9: Immunomodulatory Effect of Fasciola hepatica Excretory-Secretory Products on Macrophages
	1 Introduction
	2 Materials
		2.1 Preparation of Fasciola hepatica Excretory-Secretory Products (FhESP)
		2.2 Purification and Culture of Macrophages from Peritoneal Exudate Cells (PECs)
		2.3 Peritoneal Macrophage (pMPhi) Activation Assays
			2.3.1 Measurement of Macrophage Cytokine Production in Culture Supernatants by Enzyme-Linked Immunosorbent Assay (ELISA)
			2.3.2 Arginase Activity Assay
			2.3.3 Cytometric Analysis of Macrophage MHC-II and Costimulatory Molecule Expression
			2.3.4 Macrophage Antigen Presentation to CD4+T Lymphocytes
		2.4 Macrophage Apoptosis Assays
			2.4.1 Cytometric Analysis of Hypodiploid DNA After Propidium Iodide (PI) Staining
			2.4.2 Cytometric Analysis of Apoptotic Cells by Annexin V Staining
			2.4.3 Cytomorphologic Analysis of Macrophage Apoptosis by May-Grünwald-Giemsa Staining
	3 Methods
		3.1 Preparation of Fasciola hepatica Excretory-Secretory Products (FhESP)
		3.2 Purification and Culture of Macrophages from Peritoneal Exudate Cells (PECs)
		3.3 Macrophage Activation Assays
			3.3.1 Measurement of Macrophage Cytokine Production in Culture Supernatants by Enzyme-Linked Immunosorbent Assay (ELISA)
			3.3.2 Arginase Activity Assay
			3.3.3 Cytometric Analysis of Macrophage MHC-II and Costimulatory Molecule Expression
			3.3.4 Macrophage Antigen Presentation to CD4+T Lymphocytes
		3.4 Macrophage Apoptosis Assays
			3.4.1 Cytometric Analysis of Hypodiploid DNA After Propidium Iodide (PI) Staining
			3.4.2 Cytometric Analysis of Apoptotic Cells by Annexin V Staining
			3.4.3 Cytomorphology Analysis of Macrophage Apoptosis by May-Grünwald-Giemsa Staining
	4 Notes
	References
Chapter 10: Study of Eosinophil Apoptosis Induced by Fasciola hepatica Excretory-Secretory Products
	1 Introduction
	2 Materials
		2.1 Fasciola hepatica Excretory-Secretory Product (FhESP) Preparation
		2.2 Peritoneal Exudates Cells (PECs)
		2.3 Percoll Gradient and Eo Purification
		2.4 Eo Culture
		2.5 Apoptosis Assays
			2.5.1 Morphologic Assessment
			2.5.2 Hypodiploid DNA Quantification by Propidium Iodide
			2.5.3 Phosphatidylserine Surface Expression by Annexin V Assay
		2.6 Study of Apoptotic Mechanisms
			2.6.1 Measurement of Caspase Activity
			2.6.2 Analysis of the Mitochondrial Membrane Potential (DeltaPsim)
			2.6.3 Quantification of Malondialdehyde Levels
			2.6.4 Analysis of Reactive Oxygen Species (ROS) Production
			2.6.5 Determination of Superoxide Production
			2.6.6 Determination of Extracellular Hydrogen Peroxide Production
			2.6.7 Determination of Intracellular Hydrogen Peroxide
			2.6.8 Determination of Nitric Oxide (NO)
	3 Methods
		3.1 Fasciola hepatica Excretory-Secretory Product (FhESP) Preparation
		3.2 Peritoneal Exudates Cells (PECs)
		3.3 Percoll Gradient and Eo Purification
		3.4 Eo Culture
		3.5 Apoptosis Assays
			3.5.1 Morphologic Assessment
			3.5.2 Hypodiploid DNA Quantification by Propidium Iodide
			3.5.3 Phosphatidylserine Surface Expression by Annexin V Assay
		3.6 Study of Apoptotic Mechanisms
			3.6.1 Measurement of Caspase Activity
			3.6.2 Analysis of the Mitochondrial Membrane Potential (DeltaPsim)
			3.6.3 Quantification of Malondialdehyde Levels
			3.6.4 Analysis of Reactive Oxygen Species (ROS) Production
			3.6.5 Determination of Superoxide Production
			3.6.6 Determination of Extracellular Hydrogen Peroxide Production
			3.6.7 Determination of Intracellular Hydrogen Peroxide
			3.6.8 Determination of Nitric Oxide (NO)
	4 Notes
	References
Chapter 11: Purification of Native Fasciola hepatica Fatty Acid-Binding Protein and Induction of Alternative Activation of Hum...
	1 Introduction
	2 Materials
		2.1 Preparation of Fasciola hepatica Whole Worm Extract (FhWWE)
		2.2 Purification of Native 12 kDa Fatty Acid-Binding Protein
		2.3 Antigen Separation Through Sephadex G50 Column
		2.4 Separation by Isoelectric Focusing (IEF)
		2.5 Endotoxin Removal
		2.6 Endotoxin Detection
		2.7 Blood Sample Collection
		2.8 Isolation of Human Peripheral Blood Mononuclear Cells
		2.9 Human-Derived Macrophage and Fatty Acid-Binding Protein Treatment
		2.10 Nitrite Oxide Assay
		2.11 Arginase Activity
		2.12 Cell Viability
	3 Methods
		3.1 Preparation of FhWWE
		3.2 Purification of Native 12 kDa Fatty Acid-Binding Protein
		3.3 Antigen Separation Through Sephadex G50 Column
		3.4 Protein Separation by Isoelectric Focusing (IEF)
		3.5 Endotoxin Removal
		3.6 Endotoxin Detection
		3.7 Blood Sample Collections
		3.8 Isolation of Human Peripheral Blood Mononuclear Cells and In Vitro Culture
		3.9 Human-Derived Macrophage and Fatty Acid-Binding Protein Treatment
		3.10 Nitric Oxide Assay
		3.11 Arginase Activity
		3.12 Cell Viability Assay
	4 Notes
	References
Chapter 12: Possible Role for Toll-Like Receptors in Interaction of Fasciola hepatica Excretory-Secretory Products with Human ...
	1 Introduction
	2 Materials
		2.1 Preparation of Excretory-Secretory Products
		2.2 Antigenicity of ESPs
		2.3 Ion Exchange Chromatography and Protein Identification
		2.4 Screening System Using THP1-Blue CD14 Cell Treats with Fasciola hepatica ESPs Fractionated with an Ultrafiltration System
		2.5 NF-κB Activation in TLR4-Transfected HEK Cells Treated with Fasciola hepatica ESPs Fractionated with IEC Chromatography
	3 Methods
		3.1 Preparation of Excretory-Secretory Products (ESP)
		3.2 Antigenicity of ESPs
		3.3 Ion-Exchange Chromatography
		3.4 Screening System Using THP1-Blue CD14 Cell Treats with Fasciola hepatica ESPs Fractionated with an Ultrafiltration System
		3.5 NF-κB Activation in TLR4-Transfected HEK Cells Treated with Fasciola hepatica ESPs Fractionated with IEC Chromatography
	4 Notes
	References
Chapter 13: Evaluation of the Immune Regulatory Properties of Dendritic Cells During Fasciola hepatica Infection
	1 Introduction
	2 Materials
		2.1 Infection
		2.2 Purification of CD11c+ Cells
		2.3 Evaluation of DC Maturation
		2.4 Mixed Lymphocyte Reaction
	3 Methods
		3.1 Obtaining of Peritoneal CD11c+ Cells from F. hepatica-Infected Mice
		3.2 Evaluation of Cytokine Production and Expression of Surface Molecules by Peritoneal CD11c+ Cells from Infected Animals
		3.3 Regulatory Function in a Mixed Lymphocyte Reaction
	4 Notes
	References
Chapter 14: Design of a Peptide-Carrier Vaccine Based on the Highly Immunogenic Fasciola hepatica Leucine Aminopeptidase
	1 Introduction
	2 Materials
		2.1 Data
		2.2 Linear B-Cell Epitope Prediction Tools
		2.3 Homology Modeling Tools
			2.3.1 I-TASSER Web Server
			2.3.2 MODELLER Software
	3 Methods
		3.1 Overview
		3.2 B-Cell Epitope Prediction of the Target Protein
			3.2.1 Evaluation of Predicted B-Cell Epitopes
		3.3 Homology Modeling and Surface Accessibility
			3.3.1 Evaluation of Predicted Epitope Exposure
		3.4 Epitope-Carrier Assembly
		3.5 Final Considerations
	4 Notes
	References
Chapter 15: Liver Fluke Vaccine Assessment in Cattle
	1 Introduction
	2 Materials
		2.1 Vaccination Procedure
			2.1.1 Animal Model
			2.1.2 Antigen and Vaccine Formulation
			2.1.3 Serum Samples
			2.1.4 Oral Challenge
		2.2 Worm Recovery
		2.3 Peripheral Blood Mononuclear Cell (PBMC) Isolation
		2.4 Detection of Antibody Levels
	3 Methods
		3.1 Vaccination Procedure
		3.2 Worm Recovery
		3.3 PBMC Isolation
		3.4 Detection of Antibody Levels
	4 Notes
	References
Chapter 16: Testing Albendazole Resistance in Fasciola hepatica
	1 Introduction
	2 Materials
	3 Methods
		3.1 Egg Incubation
		3.2 Egg Development Test
	4 Notes
	References
Chapter 17: Drug Targets: Screening for Small Molecules that Inhibit Fasciola hepatica Enzymes
	1 Introduction
	2 Materials
		2.1 Cathepsin L Inhibitory Activity Screening
		2.2 Triose Phosphate Isomerase Inhibitory Activity Screening
		2.3 Compounds to Be Tested
	3 Methods
		3.1 Compound Stock Preparation
		3.2 Cathepsin L Inhibitory Activity Screening
		3.3 Triose Phosphate Isomerase Inhibitory Activity Screening
	4 Notes
	References
Correction to: Fasciola hepatica: Methods and Protocols
Index