Parasites are of global significance in terms of human an animal health and research is constantly targeted at controlling such infections. In order for this to be effective, detailed analyses of the biology of each species, particularly at the molecular level, must be carried out to promote the development of new therapeutic or diagnostic approaches. This laboratory manual, with detailed background information and practical protocols, will be a useful guide for researchers engaged in many areas of parasitology. Most techniques described can be applied to both helminthic and protozoan parasites, although protocols relevant to individual species are also included. It is largely recommended for postgraduate and postdoctoral scientists and provides procedures for some basic techniques in immunological, microscopical, and molecular analyses along with more specialized schemes to give a multidisciplinary approach to experimental parasitology. Parasite infections are very widespread, nevertheless effective therapeuticals are not yet available. The analysis of the life cycle and the parasite host interactions at the molecular level may help in the search of the "Achilles heel" of a parasite and thus promote the development of new therapeutic approaches. Parasite molecules such as surface antigens, excretory proteins or metabolic enzymes may serve as targets for new diagnostics tests, chemo- or immunotherapeutics or even as candidate vaccine.

Klappentext

Parasitic diseases still affect millions of people every year, especially in the tropics, causing considerable morbidity or death. Such infections within livestock are probably an even bigger problem, leading to poorer productivity, condemna­ tion of infected meat and considerable economic loss. Para­ sitological research has, however, helped the situation in some cases and the development of novel drugs, vaccines and diagnostics has improved our chances of controlling these diseases. Research into parasitic infections is, therefore, often goal­ orientated. However, the study of parasites and host/parasite relationships still remains one of the most exciting and in­ teresting aspects of biology. Scientists, from undergraduate students to research professors, frequently ponder over how endoparasitic organisms can survive within the most alien of environments - inside another organism. The nutritional, reproductive and survival strategies which have evolved within each group of parasites have allowed the development of highly specific host-parasite relationships and allow the successful transmission of the parasite from one host to an­ other. A considerable amount of research is therefore direc­ ted at improving our understanding of various aspects of parasite biology.

Inhalt

1 Biochemical Pathways in Parasites.- 1.1 Introduction.- 1.2 Preparation of Enzyme Fractions.- 1.3 Enzyme Assays.- 1.4 Metabolite Measurements.- 1.5 Use of Inhibitors.- References.- 2 Electrophoresis of Parasite Proteins.- 2.1 Introduction.- 2.2 Lysis of Parasites and Extraction of Proteins.- 2.2.1 Solubilisation in SDS Sample Cocktail.- 2.2.2 Detergent Solubilisation.- 2.3 SDS-PAGE.- 2.4 Modifications of Standard Method.- 2.4.1 Gradient Gels.- 2.4.2 Tricine Gels for the Analysis of Very Low Molecular Weight Polypeptides.- 2.5 Applications of SDS-PAGE to the Characterisation of Individual Polypeptides.- 2.5.1 Glycoprotein Analysis Using Specific Enzymes.- 2.5.2 Peptide Mapping.- 2.5.3 Preparative SDS-PAGE and Electroelution.- 2.6 2D Gel Electrophoresis.- 2.6.1 Non-Equilibrium pH Gradient Electrophoresis (NEPHGE).- 2.6.2 Isoelectric Focusing (IEF).- References.- 3 Molecular Techniques in Analytical Parasitology.- 3.1Introduction.- 3.2 Isolation of Parasite Nucleic Acid.- 3.2.1 Preparation of DNA.- 3.2.2 Preparation of RNA.- 3.2.3 Quantification of Nucleic Acid.- 3.3 Gel Electrophoresis and Blotting of Nucleic Acid.- 3.3.1 Gel Electrophoresis.- 3.3.2 Blotting of Nucleic Acid.- 3.4 Labelling and Hybridisation of DNA Probes.- 3.4.1 Radiolabelling of DNA Probes.- 3.4.2 Non-Isotopic Labelling of DNA Probes.- 3.4.3 Hybridisation and Signal Detection.- 3.5 Polymerase Chain Reaction (PCR).- 3.5.1 Reverse Transcriptase - PCR (RT-PCR).- 3.5.2 Touchdown PCR.- References.- 4 The Production and Analysis of Helminth Excretory-Secretory (ES) Products.- 4.1 Introduction.- 4.2 Collection of Excretory-Secretory (ES) Products.- 4.3 Acetylcholinesterase.- 4.3.1 Quantitation of Acetylcholinesterase Activity.- 4.3.2 Detection of AChE Activity Following Polyacrylamide Gel Electrophoresis.- 4.3.3 Purification of AChE.- 4.3.4 Identification and Separation of AChE Molecular Forms.- 4.3.5 Kinetic Properties of AChE.- 4.4 Proteolytic Enzymes.- 4.5 Anticoagulants, Blood Coagulation Assays.- 4.5.1 The Collection of Plasma.- 4.5.2 Recalcification Time Test (Intrinsic Pathway).- 4.5.3 Activated Partial Thromboplastin Test (Intrinsic Pathway).- 4.5.4 Prothrombin Time Test (Extrinsic Pathway).- 4.5.5 Stypven Clotting Time Test (Common Pathway).- 4.5.6 Platelet Aggregation.- 4.6 Potential Immunomodulators.- 4.6.1 Glutathione s-Transferases.- 4.6.2 Superoxide Dismutase (SODs).- 4.6.3 Detection of Immunomodulation of Antibody Production.- 4.6.4 Neutrophil Inhibitory Factor (NIF).- 4.7 Concluding Remarks.- References.- 5 Parasite Proteinases.- 5.1 Introduction.- 5.2 Sample Preparation.- 5.3 Substrates and Proteinase Assays.- 5.3.1 Optimal Conditions for Proteinase Activity.- 5.3.2 Protein and Peptide Substrates.- 5.3.3 Synthetic Fluorogenic and Chromogenic Substrates.- 5.3.4 Exopeptidase Substrates.- 5.4 Proteinase Inhibitors.- 5.4.1 Standard Procedures for Enzyme Inhibition.- 5.4.2 The Selection and Design of Specific Inhibitors.- 5.5 Electrophoretic Methods.- 5.5.1 Substrate-SDS-PAGE - General Procedure.- 5.5.2 Substrate-SDS-PAGE - Additional Techniques.- 5.5.3 Affinity Labelling.- 5.6 Purification Methods.- 5.7 Endogenous Inhibitors.- 5.8 Final Comments.- References.- 6 Neurobiology of Helminth Parasites.- 6.1 Introduction.- 6.2 Immunocytochemical Localisation of Neuroactive Substances in Helminth Parasites.- 6.2.1 Production of Antisera.- 6.2.2 Immunocytochemistry Protocol.- 6.2.3 Confocal Scanning Laser Microscopy (CSLM).- 6.2.4 Electron Microscope Immunogold Labelling.- 6.3 Characterisation and Purification of Neuropeptides.- 6.3.1 Tissue Extraction.- 6.3.2 Peptide Detection.- 6.3.3 Primary Purification Steps.- 6.3.4 Reverse-Phase HPLC.- 6.3.5 Endoproteinase Digestion.- 6.4 Physiological Action of Transmitters and Drug Assessment In Vitro.- 6.4.1 Recording of Motor Activity in Whole Animals or in Muscle Strips.- 6.4.2 Action of Transmitters and Drugs at the Cellular Level.- References.- 7 Electron Microscopy in Parasitology.- 7.1 Introduction.- 7.2 Preparation for Transmission Electron Microscopy (TEM).- 7.2.1 Fixation: General Background.- 7.2.2 Standard Fixation Methods.- 7.2.3 Standard Fixation Protocols.- 7.3 Cytochemistry and Staining Protocol in TEM.- 7.3.1 Introduction.- 7.3.2 Standard Stains.- 7.3.3 Special Cytochemical Stains.- 7.3.4 Enzyme Stains.- 7.4 Preparation for Scanning Electron Microscopy (SEM).- 7.5 Immunogold-Labelling Techniques.- 7.5.1 Basic Information.- 7.5.2 Approaches for Immunogold-Labelling.- 7.5.3 Applications of Immunogold EM Techniques.- References.- 8 Studies of the Surface Properties, Lipophilic Proteins and Metabolism of Parasites by the Use of Fluorescent and "Caged" Compounds.- 8.1 Introduction.- 8.2 Incorporation of Fluorescent Compounds into Membranes.- 8.2.1 Surface Membrane Incorporation of Fluorescent Compounds.- 8.2.2 Examination of Location of Fluorescent Probe in Membrane.- 8.2.3 Uptake of Fluorescent Fatty Acids and Cholesterol.- 8.2.4 Labelling Parasite Surfaces with a "Stable" Cell-Labelling Reagent (Sigma, PKH2 and PKH26).- 8.3 Using Fluorescent Techniques to Measure Membrane Fluidity.- 8.4 Distribution of Actin.- 8.5 Assessment of Parasite Membrane Damage.- 8.6 Localisation of Probes in Orga…