Aims and objectives

This exercise is designed to demonstrate:

1. Site selection by a parasite.

2. Niche segregation by a parasite in the absence of competitors.

3. Relationships between parasite numbers, site selection and host size.

4. The manner in which parasites are dispersed throughout a host population.

Introduction

Although we often talk loosely about parasites living in or on a host, this should not be taken to imply that they can live in all tissues or organs of a host. Indeed, all parasites show some degree of site specificity in that they are only found in a particular organ or tissue and are able to select that site. Many of the adaptations they exhibit, e.g. an attachment mechanism, are actually adaptations to this particular site rather than to the host as such.

Interpretations of site selection within an ecological framework are not generally agreed. There is a large measure of agreement that the site of a parasite can be used as an indication or measure of its niche, but far less agreement about the ecological significance of, especially, very specific site selection (i.e., narrow niche breadth) and its relationship to resource partitioning and intra-and inter-specific competition between parasites. Many species appear to increase their niche breadth at higher densities and this is often taken as a manifestation of intra-specific competition. Similarly, species packing within an organ and a narrower niche breadth is considered by many workers to be a likely indication of inter-specific competition.

Aims and objectives

This exercise is designed to demonstrate the construction of a simple genomic DNA library. It can be adapted for use with any organism including parasites. The version given here is for the construction of a Plasmodium falciparum library.

The specific objectives of this exercise are:

1. To perform a ligation reaction.

2. To prepare competent E. coli cells.

3. To transform and plate these competent cells.

4. To grow small-scale cultures from recombinant colonies.

5. To prepare plasmid DNA from recombinants.

6. To analyse recombinant plasmids by agarose gel electrophoresis.

Introduction

The work to be described is aimed at the construction of a genomic library from an isolate of P. falciparum such as might be undertaken to analyse a specific gene. This type of cloning experiment is fundamental to a whole range of analyses in molecular biology.

Laboratory equipment and consumables

(per student or group)

Equipment

P20, P200 and P1000 Gilson pipetmen or equivalents

Waste beaker for chemically contaminated plasticware

Autoclavable waste bags for bacterially contaminated waste 37°C, 42°C and 65°C waterbaths

Bench-top centrifuge capable of producing 1000g 37°C incubator, static and orbital

Vortex machine

‘Camping gaz’ and lighter

Vacuum pump and desiccator

Eppendorf microfuge or equivalent

Heater/stirrer and magnetic stirring bar

Agarose gel electrophoresis apparatus and combs

Power supply for running gels

Photographic system for recording results

Safety glasses

Aims and objectives

This exercise is designed to:

Determine the susceptibility of nematode eggs to treatment with a benzimidazole anthelmintic (thiabendazole; TBZ).

Introduction

The widespread use of anthelmintics to control gastrointestinal parasitism in sheep and goats has resulted in the emergence of strains resistant to the benzimidazole, levamisole or avermectin group of drug families. In some countries, multiple resistant strains have been selected. In the UK, the majority of cases of anthelmintic resistance are to the benzimidazole group, and an in vitro test for detection of resistant nematodes is based on the fact that benzimidazoles will inhibit the embryonation and hatching of fresh nematode eggs. The assay is based on the ovicidal properties of benzimidazoles and the ability of eggs from resistant strains to develop and hatch at higher concentrations of drug than that of susceptible strains.

The aim is to incubate fresh nematode eggs in serial concentrations of a benzimidazole anthelmintic, normally thiabendazole (TBZ). The percentage of eggs that hatch (or die) at each TBZ concentration is determined and a drug response curve plotted; i.e. drug concentration is plotted against percentage hatch (or death) of eggs, corrected for the normal mortality of untreated eggs. To obtain a linear regression from which to calculate the ED50 value (concentration of drug required to kill 50% of eggs) the data are usually transformed using arcsin or log-probit transformation.

Aims and objectives

Using commercial conjugated lectins, these two exercises aim to:

1. Detect and ascertain surface membrane carbohydrates on trypanosomes and insect vector salivary glands.

2. Determine differences in salivary gland surface carbohydrates between Glossina species and between different regions of Anopheles salivary glands.

Introduction

Lectins are proteins, usually glycoproteins, derived from both animal and plant material, that recognise and bind to specific sugar moieties (or certain glycosidic linkages) of polysaccharides, glycoproteins and glycolipids present on cell membrane surfaces or in biological fluids (Lis & Sharon, 1986; Sharon & Lis, 1989). Lectin specificity is usually defined in terms of the monosaccharides or simple oligosaccharides that inhibit precipitation or binding reactions.

In order to detect membrane-bound carbohydrates, commercial lectins have been employed either non-conjugated (for use in agglutination/precipitation reactions) or conjugated with a marker (for use in cell or tissue surface membrane studies). The labels used include fluorescein isothiocyanate (FITC) and enzymes (e.g. peroxidase). These lectin-based techniques encompass the use of light and ultraviolet microscopy (Jacobson & Doyle, 1996).

The surface of intact vector tissues to which parasites may attach in order to undergo morphogenesis during transformation from one life cycle stage to the next, or attachment sites prior to transmission, have been studied (Ingram & Molyneux, 1991). These investigations, in part, enabled detection of carbohydrate moieties on a membrane surface that might act as binding sites to facilitate parasite adhesion.

Aims and objectives

This exercise aims to:

1. Differentiate Entamoeba histolytica from E. dispar.

2. Introduce students to PCR.

Introduction

Diagnosis of infectious diseases by the detection of nucleic acids specific for the infectious agent may offer considerable advantages over traditional laboratory diagnostic methods such as microscopy and culture. One such area where this may prove very useful is in the detection and differentiation of the two morphologically similar human parasitic amoebae, E. histolytica and E. dispar. Recent work has shown that E. histolytica, the causative organism of amoebic dysentery in humans, is in fact two separate species. Prior to this work it was thought that E. histolytica generally existed in humans without disease and, in about 10% of cases, gave rise to clinical disease. It has now been shown using a combination of methods (isoenzymes, biochemical, immunological and molecular) that infection with E. histolytica, sensu stricto, is likely to lead to disease whilst infection with E. dispar results in no symptoms.

The problem in trying to diagnose which of these two organisms is present in a patient is that both organisms are morphologically identical, both in the trophozoite stage and in the cystic form. This PCR (polymerase chain reaction) method uses two pairs of discrete primers that are able to recognise and differentiate the two species. PCR provides differential diagnosis of DNA extracted from faeces. The result is detected colorimetrically in a microtitre plate, which is simpler and safer than using radio-labelled probes.

Aims and objectives

This exercise aims to perform spectrophotometric analysis of Escherichia coli DNA, followed by endonucleolytic digestion of this bacterial genome and DNA from a variety of other sources with the restriction enzyme, EcoRI.

The specific objectives of this practical are:

1. To measure the A260 and A280 of E. coli DNA.

2. To perform restriction digests with EcoRI.

3. To prepare an agarose gel.

4. To analyse the restriction digests by agarose gel electrophoresis.

Introduction

The yield and purity of isolated DNA can be estimated relatively easily using ultraviolet (UV) spectrophotometry. A further test of DNA purity is to assess the extent that it can be cut with restriction endonucleases, enzymes that recognise and make a double-stranded cut at specific nucleotide sequences. This is an essential technique used in the construction of gene libraries and the analysis of cloned genes. The digestion products are separated by size on an agarose gel and visualised under UV illumination, after staining with the fluorescent dye ethidium bromide.

Laboratory equipment and consumables

(per student or group)

Equipment

UV spectrophotometer and quartz cuvettes P20, P200 and P1000 Gilson pipetmen or equivalents 37°C waterbath; 65°C waterbath

Heater/stirrer, magnetic stirrer bar

Heat-resistant gloves

Agarose gel electrophoresis equipment and combs

Power supply for running gels

UV transilluminator and photographic system

Consumables

Latex gloves in a range of sizes

Sterile tips for pipetmen

Ice bucket and wet ice

EcoRI restriction enzyme (5 units/μ1)

Sterile distilled water and TE buffer

Aims and objectives

This exercise is designed to demonstrate:

The susceptibility of nematode larvae to treatment with levamisole. The assay is based on the paralysing property of the drug and the ability of larvae from resistant strains to migrate at a higher concentration of drug than those of susceptible strains.

Introduction

The widespread use of anthelmintics to control gastrointestinal parasitism in sheep and goats has resulted in the emergence of strains of nematodes resistant to the benzimidazole, levamisole or avermectin group of drug families. In some countries, multiple resistant strains have been selected. In the UK, the majority of cases of anthelmintic resistance are to the drugs in the benzimidazole group, although resistance to avermectins and levamisoles has also been reported. An in vitro test for resistance to avermectins is based on the fact that the paralysis resulting from treatment with this drug inhibits the migratory behaviour of infective third-stage larvae (L3).

The aim is to incubate L3s in serial concentrations of the test drug on a nylon mesh, thus simulating the gut mucosal layer through which the larvae would normally migrate. Paralysis caused by susceptibility to the test drug has the effect of preventing the migration and causing the larvae to be held by the gauze. The percentage of larvae able to migrate at each concentration is determined and a drug response curve plotted (i.e. drug concentration is plotted against percentage migrating through the membrane).

Aims and objectives

This exercise is designed to demonstrate the isolation of genomic DNA; it can, in principle, be adapted for use with any organism, including parasites. Specifically, the objectives are:

1. To perform bacterial cell lysis.

2. To extract the cell lysate with phenol/chloroform.

3. To isolate the DNA by ethanol precipitation.

Introduction

DNA encodes genetic information for all known living organisms. A basic method in molecular biology is the isolation of DNA. The version given here is for the preparation of DNA from Escherichia coli. In this context the practical is useful for demonstrating some basic methods in molecular biology as well as introducing students to the use of bacteria as hosts for gene cloning. This is a prerequisite for more complex molecular biological methods including the isolation of parasite genes and analysis of their expression products.

Laboratory equipment and consumables

(per student or group)

Equipment

Safety glasses

P20, P200, P1000 Gilson pipetmen or equivalents

Waste beaker for chemically contaminated plasticware

Autoclavable waste bags for bacterially contaminated waste

Waste container for bacterial culture supernatant

Bench top centrifuge capable of producing 1000 g

Water bath (37 °C) with suitable racks for Universal tubes

Centrifuge capable of producing 8000g at 10 °C

Use of cold room/refrigerator and freezer

Consumables

Latex gloves in a range of sizes

Sterile tips for pipetmen

Universal tube containing a 25 ml culture of E.coli grown overnight at 37 °C in L-broth

3 ml of bacterial cell lysis buffer

300 μ1 of 10% (w/v) sodium dodecyl sulphate (SDS) in a microfuge tube

Aims and objectives

This exercise is designed to demonstrate:

1. The external and internal morphology of the adult schistosome.

2. The pairing behaviour of the adult worms.

3. Destination of eggs.

4. Hatching of miracidia from eggs.

5. Some gross effects of infection on the mouse host.

Introduction

Schistosomiasis is one of the most important public health problems in the tropics. Despite intensive research, there is still no easy cure for the disease and no simple way to control transmission. It is estimated that some 200 million people are infected with schistosomes.

The human schistosomes and many of the other species in mammals belong to the genus Schistosoma, in the family Schistosomatidae. Members of this family show morphological and physiological peculiarities that set them apart from all the other trematodes. Firstly, they are dioecious, the male carrying the female in a ventral canal, the gynaecophoric canal, and secondly, they live in the bloodstream of warm-blooded hosts (See Fig. 1.13.1 for details of the life cycle).

Five species of blood flukes may infect humans, causing schistosomiasis or Bilharzia. Three of these species are very common. S. mansoni is found in Africa and South America and affects chiefly the large intestine in humans; S. haematobium is found in Africa and the Middle East and affects the urino-genital system; S. japonicum is found in parts of Asia and affects mainly the large intestine.

Aims and objectives

This exercise is designed to demonstrate:

1. Hybridoma technology, including the isolation of mouse spleen cells.

2. Hybridization of B-cells and tumour cells.

3. In vitro cultivation of fused cells.

4. Screening of monoclonal antibodies produced.

5. Cloning and cryopreservation of cell lines.

Ideally, the whole procedure consists of five 1-day practicals spread out over a period of 5 weeks (when the student actually performs all the steps of the procedure), but this can be shortened to as few as two 1-day practicals (when the student only performs some of the steps and watches a demonstration of others).

Introduction

Monoclonal antibodies have many research and clinical applications (including the detection, localisation, characterisation and purification of antigen). The technique was first described by Köhler and Milstein (1975), but has since been modified and adapted by others (see reviews by Campbell, 1984; Harlow and Lane, 1988). As a practical, the successful production of a few different monoclonal antibodies is considered a very rewarding experience by most students, but even in situations where no antibodies are actually produced during the practical, the students are given the opportunity to learn a variety of useful techniques (sterile handling of cells and centrifugation, tissue culture, cell fusion, preparation of parasite antigens, performance of ELISA and/or IFAT). In addition to the production of monoclonal antibodies itself, the practical provides the demonstrator with an opportunity to discuss (or demonstrate) immunisation, cell cloning and cryopreservation, antibody isotyping, etc.

Aims and objectives

This exercise is designed to:

1. Investigate the life history of the cestode, Schistocephalus solidus, and its effect on its stickleback host.

2. Collect and analyse quantitative data on parasite loads, stomach contents and body condition in fish.

3. Conduct behavioural experiments, and interpret the data that such experiments generate.

4. Practice summarising and synthesising conclusions from experimental studies into a report.

Introduction

Schistocephalus solidus is a cestode that matures in the intestine of piscivorous (fish-eating) birds, with the three-spined stickleback serving as an intermediate host (see Fig. 7.5.1 for details of the parasite's life cycle). While in the stickleback, the worm is in the form of a plerocercoid metacestode or juvenile stage. Plerocercoids can grow to an enormous size, regularly reaching 40% of the host's mass, and in extreme cases weighing as much as the host itself. S. solidus infection is known to alter the behaviour of both the copepod and the fish hosts (for example by suppressing the escape response) in ways that make both intermediate hosts more vulnerable to predation by the next host in the life cycle.

The aim of this exercise is to familiarise you with this common host-parasite system, and to enable you to carry out an investigation into some of the important effects infection can have on the foraging behaviour and nutrient status of hosts. In addition, you will use data from a number of recent studies examining how the parasite influences foraging and body condition in fishes.

Aims and objectives

This exercise is designed to demonstrate:

1. SDS PAGE for the resolution of proteins into their component parts.

2. Western blotting for the detection of immunogenic antigens, using serum from experimentally immunised animals.

Introduction

Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS PAGE) and Western blotting are both widely used techniques in parasitology. SDS PAGE is used to separate mixtures of proteins into individual protein bands by dissolving them in SDS (which is a mild detergent) and reducing with 2-mercaptoethanol. The protein solution is then loaded onto an acrylamide gel. When an electric field is applied to the gel, the proteins are separated according to their molecular mass, smaller peptides moving further down the gel. These protein bands can then be stained to visualise their position and therefore their molecular weight can be determined. In this exercise, however, we will not be staining the protein bands but transferring them to nitrocellulose paper where they will be immobilised. These antigens can then be probed by antibodies in order to determine the immunogenic components of the protein.

For the purposes of this practical an anti-serum to the homogenate of Heligosomoides polygyrus has been prepared (see Exercise 4.6). H. polygyrus is a trichostrongyloid parasite that was first introduced as an experimental infection in laboratory mice over 50 years ago, and has since been used extensively as an animal parasite model system. Primary infections are maintained for up to 10 months.

INTRODUCTION

In most textbooks, biofilm formation on surfaces has been depicted as a sequence of events. Depending on the particular interest of the author, these events are split up into four or more steps (Escher & Characklis, 1990; Van Loosdrecht et al., 1990). The sequence is presented in Fig. 1. Traditionally, biofilm formation is said to begin with mass transport of micro-organisms towards a substratum surface (Fig. 1b), but in almost all environments, the mass transport step is preceded by the adsorption of conditioning film components (Fig. 1a), such as: an adsorbed tear film on a contact lens (Baguet et al., 1995; Landa et al., 1998); the salivary pellicle on surfaces in the oral cavity (Busscher et al., 1989; Bradshaw et al., 1997); a film of adsorbed urinary components on urogenital surfaces (Reid et al., 1998); and adsorbed macromolecules on marine surfaces (Schneider & Marshall, 1994). Many more examples of the formation of a film conditioning a surface as the first step in biofilm formation can be given. Once transported to a substratum surface, organisms may or may not adhere, depending on the interaction forces (Rutter & Vincent, 1980). This initial adhesion (Fig. 1c) is generally reversible (Norde & Lyklema, 1989), but even in the absence of exopolymer production, becomes less reversible within minutes due to the progressive removal of water from in-between the interacting surfaces (Meinders et al., 1995). The unfolding of binding molecules and other non-metabolic mechanisms eventually lead to the strong anchoring of the initially adhering organisms (Fig. 1e). At this stage, there is often a neglected step which occurs almost simultaneously, that is, coadhesion (Fig. 1d).

MICROBIAL AGGREGATES

The vast majority of micro-organisms live and grow in aggregated forms such as biofilms, flocs (‘planktonic biofilms’) and sludges. This form of growth is lumped in the somewhat inexact but generally accepted expression ‘biofilm’. The feature which is common to all these phenomena is that the micro-organisms are embedded in a matrix of extracellular polymeric substances (EPS) which are responsible for morphology, structure, coherence, physico-chemical properties and activity of these aggregates (Wingender & Flemming, 1999). Biofilms are ubiquitously distributed in natural soil and aquatic environments, on tissues of plants, animals and man, as well as in technical systems such as filters and other porous materials, reservoirs, pipelines, ship hulls, heat exchangers, separation membranes, etc. (Costerton et al., 1987; Flemming & Schaule, 1996); biofilms may also develop on medical devices, thus initiating persistent infections in humans (Costerton et al., 1999). Biofilms develop adherent to a solid surface (substratum) at solid–water interfaces, but can also be found at water–air and at solid–air interfaces. They are accumulations of micro-organisms (prokaryotic and eukaryotic unicellular organisms), EPS, multivalent cations, inorganic particles, biogenic material (detritus) as well as colloidal and dissolved compounds. EPS are considered as the key components that determine the structural and functional integrity of microbial aggregates. EPS form a three-dimensional, gel-like, highly hydrated and locally charged biofilm matrix, in which the micro-organisms are more or less immobilized. EPS create a microenvironment for sessile cells which is conditioned by the nature of the EPS matrix. In general, the proportion of EPS in biofilms can vary between roughly 50 and 90% of the total organic matter (Christensen & Characklis, 1990; Nielsen et al., 1997).