Aims and objectives

This exercise is designed to demonstrate:

1. Some electrophysiological properties of nematode muscle cells.

2. Measurement of intracellular membrane potentials from A. suum muscle by recording from the bag (muscle cell body) region.

3. Recording the electrophysiological effects of piperazine, an inhibitory γ-aminobutyric acid (GABA)ergic anthelmintic.

4. Recording the electrophysiological effects of pyrantel, an excitatory nicotinic anthelmintic.

Introduction

Adult Ascaris suum are large parasitic nematodes that may be recovered from the intestine of pigs slaughtered at the abattoir. Mature female A. suum may be up to 35 cm in length. The number found in each pig varies dramatically from area to area and relates to the husbandry/hygiene on which the pig has been raised. Migrating A. suum larvae may cause significant scarring of the liver (milk spot) as they pass through this organ before entering the lungs and reaching the upper intestine where adults are found. After mating, many thousands of Ascaris eggs are produced each day by the mature female and are released in faeces.

Human infection with A. suum is rare. The species of Ascaris that regularly infects humans is Ascaris lumbricoides. Over half the world's human population is infected at one time during their lives with A. lumbricoides. Treatment of these infections in man or animals involves the use of anthelmintics (i.e.antihelminth drugs), which have a selective action on the nematode parasite without affecting the host.

Aims and objectives

This exercise is designed to investigate:

1. The distribution of the biogenic amine, 5-hydroxytryptamine (5-HT), in selected helminths.

2. The distribution of immunoreactivity to the neuropeptide, FMRFamide, in the nervous system of selected helminths.

Introduction

Immunocytochemistry is a method whereby antibodies, tagged with a visible fluorescent probe or fluorophore, are employed to detect antigens in tissue preparations using a specific antibody- antigen reaction. The most commonly employed immunocytochemical method is the indirect immunofluorescence technique (Coons et al., 1955) in which the primary antibody binds to the antigen in the tissue sample followed by a secondary antibody, labelled with a fluorescent tag, that binds to the primary antibody (Fig. 3.7.1A). The indirect technique allows for more than one secondary antibody to bind to the primary antibody, thereby ensuring amplification of the signal (stronger immunostaining).

The nervous system of helminth parasites is multifunctional and has been shown to be neurochemically complex (Halton & Gustafsson, 1996; Maule et al., 1996). 5-Hydroxytryptamine (5-HT) occurs extensively in the nervous systems of platyhelminth (flatworm) parasites and has been implicated as an excitatory neurotransmitter. FMRFamide is a tetrapeptide amide that was first isolated from the Venus clam, Macrocallista nimbosa, by Price & Greenberg (1977). It is now known that peptides with a similar structure to FMRFamide occur widely throughout invertebrate phyla and are commonly referred to as the FMRFamide-related peptides (FaRPs).

Aims and objectives

This exercise is designed to demonstrate:

1. Qualitative changes in body-organ systems occupied by parasites.

2. Quantitative changes in body-organ systems associated with infection.

3. Histological changes associated with infection, including the direct effects of parasites and the host response.

Introduction

Pathology is the study of the cause and effects of disease as seen in the changes in tissues and organs. The majority of infectious agents, including many parasites, cause some changes in the host that are reflected in alterations in the appearance and function of specific organs. Many parasites cause chronic infections in which pathological changes develop over a protracted period of time, during which the host's health may deteriorate gradually as a consequence of the accumulated damage to organ systems, but seldom sufficiently for the parasite itself to be directly responsible for death. Some of these changes may be sufficiently intense to affect the parasite's route of migration to its preferred site, thereby either protecting the host from further infection or resulting in the spread of the parasites to new sites. In this exercise, the pathological effects of two parasites, commonly maintained in laboratories for medical research, Mesocestoides corti and Schistosoma mansoni, will be assessed qualitatively and quantitatively. This will be achieved by close comparison of the organs and tissues of infected and uninfected animals, in order to identify changes associated with infection.

Aims and objectives

This exercise is designed to demonstrate:

1. The general morphology of the protozoan parasite Monocystis.

2. The general morphology of the parasitic and free-living stages of the nematode parasite Rhabditis.

Introduction

Earthworms are common terrestrial invertebrates from the phylum Annelida. They are exploited by a number of protozoan and nematode parasites and act as intermediate hosts for many parasites of birds and mammals. Two common parasites are Monocystis (Protozoa, Sporozoa) and Rhabditis (Nematoda); these infect earthworms only.

Monocystis is the commonest protozoan genus to infect earthworms, but another nine genera have been recorded in Britain. The growing form (trophozoite) and reproductive forms occur in the seminal vesicles of the earthworm; the reproductive forms (sporocysts – contained within larger cysts or spores) also enter the body cavity.

The nematode parasite Rhabditis also infects earthworms but only in its larval phase. The parasitic third-stage larvae occur free in nephridia of the earthworm, encysted in the body wall, or encapsulated in the coelom. Adult nematodes develop only when the worm dies, when the larvae begin feeding on the bacteria that break down the tissues. Adult Rhabditis is therefore a free-living organism. Several species of Rhabditis are involved, of which the most common is R. maupassi, but they are difficult to differentiate.

Earthworms have a body cavity (a true coelom), a well-developed blood system and the capacity to defend themselves against some types of invaders.

Aims and objectives

This exercise is designed to demonstrate:

1. The dynamic interactions occurring between host and parasite under the influence of external environmental conditions.

2. The balance that exists in the host-parasite relationship between the potential for parasite-induced pathogenic damage, including host death, and the capacity of the host response to regulate infection levels.

3. The value of numerical data with which to develop an understanding of the distribution of parasite populations within host populations (and the significance for transmission, pathology, etc).

4. Adaptation to parasitism, the skills and patience required for manipulation, and the rewards of microscopic examination.

Introduction

Assessment of infection levels in the host (and host population) provides important information on the success of the parasite life cycle, the severity of pathogenic effects on the host, and the effectiveness of host immunity. Combined with information on parasite age and developmental rate, infection levels may also enable reconstruction of the recent history of recruitment of parasites into the individual host and population: current infection levels reflect successful parasite invasion moderated by the host defensive response and other influences such as competition. Measurement of infection levels, however, represents only a ‘snapshot’ of the dynamic processes that may affect the host-parasite interaction. The illustration of dramatic fluctuations in parasite numbers within individual hosts can be observed in microparasite infections multiplying and being moderated in situ, as in the cycles of parasitaemia of trypanosomes.

Aims and objectives

This exercise is designed to demonstrate that:

1. Parasite behaviour alters with age.

2. Swimming behaviour is influenced by environmental factors.

3. Parasites have evolved sensory mechanisms that are receptive to specific stimuli encountered during their active life.

The exercise also aims to:

1. Encourage students to consider the role of the observed responses in parasite dispersal.

2. Stimulate students to reflect on behavioural strategies that may lead to successful transmission.

3. Engage students in a critical debate relating to experimental design and the limitations of laboratory-based experimental models.

Introduction

The experiments described below investigate the swimming responses of the cercariae of the digenetic trematode, Cryptocotyle lingua. The swimming behaviour of cercariae may be adapted to ensure dispersal and infection of a host species. Some species show particularly high swimming activity after being shed (Haas et al., 1994). This clearly contributes to escape from the host snail's habitat and assists dispersion. Most cercariae tend to disperse in particular habitats by responding to environmental stimuli, such as gravity, light, water currents, temperature, ionic content and physical boundaries (MacInnis, 1976). Combes et al. (1994) suggested that dispersion had both demographic and genetic consequences. It may facilitate an encounter with a host and limit inbreeding. Other non-adult stages, such as eggs, cysts and some larvae, cannot respond to environmental signals by active processes and are transmitted by passive means. Cercarial host-finding behaviour patterns and the environmental cues that stimulate them have been analysed.

Aims and objectives

This exercise is designed to demonstrate:

1. The effect of parasite population density on adult cestode size.

2. The effect of parasite population density on parasite fecundity.

3. The relationship between parasite size and fecundity.

Introduction

It has been known for a long time that there is a relationship between the number of parasites of a species in a host (infra-population density) and parasite size and fecundity. The relationship originally referred to as a crowding effect is most easily observed in parasites such as cestodes, in which growth is relatively indeterminate and adult size maybe large. This effect can be detected in other groups of parasites such as nematodes and acanthocephalans, but these are often smaller in size and growth is more determinate.

The crowding effect has been studied most intensively in cestodes, and whilst there is a measure of agreement on its manifestation, and its population consequences, there is less agreement on its causation. One school of thought believes it is an example of intra-specific competition, probably for carbohydrate resources, which become increasingly scarce as infra-population density increases. Another school believes that it is a result of a host immune response, which increases in intensity and effectiveness as infra-population density increases. A third school believes that it is caused by secretions from the parasites that suppress growth. Whatever the cause, there is no doubt that it is potentially a very effective regulator and stabiliser of parasite population density, since the effect on fecundity is density-dependent.

Aims and objectives

This exercise is designed to demonstrate the sensitivity of sporozoites, the invasive stages of coccidial parasites, to ionophorous drugs.

Specifically, the practical will examine:

1. How sporozoites may be recovered from transmission stages, the oocysts of the coccidial parasites, by a combination of physical damage and enzymatic digestion (via an intermediate sporocyst stage).

2. The lethality of the ionophore, monensin, to extracellular sporozoites by incubating the mixture of sporozoites, with remaining intact sporocysts and oocysts, in the presence of different concentrations of the drug.

Introduction

Coccidial protozoa are found wherever poultry are reared and an absolute need to prevent or control infections in commercial meat and egg-laying flocks has led to the use of many anticoccidial drugs. The most useful drugs continue to be the ionophorous antibiotics, even though widespread resistance to these drugs has been reported for many years. The drugs act by taking up residence within the cell membrane (pellicle) of the transiently extracellular stages, the sporozoites and merozoites, where they interfere with ion transport across the membrane. The test to be demonstrated in this practical is based on the viability/distortion and/or lysis of sporozoites as the drug interacts with the cell membranes of the parasite and interferes with the functioning of the ATP-dependent pumps that regulate the concentration of intracellular cations. It is thought that the drug acts as a pore in the membrane so that ions, commonly sodium, magnesium or calcium, may move from the outside milieu via the ionophore into the cytoplasm of the parasite.

Aims and objectives

This exercise is designed to demonstrate:

1. The actual transmission rates of cestode eggs to cysticercoids in a beetle host.

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

3. The influence of egg density on transmission rates and patterns of dispersion.

Introduction

Transmission of parasites from a free-living stage to a host or from one host to another is always hazardous and associated with high parasite mortality. The probability of a single egg giving rise to a cysticercoid larva in a beetle is very low, in the region of P = 10-1 or less. In nature, much of the mortality is due to beetles failing to encounter eggs, or, having encountered one, failing to become infected. Additional factors reducing transmission rates include shortage of time in which to infect and/or encounters between parasite and resistant host. Even under ideal laboratory conditions, however, the probability of successful infection is low. It can be predicted on the basis of intuition and mathematical models that the probability of a successful infection will relate to the density of parasite eggs relative to that of beetles, such that the greater the egg density, the higher the probability of transmission.

What is not always appreciated is that transmission (a birth/recruitment process) rate can also influence the dispersion pattern of a parasite in a subsequent host population. An understanding of parasite dispersion patterns within their host populations is vital to an understanding of parasite population dynamics, epidemiology and control.

Aims

This exercise is designed to demonstrate:

1. The importance of various physicochemical factors in the activation and hatching of the oncosphere/hexacanth via dis-solution of the embryophore.

2. The behaviour of freshly hatched larvae.

Introduction

Hymenolepis diminuta is a tapeworm of rodents (for life cycle, see Exercise 1.14). Hymenolepis eggs (Fig. 3.1.1) contain a single oncosphere/ hexacanth larva (six hooks). These eggs are infective to the beetles Tenebrio and Tribolium.

Sources of parasite material

A small number of infected rats held in one or two institutions of higher education can provide enough tapeworm eggs to supply large practical classes run at many other institutions. Eggs remain infective in faeces for several weeks and can be supplied by post from those universities and institutions that maintain the infections. Faeces should be refrigerated on arrival. See also Additional information in Exercise 1.14.

Safety

This exercise is entirely safe for humans since the eggs are only infectious to beetles! Tapeworm eggs will have been thoroughly washed and students will normally only touch the sides of clean microscope slides. It is good laboratory practice to ensure that hand to mouth movements do not occur and that students wash their hands and use a nail-brush before leaving the laboratory.