Aims and objectives

This practical is designed to demonstrate:

1. Part of the life cycle of a parasite of medical and veterinary significance, the common liver fluke Fasciola hepatica, from infection to maturation.

2. The general body plan of an acoelomate flatworm and its adaptations for parasitism with respect to the surface tegument, digestive, excretory and reproductive systems.

Introduction

F. hepatica, the common liver fluke, is a platyhelminth digenean trematode. The life cycle of the liver fluke (Fig. 1.6.1) is indirect and includes freshwater snails as intermediate hosts; in the UK this is the amphibious mud snail, Lymnaea truncatula. Fasciola is an example of a ‘zoonosis’; i.e. an organism that is fully infectious to humans but which is maintained in the ecosystem by a range of other animals, including rabbits, acting as definitive hosts. F. hepatica occurs widely in Eurpoe. It causes fasciolosis or ‘liver rot’, mainly in cattle, sheep and goats. It is not usually an important parasite of humans, with the exception, for example, of parts of Latin America. The related liver fluke, F. gigantica, is an important zoonosis and is found in humans and domestic and wild ruminants in Africa and the orient.

Liver fluke causes serious veterinary problems in livestock where the disease varies from year to year depending largely on climatic conditions, such as increased temperature and rainfall in June and July. Ecological studies in the UKhave enabled a valuable forecasting system to be developed to warn farmers when to expect outbreaks of the disease (Ollerenshaw & Rollands, 1959).

Aims and objectives

This exercise is designed to demonstrate:

1. The importance of various factors in the activation and excystation of the cysticercoids.

2. Changes in the appearance of the cysticercoids during excystation

3. The behaviour of freshly excysted larvae.

Introduction

Many parasites use more than a single host species during the course of their life cycle. Often the larval stages develop in one host and the adults in another. Thus, the parasite must be transmitted from one host to the next in order to complete development. The most common relationship between hosts exploited by parasites for transmission, particularly those living in the intestine as adults, is that involved in the predator-prey food chain. For example, some tapeworms develop as larvae in small flour beetles, Tribolium confusum, but parasitise rodents such as mice and rats as adults. Rodents are essentially grain feeders, but will consume insects also and therefore by depending on two host species, both of which have habitats closely associated with cereals and grain, the tapeworms ensure that their transmission cycle is completed successfully.

This practical is based on two species of tapeworm, Hymenolepis diminuta and Rodentolepis microstoma (formerly known as H. microstoma; see Exercise 1.14). The former develops successfully to the adult stage in the rat only (Fig. 3.2.1), the latter matures in mice. Both utilise the flour beetle, Tribolium confusum, as the intermediate host.

The larval (metacestode) stage developing in beetles is called a cysticercoid.

Aims and objectives

This exercise is designed to demonstrate:

1. The external and internal morphology of the adult stage of the nematode parasite Heligmosomoides polygyrus.

2. The mating behaviour of the adult worms.

3. The distribution of worms in the small intestine and the selection of a preferred site.

Introduction

Intestinal nematode parasites are very common in mammalian hosts and are responsible for human disease as well as for losses to the agricultural industries through their effects on domestic animals. H. polygyrus (Fig. 1.15.1) is an intestinal nematode parasite of mice that is very easy to maintain in the laboratory and provides convenient material to demonstrate some of the adaptations that have evolved in nematodes for survival in their hosts.

Unfortunately, this parasite has been the subject of a longstanding taxonomic debate as to the most appropriate name for the species. These problems are discussed by Behnke et al. (1991) and the reader is referred to this publication for further details. The approach used here is that the alternative name for the species, Nematospiroides dubius, is no longer used. H. polygyrus bakeri is the strain maintained in domestic/laboratory mice, Mus musculus. In Europe, however, wild field or wood mice, Apodemus sylvaticus, carry the subspecies called H.p. polygyrus and voles, Clethrionomys glareolus, H. glareoli. All of these, if available, can provide useful teaching material. This protocol is based on the laboratory passaged subspecies H. polygyrus bakeri.

Aims and objectives

This exercise is designed to demonstrate:

That a parasite can alter the behaviour of its intermediate host to make it more vulnerable to predation by its definitive host.

For students, this exercise aims to:

1. Demonstrate the influence of a parasite on the behaviour of its intermediate host in order to complete its life cycle.

2. Facilitate the design of experiments and formulation of conclusions on the strategies employed by parasites to complete their life cycle.

3. Encourage understanding about studies that might be conducted in the laboratory and in the field to study the manipulation of host behaviour by parasites.

4. Examine a facet of biology embracing the fields of parasitology and animal behaviour.

Introduction

Over the past two decades, exciting developments have been made in understanding the coevolution and coexistence of parasites and their hosts (e.g. Moore, 1995). One of the most intriguing areas of host-parasite ecology is the ability of parasites to change the appearance and/or behaviour of one (intermediate) host to facilitate transmission to another (definitive) host.

This practical exercise describes experiments applicable to an association between a parasite and its two hosts. The experiments are designed to explore morphological and behavioural changes in the intermediate host that facilitate transmission of the parasite to the definitive host. Specifically, we are concerned with the effects of an acanthocephalan parasite on the behaviour of shrimps (gammarids), and then predation by its definitive host-fish (Fig. 7.4.1).

Aims and objectives

This exercise is designed to demonstrate:

1. Utilisation of ethanol-precipitable carbohydrate (glycogen) as an energy store in an endoparasitic flatworm (trematode or cestode), using simple spectrophotometry.

2. The distribution of glycogen in the various organ systems of the parasite, using histochemical procedures.

Introduction

The bioenergetic pathways of endoparasitic flatworms generally function anaerobically, with emphasis on synthetic metabolic capacities rather than complete substrate breakdown. The worms are generally facultative anaerobes, deriving their energy largely from the catabolism of glucose and glycogen and excreting highly reduced end products. In contrast to free-living organisms, terminal oxidative pathways are either absent or largely abbreviated in parasitic flatworms, precluding the use of proteins or lipids as energy sources. Thus, cestodes and most digenean trematodes have a pronounced carbohydrate metabolism. They contain and turnover large quantities of endogenous carbohydrate, have a high rate of transport of exogenous sugars into the tissues, and are known to produce substantial amounts of fatty acids in vitro. For example, the cyclophyllidean tapeworm, Hymenolepis diminuta, may contain as much as 50% of its dry weight as glycogen. When the host (rat) is deprived of carbohydrate in its diet, the glycogen content of the worm falls rapidly, and when such starved worms are given glucose in vitro, glycogen synthesis (glycogenesis) also proceeds rapidly. Similarly, the liver fluke, Fasciola hepatica, generally contains 15–20% glycogen per dry weight and under conditions of starvation uses up some 20% of this reserve within 5 h.

Aims and objectives

This exercise is designed to demonstrate:

1. The different stages in the life cycle of Plasmodium spp.

2. How to recognise infection in a mammal and a mosquito.

Introduction

Over 40% of the world's population live at risk from infection with malaria, a parasitic protozoan of the genus Plasmodium. The malaria parasite is transmitted by its mosquito vector that feeds on blood. The typical symptoms of malaria include short episodes of severe fever, occurring every two or three days, anaemia and enlargement of the spleen. Between 300–500 million clinical cases of malaria occur each year and 1.5–2.7 million people die of the disease annually. Ninety percent of cases are in tropical Africa and half of the deaths are in children under five years. A more detailed account of malaria can be found in Knell (1991).

The life cycle of the malaria parasite (Fig. 1.11.1) has four phases: three asexual and one sexual. Sporozoites from the mosquito are injected into the blood and immediately invade the liver. Here they become trophozoites and grow and multiply to form schizonts containing many merozoites (asexual phase 1). When the liver cells burst, merozoites are released into the blood and invade red blood cells. Erythrocytic trophozoites grow and divide to form schizonts containing new merozoites. These are released when the infected red cell bursts and they invade new red cells (asexual phase 2).

Aims and objectives

Using commercial lectins, these two exercises are designed to:

1. Distinguish between different species of parasites and two strains of the same parasite species.

2. Determine the type of carbohydrate present on the parasite surface membrane.

Introduction

Lectins (often referred to as agglutinins) are proteins or glycoproteins that bind specifically to carbohydrates, usually present as glycoconjugates, on cell or tissue surfaces or in cell tissue fluids (Sharon & Lis, 1989; Jacobson, 1994). Lectin reactivity is generally designated according to the monosaccharides or oligosaccharides that cause inhibition of lectin-mediated agglutination of cells or adherence to cell membranes.

Lectins have been widely used in parasitology to detect and determine the types of saccharide moieties on the surface of trypanosomatids, e.g. Crithidia (Petry et al., 1987), Leishmania (Schottelius & Aisen, 1994) and Trypanosoma species (Maraghi et al., 1989), to demonstrate differences between parasite growth and stationary phases (Jacobson & Schnur, 1990) and to distinguish between the different stages of the trypanosomatid life cycle (Rudin et al., 1989). In addition, lectins have been employed to differentiate between parasite species (Schottelius & Aisen, 1994) and in the identification of various strains (Schnur & Jacobson, 1989) and stocks (Schottelius, 1987) of these flagellates. The following methods are applied to kinetoplastid flagellates but can be adapted to study other unicellular parasites.

Aims and objectives

This exercise is designed to demonstrate:

1. How earthworms respond to not-self matter entering their body cavity, including parasites.

2. How to generate quantitative data for subsequent interpretation by summary statistics, graphs and statistical analysis.

Introduction

All living cells need to be able to distinguish self from not-self molecules. This ability is necessary for activities as diverse as feeding (by phagocytosis/pinocytosis), fertilisation and building multicellular bodies. The more complex an animal becomes the more the integrity of its body is threatened by the invasion of infectious organisms and by the appearance of mutant cells (‘cancers’). Self and not-self recognition as a form of adaptive response is seen in all metazoa, the process becoming increasingly more complex in the higher invertebrates, and qualitatively more sophisticated, with the evolution of the immune response, in the vertebrates.

This practical will examine aspects of the ability of a common invertebrate, the earthworm, to recognise not-self molecules. Earthworms have a body cavity, the coelom, and a well-developed blood system. The fluid in the coelom contains free cells, amoebocytes, which carry out a major part of the self/not-self recognition. Amoebocytes can phagocytose material as well as encapsulate objects that are too large to ingest. Aggregations of amoebocytes around foreign bodies form the large ‘brown bodies’ that accumulate in the tail-end of the coelom.

Aims and objectives

This exercise is designed to demonstrate:

1. Methods for quantifying cellular (lymphoid) changes in body organs.

2. The morphology of the mononuclear cell types.

3. Changes in the lymphoid cell population during the course of an infection.

Introduction

The dominant cell types that are involved in an active immune response are the lymphocytes, of which there are two main classes: B-lymphocytes and T-lymphocytes. Morphologically, they are very similar and can only be distinguished by specialised staining techniques.

Lymphocytes have a limited life-span and are continuously replenished, hence the number of both circulating and organbased lymphocytes is relatively stable. If an infection becomes established, antigenic molecules derived from the invading pathogen are transported via antigen-presenting cells to the spleen and lymph nodes.

The presentation of the antigen to the T cells with specific antigen receptors (mainly T-helper and to a lesser extent T-cytotoxic) stimulates the release of a range of cytokines, interleukins and growth factors, which, in turn, promote cell proliferation (cloning) of primed lymphocytes. Hence the numbers of resident lymphocytes in both the T and B cell zones increase.

The total number of viable lymphocytes in various organs can be estimated by a simple extraction technique. This method involves identifying and dissecting out the lymphoid organs (spleen, thymus and lymph nodes) followed by extraction of the lymphocytes, staining and quantification.

This manual describes a range of well-tried practical exercises, drawn largely from the membership of the British Society for Parasitology, and currently used in the teaching of parasitology at undergraduate level. The primary aim is to promote and, hopefully, stimulate practical teaching in parasitology in institutions where levels of experience and resources devoted to the subject vary from substantial to none. For this reason, the exercises selected range from the simple, requiring little in the way of sophisticated equipment, and focusing on locally-available materials, to the more elaborate, in the fields of molecular biology and immunology; it is not intended to provide comprehensive coverage of practical parasitology. Use of the manual outside of the UK may require alternative materials and/or sources of materials.

Although the seven sections presented comprise information and exercises in different aspects of the subject, it is recognised that merging of a number of the exercises cited, or other modifications, may suit the local situation. For example, the species used in Section 1 could be substituted depending on availability; these and other adaptations are to be encouraged and, in many instances, alternative sources of material are suggested by authors. The decision on how best to produce a benchtop handout is a personal one, but readers are welcome to use the text and ideas presented here.

It is important to note that while every consideration to health and safety has been given by the authors, editors and the Society, no responsibility can be accepted if things go wrong.

Aims and objectives

This exercise is designed to demonstrate:

1. The physiological effects of classical transmitter substances on the motor activity of roundworm (nematode) parasite somatic musculature.

2. The physiological effects of classical transmitter substances on the motor activity of flatworm (platyhelminth) parasite somatic musculature.

Introduction

The common liver fluke, Fasciola hepatica, is of great economic importance, occurring worldwide, with the exception of Africa and South Asia, where it is replaced by F. gigantica. It parasitises all domestic ruminants and causes a wide range of clinical symptoms. Although not normally regarded as an important parasite of humans, there are exceptions, for example, in parts of Bolivia. The most common type of infection is chronic fascioliasis, which occurs mainly in cattle and sheep and causes anaemia, oedema (bottle j aw), digestive disturbances (constipation and diarrhoea) and general weight loss. Most of the damage is caused by juvenile F. hepatica as they migrate through liver tissue en route to the bile ducts, where they develop to maturity as sexually reproducing adult worms.

Ascariasis is the most prevalent human and livestock helminth-parasite infection and is caused by the sibling species Ascaris lumbricoides and A. suum. The latter is the large gastrointestinal parasitic roundworm of pigs and is responsible for a large economic burden on farming communities worldwide. Adult female A. suum are 20–35 cm in length, the males are somewhat smaller, 15–30 cm.

Aims and objectives

This exercise is designed to demonstrate:

1. A range of digenean parasites of Littorina spp.

2. The general morphology of the intermediate stages of Digenea.

Introduction

The larval stages of digenetic trematodes develop in intermediate hosts, almost all of which belong to the phylum Mollusca. The miracidium hatches from the egg and penetrates the molluscan host giving rise to the next larval stage (the first intramolluscan stage), the primary or mother sporocyst. In some trematodes, development of cercariae occurs directly within the secondary or daughter sporocysts; in others, cercariae develop within rediae. In both cases, development usually takes place in the gonad or digestive gland of the molluscan host.

The digestive glands of gastropod molluscs exhibit profound physico-chemical changes as a result of infection with larval trematodes. The observable manifestations of these changes are apparent in alterations in the structure, histochemistry and biochemistry of the molluscan digestive gland. Excellent reviews of the subject are provided by Wright (1971) and Smyth and Halton (1983).

During this exercise, you will be examining specimens of the marine mollusc, Littorina (the common or edible periwinkle) for infection with a variety of digenean parasites. Other species may be common locally and could be examined additionally or substituted if necessary.