1.3 Parasites and vectors - Plasmodium species and their use as animal models

Question 1

Aims

Answer 1

To introduce the more important non-humanPlasmodiumspecies and describe their life-cycles. To show the usefulness of animal models when designing and testing drugs and vaccines for human malaria

Question 2

Objectives

Answer 2

After working through this session you should be able to: Explain differences in the life-cycles ofPlasmodiumspecies in reptiles, birds,rodentsand primates. List the most important animal models for human malaria. Show ways in which information aboutPlasmodiumspecies in a variety of animals has contributed to our understanding of human malaria. Give examples of advances in malaria research made through the use of animal models. Compare the use of primate and rodent models. Evaluate the advantages and disadvantages of using animal models.

Question 3

Introduction

Answer 3

The term ‘malaria parasite’ is applied to all species of the genusPlasmodium, of which there are more than 200, in reptiles, birds and mammals. All are parasites that have similar life-cycles involving insect vectors in which a sexual cycle and sporogonic stages occur, and exoerythrocytic and erythrocytic stages in the vertebrate host.

Question 4

Introduction

Answer 4

You will sometimes see the termPlasmodiaused to cover multiple species as the plural ofPlasmodium.This is unnecessary and best not used (Plasmodia are slime moulds).

Question 5

Introduction

Answer 5

Plasmodiumspecies that infect non-human vertebrates are of interest and importance for various reasons: 1 They serve as useful models for human infections and for the design and testing of drugs and vaccines. 2 They provide clues about the biology of the species that commonly infect humans. 3 Scientists concerned with host-parasite relationships, are interested in the phylogeny,or the shared evolutionary history, of the various groups ofPlasmodiumspecies. Now test how well you remember what you’ve learned so far by answering the question that follows…

Question 6

Question 1 In Session 1.1Parasites and vectors: Introduction, you discovered that there are five species ofPlasmodiumthat are transmitted between humans,P. falciparum,P. vivax,P. ovale,P. malariae, andP.knowlesi. What approximate percentage of thePlasmodiumspecies known do they represent? a) 2% b) 12% c) 50% d) 96%

Answer 6

AlthoughP. falciparum,P. vivax,P. ovale,P. malariaeandP.knowlesiare the best-known malaria parasites, they actually constitute less than 2% of all knownPlasmodiumspecies. The rest are common blood parasites of reptiles, birds and mammals. The malaria parasites of mammals are found in rodents and non-human primates and are broadly similar to those that infect humans. The malaria parasites of animals, particularly those of mammals, have been intensively studied in their own right and have provided important clues about the nature and biology of the species that infect humans.

Question 7

Introduction A note about names and species

Answer 7

You will encounter a lot of new names in this session but you do not have to remember them all. However, you do need to familiarise yourself with those in the tables in this session as these have all been used in medically-related research. Our knowledge of malaria has been built up from observations using a number ofPlasmodiumspecies in different hosts. If you know which species infect which hosts, this will allow you to appreciate better why a particular combination has been used for experimental work.

Question 8

Introduction A note about names and species

Answer 8

Some species have been divided up into subspecies. This is mostly just a matter of convenience and is something that need not concern you too much although they will be referred to more with the rodent malarias. Also, the sub-division ofP.ovaleinto two sub-species,P.ovale curtisiandP.ovale wallikeriis one interesting example, because it is suggested that these are, in fact, separate species (Oguike & Sutherland 2015). There are also subgeneric names but these can best be avoided and left to the taxonomists, to whom they may be important. In the next topic, we’ll discuss the classification of malaria parasites…

Question 9

Classification

Answer 9

With the widespread use of sophisticated molecular methods based on DNA and RNA, the classification of single-celled, eukaryotic organisms is currently in a state of flux. It is not necessary for you to understand the taxonomy of malaria parasites in any detail so only a very broad classification is used here, enough to be able to introduce a number of organisms with which you may already be familiar or that you are likely to encounter which have some taxonomic links toPlasmodium. We’ll look at Apicomplexa first…

Question 10

Classification The phylum Apicomplexa

Answer 10

The characteristics of the Apicomplexa are: unicellular organisms possessing, at some stage, an apical complex composed of polar rings, rhoptries and micronemes, elaborate life-cycles involving a sexual process Within the Apicomplexa are two classes:

Question 11

Classification Coccidea

Answer 11

TheCoccideacontain some of the parasites you may already have encountered includingCryptosporidium

Question 12

Classification Haematozoea

Answer 12

TheHaematozoeaare defined as apicomplexans with sexual stages in the blood of a vertebrate and sporogony in a blood-sucking arthropod. There are two orders in the Haematozoea: 1 thePiroplasmidacontaining the generaBabesiaandTheileria(important parasites of cattle), and 2 theHaemosporidacontaining a number of common blood parasites including members of the generaLeucocytozoon, Plasmodium, HepatocystisandHaemoproteus. The last three of these have contributed substantially to our understanding of human malaria.

Question 13

Question 2 What is the argument for regarding the mosquito as the definitive host ofPlasmodiumspp.? a) because merozoites are found in the mosquito b) because the mosquito feeds on humans c) because gamete formation and fertilisation (the sexual stages) occur in the mosquito

Answer 13

The argument for regarding the mosquito as the definitive host ofPlasmodiumspp. is because gamete formation and fertilisation (the sexual phase) occur in the mosquito. Merozoites are not found in the mosquito but develop in the asexual blood stages in humans. Female mosquitoes take blood from vertebrates ( which in medically important cases is humans) as this is required for mosquito egg production. Transmission of malaria occurs in both directions while the blood meal is being taken.

Question 14

Question 3 A mixture of morphological and biological criteria used to distinguish species ofPlasmodiumare listed in the table below. There are four morphological and five biological criteria. Can you identify which are which?

Answer 14

Morphological criteria: gemetocyte and oocyst, nature and distribution of pigment and changes to the infected RBC, number of nuclei in exoerythrocytic or erythrocytic schizonts, shape of the trophozoites. Biological criteria: duration of different stages, host range, nature of the vector and geographical distribution, presence or absence of relapses, type of cell infected

Question 15

Why use animal models

Answer 15

Before introducing the widerangeofPlasmodiumspecies and their natural hosts, we should consider 1 Why some of them have been used as models of human malaria 2 Why we need to do this 3 How good the models are

Question 16

Why use animal models

Answer 16

Research into any aspect of human disease involves a vast amount of experimental work. Direct observations on humans can provide the most useful information but in most cases, particularly during early phases of any investigation, it is not convenient to use humans and there are ethical dilemmas that have to be addressed. Some studies can be carried out using modelling and computer simulations, andin vitroculture, but many cannot.

Question 17

Why use animal models

Answer 17

The alternative that remains is the use of animal models and, although this is not an ideal solution, it is nevertheless essential if human diseases such as malaria are to be prevented or cured or the pathology reduced. It is important to stress that use of animals for such research is regulated so that animals are treated as humanely as possible.

Question 18

Why use animal models

Answer 18

In some other infections, it is possible to maintain the species of pathogen that actually infects humans in commonly used laboratory animals, for exampleLeishmania majorin mice. Except for certain special circumstances that we shall discuss later, this is not possible with the malaria parasites. However, as we shall see, malaria research has benefited from the use of laboratory models in birds, non-human primates and rodents. Now answer the question that follows…

Question 19

Question 4 Can you think of reasons why it is difficult to develop a definitive laboratory model for malaria?

Answer 19

One reason that you might have thought of is because there are so many species and subspecies of malaria parasites that could be used and hence there is no consensus of opinion as to which species of parasite, or which host, should be used for any particular purpose. The parasite is not the one that infects humans and the hosts are different physiologically and in other ways from humans. This session is entitled ‘Animal models’, and we should be quite clear what that means. We havePlasmodiumspecies that are substitutes for the human species and the laboratory hosts used are rarely if ever the natural hosts, so substitutes again.

Question 20

Plasmodium parasites of reptiles

Answer 20

Over 90 species ofPlasmodiumare known to occur in reptiles and new species are continually being discovered. They are mostly found in agamid and iguanid lizards, geckos, skinks, chameleons and snakes.

Question 21

Plasmodium parasites of reptiles

Answer 21

ReptilePlasmodiumspecies are very host-specific and interest in the group has been largely confined to taxonomic and phylogenetic studies. Very little is known about the vectors and transmission of these parasites.

Question 22

Plasmodium parasites of reptiles

Answer 22

Most of the early descriptions were based on blood smears and it was not until 1970 that the first studies on development in their vectors was undertaken and it was found that the vector ofP. mexicanumwas not a mosquito but a sandfly. This is an unusual situation as most other species about which anything is known develop in culicine mosquitoes. However, there are also reports thatP. agamaecan develop inCulicoides(biting midges).

Question 23

Plasmodium parasites of reptiles

Answer 23

There is very little information available about the exoerythrocytic stages of reptilePlasmodiumspecies, except that they appear able to develop in the liver macrophages, endothelial cells, lungs, spleen, heart and kidneys. This makes the life-cycle different from that in mammals and more similar to that in bird malarias (see next topic). The pathology of infections caused by reptile malaria parasites can be severe.

Question 24

Plasmodium parasites of reptiles

Answer 24

Knowledge about reptile malaria parasites has added very little to our understanding of human malaria and current research on reptile malaria is focused on such topics as evolution, speciation and ecology. In the next topic, we’ll consider avian malarias…

Question 25

Avian malarias

Answer 25

The late 19th century was a period of great interest in comparative parasitology and it was not unusual for scientists to use one infection to explain another. It was against this background that Patrick Manson persuaded Ronald Ross, in the 1890s, to look for the developmental stages of a malaria parasite in mosquitoes that fed on larks and sparrows harbouring what we now know to be Plasmodium relictum, which had been newly described in 1891. Ross was lucky in choosing to use Culex pipiens for his experiments because, avian malarias are transmitted mainly by culicine, not anopheline, mosquitoes

Question 26

Avian malarias

Answer 26

This is the first record of the use of an animal model in malariology. P. relictum is common throughout the world, and has a large range of avian hosts. The distribution of avian malarias is worldwide and the host range is also wide. About 25% of all bird species are infected including a number of domesticated birds such as chickens, ducks and turkeys. As mentioned, the vectors of avian malarias are usually culicine mosquitoes although some anopheline mosquitoes and sandflies can be infected experimentally. Next you'll learn about the most used avian animal models in malariology...

Question 27

Avian malarias Avian Plasmodium-host combinations ued as models

Answer 27

The need for a larger and cheaper host that was easier to breed, had more uniform genetic characteristics, and did not suffer from any other infection led to the use of P. lophurae and P. gallinaceum in chickens. Chickens, although not easy experimental hosts ot manage, were widely-used as an animal model in the study of avian malaria.

Question 28

Avian malarias Avian Plasmodium-host combinations ued as models

Answer 28

P. gallinaceum infections of chickens, and some other avian Plasmodium-host combinations, remained the most useful laboratory models until the discovery of the rodent malaria parasites. The most commonly used avian infections are listed: P relictum in sparrows, P cathernerium in sparrows and canaries, P elongatum in canaries, P lophurae in chickens, ducks, P gallinaceum in chickens.

Question 29

Avian malarias Life-cycles of avian Plasmodium species

Answer 29

The life-cycles of all avian Plasmodium species show differences from those of mammalian forms. Following their injection by mosquitoes, the first phase of exoerythrocytic schizogony takes place in cells of the lymphoid-macrophage system and results in the production of merozoites that invade both red blood cells and other cells giving rise to erythrocytic and further exo-erythrocytic schizogonic cycles that co-exist.

Question 30

Avian malarias Life-cycles of avian Plasmodium species

Answer 30

Erythrocytic schizogony is similar to that of mammalian parasites but the merozoites produced can invade new red blood cells or other cells and can also initiate exoerythrocytic cycles. The gametocytes, gametes and sporogonic stages are similar to those in mammalian species.

Question 31

Avian malarias Life-cycles of avian Plasmodium species

Answer 31

Much of what we know about the development of exoerythrocytic schizonts of avian malarias is based on in vitro systems because of the ease of maintaining bird cells in continuous culture. Details of the biochemistry and physiology of malaria parasites in general have been derived from such studies. Next we'll review the contributions that avian malaria studies have made to the study of human malaria…

Question 32

Avian malarias Contributions made by avian malaria studies

Answer 32

For over 50 years, various avian models provided a vast amount of basic information about malaria parasites, including the first evidence of an exo-erythrocytic phase in the life-cycle (of P. elongatum and P. gallinaceum) referred to above. Some of the first experiments on immunity and immunisation were carried out in birds, particularly chicks infected with P. gallinaceum. It was in these models that the feasibility of inducing immunity using X-irradiated parasites and the possibility of inducing transmission-blocking immunity were first demonstrated.

Question 33

Avian malarias Contributions made by avian malaria studies

Answer 33

The most important contributions made by studies on avian malarias to human malaria were undoubtedly the extensive drug trials from which compounds such as proguanil, pyrimethamine, primaquine and chloroquine were developed and came onto the market. It was also clear from experiments with these avian models that different drugs acted on different stages of the life-cycle. This led to the development of drugs specific for particular stages.

Question 34

Avian malarias Contributions made by avian malaria studies

Answer 34

Following the discovery of the rodent malaria parasites, the use of avian malarias as models declined and has now virtually passed into history. As a distinguished worker in this field wrote: Avian malaria studies as models for human malaria have a distinguished past but a rather uncertain future. (McGhee 1988)  Now answer the question that follows…

Question 35

Question 5 Imagine you have discovered what looks like a malaria parasite in the blood of a bird. What facilities would be needed to set about identifying it, incriminating its vector and working out its life-cycle?

Answer 35

First, you would need to be sure that it is a malaria parasite: does it have pigment and are there any schizonts? You should check its possible identity in an authoritative book and original papers. To investigate it’s lifecycle would require finding a suitable laboratory host and trying to infect it (a parasite from a passerine bird is likely to infect a canary and so on). If you are successful, you can then try to infect suitable laboratory mosquitoes. Finding the natural vector would require going to the natural habitat to trap mosquitoes (particularly those that have fed on blood). This list is not exhaustive so you can see how difficult it is to make progress following the discovery of the parasites. Collaboration and teamwork is needed!

Question 36

Rodent malaria parasites

Answer 36

There are at least eleven species of Plasmodium known that infect rodents. The first to be identified, P. berghei, was discovered, largely by accident, in 1948. Since then, other species have been identified, isolated and intensively studied to such an extent that more is known about these parasites than about most others, including some of the human malaria species. The reason for this is that these parasites are easily maintained in laboratory rats and mice, and hence became the most used models for human infections.

Question 37

Rodent malaria parasites Species of rodent malaria parasites

Answer 37

There are four well-known species, all originally isolated in central and western Africa: P berghei, P yoelii, P vinckei, P chabaudi. Within these species are several subspecies. The natural hosts are mostly thicket rats, but the vectors are poorly known.

Question 38

Rodent malaria parasites Species of rodent malaria parasites

Answer 38

The life-cycles in both natural and laboratory hosts are similar to those of other mammalian malarias: there is an exoerythrocytic phase in the liver followed by repeated schizogony in the blood and the production of gametocytes infective to mosquitoes. The exoerythrocytic phase is short, typically 48 hours, resulting in the production of 10,000 to 18,000 merozoites, and the asexual stage is also short, about 24 hours, and the number of merozoites produced is 8-16, usually 8. As indicated, all of these parasites can be easily maintained in the laboratory; for example, P. berghei infects rats, mice and hamsters and can be blood-passaged from one host to another, transmitted in the laboratory by several mosquitoes including Anopheles stephensi, and can be kept frozen indefinitely.

Question 39

Rodent malaria parasites Species of rodent malaria parasites

Answer 39

In mice, the preferred host erythrocytes and the lethality of infection vary: P berghei prefer reticulocytes, mice die, P yoelii prefer reticulocytes, mice recover, P vinckei prefer mature erythrocytes, mice die, P chabudi prefer mature erythrocytes, mice recover

Question 40

Rodent malaria parasites Species of rodent malaria parasites

Answer 40

The patterns of parasitaemia between different laboratory hosts also varies. While rats, mice and hamsters can be infected easily, guinea pigs and rabbits are more resistant to infection. In general, mice are easier to infect and more susceptible to infection than rats. With P. berghei, young rats often die, but older rats recover because of some kind of as yet undefined age immunity. Next we'll review the uses of rodent parasites…

Question 41

Rodent malaria parasites Species of rodent malaria parasites

Answer 41

The rodent malaria parasites are used for different kinds of studies. Those that caused high parasitaemias and death were used for drug testing and the less severe ones were used for immunological studies.

Question 42

Rodent malaria parasites Species of rodent malaria parasites

Answer 42

The situation is no longer this simple, and there have been a large number of isolates of subspecies and strains that cause a range of patterns of infection in different strains of inbred mice. These strains of parasite are effectively clones derived from a single parasite, and are thus genetically identical. They can be preserved as such by freezing.

Question 43

Rodent malaria parasites Species of rodent malaria parasites

Answer 43

There are now hundreds of different clones available, and also numerous combinations of parasite and host that can be used for different purposes. In addition, it is possible to use genetically modified mice in order to investigate particular problems. For example, mice deficient in certain cytokines are often used in immunological studies.

Question 44

Rodent malaria parasites Drug testing

Answer 44

The most widely used model for testing antimalarial drugs is P. berghei in mice given a measured dose of blood-stage parasites followed by a single or multiple doses of drug. Drugs have been frequently tested against the exoerythrocytic stages of malaria in mice infected with P. y. yoelii and P. y. nigeriensis subspecies transmitted by Anopheles stephensi. These host-models, if carefully chosen, provide very useful screens for antimalarial drugs and have provided a vast amount of information about drug resistance.

Question 45

Rodent malaria parasites Immunological studies

Answer 45

Rodent malaria parasites have also been widely used in immunological studies. For example, P. chabaudi in mice is particularly useful in this respect because the infection can persist for a relatively long time thus mimicking the situation in humans. Studies on P. chabaudi in mice have been useful in attempts to understand antigenic variation in malaria parasites referred to earlier. Mouse models, particularly those using genetically engineered mice in more recent times, have also been invaluable in determining the roles of immunologically important molecules such as cytokines and in analysing the different immune mechanisms functioning in malaria.

Question 46

Rodent malaria parasites Immunological studies

Answer 46

Genetically engineered humanised mice that possess human immune cells have been developed. Human red blood cells can be introduced into these mice which can then be infected with P. falciparum to undergo asexual blood stage development and replication thereby mimicking human falciparum malaria. Chen et al. (2014) provide an example of the use of a humanised mouse model to elucidate an important early immune mechanism that controls blood stage P. falciparum infections.

Question 47

Rodent malaria parasites Immunological studies

Answer 47

Clones of rodent malaria species have been established in mice, and mosquitoes are fed on mice infected with a particular clone or a mixture of clones in order to determine how particular genetic characters are transmitted. Studies such as these have been invaluable in providing information about drug resistance and genetic diversity and variation.

Question 48

Question 6 Why do you think rodent malaria parasites have increased our knowledge of human malaria?

Answer 48

The rodent malaria parasites have made immense contributions to our knowledge of human malaria, because they are so easy to study. Hypotheses can be tested in laboratory mice and relevant findings quickly extrapolated to the situation in human malaria. Advances in chemotherapy and the development of vaccines would have been impossible without this approach but there is always the danger that what happens in a mouse is not necessarily what happens in a human.

Question 49

Question 7 Which of the following do you think rodent malaria parasites can be used for? a) to screen potential antimalarial drugs b) as a model for understanding modes of transmission c) to provide preliminary information about immune responses and possible vaccine candidates d) to develop methods for vector control

Answer 49

The correct answers are: a) To screen potential antimalarial drugs. c) To provide preliminary information about immune responses and possible vaccine candidates. For example, irradiated sporozoites were tested in mice as candidate vaccines before being used in human trials. Rodent malaria parasites cannot be used: b) As a model for understanding modes of transmission: human studies are needed to understand modes of transmission to and from mosquitoes. D) To develop methods for vector control: this is done through spraying campaigns, trials of insecticide-treated bed-nets, larviciding, and use of repellents.

Question 50

Question 8 What do you think is the main disadvantage of using these models to study malaria in humans?

Answer 50

They are most commonly used as blood-induced infections and the disadvantage then is that there is no exoerythrocytic stage. Therefore, they do not resemble the sporozoite-induced infections seen in humans. Some strains of P. yoelii and P.berghei can however be easily transmitted by Anopheles stephensi which overcomes this particular problem.

Question 51

Non-human primate malarias

Answer 51

There are at least 27 named species of Plasmodium in non-human primates that include two in New World monkes, ten in Old World monkeys, four in gibbons, four in great apes, seven lemurs. The lemur parasites are all found in the Malagasy Republic (now Madagascar); so little is known about them that they are seldom considered alongside the other primate malarias.

Question 52

Non-human primate malarias Species of non-human primate malaria parasites

Answer 52

The species that you are most likely to encounter in the literature are listed below. The first authentic description of a non-human primate malaria parasite was made in 1907 and only eight more valid species had been described by 1951, after which there was a flurry of activity that continues to the present day. P cynamolgi, P inui, P brasilianum, P simium, P knowlesi, P reichenowi

Question 53

Non-human primate malarias Species of non-human primate malaria parasites

Answer 53

The Plasmodium species of non-human primates differ from those of humans in that they are not nearly so host-specific. All the Asian primate malarias can infect Old World rhesus monkeys and several species can infect New World Aotus spp. and Saimiri spp. The natural vectors of all non-human primate malarias are various species of mosquitoes belonging to the genus Anopheles .

Question 54

Question 9 Do you think humans are susceptible to the Plasmodium species of non-human primates?

Answer 54

Yes, humans are susceptible to the Plasmodium species of non-human primates. Humans are susceptible to infection and have been accidentally or deliberately infected with P. cynomolgi and P. knowlesi. Volunteers have also been infected with P. brasilianum, P. inui and P. schwetzi (a chimpanzee parasite) with resultant low-level parasitaemias. Most of these primate malarias can infect a range of Anopheles species. P. knowlesi is now known to be transmitted from its natural monkey hosts to humans by anopheline vectors in forest areas in South East Asia. P. knowlesi malaria in humans is therefore an established zoonotic disease.

Question 55

Non-human primate malarias Use of Plasmodium species from non-human primates

Answer 55

All aspects of the biology of malaria parasites have been intensively studied using Plasmodium species of non-human primates, especially host-parasite interrelationships, parasite ultrastructure, biochemistry, physiology, immunology and chemotherapy.

Question 56

Non-human primate malarias Use of Plasmodium species from non-human primates

Answer 56

Primate models made a number of contributions to our understanding of the life-cycles of malaria parasites in humans. The most significant of these was the discovery of exoerythrocytic stages in the liver in P. cynomolgi in 1947, which led immediately to the discovery of the exoerythrocytic forms of P. vivax in humans a year later, and similarly the discovery of hypnozoites in 1980 led to the same forms being found in P.vivax.

Question 57

Non-human primate malarias Use of Plasmodium species from non-human primates

Answer 57

P. knowlesi has been widely used. It is an unusual primate malaria as it has a 24 hour erythrocytic cycle. Somewhat surprisingly P. knowlesi in rhesus monkeys provided some of the first evidence of antigenic variation in the asexual blood stages. As it is a virulent infection in the rhesus, it had to be treated several times. Each new recrudescence of infection was antigenically quite different from what had gone before.

Question 58

Non-human primate malarias Use of Plasmodium species from non-human primates

Answer 58

Antigenic variation has subsequently proved to be an important feature of immunity to malaria asexual blood stages and will be discussed later in the module

Question 59

Question 10 Can you think of the advantages and disadvantages of using non-human primate models?

Answer 59

The obvious advantage is: they resemble the human infection more closely than any other model. The disadvantages are: monkeys are expensive; monkeys are difficult to obtain and usually have to be collected from the wild and may therefore already be infected with other parasites; and in addition, the hosts are genetically diverse and cannot be used in the numbers required to give statistically significant results. For these reasons, non-human primates have been used sparingly for drug testing, but have been essential for experiments leading to the possible development of vaccines.

Question 60

Non-human primate malarias Human malaria infections in monkeys

Answer 60

Apart from P.knowlesi which is an established zoonotic infection, the other human malaria parasites are usually considered to be host-specific but there are a few important exceptions. The New World owl monkey, Aotus spp., particularly A. trivirgatus, and the squirrel monkey, Saimiri sciureus can be infected with P. falciparum, P. vivax, and P. malariae.

Question 61

Non-human primate malarias Human malaria infections in monkeys

Answer 61

Both monkey species have been used for drug testing, particularly with respect to studies on resistance. However, these hosts have to be collected from the wild and are in short supply outside South America. There is also considerable variability between the patterns of infection in individual animals because of genetic differences inherent in wild-caught animals.

Question 62

Non-human primate malarias Human malaria infections in monkeys

Answer 62

Nevertheless, owl and squirrel monkeys have been widely used to test the efficacy of candidate malaria vaccines against P. falciparum and P. vivax before performing human trials with the same vaccines. However, some promising results obtained with vaccines in these monkeys have not always been reflected in the subsequent findings in humans. These factors have recently led to the greater use of human volunteers for early stage vaccine trials.

Question 63

Human and non-human malaria parasites

Answer 63

This module is about malaria as a disease of humans but it is important to remember that four of the species that infect humans, P. falciparum, P. vivax, P. ovale, and P. malariae all have counterparts in non-human primates, and P. knowlesi is a natural parasite of both humans and old world monkeys. The life-cycles of all the mammalian Plasmodium species are similar, as are most aspects of their biology. This includes the rodent malarias, though, as we have seen, they have no obvious counterparts of the human species.

Question 64

Human and non-human malaria parasites

Answer 64

The human malaria species are very host-specific, although, as we have seen, they can infect Aotus and Saimiri monkeys experimentally, and P. falciparum can be grown continuously in culture to produce both asexual and gametocyte blood stages which the others cannot.

Question 65

Question 11 For each of the human species, list one other species that is genetically very similar.

Answer 65

P. cynomolgi and nine other species are similar to P. vivax. P. simiovale and P. fieldi are similar to P. ovale. P. brasilianum and P. inui are similar to P. malariae. P. reichenowi is similar to P. falciparum.

Question 66

Question 12 What are the relationships between the Plasmodium species in humans and other mammalian malaria parasites? What do you think is the implication of such relationships?

Answer 66

The five species in humans appear to be only remotely related to one another. P. falciparum has greater affinity with P. reichenowi in chimpanzees and other Plasmodium species in gorillas than with the other human malaria parasites. P. malariae is genetically indistinguishable from P. brasilianum and P. vivax is genetically indistinguishable from P. simiumbased on the circumsporozoite protein (CSP) gene sequences. The significance is that there has been lateral transfer of Plasmodium species between humans and New World monkeys in the case of P. vivax / P. simium and P. malariae / P. brasilianum . This most probably occurred in the post-Columbus era when migrants from Europe and Africa took P. vivax and P. malariae with them into South America. Similarly, it is now likely that P. falciparum was first acquired by humans through lateral transfer from higher apes in Africa carrying ancestral parasites related to P. reichenowi . The origin of P. falciparum and the other species is much debated and subject to molecular genetic studies. There is interesting evidence provided by Liu et al. (Nature, 2010) and Loy et al. (2016) suggesting that P. falciparum originated in African gorillas. Similarly, there is genetic evidence from Escalante et al. (Proc Natl Acad Sci USA, 2005) to suggest that human P. vivax evolved from Plasmodium species parasitising macaque monkeys in South East Asian jungles. However, more recently, there is some evidence that it too is of African origin (Loy et al. 2016).

Question 67

Human and non-human malaria parasites Spurious Plasmodium species

Answer 67

From time to time, there are records of new malaria parasites from a variety of hosts, usually based on parasites seen in blood films. These are usually misidentifications but can cause confusion if the records come from outside a recognised host range or geographical distribution.

Question 68

Question 13 What other things in the blood might be confused with malaria parasites?

Answer 68

Babesia parasites (shown earlier in the session and below), spirochaetes, Howell-Jolly and other bodies, platelets and bacteria. Note that rodent blood is frequently infected with rickettsiae that can look like malaria parasites. It is also particularly important to be aware of anything that might look like a malaria parasite when examining blood films from wild animals because the blood films themselves might be contaminated, for example, from bacteria on the hair.

Question 69

Question 14 List the main differences between avian and mammalian Plasmodium species

Answer 69

Avian malaria parasites: infect nucleated red blood cells, have exoerythrocytic stages in cells of the lymphoid-macrophage system, have exoerythrocytic stages that co-exist with erythrocytic stages, have exoerythrocytic stages that can be initiated with erythrocytic stages, and have vectors that are culicine mosquitoes. Mammalian malaria parasites: infect non-nucleated red blood cells, have sporozoites that only infect liver cells, have merozoites that only infect red blood cells and have vectors that are female Anopheles

Question 70

Question 15 What information about human malaria has been obtained from studies using avian malaria parasites? Select more than one a) the discovery of the sexual stages of malaria parasites b) information about cerebral malaria c) the elucidation of the sporogonic stages in mosquitoes d) discovery of malaria toxins e) drug resistance

Answer 70

The correct answers are:The discovery of the sexual stages of malaria parasites; The elucidation of the sporogonic stages in mosquitoes; and Drug resistance The wrong answers are: Information on cerebral malaria has not been obtained from studies using avian malaria parasites as birds do not display cerebral symptoms. There are some mouse malaria infections that display cerebral symptoms but there is divided opinion on how similar they are to cerebral malaria in humans. Studies leading to the discovery of malaria toxins have not been done in birds.

Question 71

Question 16 Can you think of some of the major contributions non-human studies have made to our understanding of the human malaria life cycle?

Answer 71

You may have come up with many answers, including that one of the major contributions was the discovery of the exoerythrocytic stages in the liver and hypnozoites. You may have also knowledge of our understanding of antigenic variation or our understanding of asexual and sexual stages of the parasite.

Question 72

Question 17 There are two Plasmodium spp. of birds, four Plasmodium spp. of non-human primates and four Plasmodium spp. of rodents. Can you match each Plasmodium species to the correct group of vertebrates?

Answer 72

Birds: P relictum, P lophurae. Non-human primates P cynomolgi, P inui, P knowlesi, P reichenowi. Rodents: P yoelii, P chabaudi, P vinckei, P berghei

Question 73

Question 18 What are the advantages and disadvantages of animal models in malaria research?

Answer 73

Advantages: Animal models make it possible to perform experiments and to make observations that would not be possible to do in humans.Disadvantages: The main disadvantage is that the infections resemble, but do not necessarily truly reflect, infections in humans.

Question 74

Question 19 What are the advantages of using rodent models over using non-human primate models?

Answer 74

Non-human primates might make good models but are difficult to obtain, expensive and cannot be guaranteed to be free from other pathogens. Laboratory mice are, or should be, free from other pathogens, can be bred as genetically pure lines and can be genetically manipulated. Humanised mice containing human immune cells can contribute to analysing immune mechanisms operative in human falciparum malaria. Furthermore, a vast amount of information about laboratory mice is easily available.

Question 75

Summary

Answer 75

There are at least 200 Plasmodium species that infect reptiles, birds and mammals. The five species of human malaria parasites evolved from ancestral Plasmodium species in primates that infected humans. Knowledge of the malaria parasites of lower animals has been important in the development of our understanding of human malaria. Animal models have provided a vast amount of useful information about the biology of malaria parasites. Animal models are invaluable for testing drugs and vaccines. Avian malarias have proved to be useful models in the past, especially for drug testing, but are no longer widely used. Malaria parasites of non-human primates, some of which resemble human species very closely, are the best models for human malaria but are neither easy nor convenient to use. Human malaria parasites in owl and squirrel monkeys provide useful models for chemotherapy and vaccine development. Four species of rodent malaria parasites infect laboratory mice and these have become the models of choice for experimental malaria research. Genetically engineered and humanised mice are used to analyse immune mechanisms in malaria. Although often very useful, no animal model is truly representative of human malaria.

Question 76

Glossary Rodents

Answer 76

Common name given to mammals belonging to the order Rodentia. The order contains a number of suborders including the Myomorpha, within which is the family Muridae containing rats and mice, Hystricomorpha containing porcupines and Sciuromorpha containing squirrels. It is important not to think of rodents only as rats and mice; the common name ‘murine rodents’ is often applied to this group.

Question 77

Glossary Anopheline

Answer 77

A common adjective applied to mosquitoes belonging to the genus Anopheles.