Control of coccidiosis: lessons from other sporozoa

International Journal for Parasitology 28 (1998) 165-179

Control of coccidiosis: lessons from other sporo;zoa F. E. G. Cox* Division

of Life Sciences,

King’s

College

London,

Campden

Hill Road, London

WS 7AH,

U.K.

Received 10 July 1997; accepted 4 August 1997

Abstract Coccidiosisis the mostimportant parasiticinfection in poultry worldwide and alsocausesprow and goats.Control is largely limited to good husbandryand prophylactic chemotherapyusinga r which resistanceis rapidly acquired.Attempts at vaccination usingconventional vaccineshave been there is now a needfor a new approach.Researchinto the immunologyof coccidiosis sporozoansand there are useful lessonsthat might be learned from studieson to theileriosisand malaria. In theseinfections the emphasishasturned to the cytokine

in eat&, sheep

towards protection. Central to these studies are the roles of interferon-gamma, int.erieuI~in-12 ~d~~v~~’ with the involvementof nitric oxidein parasitekilling. Cytotoxic T cellshavealsoincreasinglybee;m imp&W. Research hasshownthat different immune responsescan be elicited by manipulating the cytokine system and these new concepts can be appliedto the designof peptideor recombinantvaccines,and the possibi&esof develop&gsuchv&&s against coccidiosiswill be discussed. 0 1998Australian Society for Parasitology.Pubfishedby ElsevierScienceLtd Key

words: sporozoa; coccidiosis; cryptosporidiosis; theileriosis; toxoplasmosis; control strategies; vaccination

1. I-on Parasitic protozoa present some of the main threats to the wellbeing not only of humans and domesticated animals, but also of wild animals, both vertebrate and invertebrate, all over the world. If we, as humans, are threatened with any challenge to our territorial rights there are three responses: to give in, to come to live with the threat or to counteract it; in other words, to declare a state of war. Whether we like it or not, we are at war with all the parasites that threaten us, but in many cases we have not learned the first rule of warfare which is

*Tel: +44 171 333 4391; Fax: +44 171 333 4500; e-mail: [email protected].

to know the enemy. Most of our attempts to control parasitic infections are little more than limited and local skirmishes from which the emmy anqa to begin the fight again, often from a position of increased strength. If we are to win the war agtinst parasites, it is essential that we should develop appropriate defence strategies and, in order to do so, we must really understand what we are up against and be prepared to learn from the knowledge and experiences of others. Currently there are numerous battles against infectious agents going on, mo&Iy &I is&&m, but it is gradually becoming clear that there are both specific and common features in all in&&ms. It is, therefore, necessary to dissect out what is and what is not common in order to devise, at Ieast theoretically, a united approach to the design of drugs

SOfl20-7519/98 %19.00+0.00 0 1998 Australian Society for Parasitology. Published by Elsevier Science Ltd. Printed in Great Britain PIZ: SOO20-7519(97)00166-S

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and vaccines. Among the most important infections of humans and domesticated animals are several caused by sporozoans and we do have a vast amount of knowledge about the dozen or so species that are of medical or veterinary importance. In this review I shall try to consider the salient points of immunity to some of these parasites and the present state of development of vaccines against them, and point to any lessons that can be learned and applied to the control of coccidiosis.

2. Biology of parasitic sporozoa

An outline classification and list of the sporozoa to be considered here are given in Table 1. This simplified classification, best regarded as an aide mkmoire, is based on that used by Corliss [l] and cox [2].

In order to devise effective control strategies it is necessary to know something about the structure and life-cycles of the parasites involved. Sporozoa, being unicellular organisms, possess only a limited range of structural, physiological and biochemical features that are likely to be useful as targets for immunological or chemotherapeutic attack and many of these are special&d and can be correlated with the parasitic way of life. On the other hand, all have essentially similar life-cycles involving an infective stage, the sporozoite, that enters the body of the host and becomes a feeding stage (trophozoite) which typically undergoes nuclear division (schizogony) resulting in the formation of merozoites. This phase may be repeated, but eventually some merozoites develop into gameto-

Table 1 Outline classification indicating the taxonomic positions of the genera of parasites referred to in this paper Phylum Apicomplexa (Sporozoa) Class Coccidea Order Eiieriida. Genera: Cryptosporidium”, Eimeria”,

Isospora,

Sarcocystis,

Toxoplasma”

Class Haematozoa Order Haemosporida. Genus: Plasmodium” Order Piroplasmida. Genera: Babesia, TheiIeria” “The genera considered in most detail.

Cyclospora,

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cytes and gametes that fuse to produce a zygote. The rest of the life-cycle is not really relevant to the development of drugs or vaccines against coccidiosis. The various stages in the life-cycle assume distinctive degrees of importance in different groups and are vulnerable to attack in different ways. The many variations on this basic life-cycle can be categorised according to the site of infection, e.g., intracellular or extracellular, the number of schizogonies, the nature and site of gametogony and whether the life-cycle is confined to one host (homoxenous) or divided between two (heteroxenous). Again, this information is important in devising attack strategies. Within the sporozoans, members of the class Coccidea, the coccidians, form a coherent group containing some of the most advanced sporozoans, and some workers include both the haemosporidians (Plasmodium spp.) and piroplasms (Babesia spp. and Theileria spp.) in this group (see, for example, Mehlhorn, [3]). Coccidiosis, as will be discussed later, is caused by infection with certain eimeriid coccidians; a massive group containing both “primitive” and “advanced” forms, some of which have relatively simple life-cycles in invertebrates and others that have complex life-cycles involving both vertebrates and invertebrates. The basic life-cycles are all similar: infection with a sporozoite, one or more cycles of schizogony, gametogony and sporogony. The final product of the life-cycle, and the one that passes into the external environment, is usually a resistant oocyst containing sporocysts which contain sporozoites, the actual infective stages. The largest group of eimeriid coccidia is the family Eimeriidae, many of which are important pathogens of wild and domesticated animals. In most species schizogony is predominantly in the epithelial cells of the intestine, but there is a trend from superficial sites to deeper ones. The coccidia of economic importance include homoxenous species of Eimeriu that parasitise domesticated animals, particularly chickens, and a number of heteroxenous cyst-forming species. Eimeria species tend to be host- and organ-specific and infections are normally self-limiting, especially if the infective dose of sporozoites is low. However, depending on the degree of invasion of host tissues

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and under conditions that favour intensive transmission, many species of Eimeria are serious pathogens. About 15 species infect cattle and these have presumably been derived from buffalo and other wild bovids and the more important species are now present in cattle throughout the world. Similarly, the most important avian coccidia have spread worldwide and now present a continuous threat, particularly to floor-reared broiler (meat) birds or floor-reared laying hens. The success of these parasites is due in part to their efficient production of extremely resistant oocysts, the congregation of hosts and human activity in moving animals around the world. Coccidians belonging to the genus Cryptosporidium are relatively common parasites in the intestinal and respiratory tracts of mammals, birds and reptiles. Cryptosporidium parvum causes gastrointestinal disorders in cattle, sheep and humans, and is a parasite of increasing importance partly because it is a significant concomitant of human immunodeficiency virus infections [4,5]. Cyclospora cayetanensis is the most recently described coccidian of humans and has also attracted attention because of its prevalence in immunocompromised patients [6]. Toxophsma and cyst-forming eimeriid coccidia, the closest relatives of Eimeria, have been intensively investigated following the discovery that many parasites that had been difficult to classify actually represent stages in the life-cycles of members of the genus Isospora and closely related genera. Toxoplasma gondii, which occurs in humans, virtually all other species of mammals and some birds, is the best known species and represents a heteroxenous eimeriid life-cycle. Members of the family Sarcocystidae are heteroxenous with schizogony in an intermediate vertebrate host, usually a prey animal, and gametogony and sporogony in an appropriate vertebrate predator. Toxoplasma gondii is a typical intestinal eimeriid parasitic in the intestine of felids, particularly domestic cats, in which it causes little harm but in hosts other than felids a disseminated infection results [7]. Unlike most of the other eimeriids, T. gondii is capable of infecting a very wide range of hosts, over 200 species of mammals including humans and domesticated animals. Toxoplasma gondii is, therefore, a very

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successful parasite and there are obviously lessons to be learned from the fact that it rarely causes any damage to its natural hosts. The malaria parasites, members of the order Haemosporida, are the most intensively investigated of all the sporozoans. Most a&u&ies, for example, Garnham [g] and Levine [9], eensider that they represent a distinct natural assemh=lage, while others group them with other blood-inhabiting forms more closely related to eimeriid cocoidia or piroplasms (see, for example, Mehlhorn [3]). Whatever the niceties of these arguments, there is no question that the malaria parasites do have features in common with the coccidia and, therefore, can contribute to our understanding of coccidiosis. Haemosporidians are heteroxenous; the infection in the vertebrate host begins with the injer$ion of sporozoites which initiate a tissue schizogonic cycle resulting in the production of merozoites that initiate further schizogonic cycles in the blood and, subsequently, the formation of gametocytes in blood cells. These are taken up by a blood-sucking mosquito within which sporogony occurs, resulting in the production of large numbers of sporozoites that are injected into a new host when the vector feeds. There are a number of well-character&d species of malaria parasites in birds, rodents, non-human primates and four in humans, Plasmoditcm vivax, Plasmodium ovale, Plasmodbm malariae and Plasmod&m faiciparwn [lo]. All have been intensively studied, particularly with respazt to immunology and chemotherapy. The success of the malaria parasites can be attributed directly to their genetic: diversity and versatility which has permitted t&m, under natural circumstances, to adapt to different vectors and to evade the immune responses of their hosts. In addition, as a result of human-imposed selection pressure, malaria parasites have developed resistance to most antimalarial drugs [1 11. The piroplasms are heteroxenous parasites that typically undergo phases of schizogony and gametogony in a vertebrate host. Gametocytes occur in blood cells and are taken up by a blaad-sueking tick within which sporogony occurs, resulting in the production of sporozoites which infect the vertebrate host when the vector feeds. Most observers place the piroplasms close to the coccidia and the

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haemosporidians [2, 3,9]. The piroplasms of mammals include two genera, Babesia and Theileria, some of which including Babesia bigemina, Babesia bovis, Babesia major, Theileria parva and Theileria annulata, affect cattle [12, 131. In Babesia species, multiplication occurs in the form of binary fission in red blood cells, in which gametocytes are formed, and sporogony occurs in the tick. In some species there seems to be a limited schizogonic phase which precedes the phase of multiplication in the blood. In Theileria species repeated schizogony occurs in lymphocytes, and gametocytes occur in red blood cells, after which the life-cycle is essentially similar to that of Babesia.

3. Immune responses to infections The immune response to any infection, including parasites, is brought about by the interplay between a number of effector systems. Essentially, these are cytotoxic CD8 + T lymphocytes that kill target cells in a major histocompatibility complex (MHC)restricted way, cytotoxic natural killer (NK) cells that also kill target cells but are not MHC-restricted, macrophages, eosinophils and antibodies. The overall immune response is regulated by macrophages and T helper lymphocytes [14]. CD4+ Thl and Th2 lymphocytes control the cell-mediated and antibody-mediated arms of the immune response, respectively, and are characterised by the cytokines that they produce and ultimately by the immune responses they evoke [ 151. Thl cells characteristically produce interleukin (IL)-2 and interferon-gamma (IFN-y), whereas Th2 cells characteristically produce IL-3, IL-4, IL-5, IL-10 and IL-l 3. In the overall immune response, Thl cells are responsible, via IL-2, for the activation of cytotoxic CD8+ T cells, NK cells and, via IFN-y, for the activation of macrophages to produce toxic molecules of which nitric oxide and reactive oxygen intermediates are the most important. Th2 cells, on the other hand, are responsible, via IL-4, IL-10 and IL-13, for the induction of B cells to produce immunoglobulins IgM, IgG, IgA and IgE that function as antibodies and, via IL-3 and IL-5, for the activation of eosinophils. There is a considerable degree of cross-regulation between Thl and Th2

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cells, for example, IFN-y, a Thl product, inhibits antibody production and the Th2 cell products, IL4 and IL-lo, inhibit the activity of IFN-y. There are also two subsets of CD8+ cells, based on the cytokines they produce, which are essentially similar to Thl and Th2 cells [ 161. CD8 + cells are, therefore, not solely effector cells in target cell killing, but are also involved in the cytokine control of the immune response. As well as producing toxic molecules, macrophages are producers of IL-12 which has a major role in determining the outcome of the immune response. NK cells are also involved not only in cytotoxic cell killing, but also as producers of IFN-y. The nature and outcome of any immune response is determined by cytokines present at the initial stages of the immune response. IL-12 and IFN-7 direct the immune response towards the Thl pole and IL-4 directs it towards the Th2 pole. The outcome of any immune response is also determined by cross regulation between CD4+ Thl and Th2 T cells; the Thl product IFN-), downregulates Th2 responses, whereas the Th2 products IL-4 and IL10 downregulate Thl responses. In addition to these negative influences, IL-12 and IFN-1/ upregulate the activity of Thl cells and IL-4 upregulates the activity of Th2 cells. The immune response is, therefore, controlled by a network of cytokines, some driving the immune response one way and some the other and the actual outcome of any immune response is determined by the cytokine pathway that the antigen initiates. Briefly, the response can be of the Thl type, resulting in what is conventionally called cell-mediated immunity, the Th2 type, resulting in the activation of eosinophils and antibody production, or a mixture of the Thl and Th2 types. In general, Thl responses are appropriate for intracellular parasites and Th2 responses are better suited for helminths [ 141. The actual nature of the immune response is determined by a number of factors, including the nature and route of administration of the antigen, the mechanisms of processing and presentation of the antigen and the immunological environment within which the immune response operates. This information gives us the confidence to consider the rational development of vaccines against parasites. Vaccination would be the ideal solution to the

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problem of the control of a number of parasitic infections and, in this context, a vaccine can be defined as an antigen (or antigens) that, when administered artificially, induces an adaptive immune response against a particular parasite without causing disease. In some cases total protection, although ideal, may not be possible. In a previous paper [ 1’71 I discussed the possibility of designer vaccines; vaccines tailor-made in order to induce an appropriate immune response based on the totality of the immune response. In order to develop such a vaccine it is necessary to determine exactly which components of the immune response are involved in protection and how they operate. However, we know remarkably little about protective immune responses to the majority of parasites and what we do know about immunity to sporozoan parasites from which lessons about coccidiosis can be learned is discussed in the next section.

gy of sporozoan infections 4.1. Cryptosporidiosis

The group of parasitic protozoa that most closely resemble the Eimeria spp. responsible for coccidiosis, and from which something might be learned, are the intestinal coccidians of humans, C. parvum, Isospora belli and C. cayetanensis. These protozoa have received a considerable amount of detailed attenti’on, but relatively little is known about the immune responses of humans to any of them except C. pmwn. Cryptosporidiosis has been recognised as an important disease only during the last 20 years (reviewed by [4, 5, 181). Cryptosporidium spp. are smali eimeriid coccidians that typically parasitise the epithelial cells of the digestive tract. Cryptosporidium parvum and Cryptosporidium muris are mammalian forms and Cryptosporidium baileyi and Cryptosporidium meleagridis have avian hosts [4]. Cryptosporidiumparvum is the most important of these and occurs in a wide range of mammalian hosts, including humans, mainly infants, and calves and lambs in which it causes neonatal scour. The life-cycle is typically eimerian but the parasites occupy parasitophorous vacuoles in the microvillous part of the host cell, whereas in most eim-

169

erian infections parasites are found deeper within the cell. In healthy hosts the i&e&on is restricted to the intestine and is self&niting, but in immunosuppressed patients or animals the parasites spread to other epithelial cells, in&&ng those of the respiratory tract, and infections tend to be persistent, the symptoms mu& more severe and death may result [WI. This s that tkre is immune involvement. Little is known about the possible mechanisms or relevance of these immunolog&al responses in humans or domesticated animals, but in mice the immune response is essentially of the Thl type with the involvement of IFN-y [20, 211. NK cells, under the influence of IL-12, seem to be an important source of EN-y [22] but intraepithelial lymphocytes also play an important role [l8] (and see Sim [23] for a general review of the role of intraepithelial lymphocytes). CIB -t cells do not appear to be important [24]. Specific antibodies, IgM, IgG and IgA, have been de&e&d in the serum of infected individuals and experimentaf animals, but there is little evidence of any prote&ve role and AIDS patients with fulminating infections have high levels of IgA [25]. However, a role for antibody cannot be dismissed because hyperimmune bovine colostrum is protective in neonatal mice [26] and immunised hens produce eggs containing y&k with high levels of anti C. parvum antibodies which can passively transfer immunity to mice [27]. In the context of possible antibody-mediated protection, it is interesting to note that EN-~ increases the output of IgA across the epith&um into the lumen [28], thus linking the Thl and Th2 arms of the immune response. In summary, immunity to cryptosporidiosis is T cell-dependent and reliant on the Thl CD4+ response involving IL- 12, NK cells, intra-epithelial lymphocytes and IFN-7 as the key eRector molecule. In addition there is evidence that antibodies in the colostrum may be protective and that the principal of passive immunisation using hen’s eggs as a source of antibody has been established. 4.2. Toxoplasmosis Toxoplasma gondii is a common parasite of cats which can be transmitted to virtuahy all warmblooded animals [7]. One very interesting charac-

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teristic is that worldwide there seem to be only three distinct lineages of T. go&ii [29]. The life-cycle in the cat is basically similar to that of Eimeria spp., with initial development in the epithelial cells of the intestine but with subsequent multiplication in extraintestinal cells, including macrophages. The formation of sexual stages proceeds as in Eimeria spp. and oocysts are voided in the faeces. In nonfeline hosts the initial stages of the infection are followed by a disseminated infection in various cells, including macrophages in which the parasite survives by preventing phagosomelysosome fusion. Within the host cell, multiplication initially occurs rapidly but after a short time slows down and dormant cysts are found in the brain and muscle. However, if the infected host is subsequently immunocompromised in some way, the parasites in the dormant cysts may give rise to a fulminating infection and if the parasite passes across the placenta it can seriously damage the foetus. Most humans acquire the infection relatively early in life and recover, and are immune to reinfection. The immunology of toxoplasmosis has been reviewed extensively [7,30,31]. Much of what is known about immunity to toxoplasmosis has been derived from studies in mice [32], although there have also been a number of studies using sheep because toxoplasmosis is a serious veterinary problem associated with abortions. Significantly, recovery from infection is the norm in most mammalian hosts, except susceptible strains of mice, but seldom, if ever, results in the complete elimination of the parasite. The most important cells involved are macrophages and parasites are killed by IFN-yactivated macrophages [20]. The actual mechanisms involved are not clear but, from murine models, they appear to be Thl-type responses involving CD8 + lymphocytes, CD4 + lymphocytes and NK cells operating in synergy to produce IFN-y which, in combination with tumour necrosis factor, initiates macrophage-mediated killing via reactive oxygen molecules and nitric oxide [30, 331. CD8 + intra-epithelial lymphocytes appear to be important sources of IFN-y [34]. There is also evidence that IL-12 is involved in immunity to experimental toxoplasmosis and acts by inducing NK cells to produce IFN-y [20, 351. However, IL-12 on its own induces only small amounts of IFN-7 and requires

a co-factor, tumour necrosis factor, in order to produce significant amounts [36]. IFN-y plays a central role in toxoplasmosis as it induces microbicidal activity, promotes cyst formation and prevents cyst rupture. In common with other infections controlled by Thl-type responses, IL-4 causes exacerbated infections. On the other hand, IL-10 and TGF-fi ameliorate the inflammatory response and may therefore be beneficial to the host [30, 37, 381. There is also evidence that CD8 + cells are directly cytotoxic f39] as well as producing IFN-y. The relative role of antibody in immunity to toxoplasmosis is not at all clear, although all the available evidence suggests that it is important. In humans, acute infections are characterised by specific IgM and chronic or recovered infections by IgG. In experimental models, if the parasites are coated with antibody they are either prevented from invading host macrophages or are unable to prevent phagosome-lysosome fusion. There is some evidence that IgA may be protective, at least in mice [40]. The cytokine control of the immune response has attracted a considerable amount of attention and, as mentioned above, involves the typical Thl cytokines, IL-l, IL-2, IL-12 and IFN-y. In addition IL10 and TGF (transforming growth factor)+? inhibit the IL-1Zinduced production of IFN-y [41], but there are subtle shifts in the cytokine profile at different phases in the life-cycle [42] and between sexes [43]. It seems that the cytokine balance in toxoplasmosis is as important, if not more so, than in any other infections [30]. The objectives of vaccination against T. gondii are varied and include the prevention or reduction of oocyst shedding by cats and the reduction of possible foetal damage in humans and sheep [31, 441. Because the cat is the main source of infection, and the only animal in which oocyst shedding occurs, the prevention of oocyst shedding is a major vaccine objective. Currently the use of live parasites from a mutant strain of T. gondii (T-263) given orally is undergoing widespread trials, but is effective only because it arrests the infection at the stage of sexual development [31]. There is no vaccine against toxoplasmosis in humans but there a commercially available one, Toxovax, based on an attenuated, non-persistent, strain of T. gondii (S48) for use in sheep [45]. Another strain (RH) does not

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persist in pigs but, nevertheless, is protective [46] and a variant of this strain (ts-4) is persistent in animah other than cats, but is not being seriously for vaccine trials [3 11. None of these live CO?% vaccines would be acceptable for human use but there is some optimism that a more acceptable vaccine could be developed in the future, although no killed*or subunit vaccine is available at present. One of the problems with all of these vaccines is that they do not appear to elicit humoral immune re%ponsts [3@]. There have been attempts to immunise mice nasally with vaccines incorporating cholera toxin and these have reduced, but not eliminated, cyst burdens [47]. Currently the use of IL- 15 as an immunopotentiator [48] and the potential use of 2’. gorr& as a superantigen [42] are being investigated. 4.3. Theikriosis Tkileria spp., of which the two most important species are T. pama and T. unmiata in cattle, infect lymphocytes causing them to divide and lead to a potentially fatal lymphoproliferative disease. The potential targets for attack are the infective sporozoites injected through the bite of a tick and the dividing stages in the lymphocytes. Immunity against the intra-lymphocytic stages is mediated by cytutoxic CD8 + cells in a MHC restricted manner [49, 50, 511. Studies on immunity to the sporozoite stage have centred on a 67-kDa molecule that has been cloned and characterised [52] and expressed in Escherichia culi [53] and baculovirus [54]. Vaccination against T. panm based on an infectioncure regimen has been very successful (see [ 131) and has provided considerable insights into the mechanism of immunity to this parasite, but is impracticable on a large scale. Immunization against T. unnlslata can be achieved using an attenuated stain of this parasite and this is the basis of a successful commercial vaccine widely used in India, Iran, Israel and other endemic areas [55]. The 67-kDa sporozoite vaccine has also produced encouraging rest&s by protecting a proportion of cattle immunised I53,56]. However, this protection depends on the initiation of a mild infection and the generation of a specific cytotoxic T celI response. It is gradually becoming clear that immunity to theileriosis

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depends on a combination of host and parasite genetic factors and that the outcome of any infection or any vaccination cm the nature of the genetic make up of both the parasite and the host [SO]. 4.4. Malaria The malaria parasites have received more attention from immunologists than all the other parasitic protozoa together. The immunolagy of human malaria has been reviewed extensively over the past few years (see, for example 157, Sg]). What is known is that malaria i&&ions are long-lived, individuals can be reinfected after natural recovery or cure, there is a gradual build up of empty over a period of many years, immunity fa&s quickly and is largely strain specific. Immunity to malaria is, therefore, the rule but is often incomplete and may take many years of exposure to develop. The stages of the malaria parasite susceptible to immune attack are the sporozoites circulating in t&e blood, early schizonts in the liver, merozoites liberated from the liver into the bloodstream and merozoites released from red blood celis. The sporozoite is the obvious target for immune attack as it is the first stage in the infection and the parasites are free in the blood for up to 30 min. The sporazoite possesses an immunodominant protein surface coat, called the circumsporozoite protein, which is very antigenie and elicits a strong antibody response. However, when exposed to antibody the surface molecules cross-link and the sporozaite sheds the coat. Until recently, it was thought that once in the liver the parasite was safe from immune attack, but there is now evidence that there is a cytotoxic T cell response to the early stage in the liver. The erythrocytic stages, particularly the merozoites, have received the most attention as they are the easiest to study and are responsibIe for the disease (see [SS]). However, they are irrelevant to the consideration of coccidiosis and will not be discussed further here. A vaccine against malaria has proved very difficult to design [59], as its development must take into considerauon what stage of the We-cycle of the parasite to attack and what kind of antigens to use. The malaria life-cycle is complex and includes a

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number of stages that are biochemically and irnmunologically distinct and immune responses against one stage do not protect against the others. The current approach is to identify and characterise potentially protective surface antigens and to use these as subunit antigens on their own, as recombinant molecules expressed in a suitable vector or as a basis for synthetic vaccines [59--621. The circumsporozoite protein has been used as a basis for recombinant and synthetic vaccines and a number of trials with encouraging but equivocal results have been held, but a number of other more promising formulations of the sporozoite antigen are now receiving attention and have already produced some promising results [63]. Liver stages are also possible targets as it is now known that there are immune responses probably blocking the invasion of the hepatocytes. Although several liver-stage antigens have been recognised, there is currently no serious attempt to develop a vaccine using them. Blood stages are obvious targets because there are so many of them and because they cause the disease. However, they will not be discussed here as they are irrelevant to coccidiosis. 4.5. Babesiosis Babesiosis is common in wild and domesticated animals and is a rare accidental infection in humans. Infections are accompanied by raised specific antibody levels directed against the blood stages. As for the malaria parasites, these are irrelevant to coccidiosis and will not be discussed here.

5. Coccidiosis

and its control

Coccidiosis, caused by infection with Eimeria species, is probably the most important parasitic disease of veterinary importance throughout the world. Coccidiosis affects cattle, deer, sheep, goats, pigs, horses, rabbits, turkeys, ducks and poultry. However, it is among poultry that the disease is most common, particularly in conditions of intensive rearing, and the losses and potential losses are massive. Coccidiosis in poultry is caused by several species of Eimeria, of which seven are of particular importance and have received most attention. Con-

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trol of coccidiosis, like any other infection, can be brought about by good husbandry or prophylactic drug treatment [64], but totally effective husbandry is unrealistic on a large scale. The worldwide and rapid development of drug resistance [65-671, coupled with the increasing cost of developing new drugs and the public’s distrust of drug-treated meat, has led to a search for other alternatives including the breeding of disease-resistant poultry strains [68]. Vaccination is the only feasible alternative but, although there is no doubt that a vaccine is urgentiy needed, progress towards one has been very slow. Most infections with Eimeria in fowl are self-limiting, indicating an effective immune response, but the actual mechanisms involved have not been fully elucidated [69]. What is known, however, is that light infections are relatively harmless and that after recovery birds that have experienced such infections are immune to challenge with higher doses of oocysts. Infections with attenuated strains of Eimeria also generate effective immune responses which prevent the formation of oocysts and render the animals resistant to reinfection. There are, therefore, two strategies for immunising young birds: exposing them to low levels of oocysts [70] or giving them carefully controlled doses of attenuated strains [71, 721. Attempts have also been made to immunise birds with irradiation-killed parasites [73, 741 and partially characterised subunit vaccines, with little success [75]. Attention has also been given to the possibility of infecting chicks in ovo, which has proved to be unsuccessful [76], or vaccinating laying hens in order to protect their progeny, which has been more successful [77]. The immune responses to coccidial infections are complex and multifactorial, and the nature of the life-cycle presents a number of antigenically different targets including the infective sporozoite, successive generations of intracellular schizonts, merozoites and the different stages of gametogony. Immunity to coccidiosis has been reviewed by Rose [69] and, in general, it seems that immunity is T cellmediated [78,79] with the implication that vaccines that generate an antibody response might not be effective and might actually be counter-protective, given the mutual antagonism between the Thl and Th2 pathways discussed earlier. However, very little is known about the cytokine networks that operate

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in birds [80], let alone in poultry infected with Eimeria spp., although some progress is now being made in elucidating the complexities of these networks in murine coccidial infections [81]. The development of vaccines against coccidiosis has been pragmatic but remarkably successful. The use of vaccines based on small doses of oocysts [70] and attenuated parasites [82] has been evaluated under field conditions in massive trials. The use of attenuated lines has proved to be very efficacious [72] and they have been the basis of commercially available vaccines, Livacox [83], Coccivac [84] and Paracox [85]. Irradiation-killed parasites have been less successful [73] and, despite promising beginnings [86], there has been little progress towards a sporozoite-based vaccine except in combination with drug treatment as for theileriosis [87]. Progress in the identification and characterisation of antigenie peptides that might be useful as candidate vaccines has also been slow, although there have been some very promising breakthroughs such as the use of a partially defined peptide that has panspecific transmission blocking ability [75]. On the other hand, Eimeriu rhoptry proteins show variation between species [88] which would render their use less effective.

6. Discussion This comparative review of the immunology of infections caused by sporozoans clearly shows that the differences bet%een them are greater than the similarities. It also shows that attempts to develop vaccines against these parasites have been no more successful than those developed against coccidiosis. There are, however, a number of common features. Firstly, the development of vaccines in all cases has proceeded along traditional lines; the use of small initial infections, attenuated lines consisting of permanent or natural induced mutations or temporarily artificially produced using X-irradiation or drugs, poorly characterised subunit vaccines, characterised molecules, recombinant or synthetic vaccines and, more recently, the possibility of DNA vaccines. The various vaccines currently available are far from ideal. All have reached, or passed through, the stage of using living organisms and

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some of these are available commercially or semicommercially for toxoplasmosis and theileriosis and also for babesiosis. Such vaccines are not even considered for use in humans, in which this stage in the development of vaccines has been a transient one. Having moved beyond the stage of living, attenuated organisms as vaccines, the use of dead whole organisms, strikingly successful for some viral and bacterial infections, has been disappointing and might even generate irrelevant and possibly dangerous immunological responses to extraneous antigens. Similar reservations apply to uncharacterised parasite antigens, although the use of these has provided useful clues to the development of vaccines. On more rational grounds, the obvious target for the development of a vaccine is the initial stage of the infection which is, in all cases except T. gondii in its aberrant hosts, the sporozoite. Here, progress towards such a vaccine has been dramatic and led by vaccines against malaria and theileriosis. Unfortunately, following the euphoria of the first signs of success, a sporozoite-based vaccine against malaria is still not a realistic possibility and the p67 vaccine against T. parva, now undergoing field trials, is not totally effective. In both cases, the genetic make up of the host seems to determine the success or otherwise of the vaccine and, in any case, the antitheileria vaccine may depend on the subsequent generation of a CD8 + CTL (cytotoxic T lymphocyte) response which, for reasons given below. is probably not relevant to coccidiosis. Sporozoite-based vaccines are not being considered seriously for any other sporozoan infection. The next stage of the infection is the infected cell, which has proved to be a very important potential target, and here the role of the cell-mediated components of the immune response seems to be paramount. As far as coccidiosis is concerned, there seem to be few lessons from theileriosis, in which infected lymphocytes are killed by cytotoxic CD8 + cells, or from malaria in which it seems that the early infected hepatocyte is also the target for a CD8 + CTL response. The very nature of this killing is that it is MHC class I restricted, thus presenting a number of problems for the design and administration of a vaccine for wide use. In many protozoa1 infections the key molecule in protection

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is IFN-y, the key cell is the IFN-1, activated macrophage and the key effector molecules are reactive oxygen intermediates and nitric oxide acting sequentially or together. This pattern seems to hold true for toxoplasmosis and cryptosporidiosis and has a less obvious role in malaria. The generation of cell-mediated immune responses is dependent on the Thl pathway and the paradigm of protozoa1 infections is leishmaniasis, but similar patterns also apply to toxoplasmosis, listeriosis and mycobacterial infections in which Thl responses are largely protective, whereas Th2 responses are either ineffective or counter-protective. The actual roles of the various cells involved in the production of Thl molecules are also being investigated and the fact that CD8 + and NK cells produce IFN-), and that IL-12 triggers the production of IFN-y by NK cells is now a central tenet of immunity to intracellular parasites. It is now clear that the outcome of any immune response is determined by a balance between the various cytokines that control it and that this balance is affected by such factors as the nature of the antigen, the route of immunisation and the presence of certain cytokines at the time of infection [17]. Romagnani [14] has drawn attention to a number of outstanding questions relating to factors that determine the nature of any particular immune response, including the relative contributions of the antigen and the genetic background of the host in evoking Thl or Th2 responses and how the cytokine profile of ongoing Thl and Th2 responses can be changed. Such questions are now being taken into account in the design of vaccines and it is now clear that any successful vaccine will have to be one that is targeted specifically to produce a particular desired end result while damping down or eliminating other responses. The roles of other cytokines such as IL15, IL-16 and IL-18 are gradually being revealed. IL-15 functions in a similar way to IL-2 and has been shown to play a role in immunity to T. gondii [48]. IL-16 produced by mononuclear cells, is a T cell-specific attractant and proinflammatory cytokine that is also attracting attention as a potentially important molecule in immunity to infection [89] and IL-18, another macrophage-derived factor, possesses Thl T cell induction and IFN-y inducing properties [90]. It is now abundantly clear that

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understanding the cytokine network and its pattern of balances and counterbalances is central both to understanding the immune response and to designing vaccines that go along with the natural patterns of immunity instead of running counter to them. However, little attention has been paid to the manipulation of the cytokine network as a possible means of controlling the immune response in coccidiosis. The site of infection and where the immune response operates is also important. In coccidiosis the main target must be the epithelial cells of the gut and it is here that attention should be focused. In cryptosporidiosis and toxoplasmosis, it is clear that intra-epithelial lymphocytes are important in the generation of a successful immune response. The antibody involved in immunity to mucosal parasites is mainly IgA and, in mammals, IgA requires the involvement of the Th2 products IL-5 and TGF-/I [15]. It would seem that it would be necessary to take these facts into account when designing a vaccine against coccidiosis. The study of mucosal immunity is now a field in its own right [92, 93, 941 and is receiving a considerable amount of attention, especially with respect to antigen processing, the role of intra-epithelial cells, infection and possible vaccines [95]. In the development of vaccines attention has focused on antigen delivery systems and how to manipulate the immune response mainly in bacterial infections [95], but there has been very little work on sporozoan infections except for some preliminary studies with T. gondii in mice, in which the incorporation of certain T. gondii antigens and bile salts into non-ionic surfactant vesicles produced a significant mucosal immune response to the parasite antigens (J Alexander, personal communication). Another possible way of immunising animals against infection is by the oral administration of cytokines: this has been controversial but has had some success in bacterial infections, viral infections, including human immunodeficiency virus, and experimental arthritis [96]. The administration of interferon-alpha (IFN-a) to T. parua-infected cattle has also met with some success [97]. Although possibly a long shot, it might be worthwhile considering this kind of approach in coccidiosis. In this context, if immunity to coccidiosis is essentially cell

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mediated and if the targets are infected cells, then an approach to a universal vaccine such as has been proposed for LeisAmuniu and other intracellular pathogens [98] might be considered. The development of a vaccine against coccidiosis presents problems quite unlike those for any other parasite, largely because of the immensity of the problem, the fact that birds must be immunised when they are very young and the need to keep the cost down. None of the vaccines that are currently being contemplated for other sporozoans fulfils these criteria. However, birds do have one major advantage and this is that antibody produced in a hen can be transferred to the egg. A number of workers have tried to develop vaccines based on this principle and have met with considerable success [77,99, 1001. As immune cells do not pass into the egg, the generation of antibodies is the only way in which this kind of immunity can be generated [77, 99, 1001. In summary, research from other sporozoans has taught us the following lessons. o It is extremely difficult to develop a vaccine against any of these parasites and living attenuated vaccines are the best available at present. However, these have inherent problems such as cost, batch reliability and the need for a cold chain. l Vaccines against the obvious targets, the infective sporozoites, are not likely to be effective in themselves and the immune responses generated are likely to be determined by host genetic factors. l Cell-mediated responses are the key responses in acquired immunity, and macrophages and IFN3’ are the key components. a A knowledge of the actual cells involved in the immune response is essential and the role of intraepithelial lymphocytes needs to be investigated further. l The balance between Thl and Th2 responses is crucial to the outcome of the infection, and the cytokine network involved in the control of the immune response needs to be elucidated. l The roles of the nature of the antigen and the genetic make up of the host need to be considered. l The emerging roles of cytokines such as IL- 12, IL-l 5 and IL-l 8 need to be evaluated.

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a The mathematical basis of successful vaccination, e.g., the numbers of animals to be vaccinated and when, needs to be taken into account. l The potential advantages of vaccination must be weighed against potential disadvantages, e.g.. ease of vaccination procedures, costs, etc. Could all these lessons be applied to the technique of immunising hens in order to produce immune chicks? Taking all these points into consideration it is obvious that. in order to develop a rational vaccine against coccidiosis, a vast amount of additional information both about the hosts and the parasites needs to be accumulated. Our knowledge of avian immunology, particularly in the field of cytokines, has lagged behind that of mammalian immunology [80]. Similarly, little is known about the mathematical basis of immunisation compared with the vast amount of information available for human infections. Cost is another major problem and, in this context, it is interesting to note recent debates about the cost of developing a vaccine against malaria. By the beginning of this decade. this had already cost well in excess of U.S.$lOO million [ 1011. the establishment of a reagent repository for materials to give a ‘3ump start” to malaria vaccine will cost between $50 and lOOmillion 11023, and at least $15 million will be required to complete a mapping of the P. falciparwm genome [ 1031. The most important lessons that emerge from this comparative review are that it is not going to be easy to develop a vaccine against coccidiosis, that it is not going to be cheap and that it might not even be successful. However, this does not prevent us from speculating on the possible nature of such a vaccine and, from ail the evidence available, it seems that the most important compartment of the immune system that must be stimulated is the mucosal one. An anti-co&dial vaccine must. of course, have all the characteristics of any desirable vaccine in that it must be efficacious. cheap, stable and capable of being administered to newly hatched chicks, preferably orally. Some progress in this direction is already being made in the development of anti-Salmonella vaccines and it would be worthwhile tapping into this particular source given the fact that genes for parasite antigens can be inserted into Salmonella vectors and thereby induce pro-

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tective immune responses [ 1041. Assuming that the need is to stimulate intra-epithelial lymphocytes, such a vaccine would have to contain genes for cytokines that drive the immune response towards the Thl pole, for example IL-12, IL-15, IL-18 and IFN-y. In addition, there will probably be a requirement for the induction of an IgA response, in which case the genes for IL-5 and TGF-/? might also have to be included. Given the progress that is being made in the field of immunology, such a vaccine is a realistic possibility. In the meantime, the prospects of immunising hens is the most promising method currently available and the long-term possibility must be the immunisation of hens using the kind of vaccine described above.

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