Veterinary Parasitology, 42 ( 1992 ) 1-14 Elsevier Science Publishers B.V., Amsterdam
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Invited article: Richard D. Turk Memorial Lecture Parasites' Progress Lord Soulsby University of Cambridge, Department of Clinical VeterinaryMedicine, Cambridge, UK (Accepted 22 October 1991 )
ABSTRACT Soulsby, Lord, 1992. Invited article: Richard D. Turk Memorial L e c t u r e - Parasites' Progress. Vet. Parasitol., 42: 1-14. The College of Veterinary Medicine at Texas A & M University celebrated its 75th anniversary in September 1991. As a part of this celebration, Professor Lord Soulsby was invited by the faculty of the Department of Veterinary Pathobiology to deliver the annual Turk Memorial Lecture. The following is Professor Lord Soulsby's presentation.
INTRODUCTION
It is a particular honour to be asked to present this lecture, not only because I knew Dick Turk and much admired him for his work in parasitology, but also because this lecture comes at a time of the anniversary of momentous events in parasitology, particularly the discovery some 100 years ago ( 1893 ) of the life cycle ofBabesia bigemina and its transmission by the tick Boophilus annulatus (Smith and Kilbourne, 1893). The publication of the data is usually quoted as 1893 in the US Department of Agriculture Bureau of Animal Industry Bulletin, Volume 1, pp. 1-301. However, a preliminary report was published as a letter to the Veterinarian (London) in 1892 (Smith and Kilbourne, 1892). So even then the idea of a letter to the editor followed by a major article on the topic was well known and counted as two publications! From a century ago to half a century ago, to a time when along with Hubert Schmidt and C.N. Richardson, a young Richard D. Turk was publishing data on trichomonal abortion in cattle. I can trace six such reports published in the Annual Report of the Texas Agriculture Experiment Station (Schmidt and Turk, 1938; Schmidt et al., 1938a,b, 1940, 1941, 1942). Eventually the light dawned and in 1941, along with Bruce L. Warwick, Hubert Schmidt and Raymond O. Berry, Richard D. Turk published a note on disease resistance in animals (in the Annual Report of the Texas Agricultural Experiment Station ). In 1944, Warwick, Turk, Berry and Schmidt published a note on "Prog© 1992 Elsevier Science Publishers B.V. All rights reserved 0304-4017/92/$05.00
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Prof. Lord Soulsby.
ress made in breeding sheep and goats resistant to stomach worms". Fifty years later we are still working on this topic and perhaps we have made some progress, I hope to illustrate this later. The idea that animals could be resistant to parasitic infections and that one may create such resistance artificially by vaccination was, 50 years ago, somewhat too advanced thinking! Even in 1949, when ! took up the subject, immunity to parasites was a debatable issue and vaccination was considered an unrealistic conclusion. However, in the 1950s a first practical commercial
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vaccine against a parasitic helminth infection was developed, consisting of Xirradiated infective larvae of Dictyocaulus viviparus, the bovine lungworm, which prior to the development of the vaccine had been responsible for severe morbidity and mortality of calves, especially in the UK (Jarrett et al., 1958 ). Dictyocaulus viviparus vaccine has been markedly successful and several million calves have been protected by it, with very few showing any side effects. There was the hope some 30 years ago that similar approaches might be used for several other major parasitic diseases of man and animals; this included diseases such as malaria, trypanosomiasis and schistosomiasis of man and the gastrointestinal parasitisms of domestic animals (fascioliasis and the larval cestodiases). However, in no case has an effective practical vaccine been produced for a variety of reasons including: immune unresponsiveness of young animals, immunosuppression, lack of sufficient immunising stages, their preservation in a viable immunising form and perhaps most of all, the lack of a viable commercial market for a vaccine in competition with increasingly effective antiparasitic compounds. However, with the advent of molecular biological techniques, the outlook for practical field vaccines for parasitic infections has changed dramatically and there are a number of parasitic infections where vaccines constitute realistic approaches to control and in fact are in the final stages of development. However, in the early years of Richard Turk's activity as a veterinary parasitologist, drug therapy of parasitic infection was the only realistic approach and even so the drugs available were far from the highly efficacious compounds we have today. Then we believed in Stoll's (1947) "wormy world" with the concept that all members of all species are infected with parasites. While this concept brought the importance of parasitism and the losses caused by parasites into focus, what was not appreciated until later was that a parasitic population is not only that resident in the host animal but consists also of a large number of developmental stages in the environment. Indeed, the endoparasitic part of the population may be minor and may be transient in the majority of hosts except for the 'wormy animals', representing that highly aggregated situation which occurs in nature in which a minority of hosts have the majority of parasites. This represents the negative binomial distribution of parasites in their hosts (Anderson and May, 1985 ). In view of the facts that the majority of parasites are outside the host and when in the host a majority are in only a minority of hosts, one might ask: do we need to treat all animals for all their parasites all the time as we do at present, in other words, to apply mass medication? Is it logical to apply antiparasite measures to the host only? If we return to the negative binomial distribution concept, some 60-80% of animals control their own worm population, even under very heavy pressure. This situation can be modified and is done so by selective breeding in animals. Previous examples are the 'violet' factor in sheep (Whitlock and Madsen, 1958 ) so-named after a ram, and sheep
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have been separated into 'responders' and 'non-responders' on the basis of their ability to respond to an X-irradiated vaccine to Trichostrongylus colubriformis (Windon et al., 1980). This has been demonstrated to be associated with the presence of an ovine lymphocyte antigen (SY 1 ) present in high frequency - - 73% in responder rams (vs. 22% in low responder rams) and 65% in responder ewes (vs. 33% in low responder ewes). It has been concluded that the SY1 antigen is likely to be part of the sheep's major histocompatibility complex ( M H C ) (Outteridge et al., 1985 ). It is clear, therefore, that host populations can be manipulated by selective breeding. The role of i m m u n i t y on a herd basis and its implications for parasite control have been examined recently (Anderson and May, 1985 ). Studies of human parasites (hookworms, schistosomes, filarids) emphasise the points well established in the veterinary field, namely that parasite numbers per host are highly aggregated. They also recognise that in man the predisposition to heavy infection is probably genetic in origin, and as in animals, the individuals who harbour few worms are genetically 'high responders' and those with heavy burdens, 'low responders'. Mass chemotherapy at a level less than that required to eradicate the parasites can significantly reduce the level of herd immunity, and raise the average worm burden above the levels existing prior to anthelmintic 'control' (Anderson and May, 1985 ). Therefore, in answer to the theoretical question 'is mass medication the correct approach', the answer is no! However, if the logical alternative is to treat only animals which harbour the majority of parasites, these animals must be easily identifiable, but at present they cannot be readily identified other than by faecal egg counts. The long-term goal may well be to convert the flock or herd to a high responder category by selective breeding so that the highly aggregated distribution with wormy individuals is avoided. In the meantime, antiparasitic measures, relying on chemical compounds, must be applicable. However, in relying on chemotherapy for the control of parasitic infections there are a series of issues that increasingly concern both the producer and user of antiparasitic compounds. These are cost, delivery systems, drug resistance, operator safety, residues, environmental concerns and consumer pressure. In veterinary medicine, an important consideration is that of cost or cost-effectiveness. Measured in terms of economics of herd or flock production, the majority of anthelmintics are cost-effective; but is that the only criterion of costeffectiveness7 In view of consumer concerns such as the ecological aspects of disease control and increasing concern about animal welfare, cost-effectiveness may take on a different interpretation to the usual one. Part of the costeffective concern also is that of manpower costs, and as a result, there have been major advances in delivery systems with increasingly ingenious ways for the application of antiparasitic compounds. These consist of slow release, pulse-release, pour-on preparations, etc., and no doubt others are being de-
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veloped. These delivery systems also make mass medication easier but as they are not labour intensive they tend also to encourage overuse of drugs, leading to drug resistance. The problem of drug resistance is not one to be dealt with at length in this communication. Suffice it to say it is well recognised in all endo- and ectoparasitic situations, though the basis of it in parasitic helminths is as yet unclear despite intensive study of the phenomenon. One of the major pressures on the future development of parasite control will be consumer pressure. Frequently this is totally uncontrollable and totally unscientific, but nevertheless may be a very powerful force in the determination of the use of therapeutic substances in general. Examples of such consumer pressure concern the use of anabolic steroids and somatotrophins in cattle. Similarly, environmental concerns are increasingly apparent as certain compounds are shown to affect the natural fauna and flora, e.g. their effect on earthworms and dung beetles. TARGET FOR ANTIPARASITIC COMPOUNDS
Such concerns pose the question of what should be the target (s) for antiparasitic compounds? The antiparasite compounds used at present are directed against the parasites within the host and frequently against the mature developmental stages when they have developed to a pathogenic stage, though there is an increasing number effective against larval stages of parasites. However, the majority of a parasite population is outside the host, either in the form of developing or infective larvae or eggs; few, if any, antiparasitic compounds are directed against these. The nearest approach is the use of agents which will destroy intermediate hosts, such as insecticides and molluscicides. The final host is the collecting point through which this parasite population must pass in order to progress to the next generation of parasites. Present day antiparasitic compounds attack the parasite when it is established in the host and there have been few attempts to stop parasite development at the point of infection. Yet there are many triggers required to convert a free-living or an infective stage of a parasitic helminth to the parasitic stage. In gastrointestinal nematodes, exsheathment or hatching are such events. They are thus critical to the life cycle of the parasite - - no exsheathment, no parasite. Is it not time that we thought more seriously about parasite control at the point of entry into the host? The requirements for entry into the host or host cell are critical and unique to a given parasite (e.g. the exsheathment of gastrointestinal nematodes of ruminants). Would an anti-exsheathment compound be as effective as one of the compounds highly effective against an adult parasite stage? Control of the parasite outside the host is, of course, practised by pasture and grazing management with domestic animals, and the application of hy-
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gienic conditions in housed animals. The simple expedient of collecting faeces from pasture, as for the control of horse parasites, is a good example. As more is known about the ecology of non-parasitic stages of the organisms that concern us, the more effective will be our control programmes. RESPONSE TO THE CONCERNS OF CHEMOTHERAPY
A major approach must be vaccination. It has the attribute that limited handling of the animal is required, such as once or twice to inject the immunogen, or possibly the immunogen can be given in the feed. Immunisation addresses the negative binomial problem; it does not suffer from drug resistance. There are no residues or environmental concerns or consumer pressures with respect to welfare. The possible approaches to immunological control include passive immunisation (vaccination of mother), live vaccines (attenuated forms, abbreviation of infection, concealed antigens), subunit vaccines, synthetic vaccines, anti-idiotype vaccines and genetic resistance. Several parasite vaccines are now under development. Earlier attempts to produce antiparasite vaccines have been frustrated either by the inability to identify the important antigens or the limited availability of antigens responsible for resistance. Organisms were and still are not readily cultivated in vitro and in some cases the source of such material (e.g. man) made vaccine exploitation impossible. However, gene cloning techniques now offer a unique opportunity to overcome these problems. Specific monoclonal antibody probes are being developed to identify protective antigens, and these in turn are used to produce protective epitopes in unrelated microorganisms by recombinant DNA molecules coding for the protective antigens.
Passive irnrnunisation This is a feasible proposition where it has been shown that passive immunity can play a role in the control of parasitic infections. Examples are the larval cestode infections. The economically important Taenia saginata of man causing bovine cysticercosis is amenable to vaccination, and preparations of tapeworm oncosphere will immunise cattle against infection and also result in passive protection of calves born to vaccinated mothers (Lloyd and Soulsby, 1976). Because of the difficulty of obtaining human-derived tapeworm material, attention was directed to heterologous sources of immunogens, and while some useful protection was achieved with cross-reacting cestodes, none was so effective as the homologous parasite (Lloyd, 1979). Work with the rodent species Taenia taeniaeformis showed that excellent immunity could be induced with oncospheral material; the protective antibody was IgA in nature where maternal immunity was concerned, but was
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IgG based in the adult animal (Lloyd and Soulsby, 1978). Since then there have been major advances made with a vaccine for Taenia ovis, the larval stage of which is an economically important parasite of sheep in Australia and New Zealand. Antigens in the range of 47-52 kDa from T. ovis eggs were shown to be protective in sheep. Hatched and activated oncospheres of T. ovis were used as a source of m R N A for the construction of a cDNA library. This was constructed in lambdagtll and two fusion proteins fl-gal-45S and fl-gal45W purified from selected clones were tested in vaccination trials. While sheep immunised with the fl-gal fusion proteins produced antibodies which reacted with oncosphere antigens of 47-52 kDa, no protective immunity was induced. A different expression system was more successful. Using a plasmid vector which expresses antigens as fusion proteins with the enzyme glutathione Stransferase of Schistosoma japonicum (pSj 10ABam 7 stop 7, a precursor of the pGEX-1 vector), the 45W and 45S cDNA were cloned into this vector and the fusion proteins GST-45W and GST-45S proved very efficient in inducing protective immunity in sheep when injected with saponin adjuvants (Johnson et al., 1989). Why a fl-gal fusion failed to immunise while a GST fusion did, is unclear. These studies have led to the field trial of a commercial vaccine and provide the basis for similar vaccines for bovine and swine cysticercosis and also hydatid infection in man and animals.
Live vaccines These involve the use of live vaccines, either attenuated or abbreviated whole organisms or extracts of these. X-irradiation provided the basis for the first practical helminth vaccine for Dictyocaulus viviparus, but this is no longer a viable approach. Abbreviation of infections either by chemotherapy or by the deliberate selection of organisms for attenuation is practised in a number of infections. For example, a vaccine against Babesia consists of blood containing an attenuated strain of Babesia (De Vos et al., 1987), and the infect and treat approach has been used for immunisation of cattle against East Coast Fever (Theileria parva infection; Irvin and Morrison, 1987). An example of the deliberate selection of organisms for attenuation is the precocious development in coccidia which, missing out the pathogenic developmental stages but retaining the immunogenic stages, provides the basis of a vaccine. The concept of the use of concealed antigens is illustrated by the use of gut antigens to immunise against ticks (Willadsen and Kemp, 1988 ). Such antigens are those not normally 'seen' by the host i m m u n e system during normal infection. In the case of the tick vaccine, antibodies raised by concealed antigens damage the gut cells during tick feeding to the extent that host blood appears in the tick haemolymph. The immunological mechanism is antibody
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mediated and complement independent. As little as 7 ng of tick gut protein has been shown to be sufficient to immunise an animal. It should be noted with the use of concealed antigens, that it is unlikely that immune evasion mechanisms associated with salivary gland antigens of ticks will be invoked (Stewart, 1983); nevertheless, field challenge with tick infection will fail to boost the immune response to concealed antigens and regular immunising doses will be required. This indicates that expression vectors, such as the vaccinia virus, may be required to maintain an adequate level of immunity. A further example of the use of concealed antigens is in vaccination against blowfly strike, caused by Lucilia cuprina. This might be thought to be an unusual candidate for vaccination studies. Strike or cutaneous myiasis is responsible for a loss of some $150 million annually in Australia alone, and control while achievable with insecticidal drugs and sprays, suffers from the development of insecticide resistance by blowflies. Early studies demonstrated that immunisation with homogenates of maggots inhibited subsequent larval growth and excretory-secretory preparations from larvae were shown to induce significant immunity. However, a major development was the use of gut antigens of L. cuprina larvae in much the same way as with the tick Boophilus ( Sandeman, 1990).
Molecular vaccines At present there are no antiparasite subunit vaccines on the market. Nevertheless, there are reports of recombinant vaccines under development for ovine cysticercosis, coccidiosis and ticks. The development of a recombinant, molecular vaccine for parasites is beset with many more difficulties than with much simpler organisms such as viruses and bacteria. Even in the virus field, vaccination with subunits of the virus may lead to high antibody levels to a certain glycoprotein, but no protection against infection. This has led to a careful search for the essential 'T' cell epitopes, which will need to be incorporated into the molecular vaccine to provide both cell mediated as well as antibody mediated responses and immunological memory. The situation is complicated by the fact that a T cell epitope may stimulate T cells of one animal but not be effective in another strain of the same animal. Nevertheless, the optimal needs of a molecular or subunit vaccine include the identification of T and B cell epitopes of a parasite, the matching of T cell epitopes for B cell memory and CMI memory and the optimal recognition of B cells, the whole being held together by covalent bonds. An increasing amount of work is being done to identify subunits of parasites, the proteins or glycoproteins that might be immunogenic and protective. This development has been greatly helped by the use of monoclonal technology and monoclonal antibodies prepared against certain epitopes. Examples where subunit vaccines offer important opportunities for vacci-
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nation include the sporozoite antigens of Theileria parva and Theileria annulata. A 67 kDa component has been identified as important in protection and has been expressed in Escherichia coli as a C-terminal fusion protein with a Schistosomajaponicum antigen Sj26 using the pGEX expression vector system (Smith and Johnson, 1988). With the Theileria spp., the role o f T cells is particularly important, and in the case of the 67 kDa antigen, helper T-cell epitopes should be present (Good et al., 1987). The cloning of membraneassociated mid-gut derived protein from Boophilus microplus that is immunoprotective in the bovine (Opdebeeck et al., 1988 ) is a recent development which will provide the basis for a genetically engineered tick vaccine which is now under development in Australia. Synthetic vaccines Synthetic polypeptides now being examined closely in the field of virology are a natural extension of the subunit vaccine approach. It is likely, however, that these are a long way off for helminths. Initial work will entail the examination of sequences of overlapping synthetic peptides to identify T cell epitopes. If the immunising epitope is carbohydrate in nature, then recombinant approaches and peptide vaccines will not be suitable and a possible approach would be an anti-idiotype vaccine. In nematodes the cuticle is covered by mucopolysaccharides, and it is feasible that vaccine target antigens are contained in this surface material. Anti-idiotype vaccines The antigen binding site of an antibody molecule is, in its physical structure, complementary to the antigen against which the antibody is produced. Antibodies have been developed against the antigen binding site which are similarly complementary to the site and mimic the structure of the original antigen. Anti-idiotype antibodies are surrogate antigens. Anti-idiotypes could be of importance when immunising epitopes cannot be obtained by conventional methods, when an antigen is non-peptide in nature, when immunising epitope is unknown but when a monoclonal antibody possesses neutralising activity against the parasite, and can be used to induce an anti-idiotype antibody. An anti-idiotype approach has been used in schistosomiasis. An antibody which mimics a 38 kDa Schistosoma mansoni glycoprotein has been at least as effective as other approaches in vaccination (Grzych et al., 1985 ). The anti-idiotype approach may be particularly valuable where neonatal unresponsiveness occurs, e.g. in young sheep to Haemonchus contortus. Mice neonatally unresponsive to E. coli K13 polysaccharide can be immunised with an anti-idiotype (Smith, 1981 ). This area may be worthy of further study in H. contortus infection.
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There are a number of problems associated with the development of an effective vaccine, some of these are parasite in origin while others are of host origin. These include expression systems, immunosuppressive epitopes, stability, T cell epitopes, MHC, immunoglobulin characteristics, cross reactivity, adjuvants and responsiveness of young to vaccines.
Expression systems Expression systems using live recombinant vectors may be important with organisms such as Theileria, and delivery systems for cattle vaccines such as attenuated strains of Salmonella typhimurium or vaccinia virus (Smith et al., 1984). Vaccinia virus is most commonly identified as an expression carrier. Its genome is large and capable of receiving several foreign genes, but if several genes are inserted there may be a hierarchy of dominance and in some cases multiple immunisation may be necessary.
Immunosuppressive epitopes It will be necessary to ensure that immunosuppressive epitopes are not included in a vaccine. While it is recognised that immunosuppression occurs in parasitic helminth infections, the parasite components responsible for this are not known. Some are known in the virus field, e.g. the protease inhibitor in vaccinia, a homologous protein to cellular B2 microglobulin preventing presentation of virus peptides to cytotoxic T cells in CM virus.
T cell epitopes Whereas the search for T cell epitopes in a virus can be undertaken by the examination of a series of overlapping synthetic polypeptides, in helminths we are much further from this approach. We do not know the gene or the gene product responsible for immunity.
MHC The role of MHC in helminth immunity is unknown. It is of particular concern in virus infections and in protozoa. If it is of particular importance in helminths, then vaccine development with subunit vaccines will be more difficult.
Cross reactivity It is to be hoped that vaccines will have sufficient cross reactivity to permit generic vaccination rather than necessitate production of vaccines to each individual species ofhelminth.
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Responsiveness o f young to vaccines
A major handicap in the development of vaccines against gastrointestinal nematodes of ruminants is the poor immunological response of lambs and calves to parasites such as H. contortus and various other trichostrongyles. Thereby, animals are at substantial risk to infection for several months prior to the acquisition of protection, which when acquired is strong (Lloyd and Soulsby, 1987). Lambs may not respond effectively to H. contortus or T. colubriformis until they reach 3-6 months of age, and strong protection may require several more months (Smith and Angus, 1980). A similar unresponsiveness occurs in calves, but this is not well documented (as it is in sheep). Both parasite and host factors are concerned. For example, whereas young lambs are unable to develop protection against re-infection with most gastrointestinal nematodes, in the case of infection with Nematodirus battus lambs can m o u n t a prompt and effective i m m u n e response (Lee and Martin, 1976). In addition, lambs can mount an effective i m m u n e response to a wide range of bacterial and viral antigens (Lloyd and Soulsby, 1987 ). The mechanisms of this immunological unresponsiveness are not fully understood, and various hypotheses on the defect (s) include feedback inhibition by maternally transferred antibody, colostral transfer of soluble antigen or soluble suppressor factors and the generation of suppressor cell populations in the neonate. In part, the response has a genetic base and lambs may be segregated into 'responders' and 'nonresponders' as previously noted (Windon et al., 1980). Also, the infection status of the ewe may be important. Thus, immunisation with soluble antigens of 4-week-old lambs against infection with H. contortus induced a degree of protective immunity, but only if the lambs had been born of non-infected ewes. This degree of protection was abrogated if the ewes carried an infection with H. contortus (Shubber et al., 1984; Lloyd and Soulsby, 1987 ). Although there is an early failure of young ruminants to develop an effective immunity to gastrointestinal nematodes, early infection of such animals does not seem to prevent this subsequent development of protective immunity when these animals are reinfected at a later date. Recent studies have shown that adult sheep either injected with antigens of H. contortus or infected with the parasite, have antigen-reactive peripheral blood lymphocytes. More than 90% of these cells are cd4 + T cells and approximately 8% are cd8 + T cells (Haig et al., 1989). The i m m u n o d o m i n a n t third stage larval antigens had molecular weights of 15-18, 25-29, 79-80 and more than 100 kDa (Haig et al., 1989 ). Contrary to earlier studies which showed a lack of responsiveness to H. contortus antigen of peripheral blood lymphocytes of lambs, very recent, unpublished work by P. Torgerson ( 1991 ), has suggested that lambs have genetically primed antigen-reactive lymphocytes in their circulation at an early age, possibly helper T cells but also there is an unidentified plasma factor which
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inhibits their response. Higher concentrations of antigen have an inhibitory effect on lymphocytes both in the absence and particularly in the presence of plasma. This may be an important factor in the occurrence of immune unresponsiveness in an active infection. In this case the parasite would be suppressing the response of the young animal in order to gain access to and develop in the host. CONCLUSIONS
In the 50 years or so since Richard Turk undertook work in the field of veterinary parasitology, many developments have occurred in chemotherapy and immunology, and explanations have been provided for the mechanism of parasitism. These mechanisms frequently result in the survival of the parasite, the avoidance of rejection mechanisms and often a synchronisation of the biology of the parasite with that of the host: in fact, in parasites' progress. I have been privileged over the last 40 years or so, to study and admire the parasites' progress in dealing with host responses, man-made chemicals to attack them, and indeed for me to gain a little insight into the phenomenon of parasitism. I know Dick Turk was of the same mind in admiring these subtle mechanisms and it is my great honour to review some of the developments of this subject in his name.
REFERENCES Anderson, R.M. and May, R.M., 1985. Herd immunity to helminth infection and implications for parasite control. Nature (London), 315: 493-496. De Vos, A.J., Dalgliesh, R.J. and Callow, L.L., 1987. Babesia. In: E.J.L. Soulsby (Editor), Immune Responses in Parasitic Infections: Immunology, Immunopathology and Immunoprophylaxis. Vol. III, Protozoa. CRC Press, Boca Raton, FL, pp. 183-222. Good, M.F., Maloy, W.L., Lunde, M.N., Margalit, H., Cornette, J.L., Smith, G.L., Moss, B., Miller, L.H. and Berzofsky, J.A., 1987. Construction of a synthetic immunogen: use of a new T-helper epitope as malaria circumsporozoite protein. Science, 235: 1059-1062. Grzych, J.P., Capron, M., Lambert, P.H., Dissons, C., Torres, S. and Capron, A., 1985. An antiideotype vaccine against experimental schistosomiasis. Nature, 316: 74-76. Haig, D.M., Windon, R., Blackie, W., Brown, D. and Smith, W.D., 1989. Parasitic specific T cell responses following live infection with the gastric nematodes Itaemonchus contortus. Parasite lmmunol., 11: 463-477. Irvin, A.D. and Morrison, N.I., 1987. Immunopathology, immunology and immunoprophylaxis of Theileria infections. In: E.J.L. Soulsby (Editor), Immune Responses in Parasitic Infections: Immunology, Immunopathology and Immunoprophylaxis. Vol. III, Protozoa. CRC Press, Boca Raton, FL, pp. 223-274. Jarrett, W.F.H., Jennings, F.W., Martin, B., Maclntyre, W.I.M., Mulligan, W., Sharp, N.C.C.
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and Urquhart, G.M., 1958. A field trial of a parasitic bronchitis vaccine. Vet. Rec., 70:451453. Johnson, K.S., Harrison, G.B.L., Lightowlers, M.W., O'Hoy, K.L., Congle, W.G., Dempster, R.P_ Lawrence, S.B., Vinton, J.G., Heath, D.D. and Rickard, M.D., 1989. Vaccination against ovine cysticercosis using a defined recombinant antigen. Nature, 338: 585-587. Lee, D.L. and Martin, J., 1976. Changes in Nematodirus battus associated with the development of immunity to this nematode in lambs. In: H. van den Bossche (Editor), Biochemistry of Parasites and Host Parasite Relationship. Elsevier, Amsterdam. Lloyd, S., 1979. Homologous and heterologous immunisation against the metacestodes of Taenia saginata and Taenia taeniaeformis in cattle and mice. Z. Parasitenkd., 60: 87-96. Lloyd, S. and Soulsby, E.J.L., 1976. Passive transfer of immunity to neonatal calves against the metacestodes of Taenia saginata. Vet. Parasitol., 2: 355-362. Lloyd, S. and Soulsby, E.J.L., 1978. The role oflgA immunoglobulins in the passive transfer of protection in Taenia taeniaeformis in the mouse. Immunology, 34: 939-945. Lloyd, S. and Soulsby, E.J.L., 1987. Immunobiology of gastrointestinal nematodes of ruminants. In: E.J.L. Soulsby (Editor), Immune Responses in Parasitic Infections: Immunology, Immunopathology and Immunoprophylaxis. Vol. 1. Nematodes. CRC Press, Boca Raton, FL, pp. 1-41. Opdebeeck, J.P., Wong, J.Y.M., Jackson, L.A. and Dobson, C., 1988. Hereford cattle immunised and protected against Boophilus microplus with soluble and membrane associated antigens from the mid-gut of ticks. Parasite Immunol., 10:405-410. Outteridge, P.M., Windon, R.G. and Dineen, J.K., 1985. An association between a lymphocyte antigen and the response to vaccination against the parasite Trichostrongylus colubriformis. Int. J. Parasitol., 15: 121-127. Sandeman, R.M., 1990. Prospects for the control of sheep blowfly strike by vaccination. Int. J. Parasitol., 20: 537-541. Schmidt, H. and Turk, R.D., 1938. Trichomoniasis - - control and diagnosis. J. Am. Vet. Med. Assoc., 93:133. Schmidt, H., Turk, R.D. and Richardson, C.N., 1938a. Trichomonad abortion in cattle. 50th Annu. Rep. Tex. Agric. Exp. Stn., College Stn., TX, pp. 14-15. Schmidt, H., Turk, R.D. and Richardson, C.N., 1938b. Trichomonad abortion in cattle. 51st Annu. Rep. Tex. Agric. Exp. Stn., College Stn., TX, p. 13. Schmidt, H., Turk, R.D. and Richardson, C.N., 1940. Trichomonad abortion in cattle. 52nd Annu. Rep. Tex. Agric. Exp. Stn., College Stn., TX, p. 13. Schmidt, H., Turk, R.D. and Richardson, C.N., 1941. Trichomonad abortion in cattle. 53rd Annu. Rep. Tex. Agric. Exp. Stn., College Stn., TX, p. 11. Schmidt, H., Turk, R.D. and Richardson, C.N., 1942. Trichomonad abortion in cattle. 54th Annu. Rep. Tex. Agric. Exp. Stn., College Stn., TX, p. 92. Shubber, A.H., Lloyd, S. and Soulsby, E.J.L., 1984. Immunological unresponsiveness of lambs to infection with Haemonchus contortus. Effect of infection in the ewe on the subsequent responsiveness of lambs. Z. Parasitenkd., 70:219-228. Smith, B.P., Reina-Guerra, M., Hoiseth, S.K., Stocker, B.A.D., Habasha, F., Johnson, E. and Merritt, F., 1984. Aromatic-dependent Sahnonella typhimirium as modified live vaccines for calves. Am. J. Vet. Res., 45: 59-66. Smith, D.B. and Johnson, K.S., 1988. Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione-S-transferase. Gene, 67:31-40. Smith, G., 1981. Maternal regulator cells during murine pregnancy. Clin. Exp. Immunol., 44: 90-99. Smith, T. and Kilbourne, F.L., 1882. Texas fever and cattle ticks. Veterinarian, 38:351-352. Smith, T. and Kilbourne, F.L., 1883. Investigations into the nature, causation and prevention of Texas southern cattle fever. Bull. Bur. Anim. Ind., USDA, Washington, DC, pp. 1-301.
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Smith, W.D. and Angus, R.W., 1980. Haemonchus contortus: attempts to immunise lambs with irradiated larvae. Res. Vet. Sci., 29: 45-50. Stewart, C.G., 1983. In vitro immunosuppression of bovine mononuclear cells by tick saliva. S. Afr. J. Sci., 79:119-123. Stoll, N.R., 1947. This wormy world. J. Parasitol., 33:1-18. Warwick, B.L., Turk, R.D., Schmidt, H. and Berry, R.O., 1941. Disease resistance in animals. 53rd Annu. Rep. Tex. Agric. Exp. Stn., College Stn., TX, pp. 36-37. Warwick, B.L., Turk, R.D., Schmidt, H. and Berry, R.O., 1944. Progress made in breeding sheep and goats resistant to stomach worms. 55th and 56th Annu. Rep. Tex. Agric. Exp. Stn., College Stn., TX, p. 10. Whitlock, J.H. and Madsen, H., 1958. The inheritance of resistance to trichostrongylidosis in sheep. II. Observations in the genetic mechanisms in trichostrongylosis. Cornell Vet., 48: 134-145. Willadsen, P. and Kemp, D.H., 1988. Vaccination with "concealed" antigens for tick control. Parasitol. Today, 4:196-198. Windon, R.G., Dineen, J.K. and Kelly, J.D., 1980. The segregation of lambs into "responders" and "non responders": response to vaccination with irradiated Trichostrongylus colubriformis larvae before weaning. Int, J. Parasitol., 10: 65-73.
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Invited article: Richard D. Turk Memorial Lecture Parasites' Progress Lord Soulsby University of Cambridge, Department of Clinical VeterinaryMedicine, Cambridge, UK (Accepted 22 October 1991 )
ABSTRACT Soulsby, Lord, 1992. Invited article: Richard D. Turk Memorial L e c t u r e - Parasites' Progress. Vet. Parasitol., 42: 1-14. The College of Veterinary Medicine at Texas A & M University celebrated its 75th anniversary in September 1991. As a part of this celebration, Professor Lord Soulsby was invited by the faculty of the Department of Veterinary Pathobiology to deliver the annual Turk Memorial Lecture. The following is Professor Lord Soulsby's presentation.
INTRODUCTION
It is a particular honour to be asked to present this lecture, not only because I knew Dick Turk and much admired him for his work in parasitology, but also because this lecture comes at a time of the anniversary of momentous events in parasitology, particularly the discovery some 100 years ago ( 1893 ) of the life cycle ofBabesia bigemina and its transmission by the tick Boophilus annulatus (Smith and Kilbourne, 1893). The publication of the data is usually quoted as 1893 in the US Department of Agriculture Bureau of Animal Industry Bulletin, Volume 1, pp. 1-301. However, a preliminary report was published as a letter to the Veterinarian (London) in 1892 (Smith and Kilbourne, 1892). So even then the idea of a letter to the editor followed by a major article on the topic was well known and counted as two publications! From a century ago to half a century ago, to a time when along with Hubert Schmidt and C.N. Richardson, a young Richard D. Turk was publishing data on trichomonal abortion in cattle. I can trace six such reports published in the Annual Report of the Texas Agriculture Experiment Station (Schmidt and Turk, 1938; Schmidt et al., 1938a,b, 1940, 1941, 1942). Eventually the light dawned and in 1941, along with Bruce L. Warwick, Hubert Schmidt and Raymond O. Berry, Richard D. Turk published a note on disease resistance in animals (in the Annual Report of the Texas Agricultural Experiment Station ). In 1944, Warwick, Turk, Berry and Schmidt published a note on "Prog© 1992 Elsevier Science Publishers B.V. All rights reserved 0304-4017/92/$05.00
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Prof. Lord Soulsby.
ress made in breeding sheep and goats resistant to stomach worms". Fifty years later we are still working on this topic and perhaps we have made some progress, I hope to illustrate this later. The idea that animals could be resistant to parasitic infections and that one may create such resistance artificially by vaccination was, 50 years ago, somewhat too advanced thinking! Even in 1949, when ! took up the subject, immunity to parasites was a debatable issue and vaccination was considered an unrealistic conclusion. However, in the 1950s a first practical commercial
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vaccine against a parasitic helminth infection was developed, consisting of Xirradiated infective larvae of Dictyocaulus viviparus, the bovine lungworm, which prior to the development of the vaccine had been responsible for severe morbidity and mortality of calves, especially in the UK (Jarrett et al., 1958 ). Dictyocaulus viviparus vaccine has been markedly successful and several million calves have been protected by it, with very few showing any side effects. There was the hope some 30 years ago that similar approaches might be used for several other major parasitic diseases of man and animals; this included diseases such as malaria, trypanosomiasis and schistosomiasis of man and the gastrointestinal parasitisms of domestic animals (fascioliasis and the larval cestodiases). However, in no case has an effective practical vaccine been produced for a variety of reasons including: immune unresponsiveness of young animals, immunosuppression, lack of sufficient immunising stages, their preservation in a viable immunising form and perhaps most of all, the lack of a viable commercial market for a vaccine in competition with increasingly effective antiparasitic compounds. However, with the advent of molecular biological techniques, the outlook for practical field vaccines for parasitic infections has changed dramatically and there are a number of parasitic infections where vaccines constitute realistic approaches to control and in fact are in the final stages of development. However, in the early years of Richard Turk's activity as a veterinary parasitologist, drug therapy of parasitic infection was the only realistic approach and even so the drugs available were far from the highly efficacious compounds we have today. Then we believed in Stoll's (1947) "wormy world" with the concept that all members of all species are infected with parasites. While this concept brought the importance of parasitism and the losses caused by parasites into focus, what was not appreciated until later was that a parasitic population is not only that resident in the host animal but consists also of a large number of developmental stages in the environment. Indeed, the endoparasitic part of the population may be minor and may be transient in the majority of hosts except for the 'wormy animals', representing that highly aggregated situation which occurs in nature in which a minority of hosts have the majority of parasites. This represents the negative binomial distribution of parasites in their hosts (Anderson and May, 1985 ). In view of the facts that the majority of parasites are outside the host and when in the host a majority are in only a minority of hosts, one might ask: do we need to treat all animals for all their parasites all the time as we do at present, in other words, to apply mass medication? Is it logical to apply antiparasite measures to the host only? If we return to the negative binomial distribution concept, some 60-80% of animals control their own worm population, even under very heavy pressure. This situation can be modified and is done so by selective breeding in animals. Previous examples are the 'violet' factor in sheep (Whitlock and Madsen, 1958 ) so-named after a ram, and sheep
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have been separated into 'responders' and 'non-responders' on the basis of their ability to respond to an X-irradiated vaccine to Trichostrongylus colubriformis (Windon et al., 1980). This has been demonstrated to be associated with the presence of an ovine lymphocyte antigen (SY 1 ) present in high frequency - - 73% in responder rams (vs. 22% in low responder rams) and 65% in responder ewes (vs. 33% in low responder ewes). It has been concluded that the SY1 antigen is likely to be part of the sheep's major histocompatibility complex ( M H C ) (Outteridge et al., 1985 ). It is clear, therefore, that host populations can be manipulated by selective breeding. The role of i m m u n i t y on a herd basis and its implications for parasite control have been examined recently (Anderson and May, 1985 ). Studies of human parasites (hookworms, schistosomes, filarids) emphasise the points well established in the veterinary field, namely that parasite numbers per host are highly aggregated. They also recognise that in man the predisposition to heavy infection is probably genetic in origin, and as in animals, the individuals who harbour few worms are genetically 'high responders' and those with heavy burdens, 'low responders'. Mass chemotherapy at a level less than that required to eradicate the parasites can significantly reduce the level of herd immunity, and raise the average worm burden above the levels existing prior to anthelmintic 'control' (Anderson and May, 1985 ). Therefore, in answer to the theoretical question 'is mass medication the correct approach', the answer is no! However, if the logical alternative is to treat only animals which harbour the majority of parasites, these animals must be easily identifiable, but at present they cannot be readily identified other than by faecal egg counts. The long-term goal may well be to convert the flock or herd to a high responder category by selective breeding so that the highly aggregated distribution with wormy individuals is avoided. In the meantime, antiparasitic measures, relying on chemical compounds, must be applicable. However, in relying on chemotherapy for the control of parasitic infections there are a series of issues that increasingly concern both the producer and user of antiparasitic compounds. These are cost, delivery systems, drug resistance, operator safety, residues, environmental concerns and consumer pressure. In veterinary medicine, an important consideration is that of cost or cost-effectiveness. Measured in terms of economics of herd or flock production, the majority of anthelmintics are cost-effective; but is that the only criterion of costeffectiveness7 In view of consumer concerns such as the ecological aspects of disease control and increasing concern about animal welfare, cost-effectiveness may take on a different interpretation to the usual one. Part of the costeffective concern also is that of manpower costs, and as a result, there have been major advances in delivery systems with increasingly ingenious ways for the application of antiparasitic compounds. These consist of slow release, pulse-release, pour-on preparations, etc., and no doubt others are being de-
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veloped. These delivery systems also make mass medication easier but as they are not labour intensive they tend also to encourage overuse of drugs, leading to drug resistance. The problem of drug resistance is not one to be dealt with at length in this communication. Suffice it to say it is well recognised in all endo- and ectoparasitic situations, though the basis of it in parasitic helminths is as yet unclear despite intensive study of the phenomenon. One of the major pressures on the future development of parasite control will be consumer pressure. Frequently this is totally uncontrollable and totally unscientific, but nevertheless may be a very powerful force in the determination of the use of therapeutic substances in general. Examples of such consumer pressure concern the use of anabolic steroids and somatotrophins in cattle. Similarly, environmental concerns are increasingly apparent as certain compounds are shown to affect the natural fauna and flora, e.g. their effect on earthworms and dung beetles. TARGET FOR ANTIPARASITIC COMPOUNDS
Such concerns pose the question of what should be the target (s) for antiparasitic compounds? The antiparasite compounds used at present are directed against the parasites within the host and frequently against the mature developmental stages when they have developed to a pathogenic stage, though there is an increasing number effective against larval stages of parasites. However, the majority of a parasite population is outside the host, either in the form of developing or infective larvae or eggs; few, if any, antiparasitic compounds are directed against these. The nearest approach is the use of agents which will destroy intermediate hosts, such as insecticides and molluscicides. The final host is the collecting point through which this parasite population must pass in order to progress to the next generation of parasites. Present day antiparasitic compounds attack the parasite when it is established in the host and there have been few attempts to stop parasite development at the point of infection. Yet there are many triggers required to convert a free-living or an infective stage of a parasitic helminth to the parasitic stage. In gastrointestinal nematodes, exsheathment or hatching are such events. They are thus critical to the life cycle of the parasite - - no exsheathment, no parasite. Is it not time that we thought more seriously about parasite control at the point of entry into the host? The requirements for entry into the host or host cell are critical and unique to a given parasite (e.g. the exsheathment of gastrointestinal nematodes of ruminants). Would an anti-exsheathment compound be as effective as one of the compounds highly effective against an adult parasite stage? Control of the parasite outside the host is, of course, practised by pasture and grazing management with domestic animals, and the application of hy-
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gienic conditions in housed animals. The simple expedient of collecting faeces from pasture, as for the control of horse parasites, is a good example. As more is known about the ecology of non-parasitic stages of the organisms that concern us, the more effective will be our control programmes. RESPONSE TO THE CONCERNS OF CHEMOTHERAPY
A major approach must be vaccination. It has the attribute that limited handling of the animal is required, such as once or twice to inject the immunogen, or possibly the immunogen can be given in the feed. Immunisation addresses the negative binomial problem; it does not suffer from drug resistance. There are no residues or environmental concerns or consumer pressures with respect to welfare. The possible approaches to immunological control include passive immunisation (vaccination of mother), live vaccines (attenuated forms, abbreviation of infection, concealed antigens), subunit vaccines, synthetic vaccines, anti-idiotype vaccines and genetic resistance. Several parasite vaccines are now under development. Earlier attempts to produce antiparasite vaccines have been frustrated either by the inability to identify the important antigens or the limited availability of antigens responsible for resistance. Organisms were and still are not readily cultivated in vitro and in some cases the source of such material (e.g. man) made vaccine exploitation impossible. However, gene cloning techniques now offer a unique opportunity to overcome these problems. Specific monoclonal antibody probes are being developed to identify protective antigens, and these in turn are used to produce protective epitopes in unrelated microorganisms by recombinant DNA molecules coding for the protective antigens.
Passive irnrnunisation This is a feasible proposition where it has been shown that passive immunity can play a role in the control of parasitic infections. Examples are the larval cestode infections. The economically important Taenia saginata of man causing bovine cysticercosis is amenable to vaccination, and preparations of tapeworm oncosphere will immunise cattle against infection and also result in passive protection of calves born to vaccinated mothers (Lloyd and Soulsby, 1976). Because of the difficulty of obtaining human-derived tapeworm material, attention was directed to heterologous sources of immunogens, and while some useful protection was achieved with cross-reacting cestodes, none was so effective as the homologous parasite (Lloyd, 1979). Work with the rodent species Taenia taeniaeformis showed that excellent immunity could be induced with oncospheral material; the protective antibody was IgA in nature where maternal immunity was concerned, but was
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IgG based in the adult animal (Lloyd and Soulsby, 1978). Since then there have been major advances made with a vaccine for Taenia ovis, the larval stage of which is an economically important parasite of sheep in Australia and New Zealand. Antigens in the range of 47-52 kDa from T. ovis eggs were shown to be protective in sheep. Hatched and activated oncospheres of T. ovis were used as a source of m R N A for the construction of a cDNA library. This was constructed in lambdagtll and two fusion proteins fl-gal-45S and fl-gal45W purified from selected clones were tested in vaccination trials. While sheep immunised with the fl-gal fusion proteins produced antibodies which reacted with oncosphere antigens of 47-52 kDa, no protective immunity was induced. A different expression system was more successful. Using a plasmid vector which expresses antigens as fusion proteins with the enzyme glutathione Stransferase of Schistosoma japonicum (pSj 10ABam 7 stop 7, a precursor of the pGEX-1 vector), the 45W and 45S cDNA were cloned into this vector and the fusion proteins GST-45W and GST-45S proved very efficient in inducing protective immunity in sheep when injected with saponin adjuvants (Johnson et al., 1989). Why a fl-gal fusion failed to immunise while a GST fusion did, is unclear. These studies have led to the field trial of a commercial vaccine and provide the basis for similar vaccines for bovine and swine cysticercosis and also hydatid infection in man and animals.
Live vaccines These involve the use of live vaccines, either attenuated or abbreviated whole organisms or extracts of these. X-irradiation provided the basis for the first practical helminth vaccine for Dictyocaulus viviparus, but this is no longer a viable approach. Abbreviation of infections either by chemotherapy or by the deliberate selection of organisms for attenuation is practised in a number of infections. For example, a vaccine against Babesia consists of blood containing an attenuated strain of Babesia (De Vos et al., 1987), and the infect and treat approach has been used for immunisation of cattle against East Coast Fever (Theileria parva infection; Irvin and Morrison, 1987). An example of the deliberate selection of organisms for attenuation is the precocious development in coccidia which, missing out the pathogenic developmental stages but retaining the immunogenic stages, provides the basis of a vaccine. The concept of the use of concealed antigens is illustrated by the use of gut antigens to immunise against ticks (Willadsen and Kemp, 1988 ). Such antigens are those not normally 'seen' by the host i m m u n e system during normal infection. In the case of the tick vaccine, antibodies raised by concealed antigens damage the gut cells during tick feeding to the extent that host blood appears in the tick haemolymph. The immunological mechanism is antibody
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mediated and complement independent. As little as 7 ng of tick gut protein has been shown to be sufficient to immunise an animal. It should be noted with the use of concealed antigens, that it is unlikely that immune evasion mechanisms associated with salivary gland antigens of ticks will be invoked (Stewart, 1983); nevertheless, field challenge with tick infection will fail to boost the immune response to concealed antigens and regular immunising doses will be required. This indicates that expression vectors, such as the vaccinia virus, may be required to maintain an adequate level of immunity. A further example of the use of concealed antigens is in vaccination against blowfly strike, caused by Lucilia cuprina. This might be thought to be an unusual candidate for vaccination studies. Strike or cutaneous myiasis is responsible for a loss of some $150 million annually in Australia alone, and control while achievable with insecticidal drugs and sprays, suffers from the development of insecticide resistance by blowflies. Early studies demonstrated that immunisation with homogenates of maggots inhibited subsequent larval growth and excretory-secretory preparations from larvae were shown to induce significant immunity. However, a major development was the use of gut antigens of L. cuprina larvae in much the same way as with the tick Boophilus ( Sandeman, 1990).
Molecular vaccines At present there are no antiparasite subunit vaccines on the market. Nevertheless, there are reports of recombinant vaccines under development for ovine cysticercosis, coccidiosis and ticks. The development of a recombinant, molecular vaccine for parasites is beset with many more difficulties than with much simpler organisms such as viruses and bacteria. Even in the virus field, vaccination with subunits of the virus may lead to high antibody levels to a certain glycoprotein, but no protection against infection. This has led to a careful search for the essential 'T' cell epitopes, which will need to be incorporated into the molecular vaccine to provide both cell mediated as well as antibody mediated responses and immunological memory. The situation is complicated by the fact that a T cell epitope may stimulate T cells of one animal but not be effective in another strain of the same animal. Nevertheless, the optimal needs of a molecular or subunit vaccine include the identification of T and B cell epitopes of a parasite, the matching of T cell epitopes for B cell memory and CMI memory and the optimal recognition of B cells, the whole being held together by covalent bonds. An increasing amount of work is being done to identify subunits of parasites, the proteins or glycoproteins that might be immunogenic and protective. This development has been greatly helped by the use of monoclonal technology and monoclonal antibodies prepared against certain epitopes. Examples where subunit vaccines offer important opportunities for vacci-
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nation include the sporozoite antigens of Theileria parva and Theileria annulata. A 67 kDa component has been identified as important in protection and has been expressed in Escherichia coli as a C-terminal fusion protein with a Schistosomajaponicum antigen Sj26 using the pGEX expression vector system (Smith and Johnson, 1988). With the Theileria spp., the role o f T cells is particularly important, and in the case of the 67 kDa antigen, helper T-cell epitopes should be present (Good et al., 1987). The cloning of membraneassociated mid-gut derived protein from Boophilus microplus that is immunoprotective in the bovine (Opdebeeck et al., 1988 ) is a recent development which will provide the basis for a genetically engineered tick vaccine which is now under development in Australia. Synthetic vaccines Synthetic polypeptides now being examined closely in the field of virology are a natural extension of the subunit vaccine approach. It is likely, however, that these are a long way off for helminths. Initial work will entail the examination of sequences of overlapping synthetic peptides to identify T cell epitopes. If the immunising epitope is carbohydrate in nature, then recombinant approaches and peptide vaccines will not be suitable and a possible approach would be an anti-idiotype vaccine. In nematodes the cuticle is covered by mucopolysaccharides, and it is feasible that vaccine target antigens are contained in this surface material. Anti-idiotype vaccines The antigen binding site of an antibody molecule is, in its physical structure, complementary to the antigen against which the antibody is produced. Antibodies have been developed against the antigen binding site which are similarly complementary to the site and mimic the structure of the original antigen. Anti-idiotype antibodies are surrogate antigens. Anti-idiotypes could be of importance when immunising epitopes cannot be obtained by conventional methods, when an antigen is non-peptide in nature, when immunising epitope is unknown but when a monoclonal antibody possesses neutralising activity against the parasite, and can be used to induce an anti-idiotype antibody. An anti-idiotype approach has been used in schistosomiasis. An antibody which mimics a 38 kDa Schistosoma mansoni glycoprotein has been at least as effective as other approaches in vaccination (Grzych et al., 1985 ). The anti-idiotype approach may be particularly valuable where neonatal unresponsiveness occurs, e.g. in young sheep to Haemonchus contortus. Mice neonatally unresponsive to E. coli K13 polysaccharide can be immunised with an anti-idiotype (Smith, 1981 ). This area may be worthy of further study in H. contortus infection.
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There are a number of problems associated with the development of an effective vaccine, some of these are parasite in origin while others are of host origin. These include expression systems, immunosuppressive epitopes, stability, T cell epitopes, MHC, immunoglobulin characteristics, cross reactivity, adjuvants and responsiveness of young to vaccines.
Expression systems Expression systems using live recombinant vectors may be important with organisms such as Theileria, and delivery systems for cattle vaccines such as attenuated strains of Salmonella typhimurium or vaccinia virus (Smith et al., 1984). Vaccinia virus is most commonly identified as an expression carrier. Its genome is large and capable of receiving several foreign genes, but if several genes are inserted there may be a hierarchy of dominance and in some cases multiple immunisation may be necessary.
Immunosuppressive epitopes It will be necessary to ensure that immunosuppressive epitopes are not included in a vaccine. While it is recognised that immunosuppression occurs in parasitic helminth infections, the parasite components responsible for this are not known. Some are known in the virus field, e.g. the protease inhibitor in vaccinia, a homologous protein to cellular B2 microglobulin preventing presentation of virus peptides to cytotoxic T cells in CM virus.
T cell epitopes Whereas the search for T cell epitopes in a virus can be undertaken by the examination of a series of overlapping synthetic polypeptides, in helminths we are much further from this approach. We do not know the gene or the gene product responsible for immunity.
MHC The role of MHC in helminth immunity is unknown. It is of particular concern in virus infections and in protozoa. If it is of particular importance in helminths, then vaccine development with subunit vaccines will be more difficult.
Cross reactivity It is to be hoped that vaccines will have sufficient cross reactivity to permit generic vaccination rather than necessitate production of vaccines to each individual species ofhelminth.
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Responsiveness o f young to vaccines
A major handicap in the development of vaccines against gastrointestinal nematodes of ruminants is the poor immunological response of lambs and calves to parasites such as H. contortus and various other trichostrongyles. Thereby, animals are at substantial risk to infection for several months prior to the acquisition of protection, which when acquired is strong (Lloyd and Soulsby, 1987). Lambs may not respond effectively to H. contortus or T. colubriformis until they reach 3-6 months of age, and strong protection may require several more months (Smith and Angus, 1980). A similar unresponsiveness occurs in calves, but this is not well documented (as it is in sheep). Both parasite and host factors are concerned. For example, whereas young lambs are unable to develop protection against re-infection with most gastrointestinal nematodes, in the case of infection with Nematodirus battus lambs can m o u n t a prompt and effective i m m u n e response (Lee and Martin, 1976). In addition, lambs can mount an effective i m m u n e response to a wide range of bacterial and viral antigens (Lloyd and Soulsby, 1987 ). The mechanisms of this immunological unresponsiveness are not fully understood, and various hypotheses on the defect (s) include feedback inhibition by maternally transferred antibody, colostral transfer of soluble antigen or soluble suppressor factors and the generation of suppressor cell populations in the neonate. In part, the response has a genetic base and lambs may be segregated into 'responders' and 'nonresponders' as previously noted (Windon et al., 1980). Also, the infection status of the ewe may be important. Thus, immunisation with soluble antigens of 4-week-old lambs against infection with H. contortus induced a degree of protective immunity, but only if the lambs had been born of non-infected ewes. This degree of protection was abrogated if the ewes carried an infection with H. contortus (Shubber et al., 1984; Lloyd and Soulsby, 1987 ). Although there is an early failure of young ruminants to develop an effective immunity to gastrointestinal nematodes, early infection of such animals does not seem to prevent this subsequent development of protective immunity when these animals are reinfected at a later date. Recent studies have shown that adult sheep either injected with antigens of H. contortus or infected with the parasite, have antigen-reactive peripheral blood lymphocytes. More than 90% of these cells are cd4 + T cells and approximately 8% are cd8 + T cells (Haig et al., 1989). The i m m u n o d o m i n a n t third stage larval antigens had molecular weights of 15-18, 25-29, 79-80 and more than 100 kDa (Haig et al., 1989 ). Contrary to earlier studies which showed a lack of responsiveness to H. contortus antigen of peripheral blood lymphocytes of lambs, very recent, unpublished work by P. Torgerson ( 1991 ), has suggested that lambs have genetically primed antigen-reactive lymphocytes in their circulation at an early age, possibly helper T cells but also there is an unidentified plasma factor which
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inhibits their response. Higher concentrations of antigen have an inhibitory effect on lymphocytes both in the absence and particularly in the presence of plasma. This may be an important factor in the occurrence of immune unresponsiveness in an active infection. In this case the parasite would be suppressing the response of the young animal in order to gain access to and develop in the host. CONCLUSIONS
In the 50 years or so since Richard Turk undertook work in the field of veterinary parasitology, many developments have occurred in chemotherapy and immunology, and explanations have been provided for the mechanism of parasitism. These mechanisms frequently result in the survival of the parasite, the avoidance of rejection mechanisms and often a synchronisation of the biology of the parasite with that of the host: in fact, in parasites' progress. I have been privileged over the last 40 years or so, to study and admire the parasites' progress in dealing with host responses, man-made chemicals to attack them, and indeed for me to gain a little insight into the phenomenon of parasitism. I know Dick Turk was of the same mind in admiring these subtle mechanisms and it is my great honour to review some of the developments of this subject in his name.
REFERENCES Anderson, R.M. and May, R.M., 1985. Herd immunity to helminth infection and implications for parasite control. Nature (London), 315: 493-496. De Vos, A.J., Dalgliesh, R.J. and Callow, L.L., 1987. Babesia. In: E.J.L. Soulsby (Editor), Immune Responses in Parasitic Infections: Immunology, Immunopathology and Immunoprophylaxis. Vol. III, Protozoa. CRC Press, Boca Raton, FL, pp. 183-222. Good, M.F., Maloy, W.L., Lunde, M.N., Margalit, H., Cornette, J.L., Smith, G.L., Moss, B., Miller, L.H. and Berzofsky, J.A., 1987. Construction of a synthetic immunogen: use of a new T-helper epitope as malaria circumsporozoite protein. Science, 235: 1059-1062. Grzych, J.P., Capron, M., Lambert, P.H., Dissons, C., Torres, S. and Capron, A., 1985. An antiideotype vaccine against experimental schistosomiasis. Nature, 316: 74-76. Haig, D.M., Windon, R., Blackie, W., Brown, D. and Smith, W.D., 1989. Parasitic specific T cell responses following live infection with the gastric nematodes Itaemonchus contortus. Parasite lmmunol., 11: 463-477. Irvin, A.D. and Morrison, N.I., 1987. Immunopathology, immunology and immunoprophylaxis of Theileria infections. In: E.J.L. Soulsby (Editor), Immune Responses in Parasitic Infections: Immunology, Immunopathology and Immunoprophylaxis. Vol. III, Protozoa. CRC Press, Boca Raton, FL, pp. 223-274. Jarrett, W.F.H., Jennings, F.W., Martin, B., Maclntyre, W.I.M., Mulligan, W., Sharp, N.C.C.
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