Application of immunity in the control of parasitic disease

Veterinary Parasitology, 6 (1980) 2 1 7 - - 2 3 9 © Elsevier Scientific Publishing C o m p a n y , A m s t e r d a m - - Printed in The Netherlands

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Control

APPLICATION OF IMMUNITY IN THE CONTROL OF PARASITIC DISEASE

G.M. U R Q U H A R T

University of Glasgow, Veterinary School, Bearsden Road, Bearsden, Glasgow (Great Britain) ( A c c e p t e d 4 D e c e m b e r 1978)

ABSTRACT Urquhart, G.M., 1980. Application of i m m u n i t y in the control of parasitic disease. Vet. Parasitol., 6: 217--239. The application and potential of immunological m e t h o d s for the control of a n u m b e r of parasitic diseases of domestic animals is reviewed. In particuar, East Coast fever and trypanosomiasis of cattle and haemonchosis of sheep are discussed at s o m e length, while dictyocauliasis, babesiosis and C. bovis infection (all of cattle) are dealt with m o r e briefly. T o date, the only vaccines in large-scale p r o d u c t i o n are those against Dictyocaulus viviparus, the bovine lungworm, and Babesia argentina and B. bigemina in cattle. Both are a t t e n u a t e d live vaccines with quite stringent m e t h o d s o f distribution in view of their limited shelf life. Perhaps part of their success is due to the fact that by the time the clinical disease is recognised and t r e a t m e n t initiated, the hosts have usually suffered a considerable check and m a y even be very ill. If this is the case, it seems n o t unreasonable to hope, in view of the progress already achieved, that a vaccine for East Coast fever m a y b e c o m e available in the fairly near future. F o r trypanosomiasis and haemonchosis, the i m m e d i a t e o u t l o o k is less promising. With the f o r m e r perhaps the best one can strive towards is a better appreciation of the role of chemotherapy in p e r m i t t i n g the d e v e l o p m e n t of non-sterile i m m u n i t y . In the latter, apart f r o m the potential role of H. contortus-resistant breeds, there seems little o p t i o n at present but to rely on p r o p h y l a c t i c c h e m o t h e r a p y . INTRODUCTION

Rather than a t t e m p t to catalogue briefly the present state of knowledge on immune responses to all of the parasitic infections of domestic animals, I t h o u g h t it might be more interesting to review at greater length the immunology of a few of these diseases. The selection of these was based partly on the fact that I am reasonably familiar with most of them, and partly on their particular interest and importance at the present time. I would like first to discuss the practical use, if any, of immunological procedures in three diseases, East Coast fever and trypanosomiasis in cattle, and haemonchosis in sheep; and second, in less detail, to review some recent developments in dictyocauliasis, babesiosis and cysticercosis in cattle.

218 EAST COAST FEVER East Coast fever is a disease which has plagued livestock development in East Africa for many years. Because of its geographical limitations, it has inevitably attracted the attention of a relatively small number of research workers, and current progress in attempts to develop a vaccine is probably largely followed b y only a limited number of veterinarians and biologists who have seen at first hand the ravages of this disease. This is a pity, because some of the most exciting developments in the study of immunity in parasitic diseases have occurred in this field over the past 10 years, and reflect great credit on those concerned, particularly the recent UNDP/FAO project on tick-borne diseases based at the East African Veterinary Research Organisation, Muguga, Kenya, under the direction of Dr M.P. Cunningham. For this reason I will not attempt to review in detail the most recent and often highly sophisticated advances in this field, b u t will provide a more general historical account of the disease leading up to the current situation. If y o u are suitably sensitised by this account, I would recommend Purnell's recent review (1977) for a very readable in-depth study of recent research in this field. East Coast fever, so-called because of its association with the East Coast of Africa, is caused by the protozoa Theileria parva. It is transmitted by the tick Rhipicephalus appendiculatus which ingest the erythrocytic piroplasm stage of the parasite while feeding on infected cattle. After the ticks have engorged, they drop off and moult to the next instar. During this time the parasites migrate to the salivary glands of the tick, and, when the tick attaches to a new host, the parasites mature as infective particles and are inoculated into the new host during feeding. Four days later, macroschizonts are present in the lymphoblasts of the lymph nodes nearest to the tick bite and their numbers increase tenfold every 3 days until almost all the lymphoid cells in the b o d y are parasitised. Fourteen days after infection the macroschizonts give way to microschizonts, the host cells are destroyed and the micromerozoites invade the erythrocytes to become piroplasms. Up to 50% of erythrocytes may contain piroplasms. The host develops fever from the 10th day onward and almost invariably dies from 16 to 26 days after infection. Cattle reared for generations in endemic areas apparently have a low mortality rate, only a b o u t 7% dying of the disease usually in their first year of life. This resistance appears to be by evolutionary selection and not to depend on the transmission of maternal factors such as colostral antibody (Barnett, 1956, 1957). However, cattle introduced into these areas almost inevitably rapidly develop the classical disease, the mortality rate being of the order of 97%. The only practical method of controlling East Coast fever is b y the regular use of acaricide dips or sprays every 3--7 days depending on the density of tick infection. While this is possible on a routine basis in developed and fenced ranching areas, it frequently breaks down on other occasions, -- as when improved dairy cattle are introduced into smallholdings in endemic areas where

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tick control is n o t universally practiced. Also, the spread of the tick vector in advantageous climatic conditions may disseminate the disease among susceptible stock, and, as recently as the early nineteen-sixties, in one such outbreak several hundred thousands of cattle died of East Coast fever in Sukhmuland, Tanzania (Yeoman, 1966). Since it is known that the few survivors of infection with East Coast fever are immune to reinfection, at least with the same strain, it has always seemed possible that some m e t h o d of vaccination might be developed. One of the first attempts to do this was conducted b y Spreull (1914), who, using suspensions of infected lymph nodes and spleens, vaccinated 280,000 cattle in an outbreak in South Africa; of these, 25% died and the remainder developed partial or complete immunity. The project was finally abandoned in preference for dipping because, among other reasons, the technique was creating a reservoir for ticks. The use of infected lymphocytes was subsequently explored on a more modest scale by Brocklesby et al. (1965) and Pirie et al. (1970), and although the results were encouragingly successful, the collection, quantitation and preservation of macroschizonts from donor cattle on a large scale presented formidable problems. These were resolved a few years later when Purnell and J o y n e r (1967) and Cunningham et al. (1973a) showed that the infective particles in the salivary glands of ticks could be harvested in the supernatant fluid of ground-up ticks or by the collection of tick saliva. (The latter involved the immobilisation of the ticks in plasticine, the placing of a capillary tube over their hypostomes and the final indignity of an anal injection of arecoline hydrobromide; this when visualised as a vaccine production line moved Marcel Burdin, the then Director of E A V R O to remark "truly a cottage industry".) These stabilates containing thousands of infective doses were subsequently shown to be readily cryopreserved for months (Cunningham et al., 1973b). The possibility of vaccination n o w seemed feasible. A few years previously, Jarrett and his colleagues (Jarrett et al., 1969a; Pirie et al., 1970), in an elegant study of the kinetics of replication of T. parva in cattle, had shown that, for any given strain, the time elapsing between infection and piroplasm production was constant irrespective of the size of the infecting dose. The latter, however, determined the number of macroschizonts on any given day after infection and consequently the number of lymphocytes parasitised and ultimately destroyed. Thus, under natural conditions, the number of infective particles inoculated b y a tick is normally so large and the lymphocytes so rapidly infected and destroyed that an effective immune response cannot occur. However, in vaccination studies with macroschizonts it was shown that as few as 108 or 109 macroschizonts constituted a 50% immunising dose (fever occurs only after 7 × 109 macroschizonts are produced), and that an effective immunity probably takes about 3 weeks to develop. This information suggested that vaccination with a slowly replicating strain of Theileria or with a suitably small number of infective particles (Jarrett, 1969a; Radley et al., 1974)

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would allow the immune system to respond before it was overwhelmed. Unfortunately, it was n o t to be so simple. Serial dilutions of suspensions of tick-derived material failed to produce progressively attenuated reactions in groups of cattle, and uniform reactions did n o t occur in cattle given apparently identical aliquots of infective material. These results (Wilde, 1967; Cunningham et al., 1973a, 1974a) suggested that the infective particles were n o t evenly dispersed in the original material and therefore could n o t be diluted out to provide consistently the uniform small infective doses essential for the preparation of a vaccine of this type. In attempts to overcome this problem, Cunningham and his colleagues then tried to attenuate the infective particles. They first used 6°Cobalt-irradiation of infected ticks and then of infective particles, b u t found that the margin between protection on the one hand and under-or over-attenuation on the other was too narrow and sometimes unpredictable (see review by Purnell, 1977). This project was abandoned and the group then studied the value of suppressing the development of the parasite in the host b y the contemporaneous administration of the antibiotic tetracycline with the immunising infection. It was known from earlier work (Neitz, 1953; Barnett, 1956; Brocklesby and Bailey, 1965; Jarrett et al., 1969b) that the repeated administration of tetracycline throughout the incubation period could suppress the multiplication of the organisms and the subsequent clinical signs, b u t still permit the develop ment of immunity. However, little practical use had been made of these observations because of the necessity to use the drug on a dally basis for 16--28 days. It was thought that infection induced b y the injection on a single occasion of infective particles might shorten to an acceptable degree the period of drug administration; in the previous experiments when infected ticks were used, these inoculated infective particles over a period of days while feeding. This final series of experiments (Radley et al., 1975a) was remarkably successful. It was found that a single injection of infected tick stabilate and four daily injections of an oxytetracycline formulation produced a mild or inapparent reaction and that this was followed b y a solid and apparently prolonged immunity to challenge with the same strain of East Coast fever organism, whether administered b y inoculation of tick stabilates or by exposure to infected ticks. Unvaccinated cattle exposed to the same challenge invariably and rapidly succumbed. It now seemed that the way lay open to the development of a vaccine against East Coast fever, particularly if a single-shot slow-release oxytetracycline formulation could be developed which would obviate the necessity for four daily injections. However, as the poet Bums, Cunningham's compatriot and fellow Ayrshireman, observed "the best laid schemes o' mice an' men gang aft a-gley", and the group were soon wrestling with three problems, none of which could reasonably have been foreseen. First, all the previous evidence available had suggested that immunisation with any one strain of T. parva conferred protection to other strains. Although this was found to be ~enerally true in terms of

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mortality, cattle challenged with different strains from that used for immunisation sometimes developed unacceptable clinical reactions. Second, Theileria mutans, previously regarded as a ubiquitous and non-pathogenic parasite of cattle in East Africa, had shown itself, in at least one area of endemic East Coast fever, to be capable of producing severe mortality in cattle successfully vaccinated against T. parva. Finally, the occasional occurrence of a few piroplasms in the blood of artificially immunised cattle, apparently due to inadequate suppression by the drug, was observed. This raised the possibility that ticks feeding on such an animal might introduce a new strain of T. parva into a particular area. The problem of cross-immunity has been tackled b y the development of 'cocktails' of strains for immunisation. These to date have generally contained two strains of T. parva and one of T. lawrencei, a parasite c o m m o n l y found in buffaloes and transmissible to cattle. Experimental trials to date have indicated that these are likely to be an effective means of vaccinating cattle against heterologous strains of T. parva in East Africa (Radley, 1975b). The degree of involvement of T. mutans in the East Coast fever syndrome requires further definition both in terms of its pathogenic role and it geographical distribution, and Purnell (1977) has suggested that it t o o ultimately may be a c o m p o n e n t of the 'cocktail' in specific areas. The third problem, i.e. that vaccinated cattle may harbour piroplasms on occasion, may yield to further experimentation with the formulation and dosage of oxytetracycline. If it does not, Cunningham et al. (1974b) have suggested that vaccine for any particular East Coast fever are could readily be prepared from strains of the organism isolated from that area. I have dealt in this review with the most immediately promising line of research on vaccination against East Coast fever. Other possibilities explored by this group have been the development of a tissue culture vaccine derived from the cultivation of bovine spleen cells infected and transformed b y T. parva, and the use of irradiated athymic mice and cell-fusion techniques in an effort to develop a non-bovine cell infected with attenuated T. parva (Malmquist et al., 1970; Irvin et al., 1975; see review by Purnell, 1977). Any of these three may y e t be the ultimate answer, b u t even n o w it seems likely, subject to the satisfactory completion of pilot field trials, that East Coast fever vaccination could become a practical reality in East Africa. Such a development would remove the spectre of sudden financial ruin which faces many small farmers, and permit the introduction and survival of improved bulls and cows in areas where they would otherwise certainly die. TRYPANOSOMIASIS

Trypanosomiasis, transmitted b y tsetse flies, prevents any domestic animals, except the domestic fowl, being reared in almost half of Africa south of the Sahara. Despite this, the sudden prospect of completely controlling this disease would n o t necessarily be welcomed with open arms b y the governments

222 of those countries involved. Without the contemporaneous development of roads, fences, watering points and marketing arrangements -- all prodigiously expensive -- the unlimited access of domestic stock to this large area would not be beneficial, and possibly be even detrimental to the long-term prospects for f o o d production. This generalisation, however, is n o t always true, and there are many areas where the removal of the threat of trypanosomiasis would break the vicious spiral of disease, poor productivity and absence of capital for development. For example, to quote Anosa (1975) on Southern Nigeria, "In the southern parts literacy is high, the rainfall is spread o u t more evenly over the y e a r . . , and there is a wish to engage in settled f a r m i n g . . , however, heavy tsetse infestation and trypanosomiasis remain a gigantic obstacle to the establishment of dairy farming and ranching". On these grounds, it therefore seems likely to me that the control of trypanosomiasis, in the best sense of the word, is likely to come a b o u t in a piecemeal fashion throughout affected areas, and it is against this background that I would like to discuss the subject. To be fair at the outset, one must admit that any concensus of informed opinion would regard immunological control as very much a 'dark horse' among the various possibilities, and the potential of bush clearing, insecticides and trypanocidal drugs have a much more powerful advocacy. I will return to this later. Since the vast literature on immune responses to trypanosomiasis has very recently been reviewed in detail by several authors (Gray, 1967; Terry, 1976; Clarkson, 1976; Murray and Urquhart, 1977), I would like to confine m y remarks to those aspects which are relevant to the prospect of control. The fundamental problem, of course, in the development of trypanosome vaccines is the fact that the parasites have a capacity for apparently unlimited antigenic variation in the host at intervals of a few days. The immune response against each variant, although rapid and highly effective in destroying any trypanosomes which possess that particular variant, is invariably t o o late to affect that proportion of the population which has altered its antigenicity. This picture of successive waves of specific antibody chasing non-existent trypanosomes has been likened b y Goodwin (1970) to a Tom and Jerry cartoon "with a monstrously inept cat pulling the place down in his efforts to pulverise a diminutive agile and highly resourceful mouse". Indeed, were it n o t for antigenic variation, Terry (1976) has suggested that trypanosomiasis might be little more than a series of minor, short, and self-limiting infections. The complexity of the problem becomes more apparent in the light of recent research. It is n o w known that the variant antigen constitutes the major part of the surface coat of the trypanosome (Vickerman, 1969) and that it is a glycoprotein consisting of a single polypeptide chain; the different variant antigens are not merely minor modifications of this chain b u t in fact are structurally dissimilar in that their amino-acid sequences are completely different (Cross, 1975). It used to be thought th'at the trigger for antigenic variation was initiation of the specific antibody response, b u t it n o w seems that antigenic variation occurs apparently spontaneously and in a random fashion

223 within a few hours after infection with metacyclic trypanosomes (Jenni, 1977). None is this information, daunting though it is, is necessarily incompatible with successful immunisation, provided that every trypanosome after passage through a tsetse fly and before inoculation into its final host reverts to one, or at most a few, basic antigenic types. If this occurred, one could see the possibility of immunising cattle with antigens obtained from such metacyclic trypanosomes. Whether or n o t trypanosomes of the three relevant species, i.e.T, congolense, T. vivax and T. brucei, do, in fact, always revert to a very limited number of antigenic types after passage through the tsetse fly, is still unknown. However, the most recent finding (Le Ray et al., 1978) is that tsetse flies allowed to feed on a population of T. brucei, which was 99% antigenically homogeneous, subsequently developed infections with metacyclic trypanosomes which were antigenically heterogeneous. A second experiment, which might indicate that under conditions of field exposure the range of antigenic variants encountered is quite formidable, is that reported b y Wilson (1971). He first showed that t w o inoculations of the same trypanosome stabilate, each terminated b y drug therapy 3 weeks later, conferred a solid degree of immunity to subsequent challenge with the same stabilate 8 weeks after the last drug treatment. He then inoculated a second g r o u p of 10 cattle on five occasions with metacyclic trypanosomes, obtained by allowing groups of wild tsetse flies caught in the same locality to feed on blood. The resulting infections were treated with diminazine aceturate (Berenil, Hoechst, Germany). On a sixth inoculation of infected blood 6 weeks after the last of the Berenil-treated infections, all 10 cattle became parasitaemic, six of them requiring treatment to keep them alive. In summary, then, it seems rather unlikely that the production of a vaccine against bovine trypanosomiasis is imminent. However, one development which may be of great potential significance in this area, is the recent demonstration (Hirume et al., 1977} that T. brucei organisms may be cultured in vitro for an indefinite period without losing their infectivity for laboratory animals. If this can be done with the metacyclic forms of T. congolense and T. vivax it might provide some rapid answers to the problems posed by antigenic variation as well as a possible source of large quantities of antigen, hitherto a potential headache in the logistics of trypanosome vaccination. So far I have discussed immunity to trypanosomiasis in the conventional sense, i.e. the absence of trypanosomes in the blood or tissues after challenge. There is however another stage of affairs known to trypanosome aficionados as 'non-sterile immunity' or sometimes 'trypanotolerance', in which cattle, after repeated natural challenge, often become resistant to the effects of trypanosomiasis although parasites may c o m m o n l y be found in their blood. This p h e n o m e n o n has been described intermittently since Bevan (1928) introduced the first trypanocidal drug for cattle into Africa in 1909. One such account, readily available although somewhat anecdotal, is that given by Fiennes (!970}.

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More recently the significance of non-sterile immunity in the successful development of cattle husbandry has emerged from the excellent series of studies carries o u t b y Wilson and his colleagues (1975a) in East Africa. In Uganda, they studied the performance of a breeding herd introduced and maintained for 2 years in an area of heavy trypanosome challenge. Chemoprophylaxis was not attempted, and instead calves and cattle were treated on an individual basis, and then only when clinically ill or when their packed red cell volume fell below 20%. Berenil, which is essentially curative and rapidly excreted, was used throughout the study. During both the first and second years each animal required an average of 8 treatments, and became parasitaemic again a b o u t 30--40 days after each treatment. Despite this failure to develop immunity, during the second year the number of live calves born increased and the subsequent calf mortality decreased; the number of abortions, c o m m o n in infected cows, also decreased. Even more promising results were obtained in a subsequent experiment (Wilson et al., 1975b, 1976) in which steers were introduced into an area of medium trypanosome challenge in Kenya and observed for a period of 29 months, during which a regimen similar to that used in the previous experiment was practised. The period between drug treatments, initially between 50 and 60 days, increased to around 132 days b y the ninth treatment. When drugs were withdrawn from a number of the steers 6 months before the end of the experiment, all survived and gained weight at the same rate as the steers with access to therapy. In contrast, a second group of steers, all of which were treated with Berenil whenever infections were detected in the blood of one, required treatment every 26 days throughout the 29 months. When treatment was withdrawn from some of the steers for 6 months, their mean weight gains were 56 kg less than those in which treatment continued. Control animals introduced at intervals throughout the experiment developed severe anaemia and usually died. Earlier this year, through the kindness of Dr M. Scott of the British Veterinary Team in Addis Ababa, I had the opportunity to see at first-hand another example of the commercial exploitation of non-sterile immunity to trypanosomiasis. Until 1972, the Angar-Gutin valley of Ethiopia was largely uninhabited, primarily because of a dense infestation of tsetse which made stock-rearing impossible. In that year the area was selected for settlement and the cultivation of maize, cotton and peppers. For a variety of reasons it was decided to use oxen as the means of ploughing and haulage. Ininially, block treatment with Berenil was practised wherever classical signs of trypanosomiasis appeared in several of the oxen; on average this was necessary every 17--28 days. Subsequent attempts to use a prophylactic drug were unsuccessful because of the onset of drug-resistance and for the past 3 years control has been largely based on the treatment of clinically affected individuals with Berenil. Of the oxen currently working on the settlement for the past 3 years, it is now necessary to treat only approximately 20% per month. Despite this, the

225 percentage of oxen with positive blood films remains high and is of the order of 50-60%. Perhaps I should emphasise that I am not suggesting that all trypanocidal therapy should be directed towards the production of non-sterile immunity, and indeed other results of Wilson et al., (1976) illustrate the superiority of prophylactic drugs in terms of beef production. However,these workers point out that the particular advantage of encouraging the development of non-sterile immunity might lie in the development of breeding herds over periods of several years particularly in areas of low to medium trypanosome challenge. Such enterprises, inevitably associated with human settlement and the removal of trees for fuel, etc., would in time perhaps lead to such an altered environment that the tsetse flies would themselves disappear -- ultimately the only long-term solution. The events which lead to the development of 'non-sterile immunity' are unknown. It may be that experience of a great variety of variant antigens both from fly challenge and subsequent antigenic variation build up a battery of specific immune responses; alternatively, although there is no evidence for it, there may be a common antigen which only elicits a response after prolonged stimulation. The immunity, of course, may be non-specific and depend on factors such as expansion and activation of the macrophage population. It is impossible to leave the subject of 'non-sterile immunity' without a reference to the small humpless N'dama breed of cattle in West Africa, where a degree of trypanotolerance is almost legendary. In work carried out recently in The Gambia, Murray and his colleagues (1978) have confirmed that this quality seems to be an innate characteristic of the breed, although whether it is based on some kind of enhanced immunological responsiveness or particular physiological characteristics is not yet clear. It is also significant that Murray and his co-workers conclude that the commercial value of trypanotolerance in N'dama can only be fully exploited under conditions of adequate nutrition, good husbandry and chemotherapy. Whether or not this type of trypanotolerance is similar to the acquired form such as described by Wilson and his colleagues, is unknown. Finally, it is interesting to speculate why trypanotolerant cattle did not evolve, or at least have not been recognised, in East Africa. The possibility that they do, in fact, exist is supported by Cunningham's description (1966) of how thousands of Zebu cattle survive around the North East shore of Lake Victoria despite the fact that they are continuously exposed to trypanosome challenge. One interesting aside on this observation was that the incidence of infection as measured by blood parasitaemia and the presence of neutralising antibody was 30 and 90%, respectively. This in fact is similar to that encountered in game animals such as bushbuck and waterbuck, generally considered to be resistant to trypanosomiasis. Whatever it is, the scientific study of naturally acquired non-sterile immunity, should be an interesting and profitable occupation. In practical terms the technique is the only currently available means of exploiting the immune res-

226 ponse of the host, and could perhaps be of great economic significance given one essential condition, i.e. that the standard of veterinary surveillance both of trypanosomiasis and animal health generally is consistently high and that particular attention is paid to the early detection of drug resistance. HAEMONCHOSIS

In tropical and sub-tropical areas, H a e m o n c h u s c o n t o r t u s infection of sheep, and to a lesser extent of cattle and goats, is one of the most important causes of economic loss to producers of wool and mutton. Where the disease is unchecked, severe morbidity and high mortality are common; in other areas the cost of control in terms of labour and prophylactic chemotherapy is often a significant factor in production costs. Despite this, our in-depth knowledge of the immune response of sheep to this pathogen is still rudimentary. Indeed, in practical terms the situation is still best summed up b y Gordon's observations (1948, 1950) that sheep in endemic areas acquire no immunity to this parasite and remain susceptible throughout their entire lives. I must confess that it t o o k some time for me to become convinced of the validity of this observation. The fault perhaps was not entirely mine. Shortly after the development of the vaccine against the bovine lungworm with which immunity was readily stimulated against infection, m y colleagues and I carried out some initial experiments on infection and reinfection of Scottish Blackface sheep with H. contortus and found that these sheep quite quickly became almost solidly immune to infection. However, we were n o t able to extend these observations into the naturally occurring disease, since haemonchosis is a relatively rare occurrence in our Scottish environment. It was n o t until I worked in an endemic area of haemonchosis in East Africa, this time with Merino sheep, that I realised h o w repeatedly susceptible these sheep were under natural conditions. Nevertheless, the fact remains that sheep may be readily 'vaccinated' against an experimentally administered single challenge, using either priming infections of normal or X-irradiated larvae, to such an extent that the worm burdens are reduced by 98% and faecal worm egg counts are negligible (Manton et al., 1962; see review b y Urquhart et al., 1962) and it is still not quite clear h o w these findings can be reconciled with what occurs in the field. A possible explanation depends on the finding that successful experimental immunisation can be induced only in lambs over at least 7 or 8 months of age, depending on the breed, (Manton et al., 1962; Urquhart et al., 1966) a phenomenon attributed to immunological immaturity. It was therefore proposed that continuous exposure to infection under natural circumstances from the age of 3 or 4 weeks during this long unresponsive period had permanently depressed the immune system. Experimental evidence in support of this theory is still inadequate and rests mainly on two observations. First, that it was possible to vaccinate 24-month-

old East African Merino sheep reared under worm-free conditions. In contrast, adult sheep which had been reared in endemic areas could n o t be vaccinated (Lopez and Urquhart, 1967). Second, it has been shown that y o u n g ~ats up to the age of 6 weeks show a diminished immune response to infection with N. brasiliensis (Jarrett, 1971). Moreover, if they are repeatedly infected during this period and into adult life, they remain immunologically incompetent and fail to eliminate their challenge infection b e y o n d a certain degree. This theory, i.e. that acquired immunological unresponsiveness is responsible for the repeated susceptibility of sheep to haemonchosis in endemic areas, was n o t supported in a recent experiment conducted in Glasgow (BenitezUsher et al., 1977). From the age of 3 months, a group of lambs were exposed to six separate monthly doses of normal larvae, followed by an anthelmintic, vaccination and challenge. The subsequent worm burdens were reduced by 88% compared to non-vaccinated sheep reared worm-free to the time of challenge. This high degree of protection was n o t significantly different from the 95% reduction obtained in sheep reared worm-free until the time of vaccination. However, it is possible that a different result might have been obtained had the lambs been first infected before 3 months of age and with smaller and more frequently spaced doses of larvae. It is perhaps surprising that the Merino breed over the centuries has not evolved a better defence system against H. c o n t o r t u s and more comparable with the strong acquired immunity which has developed in adult sheep against ostertagiasis and trichostrongylosis. Presumably the breed evolved in an area and under conditions of husbandry where haemonchosis was n o t a constant threat. Also, because of the availability of relatively efficient enthelmintics for many years, there has perhaps been little conscious effort in selection for resistance to this disease. However, some evidence that this might have occurred to a limited extent in Australia was triggered by the observation of Evans and Blunt (1961) w h o showed that the gene frequencies of the haemoglobin types of R o m n e y Marsh and Southdown sheep in Australia were predominantly haemoglobin A, whereas the original stock in the native British environment were predominantly haemoglobin B. One possible basis for this adaptation was subsequently suggested by Evans et al. (1963) w h o experimentally infected both haemoglobin A and haemoglobin AB sheep with H. c o n t o r t u s . The trend in faecal worm counts and worm burdens at necropsy suggested that haemoglobin A sheep were more resistant to the establishment of adult infections of this nematode, although there were no significant differences in the haematological indices of the two haemoglobin types. Later, Evans and Whitlock (1964) demonstrated a significant association between the seasonal maximum haematocrit of grazing sheep and their haemoglobin type, and suggested that this might be related to a varying degree to resistance to natural infections of H. c o n t o r t u s .

In the United States of America, Jilek and Bradley (1969) and Radhakrishnan et al. (1972), in a study of the relationship of haemoglobin t y p e in Florida Native sheep to naturally acquired experimental H. c o n t o r t u s infection,

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concluded that there was " n o definitive data to support the theory that haemoglobin types in Florida sheep were indicators of resistance to H. contortus or that H b A sheep were less susceptible". Nevertheless, the HbA sheep generally had higher PCV values throughout the course of infection and appeared to undergo 'self-cure' more readily. However, recently in East Africa (Allonby and Urquhart, 1976) a preliminary study was conducted on the performance of set-stocked Merino ewes and lambs in relation to the haemoglobin phenotypes. The results provided some evidence that, in Merino sheep at least, those with haemoglobin A type show self-cure more frequently and effectively than those with haemoglobin B type. Examination of the PCV values and total b o d y weights of Merino ewes also showed a similar trend, i.e. the HbA types had the highest haematological parameters and the heaviest b o d y weights, while the haemoglobin B types were consistently lowest. Subsequently, Altaif and Dargie (1976) studied the relative resistance of two breeds of sheep, Scottish Blackface and Finn Dorset, of known haemoglobin type, to a primary infection with 350 H. contortus larvae/kg bodyweight, i.e. a single dose of between 8,000 and 9,000 larvae. The results demonstrated that sheep with haemoglobin A developed less severe clinical and haematological disturbances, passed fewer eggs and harboured fewer worms at necropsy than animals with haemoglobin B, and that Scottish Blackface exhibited similar advantages over Finn Dorsets with the same haemoglobin type. Thus, at necropsy, 32 days after infection, the mean worm burden of the Scottish Blackface HbA sheep was 1,200 while that of the Finn Dorset HbB was 3,138. The results of the radioisotopic measurements of blood loss suggested that these differences in worm burden occurred a b o u t the 10th--12th day of infection, i.e. that resistance was expressed a b o u t the time of the fourth ecdysis. It is interesting and perhaps of practical significance, that a second experiment, designed to examine the response of Scottish Blackface sheep to heavier infections (1,400 larvae/kg), failed to reveal any correlation between Hb type and worm establishment. Apart from variations within a breed there are at least two reports which indicate that certain breeds m a y be more resistant than others. In Yugoslavia, lambs and adults of three breeds of sheep (Cigaja, Merino Karkaz and Merino Prekos) were grazed together ~ o m May until December on pasture contaminated with H. contortus larvae (Cvetkovic et al., 1973). As measured by the faecal worm egg counts, the lambs all appeared to be equally susceptible. However, the mean faecal egg counts of the adult sheep of the Cigaja and Merino Prekos even after only one previous season of exposure to H. contortus were almost consistently negative throughout the year, while that of the Merino Karkaz breed flectuated between 1,000 and 3,000 eggs per gram. Whether this difference was in fact due to immune destruction of the ingested larvae, or in some degree at least merely to their inhibition until the following spring, still requires study, b u t the very different responses of the three breeds to infection during the grazing season was quite remarkable.

229 The second report (Preston and Allonby, 1979) describes some recent work in Kenya and is concerned with a comparison of the susceptibility of six breeds of sheep to H. c o n t o r t u s infection, as judged b y worm burdens and faecal egg counts when set-stocked together on a pasture endemic for H. c o n t o r t u s . Of the six breeds, the indigenous Masai was b y far the most resistant and the Merino the most susceptible. The results of a subsequent experiment indicated that the Masai was able to expel a large proportion of its worm burden during the prepatent phase of infection. It is interesting that the Masai sheep has evolved for centuries in an area of endemic haemochosis in a system of semi-nomadic husbandry in which anthelmintic therapy has played little or no part. Unfortunately, it is n o t a commercially developed breed b u t this presumably could be improved by selection. Although there might be little demand for such resistant breeds of sheep or goats in areas where husbandry is highly developed, there are many parts of the world where commercial sheep husbandry is being developed and where veterinary surveillance is inadequate; the possibility of developing H a e m o n c h u s resistant breeds of economic worth for such situations presents a fascinating challenge and one of great economic potential. Although, as noted previously, immunity of most breeds of sheep to H. contortus, as measured b y freedom from clinical disease, is n o t particularly apparent under natural circumstances, there is some evidence that it is operative at a certain level. One example is the occurrence of the 'self-cure' phenomenon whereby adult populations are expelled (vide infra). Another is that Merino sheep which have naturally acquired relatively high adult worm burdens may lose these progressively over a period of 3 or 4 months, despite the continued ingestion of infective larvae and the absence of anthelmintic treatment (Allonby and Urquhart, 1975). The possibility that the regular use of anthelmintics may affect the development of immunity to H. c o n t o r t u s infection has interested a number of parasitologists and veterinarians, particularly with the development of new and highly effective anthelmintics. Dineen and his colleagues (Dineen and Wagland, 1966; Wagland and Dineen, 1967; Donald et al., 1969) examined this subject in some detail in a series of three papers in which a series of infections of H. c o n t o r t u s were terminated with an anthelmintic and followed b y challenge. They found themselves unable to reconcile completely the results of these experiments, owing perhaps to the fact that in two of these experiments the sheep were still immunologically immature during the early months of experimental infection. Generally however, they concluded that strong resistance was associated with prolonged uninterrupted infection and that anthelmintic treatment was more likely to interfere with, than assist, the development of immunity. However, when the sensitising infection produced a persisting high worm burden of the order of 3,000 worms there was some evidence of 'immunological exhaustion', in that a very high proportion of the challenge infection developed to maturity. In contrast, when the heavy worm burdens were removed by anthelmintic 8 days before chal-

230 lenge, immunity was marked and only a small proportion of the challenge infection matured. The most recent and unexpected development in this sphere is the observation (Benitez-Usher et al., 1977) that Blackface lambs vaccinated with irradiated larvae on two occasions at 9 and 10 months of age failed to develop any immunity to subsequent challenge when thiabendazole was administered 3 weeks after each immunising dose; vaccinated b u t untreated controls were almost completely resistant. One could advance a number of explanations for this, but perhaps the most intriguing depends on the recent observations of Smith and Christie (1978) that lambs, reared worm-free until at least 7 months of age, which~are double-vaccinated with irradiated larvae and resist challenge, have high levels of both total IgA and H. contortus specific-IgA antibodies in their abomasal mucosa (Smith, 1977); in contrast, challenge control lambs or uninfected lambs have very low levels of mucosal IgA and IgA specific antibody. Since it is known that the production of IgA antibody is dependent on continuous stimulation b y antigen rather than on the presence of m e m o r y cells and their ability to evoke a secondary response (Mattioli and Tomasi, 1973). one wonders if IgA is important in the immune response to H. contortus. If it is, then the removal b y an anthelmintic of the source of antigenic stimulation, i.e. the few hundred irradiated sterile females which persist from the immunising infection, might cause the level of IgA antibody in the mucosa to fall quite rapidly. This point is currently being pursued. Relatively little is known of the role of IgA and protective responses to helminth infections, and, in view of the plethora of mechanisms already advanced as the active ingredients of the immune response to gastrointestinal helminths (IgG, IgE, mast cells, T cells, prostaglandins, macrophages and eosinophils), one is reluctant to espouse y e t another cause. Nevertheless, in rats infected with Nippostrongylus brasiliensis, the finding that mucosal specific IgA antibody in the small intestine is greatly increased around the point of worm expulsion has already prompted speculation as to its possible involvement (Poulain et al., 1976; Sinski and Holmes, 1977). More recently Lloyd and Soulsby (1978) have also shown that IgA from colostral or intestinal secretions will protect mice against infection with Taenia taeniaeformis. It is also interesting that a preliminary observation by Duncan et al. (1978) has shown that lambs 'vaccinated' with irradiated larvae while they are still immunologically unresponsive to H. contortus, and therefore susceptible to challenge, have little or no IgA. However, to end on a cautionary note, one should point out that Curtain and Anderson (1971) found little difference in the numbers of IgA producing cells in the abomasa of what they considered to be 'parasitised' (Ostertagia and Trichostrongylus spp.) and 'immune' sheep. So far I have dealt only with resistance'to infection as measured by parameters of worm burden and faecal egg production which develop from the challenge of immunised sheep with infective larvae. A second t y p e of resistance mechanism is classically associated with the ability of sheep to exhibit the

231

'self-cure' reaction, i.e. the expulsion of an existing adult H. contortus burden a few days after challenge with relatively large numbers of infective larvae (Stewart, 1953). Since the sheep usually offers no resistance to the developm e n t of the challenge infection to patency, one wonders if this ' i m m u n e ' reaction is in fact an evolved adaptation of benefit to both host and parasite, i.e. the host gets 2 or 3 weeks respite from the haematophagic activities of the adult worms but in return an old, perhaps sexually exhausted, population of adult worms is replaced by one composed of young vigorous males and females with all their reproductive life before them. A tentative conclusion that on balance the reaction favours the host is a finding, previously discussed, that haemoglobin A sheep, which are generally found to be more resistant to H. contortus infection, appear to have a heightened capacity to develop the self-cure reaction. There is now of course evidence (Allonby and Urquhart, 1973) that the self-cure reaction is n o t always an immunological reaction, but at least as a flock phenomenon is associated with the ingestion of freshly growing grass and occurs independently of the presence of H. contortus larvae. This aspect is outside the scope of this review, but the possibility of both reactions, i.e. the immunological and the dietary, utilising an ultimately c o m m o n pathway for adult worm expulsion, is an interesting one. In summary, it is unlikely in the light of available information that the immune reaction t o l l . contortus infection can be utilised or exploited in any practical fashion, unless perhaps as a primary criterion in the selection of sheep breeds in developing areas of the world. It is just possible that this conclusion could be drastically revised if one could induce immune responsiveness in lambs of 6--8 weeks of age. However, it must be borne in mind that the significance of haemonchosis is not always directly proportional to worm numbers. The finding that anaemia and severe loss of condition may occur in flocks, maintained on poor pasture, and with a very modest H. contortus worm burden (Allonby and Urquhart, 1975), imply that any m e t h o d of immune prophylaxis must be highly effective and n o t merely produce a relative, even although highly significant, degree of protection. Space does not permit a detailed review of recent progress in immunisation in other protozoal and helminth infections, but I would like to close by discussing some aspects of immunisation against three bovine parasites -- the lungworm Dictyocaulus viviparus, Babesia infection, and cysticercosis caused by C. bovis. BOVINE DICTYOCAULIASIS Vaccination using two doses of X-irradiated larvae against parasitic bronchitis ("Dictol", Allan & Hanburys Ltd., London) has now been part of the farming scene in m a n y countries in Europe and particularly the British Isles for the

232 past 18 years, and over this period millions of calves have successfully been protected against this disease. Originally the vaccine was primarily intended for use by dairy farmers where intensive methods of husbandry predisposed calves in their first year at grass to infection. Since most dairy calves are born indoors from late a u t u m n to early spring, and do not go to grass until they are 4--6 months old, it was recommended that vaccination should be delayed until the calves were 8 weeks old, since it was known from experience that calves were immunologically responsive to the vaccine at this age. It was also advised that calves should n o t go to grass until at least 2--4 weeks after the second dose of vaccine so as to allow time for an adequate degree of i m m u n i t y to develop before being exposed to challenge. In practice this has restricted to some extent the use of the vaccine, and calves suckling their dams at pasture have remained relatively unprotected. However, in a recent series of experiments (Benitez-Usher et al., 1976) it was shown that milk-fed or suckling calves could be vaccinated safely and successfully with Dictol starting at 3 weeks of age in that they acquired a degree of immunity to subsequent challenge comparable to older vaccinated calves, i.e. an 87--100% reduction in mean worm burden. This indicates that suckling beef calves may be vaccinated if necessary before the recommended age of 2 months and so acquire i m m u n i t y prior to the ingestion of lungworm infected herbage. Incidentally, Pirie et al. (1971) have shown that vaccinated calves retain a very high degree of i m m u n i t y for at least 3 m o n t h s after the completion of vaccination even in the absence of a challenge infection. The reduction in mean worm burden of such calves compared to controls was 87%, while that of calves challenged one m o n t h after vaccination was 92%. BOVINE BABESIOSIS

The worlwide veterinary importance of bovine babesiosis and the role of immune reactions, both naturally acquired and artificially stimulated, in the limitation and control of this disease are such as to justify an entire review. Fortunately for me, an excellent and comprehensive review has been prepared recently by Dr Callow {1977) of the tick fever research centre of the Department of Primary Industries in Brisbane, and will be read with great interest by all concerned with i m m u n i t y to protozoal diseases. As one who for years has asked luckless students in oral examinations to discourse on the difference between premunition and acquired immunity, with bovine babesiosis as the next item on m y mental agenda, it came as rather a surprise to find that the Australian workers consider that i m m u n i t y to babesiosis, at least to B. argentina and B. bigemina, does not depend on a carrier state and is essentially a post-infection immunity. Thus, in one experiment (Callow et al., 1974a) cattle given two artificial infections of B. argentina, 1 and 9 days apart, and then drug-sterilised with amidocarb {Imizol, Cooper & Nephews, Australia) 39 days later, were as resistant to challenge 193 days later

233 as infected but non-sterilised cattle. In another experiment, cattle drug-sterilised during or immediately after a primary infection exhibited a strong degree of immunity when challenged 190 days after chemotherapy. Even more convincing, in that the possibility of drug residues did not arise, is that a very substantial immunity to B. bigemina infection was obtained in cattle that had eliminated this parasite by their own immune response (Mahoney et al., 1973; Callow et al., 1974b). The fact that 'premunition' is not an essential condition for immune prophylaxis against bovine babesiosis perhaps improves the prospect for the development of dead vaccines. Currently these give satisfactory protection against challenge with the strain used for vaccination, but are not considered as good against different antigenic strains (Mahoney, 1971; Mahoney and Goodger, 1972; Todorovic, 1974). Recently, however, Mahoney and Wright (1976), using killed erythrocyte antigen mixed with Freund's complete adjuvant, have successfully immunised cattle against challenge with heterologous strains. The current Australian vaccine against B. argentina is produced, not from recovering or carrier cattle, but from acute infections produced in splenectomised donors. For economy the blood is collected by exchange transfusion rather than by exsanguination. It is particularly interesting that the rapid passage of the parasite by blood inoculation in splenectomised calves has fortuitously had two very desirable effects. First, the virulence of the infection in non-splenectomised calves has decreased to the extent that post-vaccination surveillance of cattle is frequently not performed. Second, the strain has lost its infectivity to ticks and thus infection cannot be spread from vaccinated to unvaccinated cattle. The parasite count of the blood determines the dilution of the latter which is dispensed in plastic bags, packed in ice and despatched in insulated containers. Each dose of vaccine contains about 10 million parasites. Most of the vaccine is used in cattle under 12 months of age living in conditions of enzootic instability. Often a second dose of vaccine, usually of a different antigenic strain, is given 6 months after the first. The degree of protection so induced is such that only 1% of vaccinated cattle subsequently develop clinical babesiosis due to field challenge, compared to 18% of unvaccinated cattle (Callow, 1976). The use of such a vaccine, with of without Anaplasma centrali, in other parts of the world, e.g. South America, The Middle East and Asia, could be of great value particularly when susceptible stock are being introduced to upgrade the local cattle. However, the success of this apparently simple technique of vaccination preparation rests on close attention to a number of complex critical considerations; the relevance of these and the preparation and use of vaccine outside Australia has been discussed by Callow (1974, 1976). In the meantime, it is encouraging that the vaccine prepared in Australia has been used successfully in South East Asia and South America in recent years.

234 BOVINE AND OVINE CYSTICERCOSIS All of the methods of inducing acquired immunity which have been discussed so far, have depended on the use of live organisms suitably attenuated by some means. Rickard and his colleagues have recently broken new ground in this respect in the field of cestode infections b y showing that a very high degree of immunity may be induced with the use of 'secretion' vaccines prepared by the in vitro cultivation of hatched and activated oncospheres of Cysticercus ovis and C. bovis. In the case of C. ovis it was found that incubation of hatched larvae for as short a period as 24--48 h in media enriched with foetal calf serum provided a highly immunogenic supernatant. When, after concentration dialysis, this material was given intramuscularly on one occasion to lambs of 12 weeks old with Freunds incomplete adjuvant, it provided complete protection against challenge with 2,000 C. ovis eggs 6 weeks later (Rickard and Adolphi, 1977); protection persisted for at least 12 months. Also, when administered to ewes 4 weeks before lambing it conferred a very high degree of transcolostral immunity to their lambs grazing on pasture heavily infected with eggs of C. ovis (Rickard et al., 1977a). A similar technique used to prepare a vaccine against C. bovis was equally successful when used in calves subjected to subsequent experimental challenge with 4,000 T. saginata eggs 4 weeks later (Rickard and Adolphi, 1976). Vaccination of pregnant dams also conferred almost solid protection to their calves when challenged 1--2 weeks after birth. This degree of passive protection did n o t interfere with subsequent active immunisation when the calves were 2 months old (Rickard et al., 1977b). There are two points which I think are particularly interesting a b o u t these results. The first is the very high degree of passive immunity conferred on calves b y the ingestion of colostrum from vaccinated dams, a fact confirmed by Lloyd and Soulsby (1976) who obtained colostral immunoglobulins by the intra-mammary injection of activated onchospheres. In contrast, most calves born of immune dams in endemic areas in East Africa were shown to have become naturally infected with C. bovis within a very few weeks of post-natal life (Urquhart, 1961). Certainly these were usually light infestations, and perhaps under conditions of natural infection the amount of protective antibody in the colostrum is insufficient to confer solid resistance and allows the development of a proportion of the challenge infection. The second point is the very high, indeed almost complete, degree of protection stimulated b y a single injection of these t w o adjuvanted 'secretion vaccines'. Based on these results, it would certainly appear that vaccination against C. ovis could be developed successfully. Unfortunately, a C. bovis vaccine depending on the availability of eggs of T. saginata, whose only final host is man, does n o t seem so imminent. However, in view of the real advantages which such a vaccine would have in beef farms in many situations, e.g. ranch-

235 es in developing countries, farms which utilise sewage or in which a C. bovis problem of embarrassing proportion has suddenly arisen, it is important that these results be confirmed in other laboratories and long-term field experiments p u t under way. If these are successful and if subsequently T. sagi~ata could be adapted to another host or to an in vitro existence, one could conceivably envisage the eradication of this parasite by vaccination. CONCLUSION It is difficult to make many generalisations about the development of vaccines against parasitic diseases except that it is likely to be fraught with difficulties. Some of these are relatively c o m m o n to most systems, e.g. the problem in helminth infections of obtaining sufficient quantities of antigen, and the fact that domestic animals are often exposed to infection in early post-natal life before vaccination could have stimulated protection. Other problems are specific to particular host--parasite relationship and in the examples reviewed here include, in trypanosomiasis, the antigenic lability of the parasite and, in haemonchosis, the prolonged period of immunological immaturity of the ovine host. Apart from the protective potential of a vaccine, price and customer acceptability are essential. Probably the most salutary example of this is the canine h o o k w o r m vaccine (see review by Miller, 1978} withdrawn from the American market after a period of only 3 years. The complex reasons why this product, of undisputed efficacy, failed commercially are discussed in detail by Miller (1978). To date, the only vaccines in large-scale production are those against D. viviparus, the bovine lungworm, and Babesia argentina and B. bigemina in cattle. Both are attenuated live vaccines with quite stringent methods of distribution because of their limited shelf life. Perhaps part of their success is due to the fact that by the time the clinical disease is recognised and treatment initiated, the hosts have usually suffered a considerable check and may even be very ill. If this is the case it seems not unreasonable to hope, in view of the progress already achieved, that a vaccine for East Coast fever ma.v become available in the fairly near future. For trypanosomiasis and haemonchosis the immediate o u t l o o k is less promising. With the former perhaps the best one can strive towards is a better appreciation of the role of chemotherapy in permitting the development of non-sterile immunity. In the latter, apart from the potential role of H. contortus-resistant breeds, there seems little option at present b u t to rely on prophylactic chemotherapy.

REFERENCES

Allonby, E.W. and Urquhart, G.M., 1973. Self-cure of Haemonchus contortus under field conditions. Parasitology, 66: 43--53.

236 Allonby, E.W. and Urquhart, G.M., 1975. The epidemiology and pathogenic significance of haemonchosis in a Merino flock in East Africa. Vet. Parasitol., 1: 129--143. Allonby, E.W. and Urquhart, G.M., 1976. A possible relationship between haemonchosis and haemoglobin polymorphism in Merino sheep in Kenya. Res. Vet. Sci., 20: 212--214. Altair, K.I. and Dargie, J.D., 1976. Genetic resistance of sheep to H a e m o n c h u s c o n t o r t u s . In: Nuclear Techniques in Animal Production and Health. I.A.E.A., Vienna, pp. 449-462. Anosa, V.O., 1975. The Effect of Splenectomy on the Anaemia and Parasitaemia of trypanosomiasis. M.V.M. Thesis, University of Glasgow, 3 pp. Barnett, S.F., 1956. East Aft. Vet. Res. Organ. Annu. Rep., 1 9 5 5 - - 1 9 5 6 . 0 0 pp. Barnett, S.F., 1957. East Aft. Vet. Res. Organ. Annu. Rep., 1956--1957.00 pp. Benitez-Usher, C., Armour, J. and Urquhart, G.M., 1976. Studies on immunisation of suckling calves with Dictol. Vet. Parasitol., 2: 209--222. Benitez-Usher, C., Armour, J., Duncan, J.L., Urquhart, G.M. and Gettinby, G., 1977. A study of some factors influencing the immunisation of sheep against H a e m o n c h u s cont o r t u s using attenuated larvae. Vet. Parasitol., 3: 327--342. Bevan, L.E.W., 1928. A method of inoculating cattle against trypanosomiasis. Trans. R. Soc. Trop. Med. Hyg., 22: 147--156. Brocklesby, D.W. and Bailey, K.P., 1965. The immunisation of cattle against East Coast fever (Theileria parva infection)using tetracyclines. Bull. Epizoot. Dis. Afr., 13: 161-168. Brocklesby, D.W., Bailey, K.P., Jarrett, W.F.H., Martin, W.B., Miller, H.R.P., Hdenito, P. and Urquhart, G.M., 1965. Experiments in immunity to East Coast fever. Vet. Rec., 77 : 512. Callow, L.L., 1974. Epizootiology, diagnosis and control of babesiosis and anaplasmosis. Anita. Quar., 3: 6--12. Callow, L.L., 1976. Australian methods of vaccination against anaplasmosis and babesiosis. In: Tick-borne Livestock Diseases and their Vectors. World Anim. Rev., 18: 9--15. Callow, L.L., 1977. Vaccination against bovine babesiosis. In: L.H. Miller, J.A. Pino and McKelvey (Editors), Immunity to Blood Parasites of Animals and Man. Adv. Exp. Med. Biol. Plenum Press, New York, N.Y., pp. 121--149. Callow, L.L., McGregor, W., Parker, R.J. and Dalgliesh, R.J., 1974a. The immunity of cattle to Babesia argentina after drug sterilisation of infections of varying duration. Anst. Vet. J., 50: 6--11. Callow, L.L., McGregor, W., Parker, R.J. and Dalgliesh, R.J., 1974b. Immunity of cattle to Babesia bigemina following its elimination from the host, with observations on antibody levels detected by the indirect fluorescent antibody test. Aust. Vet. J., 50: 12--15. Clarkson, J.J., 1976. Trypanosomes. Vet. Parasitol., 2: 9--29. Cross, G.A.M., 1975. Identification, purification and properties of clone-specific glycoprotein antigens constituting the surface coat of T r y p a n o s o m a brucei. Parasitology, 71: 393--417. Cunningham, M.P., 1966. Immunity in bovine trypanosomiasis. E. Afr. Med. J., 43: 394-397. Cunningham, M.P., Brown, C.G.D., Burridge, M.J., Jayner, L.P. and Purnell, R.E., 1973a. East Coast fever: the infectivity for cattle of infective particles of Theileria parva harvested in various substrates. Int. J. Parasitol., 3: 335--338. Cunningham, M.P., Brown, C.G.D., Burridge, J.J. and Purnell, R.E., 1973b. Cryopreservation of infective particles of Theileria parva. Int. J. Parasitol., 3: 583--587. Cunningham, M.P., Brown, C.G.D., Burridge, J.J., Musoke, A.J., Purnell, R.E., Radley, D.E. and Sernpebwa, C., 1974a. East Coast fever: titration in cattle of suspensions of Theileria parva derived from ticks. Br. Vet. J., 130: 336--345. Cunningham, M.P., Brown, C.G.D., Burridge, M.J., Irvin, A.D., Kirimi, I.M., Purnell, R.E., Radley, D.E. and Wagner, G.G., 1974b. Theileriosis: the exposure of immunised cattle in a Theileria lawrenci enzootic area. Trop. Anim. Health Prod. 6: 39--43. Curtain, C.C. and Anderson, N., 1971. Immunocytochemicai localisation of the ovine immunoglobulins IgA, IgG 1 , IgG1a and IgG 2 : effect of gastro-intestinal parasitism in the sheep. Clin. Exp. Immunol., 8: 151--162.

237 Cvetkovic, L.J., Lepojev, Olga and Vulic., 1973. Ispitivanje rasne otpornostic cigaje merino prekosa i merino kavkaz prema zeludacno-crevnim strongilidama u prirodnim uslovima infekcije. Vet. Glas., 27: 867--872. Dineen, J.K. and Wagland, B.M., 1966. The dynamics of the host--parasite relationship. V. Evidence for immunological exhaustion in sheep experimentally infected with Haemonchus contortus. Parasitology, 56: 665--677. Donald, A.D., Dineen, J.K. and Adams, D.B., 1969. The dynamics of the host--parasite relationship. VII. The effect of discontinuity of infection on resistance to Haemonchus contortus in sheep. Parasitology, 59: 497--503. Duncan, J.L., Smith, W.D. and Dargie, J.D., 1978. Possible relationship of levels of mucosal IgA to immune unresponsiveness of lambs to Haemonchus contortus. Vet. Parasitol., 4: 21--27. Evans, J.V. and Blunt, M.H., 1961. Variation in the gene frequencies of potassium and haemoglobin types in Romney Marsh and Southdown sheep established away from their native environment. Aust. J. Biol. Sci., 14: 100--108. Evans, J.V. and Whitlock, J.H., 1964. Genetic relationship between maximum haematocrit values and haemoglobin type in sheep. Science, 145: 1318. Evans, J.V., Blunt, M.H. and Southcott, W.H., 1963. The effects of infection with Haemonchus contortus on the sodium and potassium concentrations in the erythrocytes and plasma, in sheep of different haemonglobin types. Aust. J. Agric. Res., 14: 549--558. Fiennes, R.N., 1970. The pathogenesis and pathology of African trypanosomiases. In: H.W. Mulligan (Editor), The African Trypanosomiases. Allen & Unwin, London, p. 749. Goodwin, L.G., 1970. The pathology of African trypanosomiasis. Trans. R. Soc. Trop. Med. Hyg., 64: 797--812. Gordon, H. McL., 1948. The epidemiology of parasitic disease with special reference to studies with nematode parasites of sheep. Aus. Vet. J., 24: 17--45. Gordon, H. McL., 1950. Some aspects of parasitic gastro-enteritis of sheep. Aus. Vet. J., 26: 14--28, 46--52, 65--72, 93--98. Gray, A.R., 1967. Some principles of the immunology of trypanosomiasis. Bull. W. H. O., 37: 177--193. Hirume, H., Doyle, J.J. and Hirume, K., 1977. Cultivation of bloodstream Trypanosoma brucei. Bull. W.H.O., 55: 405--409. Irvin, A.D., Stagg, D.A., Kanhai, G.K. and Brown, C.G.D., 1975. Heterotransplantation of Theileria parva-infected cells to athymic (nude) mice. Nature, London, 253: 549--550. Jarrett, E.E.E., 1971. Diminished immunological responsiveness to helminth parasites. The effect of repeated reinfection of rats from an early age with Nippostrongylus brasiliensis. Clin. Exp. Immunol., 8: 141--150. Jarrett, W.F.H., Crighton, G.W. and Pirie, H.M., 1969a. Theileria parva: kinetics of replication. Exp. Parasitol., 24: 9--25. Jarrett, W.F.H., Pirie, H.M. and Sharp, N.C.C., 1969b. Immunisation against East Coast fever using tick infection and chlortetracycline. Exp. Parasitol., 24: 147--151. Jenni, L., 1977. Comparisons of antigenic types of Trypanosoma (T) brucei strains transmitted by Glossina m. morsitans. Acta Tropica, 34: 35--41. Jilek, A.F. and Bradley, R.E., 1969. Haemoglobin types and resistance to Haemonchus contortus in sheep. Am. J. Vet. Res., 30: 1773--1778. Le Ray, D., Barry, J.D. and Vickerman, K., 1978. Antigenic heterogeneity of metacyclic forms of Trypanosoma brucei. Nature, 273: 300--302. Lloyd, S. and Soulshy, 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 Soulshy, E.J.L., 1978. The role of IgA immunoglobulins in the passive transfer of protection to Taenia taeniaeformic in the mouse. Immunology, 34: 939--945. Lopez, V. and Urquhart, G.M., 1967. The immune response to Merino sheep to Haemonchus controtus infection. In: E.J.L. Souisby (Editor), The Reaction of the Host to Parasitism. Proc. W.A.A.V.P. 3rd Int. Congr., Lyon 1966. Vet. Med. Rev., pp. 153--159. Mahoney, D.F., 1971. Immunisation against Babesia argentina (Liznieres 1903). In: E.J.L. Soulsby (Editor-in-Chief), Proc. 18th World Vet. Congr., 1: 351--356.

238 Mahoney, D.F. and Goodger, B.V., 1972. Babesia argentina: immunogenicity of plasma from infected animals. Exp. Parasitol., 32: 71--85. Mahoney, D.F. and Wright, I.G., 1976. Babesia argentina: immunization of cattle with a killed antigen against infection with a heterologous strain. Vet. Parasitol, 2: 273--282. Mahoney, D.F., Wright, I.G. and Mitre, G.B., 1973. Bovine babesiosis: the persistence of immunity to Babesia argentina and B. bigemina infections in calves (Bos taurus) after naturally acquired infection. Ann. Trop. Med. Parasitol., 67: 197--203. Malmquist, W.A., Nyindo, M.B.A. and Brown, C.G.D., 1970. East Coast fever: cultivation in vitro of bovine spleen cell lines infected and transformed by Theileria parva. Trop. Anim. Health Prod., 2, 139--145. Manton, V.J.A., Peacock, R., Poynter, D., Silverman, P.H. and Terry, R.J., 1962. The influence of age on naturally acquired resistance to Haemonchus contortus in lambs. Res. Vet. Sci., 3: 308--314. Mattioli, C°A. and Tomasi, T.B., 1973. The life-span of IgA plasma cells from the mouse intestine. J. Exp. Med., 138: 452. Miller, T.A., 1978. Industrial development and field use of the canine h o o k w o r m vaccine. Adv. Parasitol., 16: 333--359. Murray, M. and Urquhart, G.M., 1977. Immunoprophylaxis against African trypanosomiasis. In: L.H. Miller, J.A. Pino and McKelvey (Editors), Immunity to Blood Parasites of Animals and Man. Adv. Exp. Med. Biol. Plenum Press, New York, N.Y., pp. 209--241. Murray, P.K., Murray, M., Morrison, W.I. and McIntyre, W.I.M., 1978. Trypanosomiasis in N'dama and Zebu cattle. Proc. 15th Conf. Int. Sci. Counc. Tryp. Res. Control. The Gambia 1977, in press. Neitz, W.O., 1953. Aureomycin in Theileria parva infection. Nature, London, 171: 34--35. Pirie, H.M., Jarrett, W.F.H. and Crighton, G.W., 1970. Studies on vaccination against East Coast fever using macroschizonts. Exp. Parasitol., 27 : 343--349. Pirie, H.M., Doyle, J.J., McIntyre, W.I.M. and Armour, J., 1971. The relationship between pulmonary lymphoid nodules and vaccination against Dictyocaulus viviparus. In: S.M. Gafaar (Editor), Pathology of Parasitic Diseases. Purdue Univ. Press, Lafayette, Ind. pp. 91--98. Poulain, J., Luffau, D. and Pery, P., 1976. Nippostrongylus brasiliensis in the rat: immune response in serum and intestinal secretions. Ann. Immunol. (Inst. Pasteur), 127 C: 215-224. Preston, J.M. and Allonby, E.W., 1979. The susceptibility of different breeds of sheep to Haemonchus contortus infection in East Africa. Res. Vet. Sci., in press. Purnell, R.E., 1977. East Coast fever: some recent research in East Africa. In: B. Dawes (Editor), Advances in Parasitology. Academic Press, London, 15: 83--132. Purnell, R.E. and Joyner, L.P., 1967. Artificial feeding technique for Rhipicephalus appendiculatus and the transmission of Theileria parva from the salivary secretion. Nature, London, 216: 484--485. Radhakrishnan, C.V., Bradley, R.E. and Loggins, P.E., 1972. Host responses of worm-free Florida Native and Rambouillet lambs experimentally infected with Haemonchus contortus. Am. J. Vet. Res., 33: 817--823. Radley, D.E., Brown, C.G.D., Burridge, M.J., Cunningham, M.P., Pierce, M.A. and Purnell, R.E., 1974. East Coast fever: quantitative studies of Theileria parva in cattle. Exp. Parasitol., 36: 278--287. Radley, D.E., Brown, C.G.D., Burridge, M.J., Cunningham, M.P., Kirimi, I.M., Purnell, R.E. and Young, A.S., 1975a. East Coast fever. I. Ghemoprophylactic immunisation of cattle against Theileria parva (Muguga) and five theileria strains. Vet. Parasitol., 1: 35--41. Radley, D.E., Brwon, C.G.D., Cunningham, M.P., Kimber, C.D., Musisi, F.L., Payne, R.C., Purnell, R.E., Stagg, S.M. and Young, A.S., 1975b. East Coast fever: 3. chemoprophylactic immunisation of cattle using oxytetracycline and a combination of theilerial strains. Vet. Parasitol., 1: 51---60. Rickard, M.D. and Adolphi, A.J., 1976. Vaccination of calves against Taenia saginata using a "parasite-free" vaccine. Vet. Parasitol., 1 : 389--392.

239 Rickard, M.D. and Adolphi, A.J., 1977. Vaccination of lambs against infection with Taenia ovis using antigen collected during short term in vitro incubation of activated T. ovis onchospheres. Parasitology, 75: 183--188. Rickard, M.D., Boddington, E.G. and McQuade, N., 1977a. Vaccination of lambs against T. ovis infection using antigens collected during in vitro cultivation of larvae. Passive protection via colostrum from vaccinated ewes and the duration of immunity from a single vaccination. Res. Vet. Sci., 23: 368--371. Rickard, M.D., Adolphi, A.J. and Arundel, J.H., 1977b. Vaccination of calves against Taenia saginata infection using antigens collected during in vitro cultivation of larvae. Passive protection via colostrum from vaccinated cows and vaccination of calves protected by maternal antibody. Res. Vet. Sci., 23: 365--367. Sinski, E. and Holmes, P.H., 1977. Nippostrongylus brasiliensis: Systemic and local IgA and IgG immunoglobulin responses in parasitized rats. Exp. Parasitol., 43: 382--389. Smith, W.D., 1977. Anti-larval antibodies in the serum and abomasal mucosa of sheep hyper-infected with Haemonchus contortus. Res. Vet. Sci., 22: 334--338. Smith, W.D. and Christie, 1978. Haemonchus contortus: local and serum antibodies in sheep immunised with irradiated larvae. Int. J. Parasitol., 8: 219--223. Spreull, J., 1914. East Coast fever inoculation in the Trankeian territories, Sout Africa. J. Comp. Pathol. Ther., 27 : 229--304. Stewart, D.F., 1953. Studies on resistance of sheep to infestation with Haemonchus contortus and Trichostrongylus species and on the immunological reactions of sheep exposed to infestation. V. The nature of the self-cure phenomenon. Austr. J. Agric. Res., 4: 100-117. Terry, R.J., 1976. Immunity to African trypanosomiasis. In: S. Cohen and E.H. Sadun (Editors), Immunology of Parasitic Infections. Blackwell Scientific Publications, Oxford, pp. 203--221. Todorovic, R.A., 1974. Bovine babesiosis: its diagnosis and control Am. J. Vet. Res., 35: 1045--1052. Urquhart, G.M., 1961. Epizootiological and experimental studies on bovine cysticercosis in East Africa. J. Parasitol., 47 : 857--869. Urquhart, G.M., Jarrett, W.F.H. and Mulligan, W., 1962. Helminth immunity. Adv. Vet. Sci., 7: 87--129. Urquhart, G.M., Jarrett, W.F.H., Jennings, F.W., McIntyre, W.I.M. and Mulligan, W., 1966. Immunity to Haemonchus contortus infection: relationship between age and successful vaccination with irradiated larvae. Am. J. Vet. Res., 27 : 1645--1648. Vickerman, K., 1969. On the surface coat and flagellar adhesion in trypanosomes. J. Cell Sci., 5: 163--193. Wagland, M.B. and Dineen, J.K., 1967. The dynamics of the host--parasite relationship. VI. Regeneration of the immune response in sheep infected with Haemonchus contortus. Parasitology, 57 : 59--65. Wilde, J.K.H., 1967. East Coast fever. Adv. Vet. Sci., 11: 207--259. Wilson, A.J., 1971. Immunological aspects of bovine trypanosomiasis. III. Patterns in the development of immunity. Trop. Anita. Health Prod., 3 : 14--22. Wilson, A.J., Paris, J. and Dar, F.K., 1975a. Maintenance of a herd of breeding cattle in an area of high trypanosome challenge. Trop. Anim. Health Prod., 7: 63--71. Wilson, A.J., LeRoux, J.G., Paris, J., Davidson, C.R. and Gray, A.R., 1975b. Observations on a herd of beef cattle maintained in a tsetse area. I. Assessment of chemotherapy as a method f or the control of trypanosomiasis. Trop. Anita. Health Prod., 7: 187--199. Wilson, A.J., Paris, J., Luckins, A.G., Dar, F.K. and Gray, A.R., 1976. Observations on a herd of beef cattle maintained in a tsetse area. II. Assessment of the development of immunity in association with trypanocidal drug treatment. Trop. Anita. Health Prod., 8: 1--12. Yeoman, G.H., 1966. Field vector studies of epizootic East Coast Fever. I. A quantitative relationship between R. appendiculatus and the epizooticity of East Coast fever. Bull. Epizoot. Dis. Afr., 14: 5--27.