Eighteenth seminar on trypanosomiasis

109 ROYAL SOCIETY OF TROPICAL

EIGHTEENTH

MEDICINE

AND HYGIENE

SElMINAR ON TRYPANOSQMIASIS

ORGANIZED BY THE TRYPANOSOMIASIS ADVISORY PANEL OF THE MINISTRY OF OVERSEASDEVELOPMENT HELD AT THE LONDON SCHOOLOF HYGIENE AND TROPICAL MEDICINE, KEPPEL STREET,

LONDONWClE 7HT, ON 22 AND23 SEPTEMBER, 1977 Dr. L. G. Goodwin, c.M.G., P.R.C.P.,F.R.s.,in the Chair

On the first day, the meeting was organized in six Discussion Groups dealing, respectively, with Protozoology, Entomology, Biochemistry, Immunology, Pathology and Epidemiology. Two Chairmen presided over each Group and an Opening Statement was presented by an invited speaker. Laboratory Demonstrations of recent research work on trypanosomiasis were presented on the second day. A list of the 56 exhibits is appended. Summary

of the ~dharir-rxsn’s

improved measures for the control of human and animal trypanosomiasis. Therefore, the Panel decided to organize this Eighteenth Seminar in the form of Discussion Groups, covering six different disciplines, in the expectation that lines of research, calculated to yield results of practical benefit to the poorest communities, would emerge.

Introductory

Dr. Goodwin explained that the present policy of the Ministry of Overseas Development is to place increasing emphasis on help for the poorest inhabitants of underdeveloped countries. By supporting research on trypanosomiasis for many years, the Government has, however, anticipated present policy because it is among the poor rural communities of Africa that sleeping sickness takes its greatest toll, at present estimated at 10,000 cases annually. There remains a constant threat of the recurrence of devastating epidemics such as have occurred in the past, most recently in the Congo basin where the number of cases was estimated at up to one million. Of even greater importance to rural communities living in tsetse-infected areas is the difficulty or impossibility of developing and maintaining an economic livestock industry. Dr. Goodwin reviewed the history of trypanosomiasis and the ways in which the British Government has supported research on trypanosomiasis in man and his homestic animals. After the Second World ..--_- War., the Colonial office aouointed the Tsetse-Fly and Trypanosomiasis Committee to advise on research projects and much was accomplished, including the establishment of major research institutes in East and West Africa. In 1960, this Committee was replaced by the Trypanosomiasis Advisory Panel of the Ministry of Overseas Development and, on the Panel’s advice, numerous research projects have been generously supported, both in the United Kingdom and overseas. In consequence, Britain has achieved a position in the forefront of trypanosomiasis research. Dr. Goodwin cited numerous examples of how fundamental _research __-~.~.~ in various disciplines had vielded results of great practical importance in the field. Recently, the Panel has paid special attention to the encouragement and support of research projects which appear to offer the best prospects of evolving new and

I. DISCUSSION GROUP ON PROTOZOOLOGY Chainjzen-Professor K. Vickerman and Professor W. H. R. Lumsden Opening

Statement

by Dr

J. R. Baker

This brief introduction is intended to outline aspects of “protozoological” research on trypanosomes which may lead to the improvement of control measures. Since most of the obviously relevant lines of research come within the purview of subsequent discussion groups at this Seminar, I have tried to think of topics related to the parasites’ basic biology which, even if not immediately applicable to the control of the organism, seem to hold some promise of contributing to the development of control measures within the foreseeable future. 1. Sex in trypamsonte

life-cycles

This topic has been raised with monotonous regularity in the past, each alleged revelation of its occurrence being followed by a convincing rebuttal. However, the World Health Organization’s Special Programme document on trypanosomiasis (WHO, 1976) suggested that ‘
110

SEMINAR ON TRYPANOSOMIASIS

mastigotes in GZossina (MSHELBWALA, 1972) following their apparently direct traverse of the peritrophic membrane and midgut epithelium (FREE&, 1973; EVANS & ELLIS. 1975). It seems unlikelv that anv conventional sexual i)rocess would have escaped observation for so long, but perhaps genetic exchange occurs in a hitherto unrecognized way. Some indirect evidence for its occurrence was provided by CULWICK et al. (1951), based on retention or loss of infectivity to man, and on morphological changes, resulting from the experimental mixing of stocks of T. brucei subspp. in r&o, but has not been generally accepted. Perhaps the question should be reinvestigated using more sophisticated genetical techniques, possibly involving drug resistance or enzymic electrophoretic mobility patterns (GODFREY, 1976; GODFREY & 1976) as markers and haematozoic KILGOUR, trypomastigotes (HIRUMI et al., 1977) or vector stages (CUNNINGHAM & HONIGBERG, 1977) cultivated in vitro. Sexual fusion of amastigotes in the vector, with associated genetic exchange, has been reported for T. cruzi (BRENER, 1972) but questioned by BAKER & PRICE (1973); perhaps this too should be reinvestigated. 2. Origin of stocks of Trypanosoma brucei infective to human beings Does T. brucei rhodesiense (or the “rhodesiense nosodeme” of T. b. brucei) arise by mutation (perhaps at a single locus) from T. b. brucei? Now that haematozoic trypomastigotes (of one stock at least) can be continuously cultivated in vitro (HIRUMI et al., 1977) and their infectivity to man assayed with some certainty by the blood incubation infectivity test (BIIT) devized by RICKMAN & ROBSON (1970), this question could perhaps be investigated in vitro by the application of mutagens to trypanosome populations known to be noninfective to man and their subsequent testing by the BIIT for acquired resistance to human serum. If this acquisition could be induced, the significance of its natural occurrence would have to be assessed in relation to eradication programmes and potential reservoir hosts. Such a mutant origin of a humaninfective stock might explain the appearance of virulent human trypanosomiasis apparently de no210 in Ethiopia in 1967 (BAKER et al., 1970; see also RICKMAN, 1977). Virulence, has been related to variant antigenic type (VAT) by MCNEILLAGE & HERBERT (1968) and VAN NEIRVENNE et al. (1975); RICKMAN (1977) reported development of human serum resistance by some clones of an originally sensitive stock of T. brucei SUtWiD.. and its loss bv some clones of a presumably originally resistant stock, during the isolation and maintenance of different VATS. Sequential BIITs on successive VATS produced by one clone in vivo (or, if the experimental induction of antigenic variation should become possible, in vitro) might indicate the stability or lability of the character of resistance to human serum and its correlation or otherwise with particular VATS. It seems unlikely that there should be a direct association between VAT and infectivity to man, as suggested by VAN MEIRVENNE et al. (1976), since, if there were, all successive VATS produced after the acquisition of human

infectivity would presumably also have to possess the character of serum resistance as the ability of a given stock to infect human beings can, sometimes at least, be retained for many years (ASHCROFT, 1959). It seems more probable that serum resistance and virulence are controlled by genes which may mutate during the laboratory maintenance of stocks (or under natural “field” conditions) independently of their changing VATS. 3. Pleomorphism and antigenic variation of Trypanosoma cruzi The significance of the well-attested pleomorphism of haematozoic trypomastigotes of T. cruzi has not been conclusively determined. Is it analogous to the apparently similar phenomenon exhibited by T. brucei, as suggested by BRENER & ALVARENGA (1976)? There is as yet no evidence of comparable respiratory changes. Solving this problem might possibly indicate ways of interrupting transmission by suppressing - the development of the putative vector-infective “broad” forms-though the nlentiful existence of non-human reservoir hosts in many endemic areas would tend to vitiate control by any Is the pleomorphism related to such means. antigenic variation? There is little direct evidence that this process occurs at all, though the long persistence-or repeated recrudescences-of parasitaemia. couuled with the report by DZBE~~SKI (1974) of the apparent non-ihentityof soluble antigens serially isolated from infected murine blood, suggest the occurrence of some kind of antigenic change. This might be associated with the intracellular reproductive phase. Prolonged observation of chronically infected rodents and perhaps study of the parasite in mammalian cell cultures might shed some light on this problem. In conclusion, I hope that this brief essay will be accepted as it was intended to be-speculative rather rather than didactic, thought-provoking than prophetic. References Ashcroft, M. T. (1959). The Tinde experiment: a further study of the long-term cyclical transmission of Trypanosoma rhodesiense. Annals of Tropical Medicine and Parasitology, 53, 137-146. Baker, J. R., McConnell, E., Kent, D. C. & Hady, J. (1970). Human trypanosomiasis in Ethiopia: ecology of Illubabor province and epidemiology in the Baro region [sic] area. Transactions of the Royal Society of Tropical Medicine and Hygiene, 64; 523-530: Baker, J. R. & Price, J. (1973). Growth in vitro of Trypanosoma cruzz as amastlgotes at temperatures below 37°C. ZnternationaZJournaZfor Parasitology, 3, 549-551. Brener, Z. (1972). A new aspect of Trypanosoma cruzi in the invertebrate host. Journal _...._. life-cvcle --~ of Protozoology, 19, 23-27. Brener, Z. & Alvarenga, N. J. (1976). Life cycle of T. cruzi in the vector. In: New Approaches in American Trypanosomiasis Research, Washington: Ban American Health Organization (Scientific Publication 318), pp. 83-86. Culwick, A. T., Fairbairn, H. &. Culwick, R. E. (1951). The genetic relationship of the poly-

TRANSACTIONS OF THER OYALSOCIETYOFTROPICALMEDICINE morphic trypanosomes and its practical implications. Annals of Tropical Medicine and Parasitology, 45, 11-29. Cunningham, I. & Honigberg, B. M. (1977). Infectivity reacquisition by Trypanosoma brucei brucei cultivated with tsetse salivary glands. Science, New York, 197, 1279-1282. Dzbenski, T. H. (1974). Exoantigens of Trypanosoma cruzi in vivo. Tropenmedizin und Parasitologic 25, 485-491.

Evans, D. A. & Ellis, D. (1975). Penetration of midgut cells of Glossina morsitans morsitans by Trvaanosoma brucei rhodesiense. Nature. I 258.I 23il233. Freeman, J. C. (1973). The penetration of the peritrophic membrane of the tsetse flies by trypanosomes. Acta Tropica, 30, 347-355. Godfrey, D. G. (1976). Biochemical strain characterization of trypanosomes. In: New Approaches in American Trypanosomiasis Research. Washington : Pan American Health Organization (Scientific Publication 318), pp. 91-97. Godfrey, D. G. & Kilgour, V. (1976). Enzyme electrophoresis in characterizing the causative organism of Gambian trypanosomiasis. Transactions of the Royal Society of Tropical Medicine and Hygiene, 70, 219-224.

Hirumi, H., Doyle, J. J. & Hirumi, K. (1977). African trypanosomes : cultivation of animalinfective Trypanosoma brucei in vitro. Science, New York, 196, 992-994.

McNeillage, Infectivity

G. J. C. & Herbert, W. J. (1968). and virulence of Trypanosoma (Trypanozoon) brucei for mice. II. Comparison of closely related trypanosome antigenic types.

Journal of Comparative Pathology and Therapeutics, 78, 345-349. Mshelbwala, A. S. (1972). Trypanosoma brucei infection in the haemocoel of tsetse flies. Transactions of the Royal Society of Tropical Medicine and Hygiene, 66, 637-643.

Ormerod, W. E. & Venkatesan, S. (1971). An amastigote phase of the sleeping sickness trypanosome. Transactions of the Royal Society of Tropical Medicine and Hygiene, 65, 736-741. Rickman, L. R. (1977). Variation in the test response of clone-derived Trypanosoma (Trypanozoon) brucei brucei and T. (T.) b. rhodesiense relapse antigenic variants, examined by a modified blood incubation infectivity test and its possible significance in Rhodesian sleeping sickness transmission. Medical Journal of Zambia, 11, 31-37. Rickman, L. R. & Robson, J. (1970). The testing of proven Trypanosoma brucei and T. rhodesiense strains by the blood incubation infectivity test. Bulletin

of the World

Health

Organization,

42,

911-916. Van Meirvenne, N., Janssens, P. G., Magnus, E., Lumsden, W. H. R. & Herbert, W. J. (1975). Antigenic variation in syringe passaged populations of Trypanosoma (Trypanozoon) brucei. II. Comparative studies on two antigenic-type collections. Annales de la Socie’te’belge de Medecine Tropicale, 55,25-30.

Van Meirvenne, N., Magnus, E. & Janssens, P. C. (1976). The effect of normal human serum on

AND HYGIENE,VOL 72, No. 2, 1978 111

trypanosomes of distinct antigenic type (ETat 1 to 12) isolated from a strain of Trypanosoma brucei rhodesiense. Annales de la Socie’te beige de Medecine tropicale, 56, 55-63. WHO (1976). Trypanosomiasis. Special Programme for Research and Training in Tropical Diseases, 1,

number 76.12, annex 2, p. 5. Geneva: World Health Organization. Summary

of Discussion

The possible occurrence of sexual processes or other means of producing gene recombination in trypanosomes was discussed. Morphological evidence of syngamy was deemed insufficient proof of such processes; experimental demonstration of genetic recombination was considered essential. So far, the experimental approach has been limited to a few attempts to show transfer of resistance to particular drugs. Genetic markers of more certain stability are needed if unequivocal demonstration of recombination is to be achieved. Isoenzyme markers are now available for this purpose. The selective advantage of having a sexual process in the life-cycle of organisms which divide rapidly by binary fission was debated. No strong argument was forthcoming for the necessity of sex in trypanosomes. Cursory dismissal of sexuality was, however, considered unwise: mechanisms of genetic exchange in bacteria have only recently been recognized and studied and, taxonomically closer, the dinoflagellates have, within the last few years, proved to have sexual processes hitherto unrecognized; dinoflagellate genetics has now become an established study. Considerable uncertainty also exists with regard to the so called “cryptic” forms of T. brucei. If multiplicative stages other than the haematozoic forms play an essential part in maintaining infection of the mammalian host, then they are obviously worthy of intensive study since plans to interrupt transmission or to improve the activity of drugs depend upon an accurate knowledge of developmental cycles. Extravascular forms require specific study to establish whether amastigote, sphaeromastigote or multinucleate forms have real significance in the life-histories of the African trypanosomes or whether they are merely aberrations of development produced by the host’s immune response or by drug treatment. The genetic control of antigenic variation and human serum sensitivity was considered worthy of further research in relation to both the design of protective vaccines and the origin of epidemics of East African sleeping sickness. Recent work showing inter-relationships between the VAT of a given trypanosome population and the resistance of that population to human serum, has upset accepted ideas on the origin of epidemics. Culture of blood stages of T. brucei offers new opportunities for the study of change of VAT and infectivity to man as assessedby BIIT. Little further progress in this field can be expected until the mechanism of the effect of human serum on the trypanosome is better understood. Finally, several theoretical approaches to control of African trypanosome infections based on advancing knowledge of trypanosome cell biology were

112

SEMINAR ON TRYPANOSOMIASIS

mentioned. These included: the design of means for “capping” the surface coat glycoprotein (variable antigen) so that natural host serum lysins could kill the trypanosome; paralysis of flagellar movement so that uptake of plasma proteins by pinocytosis was prevented and the trypanosome had reduced ability to escape from phagocytic host cells; disruption or derangement of trypanosome morphogenesis with anti-microtubule drugs; disruption of trypanosome lysosomes to result in autodigestion.

II. DISCUSSION GROUP ON ENTOMOLOGY INCLUDING INSECTICIDES Chairmen: Dr. A. M. Jordan and Dr. E. M. Thain

Opening

Statement

by Dr. J. M. B. Harley

I propose to consider first the further investigations needed to improve control methods that have already been applied or have at least been the subject of considerable discussion and experimentation and then go on to other lines that appear necessary. Insecticides BARLOW et al. (1977) have shown that new

synthetic pyrethroids, especially permethrin and NRDC 161, have considerable activity in the laboratory against tsetse. Further field and laboratory tests should be made. There has already been considerable investigation of and improvement in application techniques, but further reductions in insecticide concentration and the spraying of more limited parts of the vegetation may be possible in some areas. Observations on each tsetse species in vegetation types other than those already investigated for insecticide application, especially with regard to resting sites and other aspectsof behaviour, and further observations on the side effects of sprays are necessary. Insect growth regulators

These juvenile hormone mimics and ecdysones should be tested against Glossina. Sterile

male release

For this to be used in even a small area, vast numbers of pupae or young males are normally required; perhaps the means of keeping large numbers of flies in large cages in the laboratory or the field should be re-examined. In Rhodesia, Vale and Hargrove predict the trapping of large numbers of wild flies in the field and it is stated (MACKENZIE, 1977) that BurseII can sterilize 90% of these without detectable mortality with a chemosterilant aerosol before they are released. This merits further experimentation. Biological control

The release of pathogens, including

viruses,

bacteria, fungi, nematodes and parasitic arthropods, is also well worth further investigation. Habitat

modascation

Modification of the vegetation so that it is rendered unsuitable as a habitat for tsetse is the only way, except in small isolated belts, to get some degree of lasting control without the need for barriers. The simplest way to achieve this is by cultivation (FORD, 1971). If cattle ranches are to be set up in tsetse areas, bush control and pasture improvement will often be necessary if reasonable stocking rates are to be achieved. The extent to which measures necessary to control or eradicate the fly can be combined with those designed to improve grazing seems worth investigating as a potential means of reducing costs. Hand-clearing is unlikely to be resorted to again, but with bulldozers, arboricides, Gyromors to reslash regeneration and the judicious use of fire, we do now have some means of controlling bush. Game eradication

This seems unlikelv to be used to anv extent in future as a tsetse cbntrol measure, ahd further studies are unjustified. Detection of tsetse at low density

A sensitive method is urgently needed in all control schemes, both to delimit fly-belts before starting and, especially, to monitor population levels after the application of control measures. Traps (CHALLIER & LAVEISSI~~RE,1973; HARGROVE, 197?), gttractants and sex pherbmones come into this cateeorv. Vale and Haraove in Rhodesia have been re;oked recently (MACKENZIE, 1977) as catching 10,500 tsetse (probably G. morsitans and G. pallidipes) in one day in one trap at which the odour of several oxen was released. Clearly this method should be tested in other areas and with other tsetse species and attractants. Other lines of research

(i) Flies of the fusca group and other species rather neglected hitherto will need to be controlled as the more important speciesare dealt with; (ii) genetic methods of control, such as the use of translocations and incompatible strains or subspecies and studies being carried out on mosquitoes provide some pointers; (iii) disturbance of the metabolism or of the reproductive cycle so as to cause abortion, which is only too easy in the laboratory; (iv) factors regulating tsetse populations and their possibility of manipulation; (v) the relation between tsetse and vegetation, leading to control by vegetation modification; (vi) possibly the application of newer formulations of insecticides to cattle as a supplementary control measure; (vii) further refinement of rearing methods; (viii) the genetics and other aspects of the transmission of trypanosomes by tsetse.

Many aspects of tsetse biology are being investigated in the laboratory, which has been made relatively easy by the ready availability of tsetse flies, especially from Bristol. However, further investigations in the field are essential If control

AND HYGIENE, VOL. 72, No. 2, 19’78 113

TRANSACTIONS OF THE ROYAL SOCIETY OF TROPICAL MEDICINE

methods are to be improved, and some thought needs to be given to their promotion. References Barlow, F., Hadaway, A. B., Flower, L. S., Grose, J. E. H. & Turner, C. R. (1977). Some laboratory investigations relevant to the possible use of new pyrethroids in control of mosquitoes and tsetse flies. Pestic. Sci. 8, 291-300. Challier, A. & Laveissiere, C. (1973). Un nouveau picge pour la capture dcs glossines (Gloss&: Diptera, Muscidae) description et assais sur le terrain. Cahiers O.R.S.T.O.M., ser. Ent. med. Parasitnl. 11, 251-262. Ford, J. (1971). The role of the trypanosomiases in African ecoZogy. Oxford: Clarendon Press, 568 pp. Hargrove, J. W. (1977). Some advances in the trapping of tsetse (Glossina spp.) and other flies. Ecological Entomology, 2, 123-137. Mackenzie, P. K. I. (1977). A new method of tsetse control. Rhodesia Veterinary Journal, 8, 25. Summary of Discussion It was emphasized that entomological research aimed at the improvement of control measures should take two forms, research to improve the efficiency of current insecticidal techniques and research towards the development of the next generation of control techniques. With regard to the former, a recently initiated ODM/COPR project will be assisting aerial spray operations undertaken by the Botswana Tsetse Control authorities in the Okavango Delta G. morsitans belt; emphasis will be on experimentation in spraying techniques and studies on the ecology of the fly. Laboratory studies in the United Kingdom in co-operation with workers in Botswana aim to reach a better understanding of the effects of endosulfan on flies of different ages and physiological states with a view to developing improved strategies for insecticide application. The progress of the five-year preliminary phase of the FAO programme for the control of African animal trycanosomiasis was described. Chemical vesticides, although thev undoubtedlv will be used for many yearsto come, are only a temporary solution to a permanent problem. Some African Governments may eventually ban some of those in current use. The costs of getting a new insecticide on to the market are enormous. The effect of pesticides on target ecosystems should be studied with the objective of integrating control measures with natural mortality factors. A better understanding of tsetse behaviour in the field is essential before non-insecticidal control techniques can be fully developed. Field studies in Rhodesia have overturned iong-accepted views of how flies group behave; these studies of the G. morista?ts should be extended to include the G. palpalis group and should be supported with further laboratory studies of visual and/or olfactory attraction. The need was stressed for further studies on the ecology of triatomine bugs and for the use of those reared by the newly developed in vitro feeding system in trypanosome transmission experiments.

III.

DISCUSSION GROUP ON BIOCHEMISTRY INCLUDING CHEMOTHERAPY

Chairmen: Dr. B. A. Newton and Dr. D. G. Godfrey Opening

Statement

by Dr.

W. E. Gutteridge

Investigations of the biochemistry of trypanosomes relevant to chemotherapy fall essentially into two categories; comparative studies of host and parasite or studies of the mode of action of known anti-trypanosomal drugs. Consideringfirst comparative biochemical studies, these are aimed at uncovering differences in The metabolism between host and parasite. approach is based on the concept that although most biochemical pathways are the same in all living organisms, there are some differences and that it should be possible to exploit these by the development of drugs which specifically block the system in the parasite. Occasionally, these investigations have established the presence in the parasite of pathways with no equivalent in the host. Examples include the cc-glycerophosphate oxidase of African trypanosomes (first described by GRANT 81 SARGENT, 1960, and studied more recently in greater detail by BOWMAN & FAIRLAMB, 1976 and OPPERDOES & BORST, 1976) and a pathway for threonine catabolism (CROSS, KLEIN & LINSTEAD, 1975). More frequently, the differences are only qualitative; these, however, are still potentially exploitable, as experience with the anti-malarial drug pyrimethamine has shown. Examples are pyruvate kinase (FLYNN & BOWMAN, 1974), dihydro-orotate hydroxylase (GUTTERIDGE, 1976; DAVE, 1977) and thymidylate synthase (AL CHALABI & GUTTERIDGE, 1977). The differences need not re!ate only to general metabolism; differences in the permeability of cells can be included, since these will affect the selectivity of a drug. Recent work on pentamidine permeability and transport in Trypanosoma brucei illustrates this point (DAMPER & PATTON, 1976). Mode of action studies are concerned with attempts to expIain in biochemical terms how existing drugs bring about their effects. The reasons for seeking this information are two-fold. Firstlv. it is hoped that such information will guide the synthesis of better analogues of the existing drug. Secondly, it might be possible to find novel chemical structures even more effective against the same target. Pioneer work by one of our chairmen, Dr. Newton, went some way towards establishing the modes of action of ethidium (NEWTON, 1957) and antrycide(N~~~~~, 1966). iMore recently, WILLIAMSON, MACADAM

& DIXON

(1975)

have

shown

how

the electron microscope can aid ‘such investigations and SIMS & GUTTERIDGE (1976) have studied the mode of action of SQ 18,506, a’ nitrofuran, on T. crazi. These are but two examples of many such studies. Together, these two types of biochemical investigation have indicated a number of differences between host and trypanosome, a few of which I have mentioned already. In a recent detailed consideration of these in T. cruzi (GUTT~RIDGE, 1978), I was able to list 20 such differences; no doubt a

114

SEMINARON TRYPANOSOMIASIS

list of similar length could be made for African trypanosomes. Some of the differences discovered are probably already being exploited unknowingly. For example, suramin may act as an inhibitor of a-glycerophosphate oxidase, though there are doubts about this (BOWMAN & FAIRLAMB, 1976; OPPERDOES & BORST,1976). Others, such as that involving dihydro-orotate hydroxylase, need more investigation before we shall know if exploitation is possible (GUTTERIDGE,1976). We thus come to the first of four points for discussion today. Bearing in mind the limited funds available, should we study in depth and attempt to exploit those differences which have already been identified or should we seek further Bearing in mind the difficulties differences? involved in seeking areas of difference, even though existing drugs will no doubt continue to serve as useful probes, I believe there is some merit in concentrating on known or suspected differences. Either of these two approaches involves continuing biochemical studies and therefore we must also consider methodology. The major problem here is not the biochemical manipulations but the production of sufficient material with which to carry these out. The techniques developed by LANHAM & GODFREY(1970) and WILLIAMSON & COVER(1966) for African trypanosomes and by GUTTERIDGE,COVER& GABORAK(1978) for T. cruzi have essentially solved this problem as far as the isolation of mammalian forms from laboratory animals is concerned. However, biochemists often need to be able to grow cells in defined media under rigorously controlied conditions (e.g. the work of CROSSet al.. 1975 on threonine catabolism) and in addition, stages parasitic in the various ’ vectors need study. Thus a second point for discussion concerns how best we should attempt to grow all stages in vitro, what priority we should give to this work, how likely is it to succeed (the recent successes of HIRUMI, DOYLE & HIRIJM~, 1977 with blood forms. and CROSS& MANNING. 1973 with culture forms-are encouraging) and alsd if what is grown is likely to be the same as that which occurs in vivo. My opinion here is that from the biochemical (as opposed to the immunological) point of view, such work should be given a high priority only when it relates to the mammalian stages of the parasites. A third discussion point stems directly from the previous two-if the development of new methodology and the adoption of the correct strategy enables us to identify new targets suitable for chemotherapeutic attack, how do we produce compounds specifically active at these points? Two basic approaches seem possible-a wide range of existing chemical structures could be tested against the isolated target (the empirical approach), or attempts could be made on the basis of the known action of other drugs to design inhibitors which are specific for it (the rational approach). We need to consider which of these two approaches is likely to be the more successful and if other approaches are possible. I believe that until we know more about the mode of action of existing drugs, only the empirical approach is possible. If we can bridge the gap between biochemistry and chemistry, one final consideration remains.

Suppose we identify a suitable target in T. brucei and find an inhibitor which is able to block it both in vitro and in vivo, will we need to repeat the operation for T. congolense,T. vivax and T. cruzi? fro we have to worry also about T. rhodesiense and T. Pambiense? Undoubtedlv, there are marked diffgrences in energy metab&m (GUTTERIDGE& ROGERSON,1968) a;;h patterns of drug sensitivity (NEWTON, 1974) between the different subgroups of the genus Trypanosoma. These may well reflect fundamental metabolic differences, though insufficient comparative studies have been done for us to be certain. I believe, therefore, that for the present, we must regard each subgenus as a distinct entity. In summary, discussions of the strategy for future biochemical studies should be based on four key questions : (1) Should we concentrate on exploiting known biochemical differences or should we continue to diversify? (2) What should we do about methodology, and especially about in vitro culture? (3) How should we try to translate biochemical differences into active compounds? (4) How far must we regard each species of the genus Trypanosoma as a separate entity? References Al Chalabi, K. & Gutteridge, W. E. (1977). Presence and properties of thymidylate synthase in trypanosomatids. Biochimica et Biophysics Acta, 481, 71-79. Bowman, I. B. R. & Fairlamb, A. H. (1976). L-Glvcerol-3-nhosnhate oxidase in Trvaanosoma brucei and the ‘effeci of suramin. In : Biichemistry of Parasites and Host-Parasite Relationships, Van den Bossche, H. (Editor), Amsterdam: North Holland, pp. 501-507. Cross, G. A. M., Klein, R. A. & Linstead, D. J. (1975). Utilization of amino acids by Trypanosoma brucei in culture: L-threonine as a precursor for acetate. Parasitology, 71, 31 l-326. Cross, G. A. M. & Manning, J. C. (1973). Cultivation of Trypanosoma brucei sspp in semi-defined and defined media. Parasitology, 67, 315-331. Damper, D. & Patton, C. L. (1976). Pentamidine transport in Trypanosoma brucei-kinetics and specificity. Biochemical Pharmacology, 25, 27 l276.

Dave, D. (1977). Conversion of dihydroorotate to orotate in various species of Plasmodium and Trypanosomatidae. Ph.D. thesis, University of Kent. Flynn, I. W. & Bowman, I. B. R. (1974). The activity of trypanocidal arsenical drugs on Trypanosoma brucei and Trypanosoma rhodesiense. Comparative

Biochemistry

and Physiology,

48,

261-274. Grant, P, T. & Sargent, J. R. (1960). Properties of L-a-glycerophosphate and its role in the respiration of Trypanosoma rhodesiense. Biochemical Journal, 76, 229-237.

Gutteridge, W. E. (1976). Isolation of blood and intracellular forms of Trypanosoma cruzi and comparative aspects of nu&eic acid metabolism. In: Biochemistry of Parasites and Host-Parasite

TRANSACTIONS OF THE ROYAL SOCIETY OF TROPICAL MEDICINE

Relationships, Van den Bossche, H. (Editor), Amsterdam: North Holland. DV. 245-252. Gutteridge, W. E. (1978). Biochemical studies of intracellular, blood and culture forms of Trypanosoma cruzi: possible targets in rational chemotherapy. Pan American Health Organization Symposium, (in press). Gutteridge, W. E. & Rogerson, G. W. (1978). Biochemical aspects of the biology of Trypunosoma cruzi. In: Biology of the Kinetoplastida, Lumsden, W. H. R. & Evans, D. A. (Editors), New York: Academic Press, (in press). Gutteridge, W. E., Cover, B. & Gaborak, M. (1978). Isolation of blood and intracellular forms of Trypanosoma cruzi from rats and other rodents and preliminary studies of their metabolism. Parasitology, (in press). Hirumi, H., Doyle, J. J. & Hirumi, K. (1977). African trypanosomes : cultivation of animalinfective Trypanosoma brucei in vitro. Science, .

.I

New York, 196, 992.

Lanham, S. M. & Godfrey, D. G. (1970). Isolation of salivarian trypanosomes from-man. and other mammals using DEAE-cellulose. Exaerimental + Parasitology,

28; 521-534.

Newton, B. A. (1957). The mode of action of phenanthridines: the effect of ethidium bromide on cell division and nucleic acid synthesis. Journal of Microbiology, 17, 718-730. Newton, B. A. (1966). Effects of antrycide on nucleic acid synthesis and function. In: Biochemical Studies of Antimicrobial Drugs, Newton, B. A. & Reynolds, P. E. (Editors). Cambridge: Cambridge University Press, pp. 213-234. Newton. B. A. (1974). The chemotherauv of trypanosomiasis ~and leishmaniasis : towards a more rational approach. In : Trypanosomiasis and

AND HYGIENE,

VOL. 72, No.

2, 1978

115

empirical approach should both be encouraged in academic and industrial laboratories. The chance of a successful new drug being produced by the rational approach was slender; some compounds now being identified as selective metabolic inhibitors had been rejected as ineffective chemotherapeutic agents during empirical drug screening trials many years ago. The present day cost of development of a new drug for use in man is estimated to be between 10 and 25 million pounds sterling and it was stressed that this could not be justified commercially by the apparently low numbers of cases of African trypanosomiasis in man; however, the vast potential demand for animal treatment may encourage the pharmaceutical industry although, in the course of developing new veterinary drugs, greater attention will have to be paid in the future to the question of drug residues in meat destined for human consumption. During discussion of the possibility of making known drugs more effective, several speakers referred to recent developments in controlling leishmanial infections by drugs enclosed in liposomes which are engulfed by infected host cells. Such a method might be useful against T. cruzi, which like Leishmania can be intracellular, and other bloodstream trypanosomes can ingest lipid from the serum. The possibility of enhancing the effectiveness of drugs by giving compounds capable of stimulating the host’s defence mechanism was also discussed. Finally, because of the marked differences in metabolism between the various subgenera of trypanosomes, it was emphasized that chemotherapeutic research needs to be carried out on several representative species.

Leishmaniasis with special reference to Chagas’ Disease, Ciba Foundation Symposium 20 (New

Series), pp. 285-301. Opperdoes, F. R. & Borst, P. (1976). The effect of salicylhydroxamic acid on the glycerol-3phosphate oxidase (GPO) of Trypanosoma brucei: its influence on a T. brucei model infection and the intracellular localization of GPO. In: Biochemistrv Relationshi&,

of

Parasites

and

Host-Parasite

Van den Bossche, H. (Editor), Amsterdam: North Holland, pp. 509-517. Sims, P. & Gutteridge, W. E. (1976). Biochemical effects and mode of action of a 5-nitrofuran drug, SQ 18,506, on Trypanosoma cruzi. In: Biochemistry of parasites and Hose-Parasite Relationships, Van den Bossche, H. (Editor), Amsterdam,

North Holland, pp. 485-491. Williamson, J. & Cover, B. (1966). Separation of blood cell-free trypanosomes and malaria parasites on a sucrose gradient. Transaction of the Royal Society

of Tropical

Medicine

and Hygiene,

60,

426-427. Williamson, J., Macadam, R. F. & Dixon, H. (1975). Drug-induced lesions in trypanosome fine structure : a guide to modes of trypanocidal action. Biochemical Pharmacology, 24,147-150. Summary

of Discussion

The rational approach based on a detailed knowledge of trypanosome biochemistry and the

IV.

DISCUSSION GROUP ON IMMUNOLOGY

Chairmen-Professor Opening

Statement

M. J. Clarkson and Dr A. R. Gray by Dr

G. A. T. Targett

research on trypanosomiasis Immunological should have one of two objectives, either improvement of indirect methods for diagnosis of infection or extensive investigation of any procedures which might have an immunoprophylactic or immunoI have selected some therapeutic application. aspects that I believe require emphasis. 1. Serodiagnosis Serological diagnosis is applied primarily to mass survey studies and complements protozoological (direct) diagnosis of infection. Future requirements are already well recognized. The need is for simple tests which can be used under field conditions yet which retain adequate levels of sensitivity and specifiicity, especially where mixed in;:‘,‘?:& occur (LUCKINS, 1976). Serodiagnosis will also continue to be of special importance in Chagas’s disease so long as the absence of adequate chemo-

116

SEMINAR ON TRYPANOSOMIASIS

therapy means that emphasis must be on control measures related to the (complex) epidemiology of the disease. 2. Innate resistance and trypanotolerance

These can be considered together, even though the so-called trypano-tolerant animals are such largely by virtue of acquired immune responses (ROBERTS & GRAY, 1973). This area has received relatively little attention but has great potential in terms of control of infections. For example : Innate resistance of man, mediated through serum factors, determines whether or not infection with Trypanosoma brucei organisms will occur (RICKMAN, 1977). Attempts have been made to relate susceptibility to trypanosome infection to antigenic type (VAN MEIRVENNE et al., 1976), but there is a need to investigate closely relationships between trypanotidal effects and surface properties of the parasite. There is also renewed interest in the greater resistance to trypanosomes of certain breeds of cattle in Africa (e.g. N’dama and Muturu). We should look closely at the nature of this resistance. considering, in particular, infections in young animals and the passive transfer of immunity. 3. Antigenic variation Important advances in recent years have shown that variant antigens are glycoprotein in nature, differ markedly one from another, and are major components of the surface coat of the trypanosome (CROSS, 1975; TAYLOR & CROSS, 1977; FRUIT et al.,

1977). Modifications of the fluorescent antibody test permitting detection of variants on single organisms is another important advance, and investigations are now beginning of the antigens of metacyclic trypomastigotes (JENNI, 1977). Studies on surface antigens are crucial if progress towards immunological control is to be made. Antigenic variation as we normally see and study it is an exaggerated response of a parasite in an abnormal host (be it man, cow or experimental animal). It might well occur in a normal host but be part of a more balanced host-parasite relationship involving regulation of the parasite population. In this respect, some recent observations are of interest. Single variants can sometimes persist for a long peribd in one host and, in birds, may disappear and then reaooear (HICKS. 1977). Variants isolated from extrava&ar sites gave also been shown to be different from those occurring concurrently in the blood (SEED & EFFRON, 1973; LAMBERT & HOUBA, 1974). Also, the number of different serotvnes of T. g&biense~isolated is fewer than those from more acute T. Orucei infections (GRAY, 1975; GRAY & LUCKINS, i376). To consider what is happening at the host-parasite interface under well-adapted situations might well provide clues as to how to help bring about control where the parasite now holds sway. 4, Aberrations

qf immune functioion

This is another area OFimmunological research in which we have to consider carefully the extent to which our observations from experimental

studies apply in terms of the disease under natural Numerous aberrations of immune conditions. function during trypanosome infection have been demonstrated or proposed. Hypocomplementaemia and activation of both the classical and alternate pathways of complement occur (WOODRUFF et al., 1973; KOBAYASHI & TIZARD, 1946) and are known to have immunosuppressive effects. It is also proposed that much inununosuppression is due to release of large quantities of free fatty-acids by trypanosomes (TIZARD et al., 1977), and there is some evidence for release of a B-cell mitogen (ESURUOSO, 1976) which is responsible for polyclonal B-cell activation. secretion of IrrM of many different specifications, and clonal ex&ustion 0; paralysis (HUDSON et al., 1976). Suppressor-cell production may be either induced or i&ibited (JAYAWARDENA & WAKSMAN. 1977: CORSINI et al., 1977). The significance df all these immune responses .has still Similarly, immune-complex to be evaluated. disease seems to be a major component of the pathology of most trypanosome infections and merits greater investigation (see below, p. 118). 5. Immurwprophylaxis

It is possible to produce a fairly strong resistance to challenge by immunization against human or animal forms of African trypanosomiasis with attenuated organisms or killed parasites (see, e.g:, SEED, 1974; WELLDE et al., 1975; JAMES, 1976). This immunity is restricted, however, to the homologous variant. Future studies must continue to be largely empirical, but the suitability of metacyclic forms as a source of protective antigen should be tested, and the new procedures for partial fractionation of the parasites, especially in the analysis of surfacecoat antigens, could produce antigenic fractions to be tested for immunogenicity. Immunity has been produced against T. cruzi infection by immunizing with avirulent, attenuated, or killed and fractionated organisms (see, e.g., HANSON, 1976; GONZALEZ The special problem in CAPPA et al., 1974). attempts to immunize against the acute phase of infections is the real possibility, with live vaccines, of inducing chronic disease; killed organisms as well as live parasites might also induce damaging pathological sequelae. 6. Infection and treatment

Control by vaccination is a long-term objective, and one alternative approach, particularly applicable to cattle infections, is the use of chemotherapy in such a way that control of the infection may be effected in part by the development of immunity (WILSON et al., 1976). There is a need to investigate much more extensively all aspects of the interaction of chemotherapy and the immune response, as it might well prove more feasible to regulate trypanosome infections than to eradicate them. References

Corsini, A. C., Clayton, C., Askonas, B. A. & Ogilvie, B. M. (1977). Suppressor cells and loss of B-cell potential in mice with Trypanosoma brucei. Clinical and Experimental Inmmunology, 29, 122131.

TRANSACTIONS OF THE ROYAL SOCIETY OFTROPICAL MEDICINE AND HYGIENE,VOL. 72, No. 2, 1978 117

Cross, G. A. M. (1975). Identification, purification and properties of clone-specific glycoprotein antigens constituting the surface coat of Trypanosoma brucei. Parasitology, 71, 393-417. Esuruoso, G. 0. (1976). The demonstration in vitro of the mitogenic effects of trypanosomal antigen on the spleen cells of normal, athymic and cyclophosphamide-treated mice. Clinical and Experimental Immunology, 23, 314-317.

Fruit, J., Afchain, D., Petitprez, A., Van Meirvenne, N., Le Ray, D., Bout, D. & Capron, A. (1977). Antigenic analysis of a variant specific component of Tiypanosoma brucei brucei: localisation on the surface coat with labelled svecific antibodies. Parasitology, 74, 185-190. * Gonzalez Cappa, S. M., Pesce, U. J., Cantarella, A. I. & Schmunis, G. A. (1974). Trypanosoma cruzi: protection of mice with epimastigote antigens from immunologically different parasite strains. Experimental Parasitology, 35, 179-186. Gray, A. R. (1975). A pattern in the development of agglutinogenic antigens of cyclically transmitted isolates of Trypanosoma gambiense. Transactions of the Royal Society of Tropical Medicine and Hygiene, 69, 131-138. Gray, A. R. & Luckins, A. G. (1976). Antigenic variation in salivarian trypanosomes. In : Biology of the Kinetoplastida, Lumsden, W. H. R. & Evans, D. A. (Editors), Volume 1. London: Academic Press, pp. 493-542. Hanson, W. L. (1976). Immunology of American trypanosomiasis. In: Immunology of Parasitic Infections, Cohen, S. & Sadun, E. (Editors). Oxford: Blackwell, pp. 222-234. Hicks, B. R. (1977). Studies on the behaviour of and immunological response to Trypanosoma brucei in avian hosts. PhD Thesis, University of London. Hudson, K. M., Byner, C., Freeman, J. & Terry, R. J. (1976). Immunodepression, high IgM levels and evasion of the immune response in murine trypanosomiasis. Nature, 284, 256-258. Tames, D. M. (1976). Induction of immunity in - rodents receiving living drug-treated trypanosomes. International 70urnal for Parasitology, -- _ 6, 179-182. Jayawardena, A. N. & Waksman, B. H. (1977). Suppressor cells in experimental trypanosomiasis. Nature,

soma brucei in vitro. Parasitology,

74, 47-60.

Tizard, I. R., Nielsen, K., Mellors, A. & Assoku, R. K. (1977). Free fatty acids and pathogenesis of African trypanosomiasis. Lancet, i, 750-75 1. Van Meirvenne, N., Magnus, E. & Janssens, P. G. (1976). The effect of normal human serum on trypanosomes of distinct antigenic type (ETat 1 to 12) isolated from a strain of Trypanosoma brucei rhodesiense. Annales de la So&e’ Medecine Tropicale, 56, 55-63.

helge de

Wellde, B. T., Schoenbechler, M. J., Diggs, C. L., Longbehn, H. R. & Sadun, E. H. (197.5). Trypanosoma rhodesiense : variant specificity bf immunity induced by irradiated narasites. EXperimental Parasitology, 37, 125-l-9. Wilson, A. J., Paris, J., Luckms, A. G., Dar, F. K. & 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. Tropical Animal Health and Production, 8, 1-12.

Woodruff, A. W., Zeigler, J. Ll, Hathaway, A. & Gwata, T. (1973). Anaemia in African trypanosomiasis and ‘big spleen disease’ in Uganda. Transactions of the Royal Society of Tropical Medicine and Hygiene, 67, 329-337.

256. 539-541.

Jenni, L.-(1977). Comparisons of antigenic types of Trvpanosoma (T) brucei strains transmitted bv &&ta

Rickman, L. R. (1977). Variation in the test responses of clone-derived Trypanosoma (Trypanozoon) brucei brucei and T. (T.) b. rhodesiense relapse antigenic variants examined by a modified blood incubation infectivity test and its possible significance in Rhodesian sleeping sickness transmission. Medical Journal of Zambia, 11, 3137. Roberts, C. J. & Gray, A. R. (1973). Studies on trvpanosome-resistant cattle. II. The effect of trybanosomiasis on N’Dama, Muturu and Zebu cattle. Tropical Animal Health and P:xduccio~r, 5,220-223. Seed, J. R. (1974). Antigens and antigenic variability of the African trypanosomes. Journal of Protozoology, 21, 639-646. Seed, J. R. & Effron, H. G. (1973). Simultaneous presence of different antigenic populations of Trypanosoma brucei gambiense in ‘Mkrotus montanus. Parasitoloav, -- _ 66. 269-278. Taylor, D. W. & Cross, G. A. M. (1977). The synthesis of a variant-specific antigen by Trypano-

m. moristans. Acta Tropica, 34,35-41.

*

Kobayashi, A. & Tizard, I. R. (1976). The response ~0 Trypanosoma congolense infection in calves: determination of immunological IgG, IgG,, IaM and C3 levels and the complement fixing antibody titres during the course of infection. Troaenmedizin und Parasitolonie, 27. 41 l-417. Lamb>rt, P. H. & Houba, c (1974). Immune complexes in parasitic diseases. In : Progress in ImmunoZogy ZZ, Brent, L. & Holborrow, J. (Editors), Volume 5. Amsterdam: North Holland, pp. 57-67. Luckins, A. G. (1976). Detection of antibodies in trypanosome-infected cattle by means of a microplate enzyme-linked immunosorbent Tropical Animal Health and Production, 9, K?izgg:

Summary

of Discussion

The discussion centred largely around the problems of serological and parasitological diagnosis of trypanosomiasis. Since several different species of trypanosome were involved in animal trypanosomiasis it was necessary to use antigens from all these species to detect all infections which may be present. The ELISA test was better than the IFAT in this respect. Both these tests were proving to be useful in the serological diagnosis of T. cvansi infection in camels. In T. cruzi infection in man CFT and IFAT were good as screening techniques but IFAT was better in children for individual diagnosis. Many current serological tests required expensive equipment and a single field technique was needed. It may be possible to adapt the recently described

118

SEMINAR

ON TRYPANOSOMIASIS

technique of Ouchterlony which depended on the differing afIi.nity of antigen and antibody for water. A hydrophilic antigen could be pre-absorbed on to a plastic petri dish and the presence of antibody detected as hydrophobic spots when the dish was breathed on. It was questioned whether antigenic variation occurred as extensively in resistant cattle and game animals as in laboratory or susceptible domesticated animals.

V. DISCUSSION Chairmen-Dr

Opening

GROUP ON PATHOLOGY

L. G. Goodwin and Professor M. J. Clarkson Statement by Professor Urquhart

The speaker acknowledged the P. H. Holmes, F. W. J. Jennings and of the Veterinary College, University in the preparation of this introductory

G. M.

assistance of D. Whitelaw, of Glasgow, statement.

Pathological studies may appear to have little to contribute directly to the control of trypanosomiasis compared with other aspects of the disease, notably chemotherapy, epidemiology and immunity. In fact, the study of pathology has a very necessary role to play in the future development and evaluation of control programmes and, this short paper attempts to indicate some aspects of pathological research which should receive a measure of priority. The list is not exhaustive and you may disagree with some of the priorities; other equally valid aspects will undoubtedly be raised in the discussion. The first, and perhaps the primary problem, is the correlation of clinical signs, i.e. pathology, with the incidence of infection. In the past, it was customary, in epidemiological studies of bovine trypanosomiasis, to refer to the incidence of infection in a given area, usually based on blood examination, as being 12% or 30%, etc. This gives no indication of the economic significance of the disease-are most of the animals relatively healthy carriers or are they the emaciated survivors of previous multiple infections? It may be argued that this defect could be easily resolved if future surveys included a number of clinical criteria. This would be of some advantage if the clinical examinations were made by someone skilled in differential diagnosis and with some access to laboratory fac&ies. Even so, many diseasesof cattle in the tropics are characterized by emaciation, a degree of lymphadenopathy is common and sterility may have a multiplicity of causes. Also, in individual animals the real significance of trypanosomiasis is often particularly difficult to assess, e.g. the anaemic emaciated cow with an infrequent or scanty parasitaemia. The response to chemotheranv is often a useful guide in difficult cases, but -requires long-term surveillance and sometimes appears to be of little benefit.

The ideal would be the development of some relatively simple technique which would provide a reasonably accurate index of the amount of damage caused specifically by trypanosomes. To some extent the haematocrit fulfils this function, but the interpretation of haematological data in trypanosome endemic areas is complicated and requires a great deal more study. The type of test required should be more specific; a convenient parasitological analogy is the blood plasma pepsinogen test which gives a direct measure of the degree of abomasal damage in bovine ostertagiasis. The particular test which should be examined is outside the scope of the paper, but we suggest something which would quantitate damage to skeletal or cardiac muscle or an evaluation of the immunoglobulin (IgM) level as an index of polyclonal stimulation of the immune response by trypanosomes. A second promising aspect of pathology or immunopathology which may have an immense contribution to make to the control of trypanosomiasis is immunosuppression. This phenomenon, clearlv established but still imperfectly understood in small laboratory animals, is largely unexplored in the real hosts of this disease. Three aspects demand attention. The possible deleterious effect on vaccination against bacterial and viral diseases; the possibility that infected animals have an increased susceptibility to other diseases, e.g. helm&h infections; the efficacy of chemotherapy in restoring normal immune responses. A fourth possible pathological study is the way in which trypano-tolerant animals deal with infection. Although it is generally considered that trypano-tolerance is associated with a good humoral immune response, there is no real evidence that this is so; the differences may be physiological or cell-mediated and detectable only by pathophysiological techniques or immunopathological studieswhich might also reveal genetic markers for tolerance. Pathological studies could show unequivocally the significance of T. brucei infections in cattle in Africa, of T. evansi in Asia and of T. vivax in South America. The role of nutrition in the pathogenesis of trypanosome infections is also perhaps important, particularly in the large domestic animals. We have not stressed the self-evident need for studies on the sequential pathogenesis of the disease in the large domestic animals. These are long overdue and until they are done we shall never really understand trypanosomiasis. We should like to stress two points. Firstly, the real significance of pathological studies is to provide understanding of the mechanisms whereby disease is produced. In trypanosomiasis these are obviously complex and difficult to unravel. We have been embarrassed for too long by our utter inability to explain how this parasite, often difficult to find, can be responsible for morbidity and mortality on such a scale. Unless we can eventually do so (and the outlook is now promising) we only practise empiricism and not science. For this reason, apart from experimentation ---

-0

~~

TRANSACTIONS OF THE ROYAL SOCIETY OF TROPICAL MEDICINE AND HYGIENE, VOL. 72, No. 2, 1978 119

on large domestic animals, studies on laboratory models should be encouraged as it is from these that basic advances are most likely to be made. Secondly, the Panel should continue to encourage collaborative ventures with scientists working in endemic and enzootic areas. These need not be expensive or of extended duration. The benefits are reciprocal. The man on the spot has unique access to ideas, information, literature and techniques while the visitor can observe the disease still inadequately described and make an important individual contribution to its solution in collaboration with his colleague in the field. Unless the Panel does continue such ventures, as part of its programme, there is little justification for continuing research on small laboratory animals in this country. The disease is, after all, one of animals and man in the tropics. Summary

of Discussion

Attention was drawn to the multiple aetiology of anaemia in natural cattle trypanosomiasis. Antitrypanosome treatment does not always resolve the anaemia, especially in long-standing infections. More study is needed of the factors concerned in the removal of red cells; the anaemia of trypanosomiasis is haemolytic, but complex. Trypanosomes in the bone marrow may in some way effect the release of erythrocytes into the circulation. It has been shown by TIZAFUI et al. (1977) in Canada that phospholipases and lysophospholipases released from lysed trypanosomes may play an important role in pathogenesis of trypanosomiasis. In the microvasculature, these enzymes may release free fatty acids from host and parasite phospholipids, which cause damage to the endothelium in the capillary beds. There may be another role for lysophospholipases in the general circulation. There is evidence that freshly isolated T. congolensereleaseslysophospholipase at a high level of activity in vitro, and that this activity does not change significantly on autolysis (TIZARD. oersonal communication). This level of activity s;ggests that release of lysophospholipase from trypanosomes in the blood stream may be responsible for the marked fall in plasma concentration of lysophosphatidyl choline (LPC) which has been observed in infected ruminants (ROBERTS, 1975). Consequences of reduction in LPC concentration might be, firstly, a reduction in the plasma concentration of total phospholipids; secondly, interference with formation of high-density lipoprotein; thirdly, reduction in substrate levels for lecithin-cholesterol acyl transferase (LCAT) and lipoprotein lipase activities; and fourthly, acceleration of platelet aggregation and thrombus formation. These consequences of lysophospholipase release may interfere with energy transport mechanisms and deprive tissues of essential energy substrates, resultinrr in the develonment of debilitv which is a major fgature of chronic trypanosomia&. The sequences of events in the vertebrate host following release of phospholipases in the microvasculature and general circulation may account for several major features of the pathology of trypanosomiasis. LCAT is found in high concentration in the

baboon, which is resistant to all species of salivarian trypanosome, and in man, who is resistant to some. The enzyme is absorbed by trypanosomes and may perhaps be associated with their removal from the circulation. Our lack of understanding of the relationship between degree of infection and clinical signs was emphasized by several speakers. Pathological changes-anaemia, muscle wasting, cuffing of brain vessels-continue to advance although it may be difficult or impossible to find trypanosomes. There is a need for new serological indices to monitor pathology. References

Roberts, C. J. (1975). Ruminant lipid metabolism in trypanosomiasis. Transactions of the Royal Society of Tropical

Medicine

and Hygiene,

69,

275. Tizard, I. R., Nielsen, K., Mellors, A. & Assoku, R. K. (1977). Free fatty acids and pathogenesis of African trypanosomiasis. Lancer, i, 750-751.

VI.

DISCUSSION GROUP ON EPIDEMIOLOGY

Chairmen-Professor W. H. R. Lumsden Dr D. G. Godfrey Opening

Statement

and

by Dr A. J. Duggan

The epidemiology of African trypanosomiasis has always been rather paradoxical; until comparatively recent times there has been a plethora of doctors and few scientists, and this imbalance produced a one-sided view of the value and purpose of surveillance; today, we have a massive bulk of scientific knowledge and an ever-dwindling cadre of medical men with the opportunity to apply it. Last year the deliberations of a pre-eminent group were circulated (DE RAADT, 1976), drawing attention to 9,000 casesof sleeping sickness reported annually. But few of us believe that those reported figures are comprehensive. The prevalence is more accurately reflected by occasional publications such as that of SINA et af. (1977) referrina to 1,066 uatients treated in four years’ at a sirrile mission hospital in Cameroun, or by papers on therapy, such as that of BWST (1976). who described 78 children with Trypanosoma* brucei rhodesiense infections in Isoka over a three-year period. Such series are usually derived from the known ancient foci (DE RAADT, 1976). Within these foci the nidality of sleeping sickness is even more apparent: the chances of infection at one West African tsetse-infected water-hole may be ten times greater than at another half a mile away. If you were to choose your route carefully you could walk across Africa with less than a thousand-to-one chance of getting the disease. Yet, if you took with you a horse or an ox it is virtually certain that, without chemoprophylaxis, it would be dead of trypanosomiasis before completing the journey. This contrast between the focal nature of human

120

SEMINARON TRYPANOSOMIASIS

trypanosomiasis and the ubiquitous blanket of death which exists for domesticated animals has never been entirely explained. Neither, for that matter, have the factors responsible for the creation and maintenance of old foci been given a fair share of study. It is time that the situation was reexamined in The Gambia. Since the turn of the century there have been several excellent epidemiological studies in that country (DUTTON & TODD, 1903; NASH, 1948; DAVEY, 1948, CAMPBELL, 1950),

the last being by HUTCHINSON(i953), 25 years ago: Enidemics rarelv occurred and indeed the disease may be in biological recession there. If this is true, it would be exciting to know why. The idea of a large-scale investigation confronts us with the ardours of survey work. It is time to re-examine the dictum that each diagnosis must always rest upon the demonstration of trypanosomes. No physician likes to use the toxic and painful melarsoprol without absolute justification, but as a means of mass diagnosis the whole panoply of teams, microscopes, neck palpation, gland puncture and general blood-letting is as cumbersome, disturbing, expensive, antiquated and (I believe) inaccurate as anything could be. When combined with treatment, it is said to be an efficient control method but without simultaneous attack on local fly it has never, to my knowledge, eradicated an established focus of sleeping sickness. Too much is often claimed for it: there is no proof that an increase in surveillance always brings about a decline in the number of cases, In fact the system obeys the law of diminishing returns, and the sheer effort involved in diagnosing one case of sleeping sickness in 1970 was 96 times greater than it was in 1940 (DE RAADT, 1976). A few years ago the answer to much of this surveillance problem seemed to lie in some, or one, of the serological methods which were then being developed. But their early promise seems to have given way to disillusionment, They are evidently not as accurate as we would wish, but the real question is whether they are more or less accurate than the demonstration of trypanosomes under field conditions. igM, in the serum or having appeared in the cerebrospinal fluid, is there night and day. The significance of its quantity may be equivocal in some cases,but its constancy must surely exceed that of a chronic T. Rambiense uarasitaemia which comes and goes and cannot be *detected in a thick blood fihn until a fairly high density of parasites has been attained. In surveys such as those made by WOO (197I) in Uganda and by MCCONNELLand others on Ethiopians (MCCONNELL et al., 1970), the number of raised IgM levels and positive IFATs was considerably greater than the numbers of people in whom trypanosomes were found. But to say that these are of little value because they yield a high proportion of false positives is rather a harsh judgement. The view which I took for many years was that man became diseased whenever he received a dose of infective metacyclic trypanosomes, and that spontaneous cure never followed. This is a safe approach for the physician; it should be taught to medical students. But now I am by no means sure that it is correct. Some, at least, of the so-called

false positives might well be serological war-wounds Successful-encounters with from past-and “pathogenic” and “non-pathogenic” trypanosomes. It is time that surveillance was regarded as a means of deciding how much transmission of trypanosomes is going on, which is not the same objective as the detection of caseswhich require treatment. There is one body fluid which has never been rigorously examined in sleeping sickness, and that is saliva. It normally contains IgA rather than IgM, but is IgA output not in any way affected by trvnanosomiasis? If this surveillance problem could be-dealt with by a simple biannual test which did not require blood, we should have a continuing monitoring system for the people of endemic areas, rather than spasmodic community upheavals which come too late to save some and too early to iind others. We should regard surveillance, not only as a preliminary to treatment campaign, but as a useful tool for the scientist as well. Where anti-tsetse campaigns have been waged over large areas, the discoverv of a handful of infections at village level could bkthe first indication of re-invasion bi fly. Gland punctures and blood films have been our stock-in-trade for 72 years (THOMAS & BREINL, 1905); serology surely deserves more than a tenth of that time to show how it can assist in solving the problem of African trypanosomiasis. References

Buyst, H. (1976). Sleeping sickness in children. Paner read at the International Colloquium on Human African Trypanosomiasis at the Prince Leopold Institute of Tropical Medicine, Antwerp, December 1976 (Cyclostyled). Campbell, W. H. (1950). Trypranosomiasis and tsetse in The Gambia. Government Publication (Cyclostyled) D.S. 70476/l/933 150 2/51. R. Sam. Davey, T. H. (1948). Trypanosomiusis in British West Africa. London: H.M.S.O. De Raadt, I?. (1976). African trypanosomiasis. World Health Organization Circular TDR/WP/76.12 (Cyclostyled). Dutton, J, E. & Todd, J. L. (1903). First report of the trypanosomiasis expedition to Senegambia (1902). Memoir XI Liverpool School of Tropical Medicine. University Press of Liverpool, Hutchinson, M. P. (1953). The epidemiology of human trypanosomiasis in Rritish West Africa. II. The Gambia. Annals of Tropical Medicine and Parasitology, 47, 156-168 & 169-182. McConnell, E., Hutchinson, M. P. & Baker, J. R. (1970). Human trypanosomiasis in Ethiopia. : The Gilo River area. Transactions of the Royal Society of Tropical Medicine and Hygiene, 64, 683-691. Nash, T. A. M. (1948). Tsetse pies in British West Africa, London: H.M.S.O. Sina, G., Triolo, B., Trova, P. & Clabaut, J. M. (1977). L’enctphalopathie arstnicaie lors du traitement de la Trypanosomiase humaine Africaine & T. gambiense (a-propos de 16 cas). Annales de la Socie’td belge de Midecine Tropicale, 57, 67-74. Thomas, H. W. & Breinl, A. (1905). Report on

TRANSACTIONS OF THE ROYAL SOCIETY OF TROPICAL Mmrcm

trypanosomes, trypanosomiasis and sleeping sickness . . . Memoir XVI Liverpool School of Tropical Medicine, Liverpool : University Press, Woo, P. T. K. (1971). Evaluation of the haematocrit centrifuge and other techniques for the field diagnosis of human trypanosomiasis and filariasis. Acta Tropica, 28, 298-303.

Summary of Discussion Serological and protozoological surveying in animals should be intensified and broadened to improve understanding of the factors determining the focality of distribution of Gambian trypanosomiasis. It is now possible, using Mastomys as a laboratory animal, to isolate T. brucei gambiense-like stocks for isoenzyme typing. Pigs and possibly dogs have been shown to be involved, and other species should be sampled. The implications of the serological tests in use still require fo be defined; also that of serum and CSF IgM estimation. The time-scale of rise and fall of titres vis-a-vis infection, re-infection and cure is still insufficiently known, even for man. The significance of ‘false positives’ is uncertain. The effect of frequent inoculation by Glossina bites of non-man-infecting trypanosomes, such as T. vivax and T. congolense, on the host’s serology is unknown. Protozoological diagnosis also reauires to be improved. %enodiagnosis and culture are good diagnostic tools in Chagas’s disease; xenodiagnosis is not nracticable with T. brucei infections, but in vitro culturing should be explored as a diagnostic technique. Isoenzyme typing facilities require to be extended so that they can be used as an epidemiological tool, considering the contributions this approach is beginning to make to our understanding of the epidemiologies of sleeping sickness and Chagas’s disease in Liberia and Brazil respectively. It may well be that in Africa, as in Brazil, there are large numbers of persons sub-clinically infected. The study of the extra-vascular ‘cryptic’ forms of the organism may be important in this respect. The mechanism of the cardiac pathology in T. brucei infections is not understood. but has parallels with that of T. cruzi infechons, and comparative studies could be helpful. The process of antigenic variation in T. b. gambiense-type infections is very little known. Study of this should be intensified as it is relevant both to epidemiology and to the possibility of protection by artificial immunization.

EIGHTEENTH SEMINAR ON TRYPANOSOMIASIS List of Laboratory Demonstrations 23rd Septemter 1977 London School &fezzgcene and Tropical Glasgow University Erotozoology Group and Institute of Tropical Medicine, Antwerp-Antigenic

AND HYGIENE, VOL. 72, No. 2,197s

121

variation in Trypanosoma brucei: heterogeneity of variable antigen types in bloodstream and metacyclic clone populations. L. Tetley, J. D. Barry & K. Vickerman-Pleomorphism and antigenic variation in Trypanosoma brucei.

S. L. Hajduk, J. D. Barry & K. Vickerman-Use of trypanocidal drug DAPI in trypanosome research. S. L. Hajduk-Kinetoplast DNA dyskinetoplastic trypanosomes. J. D. Barry & D. Le Ray-In viva effects of antibodies on Trypanosoma brucei. J. D. Barry-Cloning of Trypanosome &ax. P. Borst-Maxi-circles are absent in the kDNA of T. evansi and T. equiperdum. D. J. Hammond-Anaerobic glycolysis of Trypanosoma brucei. A. H. Fairlamb-Glycerol-3-phosphate oxidase of Trypanosoma brucei: a potential site for chemotherapeutic attack? K. K. Oduro-Glycolysome: a glycolytic multienzyme complex located in subcellular organelles of Trypanosoma brucei.

I. W. Flynn-(i) The mode of action of arsenical trypanocides (ii) Isotachophoresis and the metabolism of arsenical trypanocides. G. Nogge-Aposymbiotic tsetse flies, obtained by feeding on rabbits immunized with symbionts. J. M. Scott, R. G. Pegram, P. H. Holmes, T. M. F. Pay, F. W. Jennings & G. M. Urquhart-lmmunosuppression in bovine trypanosomiasis : field studies with FMD vaccine in an area of endemic trypanosomiasis. D. I. Southern & N. D. Hillen-Interspecific variations in the fine structural surface detail of tsetse fly eggs. L. Hudson, & D. Snary-The surface of Trypanosoma cruzi: its biochemistry and immunology. F. R. Opperdoes & P. Borst-The glycosome in Trypanosoma brucei.

C. E. Clayton, B. M. Ogilvie & B. A. Askonas-The influence of host strain and parasite antigenic type on the course of infection with TrvPanosoma brucei brucei in inbred mice. C. A. Letch-Trypanosomes of small British fishes. T. Vervoort-The alvconrotein antigens of a strain if Trypanosoma br&ei brucei: rest&s of electrofocusing and polyacrylamide gradient gel electrophoresis. D. S. Ellis-The penetration of the peritrophic membrane of Glossina morsitans morsitans by Trypanosoma brucei rhodesiense.

C. J. Schofield & M. J. Lehane-Wind tunnel for the measurement of the speed and duration of flight by triatomine bugs. W. H. R. Lumsden & C. D. Kimber-Miniature anion exchanger/centrifugation technique for detection of low trypanosomaemia in mice. A. G. Gatehouse & W. H. Kwan-Some effects of endosulfan on reproduction in Glossina morsitans. A. J. Liston & J. R. Baker-Influence of immune mouse plasma on intracellular replication and morphogenesis of Trypanosoma (Schizotrypanum) dionisii in vitro. E. H. Popham & V. Chowdhry-Analysis of tsetse fly sounds.

122

SEMINAR

ON TRYPANOSOMIASIS

I. Cunningham & B. M. Honigberg-Infectivity of Trypanosoma brucei cultivated at 28°C with tsetse salivary glands. A. G. Luckins, R. Boid, M. M. Mahmoud, K. H. El Malik, P. Rae & A. R. Gray-Serodiagnosis of infections with Trypanosoma evansi in camels in the Sudan. I. Roitman-Lipids of Trypanosoma cruzi. A. Murray-Serological relationships between stocks of Trypanosoma vivax. G. C. Jenkins, P. McCrorie, C. Ramsey & J. Crosskey-Haematological and immunological observations on calves infected with Trypanosoma vivax. Y. 0. Aliu-(i) A new diluting fluid for enumerating trypanosomes in blood; (ii) Trypanosoma vivax particles in heart muscle and adrenal glands. W. E. Gutteridae & M. Gaborak-Purine and pyrimidine metabolism in Trypanosoma cruzi. A. E. R. Taylor & J. E. Williams-Polypeptide profiles of trypanosomes : an aid to characterization. K. M. Hudson, A. E. R. Taylor & B. J. ElceReversion to basic antigenic type by Trypanosoma brucei. A. R. Mews & M. G. T. Flood-Recent develbpments in in vitro rearing techniques for tsetse flies. A. R. Mews-Feeding Glossina morsitans on turkey blood. R. A. Neal-The number of trypomastigotes of Trypanosoma cruzi required to infect Rhodnius prolixus. R. A. Neal & R. A. Miles-Sensitivity of culture methods to detect experimental infections of Trypanosoma cruzi and comparison with xenodiagnosis. M. Gabarok & W. E. Gutteridge-Studies on the effects of Bayer 760AC on Trypanosoma cruzi. G. W. Rogerson & W. E. Gutteridge-Fatty acid catabolism in Trypanosoma cruzi. B. Cover & W. E. Gutteridge-Effects of drugs on different strains of Trypanosoma cruzi in mice. T. W. Jones-Immunization against metacyclic Trypanosoma brucei.

E. Beveridge-Comparative susceptibility in mice of four strains of Trypanosoma cruzi to nitrofurazone and moxipraquine. P. F. L. Boreham & M. G. Parry-Plasma kallikrein in experimental trypanosomiasis. T. Baltz, P. Degand, J. Schrevel & R. PautrizelLectin binding by Trypanosoma equiperdum variants and chemical characterization of clone-specific glycoproteins. M. S. Grundy-Precipitation of surface variant antigens of Trypanosoma brucei using flat-bed electrophoresis in a granulated gel. P. G. G. Miller & R. A. Klein-Trypanosomal metabolism : (i) acetate precursors and lipid dynthesis; (ii) ATPase inhibition and respiration. C. A. Facer-Renal pathology in experimental African trypanosomiasis : a light microscope, histochemical immunofluorescent and electron microscope study. C. A. Swindlehurst & S. G. Welch-Polyacrylamide isoelectric focusins and SDS electrophoresis of the soluble proteins from ten strains of Trypanosoma evansi. J. Williamson-New agents with trypanocidal or trypanocidal-enhancing activity. D. Rogers 8t S. Randolph-Electric traps for the study of tsetse, and some field results. M. E. Nugent & D. C. Barker-Restriction endonuclease and ultrastructural analysis of kDNA from trypanosomes and synchronous Crithidia fasciculata cultures. A. C. Corsini-Depletion of lymphoid cells and their precursors in bone marrow and peripheral blood in African trypanosomiasis. B. J. Elce-A Trypanosoma brucei contaminant of Trypanosoma vivax isolate Y486. A. A. Poltera-Cardiac histopathology of human African trypanosomiasis and immunopathology of cardiac lesions in murine trypanosomiasis (Trypanosome brucei). B. C. Castro, A. Hochmann & P. H. LambertImmunopathology of cardiac lesions in murine trypanosomiasis (Trypanosoma brucei).