Bull. Org. mond. Sante 1967, 37, 167-175 Bull. Wld Hlth Org.

Trends in Research on the Immunology of Trypanosomiasis

W. H. R. LUMSDEN 1

It was thought timely, in view of the increasingly close association between immunology and parasitology, to attempt a synthesis of research on trypanosomiasis immunology- research which may be expected both to lead to the solution ofpractical problems in this disease and to provide models for the study of basic immunological problems. The author's discussion is based onfour main applications ofimmunological knowledge: for diagnosis, for immunization of hosts, for explanation ofpathological effects, and as a contribution to the understanding of cell function. Stress is laid on the importance of determining the range and extent of antigenic variation in trypanosome material, whether with a view to the development ofreliable diagnostic tests (although quantitative estimation of IgM in serum and cerebrospinal fluid is a useful diagnostic tool circumventing the difficulties caused by antigenic variation during an ) or for assessment of the possibility of developing antitrypanosomal vaccines. Emphasis is also placed, inter alia, on the need for standardization and determination of the purity of stabilate material exchanged between research laboratories.

The history of research into immunological for the measurement of the infective potentiality, as aspects of trypanosomiasis is a long one; it is not to distinct from the organismal concentration, of be thought that interest in these aspects has only trypanosomes (Lumsden et al., 1963). These latter arisen contemporaneously with the extraordinary innovations derive directly from fields of research enlargement of interest in immunology as an indi- other than the purely protozoological-namely, vidual discipline which has occurred in the last artificial insemination and virology--which fact decade. Indeed, one is often surprised to find how emphasizes the value of fostering close contact well the immunological phenomena presented by between any particular field of research and other with these organisms were understood by scientific disciplines. The developing rapprochement workers in the first few years of the century. It is between immunology and parasitology, largely true, however, that the early work done on the stimulated by the World Health Organization (WHO immunology of trypanosome infections led to Expert Committee on Immunology and Parasitic comparatively minor advances in field practice, either Diseases, 1965), is likely, as regards trypanosomiasis, in diagnostic or in immunizing applications, so the not only to improve the rate of progress in solving subject became generally neglected. purely practical problems but also to provide useful The recent reawakening of interest can be attri- models for investigating basic problems in immuno- buted to the general development of immunological logy. studies and the introduction of powerful, yet easily The present paper is concerned practically carried out, immunological techniques: gel diffusion, exclusively with trypanosomes of the section Salivaria chromatography, immunoelectrophoresis and im- (Hoare, 1966). It surveys recent important publica- munofluorescence. Also influential have been the tions and attempts some synthesis of the subject. introduction of methods for the long-term viable For the latter purpose it will be convenient to relate preservation of standard material (Polge & Soltys, discussion to the four main possible applications of 1957; Cunningham, Lumsden & Webber, 1963) and immunological knowledge: for the diagnosis of infection, for the immunization of hosts, for the 1 Head, Protozoology Research Unit, Department of Animal Health, Royal (Dick) School of Veterinary Studies, explanation of pathological effects, and as a con- University of Edinburgh, Roslin, Midlothian, Scotland. tribution to the basic understanding of cell function.

2051 167- 168 W. H. R. LUMSDEN

Reviews of the immunology of the salivarian etc.-have contributed to an impression that special trypanosomes, to which it may be useful to refer, mechanisms operate. It is to be emphasized, there- have been provided recently by Weitz (1960, 1963a, fore, in the first place, that the mechanisms operating 1963c), Brown (1963) and Lumsden (1965a). in protozoal infections, where recognized, appear to be similar to those operative against bacteria and concepts. DEFINITIONS viruses. There is no need to invoke special For instance, premunition and sterile are Nomenclature of trypanosome materials applied to distinguish: (a) a situation of compromise Confusion has sometimes arisen from a lack of between parasite and host, in which the parasite lives clear definition of terms used. The following defini- in the host in a latent state, the host being in a state tions are therefore made for the present paper: of immunity or increased resistance to further infection by virtue of this latent infection; (b) a state Isolate: a section of a wild population separated off of immunity to further infection persisting after the by transference into artificial conditions of maintenance, complete elimination of the infecting agent. Premuni- usually by inoculation into cultures or into laboratory tion, although a valid descriptive term, does not animals (Lumsden, 1965b). stand apart as a separate immunological pheno- Strain: a population, derived from an isolate, main- menon (WHO Expert Committee on Immunology tained in captivity by inducing it to reproduce continu- and Parasitic Diseases, 1965). In any case it will ously by serial passage in cultures or in laboratory animals (Lumsden, 1965b). usually be impossible to establish with certainty that a state of specific acquired resistance is due to Stabilate: a population whose reproduction has been one or the other arrested by viable preservation on a unique occasion. situation. In stabilate material, selection of a continuously re- producing population, such as occurs in a strain, is Composition of avoided, and selection is restricted to that exerted by Williamson & Brown papers, quoted by the processes of preservation and of retrieval from (several preservation (Lumsden, 1965b; Lumsden & Hardy, 1965). Lumsden, 1965a) studied the antigens present in disintegrates of Trypanosoma brucei subgroup organ- Immunological nomenclature isms. The main precipitating antigens were 1S and The immunological terminology and definitions 4S proteins; polysaccharide and lipid antigens were used herein will in general follow those of Humphrey not found. The specificity of protein antigens is & White (1964). Additional glosses are as follows: believed to be due, not to primary structure as in the case of the carbohydrate antigens (the order of Internal antigens: antigens expressed only after the occurrence of the end-group sugars), but to the disruption of the organism; equivalent to Weitz's (1960) foldings of secondary, tertiary and even quaternary bound . structure (WHO Expert Committee on Immunology External antigens: antigens normally manifested by and Parasitic Diseases, 1965). the intact organism. Released antigens: antigens released by the organism Specificity of antigens into the ambient; equivalent to the metabolic product antigen of Thillet & Chandler (1957) and the exoantigen Internal antigens appear to be common not only of Weitz (1960). Probably the released antigen is the same to different forms of a single trypanosome species as one of the external antigens normally manifested on but also to different trypanosome species (Seed, the surface of the organism. 1963; Weitz, 1963b). The internal antigens will, of : terminology of the classes of immuno- course, comprise a wide range of enzymes, structural globulin accords with that given in Nomenclature for proteins and other substances involved in the Human Immunoglobulins (Bull. Wld Hlth Org., 1964). metabolism of the cell. They are less likely to be concerned importantly with the host-parasite rela- DISCUSSION tionship than those manifested normally externally. External and released antigens differ not only Fundamental concepts between trypanosome species but also between the Certain special terms used in describing the different physiological forms of the parasite-blood immunology of zooparasitic infections premunition forms, and insect and culture forms (Seed, 1963)- and sterile immunity, ablastin, -fastness, which forms are distinguished also by differences in TRENDS IN RESEARCH ON IMMUNOLOGY OF TRYPANOSOMIASIS 169 their oxidative metabolism (Vickerman, 1962). place on cyclical transmission. Gray (1965a, 1965b) Further, the blood forms typically vary in their designated a " basic ", relatively stable, antigenic external and released antigens during the course of type in T. brucei subgroup material; the basic type the infection in a single animal. occurred early in infections whether they were The external antigens comprise at least two types: induced by cyclical means or by syringe passage. those eliciting antibody which protects against Further, the process of antigenic variation seems not homologous challenge-PR antigens (Seed, 1963)- to be a random one; the same, or nearly the same, and those detectable by precipitation in agar gel series of antigenic variants appears in different -AG antigens (Seed, 1963). The two types are animals, sometimes even in the same order of separable by fractional precipitation in ammonium succession (Gray, 1965b). sulfate solutions. The AG antigens are of uncertain It must be admitted that much of the evidence biological function as also are the internal antigens. with regard to antigenic variation in trypanosomes, both in the mammal and in the insect host, is still Antigenic variation confused and difficult to interpret with complete Antigenic variation affects the external and confidence, mainly because much of the work has released antigens of trypanosomes and typically, in been done with insufficiently controlled techniques infections of extended duration. antigenically distinct or with material of heterogeneous antigenic speci- variants appear in succession, each corresponding, ficity. Lourie & O'Connor (1937), in a paper probably, to a particular wave of parasitaemia. classical for its precision of experimental approach Typically the serum of an infected animal contains and lucidity of argument, established clearly that antibodies against all the variants that have pre- strains were often antigenically heterogeneous. They viously appeared but not against any of those yet to used a lytic test to distinguish different antigenic materialize. The picture is probably of a rise in types; they found that lytic antisera might lyse none, population of a particular variant, its ultimate con- or all, or a proportion of the individual trypa- trol by the antibody of the host, the same process nosomes of a strain population. In an example of then being repeated with another succeeding variant, the latter result they set up several clones from variants being selected by the antibody response of individual trypanosomes and found that the clones the host. differed in antigenic type. From this they deduced How extensive is the variation possible within one that this strain population was composed of a trypanosome material is not known. Some workers mixture of two or more antigenic types. believe that the possible range is infinite or at least This result emphasizes the need to work only with very large (Gray, 1965a; Watkins, 1964). Fully the most rigorously controlled materials and comprehensive information on this point, despite methods; otherwise the interpretation of results may very painstaking studies by individual workers, is be obscure. For instance, when Glossina are found still not available, either for clones or for trypa- to transmit mixtures of antigenic types it is uncertain nosome species in the laboratory or in the field. whether this is due to failure of some variants to However, the belief that the range of variation is not revert to the " basic " type or to the ingested popula- unlimited is encouraged by the demonstration that tion containing more than one " basic " variant, or some types occur characteristically commonly, or in even to the non-cyclical transmission by the bite of a more or less stereotyped order of succession (Gray, the Glossina of variant organisms arising in the 1965b) and by the finding of Cunningham & " uninfected " animals used to maintain the flies Vickerman (1962) that several antigenic types were after their infecting blood-meals. common to 10 individual Trypanosoma brucei sub- It is clear, also, that an essential piece of informa- group materials, which although all from East tion is the extent of the range of antigenic variation Africa, comprised isolations variously from man and possible within a defined trypanosome material. from wild and domestic animals. No worker has Watkins (1964) has suggested as daunting that the reported more than 20 or so variants in the course of establishment of 100 variants by protection tests his work even though it was very extensive; examples would require 100 000 mice. But even so, precise are given by Brown (1963), Watkins (1964) and Gray information on this point would be well worth that (1965a). Also, work by Gray (1965a, 1965b) has expenditure, which would represent, after all, only a confirmed the earlier conclusions of Broom & Brown few months' consumption of many a virus labora- (1940) that reversion of variants to basic types takes tory. 170 W. H. R. LUMSDEN

Diagnosis difficult to obtain in concentrated suspension or The considerable accumulation of knowledge on which tend to auto-agglutinate, such as Trypanosoma the behaviour of the antigens of trypanosomes has, congolense. Neutralization tests, based on infectivity however, not so far found much practical application titration (Lumsden et al., 1963) have been used by for recognizing trypanosomes of particular bio- Cunningham et al. (1965). Neutralization tests have logical significance, such as infectivity to man, or the advantage that they can be applied at parasite in recognizing infection in the host. Some antigens concentrations far below those necessary for agglu- seem to repeat in man-infecting and non-infecting tination or even lytic procedures. forms (Cunningham & Vickerman, 1962) and the Precipitation in agar-gel systems has been exten- multiplicity of antigens means that failure to discover sively used for the analysis of trypanosomal antigenF antibody in a host may not be due to non-infection (Weitz, 1960; Seed, 1963) but not generally in but to failure to include the appropriate antigen diagnostic applications. Metabolic inhibition tests in the test. (Desowitz, 1956), besides being laborious, are not Immunological tests may be classified from the particularly sensitive (Thurston, 1958). Weitz diagnostic point of view as to whether they are (1963b) labelled with fluorescein antisera to the likely to discover variant-specific antigens (direct internal and to the released antigens of Trypanosoma agglutination, passive agglutination, lysis, neutral- vivax and T. brucei. He found that these organisms ization, precipitation, metabolic inhibition, immuno- did not stain differentially with antibody to the fluorescence), species- or genus-specific antigens internal antigen but that with antisera to the released (immunofluorescence, complement-fixation, skin sen- antigens immunofluorescence was variant-specific. sitivity), or simply a change in immunoglobulin Indirect fluorescence techniques have been used by content (estimation of IgM in the serum or cerebro- Williams et al. (1963) and Sadun et al. (1963) to spinal fluid). demonstrate antibody to T. cruzi, T. gambiense and The investigative tool most widely used recently T. rhodesiense. These workers found cross-reactions has been the direct agglutination test employing living between the different antigens and with T. lewisi. antigen (Cunningham & Vickerman, 1962; Watkins, They suggested that the latter species, non-infective 1964; Gray, 1965a, 1965b; Miller, 1965). Although to man, could be used for a laboratory diagnostic the method has yielded a great deal of information, it procedure. The method was applicable to blood often gives erratic results (Watkins, 1964; Miller, samples dried on paper (Anderson et al., 1961). 1965), probably because the antigens used may not Fife & Kent (1960), investigating the complement- have been pure populations of a single antigenic type fixation test for the diagnosis of T. cruzi infection, or because, prepared in animals, they may already encountered cross-reactions, e.g., with the sera of at the time of preparation have been affected by the subjects with helminthiasis. By the ether extraction hosts' immune response (the agglutination of trypa- of lyophilized antigen and its fractionation with nosomes, frequently observed in wet films of infected chloroform gel they prepared a protein antigen mouse blood, is probably related to the presence of which was specific for T. cruzi. antibody-W. J. Herbert, personal communication). Immunofluorescence and complement-fixation The direct agglutination test, besides being applicable tests deserve further study in the search for a genus- only to antigens available at high concentrations, is specific test for the diagnosis of trypanosomiasis. difficult to standardize in respect of quantities of Delayed-type skin hypersensitivity reactions, which materials used, and end-point determination with it have been developed for Leishmania and Tricho- is often extremely uncertain. Passive agglutination monas infections (Dostrovsky & Cohen, 1957; techniques offer the advantage of dispensing with Feinberg & Morgan, 1953), may also be worthy of the use of living antigen and have been applied with interest in this application. success in plasmodial infections (Stein & Desowitz, In the situation that, because of the complexity of 1964; Desowitz, 1965). They deserve attention in the the antigenic behaviour of trypanosomes, no reliable field of trypanosomiasis (Herbert, 1967). Trypanosoma genus-specific test has yet been The lytic test so informatively used by Lourie & introduced, attention has been directed to one non- O'Connor (1936) has been recently reintroduced as a specific approach with considerable success. Mattern micro-method (R. Le Page, personal communica- (1964) drew attention to the large increase which tion; Lumsden, 1967); it should have particular takes place in the IgM content of the serum and application for Trypanosoma species which are cerebrospinal fluid in trypanosomiasis in man. He TRENDS IN RESEARCH ON IMMUNOLOGY OF TRYPANOSOMIASIS 171 estimated the IgM concentration by setting up a efficient polyvalent vaccines; of the methods of series of pairs of wells in agar gel and charging one selection and incorporation of antigens for adminis- well of the pair with an antiserum specific to IgM, the tration. The matter not appearing quite unpromising, other with a dilution of the serum to be tested. The work has been begun (W. J. Herbert, personal distance to which a discernible line of precipitation communication) specifically to assess the feasibility extended was a measure of the amount of IgM in of developing trypanosomal vaccines using modern the serum. An even more convenient method is that methods of antigen extraction and of adjuvancy. of radial immunodiffusion (Mancini et al., 1965). Most thought so far has been concentrated on the The sera to be tested are introduced into wells in a more obvious antigen-antibody reactions already gel-sheet containing anti-IgM serum; the area of described. It should be remembered, also, that a precipitate developing is proportional to the amount reproduction-inhibiting antibody (ablastin) has been of IgM introduced. The serum IgM in trypanoso- postulated (Taliaferro, 1963) as controlling the miasis is characterically increased to levels clearly intensity of infections with stercorarian trypano- distinguishable from that of healthy persons. somes in rats, though not without criticism (Orme- Although, in Africa, occasional patients under rod, 1963), No evidence of antibodies of this sort treatment for other diseases-tuberculosis, leprosy, acting on the salivarian trypanosomes has been --also show increased IgM levels, in none of noted. The possibility of their occurrence should, these diseases was the IgM characteristically in- however, not be discounted; antibodies specific to creased. It may be, however, that it is characteris- purine or pyrimidine moieties attached to protein tically increased in Plasmodium nalariae infection have been shown to inhibit the development of (Lumsden, 1966). It is clear that the method, simple fertilized Echinus eggs (WHO Expert Committee in its application, offers a valuable test for field use on Immunology and Parasitic Diseases, 1965); a for the rapid recognition of suspected infected per- mechanism of this kind might be operative in sons. Whether equivalent IgM rises take place in the determining the long-term low-level parasitaemia serum of animals is yet unstudied. that is characteristic of trypanosome infection in wild animals and in some breeds of domestic cattle. Immunization Little is known of the cellular processes which It has long been known that the immunization of must be involved in destruction of trypanosomes hosts against single antigenic types of a Trypanosoma in vivo. Phagocytosis of trypanosomes has been species is easily accomplished, either passively by the reported by Dodin et al. (1962) in mice, but this inoculation of immune serum or actively by several observation does not appear to have been followed methods-the induction of an infection by the up. inoculation of virulent organisms and then cutting short the infection by chemotherapy, or by the Pathogenesis inoculation of inactivated organisms or of released Considering their importance, it is extraordinary antigen. As regards inactivating agents for the that the mechanisms of pathogenesis of trypanosome preparation of vaccines from whole living organisms, infections have been so neglected, and are still so Soltys (1965) reported on the use of ,B-propiolactone poorly understood. For instance, it is well known and considered it superior to formol and phenol. that, except with strains highly adapted to laboratory It is to be noted, however, that rather large con- rodents, death from trypanosomiasis is not typically centrations of formol and phenol were used, such as associated with a fulminant parasitaemia but rather may have destroyed the structure of the antigens. with a cachectic condition in which organisms are Although the immunization of an animal against difficult to demonstrate. The possibility of immuno- single antigenic types is straightforward, the develop- logical reactions being determinant of pathological ment of practical methods of vaccination for the effects in situations of this sort has been emphasized field demands knowledge: of the duration of immu- (WHO Expert Committee on Immunology and nity to any antigenic type following administration Parasitic Diseases, 1965) and so it is thought worth of vaccine; of the extent of the range of antigenic while to dilate on the subject here. Broadly, antigens variation of the trypanosome populations likely to may react with pre-existing antibody (leading to be encountered in nature; of the possibility of immediate-type hypersensitivity) or with sensitized cross-reactions between individual variant antigens cells of the lymphoid-macrophage system (leading to reducing the number of antigens necessary for delayed-type hypersensitivity). 172 W. H. R. LUMSDEN

Immediate-type hypersensitivity. Antibody-antigen activating them so that they also may react with the complexes in a suitable spatial orientation trigger antigen. Reactions associated with delayed-type off the sequential activation of the components of hypersensitivity include: following systemic injection complement. When complement is activated close of antigen, lymphopenia, fever or death; following to a cell membrane the membrane may be perforated intradermal inoculation, erythema, induration and and the cell destroyed. Only one or two IgM mole- necrosis; following intraperitoneal injection, dis- cules, but many IgG molecules, per cell are needed appearance of free peritoneal macrophages, which to produce the same effect. Many immunoglobulins adhere to one another and to the peritoneum, and are reversibly adsorbed to the surfaces of tissue cells enhanced capacity of macrophages for intracellular and interaction of antigen with such adsorbed anti- destruction not only of the specific sensitizing body may lead to disruption of the cells. With mast organism but of other antigenically unrelated cells the pharmacologically active substances indu- organisms. Delayed-type hypersensitivity is held to cing local or systemic anaphylactic phenomena are account for some of the pathological effects of released. Serum complement may not be necessary tuberculosis; Mycobacterium tuberculosis has little for cell disruption. It is possible, also, that, besides power to produce necrosis of tissue in the normal cell disruption, less obvious damage may occur- body but when hypersensitivity is established both acute (e.g., Arthus reaction) or subacute necrotizing the organisms and the tuberculoprotein act as inflammatory lesions, or slowly developing hyaline though they are highly toxic. How far reactions degeneration. Remembering that the serum of of this sort are of importance in trypanosomiasis is patients with trypanosomiasis contains large quanti- unknown but they merit consideration; a tuberculin- ties of IgM and that the trypanosomes release antigen like skin reaction can be obtained in leishmaniasis into the plasma, it seems rather likely that the by the intradermal inoculation of antigen. antigen-antibody complexes formed may exert patho- genic effects of the sort described. It has been sug- Cell function gested that the nephrotic syndrome associated with Ormerod (1966) discusses the trypanosome as a Plasmodium malariae infection is secondary to secretory cell and suggests, on electron microscope antiparasite antibody production on the part of evidence, that two different kinds of ribosomes the host. (protein-synthesizing cytoplasmic particles) exist in Another mechanism of pathogenesis is by anti- the cytoplasm of T. rhodesiense. One type occurs bodies against the antigens of a given tissue. In free in the cytoplasm and resembles the ribosomes in experimental models, a heterologous antikidney reticulocytes which synthesize haemoglobin; Orme- antibody will react with the glomeruli of the reci- rod suggests that these ribosomes synthesize protein pient, causing immediate damage, and then, later, materials for use within the cell-enzymes, perhaps the host's antibody reacts with the heterologous structural proteins, etc. These would comprise the immunoglobulin fixed in the glomeruli and induces internal or bound antigens. A second type of further damage. It is possible that the breakdown ribosome occurs attached to an endoplasmic reti- products either of damaged tissues or of the trypa- culum and resembles the arrangement present in nosomes themselves become localized and initiate secretory cells such as the acinar cells of the pan- processes of this sort. creas. Ormerod suggests that these ribosomes secrete Delayed-type hypersensitivity. Delayed-type hyper- the released antigen as the stage of the infection at sensitivity to antigen (characteristically slower in which this antigen is produced coincides with the onset and in decline than immediate-type hyper- endoplasmic reticulum being distended. sensitivity) does not depend on humoral antibody; As regards the mechanisms concerned in antigenic it is not transferable by serum from one animal to variation, Watkins (1964) attempted to distinguish another but only by living lymphoid cells. A working between adaptation on the one hand and mutation hypothesis of the mechanisms involved is that small and selection on the other as mechanisms by which lymphocytes become sensitized and elaborate an a change in antigen could take place under the antibody-like material which is present on their influence of antiserum. He favoured the mutation surface and which reacts with antigen so that the and selection hypothesis because large populations, sensitized cells are altered or destroyed. This mate- of the order of antilog 5 organisms, failed to adapt. rial can also be transferred to macrophages, either From comparisons of the mean survival time of before or after the lymphocytes have reacted, mice, infected with trypanosomes from 6-day-old TRENDS IN RESEARCH ON IMMUNOLOGY OF TRYPANOSOMIASIS 173 infections in mice, unprotected and protected by genetic transfer in trypanosomes either by syngamy antisera homologous to the original inoculum, he or by transformation. attempted to estimate the mutation rate. He Thivolet et al. (1965) have exploited the finding of obtained values varying from antilog 5.04 (about earlier workers that the nuclei of trypanosomes differ 1 in 90 000) to antilog 6.63 (about 1 in 230 000), with from those of the cells of higher organisms in being composed of complexes of desoxyribonucleic acid a mean value of antilog 6.83 (about 1 in 150 000). (DNA) with a protein other than histone or, like the Watkins goes on to consider the possible number of nuclei of bacteria and viruses, of DNA alone. By antigenic variants and considers that, although it immunofluorescent techniques the nuclei of trypa- might be limited, its determination would be labo- nosomes were stained only by sera containing anti- rious. He estimated that the establishment of 100 bodies against DNA. The techniques could be variants by protection tests would involve 100 000 applied to the titration of antibodies to DNA. mice. However, as he considered the antigens eliciting protective and agglutinating antibodies to be the same, an agglutination test or some equivalent CONCLUSION test should be substituted, and, further, it seems unlikely that the number of variants is as high as Recent trends in immunological research in 100, considering the reiterative occurrence of parti- trypanosomiasis have been summarized; to conclude cular variants (Gray, 1965b). it may be useful to emphasize some of the points which seem important to me. The detailed analysis of the genetic control of In the first place it is important to diminish the antigenic variation which has been made for the isolation from other biological disciplines from ciliate Paramecium (Beale, 1954) has been possible which studies on trypanosomes have suffered in the because among these organisms genetic transfer past, partly for geographical, partly for experimental takes place by syngamy. The evidence with regard and nomenclatural, reasons. A widening of the range to genetic transfer by syngamy and by transforma- of scientific disciplines interested in trypanosomes as tion in the Trypanosomatidae has been considered by experimental tools will be most advantageous in Amrein (1965a, 1965b). This author (1965a) improving the level of critical assessment of past and categorizes the morphological evidence for syngamy present studies and in orientating future ones. It is produced by previous workers as " doubtful ". He essential to attempt the explanation of the pheno- himself, in a different type of approach, used strains mena presented by trypanosomiasis in generally of T. cruzi individually marked by induced resistance acceptable immunological terms. to different drugs nitrofural (nitrofurazone) and A great deal of knowledge has been accumulated primaquine. He fed Triatoma species on mixtures by the efforts of individual workers but often now of the two strains and found that resistance to the the impediment is simply the magnitude of the work drugs was not lost in the cycle of development in the necessary. For instance, the definition of the range insect. However, none of 37 isolations made from of antigenic variation possible will inevitably daunt Triatoma faeces was resistant to both the drugs. the lone worker. Nevertheless, it appears to be an Amrein (1965b) considered unclear the results essential piece of information, and so calls for a obtained by several previous workers with regard more concerted approach. If the information is to genetic transformation. He himself prepared agreed to be essential it may be obtained and the nucleic acid extracts of normal, akinetoplastic, and need to expend 100 000 mice is not a valid impedi- Antrycide- and tryparsamide-resistant strains of ment. T. equiperdum and tested the transforming effect of It is disappointing that the implication of viably these extracts by exposing trypanosomes to them in preserved trypanosome stabilates and the value of vitro and then inoculating the trypanosomes into infectivity titration methods to attain reproducibility animals. There was no evidence for a transforma- and reliable quantitation in trypanosome experimen- tion of drug-resistance; the only observed effect was tation are so slow to be appreciated. Some recent a heightened percentage of akinetoplastic organisms papers have still described trypanosome inocula occurring after exposure to the appropriate extract derived directly from infected animals and have but this was considered of questionable significance expressed their size in numbers of organisms, thus as indicating the occurrence of transformation. raising doubts both as to the antigenic character of The sum of present evidence, therefore, is against the inocula and as to their real infective potential. 174 W. H. R. LUMSDEN

It seems essential that practices in this respect should such as infectivity or non-infectivity to particular be improved. hosts, and metacyclic trypanosomes. As long as 30 years ago the work of Lourie & As regards diagnostic procedures, improvements O'Connor (1937) established the prime need, for are needed in tests both for variant-specific and for reliable experimentation, of rigorously defined group-specific antigens. For the former application materials. It would be advantageous if these could passive haemagglutination and lytic tests appear be prepared in large quantities and made generally likely to be most promising; for the latter, comple- available to interested workers. A more rapid ment-fixation or immunofluorescence techniques. synthesis of knowledge should be possible if several The simple estimation of IgM immunoglobulin is different approaches were concentrated on well- also promising, and should be further investigated. defined and comparable materials rather than For assessment of the practical possibility of dissipated on a miscellany of materials of uncertain immunization, knowledge of the range of antigenic status and relationship and of ephemeral duration. variation possible in the laboratory and in the field, Concerting of studies in this way should offer and of the degree of cross-reactioa to be expected little difficulty as strictly comparable materials may between antigens in a polyvalent vaccine is required. now be exchanged between laboratories as stabilates. Techniques for the preparation of vaccine with The material available in this way, however, still economy of antigen will require to be developed. requires improvement as regards its definition. It The investigation of the pathological effects of should include a variety of clone populations of trypanosomiasis in terms of modern concepts of several Trypanosoma species, selections of popula- immunopathology is a wide-open and obviously tions of rigorously established biological potentiality, important field.

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Il ne faut pas s'attendre a ce que les mecanismes de complement et d'immunofluorescence paraissent pro- l'immunologie des infections a trypanosomes soient fon- metteurs comme tests specifiques de groupe. De meme, damentalement differents de ceux qui interviennent dans en cas de variation antigenique au cours d'une infection, les infections bacteriennes ou virales. Certains termes sont l'evaluation quantitative d'une classe d'immunoglobu- discutes et definis. line (IgM) dans le serum et le liquide cephalo-rachidien Les antigenes trypanosomiens se divisent en antigenes est un moyen de diagnostic utile. internes qui ne se manifestent habituellement qu'apres On envisage la mise au point de vaccins contre les desagregation du micro-organisme, en antigenes externes trypanosomiases. On immunise aisement des animaux qui se manifestent normalement alors que le micro- contre le variant antigenique homologue en leur inoculant organisme est intact, et en antigenes liberes, existant en des micro-organismes inactives ou de l'antigene libr&. dehors des corps trypanosomiens dans le milieu ambiant. Mais pour estimer les possibilites de preparer des vaccins Les antigenes internes sont typiquement specifiques de antitrypanosomiens, il faudra connaitre parfaitement groupe ou de genre et invariants. Les antigenes externes l'ampleur de la variation antigenique a prevoir. et les antigenes liberes sont typiquement covariants et se Quant a la pathogenese des infections a trypanosomes, modifient au cours de l'infection chez l'h6te, chaque type il se pourrait qu'elle depende des reactions antigene- de variant etant specifique d'une poussee particuliere de anticorps et les mecanismes possibles sont discutes. parasitemie. L'intervalle de variation antigenique possible L'elargissement de l'eventail des disciplines scientifiques a l'interieur de chaque materiel trypanosomien n'a pas applicables au domaine des trypanosomes en tant que e etabli; il est probablement etendu, mais non infini. moyen d'experimentation est considere comme souhai- L'ampleurdevariationpossible apparaitcommeunelement table et facile a realiser maintenant que les laboratoires essentiel d'information qu'il serait necessaire d'acquerir. peuvent sans difficulte echanger des stabilats conserves Des tests de determination des antigenes specifiques sous forme viable. La necessite de normaliser et de definir de genre et specifiques de variant, ainsi que des anticorps la purete des stabilats est soulignee ainsi que celle de sont discutes. Pour le materiel specifique de variant, les realiser la reproductibilite et l'evaluation quantitative de tests d'agglutination ont et ces derniers temps tres l'experimentation pour definir les inoculums de trypano- utilises, mais presentent des inconv6nients; la mise au somes en fonction de l'infectiosite determin6e par titrage point de tests d'agglutination passive, de lyse et de neu- et, eventuellement, par le nombre de micro-organismes, tralisation est recommandee. Les tests de fixation du au lieu de les definir seulement en fonction de ce nombre. TRENDS IN RESEARCH ON IMMUNOLOGY OF TRYPANOSOMIASIS 175

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