Bull. Org. mond. Sante Bull. Wld Hith Org. 1971, 44, 499-514

Immunology of Leishmaniasis*

D. HEYNEMAN 1

Knowledge of the immunological basis of the leishmaniases and of the host's response is fragmentary and largely pragmatic. This paper reviews certain conceptual and clinical aspects of the immunology of these diseases. Consideration is given to man's natural resistance and his ability to acquire resistancefrom natural infections andfrom vaccination. The age-distribution of infection in different populations is discussed in relation to the effects that interaction between the parasite and its intermediate host may have on its infec- tion characteristics and virulence. Studies in the USSR ofdifferences in virulence among 30 human strains and 39 rodent strains are reported. The rodent strains showed a broader range of virulence than did the human isolates. Serological testsfor determining species relationships among the leishmaniae are generally nonspecific, but work concerned with the development of the antiserum- culture test is reviewed. Species identification and the recognition of new forms, perhaps with different infection characteristics, is, nevertheless, of the utmost importance in the prevention and treatment of the disease. The review concludes with a discussion of functional immunity and hypotheses of the immune process in leishmaniasis.

INTRODUCTION or pathology when it is observed repeatedly in man or in experimental animals infected in a standardized Immunology is fundamentally a measure of the fashion. In unusual cases, such as in leishmaniasis living interaction between parasite and host. This tegumentaria diffusa, the response can tell us much interaction, carefully measured and compared in about the host as well. different Leishmania-host combinations, has been Immunology includes the practical application of our best clue to the biological uniqueness of the serological and skin testing procedures in diagno- parasite, its genetic identity and constancy, and sis and in studies of disease prevalence in addition to its speciation and evolution. its usefulness in distinguishing between Leishmania The key problem for a study of the host's immuno- species. Ideally, cultures of unknown Leishmania logical response is to separate invasiveness, patho- tested against known antisera should enable us genicity, and resistance into their intrinsic host and to identify the parasite and determine its antigen parasite components. A number of species and array and reaction limits; known parasite strains strains of Leishmania undoubtedly exist. Among should similarly be useful in identifying test sera. the hosts, there are differences in individual as The realization of this goal with Leishmania is only well as in species response to infection. It is often in the experimental stage. Most tests are nonspeci- difficult or impossible, therefore, to distinguish be- fic or group-specific, permitting detection of anti- tween innate virulence of the invader and immuno- gens shared by related organisms. They do not logical failure of the host. Yet we characterize distinguish antigens confined to a single species, Leishmania parasites by host susceptibility, resistance, as group reactions mask specific ones. The extreme morphological similarity among related flagellates * This study was supported by ONR Contract XYZ- YZO-1, 867,233.234, under Research Project MR005.09- is paralleled by their many shared antigenic com- 1603.1, Bureau of Medicine and Surgery, Department of the ponents. Navy, Washington, D.C., USA. One diagnostic technique often used is the delayed- I Professor of Parasitology, Department of International reaction skin test, the Montenegro (leishmanin) Health and The George Williams Hooper Foundation, University of California, San Francisco, Calif. 94122, USA. reaction. It is not highly specific and does not

2662 -499- 3 500 D. HIEYNEMAN distinguish current from past infection. Appli- (2) When infected macrophages remain in the cation of the test over large areas, however, may dermis, an island of infected cells forms. Eventually enable us to deduce migration pathways of leish- this induces a strong host cell reaction of proliferat- maniasis or at least past leishmanial foci. It is also ing infected macrophages, plasma cells, and lym- useful for the diagnosis of L. brasiliensis, especially phocytes that produces a lesion. Extension of the when parasites are often scarce or difficult to lesion, ensuing tissue destruction, and local spread demonstrate, as in espundia. of infection cause the development and charac- Prophylaxis against cutaneous leishmaniasis is a teristic ulceration of cutaneous leishmaniasis. managed infection involving inoculation of a (3) In visceral leishmaniasis, a dermal lesion living culture. Site, duration, and intensity of may develop initially, but this disease is charac- infection are carefully controlled in this induced terized by the escape of infected cells from the disease. Prophylactic infection has been best dermis into the major lymphocytopoietic and developed in the Soviet Union and its use protects macrophage-filtering centres, that is, into the thousands of workers in endemic areas, persons who spleen, the liver, the bone marrow, and the lymph otherwise would be disabled or seriously afflicted glands. A massive generalized lymphocytogenesis by disfiguring sores and painful long-lasting ulcers. in these organs leads to extreme splenomegaly and Serological tests demonstrate serum antibody, hepatomegaly. Accompanying this is an exceedingly not immunity, in contrast to the prophylactic heavy hypergammaglobulinaemia, an outpouring sore, which stimulates full protection. As with many of largely nonspecific complement-fixing antibodies tissue infections, serological titre does not necessarily into the serum, and a progressive gross lympho- measure the host's specific defensive capacity. The cytic leucopenia. two, in fact, may show an inverse relationship. Although serum antibodies are readily detected (4) Resistance to artificial or natural cutaneous by the complement fixation test (CFT) in visceral leishmaniasis is well-developed after the full course leishmaniasis, host protection does not normally of infection. Superinfection results in synchronized develop. In cutaneous leishmaniasis, on the other healing of both primary and secondary lesions, hand, no CFT response is obtained, but total varying with the host's state of resistance stimu- immunity usually follows initial infection. This lated by the first infection, as stated in the " Re- does not rule out an antibody basis for the host's inoculation Law" of Moskovskij (1937). defensive mechanism. It implies rather that such (5) Resistance to visceral leishmaniasis is essen- antibodies are cellular and locally concentrated, tially absent once the splenic phase begins, much presumably in the skin or around the infected as in the Syrian hamster. Death is the usual result phagocytes and in some way related to local infil- of untreated infection, resulting from an over- tration by lymphocytes and plasma cells. Anti- whelming lymphocyte hyperplasia. However, resis- bodies released into the blood are not effective in tance is as strong after chemotherapeutic cure of aiding this localized immune response. The particular visceral leishmaniasis as it is after the healing of pathological characteristics of the various leish- a cutaneous leishmaniasis ulcer. Presumably pro- maniae are intimately related to the degree of tection against both depends upon skin sensiti- localization of the host response and the con- zation and effective antibody concentration or tainment of infected macrophages. A reliable lymphocyte activation. immunological test capable of distinguishing cell- (6) Cure or healing of either type of infection bound antibodies and specific tissue-active protective is indicated by a delayed sensitization tuberculin- processes is needed. Perhaps the passive cutaneous type skin response (Montenegro or leishmanin anaphylaxis (PCA) test could be used. In vitro test), which is, however, rare or absent in Indian tests might be developed to measure attraction of kala azar. specifically activated lymphocytes towards infected rather than non-infected macrophages. Among the important questions still to be To emphasize poorly understood and little- answered are the following: known aspects of leishmaniasis, we can restate some (1) What is the specific role of plasmacytes and of the host-parasite characteristics of the disease: lymphocytes in the healing of cutaneous lesions? (1) It is almost exclusively a disease of the host (a) Do specific antibodies attack amastigotes macrophage (histiocyte) system. only when they are released from destroyed IMMUNOLOGY OF LEISHMANIASIS 501

macrophages? Are these antibodies, which are In few parasitic diseases are the problems, the released by sensitized lymphocytes, locally concen- challenges, and our ignorance of host-parasite trated? interactions more obvious and more in need of (b) Is there specific dissolution of infected clarification than in leishmaniasis. macrophages by activated lymphocytes? (c) Are the amastigotes attacked within the NATURAL RESISTANCE OR SUSCEPTIBILITY intact but presumably modified host macrophage or when infected macrophages are surrounded Man is one of the most susceptible hosts to all by sensitized lymphocytes? forms of leishmaniasis. The Syrian hamster (Meso- cricetus auratus) is even more susceptible, and is (d) Is there a nonspecific histamine reaction apparently unable to develop any resistance during and a walling-off of the lesion, thus isolating the course of an infection. Infections of L. tropica it during the course of an immune reaction ? are strictly limited to the skin and spontaneously (2) What is the stimulation and mechanism res- cured in man, but some strains will invade the ponsible for massive lymphocytogenesis? How is viscera in hamsters and kill them. A similar invasive this related to the splenomegaly and hepatomegaly capacity exists for certain other rodents tested in visceral infection, in which local concentration in the laboratory. On the other hand, white rats of lymphocytes is combined with leucopenia in the and rabbits will quickly eliminate an inoculum of general circulation? L. donovani that would be fatal to man, hamsters, or the chinchilla (Chinchilla panigera). White mice (3) What is the source and stimulation for specific will retain for life an L. donovani infection at a complement-fixing antibodies in visceral but not low level while the cotton rat (Sigmodon hispidus) in cutaneous infection? can sustain a massive lifetime infection without (4) What is the source and stimulation for the apparent ill-effect. The guinea-pig, the clawed jird genesis of nonspecific serum gamma-globulin in (Meriones unguiculatus), and white mice can tolerate visceral but not cutaneous infection? and eventually destroy an infection. In these cases it is a matter of the speed and effectiveness of an (5) How do parasite and host share responsi- innate host response. The capacity to resist infection in cutaneous bility for parasite localization, which or invasion of the viscera clearly varies among leishmaniasis causes restriction of infection to species and reflects more the influence of the host dermal macrophages and, in visceral leishmaniasis, than that of the parasite. Human reaction shows involves a generalized reticuloendothelial invasion? a broad range of response, although the parasites Visceral leishmaniasis follows a fairly well- do appear to differ in their inherent ability to defined progressive systemic course, essentially an invade the viscera. all-or-none reaction. The range of cutaneous Epidemic outbreaks of visceral leishmaniasis leishmaniasis, however, is from a minor non- have taken thousands of lives and destroyed whole ulcerating nodule, to an involvement of the entire villages in Assam, Kenya, and the Sudan. The speed dermis (as in leishmaniasis tegumentaria diffusa), of the outbreak and the state of acquired immunity or to the metastatic destruction of all nasal and in the population were without doubt closely buccal mucous linings and cartilage (as in espundia). related to the epidemic patterns that developed. Furthermore, except in espundia and individual Zoonotic cutaneous leishmaniasis has been repor- intractable cases, cutaneous infection is self-limit- ted from desert and semi-desert zones of Uzbekistan ing (or host-limited). How can we account for and Turkmenia in the USSR. In urban populations these differences? of Ashkabad in Turkmenia and in Kirovobad in (6) What is the relationship between degree of Azerbaidzhan the anthroponotic type of cutaneous sensitization of dermal lymphocytes and plasma leishmaniasis was once common but is now extir- cells in the skin and control of cutaneous infection? pated. The distribution of infection in these people How is this related to leishmaniasis recidivans? reflected the typical pattern of disease in an exposed To leishmaniasis tegumentaria diffusa? To espundia population: young children of the indigenous popu- of L. brasiliensis? To the kala azar dermal leish- lation and persons of all ages among newcomers. manoid of L. donovani (a post-cure skin nodular Mediterranean zoonotic kala azar caused by reaction common in India)? the infanta form of L. donovani is equally distinc- 502 D. HEYNEMAN tive. Infection is widespread among village dogs reported. This probably accounts for the failure and in children is generally limited to those under of apparently susceptible individuals to become 3, declining in frequency in older groups. A diffe- infected during mass outbreaks. Napier (1924) rent age-distribution characterizes the anthroponotic reported similar " spontaneous " cures among a form of kala azar in India, where adolescents and significant proportion of Indian kala azar cases. young adults comprise the most commonly infected Southgate & Oriedo (1967) and Southgate & Manson- group. In China, another zoonotic region, the Bahr (1967) found a number of Kenyans leishmanin- picture is more like the Mediterranean one. These positive. A group of 13 volunteers among them distinctive patterns indicate more than innate successfully withstood the challenge of L. donovani differences in the human populations. Each area from a human patient. Perhaps these Kenyans also has its specific Leishmania vector. Each vector had had asymptomatic kala azar, or an infection may interact in a specific way with the parasite's restricted to a dermal lesion, as Cahill (1964) metabolism, development, and infection charac- suggested in 4 American cases in the Sudan. On teristics. Direct or indirect influences of the inter- the other hand the local population is constantly mediate host on the promastigotes must be included exposed to natural infections with mammal or in the host-parasite complex. Presence or absence lizard leishmaniae. Such exposures over a long of a vertebrate reservoir host and the cycling rate enough period might also stimulate an immunity may also affect the virulence of the parasite in to L. donovani, or cause the organism to be restricted man, and determine the epidemiology of the result- to a dermal lesion. A significant proportion of ing disease. those Sudanese with no history of kala azar who were skin-tested by Cahill showed a positive res- ponse. As in Kenya, the percentage of positives ACQUIRED RESISTANCE FROM NATURAL INFECTION increased with age-an epidemiologically useful Resistance to cutaneous leishmaniasis following finding. Absence of cross-immunity between kala natural infection in man is generally considered azar and other disease strains sharply differentiates to be 97-98% effective. Davydov (1963) reports it from the pattern of immunity against cutaneous a record 10% reinfection rate. This is an exceptional leishmaniasis in the Soviet Union. A considerable situation that developed in Turkmenia where 80-90% degree of cross-reaction exists between L. tropica of the non-immunes were infected and as many as major and L. tropica minor, though a virulent full- 50 % of the sandfly vectors were positive. Immunity course dermal infection is required to produce is a quantitative matter; therefore several lesions complete immunity. L. t. major infection can pro- acquired during the susceptible period will confer tect against both forms, but L. t. minor protects a stronger immunity than a single lesion. Similarly, only against the relatively less virulent homologous the more severe the lesion the faster the host res- strain. ponds, the quicker the lesion heals, and the stronger the immunity induced. Immune individuals who ACQUIRED RESISTANCE FROM CONTROLLED leave an endemic area and return some years later INFECTION (VACCINATION) are still immune. Immunity to visceral leishmaniasis is apparently The resistance to reinfection produced by the absolute if the patient survives his first infection, natural disease is most effective, as stated above, that is, after chemical cure. But resistance prior if the strain is a virulent one and the infection to cure is seldom enough to control the initial is allowed to run its full course. Interruption by infection. Immunity to mucocutaneous infection is treatment, surgery (Marcinovskij & kurenkova, much less certain. As with visceral leishmaniasis, 1924), or infection with a nonulcerating strain there is probably strong resistance to a dermal produces a corresponding diminution of protection. challenge. But pockets of the original infection This is equally true of a vaccination infection. appear to escape and may metastasize years later to " Vaccination " against cutaneous leishmaniasis is produce the destructive mucocutaneous lesions that a controlled but full infection with a freshly isolated are probably never completely cured even with virulent strain of living organisms, usually a zoonotic chemotherapy. strain from gerbils or human patients. The elimination of asymptomatic or avirulent Dr 0. I. Kellina's careful laboratory studies and infections in foci of endemic leishmaniasis has been Dr N. F. Rodjakin's extensive trials with various IMMUNOLOGY OF LEISHMANIASIS 503 antigens demonstrate the need for a fully viable form has been reported to prevent cutaneous strain that will cause the patient to pass through infection. These reports require further investigation. the ulcerative and healing stages to ensure pro- Immunization against visceral leishmaniasis has tection. Although Rodjakin reported at the Semi- been attempted on a large scale only in Kenya by nar that relatively virulent L. tropica minor can Dr P. E. C. Manson-Bahr, using Dr. R. B. Heisch's protect to some degree against L. tropica major, ground-squirrel strain of Leishmania. Whereas the the more virulent zoonotic form is far more effective cross-immunity experiment worked well among and has been used in the tens of thousands of volunteers challenged with L. donovani cultures inoculations performed in the Soviet Union. An from a human case, vaccination with ground- avirulent inoculum will sensitize the skin to a squirrel Leishmania failed to prevent infection when leishmanin test, but it will not protect the individual transmitted in the normal fashion by sandflies. against a more virulent strain. However, only about 20% of the "immunized" The " Reinoculation Law " of Moskovskij (1937) population were actually converted to a positive is particularly well illustrated in cutaneous leish- leishmanin response, so the immunization proce- maniasis. If a challenge or second inoculation dure followed in this large-scale clinical experiment occurs during the course of a first infection, the was unsatisfactory. The possibility of protective second lesion quickly reaches the same stage of immunization with an immunogenic strain that ulceration as that of the first ulcer, after which does not invade the viscera therefore is questionable. both sores heal synchronously. There is apparently The extent of cross-immunity between strains no direct effect of one lesion on the other, but the varies with infection conditions. Length of exposure first causes a reaction in the host that is responsible to a primary infection appears to affect the result for a similar reaction against any subsequent of a challenge by a second strain. This may help infection. The rodent form of visceral leishmaniasis to explain some of the puzzling findings in cross- in Kenya also illustrates this principle. Reinocu- immunity tests between L. tropica, L. brasiliensis, ation of volunteers with ground-squirrel Leishmania and L. mexicana. produces a nodule that heals concurrently with the preceding nodule, after which leishmaniae disappear simultaneously from both lesions. In PASSIVE TRANSFER OF IMMUNITY Chiclero's ulcer of Mexico, however, immunity Immunity transfer from mother to child does not to a new infection develops very rapidly, so that a occur via milk-borne antibodies in cutaneous challenge inoculum may heal after the primary leishmaniasis (Gitel'zon, 1933; Rodjakin, 1957) and one has already healed. L. enriettii of guinea-pigs (Demina, 1964). Antibodies may well be transferred in this manner but, as CROSS-IMMUNITY in human visceral leishmaniasis, there is no evidence that such antibodies are functionally protective. Some degree of cross-reaction develops between Adler & Nelken (1965) demonstrated passive the strains of L. tropica, but, as noted above, transfer of delayed dermal hypersensitivity using L. t. major can break through the immunity barrier human antiserum, but they failed to show successful established by L. t. minor. Panamanian L. tropica passive transfer when they use whole blood or protects against L. mexicana, but the latter does white cells taken from hypersensitized human not provide protection against the former (Lainson donors. The whole blood or white cells were & Shaw, 1966). In rhesus monkeys, L. mexicana inoculated intradermally into a volunteer who then protects against L. brasiliensis, but the reverse was challenged with leishmanin. Dr L. A. Stauber does not occur (Lainson & Bray, 1966). Sufficiently (personal communication) indicates that in his strong dermal involvement appears to stimulate laboratory Dr Doisia has succeeded in demon- cross-protection against related forms that produce strating passive transfer of sensitivity in guinea- a less acute cutaneous infection. Thus, if infection pigs inoculated intradermally with rabbit antiserum. from L. brasiliensis could be reduced to a brief Doisia found that the inoculation technique selected, dermal one, fear of the terrible mucocutaneous the quantity of antiserum used, and the choice of after-effects that now strike about 80 % of L. host were crucial for the successful passive transfer brasiliensis victims would vanish. L. tropica does of sensitivity. Dr K. J. Ott, another student of not protect against L. donovani, though the visceral Dr Stauber, demonstrated reduction of a super- 504 D. HEYNEMAN infection in mice inoculated with guinea-pig anti- set of measurements for titrating results with this serum. Both findings are highly interesting and test. She made a valuable comparison of various point to renewed interest in the serological aspects lizard strains with rodent and human strains of of visceral leishmaniasis. L. tropica major; Dr M. de V. Coelho applied the technique to the New World forms, the results indicating that " L. pifanoi" is a form of L. brasi- SEROLOGICAL TECHNIQUES liensis. Studies are also under way at the Wellcome The standard serological tests that are available Parasitology Unit in Belem, Brazil, where Dr R. emphasize the failure of leishmaniasis to follow Lainson is investigating the difficult New World a pattern suitable for conventional microbial sero- L. tropica complex. logy. Antibodies cannot be detected in most cases Bray & Rahim (1969) have developed a promising of cutaneous leishmaniasis, as in many other intra- haemagglutination test in Iraq, using sheep cells cellular and tissue parasitic infections, such as sensitized with polysaccharide extracts of the tested leprosy, tuberculosis, and infections with various parasites. metazoan parasites. In visceral leishmaniasis, posi- Professor 0. Theodor has suggested that sen- tive serological findings are not correlated with sitive immunoelectrophoresis and diselectropho- the state of immunity. resis procedures might differentiate species of Agglutination and complement-fixation tests are Leishmania, as they have malaria parasites. relatively nonspecific in leishmaniasis, though the Schneider & Hertig (1966) used a micro agar-gel CFT is useful as a diagnostic technique, particularly immunodiffusion test at the Gorgas Laboratory in canine visceral leishmaniasis, where cross-reaction in Panama. They studied 5 human strains and 11 with Chagas' disease is not significant. sandfly strains-8 from local sandflies and 3 from The fluorescent antibody test is group-specific South America. Two distinct groups were charac- and loses its reactivity after absorption of common terized, but these were not related geographically antibodies. or with specific sandfly hosts. Common antigens Demina (1962) showed that promastigotes of again masked the results. L. tropica or L. enriettii can be kept for long periods Rodjakin & Khanmamedov (1967) developed in immune serum and do not lose their pathogenicity a promastigote immobilization test, which they feel to mice or guinea-pigs. The organisms do, however, is a very promising research technique. develop characteristic growth patterns in antiserum, There is still a need for a simple laboratory test which appear to be a sensitive measure of relation- to identify species against type colonies using ship. This phenomenon, first noted by Noguchi standardized procedures, preferably in a leishmania- (1924), was further developed as a diagnostic test sis reference laboratory. The tests mentioned here by Adler (1963, 1964). The antiserum-culture test remain primarily research techniques, difficult to is the only procedure at present available for interpret, and cumbersome or demanding to process determining species relationship among the leish- on a scale large enough for routine use. maniae. It demands great care in preparation of antisera, however, and requires the use of a number NONSPECIFIC TESTS of rabbits to prevent the serum from showing a nontypical reaction. It is necessary to observe Visceral leishmaniasis stimulates hypergamma- growth patterns over long periods to detect the globulinaemia to a degree that globulin change in precise differences among promastigote colonies serum can be detected by several simple, inexpensive, grown in various dilutions and kinds of antiserum. nonspecific tests. These have been used success- Lack of standardization of procedure and interpre- fully in India for some years, where they serve tation of results is still a major limitation to its as a rapid screening method for preliminary diag- wider adoption. Prior absorption of rabbit antisera nosis. The tests include Bramachari's reaction, against cross-reacting species of Leishmania enhances Napier's test, Chopra's test, and Sia's reaction. the sensitivity of the test. Adler was able to diffe- Hypergammaglobulinaemia, though characteristic rentiate Mediterranean from Indian L. donovani; of visceral leishmaniasis, is not confined to it and L. tropica major from L. t. minor; and L. donovani is not correlated with antibody level. Demina from L. brasiliensis, L. mexicana, and L. tropica. believes that this condition may also indicate Saf'janova (1966, 1967) has worked out a complex auto-antibodies, formed in response to the presence IMMUNOLOGY OF LEISHMANIASIS 505 of damaged host cells that are thereafter recognized A positive leishmanin response may appear very by the body as foreign substances. gujkina has soon after exposure to L. mexicana or L. brasiliensis, made an extensive paper electrophoretic and 10-20 days after a successful inoculation with L. t. refractometric study of human and guinea-pig major, 6 months after inoculation with the milder sera after subjecting the hosts to repeated doses of L. t. minor, and about 2 years after cure of visceral leptomonads. No abnormal changes could be infections in Africa. In India, conversion to a posi- detected in protein levels or in differentiated white tive skin test after kalaazar does occur, according cell counts. The fluorescent antibody test with these to Professor P. C. Sen Gupta, though the conversion sera was negative or slightly positive. According is slow to develop and variable. The dermatotropic to Vasina, Demina & Glazunova (1965) guinea-pigs characteristic of African visceral leishmaniasis is infected with L. enriettii demonstrated a sharp suggested by random surveys. A positive skin increase in plasma cells in the spleen and in lymph test response has been found in a large proportion nodes at various stages of the infection and after of the Africans in endemic regions of Kenya and reinoculation. This evidence, in addition to that the Sudan. Inability to sustain a skin hypersensiti- from delayed hypersensitivity studies, suggests that zation, the normal prelude to immunity against cell-bound antibodies are involved in the host's leishmaniasis, is indicated in individuals who do response, even in visceral infections. This aspect not show a skin test response after cutaneous is in particular need of clarification. infection (as in " diffusa" patients).

SKIN TESTS (MONTENEGRO REACTION) DEMONSTRATION OF NATURAL VARIATION IN VIRULENCE AND ITS ROLE IN IMMUNIZATION This test is a standard delayed sensitivity intrader- mal test of the allergic or tuberculin type. Killed Kellina has studied many rodent and human promastigotes of any species, even Strigomonas L. tropica nmjor strains in order to compare their or Trypanosoma, will serve as antigen, indicating virulence and antigenic effectiveness as vaccines. a common-antigen basis of the response. Tubercu- By careful control of infection procedures and losis of the lymph nodes and leprosy also react standardization oftechniques, she was able to demon- positively, but the test is nevertheless useful as a strate a remarkably broad spectrum of virulence diagnostic aid and a measure of previous exposure among many strains within a single Leishmania of residents of endemic areas. According to Mos- subspecies. The innate virulence of each strain kovskij, it was found that in people who did not was tested in white mice, golden hamsters, gerbils, contract the disease the skin test was positive only and man. The susceptibility of the hosts to infection after a sojourn of not less than 5 years in an endemic and the sensitivity of the hosts were determined. area. The reaction is probably lifelong; its duration Animals were tested for number of parasites, length has been reported as at least 35 years (Adler, 1961), of incubation, speed of development of infection, 35 years (Kellina), and 21 years years (Rodjakin). maximum intensity, and frequency of invasion of Extensive skin testing, aided by statistical age- the viscera. Human volunteers were checked for prevalence studies, might help to determine the the proportion of persons infected, the duration of initial onset ofdisease in a community or demonstrate incubation, the time to ulceration, the duration past epidemics. However, high infection densities of ulceration, the size of the scar, and the effective- may mask any such interpretation. Furthermore, ness of protection. Thirty human strains were the response of persons living in an endemic area studied (22 from Turkmenia by Kellina & Belova and to continual exposure to animal leishmaniae may 8 from Uzbekistan by Ni). All the Uzbek human add to the complications. Both sources of confusion strains were highly virulent, as were two strains appear to have taken place in Kenya (Manson- from north-east Iran studied by Ansari (1947) Bahr, 1961) and in the Sudan (Hoogstraal & Heyne- and Ansari & Faghih (1953). All human strains man, 1969). showed some degree of infectivity in mice. Use of the Montenegro reaction is particularly In all, 39 rodent strains were studied-26 from important in the diagnosis of occult L. brasiliensis Turkmenia and Uzbekistan by Kellina and 13 from infection or in cases of mucocutaneous involvement Uzbekistan by Ni (1967). They demonstrated a where it is frequently difficult or impossible to broader range of virulence than did the human demonstrate the organisms. isolates. Some of the least virulent ones, in fact, 506 D. HEYNEMAN failed to infect mice at all, although this infectivity The structure of the patient's skin appeared to be is a basic criterion for definition of the zoonotic of particular importance in determining the intensity L. t. major subspecies. All rodent strains tested of reaction. were placed in one of three groups according to The factors that Kellina considered to be due virulence: high, low, and intermediate. Subse- chiefly to the parasite strain were: quent experiments performed with many fresh strains (a) proportion of persons infected; isolated in the USSR from man and rodents fully confirmed these results. The criteria employed by (b) incubation period; Kellina for allocation of the rodent strains to the (c) speed of ulceration; and 3 groups are listed below: (d) number of promastigotes needed for infection. Group I: high virulence (9 strains that infected all In certain individuals, strains that generally 3 laboratory hosts) produced an acute infective process nevertheless caused a surprisingly prolonged development of (1) Lesions are produced in 80-100% of mice; the lesion, usually a characteristic of low virulence. (2) lesions appear after a short incubation; Kellina also distinguished 3 groups of parasite (3) lesions develop progressively from papule to strains based on the response of the human host: a non-healing ulcer; Group I: high virulence (4) invasion of the viscera is common and secon- dary foci often appear in limb joints; (1) Nearly 100% of persons exposed are infected; (5) infiltrates appear on the ears of gerbils, with (2) papule forms in 1-3 weeks; loss of hair and tissue; (3) papule softens and ulcerates in 3-6 weeks; (6) hamster infection is fulminating, with rapidly (4) ulcer heals 3-4 months later. ulcerating ear lesions and progressive invasion of the viscera, ending fatally. Group II: low virulence (1 strain) (1) Not infective to man. Group II: low virulence (28 strains) Group III: intermediate virulence (1) Only a proportion of mice become infected; (2) lesions are mild and short-lived; (1) Infection occurs only in certain hosts; (3) hamsters are more susceptible than mice; all (2) infective process is weak and brief; develop chronic nonulcerating infiltrates on the (3) no ulceration develops; ears. (4) resorption occurs in 4-6 weeks with no sub- sequent scarring. Group III: intermediate virulence (2 strains) (1) Only a proportion of mice become infected; ALTERATION OF VIRULENCE (2) infected mice develop characteristic progressive lesions; Even the highly virulent cultures tested by Kellina (3) long incubation is required and growth of lost some virulence after prolonged cultivation. papules is slow, though they eventually ulcerate. Some strains even lost virulence after maintenance in vertebrate hosts without a sandfly phase. This (Group III response was similar to that seen in loss ranged from none, as in a strain that was still a strain recovered from S. arpaklensis by Sisljaeva- virulent to man after cultivation for 31 years Matova et al., 1966.) (Adler, 1961), to a very rapid loss that occurred after In the human inoculations, Kellina considered 3-5 transfers (Rodjakin, 1961). the following to be due chiefly to the host's response: Kellina reported that she slowed the loss by (a) size of lesion; using solid NNN culture medium without anti- (b) degree of inflammation; biotics, transferred only once a month. Most strains remained unchanged for 2-3 years. One (c) depth of tissue disintegration; virulent strain withstood 30 passages during 2½/2 (d) abundance of discharge; and years, but after having been sent to London it (e) number of secondary papules. lost virulence in 10 weeks on a liquid medium IMMUNOLOGY OF LEISHMANIASIS 507 that was frequently changed (Neal, 1964). After in a covered site without disfigurement, crippling, subculture for a long time, even on solid media, or loss of working time. Rodjakin has reported the virulence of most strains fell. The loss of that no generalized spread of the inoculum has virulence was seen first in mice and later in man. ever occurred among many thousands of successful Abortive or mild chronic lesions could still develop vaccinations. Growth confined to the skin is an in man after inoculation with a strain that had intrinsic trait of L. tropica, and there is no danger become noninfective to mice. Essentially it had of visceral involvement according to the Soviet transferred from a zoonotic to an anthroponotic workers. strain. The cultures used by Rodjakin for the production Virulence was later recovered by selection of of vaccines are grown in 150-ml bottles on NNN active lesions from test mice. It could be sustained medium with 1000 units of penicillin and 1000 ,g by this means for long periods (7-8 years for the ofstreptomycin. The surface condensate is transferred virulent strain P). By artificial selection from severe to 3-5-ml ampoules after 5-6 days. Stored vacci- lesions, Kellina raised two strains from Group III nation materials can be used for 8-10 days. to Group I. In performing the vaccination, considerable care The natural heterogeneity of L. t. major seems must be exercised to ensure that the skin area selected well-established. Doubtless this fact has played an (right hip or scapula) is properly disinfected and important role in natural selection as well. The that the instruments are aseptic, in order to prevent results of tests with mice are similar to those of sepsis and ensure proper growth of the promasti- tests in man. The virulence of strains can be modi- gotes that are inoculated. Intracutaneous inocu- fied by continual selection. Since mice are more lation with a tuberculin syringe must be used and resistant to infection than man, Kellina could detect the volume of the inoculum should be 0.1-0.2 ml. the loss of virulence in mice before it was possible The stages of development of the vaccination to see any difference in human infections. This ulcer are as follows: difference proved to be useful in the repeated (1) The incubation period is from a few days standardizations required for the development and to 3 months (average 34 days); use of a leishmaniasis vaccine. (2) A hyperaemic spot appears, solidifies, and increases in size in 5 days to a nodule. Enlargement of the papule follows. It becomes flattened, thin PREPARATION OF VACCINES at the surface, and then ulcerates (average period 37 days); Kellina's work made it clear that an attenuated strain of low virulence was not suitable for human (3) Asymmetric ulceration follows decomposition protection. Cultures of sustained high virulence of the nodule. The infiltration zone is small. The must be used in order to produce a quick-ulcerating, diameter of the ulcer may reach 1-1.5 cm, but short-lived infection, able to immunize against any seldom exceeds 0.5 cm (average duration: 82 days). challenge from zoonotic L. t. major. A milder form The average total reaction time is 118 days. will induce sensitization, but will not ensure pro- The more acute the clinical manifestation is, tection from ulcer formation by a stronger pathogen. the briefer the ulceration. The vaccination ulcer, Even after proper protection, reinoculation with in the experience of the workers in the USSR, a highly virulent challenge by natural exposure or never reaches the size of a natural lesion, nor does by experimental inoculation will still produce a it induce tissue destruction and other complications mild biphasic response: (1) a delayed allergic reac- that often ensue after a naturally acquired infection. tion develops first; and then (2) papule formation Ultimate control of the disease requires control occurs in the second week, with a very small non- of the zoonosis. Immunization is employed only expanding ulceration that may last for several to reduce the risk of injury and loss of time for weeks or months before complete resorption or those who are exposed in endemic areas. Eradication formation of a small scar. It is interesting to note of the focus would require a broad-scale attack on that Kellina demonstrated live Leishmania from these reservoir hosts, vectors, and human infections. Such immune sites up to the 22nd day. multiphase measures have already eliminated urban The inoculation procedure, as developed in the leishmaniasis from the Soviet Union and have reduced USSR, produces a natural but controlled infection the zoonotic forms to a few isolated foci. 508 D. HEYNEMAN

SPECIES AND SEROLOGY strains was slight and the method required the comparison of a number of L. t. major strains Species identification among the leishmaniae is of rather than with only one and also required inti- considerable interest and causes immense frustration. mate familiarity with the technique. Are the various Morphological criteria are unreliable and essentially strains we study in the laboratory simply different useless. Epidemiological factors, host and vector expressions of a genetic continuum, or do they species, and the range and cycling rate of different constitute genetically distinct or nascent types ? strains are all without doubt very important in We assume that L. t. major changed to a mild the process of species formation. But these criteria pathogen, L. t. minor, when after sufficient selection are difficult to quantify and compare, especially and isolation, it became part of an urban focus with new strain isolates whose life cycles are un- with a new vector and the loss of its rodent reservoir. known. In vitro serological tests remain the most On the other hand, might not L. t. minor increase reliable index, but these are far from satisfactory. its virulence again if it were passed long enough Yet, new forms of Leishmania continue to appear, through dogs or urban rodents? If the patterns to be described, and to require clarification. Are of pathogenicity now recognized in man are a re- they genetically distinct? Do their disease charac- flection of genetically distinct parasite complexes, teristics reflect that uniqueness? Are these charac- we would be justified in a fairly rigid taxonomic teristics constant ? and clinical codification. But separation between The answer is of more than biological interest. such units tends to break down, particularly at the It will help to govern our handling of the disease subspecific level. Intermediate forms are found, or to prevent the risk of exposure of nonimmune criteria lose their clear borders, and host effects populations. The possibility of invasion of the confuse the distinctions. The mid-grouping of viscera or mucocutaneous destruction greatly modi- each cluster of geographical strains is usually fies the control or clinical measures to be selected. clear, nevertheless, and the division is epidemio- A change of parasite strain often results in a change logically useful, as in the New World agents of of pathogenesis. The identification of such a cutaneous leishmaniasis. However, the question change is a function of our knowledge of the species of biological (i.e., genetic) distinction among the involved. The validity of L. tropica major and many forms of Leishmania found in man is no L. t. tropica (= minor) as subspecies is a good example closer to solution today than when it was argued and is the subject of much discussion. Uncertainty 50 years ago. over this division has been reflected in an uncertainty Similar questions will continue to arise as still over the proper handling of individual infections more variants and intermediates are described. L. and of exposed human populations in endemic mexicana can be separated on serological grounds. areas of cutaneous leishmaniasis outside the Soviet The relatively nonvirulent L. peruana and L. guyan- Union. Even within the USSR, Kellina's discovery ensis can be separated on geographical and epidemio- of heterogeneity in the zoonotic complex of rodent logical grounds, and should be retained as a matter strains of L. t. major emphasizes the great speciation of clinical and biological usefulness pending sero- potential in this complex, ranging from strains that logical review. The Panamanian strains discovered infect mice at a very low level if at all to those that by Hertig and co-workers (which may include rapidly kill all mice tested. The loss of virulence in non-mammalian flagellates), and those isolated culture and its subsequent recovery by passage by Strangeways-Dixon & Lainson in Belize, Bri- through laboratory rodents emphasize the fact that tish Honduras, must also be evaluated. these organisms retain a considerable degree of L. chagasi, in contrast to the species mentioned evolutionary plasticity. above, stands in doubt, even though no type of The considerable degree of cross-immunity that visceral leishmaniasis other than that caused by exists between anthropophilic and zoonotic strains this organism has been found in South America. illustrates their basic similarity. Clinical variations It appears to be an introduced form with an epid- show a broad and confusing overlap that prevents demiology closely tied to the domestic dog, whose clear-cut separation of strains on clinical grounds. infections may have spilled over into the adjacent Yet Adler (1963, 1964) could distinguish L. t. wild fox population (Lycalopex vetulus). This major from L. t. minor by his culture-antiserum view suggests that the domestic dog is the source method. Admittedly the difference between the and not the recipient of the disease from wild IMMUNOLOGY OF LEISHMANIASIS 509 reservoirs. According to Garnham the matter is agent in man is quite convincing. " Live " scars have not resolved and an intensive search for a wild been described by Kozevnikov (1941) and Kozev- animal reservoir is required before we can dismiss nikov et al. (1947), Subarova (1957), and Latysev, the view that an endemic New World form of who found living organisms in " healed " cutaneous visceral leishmaniasis exists, one that antedates lesions 6 months, 4 years, and 13 years, respectively, human occupation of Brazil. Infection is trans- after cure. Fluctuating erythema, peeling, oedema, mitted chiefly if not entirely by Lutzomyia longi- and nodule formation around healed ulcers indicate palpis. Serological separation of L. chagasi from survival of infection following the supposed cure L. donovani is not possible by the Adler technique, of a cutaneous lesion. Parasites have been cultured which suggests that L. chagasi is a recent genetic from the tissues of dogs (Vavilova, 1960) and variant of L. donovani, or one that is strikingly gerbils (Krjukova, 1941) presumed to be cured similar to it if L. chagasi is a truly endemic species. of infection, and similar cultures have been obtained Based on present evidence, however, specific status from laboratory rodents presumed to be cured of is not yet warranted. visceral infections (Stauber, personal communica- Adler et al. (1966), using Adler's culture technique, tion). The clinical history of espundia demonstrates also confirmed the serological (hence genetic) cross- long parasite survival and eventual metastasis from specificity of the various strains of L. donovani found foci presumably hidden in the skin, possibly hidden by Hoogstraal and co-workers in the Sudan. Strains in nasal mucosa as well. Buccal and nasal tissues were isolated from man, from the roof rat (Rattus may be attacked by L. brasiliensis 7 or more years rattus), from the Nile rat (Arvicanthus nyloticus after the original lesion has healed. Post-kala-azar luctuosus), from the spiny rat (Acomys albigena), dermal leishmanoid often develops several years from the serval (Felis servalphilippsi), and from the after the cure of visceral leishmaniasis, as has been genet (Genettagenetta senegalensis). This broad host reported recently by Dr P. C. Sen Gupta. This spectrum suggests a relatively non-host-specific strain. creates an enigma. Visceral " cure" is followed It also leaves unanswered the question of its origi by dermal resistance to a new infection. But, after and status as an anthropozoonosis or a zooanthro- a varying period, skin resistance is lost, and a ponosis. It could be a fairly recent immigrant, resurgence of the old infection develops as a papular presumably from India via Arabia, Ethiopia, and dermal outbreak, while the original visceral organs Kenya, which then infected a large number of remain uninfected. These nodules, which are varied vertebrates, including man (hence it would be a in form and frequently highly resistant to treatment, zooanthroponosis). Or it might be a truly endemic contain numerous parasites. Dermal leishmanoid zoonosis, found in many wild hosts and accidentally sometimes spreads throughout the dermis, much as encountered by man (hence an anthropozoonosis). in " diffusa" cases of cutaneous leishmaniasis. The question may not be answerable. The view Similarly, the dermal infection known as the favouring immigration is suggested by the recent " recidivans " type is a recrudescence of the active history of kala azar epidemics in Kenya and the spread of infection at the margins of old " healed " Sudan and the presence of the disease in Ethiopia, leishmanial scars. Frequently these infections resist with epidemiological characteristics suggestive of all attempts at chemotherapy, smouldering on for Indian kala azar. Yet the large number of host decades. species incriminated in the Sudan, involving both Reports of asymptomatic visceral infection (Arm- rodents and rodent-eating carnivores, is equally strong, 1945; Prata, 1957; Sen Gupta, 1962), as suggestive of a complex, widespread zoonosis. well as positive leishmanin tests in East African Evidence of a nonhuman cycle of infection was and Sudanese endemic centres of visceral leish- found in Malakal, in the central Sudan (Hoog- maniasis, add to our uncertainty over the existence straal & Heyneman, 1969). of a true sterile immunity. Premunition may there- fore exist in many individuals, and constitute a PREMUNITION VERSUS IMMUNITY continuous source of antigen stimulation in the dermis. The presence of functional immunity The question of a premunition, or non-sterile pro- in many persons free of parasites is nevertheless tection (the occult presence of a primary infection), equally clear. as opposed to a true residual (sterile) immunity is Patients who have recovered from kala azar, not yet solved. Evidence of long persistence of the or who have been cured by chemotherapy, appear 51 0 D. HEYNEMAN to possess a solid immunity against reinfection, for man, but the implication would nevertheless be even in hyperendemic areas where re-exposure is strong. frequent. But this protection is not related to the The relative effectiveness of an immunizing high CFT titres and hypergammaglobulinaemia infection in stimulating rejection of a subsequent that develops during the course of infection. These challenge exposure appears related to the speed and serological manifestations seem unable to serve any degree of skin reaction. The host's capacity to protective role against the course of visceral disease respond, at least in cutaneous leishmaniasis, requires in man. specific, intense, and prolonged stimulation. Kellina In animal experiments with L. donovani, chiefly has emphasized that only a highly virulent infection by Stauber and his students (Stauber, 1955, 1958), allowed to develop through its full course would an immunological element in resistance is inferred provide protection against an equally virulent chal- rather than proved directly. Rabbits, white rats, lenge. Attenuated or less naturally virulent forms of guinea-pigs, gerbils, and white mice show a wide L. tropica that fail to produce an ulcer do not range of innate resistance. Some host species stimulate full protection, despite earlier reports of tolerate infection only at low levels, later arresting success with killed leptomonads by Pessoa (1941) the infection and rejecting it; others cannot even and others. Rodjakin's extensive practical experience be infected. At the other extreme, hamsters and in developing and using the L. t. major vaccine chinchillas die of the infection, and the cotton-rat corroborates Kellina's experimental findings. survives with a level of parasitism that is lethal Yet Manson-Bahr (1961, 1963) demonstrated in a to the hamster. Mice, guinea-pigs, and gerbils lose small number of soldier volunteers that protection a challenging infection more quickly than they do is possible against the East African strain of L. the initial one. This rapid loss can be interpreted donovani that invades the viscera using an intra- either as a degree of acquired resistance or as a dermal inoculation of the relatively nonvirulent premunition effect, as the initial infection was ground-squirrel strain that does not invade the still present at the time of challenge. Comparative viscera. studies on reinfection after chemical cure remain Subsequent field trials with 1500 persons in a to be carried out. Interference with the host's Tana River endemic focus unfortunately failed protein metabolism and reduction of antibody to protect them against natural kala azar infection, synthesis by elimination of pyridoxine in the diet owing to failure of the inoculation procedure drastically reduce both innate and acquired resis- employed. This may have been because the ground- tance to L. donovani in the white mouse (Actor, 1960). squirrel strain used was impaired by long in vitro cultivation. Southgate has reported that he had FUNCTIONAL IMMUNITY AND HYPOTHESES OF THE repeated the immunization with the original ground- IMMUNE PROCESS IN LEISHMANIASIS squirrel strain in Kenyan volunteers and that it did protect them against L. donovani. Once human infection with kala azar passes the The role of the skin as the major defence organ dermal barrier, the concentration of cellular anti- against leishmaniasis suggests some comparative body in the dermis is apparently useless in retarding observations between Indian kala azar and other infection. However, the host is protected against forms of visceral leishmaniasis: subsequent sandfly exposure following cure, as was noted in the preceding section. The hypothesis, (1) In Indian kala azar, evidence of a primary therefore, seems warranted that immunity to vis- lesion is rare or absent, whereas it is common in ceral leishmaniasis resides largely in the skin, where Sudanese and East African foci and is probably cell-bound antibodies, plasma cells, and sensitized present in other areas as well (e.g. in Uzbekistan). lymphocytes are concentrated. Experimental proof (2) Demonstration of parasites by direct micro- of this view is lacking, but it might be tested by scopical blood examination is common only in comparing intradermal with intraperitoneal or Indian kala zaar. In Kenya and the Sudan, para- intracardiac infection in a chemically cured animal. sitaemia during early stages of infection is equally The animal of choice would be one that demon- common but less intense, and is detectable only strates some degree of innate resistance, such as the in cultures of blood. On the other hand, the iso- guinea-pig or Meriones, as shown by Stauber (1958). lation of parasites from skin snips or nasal mucosa Positive results would not necessarily prove the case is common in African and rare in Indian cases. IMMUNOLOGY OF LEISHMANIASIS 511

(3) Post-kala-azar dermal leishmaniasis is com- the body to isolate and destroy them in spite of mon following Indian kala azar (5-10%), but rare the massive proliferation of lymphocytes. Ob- or absent in other areas with the possible exception struction and enlargement of the liver and spleen of China. Sen Gupta has said that the numerous result from this proliferation, inducing conditions cases reported from the Sudan and the few from that lead to profound hepatosplenomegaly and the Kenya are concurrent cutaneous manifestations, death of the patient. Extreme hypergammaglobulin- rather than true post-kala-azar dermal leishmaniasis aemia appears to be a systemic recognition that as he defines it. something is wrong, but the exact nature of the (4) Response to the Montenegro test is immediate disturbance is hidden from the immunologically and strong following the cure of African or Mediter- competent cells-hidden, of course inside the ranean kala azar. Sen Gupta has stated that it does phagocytic histiocytes. This undirected and in- occur after treatment for Indian kala azar, but that effectual emergency defence response may be further it is weak and slow. increased if antigens from destroyed macrophages stimulate an auto-immune response. Perhaps this (5) Indian kala azar responds readily to treatment is what occurs late in the infection sequence in with antimony (III) sodium dimercaptosuccinate espundia, the final self-destructive stage of mucocu- (stibocaptate), which is ineffective against African taneous leishmaniasis. Parasites are seldom seen forms and relatively so against the forms found in at this stage, but nasal and buccal tissues are des- Mediterranean, Soviet, and Chinese foci. troyed nevertheless. The body's defensive cells (6) Spontaneous cure is reported to be common appear to have lost the vital characteristic of self- in Indian kala azar, averaging 10% and ranging up recognition. Destruction of the body's own tissues to 25 %, according to Napier (1924). It is thought to may occur rapidly once the process begins, as in be significantly less, perhaps 5-10%, in other areas. other auto-immune wasting diseases. Damage to Could these conflicting characteristics of what infected macrophages may have caused these cells is considered to be the same infective agent be to become antigenic, stimulating cell-bound anti- caused by a weaker dermal sensitization and bodies or activated lymphocytes to attack other consequently a lowered functional immune res- macrophages and ultimately the nasal mucosa and ponse in Indian kala azar? In the latter infection, cartilage as well. We have no idea, however, why the lack of a primary dermal ulcer and the abun- nasal and buccal mucosa should become the pri- dance of parasites in peripheral blood is in contrast mary target, nor do we understand the role of the to the characteristically strong initial dermal res- parasites in stimulating or directing the process. ponse and lower parasitaemia in kala azar from The frightfully disfiguring results are evidence other areas such as Africa. The greater number of enough of its importance. parasites in the blood in Indian kala azar presumably Perhaps the key questions remain unasked for renders it more amenable to blood-borne chemo- lack of a suitable experimental design. Why do therapy; the frequent appearance of post kala azar infected macrophages remain in a cutaneous sore dermal leishmanoid and a less intense leishmanin in one case, and a histiocytoma in another, and why response suggest a failure of the skin response. are they not at all confined in a third? What deter- Manson-Bahr (1961) noted that though the leish- mines blood- or lymph-borne metastasis to the manin reaction develops only after cure of East nasal mucosa in one disease, or filtration within African kala azar, the reaction is positive much the liver or spleen in the other? Is this migration earlier if the infection does not invade the viscera. undertaken by freed parasitic cells or by infected Similarly, non-rodent strains that do not invade histiocytes? These are all restatements of the same the viscera induce a rapid leishmanin response, basic question: to what degree is the clinical form as does infection with L. t. major or L. mexicana. of leishmaniasis an expression of the parasite's L. t. minor, which causes a less intense skin infection, genetic constitution (virulence) or of the host's requires about 6 months to induce a positive leish- innate capacity to respond (immunity)? The un- manin response. These observations reinforce the solved problems and unasked questions about this view that it is in the skin that the most effective complex of diseases demand our continued interest protection of man probably occurs against any and attention. The discussion of immunological form of leishmaniasis. Once infected macrophages aspects in these hypothetical terms is imposed by our escape from the skin, there is little chance for ignorance and is an expression of that challenge. 512 D. HEYNEMAN

RltSUMIt IMMUNOLOGIE DE LA LEISHMANIOSE

La leishmaniose humaine resulte d'une interaction complement sont des signes de kala-azar, mais ils entre l'h6te et le parasite: le pouvoir envahissant intrin- paraissent inddpendants de la gravite de la maladie ou seque du parasite et la virulence telle qu'elle est modifi6e de l'etat d'immunite fonctionnelle. Le test cutane de par la reponse individuelle et l'espece de l'h6te. Le pre- Montenegro est utile dans le diagnostic de l'infection sent article passe en revue les aspects conceptuels et cli- cutan&e et il est important du point de vue epidemiolo- niques de l'immunologie de la leishmaniose humaine. gique dans certaines regions oui le kala-azar est ende- Nos connaissances sur les aspects immunologiques de mique, mais ses possibilites d'utilisation dans la pratique cette affection restent fragmentaires et surtout pragma- sont tres limitees. Des chercheurs sovietiques ont etudie tiques, comme dans le cas de la vaccination anti-L. tro- les differences de virulence entre les souches de L. tropica pica. La variabilite de la reponse d'une meme souche major, la modification de la virulence par culture in vitro, parasitaire chez differents hotes est revelee par l'exposi- et l'application des resultats de ces etudes a la preparation tion de divers hotes de laboratoire a des infections expe- de vaccins. La base serologique de la separation des especes rimentales normalisees. Chez l'homme, des reponses est encore loin d'etre satisfaisante. La methode d'Adler individuelles extremement variables masquent et com- de culture par antiserum est peut-etre la plus prometteuse, pliquent la reponse de l'espece. Celle-ci se produit, mais les procedes de culture, les souches, les antis6rums toutefois, ainsi que le montre la distribution regionale et les criteres d'interpretation des resultats ne sont pas des formes cliniques de la maladie et les poussees epide- completement normalises, d'o'u la difficulte de repeter les miques de kala-azar en Inde et en Afrique. On ne sait experiences dans d'autres laboratoires. pratiquement rien de la mesure dans laquelle vecteur et Malgre de nombreuses d'annees d'efforts, personne n'a reservoir contribuent a la maladie humaine. encore mis au point une definition satisfaisante de 1'espece La resistance acquise naturellement et les resultats de Leishmania. Ce sont les criteres epidemiologiques qui sont la vaccination mettent en evidence que seul le deroule- les plus utiles et les plus acceptables, car ils comprennent ment complet de l'infection par une souche virulente de la variete maximale d'indices des relations parasitaires: L. tropica confere la protection contre une reinfection facteurs geographiques, vecteur, reservoir et caracteris- par une forme d'une virulence comparable. D'apres des tiques cliniques chez l'homme et chez les animaux chercheurs sovietiques, seules des souches zoonotiques d'experience. Le complexe d'especes d'Amerique du Sud vivantes, recemment isol&es, sont assez fortes pour et d'Amerique centrale a tout particulierement besoin induire une protection totale. L'evolution dermique de d'etre elucide. Les observations effectuees donnent a l'infection immunisante doit etre rapide, aigue et ininter- penser qu'il existe un certain nombre de formes de rompue par un traitement. Une fois que l'immunitd Leishmania dans le Nouveau Monde, principalement des contre la leishmaniose cutanee a commence d'apparaitre, zoonoses en associations h6te-vecteur-parasite dans des les inoculations d'epreuve en d'autres points de la peau regions forestieres tropicales ecologiquement isolees; il provoquent des ulcerations qui guerissent en meme s'agit, semble-t-il, d'esp&es en existence ou en formation. temps que la lesion primaire (R loi de reinoculation * de Moskovskij). Dans la leishmaniose viscerale, l'immunite I1 importe de prevenir la leishmaniose humaine. Les n'apparait qu'apres une guerison spontanee ou resultat crypto-infections paraissent repandues et pourraient etre d'un traitement medical. partiellement responsables de la resistance contre la Au cours d'etudes sur la protection croisee, on n'a pas reinfection, que l'on constate parmi certaines populations reussi 'a juguler l'infection due aux especes les plus des regions d'endemicite. La survie a long terme des pathogenes telles que L. brasiliensis et L. donovani. I1 se parasites s'observe dans la leishmaniose recidivante pourrait qu'il y ait une exception importante, 'a savoir (inflammation marginale d'une ancienne lesion), dans la souche qui affecte les rongeurs, ne provoque pas la la leishmaniose tegumentaire diffuse (extension anarchique leishmaniose viscerale et existe au Kenya, souche que de lesions dans le derme) et dans la leishmaniose ameri- l'on croit etroitement apparentee a L. donovani et qui a caine ou R espundia)) (destruction etendue de la muqueuse protege des volontaires contre une inoculation d'dpreuve bucco-nasale et des tissus adjacents). Cette derniere peut par une culture de laboratoire d'une souche humaine impliquer une auto-immunite et les autres peuvent etre virulente de L. donovani. Des etudes sur le transfert passif dues a une allergie ou 'a quelque autre echec de l'immunite de l'immunite ont montre combien il est difficile de mesu- de l'hote. Il est egalement probable que dans les regions rer, ou simplement d'evaluer la resistance a l'infection d'endemie de nombreux individus hdbergent, au niveau cutanee. Bien qu'ils ne soient pas specifiques, les procedes du derme, des colonies de parasites non pathogenes qui fondes sur l'etude du serum sont utilisables pour le dchappent a l'observation. Ces colonies peuvent prdvenir depistage preliminaire. L'elevation de la gammaglobuli- la reinfection, mais etre capables d'une recrudescence ndmie et des titres d'anticorps non specifiques fixant le d'activite pathogene ulterieure. IMMUNOLOGY OF LEISHMANIASIS 513

Le role de la peau est important dans toutes les leish- viscerales et mucocutanees sont des echecs de la reponse manioses et non seulement dans les infections cutanees. dermique, mais qui sont Mies aux caracteristiques enva- II est evident, par exemple, dans la forme leishmanoide hissantes propres des pathogenes en cause, peut-etre dermique qui succede au kala-azar et qui apparaft chez modifiees par certains vecteurs ou h6tes-reservoirs. Notre les malades indiens de 1 a 3 annees apres la guerison d'une comprehension de cet equilibre ou, bien trop souvent, de infection viscerale. La reponse cellulaire de F'h6tel a ce desequilibre, depend de la mise au point d'une methode l'infection (principalement cellules plasmatiques et lym- suire (ou de l'adaptation de l'un des procedes modernes phocytes) determine le taux de guerison et d'immunite que l'on perfectionne actuellement au cours d'autres ulterieure, mais la souche de Leishmania regit le tableau etudes) qui permette de mesurer la reponse immunitaire de l'infection et le degre de la virulence. Les formes cutanee cellulaire de 1'h6te a la presence de Leishmania.

REFERENCES

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