New prospects for the study of leprosy in the laboratory

Bull. Org. mond. Sant| 1969, 40, 785-800Bull. Wid Hith Org. New Prospects for the Study of Leprosy in the Laboratory R. J. W. REES 1 Although Mycobacterium leprae was identified earlier than Myco. tuberculosis, it has still not been cultured in vitro and only in 1960 was an infection obtained in laboratory animals. However, important advances have been made in the field ofexperimental leprosy in the last decade due to the development ofnew techniques and models for studying Myco. Ieprae in vivo, thus overcoming the limitations imposed by a non-cultivable mycobacterium. Quantitative techniques using Myco. lepraemurium provided the first modelfor developing an indirect method for distinguishing dead (non-infectious) from living Vinfectious) bacilli, based on morphological differences in organisms stained by the Ziehl-Neelsen method. However, the most important advances resulted from the limited and localized growth of Myco. leprae when inoculated into the foot pads of mice and, later, the more substantial and generalized multiplication of Myco. leprae in immunologically deficient mice (thymectomized and irradiated with a dose of 900 r). Moreover, in the immuno- logically deficient animals, the infection eventually resulted in a disease replicating that of lepromatous type leprosy in man, including the involvement of peripheral nerves. The results from these studies and the future prospects for the study of leprosy in the laboratory are reviewed in this article. Of the mycobacterial diseases affecting man, leprosy is second only to tuberculosis in presenting a world health problem affecting more than 10 mil- lion people, most of whom live in newly developing tropical and subtropical countries. Leprosy is a chronic infectious disease caused by Mycobacterium leprae, an obligatory intracellular parasite often giving rise to virtually no symptoms. Clinically, the disease manifests itself most commonly in the skin, nose, upper respiratory tract and peripheral nerves and its form is related to the capacity of the individual to destroy the organism. The effect on nerves is responsible for the serious disabilities and deformities of the feet, hands and face, and a Scientific Meeting on Rehabilitation in Leprosy (1961) estimated that in at least 25% of all cases there is some degree of deformity. It is of interest that Myco. leprae is the only species of mycobacterium which affects nerves in either man or animals. This unique property 1 Head, Laboratory for Leprosy and Mycobacterial Research, National Institute for Medical Research, London, England. This report received financial support from the World Health Organization. deserves special study, for it is likely to increase our knowledge both of the organism and of peripheral nerve fibres. Patients with leprosy present a wide range of symptoms and signs, ranging between one or other of the 2 polar forms of the disease; namely, tuber- culoid leprosy, in which there are few bacilli, and lepromatous leprosy, in which the number of bacilli is extremely high. Thus, a study of the pathogenesis of leprosy requires knowledge drawn from a wider range of scientific disciplines than for any of the other mycobacterial diseases. Hitherto, progress has been severely restricted because Myco. leprae cannot, as yet, be grown in vitro and has only been grown in vivo to a limited extent since 1960 (Shepard, 1960a, 1960b). This is unfortunate because the bacterium was one of the first to be linked specific- ally to a human disease. It is obvious that, if the causative organism cannot be cultivated or the disease transmitted to experi- mental animals, neither the bacteriology nor the pathology of the infection can be studied in the laboratory. The lack of success with these techniques has seriously limited the scope of fundamental and 2335 - 785 - R. J. W. REES applied research in the leprosy field since the isola- tion and identification of causative organisms, and their subsequent cultivation in vitro and in vivo has resulted in the control of most of the bacterial diseases of man. Failure to cultivate and transmit leprosy in the laboratory has not been due to lack of effort. The first attempts were made by Hansen and since then by many bacteriologists and pathologists. From time to time, claims to success have been made but, until recently, none has been upheld although, between 1874 and 1930, all the other important human pathogens were successfully cultured by one means or another. Since 1960, however, techniques have been developed for the transmission of leprosy to animals, and the situation has been transformed. This review is confined to a consideration of the experimental models developed during the last 10 years which have now reached a stage that permits Myco. leprae to be studied on a sound experimental basis. Although it is still impossible to grow the bacilli in vitro, these models have already contributed significantly to the study of leprosy in man and are likely to contribute even more during the next 10 years. Although the most important advances arose from the development in 1960 of techniques for transmitting leprosy to animals, it was a prerequisite that experimental models using other species of mycobacteria were developed before the laboratory study of human leprosy was started. The study of models, particularly those using Myco. lepraemurium, established the method for deter- mining, indirectly, the infectivity of Myco. leprae and the techniques of tissue culture adapted to the cultivation of human leprosy bacilli. MODELS BASED ON COMPARATIVE STUDIES WITH MYCO. LEPRAEMURIUM An indirect method for determining the viability of leprosy bacilli Most species of bacteria are completely destroyed when they die within an infected host, but myco- bacteria are an exception since they retain both their bacillary form and the property of staining with carbol fuchsin when they are no longer alive. However, in a Myco. keprae infection it was not possible to determine directly whether bacilli in the patient were alive or dead because they could not be cultured. Therefore, indirect methods for measuring their viability had to be used. For these studies the closely related Myco. kepraemurium proved to be an ideal model since, although the rat leprosy bacillus also failed to grow in vitro, its viability could be assessed in terms of infectivity by determining its ability to produce disease following its reinoculation into animals. Early studies using electron micro- scopy showed that viable Myco. lepraemurium, that is, bacilli that infected animals, could readily be distinguished from non-viable bacilli, that is, bacilli that failed to infect animals (McFadzean & Valen- tine, 1959; Rees, Valentine & Wong, 1960). The essential differences were that the cell wall of the non-infectious (i.e., dead) bacillus, though still intact, was no longer uniformly filled but contained only electron-dense aggregates of disorganized proto- plasmic material, whereas the infectious form was uniformly electron-dense. Evidence that this was a gpneral phenomenon was provided by using an entirely different species of bacterium, Escherichia coli, which had the added advantage that viability could quickly be tested by means of conventional viable colony counts on solid media. The results of such studies showed that there was a close correla- tion between the decrease in the number of colonies and the increase in the proportion of degenerate forms of E. coli seen under the electron microscope. Furthermore, the degenerate changes which took place in E. coli resembled those seen in Myco. lepraemurium (Rees, Valentine & Wong, 1960). Once it had been established that living and dead forms of bacilli could be distinguished, a new set of comparisons was undertaken to see how far the morphological changes seen under the electron microscope were correlated with those shown by the light microscope when Myco. lepraemurium was stained with caibol fuchsin using the routine Ziehl- Neelsen method. Rees & Valentine (1962) developed a new technique that allowed individually identified and stained bacilli to be examined first under the light microscope and then under the electron micro- scope. Close agreement was found between the proportion of degenerate forms of Myco. leprae- murium seen under the electron microscope and the proportion of bacilli showing irregular staining with carbol fuchsin under the light microscope. From these studies it appeared that the electron-dense material within the cell wall of the organism corre- sponded exactly to the part staining with carbol fuchsin. It was therefore concluded that all forms of Myco. lepraemurium showing irregular staining were dead and only those showing uniform or " solid " staining were likely to be viable. It was reasonable to expect that this assumption could be extended to the human leprosy bacillus since the 786 NEW PROSPECTS FOR THE STUDY OF LEPROSY IN THE LABORATORY same morphological changes were likely to be shared by all species of mycobacteria. This expectation was fully confirmed when, using the same technique, suspensions of Myco. leprae were examined by both light and electron microscopy (Rees & Valentine, 1962). More recently, it has been possible to confirm this assumption by demonstrating experimentally that only the uniformly staining forms of Myco. keprae are capable of multiplying in the mouse foot pad (Rees, 1965b; Shepard & McRae, 1965). Rate of multiplication of leprosy bacilli in vivo " One explanation for the chronicity of human leprosy might be that Myco. leprae divides more slowly than other bacteria or even other myco- bacteria; this hypothesis could be tested in murine leprosy since the natural and experimental infections are known to be chronic. By applying quantitative techniques it has been possible to follow the total number of stained acid-fast bacilli in the tissues of animals during the infection and therefore to deter- mine the rate of multiplication or the generation time of Myco. kepraemurium. Several different groups of workers (Hilson & Elek, 1957; Hobby et al., 1954; Rees, 1957) have found that even in sus- ceptible animals, the bacilli divide only every 10-14 days. This very long generation time is unique for micro-organisms and even for other species of mycobacteria. It is most unlikely that Myco. leprae would have a shorter generation time than that of Myco. lepraemurium. The long genera- tion time must be considered when claims of success- ful cultivation or transmission of Myco. leprae are being assessed particularly if the claim suggests that the organism is multiplying rapidly. It is even more important to accept a long generation time when methods for attempting to cultivate or trans- mit Myco. leprae are being planned. The model has been shown to be applicable by Shepard & McRae (1965), who demonstrated that Myco. leprae has a generation time of 13-25 days in the mouse foot pad (see p. 791). Time taken to kill leprosy bacilli and the fate of dead organisms following chemotherapy It is known that in patients receiving successful chemotherapy, in particular after treatment with diaminodiphenylsulfone, a very long time elapses before bacilli disappear from the lesions. Whether this was due to the slow rate at which the drug killed the bacilli or to the inability of the host to dispose of the dead organisms could only be a matter of conjecture in the absence of direct or indirect evidence. However, when murine leprosy was used as a model to study the effect of chemo- therapy on the rate of kill (as determined by the proportion of irregularly stained, " dead ", bacilli) and the ability of the host to dispose of the bacilli killed by chemotherapy in vivo (Rees & Waters, 1963), indirect evidence on this point became available. Mice heavily infected with Myco. leprae- murium were treated with isoniazid and the total number of bacilli and the total numbers of viable (solidly staining bacilli) were determined in the treated and untreated groups at regular intervals for more than a year. The results showed that isoniazid was very effective since the untreated animals all died of gross infection within 100 days, whereas the treated mice lived for more than a year. Furthermore, 73% of the bacilli in the treated mice showed irregular staining after only 28 days and the proportion had reached 93% by the 63rd day of treatment. There was, therefore, a 90% kill within 50 days of treatment. On the other hand, the fall in the total number of stained bacilli in the tissues of the treated mice was much slower; it took 250 days for a 90% fall in numbers to occur (Fig. 1). Thus, although isoniazid was effectively bactericidal within a relatively short time, dead Myco. lepraemurium FIG. 1 "VIABLE" AND TOTAL POPULATIONS,OF MYCO. LEPRAEMURIUM IN THE LIVER AND SPLEEN OF UNTREATED AND ISONIAZID-TREATED MICE a, b X' 13 <D 1.> CA IQ112 D l _11 Iso1I0 IZ ---I--_ __ d 50 100 150 200 250 Time (d.v_-A a All untreated mice died with gross infections by day 100. b Reproduced, by permission, from Rees & Waters (1963). A = Untreated, total count; B = Untreated, viable count; C = Isoniazid-treated, total count; D = Isoniazid-treated, viable count. 787 R. J. W. REES were not nearly so quickly removed from the infected tissues. After continuous treatment with isoniazid for 1 year the proportion of solidly staining acid-fast bacilli started to rise again; in other words, viable organisms were reappearing. The total num- ber of such organisms steadily increased and within 3 months the animals died of Myco. leprae- murium infections. Bacilli recovered from animals that had relapsed under treatment with isoniazid were inoculated into healthy mice which were also treated with isoniazid. These animals showed no response to the drug and the infections took the same course as in untreated controls (Hart, Rees & Valen- tine, 1962). This indicated, without doubt, that the recurrence of active disease in the mice used in the first experiment was due to the appearance of isoniazid-resistant organisms and that the relapse was heralded by the reappearance of bacilli which were morphologically viable. Use of tissue culture systems for the cultivation of Myco. lepraemurium in vitro Myco. lepraemurium, like Myco. leprae, is pre- dominantly an intracellular parasite and so far neither organism has been cultured in bacterio- logical media. The dependence on an intracellular environment strongly suggested that tissue-culture methods might offer a means ofgrowing Myco. leprae- murium in vitro. The first successful claims of limited multiplication of Myco. lepraemurium in tissue culture systems were made by Wallace, Elek & Hanks (1958) and by Rees & Wong (1958). Since then, techniques have been developed using an established strain of rat fibroblast cells (Rees & Garbutt, 1962) or cultures of mouse macrophages (Chang, 1960) in which indefinite and continuous multiplication of Myco. lepraemurium has been obtained. The success of these tissue-culture methods for Myco. leprae- murium provides a wealth of knowledge which can be applied directly to the problems concerned with the very similar, slow-growing organism Myco. leprae. In particular, the studies have stressed the importance of using quantitative methods for determining the total number of acid-fast bacilli present at the beginning and at the end of each culture period, and therefore basing multiplication on absolute increases in the bacillary population. The value of examining the morphology of the bacilli as a sensitive means of determining their survival in tissue culture has also been made clear. It is significant that the charactel istics of Myco. lepraemurium maintained in continuous cultivation in tissue-culture systems for more than 3 years have not changed (Rees & Garbutt, 1962). Myco leprae- murium grown in tissue culture do not multiply in bacteriological media, still have a generation time of 10-12 days and retain their pathogenicity for mice and rats. These observations are of considerable importance since they confirm the generally accepted view of the stability of bacterial populations, which is in sharp contrast to so many of the acid-fast bacillary strains, claimed to be Myco. leprae, which were isolated from leprosy patients. These strains had a wide and variable range of characteristics which were, unjustifiably, explained on the basis of adaptation or mutation. Application of the results of studies on murine leprosY to human leprosy Myco. kepraemurium has been extensively used as a model in leprosy research, although more recently its value has been criticized. Such criticism is justifiable only when murine leprosy has been used uncritically as a model for the human infection- namely, for screening potential antileprosy drugs and in comparative pathological and histological studies. Where murine leprosy has been used to answer specific questions or to test hypotheses directly related to Myco. leprae, these studies have provided valuable information. It is clear from the results of these studies that a new and valuable technique was available that could now be used in man to deter- mine the viability of Myco. leprae in patients and thus to assess precisely their probable response to chemotherapy, the infectivity of the various types of leprosy, and, by histological studies, the viability of Myco. leprae in different tissues. Thus the morphological index (MI), that is, the percentage of solidly staining bacilli seen in smears or sections from leprosy patients, could now be used as the measure of viability. The routine bacteriological examination of patients is based on smears prepared from diseased skin or a nasal scraping and, after staining the smear by the carbol fuchsin method, the density of bacilli (this is known as the bacteriological index (BI)) is scored, irrespective of the morphological appearance of the bacilli. The progress of the patient under treatment is then judged by the rate of disappearance of bacilli from these smears, that is, by the fall in the BI. It has long been considered that even the most active antileprosy drugs, including diaminodiphenylsulfone, leave much to be desired since in patients with the more severe lepromatous type of leprosy many years 788 NEW PROSPECTS FOR THE STUDY OF LEPROSY IN THE LABORATORY elapse before negative smears give evidence of a ' cure ". Hitherto, it has been assumed that more active drugs are required to kill Myco. leprae more rapidly than diaminodiphenylsulfone, an assumption that was based on the fact that the BI falls so slowly. Now that is has been demonstrated conclusively that the viability of Myco. leprae can be assessed on the basis of their morphology rather than on the total number of the bacilli, it has been possible to show, in carefully controlled studies, that a very high proportion of bacilli are killed in 3 months in patients receiving standard treatment with diamino- diphenylsulfone (Waters & Rees, 1962). This obser- vation suggests that persisting lesions and many of the manifestations of leprosy, including reactions of the erythema nodosum leprosum type, that follow the initial phase of chemotherapy must be due, in part, to the presence of dead bacilli. It implies that a more rapid cure will be achieved only if other drugs or methods are found that could be used, after the initial killing of leprosy bacilli with standard anti- leprosy drugs, to enhance the host's ability to dispose of dead, but still intact, leprosy bacilli. Thus the introduction of the MI as an index of viability has not only provided a rapid method for determining, within a period of only 3-6 months (Fig. 2), the activity of potential antileprosy drugs (Waters, Rees & Sutherland, 1967) but has led to an entirely new approach to the problem of leprosy chemotherapy. Furthermore, an increase in the MI during treament provides a sensitive measure of the patient's deterioration, whether this is due to FIG. 2 EFFECT OF TREATMENT WITH DIAMINODIPHENYL- SULFONEa FOR 6 MONTHS ON THE MORPHOLOGICAL INDEX (MI) IN 6, PREVIOUSLY UNTREATED, LEPROMATOUS PATIENTS 40r 10 G.) G <, = EA _& a = a o6 no C2 9 CS a 100 mg daily. failure to take the drug or to the emergence of drug resistance (Pettit & Rees, 1964; Pettit, Rees & Ridley, 1966) (see p. 793). EXPERIMENTAL LEPROSY IN ANIMALS Before describing advances that have been made in the field of experimental transmission of leprosy to animals since 1960, it is pertinent to review briefly the general problems and methods used in animal transmission experiments and the criteria for assess- ing successful claims. These were defined in detail by the Technical Committee on Pathology and Experimental Transmission (1963) at the Eighth International Congress of Leprology in Rio de Janeiro, Brazil. Bacilli for inoculating laboratory animals should be obtained from patients with untreated leprosy and with a high MI and, because of the possible contamination of skin by other cultivable mycobacteria, biopsy specimens should not be taken from ulcerated lesions. Moreover, all suspensions prepared for purposes of inoculation should be cultured on a variety of media suitable for isolating mycobacteria. In addition to each group of animals inoculated with fresh suspensions of bacilli, there should be a -group inoculated with heat-killed organisms and an uninoculated group of animals. Quantitative bacteriological methods should be used in order to determine the number of orga- nisms inoculated and subsequently to determine the number of organisms present in the animals. These rigorous methods and checks were introduced in order to exclude as far as possible the inoculation and subsequent development of infections with contaminating strains of mycobacteria, and also the possibility that the animals themselves might be carriers of mycobacteria. This latter possibility has been demonstrated very clearly by the elegant work of Nishimura and his colleagues working at Osaka in Japan, showing that a proportion of apparently healthy mice and other rodents can be carriers of a murine leprosy-like infection (Nishimura et al., 1964). The criteria for assessing successful claims for transmission of leprosy were also rigorously defined. In addition to counting the number of bacilli isolated from the animal tissues, the organisms should be cultured, using again media suitable for growing mycobacteria. Successful transmission of an infec- tion from one patient should be reproduced from others, using the same experimental conditions. A standard type lepromin should be prepared from the bacilli harvested from the animals and compared 789 6 W"D 908 10 R. J. W. REES with a similarly prepared human lepromin from patients with tuberculoid and lepromatous leprosy; these tests should be carried out and read blindly. Finally, a histopathological examination should be made of the infected tissues including, in particular, a careful examination of the nerves, using the requi- site staining methods. Experimental leprosy in normal animals Undoubtedly the most important direct contribu- tion to the study of leprosy since the identification of Myco. leprae by Hansen has been the transmission of experimental leprosy to animals. This was achieved first by Shepard (1960a, 1960b), at the Communicable Disease Center, Atlanta, Ga., USA, who showed that a reproducible and limited infection could be produced when the foot pads of mice were inoculated with Myco. leprae. Moreover, an identi- cal type of infection has now been produced in other centres throughout the world. General features The infection obtained in the mouse foot pad is a local one and is dependent upon the number of bacilli inoculated. Thus inocula of 5 x 103-104 Myco. keprae multiply 100-fold in 6-8 months, whereas larger inocula fail to give a higher yield, and inocula of 106 bacilli fail to show any multiplica- tion. Moreover, having multiplied as far as they are able, the bacilli gradually die (Rees, 1964). A similar type of infection has been obtained subse- quently in the ears of mice and in the ears and foot pads of hamsters (Waters & Niven, 1965, 1966) and in the foot pad of the rat (Hilson, 1965). Although these experimental infections give only limited multiplication of Myco. leprae, they are reproducible and can be maintained indefinitely in the laboratory by passage and are adaptable to quantitative anal- ysis. It is probable that more than 200 strains of Myco. leprae derived from individual patients with active disease originating from nearly every part of the world have produced an identical type of infection when inoculated into foot pads of mice. For example, in our own series we obtained success- ful transmission with all 89 strains of bacilli isolated from individual patients with active leprosy from different parts of the world and with different types of the disease (79 patients with lepromatous leprosy and 10 with borderline tuberculoid leprosy). The sources of the 89 strains is shown in the following tabulation. Origin Africa Central East West Burma India Malaysia Malta Pakistan Samoa West Indies No. of strains 3 6 8 66 1 1 l These results are important since they indicate that the virulence of strains of Myco. leprae for the mouse is similar, irrespective of whether the strains were derived from patients with the more tuberculoid or more lepromatous form of the disease, thus sug- gesting that the pattern of disease in man is deter- mined by the host and not by the parasite. However, these findings do not exclude the possibility of strain difference in Myco. leprae of the type, for example, that has been shown with Myco. tuberculosis in animals where both Myco. tuberculosis hominis and Myco. tuberculosis b,vis are virulent in the guinea-pig but only the latter is virulent in the rabbit. The evidence that the infection produced in the foot pads or ears of various rodents, including the mouse, rat 'and hamster, is overwhelmingly in favour of the infection being due to the human leprosy bacillus. Although this is now universally accepted, it is important to recapitulate the criteria which were used to establish this evidence and the care that was taken by those working in the field to satisfy all the criteria that are outlined in the fol- lowing section. The criterion of bacterial multiplica- tion was based on precise quantitative methods so that the number of bacilli inoculated could be com- pared with the number finally harvested in the foot pads of mice. The pattern and rate of multiplication were uniformly reproducible with all strains of Myco. leprae derived from untreated patients. All the inocula and harvests were cultured on media suitable for isolating mycobacteria, in order to exclude the possibility that the infection was caused by a cultivable organism. Lepromin prepared from bacilli harvested from the mouse foot-pad infections was compared with standard Mitsuda-type lepromin prepared from man in a series of patients with different types of leprosy and the pattern of response was identical (Shepard & Guinto, 1963). The histology of the foot-pad infection in mice was also studied and the pattern of response was shown to be similar for all strains of Myco. leprae and, although 790 NEW PROSPECTS FOR THE STUDY OF LEPROSY IN THE LABORATORY the cellular changes were indeterminate and not characteristic of the polar types of leprosy as seen in man, they were not characteristic of those pro- duced by any other known species of mycobacteria (Rees & Weddell, 1968). Moreover, in a proportion of the infected animals acid-fast bacilli were found, occasionally late on in the infection, within nerves of the foot pad or in the sciatic nerve (Wiersema et al., 1965; Rees & Weddell, 1968) and therefore showed a selectivity for peripheral nerves shared by no other species of mycobacteria. Although the infection produced by the local inoculation of Myco. leprae into the ears or foot pads of rodents was a limited one and multiplication only occurred when the number of bacilli inoculated was less than 106, bacilli could be harvested from these infections and reinoculated into animals, where again they multiplied and reproduced the same bacteriological and histological patterns of response. Therefore, because the characteristics of Myco. leprae were not changed by serial passage, this method could be used for maintaining indefinitely experimental infections with Myco. leprae for study in the laboratory (Rees, 1965b; Shepard, 1965b). Moreover, a similar pattern of infection has been obtained in the many different strains of mice used although more detailed comparisons suggest that the CBA and BALB/C strains of mice are the most susceptible (Shepard & Habas, 1967). RECENT APPLICATIONS OF INFECTIONS IN THE FOOT PADS OF MICE FOR THE STUDY OF MYCO. LEPRAE Once it was established that Myco. leprae could be transmitted to animals it was hoped that this experimental infection would provide the first opportunity for studying Myco. leprae in the labor- atory. These hopes have been fully justified; within less than 10 years, despite the limited nature of experimental human leprosy in normal animals, this in vivo technique has provided new information of great importance concerning the properties of Myco. leprae. For practical reasons the mouse foot-pad infection with Myco. leprae has been chosen by most laboratories working in this field. Generation time of Myco. leprae During the logarithmic phase of multiplication in the mouse foot-pad a generation time of 13-25 days has been observed (Shepard & McRae, 1965). This very long generation time is consistent with the chronicity of the disease and the long incubation period observed in man and, although comparable to the generation time of Myco. lepraemurium it is otherwise unique, even for the slowly multiplying Mycobacteriaceae. Drug sensitivity testing The foot-pad infection, in spite of its limited nature, has been used successfully for testing drugs for antileprosy activity. Thus it has been shown that when Myco. leprae is injected into the foot pads of mice treated with known antileprosy drugs the organisms fail to multiply. Therefore, the mouse foot-pad infection provides, for the first time, a specific test for screening new antileprosy drugs and the search is no longer restricted to drugs that are known to be active against Myco. tuberculosis (Table 1). Hitherto, chemotherapy in leprosy had necessarily evolved from an entirely empirical basis and even the regimen of treatment with diamino- diphenylsulfone, which has been the standard used for leprosy since 1943, was established by trial and error. This was due to the fact that although diaminodiphenylsulfone has slight in vitro and in vivo activity against Myco. tuberculosis in guinea- pigs, it is ineffective against tuberculosis in man. Thus on an empirical basis it has been accepted that the standard dose of diaminodiphenylsulfone should be 100 mg daily; however, there are important practical and clinical advantages to be gained by modifying this regimen. For example, intermittent treatment with diaminodiphenylsulfone could more easily be supervised than daily treatment. Further- more, leprologists are now tending to advocate smaller doses of diaminodiphenylsulfone because these appear to reduce the frequency and severity of acute reactions (exacerbation) with their attendant nerve and eye complications but are equally effective in controlling the disease. On account of these trends, the mouse foot-pad technique is used not only for screening drugs but has also been specific- ally applied to determine, for the first time, the minimal inhibitory concentration (MIC) of diamino- diphenylsulfone in vivo against strains of Myco. leprae from previously untreated patients (Shepard, 1967b; Rees, 1967a, 1967b). The sensitivities of 5 " wild " strains of Myco. leprae from patients in Malaysia were tested in this way using concentrations of 0.0001 % and 0.00001 % of diaminodiphenyl- sulfone in the diets of the mice. The results are shown in the following tabulation: 791 R. J. W. REES Strain 1 2 3 4 5 0.0001 % Sensitive Sensitive Sensitive Sensitive Sensitive 0.00001 / Sensitive Resistant Resistant Resistant Resistant The MIC was determined by feeding groups of mice with diminishing concentrations of diamino- diphenylsulfone in their diet and determining the concentrations of diaminodiphenylsulfone in the sera of each group. The results of these studies are shown in Table 2, from which it is clear that the MIC for diaminodiphenylsulfone against wild strains of Myco. leprae is approximately 0.015 ,ug/ml. Thus, Myco. leprae in the mouse is exquisitely sensitive to diaminodiphenylsulfone; an unexpected finding, because all other species of mycobacteria are relativ- ely insensitive to sulfones and even the diamino- diphenylsulfone-sensitive micro-organisms such as group A streptococci (Francis & Spinks, 1950) and Plasmodium berghei (Thompson, Oleszewski & Waitz, 1965) are 3-100 times less sensitive than Myco. leprae to diaminodiphenylsulfone. The standard treatment of 100 mg of diaminodiphenylsulfone per day for man gives serum concentrations of approx- imately 1.5 ,ug/ml. Assuming that it is permissible to extrapolate these findings from mouse to man, they suggest that a daily dose of 1 mg of diaminodiphenyl- TABLE 1 TESTS OF ACTIVITY OF DRUGS AGAINST MYCO. LEPRAE USING THE MOUSE FOOT-PAD TECHNIQUE Dose No. of strains IAtvt in diet) tested Activity Reference Diami nodiphen ylsulfone Sulfadimethoxine Sulformethoxi ne Sulformethoxi ne Diphenylthioureas Thiambutosi ne Thiambutosi ne Ba-22'330 b Ba-36'223 c SU-2079 d Thioacetazone Thioacetazone Clofazimine Clofazimine Streptomyci n Isoniazid p-Aminosalicylic acid Cycloserine Ethambutol Ditophal Pyrazinamide Capreomycin 0.1-0.0001 0.1 0.04 0.1 (3 times weekly) 0.1 0.1 0.1 0.1 0.1 0.1 0.2 0.01 0.006 2 mg/day e 0.01 0.6 0.5 0.25 0.5 0.5 10 mg/dayf 2 6 6 + p 0 Rees (1965b), Shepard (1964), Shepard & Chang (1962, 1964) Rees (1965b) Rees (1965b) Rees (1965b) Shepard & Chang (1964) Rees (1965b) Rees (1967a) Rees (1967a) Rees (1967a) Shepard & Ct Rees (1965b) Shepard & Ch Rees (1965b) iang (1964) iang (1964) Shepard & Chang (1964) Shepard & Chang (1962, 1964) Shepard & Chang (1962, 1964) Shepard & Chang (1962, 1964) Shepard & Chang (1964) Shepard & Chang (1964) Shepard & Chang (1964) Shepard (1964) a + = Full activity; P = partial activity; 0 = inactive. b Ba-22'330 = 4-(3-carboxypropoxy)-4'-dimethylami no-diphenylthiourea c Ba-36'223 = 4-dimethylami no-4'-(4-hydroxybutoxy)-diphenylthiourea d SU-2079 = 4-butoxy-4'-diethylaminoethoxy-diphenylthiourea e Once-daily injections. thiambutosine metabolites. Drug 792 NEW PROSPECTS FOR THE STUDY OF LEPROSY IN THE LABORATORY TABLE 2 CONCENTRATION OF DIAMINODIPHENYSULFONE IN THE SERA OF MICE FED DIFFERENT LEVELS OF DRUG IN THE DIET ,~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ Dose of diaminodiphenylsulfone Percentage in diet a 0.1 0.025 0.01 (Man: 100 mg/day) 0.001 0.0001 (Man: 1 mg/day) 0.00001 Mg per kg of body-weight 200.0 50.0 20.0 (2.0) 2.0 0.2 (0.02) 0.002 Concentration of diaminodiphenyl- sulfone in serum or plasma 12.5 3.3 1.0 b (1.5) 0.15 b 0.015 b (0.018) b (0.0015) e a g/100 g. b Estimated by Glazko's method. c Calculated concentration. Serum concentration varies linearly with doses between 0.1% and 0.0001%. Actual value is below level of detectability. sulfone would be effective in man. This means that there is strong support from the animal studies for encouraging trials, using lower doses of diamino- diphenylsulfone in man, to reduce the incidence of reactions without the fear of diminishing the therapeutic efficacy of the drug. Drug-resistant strains of Myco. leprae The result which has emerged from the systematic investigation of foot-pad infections has been direct proof of the existence of drug-resistant strains of Myco. leprae. Carefully controlled investigations on specially selected patients who showed active disease despite treatment with diaminodiphenyl- sulfone for at least 10 years, combined with studies on the diaminodiphenylsulfone sensitivity of bacilli from these patients using the mouse foot-pad technique, have shown that a proportion of such patients are infected with diaminodiphenylsulfone- Man Mouse Man Man/mouse Man/mouse Mouse/man resistant strains of bacilli (Pettit & Rees, 1964; Pettit, Rees & Ridley, 1966; Adams & Waters, 1966; Rees, 1967b). To date, 19 diaminodiphenylsulfone- resistant strains from individual patients in different parts of the world have been detected using the mouse foot-pad test (Table 3). From the data presented in Table 2 on the serum levels obtained in patients on regimens of 100 mg of diamino- diphenylsulfone daily (1.5 jug/ml), and from the MIC of diaminodiphenylsulfone for wild strains of Myco. leprae calculated from the foot-pad test (0.015 ,ug/ ml), relapses under such doses of diaminodiphenyl- sulfone in man due to the emergence of drug-resistant strains, gives a resistance ratio of 100. The results from Table 3 show clearly that the degree of resis- tance developed by all 19 strains had a resistance ratio of not less than 100. The relevant data on resistance studies which show that the correlation between studies in man and mouse are satisfactory, are summarized below: 100 mg of diaminodiphenylsulfone/day; serum level = 1.5 ytg/ml MIC of diaminodiphenylsulfone; serum level = (0.015 ,ug/ml) I mg of diaminodiphenylsulfone/day; serum level = (0.018 jug/ml) (103 mg of diaminodiphenylsulfone/day; serum level in man) / (MIC in mouse) = "therapeutic ra- tio" = 1.5/0.015 = 100 Similarly, strains of Myco. leprae resistant to treatment with 100 mg of diaminodiphenylsulfone/day in man would be expected to have a resistance ratio of not less than 100 This expectation was confirmed: diaminodiphenylsulfone-resistant strains of Myco. leprae from patients receiving 100 mg of diaminodiphenylsulfone/day multiplied in mice fed doses of diaminodiphenylsulfone resulting in serum levels from 1 Lg-12.5 yg of diaminodiphenylsulfone ml, giving a resistance ratio of 100-1250 793 R. J. W. REES TABLE 3 SENSITIVITY OF STRAINS OF MYCO. LEPRAE FROM RELAPSED DIAMINODIPHENYLSULFONE-TREATED PATIENTS a TO DIAMINODIPHENYLSULFONE b Strain Country ofStan origin 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 I Malaysia India Malaysia I ndia West Africa India Sensivity to diaminodiphenylsulfone: c percentage of drug in the diet 0.1 0.025 0.01 e 0.001 0.0001 0.00001 R R R R S S R Malaysia R R R R R S S S S !s s R R R SI R R R R R R R R R R R I R R R R R R R R R R R R R R R R R R R R R R R R R R R R R R a All these patients failed to respond bacteriologically, histologically or clinically to a rigorously controlled period of not less than 6 months on a supervised dose of 600 mg of diaminodiphenyl- sulfone per week. b A dosage of 0.01% of diaminodiphenylsulfone in the diet of mice gives a serum concentration of I iAg/ml and is therefore of the same order as the concentration in man on a regimen of 100 mg/ day. Thus the level of resistance in the 19 strains of Myco. Ieprae determined in mice is consistent with the therapeutic failure of diaminodiphenylsulfone in the patients. CS = Sensitive; R = resistant. Although the mouse foot-pad test has provided the first direct evidence of the emergence of resistance to diaminodiphenylsulfone, the phenomenon appears to be rare, and may have resulted from the use of excessively high doses of diaminodiphenylsulfone. It has to be admitted that the use of smaller doses of diaminodiphenylsulfone, resulting in concentration of diaminodiphenylsulfone in the serum and tissues nearer to the MIC of diaminodiphenylsulfone for Myco. leprae, could lead to the emergence of a greater number of resistant strains. This possibility must be weighed against the advantages to be gained by a significant diminution in the incidence of serious reactions that are believed to be directly related to high doses of diaminodiphenylsulfone. Similar but less extensive studies have demon- strated the emergence of thiambutosine-resistant strains of Myco. leprae- (Rees, 1967a, 1967b); such strains show cross-resistance to thioaceta- zone, a feature shared by thiambutosine-resis- tant strains of Myco. tuberculosis (Konopka et al., 1955). .~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 794 NEW PROSPECTS FOR THE STUDY OF LEPROSY IN THE LABORATORY Effect of BCG vaccination against Myco. leprae The foot-pad infection technique provides an experimental model for investigating the prophylac- tic effects of various vaccines and has already demonstrated that vaccination by BCG significantly inhibits the multiplication of Myco. leprae (Shepard, 1965a, 1966). This finding is important although it is not unique since it has already been shown experimentally that vaccination by BCG can produce protective immunity against species of mycobacteria other than Myco. tuberculosis (Fenner, 1957). Still more recently. Shepard & Ribi (1968) have shown that vaccination with the cell-wall fraction of BCG incorporated in an oily base is as protective as living BCG, weight for weight, against infections with Myco. leprae in the foot pads of mice. Thus, mouse foot-pad infections with Myco. leprae provide an important model for investigating the value of prophylactic vaccinations against human leprosy which, if they could eventually be applied to man, would be expected to play a major role in the eradication of the disease. The current importance of these experimental observations that BCG pre- vents the multiplication of Myco. leprae in the mouse foot-pad is that they support the preliminary results in man that BCG vaccination significantly reduces the incidence of early type leprosy in child contacts in Uganda (Brown & Stone, 1966; Brown, Stone & Sutherland, 1968). EXPERIMENTAL LEPROSY IN MICE WITH REDUCED IMMUNOLOGICAL CAPACITY The successful transmission of human leprosy to animals in 1960 provided the first and only means of studying Myco. leprae in the laboratory. Although this experimental model has, in less than a decade, provided more information on the human leprosy bacillus than was available previously, progress in the field of leprosy research was still restricted by the limited nature of the infection. Clearly, the next step was to determine the factor or factors pre- venting Myco. leprae from multiplying freely in mice or in other rodents. On the assumption that the infection was limited by the development of immunity, various methods for reducing the immuno- logical capacity of mice were investigated in an attempt to enhance the infection. The assumption was confirmed when it was demonstrated that enhanced infections with Myco. leprae could be obtained in mice following thymectomy and whole- body irradiation (with 900 r) as a means of reducing their immunological capacity. General features of the infection in thymectomized, irradiated mice inoculated with Myco. leprae It has now been established (Rees, 1965a, 1965b; Rees & Weddell, 1968; Rees et al., 1967) and confirmed (Gaugas, 1967; Shepard & Congdon, 1968) that when the immunological capacity of mice is. reduced by thymectomy plus irradiation (with 900 r), Myco. leprae inoculated locally into the foot pads or ears multiply more freely and yield 100-1000 times more bacilli per site than in normal animals (Fig. 3), and that in due course the infection spreads to other sites. Moreover, similarly treated mice become heavily infected in specific sites when inoculated intravenously with Myco. leprae (Rees & Weddell, 1968; Rees et al., 1967). Although, in these animals, the generation time is not reduced, the bacilli, continue to multiply for a longer period. The spread of infection in locally inoculated animals is also highly selective. The sites of predilection are in the skin of the ears, hind and fore paws, the tail and also the nose (Table 4). The same rigorously controlled criteria have been used to identify Myco. leprae in the enhanced infec- FIG. 3 GROWTH CURVES OF MYCO. LEPRAE IN THE FOOT PADS OF NORMAL AND THYMECTOMIZED, IRRADIATED a MICE INOCULATED WITH 104 BACILLI b i 7~~~7 2 4 6 11 1 14 1 Time (months) aT + I; dose, 900 r. b Data taken, by permission, from Rees et al. (1967). =- Total number of Myco. Ieprae. - ...... Number of viable Myco. Ieprae. II 795 R. J. W. REES TABLE 4 LOCALIZATION AND YIELD OF MYCO. LEPRAE IN A THYMECTOMIZED, IRRADIATED a MOUSE 19 MONTHS AFTER THE INTRAVENOUS INJECTION OF3x107 BACILLI Estimated yield of bacilli Degener- Organ ortissue Per site Percentage of (0/) (x106) total yield _ Foot pads: Hind 1 740 18 56 Fore 960 10 60 Total 2 700 28 95 59 Ears 4 800 49 50 Nose 1800 18 61 Muscle of leg 76 0.8 30 Muscle of body 160 1.6 31 Skin of tail 21 0.2 89 Skin of body 5 0.05 55 Liver 130 1.3 82 Spleen 53 5.0 87 Lung 10 0.1 70 Total 9 755 a Dosage: 900 r. tions that were used in normal animals and have included testing the sensitivity of the organisms to diaminodiphenylsulfone (Rees & Weddell, 1968; Rees et al., 1967) and the production of lepromin (Draper, Rees & Waters, 1968). In addition to the higher yields of bacilli from such animals it has been shown that later in the infection there is frequently nodular swelling of the foot pads (Fig. 4) and the histology of the lesions replicates that seen in patients with lepromatous or borderline type leprosy (Fig. 5) (Rees & Weddell, 1968; Rees et al., 1967). Thus there is heavy infection of the peripheral nerves in the skin sites referred to (Fig. 6). Positive smears can be obtained from nasal swabs and the histological picture shows typical foam cells and degenerative changes in some of the infected peripheral nerves (Rees & Weddell, 1968). There is increasing evidence that these changes occur with the slow and partial recovery of the immunological capacity of the animals and can be enhanced in animals with established infections by the donation of immunologically competent syngeneic lymphoid cells from normal mice (Fig. 7) (Rees & Weddell, 1968). FUTURE PROSPECTS The successful transmission of human leprosy to animals (with the prospect of maintaining the infec- IG. 4 NODULAR SWELLING OF HIND FOOT PAD OF A THYMECTOMIZED, IRRADIATED MOUSE INOCULATED LOCALLY WITH 10' MYCO. LEPRAE 9 MONTHS PREVIOUSLY 796 FIG. 5 SKIN FROM THE NODULAR, SWOLLEN FOOT PAD OF A THYMECTOMIZED, IRRADIATED MOUSE INOCULATED 8.5 MONTHS PREVIOUSLY WITH 108 MYCO. LEPRAEa - %-''2Xt. ..45M$3_FIG. 's~~~~IFCE WIT YO LEPRAE !y E714~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~. a Globi loaded with bacilIli and foam (Virchow's cells) are seen in the dermis that is sepa- rated by a clear zone from the epidermis. These features replicate those seen in lepro- matous leprosy in man. Stained: haematoxylin and cold carbol fuchsin; magnification: FIG. 6 MEDIAL PLANTAR NERVE FROM THE SAME MOUSE AS IN FIG. 5 TO SHOW SCHWANN CELLS INFECTED WITH MYCO. LEPRAEa *l~r.,ccWM a Stained: haematoxylin and cold carbol fuchsin; magnification: x 5000. FIG. 7 ELECTRON PHOTOMICROGRAPH OF DERMIS FROM EAR OF THYMECTOMIZED, IRRADIATED MOUSE INOCULATED WITH 107 MYCO. LEPRAE LOCALLY IN BOTH EARS AND FOOT PADS 15 MONTHS PREVIOUSLY AND GIVEN SYNGENEIC LYMPHOID CELLS FROM A NORMAL MOUSE 5 MONTHS BEFORE THIS PREPARATION WAS MADEa A hedgeertonofnrvaos, i S...cwn cl in 2mene n fs en 4 a The degeneration of nerve axons, myelin rings, and Schwann cells in 2 myelinated nerve fibres can be seen: x 45 000. r N.- F" I1110011- idi,: V"' 4 .. f. -1 4 mk. "p- 4i k-4 NEW PROSPECTS FOR THE STUDY OF LEPROSY IN THE LABORATORY tion indefinitely by serial passage) and the reproduc- tion of some of the major characteristics of the human disease in laboratory animals by reducing their immunological capacity, provide, for the first time, a means of studying Myco. leprae in the laboratory. Chemotherapeutic studies The enhanced infection provides a more rapid method for screening new drugs against Myco. leprae and for applying the more quantitative and kinetic methods already developed in normal mice for studying the action of antileprosy drugs (Shepard, 1967a). In particular, the larger infections obtained in thymectomized, irradiated mice will provide a more sensitive method for determining and com- paring the bacteriostatic, as well as the bactericidal, effects of drugs on Myco. keprae. Although the application of the MI to chemotherapeutic studies in man indicates that a very high proportion of bacilli in the skin are killed, by diaminodiphenyl- sulfone, for example, within a period of 3-6 months, there is strong clinical evidence that patients relapse unless diaminodiphenylsulfone treatment is main- tained for several years (Quagliato, Berquo & Leser, 1961). One possible explanation is that there are specific sites in the body in which diaminodiphenyl- sulfone and other antileprosy drugs are ineffective, or less effective, and that such sites provide a source of viable organisms. There are suggestions from histological studies on human tissues that arrectores pili muscles and Schwann cells may still harbour healthy bacilli when those in surrounding tissues are very degenerate. The heavy and generalized infec- tion which can be obtained in intravenously inocu- lated thymectomized, irradiated mices provides an ideal model for investigating these possibilities since the cellular pattern of infection exactly mimics that seen in man, including parasitization of both muscle and Schwann cells. By treating such animals with antileprosy drugs, including drugs labelled with radioactive isotopes, it should be possible to deter- mine both the distribution of the drugs and of degenerate and normal bacilli, at an intracellular level. Emergence of drug-resistant strains of Myco. leprae The foot-pad infection technique in normal and thymectomized, irradiated mice is the only method available at present for detecting the emergence of drug-resistant strains of Myco. leprae and the method could now be used to survey the importance of drug resistance on a world-wide basis. Because of the increasing use of low-dose regimens of diamino- diphenylsulfone, there is obviously a danger of diaminodiphenylsulfone resistance emerging. Under field conditions, however, if such resistance emerged it might remain undetected by the routine clinical and bacteriological methods until it had reached serious proportions. Because diaminodiphenyl- sulfone is the standard form of treatment through- out the world, such an occurrence would be dis- astrous to the control of leprosy. Every effort should therefore be made now to devise suitable experimental models which might be used to predict the rate of emergence of diaminodiphenylsulfone- resistant strains of Myco. leprae in animals receiving decreasing doses of the drug. The bacterial popula- tions in an established infection in intravenously inoculated, thymectomized, irradiated mice, would be large enough to detect resistance since such mice have a bacterial population of 1010 and drug- resistant mutants could be expected in a proportion of 1: 107. Routes of infection The routes of infection for leprosy in man are still unknown and the much more susceptible, thymect- omized, irradiated mice provide an experimental model for investigating this important problem. The nose, upper respiratory tract, alimentary tract and skin are all routes of infection that should be studied. Moreover, because it is known that thymectomized, irradiated mice heavily infected with Myco. leprae excrete bacilli from their nasal mucosa (Rees & Weddell, 1968), they could be used as " open cases" to determine their infectious- ness for highly susceptible but non-infected thymec- tomized, irradiated mice, housed in the same cages. Sources of infection other than man There has been much discussion on whether vectors, either insect or mammalian, could be involved in the transmission of leprosy from man to man and whether any domestic or wild animals can be infected with Myco. leprae and therefore be a source of bacilli to infect man. The successful transmission of human leprosy to normal or thymec- tomized, irradiated mice might be used in 2 ways. to study these possibilities. The mouse foot-pad technique could provide, for the first time, a reliable method of identifying as Myco. leprae any non- cultivable, acid-fast bacilli isolated from potential vectors. On the other hand, and perhaps even more 797 R. J. W. REES important, there is the observation that the intra- venous inoculation of Myco. leprae into normal mice can produce, towards the end of their life, an infec- tion of the nose and the paw skin. Because the inoculation of Myco. leprae into the foot pads of rats results in a pattern of infection similar to that in mice, it is likely that both species are equally susceptible to the human leprosy bacillus. It is possible, therefore, that wild mice and rats in leprosy endemic areas could be infected with Myco. leprae and thus be a source of human infection. Since both rats and mice are present in large numbers in all epidemic and endemic leprosy areas throughout the world, it is suggested that this possibility should be investigated by sample surveying. Application of enhanced infection for studying the pathogenesis of human leprosy Leprosy in man presents a wide clinical spectrum ranging from the tuberculoid type, where there are few bacilli and the patient has a high degree of immunity, to the lepromatous form, where there are many bacilli and the patient has little or no resistance. Superimposed on these very variable clinical forms is the common feature that peripheral nerves are infected. Myco. leprae is the only species of myco- bacterium known to infect nerves in either man or animals and the extent of damage to the infected nerves appears to depend on the immunological capacity of the host. Thus, in tuberculoid type leprosy infection of nerves results in the destruction of axons, whereas in lepromatous leprosy, the nerves can be heavily infected with bacilli without damage to the axons. The " target cell " for parasitization by Myco. leprae within nerves is the Schwann cell. In addition to the variable clinical picture " reac- tional" episodes may occur and on these occasions the existing lesions, or new ones, present as sites of acute inflammation. These episodes are likely to have an immunological basis; certainly, an increase in the immunological capacity of the patient must play a major role in one type of reaction since it is followed by a shift in the clinical picture from the lepromatous towards the tuberculoid form of the disease. The term "reversal reaction" has been applied, to such a shift. The importance of nerve involvement in all forms of leprosy, together with the very wide range of clinical, bacteriological and histological forms, and the way in which each seems to be dependent upon fine differences in the immunological capacity of the patient, have been stressed in order to illustrate the complexity of leprosy in man. On account of the chronicity and complex nature of the disease in man, it is probable that the final elucidation of the pathogenesis of human leprosy will be achieved only if contributions are made from the experimental studies and such contributions can only be made if the human disease can be duplicated in experimental animals. This prerequisite seems likely to be achieved because already the inocultation of Myco. leprae into mice subjected to thymectomy and total body irradiation has reproduced completely the lepromatous type of disease seen in man (Rees & Weddell, 1968). These two areas of immunological research in leprosy are, of course, importantly related. Mice have been shown to develop lepromatous-type infection when they are subjected to procedures (thymectomy and irradiation) that produce a pro- found and long-lasting immunological depression, and the immunological depression in lepromatous patients has been made clear by well tried immuno- logical procedures (Int. J. Leprosy, 1968). Fortunate- ly, it is possible to increase the immunological capacity of treated mice at will (by the intraperi- toneal inoculation of syngeneic lymphoid cells from normal mice) and already preliminary results of such manipulations show that the progressive form of lepromatous leprosy can be halted or that reversal reactions can be produced which result in a shift from the lepromatous form of the disease to the tuberculoid form seen in man (Rees & Weddell, 1968). Therefore, there is every reason to believe that these models can and should be developed in order to provide means to study the pathogenesis of the different forms of human leprosy, to study the etiology of nerve involvement and damage and to study the role of immunology in these processes. 798 NEW PROSPECTS FOR THE STUDY OF LEPROSY IN THE LABORATORY' 799 RISUMt PERSPECTIVES NOUVELLES POUR L'tTUDE DE LA LE-PRE AU LABORATOIRE On n'est pas encore parvenu a cultiver Mycobacterium leprae in vitro et il a fallu recourir a l'experimentation in vivo afin de pouvoir 6tudier l'infection l6preuse. Par ailleurs, ce n'est qu'en 1960 qu'on a r6ussi a transmettre la 1lpre a des animaux. Cela explique que les progres de nos connaissances relatives a cette affection soient restes bien en de,a de ceux obtenus dans d'autres secteurs de la pathologie et que l'essentiel de la recherche en matiere de lepre ait porte sur l'obtention de cultures du micro-organisme in vitro et in vivo. Le probleme a pu etre aborde indirectement grace a l'elaboration de modeles exp6rimentaux. Une des pre- mieres realisations de ce genre a ete, il y a une dizaine d'ann6es, I'application de techniques quantitatives 'a 1'etude de Myco. kepraemurium qui a permis de definir une methode indirecte pour evaluer la vitalite des bacilles de la lepre en l'absence de cultures in vitro. On parvient actuellement a distinguer les bacilles vivants des bacilles morts, sur la base de criteres morphologiques, apres une simple coloration par la m6thode courante de Ziehl- Neelsen. Cette decouverte a beaucoup facilite l'etude de l'e'volution de l'infection lepreuse chez l'homme et de l'infection experimentale chez l'animal. L'indice mesu- rant la vitalite des bacilles, appele #indice morpholo- gique)) fournit un moyen tres sensible d'apprecier la reponse des malades a la chimiotherapie. C'est vers la meme epoque qu'on a obtenu pour la premiere fois la croissance de Myco. lepraemurium en cultures cellulaires et on peut esperer que ce mode de culture in vitro pourra ulterieurement etre applique a Myco. leprae. Un nouveau progres, plus important encore, a et realise en 1960 lorsqu'on a demontre que Myco. leprae pouvait se multiplier dans le coussinet plantaire de la souris. Cette infection experimentale a permis de calculer le temps de g6neration du bacille de la lepre (13-25 jours), d'evaluer l'efficacit6 des m6dicaments antil6preux, de mesurer la sensibilite des souches de Myco. leprae d'ori- gine humaine a la diamino4-4'diph6nylsulfone (DDS), d'identifier les mutants r6sistants a la DDS ou A d'autres medicaments en cas de rechute en cours de traitement, et enfin d'6tudier l'effet protecteur de la vaccination par le BCG. Ce materiel d'etude, bien qu'ayant permis d'6largir notre connaissance de la maladie, ne donnait pas entiere satisfaction, car l'infection experimentale ainsi realis6e restait limitee et localis6e et ne pouvait pretendre repro- duire tous les aspects de la lepre humaine, et notamment l'atteinte nerveuse. Cette lacune a e recemment com- blee: on a montre qu'une infection progressive et genera- lisee succede a l'inoculation intraplantaire ou intravei- neuse de Myco. leprae chez la souris dont les defenses immunitaires ont ete affaiblies par thymectomie et irra- diation totale de l'organisme. Bien plus, lorsque l'infec- tion ainsi favorisee est bien etablie, des le 9e mois, 1'evolution de la maladie chez l'animal presente des caracteristiques histologiques identiques a celles de la lepre Ilpromateuse chez l'homme, avec infection et l6sion des nerfs peripheriques. Les resultats obtenus grace a ces modeles experimen- taux sont particuli6rement importants, car pour la pre- miere fois on dispose de methodes permettant 1'etude directe de Myco. leprae en laboratoire. On doit naturelle- ment continuer a mettre tout en aeuvre pour obtenir la multiplication de Myco. leprae in vitro. En atten- dant, il n'est pas impossible que les progres de nos connaissances concemant la pathogenie de la lepre humaine depassent nos esperances, maintenant que de nombreux aspects de la maladie peuvent 8tre reproduits experimentalement chez la souris privee de ses d6fenses immunitaires. REFERENCES Adams, A. R. D. & Waters, M. F. R. (1966) Brit. med. J., 9, 872 Brown, J. A. K. & Stone, M. M. (1966) Brit. med. J., 1, 7 Brown, J. A. K., Stone, M. M. & Sutherland, 1. (1968) Brit. med. J., 1, 24 Chang, Y. T. (1960) Fed. Proc., 19, 385 Draper, P., Rees, R. J. W. & Waters, M. F. R. (1968) Clin. exp. Immunol., 3, 809 Fenner, F. (1957) Amer. Rev. 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