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Environmental mycobacteria and BCG vaccination
Tubercle 67 (1986) l-4 0 Longman Group Ltd.
LEADING
ENVIRONMENTAL
ARTICLE
MYCOBACTERIA
AND BCG VACCINATION
Reflecting upon the prevalence and antiquity of tuberculosis, Bankoff [II remarked that ‘When the Lord created man on the sixth day, one is forced to believe that He must have used clay of ancient Asia abounding in tubercle microbes’. Although this claim might not be accepted unreservedly, there is no doubt that the clay of that continent is abounding in many saprophytic species of mycobacteria 121. In 1966 Palmer and Long [3] postulated that ‘natural vaccination’ due to contact with environmental mycobacteria (EM) accounted for the striking differences in the efficacy of BCG from trial to trial. Interest in this theory was reawakened when a major and well-executed BCG trial at Chingleput in South India, where tuberculosis is rife, indicated that the vaccine had no protective effect in that region [41. Using guinea pigs, Palmer and Long found that BCG could increase the partial degree of immunity imparted by prior contact with an environmental mycobacterium but never to a level above that achieved by BCG alone. It was therefore concluded that BCG only achieves its full potential in regions where the population is not exposed to EM but that elsewhere the observed effect of the vaccine is the difference between the potential effect and that of the ‘natural vaccination’. An explanation of the Chingleput findings, on the basis of the hypothesis of Palmer and Long, is that BCG added nothing to a population already maximally protected by prior contact with EM. It is, however, also necessary to consider whether the vaccine could fail in an unprotected population and whether excessive contact with EM, or certain species thereof, may result in a paralysis of the protective immune response or the generation of a reaction that antagonises it. In 1929 Rich [5] questioned the then widely accepted dogma that tuberculin reactivity was a measure of resistance of the individual and suggested that excess sensitivity (i.e. hypersensitivity) might be harmful. It was subsequently confirmed both in animals [6] and in man [7] that individuals with a small degree of tuberculin sensitivity are protected, but that those with large reactions are less protected than those with no sensitivity at all. One school of thought (the ‘one pathway’ school) argues that a moderate degree of cell-mediated reactivity is indeed protective whereas an excessive reaction, like many excesses in life, is harmful. Thus the difference between ‘prophylactic’ cell mediated immunity and tissue-damaging ‘anaphylactic’ hypersensitivity is seen only as 3 quantitative one. On the basis of this theory it may be predicted that BCG vaccination would be effective in children, whose sensitisation by EM would be minimal, but could be worse than useless in hypersensitive adults by further enhancing such tissue-damaging reactivity. Both these predictions were, in fact, fulfilled in the Chingleput study. An important alternative explanation which also leads to the same predictions was advanced by Stanford and Rook and their coworkers 18, 91. These investigators postulate a fundamental qualitative difference between the protective and antagonistic immune pathways. The former, that of true protective immunity, was considered analogous to that elucidated by Mackaness [IO] in mice infected with Listerie monocytogenes. The latter was equated with the necrotic reaction in guinea-pigs known as the ‘Koch phenomenon’.
2
Grange
Accordingly
the two patterns of responses were termed the Listeria-type and Koch-type It was postulated that some species or populations of EM induce the former response while others, or infection with M. tuberculosis, induce the latter. It was further postulated that the induction of one of these responses blocks the subsequent induction of the other. (The idea that early mycobacterial contacts influence the individual’s future pattern of reactivity was first advanced by Abrahams [II] in a paper entitled ‘Original Mycobacterial Sin’.] According to this, the ‘two pathway’ theory, BCG affords protection when it elicits or boosts Listeria-type reactivity but proves ineffective or even harmful when it boosts Koch-type reactivity. The Chingleput findings are thus accounted for by a high prevalence of the latter type of reactivity amongst those vaccinated. At present, the effect of ‘immunological effective contact’ with EM on immunity to mycobacterial disease is the subject of controversy, as is evident from a number of publications in Tubercle and elsewhere [12-161. A critical appraisal of the arguments for and against both theories and of the data used to formulate the arguments is therefore required. It is important to realise that the proponents of the ‘two pathway’ theory accept that EM do indeed induce a protective response in many situations and that the antagonistic response only occurs in certain regions where contact with EM is excessive or where particular species or combinations of species occur. Thus Stanford and Rook [17] accept that ‘natural vaccination’ probably accounted for differences between the British and Ugandan BCG trials on the one hand and the Alabama and Georgia trials on the other. It is also necessary to restate a well known scientific principle that it is impossible to prove that a phenomenon does not occur. Accordingly, the fact that several animal experiments support the hypothesis that contact with EM induces immunity cannot, be used as a primary argumentagainst the occurrence of other effects in man under quite different circumstances. It is also, of course, necessary that any animal model should, as closely as possible, resemble the natural situation. Thus an animal given a single large dose of an EM by injection or inhalation may react quite differently to one following the more natural course of ingesting small numbers of bacilli in food or drink over a period of time. The latter method of sensitisation was used by Brown and her colleagues [I61 to demonstrate that, depending on the dose and timing, such exposure could potentiate, mask or interfere with the protective effect of BCG in mice. In considering the validity of the two-pathway theory, one must look at the evidence for a dissociation between protective and non-protective immune reactions, at the claim that the induction of one of these reactions can block the subsequent induction of the other and, most importantly from the practical point of view, at the claim that these 2 pathways can be distinguished in man by qualitative differences in the dermal reaction to mycobacterial antigens. The first of these 3 points has been hotly debated since the observations of Rich j5l in 1929 and was discussed in a series of editorials in the American Review of Respiratory Disease in 1975 [l&20]. More recently, a number of distinctions between the Listeria- and Koch-type reactions has been demonstrated in the mouse [21]. Nevertheless much more needs to be discovered at the cellular and molecular level, particularly about the mechanism of the necrotic Koch-type reactions, before the debate can be resolved. Hopefully, modern techniques for cloning cells in vitro and for characterizing the relevant mediators will soon clarify the issue. The concept that the induction of one immune pathway can block the development of the other arose from 2 main sources of evidence. First, the induction of necrotic Koch-type reactions in experimental animals by certain mycobacterial species, such as Mycobacterium kansasii, was found to be blocked by prior exposure of the animals to species, such as M. vaccae, that induce Listeria-type responses [9]. Second, multiple skin testing in Burma reSPeCtiVely.
Environmental
mycobacteria
and BCG vaccination
3
revealed that strong Koch-type reactivity to M. scrofulaceum (a species common in that environment) was associated with an inhibition of reactivity to the soluble antigens of reactions [21. M. leprae which are only able to elicit Listeria-type The major stumbling block to the acceptance of the concepts of Stanford and Rook is the fact that they have been based on the use of a set of reagents, the ‘New Tuberculins’ [221, which are not commercially available. It has been claimed that these reagents enable two immune pathways to be differentiated in man by a single examination of the reaction at 48 hours, and that the type of reactivity thus determined after BCG vaccination can be used to assess the protective efficacy of that vaccine in a given region [231. The acceptance of these claims depends on the strength of the evidence that two observed patterns of dermal reactivity in man correspond respectively to the macrophage-activating protective Listeriatype responses and to the necrotic Koch-type reactions demonstrable in animals. Critics may argue that the two reactions merely differ in extent and that, in any case, the subjective nature of the examination compromises the comparisonof studies by different workers. The problem of the availability of the New Tuberculins could be rectified yet, whatever reagent is used, more objective methods for analysing patterns of skin test reactivity and for relating them to underlying immune mechanisms will be essential for a resolution of the debate. Recent developments in techniques for identifying functional subsets of lymphocytes and macrophages and for the assay of mediators should, however, soon enable definitive studies on biopsies of the various types of tuberculin reactions to be undertaken. In fine, the disciples of Palmer and Long claim that individuals in those areas where BCG shows no protective effect are already maximally protected. If this theory is accepted, health authorities might abandon the costly procedure of BCG vaccination in the belief that the environment provides the same service free! By contrast, Stanford and Rook postulate that the vaccine may fail to imrnunise an unprotected population owing to the presence of an antagonistic immune reaction that cannot be overridden by vaccination. If this theory is confirmed it will be of great importance, in the relevent regions, to give BCG (or some improved vaccine) early in life so as to induce protective immunity before the environment can have the adverse effect. Further, some form of immunotherapy that could ‘reprogram’ the immune system of inappropriately sensitized individuals would be of great value. The importance of resolving the controversy surrounding the two theories cannot be overstated: the lives of millions of potential victims of tuberculosis, and perhaps leprosy also, depend upon it. Cardiothoracic Institute, University of London, Fulham Road, London
J. Ibl. Grange SW3
6HP. References
1 Bankoff, G. (1946). The Conquesf of Tuberculosis. London: MacDonald & Co. p. 11. 2 Shield, M. J. (1983). The importance of immunologically effective contact with environmental mycobacteria. In: The Biology of the Mycobacteria. Vol. 2. Edited by C. Ratledge & J. L. Stanford. London & New York: Academic Press. p. 343. 3 Palmer, C. E. & Long, M. W. (1966). Effects of infection with atypical mycobacteria on BCG vaccination and tuberculosis. American Review of Respiratory Disease, 94, 553. 4 Tuberculosis Prevention Trial (1979). Trial of BCG vaccines in South India for tuberculosis prevention: first report. Bulletin of the World Health Organization, 57, 819. 5 Rich, A. Ft. (1929). The role of allergy in tuberculosis. Archives of Internal Medicine, 43, 691. 6 Wilson, G. S., Schwabacher, H. & Maier, I. (1940). The effect of the desensitisation of tuberculous guinea-pigs. Journal of Pathology and Bacferiology, 50, 89. 7 Turner, H. M. (1953). Correlation of quantitative Mantoux reactions with clinical progress in pulmonan/ tuberculosis. Tubercle, 34, 153.
4
Grange
8 Stanford, J. L., Shield, M. J. & Rook, G. A. W. (1981). Hypothesis 1. How environmental mycobacteria may predetermine the protective efficacy of BCG. T&em/e, 62, 55. 9 Rook, G. A. W., Bahr, G. M. &Stanford, J. L. (1981). Hypothesis 2. The effect of two distinct forms of cell-mediated response to mycobacteria on the protective efficacy of BCG. Tubercle, 62, 63. 10 Mackaness, G. B. (1968). The immunology of antituberculous immunity. American Review of Respiratory Disease, 97. 337. 11 Abrahams, E. W. (1970). Original mycobacterial sin. Tubercle, 51, 316. 12 Edwards, M. L., Goodrich, J. M., Muller, D., Pollack, A., Ziegler, J. E. & Smith, D. W. (1982). Infection with Mycobacterium avium-intracehlare and the protective effects of bacille Calmette Guerin. Journal of Infectious Disease, 145, 733. 13 Orme, I. M. & Collins, F. M. (1984). Efficacy of Mycobacferium bovis BCG vaccination in mice undergoing prior pulmonary infection with atypical mycobacteria. infection and Immunity, 44, 28. 14 Smith, D., Reeser, P. & Musa, S. (1985). Does infection with environmental mycobacteria suppress the protective response to subsequent vaccination with KG? Tubercle, 66, 17. 15 Orme, I. M., Roberts, A. R. &Collins, F. M. (1986). Lack of evidence for a reduction in the efficacy of subcutaneous BCG vaccination in mice infected with nontuberculous mycobacteria. Tubercle, 67, 41. 16 Brown, C. A., Brown, I. N. & Swinburne, S. (1985). The effect of oral Mycobacterium vaccae on subsequent responses of mice to BCG sensitization. Tubercle, 66, 251. 17 Stanford, J. L. & Rook, G. A. W. (1983). Environmental mycobacteria and immunization with BCG. In Medical Microbiology Vol. 2. Edited by C. S. F. Easmon & J. Jellaszewicz. London and New York: Academic Press. 18 Youmans, G. P. (1975). Relation between delayed hypersensitivity and immunity in tuberculosis. American Review of Respiratory Disease. 111, 109. 19 Lefford, M. J. (1975). Delayed hypersensitivity and immunity in tuberculosis. American Review of Respiratory Disease, 111, 243. 20 Salvin, S. B. & Neta, R. (1975). A possible relationship between delayed hypersensitivity and cell-mediated immunity. American Review of Respiratory Disease, 111, 373. 21 Rook, G. A. W. & Stanford, J. L. (1979). The relevance to protection of three forms of delayed skin test response evoked by M. leprae and other mycobacteria in mice. Correlation with the classical work in the guinea-pig. Parasite Immunology, 1, 111. 22 Editorial (1984). New tuberculins. Lancer, i, 199. 23 Stanford, J. L. & Lema, E. (1983). The use of a sonicate preparation of Mycobacterium tuberculosis in the assessment of BCG vaccination. Tubercle, 64, 275.
LEADING
ENVIRONMENTAL
ARTICLE
MYCOBACTERIA
AND BCG VACCINATION
Reflecting upon the prevalence and antiquity of tuberculosis, Bankoff [II remarked that ‘When the Lord created man on the sixth day, one is forced to believe that He must have used clay of ancient Asia abounding in tubercle microbes’. Although this claim might not be accepted unreservedly, there is no doubt that the clay of that continent is abounding in many saprophytic species of mycobacteria 121. In 1966 Palmer and Long [3] postulated that ‘natural vaccination’ due to contact with environmental mycobacteria (EM) accounted for the striking differences in the efficacy of BCG from trial to trial. Interest in this theory was reawakened when a major and well-executed BCG trial at Chingleput in South India, where tuberculosis is rife, indicated that the vaccine had no protective effect in that region [41. Using guinea pigs, Palmer and Long found that BCG could increase the partial degree of immunity imparted by prior contact with an environmental mycobacterium but never to a level above that achieved by BCG alone. It was therefore concluded that BCG only achieves its full potential in regions where the population is not exposed to EM but that elsewhere the observed effect of the vaccine is the difference between the potential effect and that of the ‘natural vaccination’. An explanation of the Chingleput findings, on the basis of the hypothesis of Palmer and Long, is that BCG added nothing to a population already maximally protected by prior contact with EM. It is, however, also necessary to consider whether the vaccine could fail in an unprotected population and whether excessive contact with EM, or certain species thereof, may result in a paralysis of the protective immune response or the generation of a reaction that antagonises it. In 1929 Rich [5] questioned the then widely accepted dogma that tuberculin reactivity was a measure of resistance of the individual and suggested that excess sensitivity (i.e. hypersensitivity) might be harmful. It was subsequently confirmed both in animals [6] and in man [7] that individuals with a small degree of tuberculin sensitivity are protected, but that those with large reactions are less protected than those with no sensitivity at all. One school of thought (the ‘one pathway’ school) argues that a moderate degree of cell-mediated reactivity is indeed protective whereas an excessive reaction, like many excesses in life, is harmful. Thus the difference between ‘prophylactic’ cell mediated immunity and tissue-damaging ‘anaphylactic’ hypersensitivity is seen only as 3 quantitative one. On the basis of this theory it may be predicted that BCG vaccination would be effective in children, whose sensitisation by EM would be minimal, but could be worse than useless in hypersensitive adults by further enhancing such tissue-damaging reactivity. Both these predictions were, in fact, fulfilled in the Chingleput study. An important alternative explanation which also leads to the same predictions was advanced by Stanford and Rook and their coworkers 18, 91. These investigators postulate a fundamental qualitative difference between the protective and antagonistic immune pathways. The former, that of true protective immunity, was considered analogous to that elucidated by Mackaness [IO] in mice infected with Listerie monocytogenes. The latter was equated with the necrotic reaction in guinea-pigs known as the ‘Koch phenomenon’.
2
Grange
Accordingly
the two patterns of responses were termed the Listeria-type and Koch-type It was postulated that some species or populations of EM induce the former response while others, or infection with M. tuberculosis, induce the latter. It was further postulated that the induction of one of these responses blocks the subsequent induction of the other. (The idea that early mycobacterial contacts influence the individual’s future pattern of reactivity was first advanced by Abrahams [II] in a paper entitled ‘Original Mycobacterial Sin’.] According to this, the ‘two pathway’ theory, BCG affords protection when it elicits or boosts Listeria-type reactivity but proves ineffective or even harmful when it boosts Koch-type reactivity. The Chingleput findings are thus accounted for by a high prevalence of the latter type of reactivity amongst those vaccinated. At present, the effect of ‘immunological effective contact’ with EM on immunity to mycobacterial disease is the subject of controversy, as is evident from a number of publications in Tubercle and elsewhere [12-161. A critical appraisal of the arguments for and against both theories and of the data used to formulate the arguments is therefore required. It is important to realise that the proponents of the ‘two pathway’ theory accept that EM do indeed induce a protective response in many situations and that the antagonistic response only occurs in certain regions where contact with EM is excessive or where particular species or combinations of species occur. Thus Stanford and Rook [17] accept that ‘natural vaccination’ probably accounted for differences between the British and Ugandan BCG trials on the one hand and the Alabama and Georgia trials on the other. It is also necessary to restate a well known scientific principle that it is impossible to prove that a phenomenon does not occur. Accordingly, the fact that several animal experiments support the hypothesis that contact with EM induces immunity cannot, be used as a primary argumentagainst the occurrence of other effects in man under quite different circumstances. It is also, of course, necessary that any animal model should, as closely as possible, resemble the natural situation. Thus an animal given a single large dose of an EM by injection or inhalation may react quite differently to one following the more natural course of ingesting small numbers of bacilli in food or drink over a period of time. The latter method of sensitisation was used by Brown and her colleagues [I61 to demonstrate that, depending on the dose and timing, such exposure could potentiate, mask or interfere with the protective effect of BCG in mice. In considering the validity of the two-pathway theory, one must look at the evidence for a dissociation between protective and non-protective immune reactions, at the claim that the induction of one of these reactions can block the subsequent induction of the other and, most importantly from the practical point of view, at the claim that these 2 pathways can be distinguished in man by qualitative differences in the dermal reaction to mycobacterial antigens. The first of these 3 points has been hotly debated since the observations of Rich j5l in 1929 and was discussed in a series of editorials in the American Review of Respiratory Disease in 1975 [l&20]. More recently, a number of distinctions between the Listeria- and Koch-type reactions has been demonstrated in the mouse [21]. Nevertheless much more needs to be discovered at the cellular and molecular level, particularly about the mechanism of the necrotic Koch-type reactions, before the debate can be resolved. Hopefully, modern techniques for cloning cells in vitro and for characterizing the relevant mediators will soon clarify the issue. The concept that the induction of one immune pathway can block the development of the other arose from 2 main sources of evidence. First, the induction of necrotic Koch-type reactions in experimental animals by certain mycobacterial species, such as Mycobacterium kansasii, was found to be blocked by prior exposure of the animals to species, such as M. vaccae, that induce Listeria-type responses [9]. Second, multiple skin testing in Burma reSPeCtiVely.
Environmental
mycobacteria
and BCG vaccination
3
revealed that strong Koch-type reactivity to M. scrofulaceum (a species common in that environment) was associated with an inhibition of reactivity to the soluble antigens of reactions [21. M. leprae which are only able to elicit Listeria-type The major stumbling block to the acceptance of the concepts of Stanford and Rook is the fact that they have been based on the use of a set of reagents, the ‘New Tuberculins’ [221, which are not commercially available. It has been claimed that these reagents enable two immune pathways to be differentiated in man by a single examination of the reaction at 48 hours, and that the type of reactivity thus determined after BCG vaccination can be used to assess the protective efficacy of that vaccine in a given region [231. The acceptance of these claims depends on the strength of the evidence that two observed patterns of dermal reactivity in man correspond respectively to the macrophage-activating protective Listeriatype responses and to the necrotic Koch-type reactions demonstrable in animals. Critics may argue that the two reactions merely differ in extent and that, in any case, the subjective nature of the examination compromises the comparisonof studies by different workers. The problem of the availability of the New Tuberculins could be rectified yet, whatever reagent is used, more objective methods for analysing patterns of skin test reactivity and for relating them to underlying immune mechanisms will be essential for a resolution of the debate. Recent developments in techniques for identifying functional subsets of lymphocytes and macrophages and for the assay of mediators should, however, soon enable definitive studies on biopsies of the various types of tuberculin reactions to be undertaken. In fine, the disciples of Palmer and Long claim that individuals in those areas where BCG shows no protective effect are already maximally protected. If this theory is accepted, health authorities might abandon the costly procedure of BCG vaccination in the belief that the environment provides the same service free! By contrast, Stanford and Rook postulate that the vaccine may fail to imrnunise an unprotected population owing to the presence of an antagonistic immune reaction that cannot be overridden by vaccination. If this theory is confirmed it will be of great importance, in the relevent regions, to give BCG (or some improved vaccine) early in life so as to induce protective immunity before the environment can have the adverse effect. Further, some form of immunotherapy that could ‘reprogram’ the immune system of inappropriately sensitized individuals would be of great value. The importance of resolving the controversy surrounding the two theories cannot be overstated: the lives of millions of potential victims of tuberculosis, and perhaps leprosy also, depend upon it. Cardiothoracic Institute, University of London, Fulham Road, London
J. Ibl. Grange SW3
6HP. References
1 Bankoff, G. (1946). The Conquesf of Tuberculosis. London: MacDonald & Co. p. 11. 2 Shield, M. J. (1983). The importance of immunologically effective contact with environmental mycobacteria. In: The Biology of the Mycobacteria. Vol. 2. Edited by C. Ratledge & J. L. Stanford. London & New York: Academic Press. p. 343. 3 Palmer, C. E. & Long, M. W. (1966). Effects of infection with atypical mycobacteria on BCG vaccination and tuberculosis. American Review of Respiratory Disease, 94, 553. 4 Tuberculosis Prevention Trial (1979). Trial of BCG vaccines in South India for tuberculosis prevention: first report. Bulletin of the World Health Organization, 57, 819. 5 Rich, A. Ft. (1929). The role of allergy in tuberculosis. Archives of Internal Medicine, 43, 691. 6 Wilson, G. S., Schwabacher, H. & Maier, I. (1940). The effect of the desensitisation of tuberculous guinea-pigs. Journal of Pathology and Bacferiology, 50, 89. 7 Turner, H. M. (1953). Correlation of quantitative Mantoux reactions with clinical progress in pulmonan/ tuberculosis. Tubercle, 34, 153.
4
Grange
8 Stanford, J. L., Shield, M. J. & Rook, G. A. W. (1981). Hypothesis 1. How environmental mycobacteria may predetermine the protective efficacy of BCG. T&em/e, 62, 55. 9 Rook, G. A. W., Bahr, G. M. &Stanford, J. L. (1981). Hypothesis 2. The effect of two distinct forms of cell-mediated response to mycobacteria on the protective efficacy of BCG. Tubercle, 62, 63. 10 Mackaness, G. B. (1968). The immunology of antituberculous immunity. American Review of Respiratory Disease, 97. 337. 11 Abrahams, E. W. (1970). Original mycobacterial sin. Tubercle, 51, 316. 12 Edwards, M. L., Goodrich, J. M., Muller, D., Pollack, A., Ziegler, J. E. & Smith, D. W. (1982). Infection with Mycobacterium avium-intracehlare and the protective effects of bacille Calmette Guerin. Journal of Infectious Disease, 145, 733. 13 Orme, I. M. & Collins, F. M. (1984). Efficacy of Mycobacferium bovis BCG vaccination in mice undergoing prior pulmonary infection with atypical mycobacteria. infection and Immunity, 44, 28. 14 Smith, D., Reeser, P. & Musa, S. (1985). Does infection with environmental mycobacteria suppress the protective response to subsequent vaccination with KG? Tubercle, 66, 17. 15 Orme, I. M., Roberts, A. R. &Collins, F. M. (1986). Lack of evidence for a reduction in the efficacy of subcutaneous BCG vaccination in mice infected with nontuberculous mycobacteria. Tubercle, 67, 41. 16 Brown, C. A., Brown, I. N. & Swinburne, S. (1985). The effect of oral Mycobacterium vaccae on subsequent responses of mice to BCG sensitization. Tubercle, 66, 251. 17 Stanford, J. L. & Rook, G. A. W. (1983). Environmental mycobacteria and immunization with BCG. In Medical Microbiology Vol. 2. Edited by C. S. F. Easmon & J. Jellaszewicz. London and New York: Academic Press. 18 Youmans, G. P. (1975). Relation between delayed hypersensitivity and immunity in tuberculosis. American Review of Respiratory Disease. 111, 109. 19 Lefford, M. J. (1975). Delayed hypersensitivity and immunity in tuberculosis. American Review of Respiratory Disease, 111, 243. 20 Salvin, S. B. & Neta, R. (1975). A possible relationship between delayed hypersensitivity and cell-mediated immunity. American Review of Respiratory Disease, 111, 373. 21 Rook, G. A. W. & Stanford, J. L. (1979). The relevance to protection of three forms of delayed skin test response evoked by M. leprae and other mycobacteria in mice. Correlation with the classical work in the guinea-pig. Parasite Immunology, 1, 111. 22 Editorial (1984). New tuberculins. Lancer, i, 199. 23 Stanford, J. L. & Lema, E. (1983). The use of a sonicate preparation of Mycobacterium tuberculosis in the assessment of BCG vaccination. Tubercle, 64, 275.