International Journal of Mycobacteriology 1 (2012) 3– 12

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Review Article Mycobacteria and their world

John Stanford *, Cynthia Stanford

Department of Medical Microbiology, Royal Free and University College Medical School, London, UK BioEos Ltd., Mill House, Claygate, Marden, Near Tonbridge, Kent TN12 9PE, UK

Contents

Introduction ...... 3 Mycobacteria and the natural environment...... 4 Investigational aspects ...... 4 Skin-testing with tuberculins ...... 4 The antigens of mycobacteria ...... 4 and ...... 5 Buruli ulcer—M. ulcerans disease...... 6 Other mycobacterial diseases occurring in man ...... 6 The Immuno-pathological spectrum of mycobacterial infections ...... 6 The Bacillus of Calmette and Guerin (BCG) ...... 6 vaccae—an immunomodulator for prophylaxis and therapy...... 7 Mycobacterium vaccae in prophylaxis ...... 7 Mycobacterium vaccae in therapy for leprosy and tuberculosis ...... 7 Unexpected findings with M. vaccae ...... 8 Mycobacteria and ...... 8 Immune modulation and genera related to mycobacteria...... 8 Tsukamurella inchonensis ...... 8 Gordonia bronchialis...... 9 Conclusions...... 9 References ...... 9

Introduction external environment and the other in animal tissues, where they live in complicated relationships with the immune sys- Mycobacteria are ubiquitous, occurring everywhere in the tem. It is their pathogenic species causing leprosy, tuberculo- world, except perhaps the Polar Regions. In deserts they can sis and Buruli ulcer that have made the mycobacteria be found under rocks and among the dried roots of vegeta- notorious. The environmental species, seldom causing dis- tion. Their favoured environmental habitat is close to, and ease, have the greatest effects on the majority of healthy peo- at the edges of, fresh water both flowing and static. Besides ple. They were probably at least part of the driving force their natural habitat in the environment, a few of their hun- leading to the development of cell-mediated immunity during dred or so species have become strict or obligate pathogens, evolution in all animals. Environmental contact regulates sus- and a number of species are opportunist pathogens, normally ceptibility to many conditions other than those of mycobacte- living in the environment but with the capacity to invade ani- rial aetiology and offers an unexpected means of protection mal tissues. Thus, mycobacteria have two worlds: one in the from, and treatment for, humanity’s most important diseases.

* Corresponding author at: BioEos Ltd., Mill House, Claygate, Marden, Near Tonbridge, Kent TN12 9PE, UK. Tel.: +44 (0)1892 730298. E-mail address: [email protected] (J. Stanford). 2212-5531/$ - see front matter Ó 2012 Asian-African Society for Mycobacteriology. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijmyco.2012.01.001 4 International Journal of Mycobacteriology 1 (2012) 3– 12

Their potentially beneficial effects are also exerted by the immune responses in those living in the environment under other aerobic genera of the Actinomycetales, the order of bac- investigation [6,7]. teria to which the mycobacteria belong. Skin-testing with tuberculins Mycobacteria and the natural environment Skin-testing with tuberculins is carried out by intradermal Although mycobacteria, except for the strict pathogens, all injection of soluble reagents prepared from various mycobac- live in the environment of soil and water, their distribution terial species. Reactions are read as diameters of induration is dictated by physical and chemical conditions. There is evi- after 48–72 h. When dealing with reagents made from patho- dence that temperature, moisture, ultra-violet light, pH and gens, qualitative differences in positive responses can also be geography affect this. In deserts the predominant species distinguished [8]. Safe and minimally invasive, these reagents are chiefly of pigmented organisms, scotochromogens or pho- are no longer available under current drug-regulatory condi- tochromogens, of the slow-growing sub-. Their yellow tions. However, in former times, enormous amounts of infor- carotinoid pigments protect them from sunlight, the ultra- mation were obtained [6–12]. violet bands of sunlight being lethal to non-pigmented There are interpretational difficulties owing to interfer- species. ence by previous vaccination with BCG, and to individuals Mycobacteria as a genus, by virtue of their thick cell walls, responding to different groups of mycobacterial antigens, generally survive drying and the effects of strong acids and allowing a categorisation of responders [13,14]. Nonetheless, alkalis, enabling them to pass through animal gut and be de- through these tests the interaction between man and his tected in faeces and in city sewerage outlets. The great major- mycobacterial environment became apparent. ity of tubercle bacilli which become extracellular in the natural degradation of pulmonary lesions are either aeroso- The antigens of mycobacteria lised or swallowed, thus becoming widely distributed in soil where they survive for long periods if protected from ultra- Immunodiffusion analysis of sonicate preparations of myco- violet light [1,2]. bacterial species against high titre antibodies raised in rabbits In fresh-water situations, fast-growing species exceed the gives rise to the following definitions of four antigenic groups slow-growers and there is further selection by pH, some spe- [15,16] (see Fig. 1): cies preferring acidic conditions and others alkaline condi- tions. For example, Mycobacterium vaccae amongst fast- (1) Group i antigens are shared between all mycobacterial growers and M. ulcerans amongst slow-growers are more com- species and some of them are shared by all , mon in neutral to acid conditions than they are in alkaline including mitochondria in animal cells [17]. ones. (2) Group ii antigens are limited to the slowly growing species. Investigational aspects (3) Group iii antigens are shared by many fast-growing species. In the early 1970s in an attempt to isolate M. ulcerans (4) Group iv antigens are species-specific and limited to from the Ugandan environment, an extensive geographical single species, or to small groups of closely related survey of cultivable environmental species was carried species [18]. out [3]. There are three ways of assessing mycobacterial distribu- In relation to these groups of bacterial antigens are the dif- tion in the environment: ferent categories of skin-test responders [13,14]:

(1) Bacteriological culture of environmental samples. (1) Category 1 responders react to group i antigens, respond- (2) Polymerase chain reaction (PCR) probing of environ- ing positively to tuberculins prepared from any myco- mental samples. bacterial species. (3) Differential skin-testing of the resident human popula- (2) Category 2 responders do not produce positive tion. responses to any of the tuberculins at their standard test concentrations; 10–20% of individuals fall into each All of these have been valuable. Culture sounds simple, but of categories 1 and 2. the methods required to isolate organisms, generally more (3) Category 3 responders react positively to the species- slow-growing than much of the other environmental bacterial specific antigens of the species encountered. This cate- flora, are time-consuming and not always effective. Nonethe- gory consisting of around 70% of individuals can be less, culture of an organism, the identity of which can be con- used to determine which species are present in an firmed by phenetic, antigenic or genetic analysis, is the only environment. absolute proof of its presence in an environment. PCR is gen- (4) Category 4 is a minor category of those reacting to the erally reserved for searches for particular species, such as group ii antigens of slow-growers [14]. M. ulcerans [4], and for non-cultivable species, such as the lep- rosy bacillus [5]. Easier and faster is multiple skin-testing, Analysis of the results from category 3 responders reveals depending upon the detection of remembered cellular the immunological significance of environmental contact, International Journal of Mycobacteriology 1 (2012) 3– 12 5

Fig. 1 – The antigens of mycobacteria. The groups of antigens shown are those demonstrable in immunodiffusion studies of sonicate preparations of mycobacterial species against high titre antibodies raised to them in rabbits. Similar groups of antigens, and possibly others, can be demonstrated in tests of cellular immunity based on lymphocyte transformation and multiple skin-testing. Group i antigens (common mycobacterial) are shared by all mycobacterial species, and other aerobic Actinomycetales. Some of them are also shared with other bacterial families and with the ancient bacteria, known as mitochondria, present in almost all animal tissue cells. These include the heat-shock-proteins of modern bacteria and the stress proteins of mitochondrial origin expressed in stressed animal cells. Group ii antigens (slow-grower associated) are shared by tubercle bacilli, and all the slowly-growing species of mycobacteria. Group iii antigens (fast- grower associated) are shared by most fast-growing mycobacterial species. These antigens are not detectable in leprosy bacilli, M. vaccae and some closely related species. Group iv antigens (species-specific) are limited to individual species, but may show differences in distribution between sub-species and some sharing between closely related species, e.g. M. avium, M. intracellulare, M. paratuberculosis and M. leprae-murium. In cell-mediated immunity, group i antigens are recognised through Th1 mechanisms, whereas groups ii and iv antigens are best recognised by Th2 mechanisms. the species present, their immunising capacity and their is perturbed, as with human immunodeficiency virus (HIV) frequency. infection, endogenous activation of tuberculosis persisters By skin testing with combinations of tuberculins prepared occurs and susceptibility to exogenous infection is increased. from four different mycobacterial species (the quadruple In both leprosy and tuberculosis the question remains open skin-testing system), the complications in interpretation can as to whether persisters are complete, cell-walled organisms, be in part elucidated [6–12]. or cell-wall free [22–24]. Whereas infectious particles of tuberculosis are dried Leprosy and tuberculosis droplet nuclei of aerosols exhaled from the lungs, those of leprosy are from the nose. They are considered the major These two mycobacterioses share many characteristics, yet source of new infections, with fomites and environmental are also quite distinct. As pathogens, their world is the inter- aerosols occasionally giving rise to disease. These latter are nal environment of animal tissues, with the complication of thought to be the source of many opportunist pulmonary the immune response. They share an immunological spec- infections with water-borne, slow-growing species [25,26]. trum [19], of which each disease covers a part. Both leprosy The site of primary infection in tuberculosis is generally and tubercle bacilli (M. leprae and M. tuberculosis, respectively) the periphery of the lung, whereas that of leprosy is thought are intracellular pathogens, becoming extra-cellular when to be the nasal mucosa [27]. Both diseases are associated with bacilliferous lesions break down; both exhibit persistence, derangement of cellular immunity, and antibodies probably the continued presence of living bacilli quiescent in the tis- have little part to play beyond the establishment of infection. sues in the absence of clinical disease [20,21]. These persisters Infective particles of tubercle bacilli are thought to be small may precede or follow active disease and can activate when- clumps of fewer than five bacilli, larger clumps being caught ever the immunological situation is suitable for them. It is in the mucus flow of the airways and transported to the estimated that three quarters of the world’s human popula- gut, a site much less susceptible to infection [28]. The same tion carry tubercle bacilli without ever having had the clinical principles are likely to be true for leprosy bacilli. Good disease. Skin testing with leprosin (soluble reagent from lep- evidence exists proving that healthy, close contacts of leprosy rosy bacilli harvested from experimentally infected armadil- patients may temporarily carry leprosy bacilli in their nasal los) suggests that many people in leprosy-endemic mucosa that can be released as potentially infective particles countries are infected with leprosy bacilli. When immunity [29,30]. 6 International Journal of Mycobacteriology 1 (2012) 3– 12

Buruli ulcer—M. ulcerans disease progressive or static disease as a specific cellular response supersedes. The worst tissue destruction is usually associated A disease of perturbed cellular immunity following the immu- with the change from immune suppression to recognition. nopathological spectrum of the mycobacterioses (see below), Leprosy patients tend to remain either in a multi-bacillary Buruli ulcer is unlike leprosy and tuberculosis. It is almost lepromatous state or in a paucibacillary tuberculoid state. never passed from person to person, but is rather caught from Those with the borderline types of disease, where most tissue the environment [31]. Very slow growing, even when the inoc- damage occurs, are associated with movement, up or down, ulum is taken from grossly bacilliferous lesions, it has only from suppressed to expressed cellular immune response very recently been cultured from the environment [32]. None- [43]. Tuberculosis fits less easily than does leprosy into the theless, much can be learned from careful epidemiology. spectrum, but can generally be compared with the borderline Unlike leprosy and tuberculosis, where virulence and patho- states in leprosy according to the activity of tissue destruc- genicity are thought to be direct immuno-perturbing charac- tion. The most tuberculoid form of tuberculosis progresses teristics of the bacilli, M. ulcerans causes the ulcerative and very slowly with few visible bacilli and limited tissue necrotising characteristics of Buruli ulcer by its production destruction. of a toxin [33,34]. This toxin, called ‘‘mycolactone’’ [35], is the Patients developing M. ulcerans infection (Buruli ulcer) pass result of the bacilli themselves being infected with a transmis- through a latent phase of several weeks, into the clinical sible plasmid [36]. Non-toxigenic strains of M. ulcerans can phase when ulceration begins under the immunosuppressive arise during prolonged artificial culture and can be recognised effects of mycolactone [35] and bacilli are present in millions. by their inability to cause disease when injected experimen- For unknown reasons, perhaps owing to production of anti- tally into mice—animals very susceptible to the toxin [37].In bodies that neutralise the toxin, the disease changes from the absence of the occurrence of clinical Buruli ulcer, the envi- multibacillary and necrotising to paucibacillary, tuberculoid ronmental distribution of non-toxigenic M. ulcerans is hard to and fibrosing [44,45] (see Fig. 2). determine. An assessment of the role contact with mycobacteria may The Bacillus of Calmette and Guerin (BCG) play in susceptibility to juvenile asthma was carried out in Crete [38], an island where clinical cases of Buruli ulcer have Following Jenner’s observations on vaccination, the discov- not been recorded. More than the expected number of chil- ery and development of vaccines effective against a number dren coming from a village close to a freshwater lake re- of infections took place in the second half of the 19th cen- sponded positively to Burulin—a tuberculin made from tury. Attempts were made in the 1870s to develop a vaccine cultured M. ulcerans [39], indicating the probable presence of for tuberculosis, and Robert Koch’s work in the 1880s led to non-toxigenic M. ulcerans in their surroundings. real progress [46,47]. Calmette and Guerin developed a sus- pension of live, attenuated bacilli isolated from a calf be- Other mycobacterial diseases occurring in man lieved to be infected with the bovine form of tubercle bacillus, which could protect children from active tuberculo- These are opportunist diseases in which the tissues of sus- sis [48]. BCG is an enhancer of cellular immunity, as were the ceptible individuals are invaded from environmental sources. earlier, less well-known products. These include Spahlinger’s They include swimming-pool granuloma caused by M. mari- vaccine produced from living, but dormant, tubercle bacilli num, mycobacterial injection abscesses [40], frequently [49] and Friedman’s preparation of live M. chelonae [50]. BCG caused by M. chelonae or M. fortuitum, and mycobacterial cervi- gives a variable degree of protection from tuberculosis, lep- cal adenitis caused by a range of slow-growing species, rosy [51,52], Buruli ulcer [53], malignant melanoma [54] and including M. scrofulaceum, M. avium and M. malmoense [41]. probably other . We can only guess at the contribu- Chronic lung disease, usually of the elderly, can be caused tion that the widespread application of BCG vaccination by M. avium, M. intracellulare, M. kansasii and M. xenopi [42]. has had on the changing patterns of many other diseases. BCG demonstrates that there is an immune response other The Immuno-pathological spectrum of than that expressed in the Ridley–Jopling spectrum, and that mycobacterial infections its efficacy is not dependent upon responses to species-spe- cific antigens but to primitive antigens common to all bacte- The concept of an immunopathological spectrum proposed ria and to the stress proteins of animal tissues [55,56]. Once for leprosy by Ridley and Jopling [19,43] has been very influen- this essential point has been grasped, progress can be made tial. The spectrum embraces a series of phases related to bac- towards the recognition and development of immuno-pro- terial growth. Prior to the spectrum, there is a latent phase phylaxis and . BCG is the only survivor of which may vary from weeks to years in length. There is then the early 20th century preparations of live organisms a phase of uncontrolled bacterial growth when lesions are [49,50], though Friedmann’s ‘‘Anningzochin’’ was claimed to bacilliferous or multi-bacillary and antibody levels are high. be effective against asthma, and some other non- As cellular immune reaction increases and antibody levels mycobacterial diseases as well as tuberculosis. These are fall, bacillary numbers are reduced and a tuberculoid state claims revisited in the late 20th century and today with supervenes. From the hosts viewpoint there is a stage of im- the recognition of the immunomodulatory powers of M. vac- mune suppression with rapid development of disease associ- cae, M. obuense and of selected species from other genera of ated with little cellular response and a stage of slowly the aerobic Actinomycetales. International Journal of Mycobacteriology 1 (2012) 3– 12 7

Fig. 2 – The immuno-pathological spectrum of the mycobacterioses. The basis of the spectrum for leprosy described by Ridley and Jopling [19] is shown in the upper section of the figure. To the left of the clinical spectrum is the period of latent disease and to the right of it is the period of persistent disabilities. Patients with clinical leprosy, except for those with borderline presentations, do not shift from multibacillary, lepromatous to paucibacillary, tuberculoid forms, without the application of immunotherapy. Untreated lepromatous disease can be life-long, whereas tuberculoid disease can be self-limiting. The middle section shows the situation of pulmonary tuberculosis in relation to the leprosy spectrum. Active tuberculosis is more analogous to lepromatous to borderline leprosy, and only healed cases correspond with tuberculoid leprosy. The bottom section shows Buruli ulcer (M. ulcerans disease) in relation to the leprosy spectrum. Coming from the environment there is a latent phase of several weeks’ duration, following which the disease passes through a multibacillary phase, analogous to lepromatous leprosy, when rapid extension of ulceration occurs. This is followed by a very rapid reduction of bacilli with the onset of cellular immune recognition, finishing with extensive disabilities in many cases owing to massive fibrosis.

Mycobacterium vaccae—an immunomodulator for combined vaccine of live BCG plus killed M. vaccae was about prophylaxis and therapy 50% more effective than BCG alone in the induction of corre- lates of protective immunity, and the effect of killed M. vaccae In a search for an explanation of why BCG was so effective alone was little different from the combination. A recent against leprosy in Uganda, but had little effect in Burma, study has shown the benefits of repeated doses in preventing the influence of priming by environmental species was tuberculosis in HIV-positive patients [67]. investigated and it was found that the fast-grower M. vaccae enhanced protective post-BCG immune responses in Ugan- Mycobacterium vaccae in therapy for leprosy and tuberculosis da, and slow-growers including M. scrofulaceum blocked them in Burma [57]. In London this could be demonstrated in lab- The potential for M. vaccae as an immuno-therapeutic for lep- oratory mice [58], and it was shown that priming with killed rosy was first investigated in Spain [68,69], Iran [64,70], India M. vaccae alone also enhanced protective immune responses. [71] and Vietnam, and for tuberculosis in London [72], the This discovery of the potential value of sensitisation to Gambia, Kuwait [73,74], Nigeria [75], Romania [76,77] and Viet- M. vaccae came in the 1970s at the time of a great explosion nam [78]. Subsequent studies found a single injected dose to of knowledge about immunology. The old idea that protec- be inadequate in some places, especially in HIV-infected per- tive immunity and tissue-damaging allergy were different sons [79]. presentations of cellular immunity demonstrated in the A series of studies of tuberculosis were carried out in tuberculin response was proven. At first these were desig- Argentina, with single injections [80], three injections [81] or nated ‘‘Listeria-like’’ and ‘‘Koch-like’’ responses, respectively 10 oral doses [82]. The principles of this type of immunother- [59]. These dubious terms were replaced by Th1 and Th2 apy, affecting host immunity and not directly the bacilli as different pathways of maturation of CD4+ cells were themselves, means that it would be equally effective against recognised [60]. drug-resistant bacilli, and this has proved to be the case [73,76,83–85]. This was well demonstrated in a study at Ma- Mycobacterium vaccae in prophylaxis shad in Iran. A combination of anti-bacterial chemotherapy plus immunotherapy with M. vaccae should lead to less devel- Transferred to the children of leprosy patients in Bombay [61], opment of drug resistance and to substantially shorter treat- Iran [62–65] and later Vietnam [66], it was shown that a ment regimens. 8 International Journal of Mycobacteriology 1 (2012) 3– 12

The laying down of fibrous tissue is associated with as an immunotherapeutic [94]. Some benefits were claimed, type 2 activity and reports of M. vaccae immuno- but the only successful application of BCG is by instilling it therapy for pulmonary tuberculosis record a reduction in into the bladder of patients with carcinoma in situ [95], and radiological opacities and improved closure of cavities currently this is the treatment of choice. The problem with [75,77,80]. BCG is that it enhances the cellular immune response for which the patient is already primed and does not modulate Unexpected findings with M. vaccae from Th2 towards Th1 maturation of T cells, an essential step in effective immunotherapy for cancer [96]. An exten- During the period of investigation of immunotherapy for sive survey has shown that past BCG vaccination reduces mycobacterial disease, patients and their physicians were susceptibility to malignant melanoma by about 50% and in- asked to note effects on concurrent conditions. The first to creases the successful outcome to treatment also by 50% be reported and investigated was psoriasis in a leprosy pa- [54]. tient in South India [86], and this was confirmed in a study With the development of M. vaccae and its use in patients from Argentina [87]. Following this observation other autoim- with various conditions, remarkable effects have been ob- mune and related conditions have been investigated. The first served in those with cancers [97–99]. Direct studies were of these was Raynaud’s disease and blood flow in the fingers performed in which patients with a variety of different of leprosy patients [70], from which success with M. vaccae malignancies received repeated intradermal injections, and led to studies of myointimal hyperplasia following balloon a significant improvement in quality of life [100], better toler- damage to the carotid arteries of rats [88]. Experimental peri- ance of the side effects of chemotherapy [99] and increased odontal disease in rats also responded to treatment with survival were achieved [98]. More recently, a combination of M. vaccae [89]. injections and oral capsules containing the same dose of M. There have been conflicting reports from various countries vaccae have been used. An extensive and comprehensive list suggesting that BCG vaccination has an effect on bronchial of patients with many different kinds of cancer has been asthma, but this has been difficult to confirm [38]. Studies progressing for several years and has produced some striking with M. vaccae in asthma and bronchial allergy in hay fever results. showed that its use is drug sparing so far as steroids and sal- Another mycobacterial species, M. obuense closely related butamol are concerned and that the effect is on the delayed to M. vaccae [101], was found to have similar immunothera- part of the immune response of airways, rather than their peutic activity and is being developed by Immodulon Thera- immediate responses [90]. peutics Ltd. in London [102]. It is undergoing clinical trials Patients treated with M. vaccae have reported a reduction in malignant melanoma, carcinoma of the head of the pan- in susceptibility to the common cold and an observation creas and bowel cancer with liver metastases. made during studies on the prevention of tuberculosis in HIV-positive patients in Africa recorded an interesting reduc- Immune modulation and genera related to tion in malaria [67], which still has to be followed up. Atopic mycobacteria dermatitis in children responded well to a single injected treatment with M. vaccae, providing it was prepared in borate Among the aerobic, near-mycobacterial genera, within the buffer [91,92], though a small effect was found on chronic se- Actinomycetales, are some species with adjuvant activities vere dermatitis in adults, using the phosphate-buffered prep- and antigens very similar to those of M. vaccae, but with sub- aration [93]. Borate buffer appears to result in a more stable tle differences. Some of these species have been investigated product than does the phosphate buffer, and discussions with for therapeutic activities in human, veterinary and agricul- a glycobiologist disclosed that autoclaving in borate breaks tural medicine. Made in the same way as the M. vaccae prep- down proteins to short amino acid chains suitable for T cell aration, useful results have been obtained. activation, and better preserves the bacterial sugars. A com- parative study in Kuwait found that killing M. vaccae by auto- Tsukamurella inchonensis claving produces a more effective product than does exposure In animal models, a preparation of this species has been par- to Co60 [74]. ticularly successful in preventing inflammation of the intima Two important properties of the M. vaccae preparation are of arteries damaged with a balloon catheter [88] and for use in expressed in immuno-prophylaxis and immunotherapy. The the prevention and treatment of spontaneous type 2 diabetes first is the immuno-modulating adjuvant activity of the cell mellitus. It was found that M. vaccae and T. inchonensis were wall complex down-regulating Th2 mechanisms [64]. The the most effective, followed by Gordonia bronchialis and with other is the content of antigens cross-reactive with invading Rhodococcus coprophilus being the least effective. Both of these bacteria and stressed host tissue [56], explaining the very are models of important human diseases. In man, anecdotal wide therapeutic activities of the preparation. cases of asthma and periodontal disease treated with oral T. inchonensis have shown benefits similar to those achieved Mycobacteria and cancer with M. vaccae. A marked reduction in frequency of common colds has been observed in volunteers taking oral therapy At one time mycobacteria were considered as possible causes with T. inchonensis and patients with recurrent cold sores ow- of cancer, but this has never been substantiated. However, ing to Herpes simplex virus infection have found that the sores mycobacteria do have the capacity to prevent and treat can- are of much shorter duration, less frequent and less severe. cer. During the 1970s attempts were made to use BCG vaccine Currently, T. inchonensis is being investigated for potential International Journal of Mycobacteriology 1 (2012) 3– 12 9

introduction into human medicine by a new company, [4] F. Portaels, J. Agular, K. Fissette, et al, Direct detection and Actinopharma Ltd. In veterinary medicine, T. inchonensis has identification of Mycobacterium ulcerans in clinical specimens been found to be effective in the treatment of ‘‘sweet-itch’’, by PCR and oligonucleotide-specific capture plate hybridization, Journal of Clinical Microbiology 35 (1997) a seasonal dermal allergy of horses [103], for which there is 1097. no other effective treatment, and in ‘‘heaves’’, an allergic [5] M. Lavania, K. Katoch, P. Sachan, et al, Detection of respiratory airways obstructive disease of equines. DNA from soil samples by PCR targeting RLEP sequences, Journal of Communicable Diseases 38 Gordonia bronchialis (2006) 69–73. Rats treated with G. bronchialis or R. coprophilus, but not with [6] J.L. Stanford, R.C. Paul, A. Penketh, S. Thurlow, J.W. Carswell, T. inchonensis, and challenged with live Trypanosoma cruzi show D.J.P. Barker, et al, A preliminary study of the effect of significantly reduced parasitaemias and less chronic myocar- contact with environmental mycobacteria on the pattern of sensitivity to a range of new tuberculins amongst Ugandan ditis [104]. Pregnant rats treated with G. bronchialis, but not R. adults, Journal of Hygiene (Cambridge) 76 (1976) 205–214. coprophilus gave birth to offspring protected from subsequent [7] J.L. Stanford, M.J. Shield, R.C. Paul, A. Khalil, R.S. Tobgi, A. challenge with T. cruzi [105]. Potentially, these are models of Wallace, The effect of desert conditions on the reactivity of the consequences of Chagas’ disease in man, and may have Libyan schoolchildren to a range of new tuberculins, Journal relevance in other causes of myocardial disease. In dogs, a of Hygiene (Cambridge) 77 (1976) 63–75. preparation of G. bronchialis has been particularly effective [8] J.L. Stanford, E. Lema, The use of a sonicate preparation of Mycobacterium tuberculosis (new tuberculin) in the against allergy to flea-bites [106] and is likely to be developed assessment of BCG vaccination, Tubercle 64 (1983) 275–282. as veterinary medicine. In agricultural medicine, G. bronchialis [9] E. Lema, J.L. Stanford, Skin-test sensitization by tubercle induces improved survival and weight gain in shrimp and bacilli and by other mycobacteria in Ethiopian school- fish hatchlings and is improving growth rate and colour and children, Tubercle 65 (1984) 285–293. reducing skeletal abnormalities in koi carp. Treatment [10] G.M. Bahr, J.L. Stanford, G.A.W. Rook, R.J.W. Rees, G.J. Frayha, with the same organism has resulted in improved growth A.M. Abdelnoor, Skin sensitization to mycobacteria amongst rates in pigs and , which is now being further school children prior to a study of BCG vaccination in North Lebanon, Tubercle 67 (1986) 197–203. investigated. [11] J.L. Stanford, M.J. Shield, G.A.W. Rook, Research on a new tuberculin series: Part 1. Effects of age, residence, BCG Conclusions vaccination and tuberculosis, Bulletin of the International Union against Tuberculosis 56 (1981) 31–37. Among the 100 or more species of mycobacteria, M. tuberculo- [12] P.M. Nye, J.E. Price, C.R. Revankar, G.A.W. Rook, J.L. Stanford, sis and M. leprae are important pathogens and amongst the The demonstration of two types of suppressor mechanism in leprosy patients and their contacts by quadruple skin- remainder are a number of species capable of becoming testing with mycobacterial reagent mixtures, Leprosy opportunist pathogens, including the toxin-producing species Review 54 (1983) 9–18. M. ulcerans. Within the environment, moisture, temperature, [13] D.N.J. Lockwood, I.C. McManus, J.L. Stanford, A. Thomas, pH, exposure to ultra-violet light and geography all have sig- D.V.P. Abeyagunawardana, Three types of human response nificant influences on mycobacterial distribution. Their pres- to mycobacterial antigen: population studies in India and Sri ence probably provides an evolutionary drive towards the Lanka, European Journal of Respiratory Diseases 71 (1987) development of the immune systems of all animals. 348–355. [14] I.C. McManus, D.N.J. Lockwood, J.L. Stanford, M.A. Shaaban, Sensitisation of human and animal populations with locally M. Abdul Ati, G.M. Bahr, Recognition of a category of common species offers a method of detecting their presence responders to group ii, slow-grower associated antigens and provides a means by which susceptibility or protection amongst Kuwaiti senior schoolchildren, Tubercle 69 (1988) from mycobacterial and other diseases can be determined. 275–281. Killed preparations of selected mycobacteria and of species [15] J.L. Stanford, A. Beck, An antigenic analysis of the from other genera within the order Actinomycetales are potent mycobacteria, , M. kansasii, M. smegmatis M. tuberculosis immune modulators and enhancers useful in the prevention and , Journal of Pathology and Bacteriology 95 (1968) 131–139. and treatment of many diseases in which cellular immunity [16] J.L. Stanford, J.M. Grange, The meaning and structure of plays a large part. Between them they provide products likely species as applied to mycobacteria, Tubercle 55 (1974) 143– to transform the face of modern medicine. 152. [17] J. Stanford, C. Stanford, G. Stansby, et al, The common mycobacterial antigens and their importance in the treatment of disease, Current Pharmaceutical Design 15 REFERENCES (2009) 1248–1260. [18] G. McIntyre, E. Belsey, J.L. Stanford, Taxonomic differences between Mycobacterium avium and Mycobacterium [1] B.J. Duffield, D.A. Young, Survival of in intracellulare elucidated in man by skin tests with three new defined environmental conditions, Veterinary Microbiology tuberculins, European Journal of Respiratory Diseases 69 10 (1985) 193–197. (1986) 146–152. [2] K.V. Desikan, Viability of Mycobacterium leprae outside the [19] D.S. Ridley, W.H. Jopling, Classification of leprosy according human body, Leprosy Review 48 (1977) 231–235. to immunity: a five-group system, International Journal of [3] J.L. Stanford, R.C. Paul, A preliminary report on some studies Leprosy 34 (1966) 255–273. of environmental mycobacteria, Annals de la Societe Belge [20] R. Hernandez-Pando, M. Jeyanathan, G. Mengistu, et al, de Medecine Tropicale 53 (1973) 389–393. Persistence of DNA from Mycobacterium tuberculosis in 10 International Journal of Mycobacteriology 1 (2012) 3– 12

superficially normal lung tissue during latent infection, [39] J.L. Stanford, W.D. Revill, W.J. Gunthorpe, The production Lancet 356 (2006) 2133–2138. and preliminary investigation of Burulin, a new skin test [21] A.G. Khomenko, V.V. Muratov, Epidemiological risks of reagent for Mycobacterium ulcerans infection, Journal of tuberculosis infection foci in patients discharging L forms of Hygiene (Cambridge) 74 (1975) 7–16. Mycobacterium tuberculosis, Problemy Tuberkuleza 2 (1993) 2– [40] J.G. Borghans, J.L. Stanford, Mycobacterium chelonei in 5. abscesses after injection of diphtheria–pertussis–tetanus– [22] H.C. Much, U¨ ber die granula¨re, nach Ziehl nicht fa¨rbbare polio vaccine, American Review of Respiratory Diseases 107 Form des Tuberkulosevirus, Beitra¨ge zum Klinik der (1973) 1–8. Tuberkulose 8 (1907) 86–99. [41] A. Yildirim, N. Bayazit, M. Namiduru, Mycobacterial cervical [23] W.C. Minchin, The Tubercle Virus, 3rd ed., Balliere, Tindall lymphadenitis, ORL 66 (2004) 275–280. and Cox, London, 1927. [42] J.J. Erasmus, H.P. McAdams, M.A. Farrell, E.F. Patz, Pulmonary [24] L. Mattmann, Mycobacterium tuberculosis and the nontuberculous mycobacterial infection: radiologic atypicals, in: Cell Wall Deficient Forms: Stealth Pathogens, manifestations, Radiographics 19 (1999) 1487–1503. CRC Press, USA, 2001. [43] D.S. Ridley, Pathogenesis of Leprosy and Related Diseases, [25] J. Falkingham III, B.C. Parker, H. Gruft, Epidemiology of Wright, London, 1988. infection by non-tuberculous mycobacteria. Geographic [44] J.L. Stanford, M.S.R. Hutt, I. Phillips, W.D.L. Revill, Antibiotic distribution in the Eastern United States, American Review treatment in Mycobacterium ulcerans infection, Ain Shams of Respiratory Disease 121 (1980) 931–937. Medical Journal 25 (Suppl.) (1973) 258–261. [26] I. Szabo, K.K. Kiss, I. Varnai, Epidemic pulmonary infection [45] T.M. Gooding, P.D.R. Johnson, D.E. Campbell, et al, Immune associated with Mycobacterium xenopi indigenous in sewage- response to infection with Mycobacterium ulcerans, Infection sludge, Acta Microbiologica Academiae Scientiarum and Immunity 69 (2001) 1704–1707. Hungaricae 29 (1982) 263–266. [46] R. Koch, An address on bacteriological research delivered [27] A.C. McDougall, R.J. Rees, A.G. Weddell, M.W. Kanan, The before the International Medical Congress, held in Berlin, histopathology of lepromatous leprosy in the nose, Journal August 1890, British Medical Journal 2 (1890) 380–383. of Pathology 115 (1975) 215–226. [47] R. Koch, A further communication on a remedy for [28] K.P. Fennelly, J.W. Martyny, K.E. Fulton, et al, Cough- tuberculosis, British Medical Journal 1 (1891) 125–127. generated aerosols of Mycobacterium tuberculosis, American [48] J.M. Grange, J. Gibson, T.W. Osborn, et al, What is BCG?, Journal of Respiratory and Critical Care Medicine 169 (2004) Tubercle 64 (1983) 129–139. 604–609. [49] L. MacAssey, C.W. Saleeby (Eds.), Spahlinger Contra [29] P.R. Klatser, S. van Beers, B. Madjid, et al, Detection of M. Tuberculosis, 1908–1934, John Bale, Sons and Danielsson, leprae nasal carriers in populations for which leprosy is London, 1934. endemic, Journal of Clinical Microbiology 31 (1993) 2947– [50] F. Friedmann, Zur frage der skitien immunisiering gegen 2952. Tuberculose, Deutsche Medizinische Wochenschrift 5 (1904) [30] D. Beyene, A. Aseffa, M. Harboe, et al, Nasal carriage of 166. Mycobacterium leprae DNA in healthy individuals in Lega Robi [51] M.S. Setia, C. Steinmaus, C.S. Ho, G.W. Rutherford, The role village, Ethiopia, Epidemiology and Infection 131 (2003) 841– of BCG in prevention of leprosy: a meta-analysis, The Lancet 848. Infectious Diseases 6 (2006) 162–170. [31] Uganda Buruli Group, Epidemiology of Mycobacterium [52] P.E.M. Fine, BCG vaccination against tuberculosis and ulcerans infection (Buruli ulcer) at Kinyara, Uganda, leprosy, British Medical Bulletin 44 (1988) 691–703. Transactions of the Royal Society of Tropical Medicine and [53] The Uganda Buruli Group, BCG vaccination against Hygiene 65 (1971) 763–775. Mycobacterium ulcerans infection (Buruli ulcer), Lancet 293 [32] F. Portaels, W.M. Meyers, A. Ablordey, et al, First cultivation (1969) 111–115. of Mycobacterium ulcerans from the environment, PLoS [54] K.F. Ko¨ lmel, J.M. Grange, B. Krone, et al, Prior immunization Neglected Tropical Diseases 2 (2008) 178–184. of patients with malignant melanoma with vaccinia or BCG [33] M. Pimsler, T.A. Sponsler, W.M. Myers, Immunosuppressive is associated with better survival. A European Organization properties of the soluble toxin from Mycobacterium ulcerans, for Research and Treatment of Cancer cohort study on 542 Journal of Infectious Diseases 157 (1988) 577–580. patients, European Journal of Cancer 41 (2005) 118–125. [34] K.M. George, D. Chatterjee, G. Gunawardana, et al, [55] J.L. Stanford, G.A.W. Rook, Environmental mycobacteria and Mycolactone: a polyketide toxin from Mycobacterium ulcerans immunization with BCG, in: C.S.F. Easmon, J. Jeljaszewicz required for virulence, Science 283 (1999) 854–857. (Eds.), Medical Microbiology, vol. 2, Academic Press, London, [35] G. Gunawardana et al, Characterization of novel macrolide 1983, pp. 43–69. toxins, mycolactones A and B from a human pathogen, [56] I.R. Cohen, D.B. Young, Autoimmunity, microbial immunity Mycobacterium ulcerans, Journal of the American Chemical and the immunological homunculus, Immunology Today 12 Society 121 (1999) 6092–6093. (1991) 105–110. [36] T.P. Stinear, A. Mve-Obiang, P.L. Small, et al, Giant plasmid- [57] J.L. Stanford, M.J. Shield, G.A.W. Rook, How environmental encoded polyketide synthases produce the macrolide toxin mycobacteria may predetermine the protective efficacy of of Mycobacterium ulcerans, Proceedings of the National BCG, Tubercle 62 (1981) 55–62. Academy of Sciences of the United States of America 101 [58] G.A.W. Rook, J.L. Stanford, The relevance to protection of (2004) 1345–1349. three forms of delayed skin-test response evoked by [37] M.S. Oliveira, A.G. Fraga, E. Torrado, et al, Infection with Mycobacterium leprae and other mycobacteria in mice. Mycobacterium ulcerans induces persistent inflammatory Correlation with the classical work in the guinea-pig, responses in mice, Infection and Immunity 73 (2005) 6299– Parasite Immunology 1 (1979) 111–123. 6310. [59] R.C. Potts, J.S. Beck, J.H. Gibbs, et al, Measurements of blood [38] I. Bibakis, C. Zekwald, I. Dimitroulis, et al, Childhood atopy flow and histometry of the cellular infiltrate in tuberculin and allergic disease and skin test responses to skin test responses of the typical Koch type and the non- environmental mycobacteria in rural Crete: a cross- turgid variant form (Listeria-type) in pulmonary sectional survey, Clinical and Experimental Allergy 35 (2005) tuberculosis patients and healthy controls, International 624–629. Journal of Experimental Pathology 73 (1992) 565–572. International Journal of Mycobacteriology 1 (2012) 3– 12 11

[60] T.R. Mosmann, R.L. Coffman, TH1 and TH2 cells: different [77] E. Corlan, C. Marica, C. Macavei, et al, Immunotherapy with patterns of lymphokine secretion lead to different Mycobacterium vaccae: 2. In the treatment of chronic or functional properties, Annual Review of Immunology 7 relapsed tuberculosis in Romania, Respiratory Medicine 91 (1989) 145–173. (1997) 21–29. [61] R.P. Ganapati, C.R. Revankar, D.N.P. Lockwood, et al, A pilot [78] G.A. Rook, P. Onyebujoh, E. Wilkins, et al, A longitudinal study of three potential vaccines for leprosy in Bombay, study of per cent agalactosyl IgG in tuberculosis patients International Journal of Leprosy 57 (1989) 33–37. receiving chemotherapy, with or without immunotherapy, [62] K. Ghazi-Saidi, J.L. Stanford, C.A. Stanford, et al, Vaccination Immunology 81 (1994) 149–154. and skin test studies on children living in villages with [79] A. Mwinga, A. Nunn, B. Ngwira, et al, Mycobacterium vaccae differing endemicity for leprosy and tuberculosis, (SRL172) immunotherapy as an adjunct to standard International Journal of Leprosy 57 (1989) 45–53. antituberculosis treatment in HIV-infected adults with [63] J.L. Stanford, C.A. Stanford, K. Ghazi-Saidi, et al, pulmonary tuberculosis: a randomized placebo-controlled Vaccination and skin test studies on the children trial, Lancet 360 (2002) 1050–1055. of leprosy patients, International Journal of Leprosy 57 [80] D. Dlugovitzky, O. Bottasso, J.C. Dominino, et al, Clinical and (1989) 38–44. serological studies of tuberculosis patients in Argentina [64] J.L. Stanford, G.A.W. Rook, G.M. Bahr, et al, Mycobacterium receiving immunotherapy with Mycobacterium vaccae (SRL vaccae in immunoprophylaxis and immunotherapy of 172), Respiratory Medicine 93 (1999) 557–562. leprosy and tuberculosis, Vaccine 8 (1990) 525–530. [81] D. Dlugovitzky, G. Fiorenza, M. Farroni, et al, Immunological [65] K. Ghazi-Saidi, J.L. Stanford, C.A. Stanford, Y. Dowlati, consequences of three doses of heat-killed Mycobacterium Vaccines containing Mycobacterium vaccae and their use in vaccae in the immunotherapy of tuberculosis, Respiratory the children of leprosy patients in Iran, Medical Journal of Medicine 100 (2006) 1079–1087. the Islamic Republic of Iran 8 (1994) 87–91. [82] D. Dlugovitzky, D. Diego Martinel-Lamas, G. Fiorenza, et al, [66] T. Le Van, M. Ho, T. Nguyen, et al, Vaccination against Immunotherapy with oral, heat-killed Mycobacterium vaccae leprosy at Ben San Leprosy Centre, Ho Chi Minh City, in patients with moderate to advanced pulmonary Vietnam, Vaccine 19 (2001) 3451–3458. tuberculosis, Immunotherapy 2 (2010) 159–169. [67] C.F. Von Reyn, L. Mtei, R.D. Arbeit, et al, Prevention of [83] A. Etemadi, R. Farid, J.L. Stanford, Immunotherapy for drug- tuberculosis in BCG-primed, HIV-infected adults boosted resistant tuberculosis (letter), Lancet 340 (1992) 360–361. with an inactivated whole cell mycobacterial vaccine, AIDS [84] J.L. Stanford, C.A. Stanford, J.M. Grange, et al, Does 24 (2009) 675–685. immunotherapy with heat-killed Mycobacterium vaccae offer [68] P. Torres, J. Gozalbez, J. Terencio de las Aguas, J.L. Stanford, A hope for the treatment of multi-drug-resistant pulmonary potential immunotherapeutic agent for leprosy tested in tuberculosis?, Respiratory Medicine 95 (2001) 444–447 healthy volunteers, in: Health Cooperation Papers 7, [85] R. Farid, A. Etemadi, M. Mehvar, et al, Mycobacterium vaccae Proceedings of the Fourth European Leprosy Symposium, immunotherapy in the treatment of multi-drug-resistant 1987, pp. 195–199. tuberculosis: a preliminary report, Iranian Journal of [69] J.L. Stanford, J. Terencio de las Aguas, P. Torres, et al., Studies Medical Sciences 19 (1994) 37–39. on the effects of a potential immunotherapeutic agent in [86] G. Ramu, G.D. Prema, S. Balakrishnan, et al, A preliminary leprosy patients, in: Health Cooperation Papers 7, report on the immunotherapy of psoriasis, Indian Medical Proceedings of the Fourth European Leprosy Symposium, Gazette 124 (1990) 381–382. 1987, pp. 201–206. [87] A. Lehrer, A. Bressanelli, V. Wachsman, et al, [70] N.C. Abbot, J.S. Beck, F. Feval, et al, Immunotherapy with Immunotherapy with Mycobacterium vaccae in the treatment Mycobacterium vaccae and peripheral blood flow in long- of psoriasis, FEMS Immunology and Medical Microbiology 21 treated leprosy patients, a randomized, placebo-controlled (1998) 71–77. trial, European Journal of Vascular and Endovascular [88] M. Hansrani, J. Stanford, G. McIntyre, et al, Immunotherapy Surgery 24 (2002) 202–208. for the prevention of myointimal hyperplasia after [71] G. Ramu, J. Grange, J. Stanford, Evaluation of adjunct experimental balloon of the rat carotid artery, Angiology 61 immunotherapy with killed Mycobacterium vaccae in the (2010) 437–442. treatment of multibacillary leprosy and erythema nodosum [89] T. Breivik, G.A.W. Rook, Prevaccination with SRL 172 (heat- leprosum, in: M. Casal (Ed.), Clinical Mycobacteriology, killed Mycobacterium vaccae) inhibits experimental Prous Science, Barcelona, 1998, pp. 393–402. periodontal disease in Wistar rats, Clinical and [72] A. Pozniak, J.L. Stanford, N.M. Johnson, G.A.W. Rook, Experimental Immunology 120 (2000) 463–467. Preliminary studies of immunotherapy of tuberculosis in [90] L. Camporota, A. Corkhill, H. Long, et al, The effects of man. Proceedings of the International Tuberculosis Mycobacterium vaccae on allergen-induced airway responses Congress, Singapore 1986, Bulletin of the International in atopic asthma, European Respiratory Journal 21 (2003) Union against Tuberculosis 62 (1987) 39–40. 287–293. [73] J.L. Stanford, G.M. Bahr, G.A.W. Rook, et al, Immunotherapy [91] D.D. Arkwright, T.J. David, Intradermal administration of a with Mycobacterium vaccae as an adjunct to chemotherapy in killed Mycobacterium vaccae suspension (SRL172) is the treatment of pulmonary tuberculosis, Tubercle 71 (1990) associated with improvement in atopic dermatitis in 87–93. children with moderate to severe disease, Journal of Allergy [74] G.M. Bahr, M.A. Shaaban, M. Gabriel, et al, Improved and Clinical Immunology 107 (2001) 531–534. immunotherapy for pulmonary tuberculosis with [92] J. Berth-Jones, P.D. Arkwright, D. Marasovic, et al, Killed Mycobacterium vaccae, Tubercle 71 (1990) 259–266. Mycobacterium vaccae suspension in children with moderate- [75] P. Onyebujoh, T. Abdulmumini, S. Robinson, et al, to-severe atopic dermatitis: a randomized, double-blind, Immunotherapy for tuberculosis in African conditions, placebo-controlled trial, Clinical and Experimental Allergy Respiratory Medicine 89 (1995) 199–207. 36 (2006) 1115–1121. [76] E. Corlan, C. Marica, C. Macavei, et al, Immunotherapy with [93] M.G. Davies, C. Symons, S. Shaw, et al, An open study to Mycobacterium vaccae: 1. In the treatment of newly diagnosed assess the efficacy, clinically and immunologically of M. pulmonary tuberculosis in Romania, Respiratory Medicine vaccae vaccine in patients with atopic dermatitis, The 91 (1997) 13–19. Journal of Dermatological Treatment 17 (2006) 74–77. 12 International Journal of Mycobacteriology 1 (2012) 3– 12

[94] J.M. Grange, J.L. Stanford, BCG vaccination and cancer, [101] M. Tsukamura, S. Mizuno, Mycobacterium obuense, a rapidly Tubercle 71 (1990) 61–64. growing Mycobacterium capable of [95] A.B. Alexandroff, A.M. Jackson, M.A. O’Donnell, K. James, forming a black product from p-aminosalicylate and BCG immunotherapy of bladder cancer: 20 years on, Lancet salicylate, Journal of General Microbiology 68 (1971) 129–134. 353 (9165) (1999) 1689–1694. [102] J. Stebbing, A. Dalgleish, A. Gifford-Moore, et al, An intra- [96] J.M. Grange, J.L. Stanford, G.A.W. Rook, Tuberculosis and patient placebo-controlled phase I trial to evaluate the cancer: parallels in host responses and therapeutic safety and tolerability of intradermal IMM-101 in approaches?, Lancet 345 (1995) 1350–1352 melanoma, Annals of Oncology 12 (2011) 873–874. [97] J.L. Stanford, C.A. Stanford, B. Baban, J.M. Grange, [103] J.D. Littlewood, J.K. O’Brien, J.L. Stanford, et al., Management Therapeutic vaccination for cancer: the potential value of of Culicoides hypersensitivity (sweet-itch) in equids using mycobacterial products, International Journal of killed bacterial preparation BE-T101: a randomised, double- Pharmaceutical Medicine 13 (1999) 191–195. blind, placebo-controlled study, in: Proceedings of the 17th [98] J.L. Stanford, C.A. Stanford, M.E.R. O’Brien, J.M. Grange, World Association of Veterinary Dermatology Congress, Successful immunotherapy with Mycobacterium vaccae in the Hong Kong, Abstract A71, 2008. treatment of adenocarcinoma of the lung, European Journal [104] G.H. Fontanella, M.F. Pascutti, L. Daurelio, et al, Improved of Cancer 44 (2008) 224–227. outcome of Trypanosoma cruzi infection in rats following [99] P.M. Patel, S. Sim, D.O. O’Donnell, A. Protheroe, D. Beirne, A. treatment in early life with suspensions of heat-killed Stanley, et al, An evaluation of a preparation of environmental Actinomycetales, Vaccine 25 (2007) 3492–3500. mycobacterium vaccae (SRL172) as an immunotherapeutic [105] H. Davila, G. Didoli, O. Bottasso, J. Stanford, Maternal agent in renal cancer, European Journal of Cancer 44 (2008) immunization with Actinomycetales immunomodulators 216–223. reduces parasitaemias in offspring challenged with [100] M.E. O’Brien, H. Anderson, E. Kaukel, et al, SRL172 (killed Trypanosoma cruzi, Immunotherapy 3 (2011) 577–583. Mycobacterium vaccae) in addition to standard chemotherapy [106] A. Marro, M. Pirles, L. Schiaffino, et al, Successful improves quality of life, without affecting survival in immunotherapy of canine flea allergy with injected patients with advanced, non-small-cell lung cancer: phase Actinomycetales preparations, Immunotherapy 3 (2011) III results, Annals of Oncology 15 (2004) 906–914. 971–978.