Isolation of cultivable mycobacteria from feces and lungs of armadillos infected with Mycobacterium leprae.

In the past, no cultivable mycobacteria were isolated from armadillos captured in the state of Florida, U.S.A. But recent findings of acid-fast bacilli (AFB) in the lungs of armadillos infected with Mycobacterium leprae prompted us to undertake this study to determine the correlation between systemic leprosy infection and the occurrence of cultivable mycobacteria in the lungs and stools of these animals. No AFB could be isolated from noninfected animals. Seventy percent of the infected animals developed disseminated infection, but no cultivable mycobacteria were isolated from their livers and spleens. However, cultivable mycobacteria were isolated from the lungs and stools of a large number of armadillos showing disseminated infection. The most common among these were M. gordonae, M. fortuitum, and M. avium. There was a close correlation between the development of disseminated leprosy infection and the occurrence of cultivable mycobacteria in their lungs and stools, perhaps due to the decline in the immune system in these animals in the later stages of infection.     

Effects of freezing and thawing on the viability and the ultrastructure of in vivo grown mycobacteria

The influence of different frequencies of freezing-thawing cycles on the viability of in vivo grown mycobacteria was investigated. Pieces of armadillo tissues naturally or experimentally infected with Mycobacterium leprae were analyzed. The viability of M. leprae was determined by mouse foot pad titration. The viability of cultivable mycobacteria, sometimes present in armadillo tissues, was determined by culture. Electron-microscopic studies were performed on fresh or frozen-thawed armadillo tissues with natural leprosy and on livers of C57BL/6 mice experimentally infected with M. avium or M. lepraemurium. We found that the percentage of viable M. leprae bacilli is identical for naturally infected and experimentally infected tissues, frozen and thawed once. When the tissues were subjected to a second freezing-thawing cycle, a considerable loss of viability was observed (65%-97%). A third freezing-thawing cycle was lethal for most of the M. leprae cells, and after four freezing-thawing cycles no viable bacilli were found. The cultivable mycobacteria present in some armadillo tissues were found to be more resistant than M. leprae to freezing-thawing since these mycobacteria could still be cultivated after four freezing-thawing cycles. The results of the electron-microscopy study support the conclusion that M. leprae is more sensitive to freezing-thawing than the cultivable mycobacteria and show that the cytoplasmic membrane appears to be the target for the lethal action of freezing-thawing on mycobacterial cells. These results emphasize the importance of avoiding repeated thawing and refreezing of M. leprae-infected tissues when viable M. leprae cells need to be studied.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mycobacterial Esx-3 is required for mycobactin-mediated iron acquisition

The Esx secretion pathway is conserved across Gram-positive bacteria. Esx-1, the best-characterized system, is required for virulence of Mycobacterium tuberculosis, although its precise function during infection remains unclear. Esx-3, a paralogous system present in all mycobacterial species, is required for growth in vitro. Here, we demonstrate that mycobacteria lacking Esx-3 are defective in acquiring iron. To compete for the limited iron available in the host and the environment, these organisms use mycobactin, high-affinity iron-binding molecules. In the absence of Esx-3, mycobacteria synthesize mycobactin but are unable to use the bound iron and are impaired severely for growth during macrophage infection. Mycobacteria thus require a specialized secretion system for acquiring iron from siderophores.     

Electron microscopic study of Mycobacterium leprae membrane

We report the results of the study by transmission electron microscopy of normal Mycobacterium leprae in the tissues of experimentally infected armadillos. Several fixation procedures were used and compared to those previously employed in the study of M. leprae in lepromatous leprosy patients. The results show that the ultrastructure of M. leprae is identical in both hosts. The demonstration of a symmetric membrane profile in M. leprae in armadillos confirms our previous results. This characteristic of the M. leprae membrane is peculiar in that it is not shared by any of the easily cultivable species of mycobacteria we have studied so far.     

Influence of environmental mycobacteria on the prevalence of leprosy clinical type

A comparison was made of antibody levels to 16 environmental mycobacteria in leprosy patients and healthy controls. Significant differences in response were found between patients and controls from an area of Zimbabwe with predominantly lepromatous leprosy when compared to an area where more tuberculoid cases were found. The results obtained suggest that exposure to some environmental mycobacteria may influence the type of leprosy developed by susceptible individuals.     

Use of gas chromatography to differentiate Mycobacterium leprae from cultivable armadillo-derived mycobacteria, M. avium/intracellulare, and M. lepraemurium by analysis of secondary alcohols

Two long-chain secondary alcohols, 2-octadecanol and 2-eicosanol, were demonstrated by gas chromatography in hydrolysates of Mycobacterium avium/intracellulare, in cultivable, armadillo-derived mycobacteria, and in M. lepraemurium grown in vivo, but they were not found in purified suspensions of M. leprae isolated from experimentally infected armadillos. Gas chromatographic analysis of these alcohols constitutes a method for rapid detection and quantification of contaminating mycobacteria in preparations of M. leprae intended, for example, for vaccine use. The technique may also be of value for critical evaluation of cultures of "in vitro-grown" M. leprae.     

Mycobacterial flora of the skin in leprosy.

The presence of mycobacteria on the skin of healthy people and in leprosy lesions has been documented previously. The present study observed the mycobacterial flora on the hands (by the hand-washing method) and fingers (by the inoculated culture medium using scraped material obtained during the preparation of slit-skin smears) in 89 untreated leprosy patients. We also evaluated the slit-skin smears from fingers for the diagnosis of leprosy. In 16 patients (17.9%) mycobacteria were cultured from scrapings and hand washings. The frequency of isolates from lepromatous (LL) leprosy cases (52.9%) was significantly higher than from tuberculoid (TT) leprosy cases (5.2%). It was observed that Mycobacterium avium and M. scrofulaceum were the only opportunistic mycobacteria isolated from multibacillary patients, and two hypotheses are discussed to explain these findings. The slit-skin smears from fingers were as satisfactory as smears from other sites for the diagnosis of leprosy, but they were less satisfactory for estimating the morphological index.     

An electron microscopic study on macrophages and lymphocytes in lepromatous and borderline leprosy

We describe how macrophages are activated and phagocytose mycobacteria in lepromatous leprosy. The differentiation of macrophages into epithelioid cells and into giant cells in borderline leprosy is shown. Close apposition between macrophages and lymphocytes is seen in those areas where mycobacteria disintegrate inside macrophages.     

Immunodiffusion analyses of some diphtheroid organisms isolated from patients with leprosy

Eight strains of diphtheroid bacteria isolated from patients with leprosy were analyzed by immunodiffusion, using precipitation systems representing various species of Corynebacterium, Mycobacterium, Nocardia, Rhodococcus, and related organisms. The analyses showed that six of the eight strains shared several antigens with representatives of these four genera. The largest number of shared precipitinogens was revealed when the corynebacterial precipitation systems were used, thus indicating that these organisms either belong to, or are closely related to the genus Corynebacterium. This assumption was further supported by the fact that the ribosomal precipitinogen beta--earlier demonstrated in mycobacteria but not in corynebacteria--was not found in the diphtheroid strains. Other ribosomal antigens were, however, revealed to be common to the diphtheroid organisms and mycobacteria. Further, the reaction between sera from patients with lepromatous leprosy and the diphtheroid strains was analyzed, very few and faint precipitates being demonstrated. It is concluded that the presence of anti-beta antibodies in leprosy sera is, most likely, not a result of the presence of diphtheroid organisms in the patients.     

Adoptive transfer of tolerance induced by ICRC bacilli against Mycobacterium leprae in mice

ICRC bacilli, the cultivable leprosy-derived mycobacteria, isolated from lepromatous nodules of leprosy patients were found to be immunogenic in BALB/c mice at a dose of 2 X 10(7) acid-fast bacilli when injected by the intradermal (i.d.) route. The sensitization to lepromin and ICRC antigens was measured by the foot pad enlargement (FPE) method. The same dose of bacilli when injected by intravenous (i.v.), intraperitoneal, and subcutaneous routes induced immune tolerance in mice as indicated by reduction in the FPE to the test antigens. The spleen cells obtained after i.v. injection of ICRC bacilli/Mycobacterium leprae after adoptive transfer brought about suppression of delayed-type hypersensitivity in sensitized as well as nonsensitized recipients, indicating production of suppressor cells after i.v. injection. Similarly, the tolerance induced by i.v. injection of M. leprae in mice could be partially converted to immunity by i.d. sensitization with live BCG and two strains of ICRC bacilli (C-44 and C-75).     

Morphologic and biologic properties of diphtherroid organisms (LDC) isolated in leprosy

Acid-alcohol-fast bacteria are not always detectable in all leprosy lesions. Non acid-fast microorganisms may be associated with acid-fast bacteria. The most frequently isolated strains from leprosy lesions are non acid-fast bacteria, morphologically related to C. diptheirae. Hence their designation as diphthero?ds or LDC (leprosy derived corynebacteria). Their antigenic structure is more closely related to M. leprae and other mycobacteria than to classical corynebacteria. This leads to the hypothesis of a potential role in the pathogenesis of leprosy and their use as an antigen for skin tests by leprosy patients.     

Occurrence of antigen BCG 60 in leprosy derived corynebacteria and other coryneforms

Previous studies have shown that anti BCG 60 monoclonal antibody could recognize major antigen A 7 of M. leprae. In the present investigation we attempted to search the presence of the same antigen from the strains of Leprosy Derived Corynebacteria (LDC+), which were isolated from leprosy lesions and were supposed to be the cultivable form of the leprosy bacillus. A comparative study was equally performed on 7 strains of Corynebacteria which were isolated in France and had no relation with leprosy patients (LDC-). Utilizing the ELISA technique with an anti BCG 60 monoclonal antibody, it was found that all the three LDC+ strains contained an antigenic determinant common to M. leprae, however no LDC+ specificity was found as this antigenic determinant was also revealed on two LDC- strains.     

Characterization of Mycobacterium leprae by lipid analysis

The lipid composition of the leprosy bacillus, harvested from experimentally infected nine-banded armadillos, strongly supports it status as a distinct species of the genus Mycobacterium. Phthiocerol dimycocerosate waxes and glycosylated phenophthiocerol dimycocerosates are distinct from those characterised from a number of other mycobacteria. The polar lipids of a single isolate lack diacylated forms of phosphatidylinositol di- and pentamannosides, lipids usually found in most mycobacteria. A simple mycolic acid pattern composed of alpha-mycolates and ketomycolates is characteristic of most preparations of M. leprae.     

Effect of presensitization with BCG and Mycobacterium leprae on granuloma formation to M. leprae

Granulomas which develop in draining lymph nodes, following the intradermal injection of cobalt-irradiated Mycobacterium leprae into the ear of the guinea pig 2 and 5 weeks earlier, were studied in animals which had been presensitized with BCG vaccine or M. leprae and compared with granulomas that developed in previously unsensitized guinea pigs. Presensitization with mycobacteria accelerated the development of the granulomas. Granulomas in previously unsensitized guinea pigs were found ultrastructurally to contain phagocytosing macrophages similar to those in lepromatous leprosy, and M. leprae presensitization did not alter the type of granuloma found. Those in BCG-presensitized guinea pigs contained secretory epithelioid cells with rough endoplasmic reticulum similar to those found in borderline tuberculoid leprosy or reversal reactions. The significance of these findings in relation to the current use of vaccines in leprosy is discussed.     

Iron, mycobacteria and tuberculosis

The role of iron in the growth and metabolism of M. tuberculosis and other mycobacteria is discussed in relation to the acquisiton of iron from host sources, such as transferrin, lactoferrin and ferritin, and its subsequent assimilation and utilization by the bacteria. Key components involved in the acquisition of iron (as ferric ion) and its initial transport into the mycobacterial cell are extracellular iron binding agents (siderophores) which, in pathogenic mycobacteria, are the carboxymycobactins and, in saprophytic mycobacteria, are the exochelins. In both cases, iron may be transferred to an intra-envelope, short-term storage molecule, mycobactin. For transport across the cell membrane, a reductase is used which converts FeIII-mycobactin to the FeII form. The ferrous ion, possibly complexed with salicylic acid, is then shuttled across the membrane either for direct incorporation into various porphyrins and apoproteins or, for storage of iron within the bacterial cytoplasm, bacterioferritin. The overall process of iron acquisition and its utilization is under very genetic tight control. The importance of iron in the virulence of mycobacteria is discussed in relationship to the development of tuberculosis. The management of dietary iron can therefore be influential in aiding the outcome of this disease. The role of the old anti-TB compound, p-aminosalicylate (PAS), is discussed in its action as an inhibitor of iron assimilation, together with the prospects of being able to synthesize further selective inhibitors of iron metabolism that may be useful as future chemotherapeutic agents.     

Crossreactivity between Mycobacterium leprae and various actinomycetes and related organisms

Serological crossreactivity was analyzed between M. leprae and strains of various species of Corynebacterium, Mycobacterium, Nocardia, Rhodococcus, Streptomyces, and related organisms. M. leprae shares antigens with most of these organisms, and sera from patients with lepromatous leprosy contain antibodies against them. The results demonstrate that M. leprae shares more antigens with the mycobacteria than with strains of the other tested genera, thus supporting the view that the leprosy organism belongs to the genus Mycobacterium. One precipitinogen (designated p beta) was found to be common to M. leprae and the streptomycetes, and sera from patients with lepromatous leprosy contain antibodies against this antigen.     

Serum amyloid protein SAA, C-reactive protein and lysozyme in leprosy

Serum amyloid protein (SAA) appears to be the precursor of amyloid protein AA, the non-immunoglobulin fibril protein of secondary amyloidosis. Since amyloidosis is known to occur in high frequency associated with lepromatous leprosy (LL), we have examined the SAA levels in untreated LL patients and compared them to the levels observed in patients with tuberculoid leprosy (TT) and a large number observed in healthy controls. We found that SAA is markedly elevated in LL when compared to TT and controls. No clear correlation could be established with C-reactive protein, a well-documented acute phase reactant, or serum lysozyme levels that reflect the presence of monocyte activity. This study showed that SAA levels in leprosy do not appear to be a reflection of inflammatory activity or monocyte turnover. Whether amyloidosis will be more prevalent in patients who have higher SAA levels remains to be determined.     

New data on the ultrastructure of the membrane of Mycobacterium leprae

In previous reports on the ultrastructure of Mycobacterium leprae, we described the occurrence of symmetric membranes in normal-looking bacilli from fresh or frozen samples primarily fixed with aldehydes. In those reports we admitted that such a symmetric profile, which is not found in the other normal mycobacteria, would not represent the structure of the normal membrane of the leprosy bacillus. We, therefore, re-analyzed the ultrastructure of the membrane of M. leprae. In the present work the micromorphology of the M. leprae membrane was studied by transmission electronmicroscopy after the fixation of fresh samples by OsO4 plus calcium followed by glutaraldehyde plus formaldehyde and calcium followed by uranyl acetate. The study of samples from two patients with lepromatous (LL) leprosy, three armadillos with natural leprosy, and one nude mouse with experimental leprosy showed that normal-looking bacilli present in lead-stained sections had asymmetric membranes with a thickness of 6.49 +/- 0.36 nm. These membranes showed periodic acid-Schiff (PAS)-positive components exclusively located in the outer half of the bilayer. We demonstrated that the symmetric profile of the M. leprae membrane described in our previous reports corresponds, as admitted in those reports, to an abnormal membrane structure. Such an abnormality was now found to result from the use of primary fixation with aldehydes or of samples stored frozen before fixation. These results indicate that, although ultrastructurally similar to that of the other mycobacteria, the membrane of M. leprae has a peculiar sensitivity to fixation by aldehydes. Such a characteristic, which was not found in M. lepraemurium, M. aurum, M. avium, and M. tuberculosis H37Ra, must reflect a unique membrane molecular structure, which is presently unknown.     

Pyridine extractability of acid-fastness from Mycobacterium leprae.

Various mycobacteria were tested for their ability to retain acid-fastness after treatment with pyridine: a) Mycobacterium leprae separated from organs of 20 experimentally infected armadillos (which were sacrificed); b) M. leprae separated from a biopsy of a lepromatous patient; c) direct smears of lepromatous tissues from armadillos; d) eighteen cultivable mycobacteria obtained from the American Type Culture Collection (ATCC); E) cultivatable mycobacteria separated from the lymph nodes of a wild-caught armadillo and also the same organism grown in culture and Skinsnes' alleged M. leprae culture. A loss of acid-fastness was observed microscopically from M. leprae separated from experimentally infected armadillo tissues, M. leprae separated from a lepromatous patient biopsy, and M. leprae found in direct smears prepared from infected armadillo tissues. The eighteen cultivatable mycobacteria from ATCC, cultivatable mycobacteria separated from the tissue of a wild-caught armadillo (and also grown in culture) and Skinsnes' alleged M. leprae culture retained their acid-fastness. Testing of pyridine extractability of acid-fastness combined with those of D-DOPA oxidase testing proved to be extremely reliable in our laboratory in differentiating M. leprae from other mycobacteria.     

The pathology and pathophysiology of mycobacterial infections

Classic tuberculosis is the result of infection with the human strain of Mycobacterium tuberculosis, and atypical tuberculosis is the result of infection with atypical mycobacteria. The pathology and course of the disease depend on the sensitivity of the host. Primary tuberculosis is the first infection in an unsensitized host, and secondary, postprimary, or chronic tuberculosis results from reactivation of previously acquired infection or, rarely, reinfection of a sensitized host. The pathology of infection with M avium-intracellulare in patients with acquired immunodeficiency syndrome (AIDS) is different from that of tuberculosis; formation of noncaseating lesions and a marked macrophage response resemble changes seen in lepromatous leprosy. These infections in patients with AIDS are predominantly extrapulmonary.