Intracellular parasitism of parenchymal cells by Mycobacterium leprae.

The liver, skeletal muscle, and adrenal gland obtained from two nine-banded armadillos infected with Mycobacterium leprae were studied using an electron microscope. M. leprae were found in varying numbers inside hepatocytes, Kupffer's cells, striated muscle cells, adrenal cortical and adrenal medullary cells, endothelial cells, and macrophages. There was evidence to suggest that M. leprae were actively phagocytosed by the liver and skeletal muscle cells. The inert nature of M. leprae and its behavior as an almost ideal parasite of parenchymal cells are emphasized. The question of whether this unique parasitism of parenchymal cells and the possible processing and presentation of M. leprae antigens by these cells could be responsible for aberrant immune responses is raised.

An electron microscopic study of alterations in the morphology and permeability of purified Mycobacterium leprae.

This communication reports the association of changes in ultrastructure of Mycobacterium leprae with alterations in its permeability. To study morphologic changes of the organisms under different conditions (of temperature and exposure to NaOH and trypsin), ultrathin sections of the bacteria were cut and examined in an electron microscope. In the untreated bacilli and those washed with trypsin, the cytoplasmic membrane and the cell wall (peptidoglycan layer) remained intact; dapsone showed little effect on diphenoloxidase of the bacteria. M. leprae is unique among mycobacteria in possessing an unusual form of the enzyme diphenoloxidase. The antileprosy drug dapsone is a potent inhibitor of the enzyme, but it does not readily penetrate the bacteria where the cell envelope remains intact. The cell wall of M. leprae exposed to -80 degrees C or washed with NaOH was partially detached from the cell membrane; dapsone readily penetrated these organisms and inhibited the bacterial enzyme. In the above preparations, the cytoplasmic membrane appeared undamaged and the bacteria remained viable, as evidenced by multiplication in mouse foot pads. At 50 degrees C, the peptidoglycan layer became completely separated from the membrane and the cytoplasm was partially denatured. These organisms were permeable to dapsone, but were no longer viable. At 100 degrees C, the structural organization of the bacilli was completely destroyed, and of course, they lost their enzyme activity as well as viability. Evidently, the intact cell wall layer mediates the exclusion of dapsone from M. leprae, and there is no correlation between its viability and permeability. The ultrathin sections also reveal the internal organization and cytoplasmic inclusions of M. leprae, as never before seen.

Ability of the phenolic glycolipid-I antigen of M. leprae to elicit a positive Mitsuda response in the armadillo (Dasypus novemcinctus).

Three lepromin-positive armadillos and three lepromin-negative armadillos were tested intradermally with 100 micrograms of phenolic glycolipid-I (PGL-I) in 0.1 ml of normal saline. Positive delayed-hypersensitivity granulomas at 21 days in the lepromin-positive animals and negative responses in the lepromin-negative animals were obtained. These observations suggest that purified PGL-I is capable of eliciting cell-mediated immune or delayed-hypersensitivity responses in animals sensitized to Mycobacterium leprae.

First lesion in experimental armadillo leprosy.

Eighteen armadillos were infected intravenously with 10(8) M. leprae and 10 intracutaneously with 10(7) M. leprae. Among those which developed disseminated disease, a nodule at the site of inoculation was the first lesion noticed in 14 of the 16 infected intravenously and 4 of the 4 infected intradermally. It is possible that in human leprosy the first sign of infection is localized proliferation of M. leprae at the site of entry, and even nodule formation in lepromatous patients. It may be important to search for asymptomatic swelling or keloid-like lesions in skin or in nasal mucosa while screening a population for early leprosy.

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.