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Anergy, or contrarily, Mitsuda-type responses towards 4 chemoautotrophic nocardioform antigens (CAN-Ags) and a control standard lepromin were tested in 73 LL, TT and borderline cases of leprosy. The antigens injected per patient varied from a maximum of 5 to a minimum of 2. Complete anergy to CAN-Ags was seen in 92/92 instances tested on 24 LL cases. The anergy was weakly modified or unmodified in 3 other LL cases which had been vaccinated before. Concurrent studies with the same antigens tested on 33 TT cases showed clear-cut, dose-dependent, Mitsuda-type late responses in 80/81 instances. The CAN bacteria, therefore, despite their origin from different unrelated leprous human, mouse footpad (MFP) and armadillo tissues, appeared to be identical with each other and also probably related to the leprosy bacillus, on the basis of these parameters.

The transmission electron microscopic (TEM) studies of the human leprosy derived chemoautotrophic nocardio-form (CAN) bacteria and EUS derived CAN bacteria showed presence of double contoured cell-walls consisting of an electron transparent and a dense layer. The fibrillar structures on the surface of these CAN bacterial cells also suggested their similarity to the human tissue derived Mycobacterium leprae cells. These EM studies further revealed mycelial and coccoid bodies in all these bacteria as was observed originally.

On the basis of thin layer chromatography and gas-chromatography-mass spectrometric studies, the lipid profiles of all the chemoautotrophic nocardioform (CAN) bacteria derived from human and animal leprosy tissues appear to be identical with each other, and closest to or identical with the most probable profile of M. leprae.

Nocardioform actinomycetic organisms were present regularly in, and isolated repeatedly from, different varieties of fish affected with epizootic ulcerative syndrome lesions of dermis, muscle, subcutaneous tissues and internal organs. These acid-fast bacilli, resembling human and rat leprosy bacilli, together with other actinomycotic bodies, appeared to explain the characteristic macrophage granuloma observed in such lesions, similar to those of humans. These isolates possessed fundamental similarities to the human isolates of chemoautotrophic nocardioform bacteria reported earlier.

In vitro cultures of the nocardioform bacteria from leprosy-infected tissues consisted of granules and bacilli. Inoculation of these granules into mouse footpads (MFP) produced a mild, localised, inflammation for 4-6 weeks. The granules evoked typical granulomatous response in the subcutaneous tissue and showed gradual disintegration. Infiltration of muscles, connective tissue and epithelial cells by bacillary/mycelial masses was seen very frequently, and that of nerve bundles occasionally. Plenty of mycelial tufts emanated from many 'macrophage globi'. By 6-8 months, the granules disintegrated nearly completely releasing a large number of acid-fast bacilli (AFB), single layered rings of AFB, small globi and some residual mycelia. These AFB, harvested from the MFP, were similar to or indistinguishable from the bacillary preparations from the in vitro cultures and from the leprosy bacillus obtained directly from humans or as passaged into the MFP, on the basis of many criteria studied, including the 36k gene positivity.

Acid fast rods, constituting chemoautotrophic nocardioform bacteria, could be repeatedly cultivated and isolated and propagated indefinitely in vitro from fish actinomycotic macrophage granuloma from the massive epizootics of ulcerative disease syndrome of fish in eastern India during 1988-90. The possible human hazards of zoonotic actinomycotic infections as a result of handling and consumption of such fish are assessed.