PCR studies of feline leprosy cases.

16S rRNA gene sequence analysis provided evidence for two different mycobacterial species, Mycobacterium lepraemurium and a potentially novel species, as causative agents of 'feline leprosy'. Comparison of 16S rRNA gene sequence data obtained for M. lepraemurium and the potentially novel species indicated 12 nucleotide differences over a 446 bp region encompassing the V2 and V3 hypervariable regions. From available 16S rRNA gene sequence data, M. lepraemurium shared greatest nucleotide identity with M. avium subsp paratuberculosis and M. avium. The novel species had a long helix 18 in the V3 region and shared greatest nucleotide identity with M. leprae, M. haemophilum and M. malmoense. The novel species had an additional 'A' nucleotide at position 105 of the aligned 16S rRNA gene sequence, the only other mycobacterial database sequence having this same extra nucleotide being M. leprae. This nucleotide variation was exploited to develop specific PCR assays for the two species. These were found to be effective and specific when tested against a panel of mycobacteria including species found in feline leprosy lesions and closely related mycobacteria and also when applied directly to formalin-fixed, paraffin-embedded tissues from feline leprosy cases.

Serological approaches for the characterization of catalase in tissue-derived mycobacteria.

Cell-free extracts of Mycobacterium lepraemurium from mouse liver and M. leprae from armadillo liver were analysed for the presence of any mycobacterial catalase by using the specific inhibitor 3-amino-1,2,4-triazole and seroprecipitation titrations. These studies clearly demonstrated the presence of a "T" type of mycobacterial catalase in M. lepraemurium and placed it, in terms of immunological distance, in a position between M. tuberculosis and M. avium. The results did not reveal any detectable "T" catalase activity in the M. leprae preparations. The "M" type catalase activity which was observed did not bind to antisera against "M" catalase of M. kansasii, but was bound to the extent of 80% to antisera against normal armadillo liver catalase. The significance of the component of the "M" catalase in M. leprae preparations which did not react against antibodies to normal liver remains to be determined.

Electron microscopy of Mycobacterium leprae murium in ultra-thin sections of murine leprosy lesions

1- Lepromas of murine leprosy were studied in ultra-thin sections with the electron microscope. 2- The cell wall, cytoplasm and nuclear elements of M. leprae murium are described. 3- Around each bacillus there is a distinct electron-transparent zone which distinguishes them from other cytoplasmic components of the leproma cells. 4- Foamy structures as are seen in human lepra cells were not observed.

Intratesticular multiplication of Mycobacterium leprae murium in normal and suramin-treated animals

A technique is described for the intratesticular inoculation in mice and rats of suspensions of M. leprae murium, and for subsequent quantitative assessment of bacterial multiplication by counting the numbers of stained bacilli in homogenates of testes from animals killed at inervals. The method is convenient to carry out, and a statistical assessment shows that considerable accuracy is attainable. Bacilli from an 18-month-old rat leproma, transferred to a different host species, the mouse, showed a "lag" phase of 2-5 weeks in its multiplication, succeeded by a logarithmic phase which ciontinued for a further 2-3 months. On the other hand, when rat testes were inoculated with bacilli from a 7-month-old rat-testis leproma, the logarithmic phase was entered at once. The mean generation time in this phase was 7-8 days in rats, and 10 days in mice. During the "lag" phase a transient but significant increase in average bacillus length occurred. Suramin treatment of mice enhanced the growth of the bacilli in them. In rats, however, its effect was to decrease the rate of multiplication.

The infectivity of M. leprae murium kept for various periods on the bench snd in the refrigerator

M. leprae murium in a Tween-saline suspension stored for 1 to 6 days, and in lepromatous tissue kept for 1, 2 and 3 weks, whether on the bench or in the refrigerator, remmained alive and were capable of producing infection. This was also true of a suspension prepared from the 3-week-old leproma and heated for 2 hours at 60°C. before inoculation, although the latent period was greatly prolonged. These materials were tested by intracorneal inoculation in mice.

Histological reactions produced by experimental inoculation of mycobacterium leprae murium into the golden hamster (Cricetus Auratus)

The intraperitoneal inoculation of about 6 mgm. of M. leprae murium into the golden hamster causes a slowly-developing disease which ends with the death of the animal. The disease during its evolution shows two phases: (1) an initial phase, with small macroscopic lesions histologicallyy of retrogresssive character, and (2) a late phase, observed after the 150th day after inoculation, with larger lesions which histologically are of progressive nature. In the early lesions the bacilli are relatively few and show morphological alterations. This seems to be due to lysis of the bacilli in the tissue, probably with active partiicipation of the macrophages. The bacilli seem not to multiply in the lesions, which undergo slow involution. The late lesions, on the other hand, do not show lysis of the mycobacteria and they multiply actively. The appearance and numbers of the bacilli in the lesions are related to the cells which constitute these lesions. In the initial lesions the cells have morphologic and physiologic characters which place them between the epithelioid cell of the guinea-pig on one hand the lepra cell of the rat on the other hand. In the late lesions, however, the cells have only the characters of rat lepra cell. The differences between these two kinds of lesions seems to be due more to the tissue reaction of the hamster than to the adaptation of the mycobacteria to the animal's organism. During the initial phase, secondary lesions occur in various organs caused by the deposition of a "hyalin substance". In the late phase, asteroid bodies may be seen in the lesions.

Mycobacterium leprae murium: preservation by freeze-drying

A method is described for freeze-drying clean suspensions of Mycobacterium leprae murium in horse serum and in glucose-digest broth Judging by the virulence of the reconstituted specimens when injected into susceptible mice, the viability of the organisms was preserved. It appears from these experiments that viable bacilli can be satisfactorily preserved by freeze-drying, at least for periods up to six months.

Relationship between the metabolic capacity and the infectiousness of M. leprae murium; refrigeration studies

1- The unfavorable affect of serum from rats and other animals on the infectiousness of M. leprae murium was first observed in very dilute solutions. The adverse effect is here shown to occur within the time during which it is known that inoculated mycobacteria lie in serous exudate prior to phagocytosis. Because of the progressive, seriuos damage to the endogenous metabolism of the bacilli, it is concluded that this exposure to natural extracellular inhibitors lowers significantly both their viability and their infectiousness when inoculated into animals. 2- Similar inhibitors...