Lytic potency against various mycobacterial strains of the phage isolated from Mycobacterium leprae murium "Douglas".

The lytic potency of a newly isolated phage Al-1 obtained from the laboratory strain M. leprae murium "Douglas" was examined. The phage was multiplied on the laboratory strain M. smegmatis ATCC 607 and for the lytic test 0.1 ml of suspension containing PFU 10(5) was used. In the whole 18 mycobacterial strains both slowly and fast growing multiplied in liquid Sula's medium were tested. For phage lytic tests two simple agar media and standard Redmond's medium RVA-24 were used. The examined slowly growing mycobacteria (H37Rv. M. bovis "Ravenel", M. avium "Kirchberg", M. kansasii "Svizenský", M. tbc INH resist., M. tbc INH, STM, PAS resist.) were resistant to the tested phage similarly as M. phlei from the group of fast growing strains. The results of phage tests on all three used media were characterized by a confluent phage lysis with the exception of the strain M. butyricum "Rabinovic", in which even on a very rich media an incomplete lysis with countable plaques was found out. The use of the phage Al-1 for the phage typification also of the strain M. leprae murium is considered on the basis of the inhibition growth tests on Ogawa's egg media.

Isolation of mycobacterial phage from the laboratory strain Mycobacterium leprae murium "Douglas".

The colony microstructure of the laboratory strain Mycobacterium leprae murium "Douglas" cultivated on Ogawa's egg medium was examined. A bioptical sample from the liver of a white mouse subcutaneously infected and observed for ten months was used as inoculum. The inoculum contained 5.2 X 10(9) acidfast rods. The Ogawa's media were incubated in 5% atmosphere of CO2 at 33 degrees C to 37 degrees C for 6 to 10 months. The outgrown colonies were killed with a formol solution, then embedded into the agarparaffin and cut out with the aid of Reichert's microtom. In thin sections there was an apparent vacuolisation of colonies proving the presence of the temperate phage, which was isolated from the bacterial suspension inoculated on the host non lysogenic strain Mycobacterium smegmatis ATCC 607. On the simple agar medium N-4 the number of 2.4 X 10(9) living particles was achieved, which shows the possible use of this phage for differential diagnostic purposes in the taxonomy studies of mycobacteria.

[Incidence of phagotypes of Mycobacterium tuberculosis in Czechoslovakia and East Germany and of Mycobacterium bovis in Czechoslovakia and Argentina]. / Untersuchungen über das Vorkommen von Phagotypen des Mycobacterium tuberculosis in der CSSR und der DDR sowie des Mycobacterium bovis in der CSSR und Argentinien.

The following study gives review of the results of the phagetyping of M. tuberculosis strains in different localities of Czechoslovakia and German Democratic Republic (165 strains from Czechoslovakia and 102 strains from GDR) and the comparison of the occurrence of phage subgroups of M. bovis strains in two different localities of Czechoslovakia (62 strains) and Argentina (93 strains). The results of the phagetyping of the Czechoslovak M. tuberculosis strains show that this group comprises all the 6 phage subgroups and agree very well in subgroups Ax, A2, B and C with the M. tuberculosis strains from GDR. Both groups of strains differ in subgroups A2 (9) and D. The results with the fresh isolated Argentinian M. bovis strains show that this group has only subgroups Ax and D in comparison with the old laboratory strains of M. bovis which have subgroups Ax, A2(9), and D. The phagetyping of the M. tuberculosis and M. bovis strains was undertaken with the help of 11 international mycobacterial phages. The use of mycobacterial phages in 1 RTD (Routine Test Dilution) and 10 RTD concentrations (in contrary to the 1 RTD and concentrated phage suspensions used previously) decreased the number of the "unspecific" reactions.

Stimulating effects of a weak constant magnetic field (0.8 Gauss) on the growth of two mycobacterial phages no 308 and 33d.

A liquid culture of ATCC 607 infected with two mycobacterial phages and exposed to constant magnetic fields 0.8 G for 48 hours was replanted on simple agar media without oleic acidalbumin enrichment. In both phages a pronounced stimulating effect was observed demonstrated in the phage No. 308 by almost confluent lysis in magnetized culture and in the phage 33 D by uncountable lytic plaques. Magnetized phage cultures could be preferably used for preparing rich stock suspensions also from poorly growing phages needed for phage classification of mycobacteria.

The microscopical pattern of growth of Mycobacterium leprae-murium "Douglas" in microcolonies.

A thin section technique of Ogawa egg yolk culture medium inoculated with Mycobacterium leprae-murium was found to reveal microscopical growth of the strain which could not be demonstrated by macroscopical examination. A peculiar structure of the growth, characterized by many lytic spots different in size, was observed indicating the possible presence of a temperate phage which may interfere with the synthesis of nucleic acids needed for the active multiplication of M. leprae-murium.

[The survival of D-29 and MyF2 P/59-mycobacteriophages in continuous cultures]. / Das Uberleben von D-29- und MyF2 P/59-Mykobakteriophagen in kontinuierlichen Kulturen.

The cultivation technique of two mycobacteriophages (D-29 and MyF2 P/59) in continuous cultures with a simple synthetic medium is described. The ATCC-607-strain (M. smegmatis) was used as a host strain. The medium was exchanged every 24 hours for 14 days, then the whole cultivation equipment including waste bottle was hermetically closed and preserved in a thermostat for seven years. Every six months about 250 ml of new liquid medium was added into the cultivation container after finishing the passage cultivation. Both mycobacteriophages could be found out still after 6 years in the mixture of the mycobacteriophages preserved in the waste bottle and in the culture container even after seven years. The possibility of using the technique of continuous cultivation of phages for preparing fresh 24 hours old suspensions necessary for phage typing of the mycobacteria is discussed.

Therapy of experimental tuberculosis in guinea pigs with mycobacterial phages DS-6A, GR-21 T, My-327.

Guinea pigs, weighing 250-350 g, were infected with approximately 5,000 of live germs M- tuberculosis H 37 Rv grown 10 days in deep culture of liquid semisynthetic medium according to Sula. The infection was performed subcutaneously in inquinal region. For the therapy following phages were used: DS-6A, GR-21/T, My-327 injected twice a week subcutaneously in the dose of 10(6)/1 ml of live particles for 10 weeks. The therapeutic effect was expressed by spleen and hilus index. Out of the phages used, phage DS-6A had the highest therapeutic effect with mean spleen index of 0.19, corresponding approximately to the spleen index reached with the most effective tuberculostaticum INH. The exact explanation of the phage therapeutic effect in given experimental conditions, when the phages are not applied locally in order to gain the direct contact with infectious antigens, is not known. It is suggested that there presumably exists an interaction between the released phage nucleic acid and the nucleic acid synthesis needed for the growth of mycobacteria in vivo.

The colonial microtexture of human mycobacterial strains isolated in Burma.

Sixteen strains of Mycobacterium tuberculosis isolated in Burma were investigated for microcolonial texture using the thin section technique developed at the WHO Collaborating Centre in Prague. The strains were grown in deep (Sula's liquid medium) and surface cultures (Loevenstein-Jensen medium). Their colonies killed by 10% formol were embedded in 2% agar and paraffin, cut by a Reichert microtome and stained by the Ziehl-Neelsen technique. According to the surface texture and deep growth pattern, the Burmese strains were divided in three different groups. The European type was characterized by a growth strictly confined to the surface of the Loevenstein-Jensen medium, the African type by combined surface and subsurface growth, and the Burmese type not seen in Europe and Africa by non acid-fast granular and acid-fast colonies with distinct formation of strongly acid-fast cords both in surface and subsurface growth.

Morphological and cytoenzymatic characteristics of peripheral zone of lytic plaques induced by mycobacterial phages.

Morphology and cytoenzymatic characteristics were studied using potassium tellurite, of peripheral zones of lytic plaques in fast growing mycobacteria (ATCC 607, Redmond No 521, Penso S1P) exposed to various mycobacterial phages and slowly growing M. bovis BCG, H37RV 50 gamma INH resistant strains. The latter revealed on borders of lytic plaques a bright tellurite-negative zone of variable width, macroscopically undemonstrable in culture free from potassium tellurite. Microscopically only single mycobacteria of various shapes, whereas in the tellurite-positive zone typical arrangement of BCG rods in compact, strong acid-fast cords present also in the so-called grey zone contacting the tellurite-negative zone, were found. The said tellurite zonal phenomen is explained by abortive infection with phages under study, which results in killing the mycobacterial cells without phage multiplication. The microscopical examination of surface pellicles by thin section showed the phage abortive infection to form a much broader zone than demonstrable in macroscopical examination, since under the black pellicle containing reduced metallic tellurite another tellurite-negative layer is hidden. The quickly growing mycobacteria in the peripheral zones of lytic plaques were either fully tellurite-positive or showed on the inside of the plaque a very narrow, hardly visible tellurite-negative border, microscopically similar to M. bovis BCG and H37RV. The possible utilization of tellurite cytoenzymatic reaction in differential diagnostics both of mycobacteria and mycobacterial phages is discussed.

Accidental contamination of diagnostic cultures of mycobacteria and phage identification of the contaminating strain.

Phage typing of mycobacteria is a new technique not yet widely used for classification and intraspecies differentiation. Systematic long-term studies organized by the WHO have succeeded, using a battery of 11 different mycobacterial phages, in dividing M. tuberculosis into 3 different phage sub-groups, preliminarily labelled as A, B and C. A special case of phage-typing is presented which enabled identification of a virulent mycobacterial strain causing accidental contamination of diagnostic mycobacterial cultures. The strain was an old laboratory one, H37Rv, belonging to phage subgroup B.

Self-inoculation with Mycobacterium tuberculosis Weiszfeiler W-115.

After intracutaneous self-inoculation with 0.05 mg (wet weight) of Mycobacterium tuberculosis W-115 containing approximately 600 000 viable units, the local reaction was observed during a period of 9 weeks. No adverse effects or general reaction were observed. From the lesion the W-115 strain was recultivated. It was labelled W-115(78)ST vaccine strain and is used for further studies. On the basis of experiments on laboratory animals, monkeys, human new-borns and adults, the strain W-115 is considered as safe for antituberculous vaccination as the strains Mycobacterium bovis BCG and the attenuated "MP" strain of Mycobacterium microti OV 166.

Use of pyridine for differentiating Mycobacterium leprae from other mycobacteria in direct microscopy.

The loss of acid-fastness by M. leprae after two-hour pyridine extractions, reportedly a specific test for differentiating M. leprae from all other mycobacteria, was verified on different materials obtained from leprosy patients, histologic sections from a fatal post-BCG vaccination case and smears prepared from pure cultures of 32 strains of 18 different mycobacterial species. Under the conditions used, pyridine extraction led to complete loss of acid-fastness in M. leprae only in histologic sections of biopsy specimens from leprosy patients, whereas in direct smears from skin lesions containing M. leprae the number of acid-fast rods after pyridine extraction was either equal to or only slightly smaller than in control preparations. Moreover, since smears from pure cultures of M. avium, M. diernhoferi, M. fortuitum, M. scrofulaceum, M. vaccae and especially M. phlei displayed a smaller or greater number of nonacid-fast cells as well (in some instances only 10% to 20% of cells were found stained whereas control slides contained 90% to 100% acid-fast rods), loss of acid-fastness after two-hour pyridine extraction cannot be considered a property typical of M. leprae only.

Fatal generalized BCG histiocytosis.

An infant was vaccinated at the age of 3 days with BCG vaccine. At the age of 3 years 10 months he developed an infection by Salmonella typhimurium. The infection persisted with recurrent episodes of fever, peri-nephritic abscess, abscesses of lymph nodes, hepatomegaly, splenomegaly and paravertebral and retro-peritoneal abscesses, from which Salmonella were isolated. At the age of 6 years and 2 months later. At post-mortem examination there were widespread histiocytic nodules in many organs, from which Mycobacterium bovis BCG were cultured. One previous case has been reported from Czechoslovakia. The mother of that child was the sister of the father of the child reported here. It was not possible to investigate the possibility of abnormalities of humoral or cellular immunity in the family.