Mycobacterium intracellulare Reference Precipitation System

Abstract

Characteristics of a Mycobacterium intracellulare reference precipitation system for immunodiffusion and immunoelectrophoresis analyses are presented. The system was produced by the initiative of the International Working Group on Mycobacterial Taxonomy, and the purpose is to permit comparisons of precipitinogenic patterns of mycobacteria obtained in different laboratories. The reference material, consisting of an antigen preparation and a corresponding antiserum, is available for students of mycobacterial antigens in accordance with guidelines adopted by the Working Group.Immunodiffusion (ID) and immunoelectrophoresis (IE) are used by many investigators for taxonomic analyses and other research studies of mycobacteria (for a review, see reference 4). The knowledge concerning mycobacterial antigens is, however, small and scattered compared with that for many other genera. The need for mycobacterial reference systems that are internationally available for comparison of results from different laboratories has been recognized. Thus, in 1971, such a system was presented for Mycobacterium tuberculosis by the Mycobacterial Antigen Subcommittee of the U.S.-Japan Cooperative Medical Science Program (3). In 1980, Closs et al. (2) introduced a commercially available antiserum against Mycobacterium bovis BCG. These two systems were recently compared (18). The International Working Group on Mycobacterial Taxonomy has found it advantageous to establish reference systems for additional mycobacterial species. Accordingly, an antigen preparation and corresponding antiserum for a serological reference system representing Mycobacterium intracellulare were produced, and the present report describes this system. MATERIALS AND METHODSBacterial strains. The Boone strain of M . intracellulare (TMC 1403) isolated at the Battey State Hospital was used. The strain was obtained from the Trudeau Mycobacterial Culture Collection, currently housed at the American Type Culture Collection, Rockville, Md. In addition, 25 strains of Mycobacterium species and related genera were included for comparison (Table 1).Antigen preparation for serological analyses. M . intracellulare cells were grown on Watson Reid medium (17) as surface growth, Thirty flasks, each containing 1,000 ml of medium, were inoculated from one seed culture. The cultures were incubated at 37°C for 6 weeks, harvested by filtration, and washed three times in sterile distilled water. The washed bacilli were suspended in an equal volume of sterile distilled water and disrupted by sonication six times for 5 min each time in an ice bath, at 150 W, at an output frequency of 23 kHz. An ultrasonic disintegrator (Soniprep * Corresponding author 150; MSE Scientific Instruments, Manor Royal, Crawley , Sussex, England) was used. The extract was centrifuged at 50,000 X g for 60 min at 4"C, and the supernatant was filtered through a Millipore filter (pore size, 0.45 pm). The sterile 1,500 ml of antigenic material was stored at -20°C.Antigen preparations from 25 other strains were described ear...

Agents of newly recognized or infrequently encountered mycobacterial diseases

“…Furthermore, plasmids have also been implicated in the variability of catalase production by members of the complex (140) and may yet be found to influence other properties that are important in defining the overall phenotypic relationships within this group of organisms. Further information on the relationships within this complex may emerge from the comparative use of an IWGMT M. intracellulare reference antigen and antiserum that have been made available to interested investigators (151).…”

“…In 1967 the International Working Group on Mycobacterial Taxonomy (IWGMT) was organized and a series of cooperative studies was undertaken, using the recently developed principles of numerical taxonomy (172) to incorporate laboratory data from diverse sources into a unified taxonomic scheme. Over the next 20 years, the results of those cooperative efforts helped to consolidate the phenotypic taxonomy of mycobacteria (26,66,99,125,151,161,207,(215)(216)(217)(218)(219)(220). During that same period, and especially over the last decade, technologies emerged for the analysis of semantides, large information-bearing molecules such as nucleic acids and proteins (244), that permitted comparisons to be made between phenotypic classification and evolutionary relationships.…”

Agents of newly recognized or infrequently encountered mycobacterial diseases.

“…Furthermore, plasmids have also been implicated in the variability of catalase production by members of the complex (140) and may yet be found to influence other properties that are important in defining the overall phenotypic relationships within this group of organisms. Further information on the relationships within this complex may emerge from the comparative use of an IWGMT M. intracellulare reference antigen and antiserum that have been made available to interested investigators (151).…”

“…In 1967 the International Working Group on Mycobacterial Taxonomy (IWGMT) was organized and a series of cooperative studies was undertaken, using the recently developed principles of numerical taxonomy (172) to incorporate laboratory data from diverse sources into a unified taxonomic scheme. Over the next 20 years, the results of those cooperative efforts helped to consolidate the phenotypic taxonomy of mycobacteria (26,66,99,125,151,161,207,(215)(216)(217)(218)(219)(220). During that same period, and especially over the last decade, technologies emerged for the analysis of semantides, large information-bearing molecules such as nucleic acids and proteins (244), that permitted comparisons to be made between phenotypic classification and evolutionary relationships.…”