THE JOURNAL OF INFECTIOUS DISEASES. VOL. 135, NO.5. MAY 1977 © 1977 by the University of Chicago. All rights reserved.
Immunochemical Analyses of a Major Antigen of Mycobacterium szulgai Fr01l1 the Department of Medicine, Case Western Reserve University School of Medicine, and the Department of Medicine, University Hospitals, Cleveland, Ohio
Thomas M. Daniel and Richard W. DeMuth
Mycobacterium szulgai is a recently described human pathogen of apparently worldwide distribution but relatively uncommon occurrence [1, 2]. To date the organism has not been reported in a context that suggests human-to-human transmission and has not been isolated from environmental sources; its epidemiology is completely unknown. Our interest in this organism was aroused when an individual who resided in northeastern Ohio came under the care of one of us (T.~f.D.) for advanced pulmonary disease due to M. szulgai. In the report of Schaefer et al. [2], this patient was described as case 3. As noted in that report, she did not give a dermal reaction to purified protein derivative (PPD) but did respond to an unheated culture filtrate prepared from M. szulgai. Sensitized guinea pigs tested in preliminary experiments with the same filtrate of M. szulgai gave reactions that differed significantly in size from those of animals sensitized with filtrates of Mycobacterium tuberculosis. It seemed likely that significant antigenic dif-
ferences existed between M. szulgai and M. tuberculosis. For this reason studies aimed at partial purification of species-specific antigens from M. szulgai were planned. It was hoped that such materials might provide additional information pertinent to the antigenic constitution of M. szuZgai and other mycobacteria. Materials and Methods
Isolates of M. szulgai from the patient were grown on the totally synthetic, liquid medium of Proskauer and Beck for the production of unheated culture filtrate as described [3]. A total of 2,000 ml of liquid culture was grown. After successive filtration through Whatman no. I filter paper (Fisher Scientific Co., Pittsburgh, Pa.), 0.45-,um cellulose acetate, and 0.22-,um cellulose acetate, the culture was merthiolated in a concentration of 0.01 % and stored at 4 C in sterile bottles. The wet bacillary mass was washed twice in isotonic saline containing 1% phenol and then resuspended in 1% phenolized saline. Prior to use these cells were washed and resuspended in saline and quantitated by oven drying of measured aliquots in tared tubes. Similar preparations of culture filtrate and phenolized bacteria were prepared with M. tuberculosis strain H 37 Ra (Trudeau Institute, Saranac Lake, N.Y.). Prior to fractionation the filtrate was dialyzed against starting buffers. For comparison with reference materials, an aliquot of culture filtrate was dialyzed against distilled water, lyophilized to dryness, and reconstituted to 10 mg of dry solids/ml.
Received for publication July 26, 1976, and in revised form October 7,1976. This paper was presented in part at the annual meeting of the American Thoracic Society, New Orleans, Louisiana, May 17, 1976. This work was supported by research grants from the American Lung Association of Lake County, Ohio, and the Ohio Lung Association. Please address requests for reprints to Dr. Thomas M. Daniel, Department of Medicine, University Hospitals, Cleveland, Ohio 44106.
778
Downloaded from http://jid.oxfordjournals.org/ at University of Iowa Libraries/Serials Acquisitions on July 13, 2015
Unheated culture filtrate of iYlycobacterium swlgai and a homologous goat antiserum were prepared. Immunoelectrophoretic analysis demonstrated a dominant anodal antigen in the culture filtrate. By the use of diethylaminQethyl-cellulose chromatography, a fraction designated l\JSP, which was rich in this anodal antigen, was recovered. The major antigen of MSP was demonstrated to have partial identity with reference mycobacterial antigen 6, and evidence was obtained for separate shared and specific antigenic determinants. MSP was found to be a potent, delayed skintest antigen of considerable specificity when used in sensitized guinea pigs. Arthus reactions were also observed and were not specific.
779
Analyses of M. szulgai Antigen
g of glycine per liter with the addition of 0.1
Immunochemical Analyses of a Major Antigen of Mycobacterium szulgai Fr01l1 the Department of Medicine, Case Western Reserve University School of Medicine, and the Department of Medicine, University Hospitals, Cleveland, Ohio
Thomas M. Daniel and Richard W. DeMuth
Mycobacterium szulgai is a recently described human pathogen of apparently worldwide distribution but relatively uncommon occurrence [1, 2]. To date the organism has not been reported in a context that suggests human-to-human transmission and has not been isolated from environmental sources; its epidemiology is completely unknown. Our interest in this organism was aroused when an individual who resided in northeastern Ohio came under the care of one of us (T.~f.D.) for advanced pulmonary disease due to M. szulgai. In the report of Schaefer et al. [2], this patient was described as case 3. As noted in that report, she did not give a dermal reaction to purified protein derivative (PPD) but did respond to an unheated culture filtrate prepared from M. szulgai. Sensitized guinea pigs tested in preliminary experiments with the same filtrate of M. szulgai gave reactions that differed significantly in size from those of animals sensitized with filtrates of Mycobacterium tuberculosis. It seemed likely that significant antigenic dif-
ferences existed between M. szulgai and M. tuberculosis. For this reason studies aimed at partial purification of species-specific antigens from M. szulgai were planned. It was hoped that such materials might provide additional information pertinent to the antigenic constitution of M. szuZgai and other mycobacteria. Materials and Methods
Isolates of M. szulgai from the patient were grown on the totally synthetic, liquid medium of Proskauer and Beck for the production of unheated culture filtrate as described [3]. A total of 2,000 ml of liquid culture was grown. After successive filtration through Whatman no. I filter paper (Fisher Scientific Co., Pittsburgh, Pa.), 0.45-,um cellulose acetate, and 0.22-,um cellulose acetate, the culture was merthiolated in a concentration of 0.01 % and stored at 4 C in sterile bottles. The wet bacillary mass was washed twice in isotonic saline containing 1% phenol and then resuspended in 1% phenolized saline. Prior to use these cells were washed and resuspended in saline and quantitated by oven drying of measured aliquots in tared tubes. Similar preparations of culture filtrate and phenolized bacteria were prepared with M. tuberculosis strain H 37 Ra (Trudeau Institute, Saranac Lake, N.Y.). Prior to fractionation the filtrate was dialyzed against starting buffers. For comparison with reference materials, an aliquot of culture filtrate was dialyzed against distilled water, lyophilized to dryness, and reconstituted to 10 mg of dry solids/ml.
Received for publication July 26, 1976, and in revised form October 7,1976. This paper was presented in part at the annual meeting of the American Thoracic Society, New Orleans, Louisiana, May 17, 1976. This work was supported by research grants from the American Lung Association of Lake County, Ohio, and the Ohio Lung Association. Please address requests for reprints to Dr. Thomas M. Daniel, Department of Medicine, University Hospitals, Cleveland, Ohio 44106.
778
Downloaded from http://jid.oxfordjournals.org/ at University of Iowa Libraries/Serials Acquisitions on July 13, 2015
Unheated culture filtrate of iYlycobacterium swlgai and a homologous goat antiserum were prepared. Immunoelectrophoretic analysis demonstrated a dominant anodal antigen in the culture filtrate. By the use of diethylaminQethyl-cellulose chromatography, a fraction designated l\JSP, which was rich in this anodal antigen, was recovered. The major antigen of MSP was demonstrated to have partial identity with reference mycobacterial antigen 6, and evidence was obtained for separate shared and specific antigenic determinants. MSP was found to be a potent, delayed skintest antigen of considerable specificity when used in sensitized guinea pigs. Arthus reactions were also observed and were not specific.
779
Analyses of M. szulgai Antigen
g of glycine per liter with the addition of 0.1
