ABSTRACT IMMUNOLOGIC RESPONSES OF RABBITS TO MYCOBACTERIUM BOVIS; CHEMICAL, ELECTROPHORETIC AND ANTIGENIC ANALYSES OF BACILLARY EXTRACTS by Gary F. Dardas Antibody production was studied in experimental models designed to simulate (a) active tuberculosis, (b) closed tuberculosis with tuberculoimmunity and tuberculin hyper- sensitivity, (c) no tuberculosis with tuberculoimmunity and tuberculin sensitivity, and (d) no tuberculosis with tuber— culoimmunity and reduced tuberculin sensitivity. Passive hemagglutination and bacterial agglutination tests were used for antibody determinations. Rabbits infected with virulent fl, bgygg (strain 310) (a) produced negligible amounts of antibody. The antibody response of rabbits infected with attenuated.fl, boyig (strain BCG)(b) was comparable to that elicited by killed cells. Antibody elicited by injections of heat or acetone- killed cells was greater than was elicited by inoculation with betapropriolactone-killed cells (c) or when subsequent- ly extracted with methanol and acetone (d). A sequential production of mercaptoethanol-sensitive followed by mercaptoethanol-resistant antibody was detected Gary F. Dardas in all rabbits regardless of the antigen preparation. Mercaptoethanol-sensitive and resistant antibody was detected in sera from most rabbits for the duration of the experi- mental period, up to 25 weeks. Skin testing with PPD-S 14 weeks post-inoculation stimu- lated antibody production in approximately 50% of the rabbits tested. Both the absolute and relative amounts of the two types of antibody produced were changed as a result of skin testing. Extraction of betapropiolactone-killed cells of g, bovis with methanol and acetone considerably reduced their ability to sensitize rabbits to tuberculin (d). The antigenic and chemical composition of ultrasonic extracts of viable cells of g, bgyig varied with the length of incubation of the cells, prior to insonation, and the intensity of insonation used for extraction. Disc electro- phoresis effectively separated the greatest number (24) of constituents in ultrasonic extracts. Both carbohydrate and protein components were detected using disc electrophoresis. Antigens were detected in ultrasonic extracts by Ouchterlony immunodiffusion and immunoelectrophoresis. Ultrasonic ex- tracts of cells from two and one-half month-old cultures contained 20-22 separate antigen-antibody systems detected by immunoelectrophoresis. Chemical extraction of viable cells with Triton X-100, sodium desoxycholate, urea, guanidine and phOSphate buffer Gary F. Dardas containing ethyl ether yielded antigen preparations of varying complexity. From 4-15 components were detected in chemical bacillary extracts by disc electrophoresis. The number of antigens detected by Ouchterlony immunodiffusion was usually less than was detected by immunoelectrophoresis. IMMUNOLOGIC RESPONSES OF RABBITS TO MYCOBACTERIUM BOVIS: CHEMICAL. ELECTROPHORETIC AND ANTIGENIC ANALYSES OF BACILLARY EXTRACTS a. I}; Gary F: Dardas A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Microbiology and Public Health 1967 é L+ 3 tic-“H? 3-7vé3 ACKNOWLEDGEMENTS The author wishes to express his appreciation and thanks to Dr. V. H. Mallmann for her interest and guidance throughout this investigation. Dr. Mallmann‘s fine example of high ethical and academic standards will always be ad- mired and appreciated. Appreciation is also extended to Dr. W. L. Mallmann for his wise counsel on many aSpects of this study. To the author's brother, Dr. Terry Dardas, goes my special thanks for many fine suggestions and contributions to this research. A special expression of appreciation goes to the author's wife, Patricia, and family for their great understanding afifi inspiration without which this work would not have been possible. The author is also indebted to the many members of the tuberculosis project who have contributed so much directly and indirectly to this investigation. ii TABLE OF CONTENTS Page INTRODUCTION . . . . . . . . . . . . . . . . . . . . 1 HISTORICAL REVIEW. . . . . . . . . . . . . . . . . . 5 Immunogenic and Allergenic Constituents of Mycobacteria . . . . . . . . . . . . . . . 9 Antigens of Mycobacteria. . . . . . . . . . . . 16 Cell Disruption by Ultrasound . . . . . . . . . 26 MATERIALS AND METHODS. . . . . . . . . . . . . . . . 51 Mycobacterial Inoculums for Rabbits and Inocu- lation Protocol. . . . . . . . . . . . . . 51 Antibody Titrations . . . . . . . . . . . . . . 54 Statistical Analyses. . . . . . . . . . . . . . 56 Tuberculin Tests. . . . . . . . . . . . . . . . 57 Cultures for Extraction of Cellular Components. 57 Extraction of Cells with Ultrasound . . . . . . 38 Chemical Extraction of Mycobacterial Cells. . . 4O Acetone, Ethanol and Trichloracetic Acid Ex- traction of Ultrasonic Extract C . . . . . 42 Production of Ultrasonic Extract-Specific Antisera . . . . . . . . . . . . . . . . . 42 Zone Electrophoresis in Cellulose Acetate Mem- branes . . . . . . . . . . . . . . . . . . 43 Disc Electrophoresis. . . . . . . . . . . . . . 44 Immunoelectrophoresis . . . . . . . . . . . . . 47 Ouchterlong Immunodiffusion . . . . . . . . . . 49 Chromatography. . . . . . . . . . . . . . . . . 50 Chemical Analyses . . . . . . . . . . . . . . . 55 Dialysis. . . . . . . . . . . . . . . . . . . . 55 RESULTS. . . . . . . . . . . . . . . . . . . . . . . 56 Antibody ReSponses Elicited by Cells of Myco- bacterium bovis. . . . . . . . . . . . . . 56 Chemical Analyses of Bacillary Extracts Pre- pared by Ultrasound. . . . . . . . . . . . 78 Chromatography of Ultrasonic Extracts . . . . . 78 iii TABLE OF CONTENTS - Continued Immunodiffusion Analyses of Ultrasonic Extracts. . . . . . . . . . Electrophoretic Analyses of Ultrasonic Extracts. . . . . . . . . . Immunoelectrophoretic Analyses of Ultrasonic Extracts. . . . . . . . . . Immunodiffusion, Electrophoretic electrophoretic Analyses of Bacillary Extracts. . . . . Skin Testing with Ultrasonic and Bacillary Extracts. . . . . DISCUSSION. . . . . . . . . . . . . . SUMMARY . . . . . . . . . . . . . . . LITERATURE CITED. . . . . . . . L . . iv and Immuno- Chemical Chemical Page 102 112 122 128 140 154 174 177 LIST OF TABLES TABLE 1. 2. 10. Protocol for rabbit inoculations of live and killed Mycobacterium bovis. . . . . . . . . . . Ultrasonic extracts obtained from viable cells of Mycobacterium bovis. . . . . . . . . . . . . Stock and working solutions used for disc electrophoresis . . . . . . . . . . . . . . . . Mean hemagglutinin titers in sera from rabbits inoculated with viable or killed cells of Myco- bacterium bovis . . . . . . . . . . . . . . . . Mean bacterial agglutinin titers in sera from rabbits inoculated with viable or killed cells of Mycobacterium bovis. . . . . . . . . . . . . Titers of mercaptoethanol-sensitive and mercap- toethanol-resistant hemagglutinins in sera from rabbits inoculated with viable or killed cells of Mycobacterium bovis. . . . . . . . . . . . . Titers of mercaptoethanol-sensitive and mercap- toethanol-resistant bacterial agglutinins in sera from rabbits inoculated with viable or killed cells of Mycobacterium bovis . . . . . . Ratios of mercaptoethanol—sensitive/mercapto- ethanol-resistant hemagglutinins in sera from rabbits inoculated with viable or killed cells of Mycobacterium bovis. . . . . . . . . . . . . Ratios of mercaptoethanol-sensitive/mercapto- ethanol-resistant agglutinins in sera from rabbits unoculated with viable or killed cells of Mycobacterium bovis. . . . . . . . . . . . . Relative percentage of gamma-globulin in sera from rabbits inoculated with viable or killed cells of Mycobacterium bovis. . . . . . . . . . Page 55 38 45 67 68 69 7O 71 72 77 LIST OF TABLES - Continued TABLE 11. 12. 15. 14. 15. 16. 17. 18. 19. Amounts of protein, carbohydrate and nucleic acid in ultrasonic extracts obtained from viable cells of Mycobacterium bovis. . . . . . Distribution of protein, carbohydrate and nucleic acid in the major chromatographic fractions of ultrasonic extract A eluted from Sephadex G-25. . . . . . . . . . . . . . . . . Distribution of protein, carbohydrate and nucleic acid in the major chromatographic fractions of ultrasonic extract B eluted from Sephadex G-25. . . . . . . . . . . . . . . . . Distribution of protein, carbohydrate and nucleic acid in the major chromatographic fractions of ultrasonic extract C eluted from Sephadex G-25. . . . . . . . . . . . . . . . . Distribution of protein, carbohydrate and nucleic acid in the major chromatographic . fractions of ultrasonic extract D eluted from Sephadex G-25. . . . . . . . . . . . . . . . . Distribution of 280 mu—absorbing material in chromatographic fractions from BioGel media following rechromatography of Fraction IG_25 from ultrasonic extracts . . . . . . . .'. . . Distribution of 280 mu-absorbing material in fractions obtained by molecular exclusion chromatography of ultrasonic extracts obtain- ed from viable cells of Mycobacterium bovis. . Number of mycobacterial components detected in ultrasonic extracts by disc electrophoresis, Ouchterlony immunodiffusion and immunoelectro— phoresis . . . . . . . . . . . . . . . . . . . Number of mycobacterial components detected in chromatographic fractions of ultrasonic ex- tracts C and D by Ouchterlony immunodiffusion analysis . . . . . . . . . . . . . . . . . . . vi Page 79 85 86 87 88 95 100 104 110 LIST OF TABLES — Continued TABLE Page 20. Distribution of amido black-positive compon- ents in disc electrophorograms of ultrasonic extracts obtained from viable cells of Myco- bacterium bovis. . . . . . . . . . . . . . . . 119 21. Distribution of PAS-positive components in disc electrophorograms of ultrasonic extracts obtained from viable cells of Mycobacterium bovis. . . . . . . . . . . . . . . . . . . . . 121 22. Number of mycobacterial components detected in chemical bacillary extracts by disc electro- phoresis, Ouchterlony immunodiffusion and immunoelectrophoresis. . . . . . . . . . . . . 129 25. Distribution of amido black-positive compon- ents in disc electrophorograms of chemical bacillary extracts obtained from viable cells of Mycobacterium bovis . . . . . . . . . . . . 145 24. Distribution of PAS-positive components in disc electrophorograms of chemical bacillary extracts obtained from viable cells of Myco- bacterium bovis. . . . . . . . . . . . . . . . 146 vii LIST OF FIGURES FIGURE 1. 2. 10. 11. 12. 15. 14. Hemagglutinins produced by rabbits in Group II(_Table1).................. Bacterial agglutinins produced by rabbits in Group II (Table 1). . . . . . . . . . . . . . . Hemagglutinins produced by rabbits in Group III (Table1)................... Bacterial agglutinins produced by rabbits in Group III (Table 1) . . . . . . . . . . . . . . Hemagglutinins produced by rabbits in Group IV (Table1)................... Bacterial agglutinins produced by rabbits in Group IV (Table 1). . . . . . . . . . . . . . . Hemagglutinins produced by rabbits in Group V (Table 1) . . . . . . . . . . . . . . . . . . . Bacterial agglutinins produced by rabbits in Group V (Table 1) . . . . . . . . . . . . . . . Hemagglutinins produced by rabbits in Group VI (Tablel)................... Bacterial agglutinins produced by rabbits in Group VI (Table 1). . . . . . . . . . . . . . . Skin test reactions elicited in rabbits of Groups II, IV and V (Table 1) elicited by an intradermal injection of PPD 14 weeks post- inoculation of antigen. . . . . . . . . . . . . Representative densitometric recording of a cellulose acetate electrophorogram of normal rabbit serum. . . . . . . . . . . . . . . . . . Molecular exclusion chromatography of ultra— sonic extract A (Table 2) in Sephadex G-25. . . Molecular exclusion chromatography of ultra- sonic extract B (Table 2) in Sephadex G-25. . . viii Page 57 58 59 6O 61 62 65 64 65 66 75 76 80 81 LIST OF FIGURES - Continued FIGURE Page 15. 16. 17. 18. 19. 20. 21. 22. 25. 24. 25. Molecular exclusion chromatography of ultra- sonic extract C (Table 2) in Sephadex G-25. . . 82 Molecular exclusion chromatography of ultra- sonic extract D (Table 2) in Sephadex G—25. . . 85 Ultraviolet absorption spectra of ultrasonic extract B (USE-B) (Table 2) and chromatographic fraction I (IG_25) obtained from Sephadex 6-25. 92 Ultraviolet absorption Spectra of chroma- tographic fractions II (FII _ 5) and III . (FIIIG_ ) from ultrasonic gxfiract B (Table 2) obtaineasfrom Sephadex G-25 . . . . . . . . . . 95 Rechromatography of fraction I _ from ultra- sonic extract B (Table 2) in 3206815 P-100, P-150 and P-ZOO . . . . . . . . . . . . . . . . 94 Molecular exclusion chromatography of ultra- sonic extract B (Table 2) in BioGels P-1OO (A), P-150 (B), and 9-200 (c). . . . . . . . . . . . 97 Molecular exclusion chromatography of ultra- sonic extract C (Table 2) in BioGels P-1OO (A), P—iso (B), and P-200 (c). . . . . . . . . . . . 98 Molecular exclusion chromatography of ultra- sonic extract D (Table 2) in BioGels P—1OO (A), 9-150 (B), and 9-200 (c). . . . . . . . . . . . 99 Molecular exclusion chromatography of dialyz- able and nondialyzable fractions from ultra- sonic extract A (Table 2) in Sephadex G-25. . . 101 Ion exchange chromatography of ultrasonic extract B (Table 2) in DEAE-cellulose . . . . . 105 Schematic immunogram of ultrasonic extract A (USE-A) (Table 2) and chromatographic fractions I (I), II (II), and III (III) from Sephadex G-25. AS = Antiserum . . . . . . . . . . . . . 106 ix LIST OF FIGURES - Continued FIGURE 26. 27. 28. 29. 50. 51. 52. 55. 54. 55. 56. Schematic immunogram of ultrasonic extract B (USE-B) (Table 2) and chromatographic fractions I (I), II (II), and III (III) from Sephadex G-25. AS = Antiserum. . . . . . . . . . . . . . Schematic immunogram of ultrasonic extract C (USE-C) (Table 2) and chromatographic fractions I (I), II (II), and III (III) from Sehpadex G-25. AS = Antiserum. . . . . . . . . . . . . . Schematic immunogram of ultrasonic extract D (USE-D) (Table 2) and chromatographic fractions I (I), II (II), and III (III) from Sephadex G-25. AS = Antiserum. . . . . . . . . . . . . . Schematic immunogram of ultrasonic extract D (USE-D) (Table 2) and filtrate from two and one- half (CFg) and six-month-old (CPS) cultures of Mycobacterium bovis. AS ; Antiserum . . . . . . Comparative antigenic analyses of ultrasonic extract C (USE-C) (Table 2) and ethanol (EP) and acetone (AB) precipitates obtained from ultrasonic extract C (USE-C) . . . . . . . . . . Comparative antigenic analysis of ultrasonic extract C (USE-C) (Table 2) and a trichloro- acetic acid precipitate (TCA-P) obtained from ultrasonic extract C . . . . . . . . . . . . . . Schematic disc electrophorograms of ultrasonic extract A (Table 2). . . . . . . . . . . . . . . Schematic disc electrophorograms of ultrasonic extract B (Table 2). . . . . . . . . . . . . . . Schematic disc electrOphorograms of ultrasonic extract C (Table 2). . . . . . . . . . . . . . . Schematic disc electrophorograms of ultrasonic extract D (Table 2). . . . . . . . . . . . . . . Cellulose acetate electrophorograms of ultra- sonic extracts A (A), B (B), C (C), and D (D) (Table 2). . . . . . . . . . . . . . . . . . . . Page 107 108 109 111 115 114 115 116 117 118 125 LIST OF FIGURES - Continued FIGURE 57. 58. 59. 40. 41. 42. 45. 44. 45. 46. 47. 48. 49. Diagramatic immunoelectrophorogram of ultra- sonic extract A (Table 2). . . . . . . . . . . Diagramatic immunoelectrophorogram of ultra- sonic extract B (Table 2). . . . . . . . . . . Diagramatic immunoelectrophorogram of ultra- sonic extract C (Table 2). . . . . . . . . . . Diagramatic immunoelectrophorogram of ultra- sonic extract D (Table 2). . . . . . . . . . . Comparative antigenic analysis of Triton (TE) extract and ultrasonic extract B (USE-B) (Table 2) o o o o o o o o o o o o o o o o o o 0 Comparative antigenic analyses of ether ex- tract (EC) and ultrasonic extract B (USE-B) (Table 2). As = Antiserum . . . . . . . . . . Comparative antigenic anal ses of sodium desoxycholate extract (SDE and Ultrasonic extract B (USE-B) (Table 2). AS = Antiserum . Comparative antigenic analyses of urea extract (UE) and ultrasonic extract B (USE-B) (Table 2) AS = Antiserum . . . . . . . . . . . . . . . . Comparative antigenic analyses of guanidine extract (GE) and ultrasonic extract B (USE-B) (Table 2). AS = Antiserum . . . . . . . . . . Comparative antigenic analyses of urea (UE) and Triton extracts (TE) obtained from viable (V) and heat—killed (h) cells. AS = Antiserum Diagramatic immunoelectrophorogram of Triton extract of viable cells of Mycobacterium bovis Diagramatic immunoelectrophorogram of sodium desoxycholate extract of viable cells of Mycobacterium bovis . . . . . . . . . . . . . Diagramatic immunoelectrophorogram of urea ex- tract of viable cells of Mycobacterium bovis . xi Page 124 125 126 127 150 151 152 155 154 155 156 157 158 LIST OF FIGURES - Continued FIGURE 50. 51. 52. 55. 54. 55. 56. 57. 58. 59. 60. Diagramatic immunoelectrophorogram of guanidine extract of viable cells of Mycobacterium bovis. Diagramatic disc electrophorograms of Triton extract of viable cells of Mycobacterium bovis. Diagramatic disc electrophorograms of sodium desoxycholate extract of viable cells of Mycobacterium bovis . . . . . . . . . . . . . . Diagramatic disc electrophorograms of urea extract of viable cells of Mycobacterium bovis. Diagramatic disc electrophorograms of guanidine extract of viable cells of Mycobacterium bovis. Cellulose acetate electrophorograms of chemical bacillary extracts from viable cells of Myco- bacterium bovis. TE = Triton extract; SDE = Sodium desoxycholate extract; GE = Guanidine extract; UE = Urea extract. . . . . . . . . . . Comparative antigenic analyses of chemical bacillary extracts. EE = Ether extract; TE = Triton extract; UE = Urea extract; SDE e Sodium desoxycholate extract; AD = Antiserum . . . . . Comparative antigenic analyses of chemical bacillary extracts. EE = Ether extract; SDE = Sodium desoxycholate extract; GE = Guanidine extract; AS = Antiserum . . . . . . . . . . . . Comparative antigenic analyses of chemical bacillary extracts. UE = Urea extract; TE = Triton extract; SDE = Sodium desoxycholate ex- tract; AS = Antiserum . . . . . . . . . . . . . Comparative antigenic analyses of chemical bacillary extracts. TE = Triton extract; GE = Guanidine extract; EE = Ether extract; AS = Antiserum . . . . . . . . . . . . . . . . . . . Comparative antigenic analyses of Triton (TE) and desoxycholate extracts (SDE) and filtrate (CF) obtained from six-month-old cultures of Mycobacterium bovis. AS = Antiserum. . . . . . xii Page 159 141 142 145 144 147 148 149 150 151 152 INTRODUCTION Tuberculosis is the single major bacterial disease for which serologic procedures fail to yield reliable informa- tion of diagnostic or prognostic significance. Isolation and identification of the causative agent is troublesome and time consuming but remains the only reliable proof of active disease. The tuberculin test fails to differentiate between infection and disease but serves as a major tool in the detection of tuberculosis. Understanding of the role of atypical mycobacteria in infection and heterologous sensitization to tuberculin necessitates the use of more specific mycobacterial sensitins for skin testing (159,227,112). Very few investigations have been concerned with the fundamental properties of the antibody response elicited by mycobacterial antigens. Only recently have such studies been undertaken (40,42,156). A better understanding of the anti- body response to tuberculosis may eventually lead to the development of meaningful serological procedures useful in diagnosis. The antigenic composition of different mycobacteria is largely unknown deSpite much investigation. Mycobacterial preparations currently available for skin tests, including “purified protein derivatives" (PPD), are crude and anti- genically complex mixtures. A need exists for specific antigens and sensitins from the classical and atypical myco- bacteria which are of pathogenic and epidemiologic signifi- cance. This is a report of studies of antibody production and induction of hypersensitivity in rabbits to constituents of M. bovis. Passive hemagglutination and bacterial agglutina- tion were used to detect antibodies produced in rabbits ino- culation with virulent, attenuated, or killed M, bovis. Disc electrophoresis, Ouchterlony immunodiffusion and immuno- electrophoresis were used to study the chemical and antigenic composition of bacillary extracts of viable cells of M, bovis prepared by ultrasonic disruption or chemical treatment. HICTORICAL REVIEW The need for a reliable diagnostic test for tubercu- losis still exists. A prime objective of such a test would be to differentiate between infection and disease and be- tween active and closed cases of the disease if it existed. Despite much investigation, no such test or combination of tests have been found. The tuberculin test is the most widely used test for the detection of tuberculosis. This test also fails to distinguish between infection and disease, or between present and past infections. Furthermore, it does not indicate conclusively which species of mycobacteria has induced sensitivity. The tuberculin reaction has been reviewed as to the specific and nonspecific immunologic factors involved (6,157); the general mechanisms (122) and epidemiologic significance (54). It is an area of consider- able research activity today but fundamental knowledge is meager. Sensitization by many of the well-defined mycobacterial Species as well as the anonymous (unclassified, atypical) mycobacteria has been well documented. This imposes many complications on diagnosis and epidemiology (54,158,112,159, 227,226). The need exists for skin test preparations (sensitins) with which the causative agent of sensitization, with or without disease, can be determined more reliably. Antibodies with specificities for various antigenic constituents of mycobacteria may be present in sera from infected individuals. A number of serologic tests for the detection of antibody in serum from tuberculous individuals have been used. These tests include agglutination, passive hemagglutination, passive hemolysis, precipitation and complement fixation reactions and many modifications. Middlebrook and Dubos deve10ped a passive hemagglutination test with which antibodies specific for tuberculopolysaccharide can be detected (117). Many modifications and conflicting results on the reliability of the test have been reported (157,174,98,180,147,195,70,160.88). Treatment of erythro- cytes with tannic acid (19) decreases their adsorption of polysaccharides and increases the adsorbtion of protein. It has been used to detect tuberculoprotein-specific anti- bodies (20,195). Protein antigens can be coupled to formalin- ized erythrocytes with bis-diazotized benzidine (50) and has been used to detect tuberculoprotein-specific antibodies (28,29). Turcotte and co-workers used the bis-diagotized modification to examine serums from tuberculous individuals (205,204,64). Serum to be tested was fractionated by chroma- tography on DEAE-cellulose or treated with mercaptoethanol to destroy the activity of macroglobulin antibody, IgM. The results obtained suggested that only the presence of 7 8 antibody, IgG, was indicative of active disease. Tuberculophosphatides have been adsorbed to kaolin particles to detect antibodies specific for the phosphatide fractions (196,195,194,195). The specific agglutination of the tuberculophosphatide-kaolin particles are independent of specific reactions with tuberculoprotein or tuberculo- polysaccharide (196) . The kaolin-phosphatide test was of no value in detecting disease or differentiating causative agents in calves inoculated with different mycobacteria, and phos— phatides extracted from the different mycobacteria did not improve the test (155). Takahashi and his associates described the occurrence of antibodies of more specificities in patients with tubercu- losis, antiprotein, polysaccharide and phOSphatide (192). The sequence of production and the titers obtained of antiphOSphatide antibody most faithfully reflected the degree of development of the infection (192). Anti-protein and anti-polysaccharide antibody was produced for long periods without regard to the virulence of the infecting organism. There was no apparent relationship between the occurrence of antibodies and the presence of delayed type hypersensitiv- ity to mycobacterial constituents (192). A combination of an immediate-type reaction and pre- cipitins (agar double diffusion test) has been used for the diagnosis of active pulmonary tuberculosis. The antigens used for both of these tests were either crude extracts or chromatographic fractions of extracts of mechanically dis- rupted mycobacteria (71,72,75). A double diffusion test was developed (152,155,154,155), which clinical evaluation indicates is a diagnostic aid (2,119,67,200,108). Our knowledge of many fundamental properties of anti- bodies and the immune response has been greatly expanded dur- ing the past decade. The development of refined techniques for biochemical analyses has facilitated investigations of the physical and chemical properties of immunoglobulins (62). Stelos (184,185,186) detected two molecular sizes of antibody elicited in rabbits by injections of ovine or bovine erythrocytes. The different molecular species of antibody had differences in their serological activity. The production of at least two different molecular species of antibodies by rabbits was confirmed and extended (11,12,15,15). There was a sequential production of the two different antibodies. The high molecular weight antibody with a sedimentation con- stant of approximately 19 S (macroglobulin, gammal, IgM) was produced first and subsequently, a lower molecular weight antibody (gammag-globulin, IgG) with a sedimentation constant of approximately 7 S (gammag-IgG). Human serum macroglobu- lins were sensitive to reductive cleavage by mercaptoethanol (50) and the reduction was used to distinguish between the immunoglobulins. Reductive cleavage by mercaptoethanol and fractionation of serum proteins has aided in determining the temporal sequence of the kinds of immunoglobulins elicited by a variety of antigenijldifferent species of animals (25,205, 208.158.206.17.211,121). When viral antigens were injected into rabbits, the IgM was first detected four-five hours after a single intra- venous injection of virus; IgG was detected one-two days after antigen injection (189,190,191). The rate of formation, amount and persistence of antibody were antigen-dose depend— ent. Prolonged production of IgG and transient responses of only IgM were produced. Approximately 50 times more antigen was required to induce production of IgG than to induce production of IgM. Gamma M and gamma G antibody responses differed in four fundamental properties; (1) the amount of antigen required for induction and maintenance of antibody synthesis, (2) the kinetics of antibody synthesis, (5) specific anamnestic reSponse, and (4) in sensitivity to irradiation. There have been extensive studies on the effect of route of injection and dosage of antigen upon the gross properties of the antibody response (100,156,207,140,189,190, 191,210); variations in antibody response to soluble or particulate antigens (120,202,155); and variations in the electrophoretic mobility of the immunoglobulins involved (218,125,202,142). ' Results of recent investigations using sensitive methods for antibody detection have indicated that IgG and IgM may be synthesized simultaneously and not sequentially as had been previously observed (210,162,5,65). There is evidence that specific agglutination reactions may be more sensitive for the detection of IgM than for IgG (15,76,16,210) and that differences exist between the binding constants of IgG and IgM (125,75). The IgM is reportedly 750 times more efficient than IgG on a molar basis as hemolysins or hemag- glutinins (75); 60-180 times as hemolysins and hemagglutinins using an anti-azobenzenearsonate antibody system (125); 22 times as bacterial agglutinins, 120 times more potent in sensitizing bacteria for complement lysis and 500-1000 times more efficient as an opsonin (152). The production of IgM and IgG may be independent responses and, depending on the nature of the antigens, the amount of antigen and the ino- culation schedule of the antigen, the two antibody types may be synthesized simultaneously or sequentially (210). There have been few studies on the kind of antibodies elicited by the mycobacteria which cause natural or experi- mental disease. Chronicity and the inconsistency of anti- body production of relationship to diagnosis or prognosis is well-established. The mechanism of enhanced resistance to tuberculosis in vaccinated individuals inoculated with BDG (Bacillus of Calmette-Geurin; attenuated, viable M, ngis) has been and continues to be of.major academic and prac- tical importance. Theories have been proposed but to date none are universally accepted (52,144,22,172,146,51). The role of antibodies is controversial. Seibert (172) proposed a complex and changing balance in the host of tuberculopoly- saccharides, antibodies and lysozyme (171,172). Generally, viable cells are necessary to induce tuberculoimmunity. Delayed sensitivity is also induced. Its role is unknown. Skin testing has, however, been reputed to stimulate in- creased antibody production (111,155,174). The rabbit antibody responses to mycobacteria do not seem to differ significantly from the responses to other particulate antigens (40,41,42,45,156,45). Results from very recent studies with mycobacterial antigens indicate that the route of inoculation, antigen concentration and use of adjuvants influence the nature of the antibody response (40,45). Both IgM and IgG have elicited in rabbits by viable BCG. The detection of IgG was more indi- cative of active disease in rabbits than detection of IgM but was not consistent with active disease in humans (45). Parlett and Chu (156) detected antibody production earlier with antigens in cell extracts and culture filtrates than to viable cell suspensions. They suggested that the availabil- ity of antigen or the relative dosage received was respons- ible. The present difficulty experienced in interpreting the results of serological tests may be due to our lack of knowl- edge of temporal patterns of antibody production in infected tuberculous individuals (156). Immunogenic and Allergenic Constituents Q§_Mycobacteria The basis of tuberculoimmunity (enhanced resistance to tuberculosis) is unknown. The relationship between tubercu- lin hypersensitivity and antibody production in tuberculosis 10 is controversial. Mycobacterial components can be antigenic (stimulate antibody production), allergenic (induce hyper- sensitivity), and immunogenic, although these conditions do not necessarily coexist. Suspensions of particulate material from mycobacteria have been shown to induce delayed type hypersensitivity and induce enhanced resistance in experimental animals to viru- lent organisms. Ribi and co-workers studied cell wall and cytoplasmic fractions from mechanically disrupted cells of various species of mycobacteria (150,99,151). Cell wall constituents induced delayed type hypersensitivity and caused dermal lesions in normal rabbits (150,99). Protoplasmic fractions failed to cause dermal lesions or to induce de— layed hypersensitivity. A delayed hypersensitivity reaction was elicited by protoplasmic fractions when injected intra- dermally into rabbits which had previously been sensitized with intact cells or a cell wall fraction. A cell wall constituent other than "wax D" was responsi- ble for eliciting hypersensitivity (99). This factor con- sisted of a firmly bound lipid and a peptide composed of alanine, glutamic acid and diaminopamilic acid. The specifi- city of the delayed hypersensitivity reaction provoked in sensitive animals by intradermal injections of cell walls or protoplasm differed significantly. Cell wall preparations from different species of mycobacteria elicited reactions in animals sensitized with heterologous species. In contrast, 11 delayed reactions using protoplasmic fractions were greater in intensity with animals sensitized with the homologous species. Cell walls from a variety of mycobacterial species contained closely related constituents whereas protoplasmic fractions contained more species or strain-specific con- stituents. Mice immunized with a killed vaccine consisting of cells mechanically disrupted in mineral oil were as resistent to virulent organisms as mice which had been vaccinated with the Bacillus of Calmette-Geurin (ECG). The potency of this vaccine was correlated with the presence of cellwall frac- tions from the disrupted cells. Only oil disruption products from either ECG or M, tuberculosis (H57Ra) were immunogenic. Cell walls from several strains of atypical mycobacteria and ECG had a common factor which was correlated with the protective potency and virulence of the organism. Cell wall preparations from various mycobacteria have been shown to induce enhanced resistance to infection with other species and genera of bacteria (59,60,61). Myco- bacterial cell walls extracted with 20% urea yielded a re— sistance-enhancing factor which represented 15% of the dry weight of the cell wall (61). The extract was chemically complex and consisted of protein, carbohydrate, lipid and nucleic acid. It was not antigenic and when injected into experimental animals, induced resistence against a variety of viral and bacterial agents (60). 12 Enhanced resistance was induced in animals inoculated with either whole cells or chemical fractions from various mycobacteria (215). Vaccination of mice with live or in- activated acetone, methanol, NaOH-extracted BCG induced resistance to subsequent infections with virulent staphys lococci (51). Methanol extracts from live or killed BCG induced enhanced resistance in mice to subsequent infection with either Staphylococcus aureus or M, fortuitum. Moreover, if methanol extracts of BCG or the residue fraction from methanol extracts were injected simultaneously or shortly after injection of mice with Staphylococcus aureus or M, fortuitum, their mean survival time was shortened (161). Enhanced resistance was attributed to a polysaccharide present in the methanol extract and was described as being broadly specific rather than nonspecific (217). Weiss and co-workers (212,215) found that methanol-soluble fractions from BCG had little protective effect whereas vaccination with phenol killed cells extracted with chloroform, methyl- chlorobenzene or methanol provided enhanced resistance (215). A highly immunogenic particulate fraction was isolated from the cytoplasm of mechanically, chemically, or enzy- matically disrupted mycobacteria (225). Neither soluble cytoplasmic constituents nor the intracellular particulate fraction from disrupted organisms induced hypersensitivity in normal animals although both provoked delayed hypersensi- tivity reactions in animals which had been previously sensi- tized with cell walls. Ribosomes obtained from young cells 15 by extraction with sodium dodocyl sulfate were immunogenic and resembled the intracellular particulate fractions. The immunogenecity of this preparation was destroyed by treatment with RNAase. Crowle (51) reviewed the subject of immunizing constit- uents from mycobacteria. Of the many chemical fractions of tubercle bacilli tested for immunogenecity, the lipid components were thought to be most active. "Antigen methyleque" obtained from acetone washed, autoclaved, air- dried cells, increased reSistance in animals to virulent organisms. Bacillary extracts containing "antigen methyleque" are antigenic and contain tuberculophosphatide in relatively pure form although other lipid and nitrogenous components have been detected (51). Tuberculophosphatide is the single, chemically defined mycobacterial constituent which has been shown to be immunogenic. The wax fractions from tubercle bacilli do not appear to be immunogenic although wax B has not been thoroughly investigated (51). Water soluble cell- free extracts from acetone washed, trypsin digested M, EEQEEP culosis were studied for their immunogenicity (55,54,55,56). Immunization with the trypsin extract was as effective as with whole bacilli (55). The level of resistance was great- est when the homologous strain or Species was used to chal— lenge the immunity of the animals. Immunization of mice or guinea pigs with the trypsin extract did not induce hyper- sensitivity to tuberculin, did not elicit antibody formation 14 and did not precipitate with antiserum (56). Chemical analyses of the extract indicated the active component was carbohydrate and was normally present in the cell wall (57). A major disadvantage of vaccination with mycobacteria or products derived from mycobacteria is the possibility of inducing delayed type hypersensitivity which eliminates the use of skin tests for the detection of tuberculosis. Protein-lipid complexes may be responsible for both immuno- genicity and allergenicity (214). Components responsible for these two possibly related phenomenon have not been chemically separated (175). Extraction of mycobacteria with neutral solvents have been reported on the one hand to in- crease their immunogenicity, and on the other hand not to increase their immunogenicity (65). Progressive removal of the lipid components from mycobacteria was found to decrease both the immunogenicity and allergenicity of defatted organ- isms (65). Lipopolysaccharide fractions from paraffin oil extracts of killed cells did not induce a Significant level of resistance in mice to virulent organisms (154). Raffel (145) demonstrated that neither tuberculo-protein nor wax constituents alone were capable of eliciting hyper- sensitivity but that a combination of the two constituents was necessary. The protein-containing wax fractions would induce hypersensitivity to several materials including oval- bumin and picryl chloride. Tuberculopolysaccharides either alone or with other bacillary components were not immunogenic. 15 Hypersensitivity was induced in guinea pigs with tuberculo- protein but resistance was not enhanced. Raffel concluded that acquired resistance and delayed sensitivity could not be attributed to any chemically pure substance of the tubercle bacillus. Guinea pigs inoculated with crude protein-containing fractions from borate buffer extracts of mechanically dis- rupted cells did not have increased resistance although they developed hypersensitivity to tuberculin and produced anti- body specific for tuberculoprotein (51). Freund and Stone (66) isolated a component from M, Egbegf culosis which was responsible for the adjuvant effect and designated it wax D. White and Marshall (216) reported that either wax D or a cell wall fraction consisting of firmly bound lipids and a peptide containing glutamic acid, alanine and diaminopamilic acid was necessary for the development of hypersensitivity. A basic failure of much of the research on immuno~ genicity and allergenicity of fractions of mycobacteria is that these fractions have not been chemically defined and purified. In order for immunogenicity, antigenicity and allergenicity to be fully understood, more purified com- ponents from mycobacteria must be used. Procedures for evaluating immunogenicity must be standardized. 16 Antigens of Mycobacteria The chemical composition of mycobacteria has been re- viewed by a number of authors (168,5,185). Despite much investigation, the chemical and antigenic nature of myco— bacterial constituents are poorly understood. A character- istic of mycobacteria is the high percentage of lipid, up to 40% of the dry weight of the cell (7). Lipids may be re- sponsible for many of the biological and chemical prOpertieS of the mycobacteria including immunologic adjuvant activity, induction of antibody formation, induction of delayed hyper— sensitivity, acid-fastness, their hydrophobic nature and possibly virulence (7). Although the polysaccharide com- ponents of mycobacteria are diverse and chemical complex, there is little or no evidence that strain-Specific poly- saccharides exist (181). Crowle (51) noted that tubercle bacilli contain at least nine different proteins, at least two distinct polysaccharides, three different lipids and a variety of yet undetermined constituents. Cell walls from certain Species of Mycobacterium, Nocardia and Corynebacterium have been found to be similar in chemical composition suggesting that these three genera are closely related taxonomically (58,59). The principle components detected were galactose, arabanose, hexosamine, glucose, alanine, glutamic acid and diaminopamilic acid. Tuberculopolysaccharide antigens have been found within the cell wall (59). Meynell (116) suggested that the outer- most superficial layers consisted of polysaccharide while 17 those more deeply Situated in the cell envelope were protein. From defatted mechanically disrupted cells, three anti— genic components were isolated by salt fractionation from aqueous extracts and two were isolated by extraction of the cell debris with neutral salts (114,115). Three different antigenic proteins were obtained from frozen or vacuum dried mycobacteria by extraction with acetone and alkaline solu- tions of different pH (114). The components responsible for the specificity of mycobacteria resided in the protein con- stituents of the cells rather than in the carbohydrates. A protein fraction was obtained from live tubercle bacil— li by extraction with solid urea for several days at 57°C (87). The antigenic fraction was 95% homogenous as determined by electrophoresis, yielded positive complement fixation reactions with serums from tuberculous patients, and elicited hypersensitivity reactions in sensitized individuals. A poly- saccharide component was extracted from the moist, steam- killed tubercle bacilli by extraction with solid urea (182). The extract had a single electrophoretic component and reacted.ig_vitro with sera from tuberculous patients. A single antigenic protein fraction was extracted with urea which had an electrophoretic mobility Similar to the C protein isolated from culture filtrates (196). It was more potent than C protein in eliciting skin reactions in sensitized animals. The fraction was pyrogenic but not immunogenic. Injections of the urea extract in the normal guinea pigs in- duced delayed hypersensitivity to tuberculin. An interesting 18 property of the urea extract was the ability to inhibit the ig.yi££g migration of polymorphonuclear neutrophils from tuberculous guinea pigs (196). Analyses of urea extracts by immunodiffusion detected two or more precipitinogens (170). Serologic relationships among mycobacteria and related genera were studied using chemical fractions from intact or disintegrated cells (95,96,97). Three serologic groups of mycobacteria were distinguished with the chemical extracts from the disrupted cells; Group I--Mycobacte;ium bovis, M, fortuitum, M, avium; Group II--atypical mycobacteria, and Group III--saprophytic mycobacteria. Seventy-two chemi— cal fractions from several mycobacteria, actinomycetes and streptococci were compared with antisera (95). Antigens ob- tained from the cytoplasmic fractions of the mechanically disrupted cells of the different groups were related and cross reacted among various members. However, polysaccharide antigens from the cell walls of the different organisms possessed some degree of specificity. Among the myco- bacteria there were a large number of cross reactions among the polysaccharides from the cell walls. Two explanations for the lack of serologic Specificity among the mycobacteria were offered: (1) the specific components could not be separated from the nonSpecific or cross reacting materials, or (2) the specific components, if present, were too low in concentration to stimulate antibody production. 19 Attempts to isolate pure, undenatured specific antigens from bacillary extracts of tubercle bacilli are not new (77). Bacillary extracts were chosen for antigen preparations in preference to concentrated culture filtrate because of the various stages of degradation in the culture filtrate pro— teins. Therefore, more homogenous antigen preparations could be obtained from whole extracts. Mechanically disrupted mycobacteria were extracted with borate buffer and phosphate buffer containing ether (80,81). Both removed the same cell constituents but in different proportions. Phosphate buffer extracts contained nucleic acid and less protein and carbo- hydrate than the borate buffer extract. Extracts of young cells had more tuberculin activity than extracts of cells from old cultures. Antigens obtained from mycobacteria by aqueous and saline extractions of intact viable cells were more Specific (than those in O.T. (97,98,99). Mycobacterium tuberculosis and M, bovis were closely related antigenically. There was considerable cross reactivity among the different Species of mycobacteria. It was suggested that antigens were stratified in the cell with cross reacting polysaccharide antigens lo- cated in the outermost surface of the cell. Antigens with more strain Specificity were located deeper within the cell and were only removed by subsequent extractions. Serologic Specificity was attributed to the protein moiety of carbo- hydrate-protein complexes. The aqueous extracts contained precipitinogens. 20 Glenchur and his associates examined bacillary extracts to find a tuberculospecific antigen for serodiagnostic tests (71,72,75). Chemical and antigenic differences between bacil- lary extracts and PPD were found using chromatography on ion exchange resins and gel filtration. Bacillary extracts were more complex chemically than PPD and contained several addi- tional components. Stepwise elution of proteins from DEAE- cellulose with phosphate buffers yielded seven separate protein- containing fractions from bacillary extracts and only three fractions from PPD. Fractions from ion exchange chroma- tography were tested for their ability to elicit delayed hyper- sensitivity reactions in sensitized animals. A fraction from bacillary extracts was chosen for testing sera from tuber- culous humans. A total of seven precipitating antigen-antibody systems were detected in the bacillary extracts. Pepys and c0dworkers (158) found antigenic differences between bacillary extracts and culture filtrates of different mycobacteria. Antibody elicited in experimental animals by inoculation with products of the tubercle bacilli were specific for poly- saccharide but not protein. They suggested that the so-called chemical impurities in tuberculin may actually be responsible for potentiating the tuberculin reaction. Tissues used for the Schultz-Dale test were reciprocally desensitized with culture filtrate antigens or lipopolysaccharide extracts from homologous mycobacterial cells. Culture filtrates obtained from mycobacteria after dif- ferent growth periods and bacillary extracts prepared by 21 mechanical disruption of mycobacterial cells in a pressure cell were compared (25). The antigenic composition of bacillary extracts from a single species remained constant regardless of the age of the culture from which the cells were obtained. More variation in the antigenic content was observed with culture filtrates of different ages. Thus, bacillary extracts were again reportedly superior to culture filtrates for analyses of the antigen constituents of dif- ferent strains and Species of mycobacteria. Precipitinogens in chemical fractions from mechanically disrupted cells were examined chemically. A polysaccharide preparation contained two distinct precipitinogens, a lipid fraction contained only one. In a similar study, bacillary extracts prepared from meChanically disintegrated cells con- tained more than six distinct precipitinogens (78). Yamaguchi (219) isolated protein, carbohydrate and lipid fractions from both culture filtrates and bacillary extracts. Poly- saccharide antigens were precipitated in two discrete zones in immunodiffusion reactions. Lipid and protein antigens were difficult to detect. The protein fractions from bacil- lary extracts and the culture filtrates were different. Bacillary extracts from sonic disruption of tubercle bacilli with glass powder were fractionated with ion exchange chromatography on DEAE-cellulose (84,85,86,228). Five major antigen-containing peaks were obtained. Ion exchange chroma- tography separated protein components from the nucleic acid 22 and carbohydrate components of the extract. Three protein- containing fractions elicited skin reactions in sensitized animals equal to protein fractions from homologous culture filtrates. The tuberculin activity was associated with the tuberculOprotein which was retained even if the tuberculo- proteins were denatured. Few attempts have been made to obtain antigen-containing bacillary extracts from mycobacteria using ultrasonic dis- ruption. Serologically active antigens were obtained by this method which were reportedly not chemically or anti- genically altered by this extraction procedure (201,187,18). Soluble and particulate fractions from BCG contained 61.1% lipids, 21.2% reducing substances, 5% total nitrogen and 0.56% phosphorus (95,94). The principle chemical components were arabinose, galactose, hexosamine, alanine, glutamic acid and diaminopimilic acid. Cell walls induced delayed hypersensitivity. Cell wall and intracellular particulate fractions were most antigenic but many cross reactions occurred. Disc electrophoresis of ultrasonic extracts detected 10 or more discrete components of which four were considered to be of major taxonomic importance (48,49). Several disc bands were capable of provoking delayed type hypersensitivity reactions in sensitized animals without eliciting antibody production. The skin activity was associated with polysac- charide-protein complexes. Three disc components were 25 detected in extracts which were common to a number of repre- sentative strains of atypical mycobacteria. Tuberculin-active peptides have been isolated from mycobacteria by precipitation with picric acid (177,178). The peptide preparation did not induce delayed hypersensi- tivity but provoked delayed reactions in sensitized guinea pigs. Culture filtrates have been used as a major source of mycobacterial antigens. Seibert and co-workers developed methods for obtaining and standardizing a purified protein derivative (PPD) to be used as tuberculin (165,164,165,167). From these classical studies the international standard for PPD, PPD-S, was developed. Using an alcohol fractionation procedure, Seibert separated PPD into three protein contain- ing fractions, designated A, B, and C and two polysaccharide fractions, I and II. Analyses of these fractions proved that they were chemically complex (107,166,21,1). Culture filtrates were fractionated by dialysis and molecular exclusion chromatography (8). Thirteen distinct precipitating antigen antibody systems were detected in a nondialyzable fraction from culture filtrates of BCG (8). Both nondialyzable and dialyzable fractions had tuberculin activity in sensitized animals. The different components varied in their chemical composition, sedimentation constants and electrOphoretic mobility. Tuberculin activity was associated with precipitating antigens and carbohydrate was also found to play a role in the delayed type reaction (26,27). 24 Identical or closely related antigenic determinants were shared by molecules of different sizes. Yoneda and co-workers employed several different pro- cedures to fractionate culture filtrates from a virulent strain of M, tuberculosis (221,222,68,69). Salt fraction- ation with ammonium sulfate separated tuberculoproteins into three fractions: 0-50% (NH4)ZSO4-insoluble fraction; 50-50% (NH4)ZSO4-insoluble fraction and 50-80% (NH4)ZSO4-insoluble fraction. Starch block electrophoresis of these fractions yielded four apparently homogenous protein components. Ultracentrifugation and antigenic analyses by immunodiffusion indicated that these fractions were not homogenous. Ion ex— change chromatography on DEAE-cellulose improved the purity of several fractions and freed them from contaminating nucleic acids and carbohydrates. Two major protein fractions, desig- nated alpha and beta antigens, comprised approximately 70% of the total protein released by cells into the culture medium. Alpha and beta antigens were shown to be type speci— fic, heat labile, protein antigens representing a major portion of the protein complement of the cell. Both antigens could be removed from the cell surface without apparent damage to the cell. Ouchterlony immunodiffusion was used to determine the distribution of the alpha and beta antigens in culture filtrates from 120 strains in mycobacteria (69). Four serologic groups were established on the presence or absence of one or both of the antigens. 25 Twelve fractions were separated from culture filtrates of four strains of Mycobacterium tuberculosis by ion ex- change chromatography on DEAE-cellulose (91,92). Antigenic analyses by Ouchterlony immunodiffusion indicated that no fractions were antigenically pure. As many as eight discrete antigen-antibody systems was detected in a Single fraction. Twenty distinct antigen-antibody systems were detected in the fractions from culture filtrates from a single organism. Twenty percent of the antigens were reported to be strain specific and of potential diagnostic significance. A tuberculoprotein fraction from culture filtrates was separated into four components by column chromatography on DEAE—cellulose and into three fractions by paper electro- phoresis (148,149). These fractions differed significantly in their ability to provoke delayed type hypersensitivity reactions in sensitized animals. Ion exchange chromatog- raphy on carboxymethyl cellulose did not provide adequate separation of antigens from unheated culture filtrates (101). However, optimal conditions of pH and ionicity of the eluting buffers may not have been used. Various other electrophoretic and chromatographic procedures have been used for the separa- tion and purification of individual components of culture filtrates of M, tuberculosis, M, bovis, and other myco- bacteria (148). Antigens from culture filtrates have been used to study the antigenic relationships among different strains and 26 Species of mycobacteria (151,152,156,222,188,101,102,105, 104). The results of these studies have contributed greatly to our understanding of the antigens of mycobacteria and have supported the idea that different mycobacteria may con- tain specific antigens. Dardas (44) found that the number of antigenic components in culture filtrates from M, ngis was dependent upon the age of the culture from which the fil- trate was obtained. Disc electrophoresis is a relatively new method which has been used for the separation of mycobacterial components on the basis of electrophoretic mobility and molecular size (128). Affronti (1) found up to 19 components in various fractions. Roszman (156) separated 18 to 24 protein compon- ents and 5 to 8 polysaccharides in unheated culture filtrates from M, nglg, M, gyigg_and two Group III mycobacteria. The preparative disc electrophoretic technique was developed by which larger quantities of components from the disc bands could be obtained. A Single band after being eluted, and re- electrophoresed on a different concentration of gel in disc electrophoresis, contained more than one component. However, what had appeared to be the original band was still present in the greatest quantity and the reelectrophoresis may pro- vide a method of obtaining relatively pure fractions. Cell Disruption by Ultrasound The application of ultrasound in bacteriology, immunol- ogy and biochemistry was initiated by the observation that 27 cellular structures could be disrupted by this method of treatment (79). Grabar and Royer (74), showed that sensi- tivity to the destructive effects of ultrasound varied among different species of bacteria and among strains of the same Species. Mycobacterium tuberculosis was more resistant to ultrasound than any of the other pathogenic bacteria tested. The sensitivity of different species and strains of myco- bacteria to ultrasound differed greatly. Exposure to ultrasound of sufficient intensity and dura- tion to kill all the cells of a BCG culture only inactivated 15% of a strain of M, tuberculosis isolated from a clinical specimen (74). Varying degrees of morphologic disorgani- zation were observed in mycobacterial cells exposed to ultra- sound (209). The envelope of some cells was fractured and cytoplasmic material was released into the medium; in other cells the damage was at the end or center of the cell. The effects of ultrasound upon bacteria depends upon the intensity and duration of insonation as well as the density of the suspension of cells used (46). Increasing cell densities (number of cells/unit volume of medium) reduced the bacterio— cidal effect on various strains of mycobacteria (110). The effect of ultrasound on bacteria also depends to a certain extent upon their morphological features and physiological state (57). The mode of action by ultrasound on microorganisms is controversial. Cavitation undoubtedly is a principal agent 28 causing the bacteriocidal effect. Altering the conditions of the medium to suppress cavitation decreases the bacterio— cidal action. Although the medium absorbs a substantial amount of ultrasound energy as heat, temperature is thought to play a secondary role (58). Exposure of microorganisms to ultrasound causes damage to the cell wall and membranes. Since the disruptive forces of ultrasound are thought to be caused by cavitation, the distance between a cavitation bubble and its object are im- portant. The intensity of the shock wave varies inversely with the square of the distance and acts over a distance of several microns (55). The nature of the cell wall and mem- branes are important in this respect. If the cell envelope is hydrOphobic the formation of cavitation bubbles at the cell surfacedwater interface is favored and the organisms should be sensitive to the effects of ultrasound. If the cell surface is hydrOphilic the formation of cavitation bubbles at the cell surface-medium interface will be inhibited and the organisms should be more resistant to the effects of cavitation. Alterations in sensitivity to bacteriophage, sensitivity to antibiotics, and disinfectants or ultraviolet light and altered cell morphology are probably due to sur- face effects (58). Cavitation is not thought to occur within the cytoplasm of cells due to its high viscosity (57). However, a number of changes are induced within the cytoplasm of cells (56). 29 Alterations in the physicochemical state of the cytoplasm may lead to various degree of degradation or death. An important aspect of the application of ultrasound to microbiology is the possibility of extracting biologically or chemically active soluble or particulate constituents from cells. Biomacromolecules are extracted from viable cells by ultrasound with very little alteration in their physical or chemical state (57). Live and co-workers (106) obtained protective antigens in soluble extracts from ultra- sonically disrupted cells of Brucella s3. The sedimentable fraction from ultrasonically disrupted Brucella cells con- ferred protection to mice against subsequent infection with virulent organisms (115). Ultrasonic extracts from various microorganisms have been used as a source of antigens for serological tests and antigenic analyses (225,118,89). Nine distinct precipitat— ing antigen were detected by immunoelectrophoresis in ultrasonic extracts of Brucella abortus (22). Thirteen antigen were detected in ultrasonic extracts of intact cells of Brucella Eggs, Ten of the antigens were precipitated with antiserum elicited with intact cells (14). Three additional antigens were detected using spheroplast-specific antiserum. Four antigens were associated with the cell surface. Highly specific and very sensitive allergins were isolated from extracts of Brucella brucei and Pasteurella tularensis prepared by ultrasonic disruption (150). Cells of the yeast stage of Histoplasma capsulatum were disrupted by ultrasound 50 for the purpose of obtaining a specific antigen for sero- logic testing (141). The soluble extract contained constituents which were more antigenic than components found in the sedimentable fraction of the disrupted cells. Two antigens were detected in ultrasonic extracts from cells of Corynebacterium hofmanii (10). A study was made of the antigenic changes which accompanied sporulation in Bacillus cereus (9). Immunoelectrophoresis detected seven thermal- resistant and 8-10 thermal sensitive antigens in ultrasonic extracts from intact vegatative cells. Five thermal- resistant antigens present in Spore extracts were absent in extracts of the vegetative cells. Changes in the antigenic composition of different developmental stages of slime molds were detected using sonic extracts of cells at different ages (179). Ultrasonic extracts from cells of Mycoplasma pneumoniae were fractionated by gel filtration (176,145). One lipid fraction, three protein fractions and four poly- saccharide-containing fractions were obtained. Antigens detected by component-fixation tests were associated with the lipid components of the extract; precipitnogens were associated in polysaccharide-containing fractions (145). Ultrasonic disruption has been Shown to be an effective means of obtaining antigens from a variety of bacterial Species. Thus far, however, very few attempts have been made to prepare antigens and sensitins from mycobacteria. This technique has great promise for obtaining undenatured mycobacterial constituents. MATERIALS AND METHODS Mycobacterial Inoculums for Rabbits and Inoculation Protocol Mycobacterium bovis, 510-2, was isolated from a natural- ly infected, tuberculin-positive, gross lesion cow. It was identified by morphologic, cultural, biochemical and patho- genicity tests. Mycobacterigm bovis (510-2) was grown three weeks at 55%2in 20 ml tubes containing 1 ml of modified Proskauer Beck medium (224). The supernatant fluid was removed after centri- fugation (1500 x g, 15 min). The cells were washed with 0.15 M phosphate buffered saline (PBS) solution, pH 7.2. Portions of the cells were resuspended in PBS-solution to contain 0.1 mg wet weight/ml. The remainder of the cells were killed by the procedures indicated below. Cells were killed with betapropriolactone (BPL)1 (126). Washed cells were suspended in triple distilled water, 1.0 mg wet weight/ml. The pH was adjusted to 8.4 with 0.5 M NagHPO4. A 250 ml centrifuge bottle containing the suspension was placed in an ice bath and cold BPL added Slowly with constant agitation to a concentration of 0.4%. The mixture was incubated in a constant-shaker water bath at 570C for two hours. The pH was adjusted to 7.6 at 15 minute intervals 1Betaprone, Testagar and Co. Inc. 51 52 with 0.5 M NagHPO4. After incubation, the mixture was centrifuged and the supernatant fluid removed. The cells were washed four times with sterile distilled water and suspended in isotonic saline solution to a concentration of 1.5 mg wet weight/ml. Acetone-killed cells were prepared by adding acetone to washed, packed cells, one mg wet weight cells/ml acetone, and incubating at 4°C for seven days with daily changes of acetone. The cells were washed and resuspended in 0.15 M PBS-solution pH 7.2. Cells were killed by moist heat, 100°C for 50 minutes. The heat-killed cells were resuspended in 0.15 M PBS-solu- tion pH 7.2. A portion of the BPL-killed cells were extracted with methanol and acetone. Ten mg of BPL-killed cells were sus- pended in 10 ml of acetone and shaken for three hours at 570C. The mixture was centrifuged and the supernatant fluid removed. The cells were suspended in methanol overnight. This procedure was repeated three times, after which the cells were washed and resuspended in 0.15 M PBS-solution pH 7.2. The attenuated strain of M, ngig, BCG, was prepared as described for M, bovis, 510. Six groups of adult Dutch rabbits, three rabbits per group, were inoculated with preparations of mycobacterial cells according to the protocol in Table 1. 55 Table 1. Protocol for rabbit inoculations of live and killed Mycobacterium bovis.1 Rabbit Preparation Cells Group of M. bovis mg/ml Adjuvant2 Schedule I Live M, ngis 0.01 - Single3 II Live BCG 0.6 + Multiple4 III A-killed M, ppgigé 9.0 + Multiple IV BPL-killed M, ppyggs 9.0 + Multiple v BPL-killed Ma7 9.0 + Multiple extracted M, bovis VI Acetone killed M, bovis 1Virulent strain of Mycobacterium bovis 510-2 except Group III which received BCG, attenuated M, bovis. 2Adjuvant--incomplete Freunds adjuvant (Difco) 3Single-—a single subcutaneous injection. 4Multiple--six sub cutaneous injections given at separate sites on the first day. sMoist heat--100 C, 50 min. 6BPL--betapropriolactone (Betaprone, Testagar Co.) 7Ma—extracted--BPL-killed cells extracted with methanol and acetone. 54 Each rabbit in Group I received a Single subcutaneous injection of 0.01 mg wet weight of live M, ngig (510-2) without adjuvant. Each rabbit in Group II received multiple injections containing a total of 0.6 mg (wet weight) of attenuated, viable M, ngig (BCG) emulsified in adjuvant. On the same day, each rabbit in Groups III-VI received Six subcutaneous injections of killed cell preparations, 9.0 mg (wet weight) of cells in adjuvant. The inocula were prepared by emulsifying equal parts of killed cells in 0.15 M PBS-solution pH 7.2 with incomplete Freunds adjuvant. Antibody Titrations Blood was collected from the marginal ear vein from each rabbit in Groups I-VI prior to and weekly for at least eight weeks post-inoculation. Sera were decanted from the clotted blood, centrifuged, and decanted. Sera from rabbits in Group I were filtered1 before being centrifuged. Approximately three ml of each serum were dispensed into sterile brucella tubes, merthiolate was added (1:10,000), and the sera were stored at -70°C. Sera to be tested by passive hemagglutination were inactivated and adsorbed twice for 50 min each at 57°C with sheep red blood cells prior to storage. Polysaccharide-specific antibody was measured by a modi- fication of the Middlebrook-Dubos passive hemagglutination 1Seita pad supported on a Swinney filter. 55 test (HA) (111). Sheep blood was collected aseptically in an equal volume of modified Alsevers solution and stored at 4°C for not more than ten weeks. Erythrocytes were washed three times in PBS-solution before being used. One tenth ml of packed, washed erythrocytes were mixed with 6.0 ml of Old Tuberculin,l diluted 1:15 with buffered saline-solution pH 7.2 and incubated in a water bath at 57°C for two hours. The suSpenSion was centrifuged at 550 x g for four minutes and the supernatant fluid discarded. The sensitized cells were washed three times and resuspended in 0.5% in PBS- solution. The cell suSpenSion was used within 24 hours. Serial dilutions of serum were made in 0.15 M PBS-solu- tion pH 7.2 beginning with 1:10 dilution. Three drops of the 0.5% sensitized erythrocyte suspension were added to one ml of the serum dilutions in 12 x 75 mm tubes and shaken. The tubes were incubated in a water bath at 57°C for two hours, at room temperature for two hours and at 4°C over- night. Tubes were observed for settling patterns and visible clumping of erythrocytes when the tubes were shaken gently. The titer was recorded as the reciprocal of the highest dilution of serum which caused hemagglutination. Bacterial agglutinins were measured with BPL-killed M, ngig, A uniform suspension of the cells in PBS-solution pH 7.2 was prepared by dispensing cells in a tissue grinder, 1Agriculture Research Service, U. S. Dept. Agriculture. U. S. Vet Licence No. 107. 56 and centrifuging at approximately 250 x g for four minutes. The supernatant fluid was removed and diluted to an absorb- ency of 0.5 at 525 mu.1 Twenty-five hundredths ml of the antigen suspension was mixed with an equal volume of twofold serial dilutions of antiserum. The mixtures were incubated 12 hours at 57°C. The titer was recorded as the reciprocal of the highest dilution of serum which caused agglutination. Bacterial agglutinin and hemagglutinin titers were determined before and after treatment of the sera with 2-mercaptoethanol (ME). Equal volumes of 0.2 M ME and serum were mixed and incubated for 8-12 hours at room temperature. The difference between titers obtained before and after treatment of the serum with ME were recorded as the titer of ME-sensitive antibody. Statisticalggpalyses The results of antibody titrations were analyzed using the multiple range test developed by Duncan (55). The analyses were made with the number of the dilution tube of the titer as follows: 1:10 = 1 1:80 = 4 1:640 = 7 1:20 = 2 1:160 = 5 1:1280 = 8 1:40 = 5 1:520 = 6 1:5120 = 9 The significance of the variance between treatment means was determined at the 95% level. 1Bausch and Lamb "Spectronic 20" Spectrophotometer. 57 Tuberculin Tests Rabbits in Group I, IV and V were tested intradermally 14 week post-inoculation with 0.1 ml of second strength PPD-S.l- The diameters of palpable induration at the Site of injection were observed and recorded at 50 minutes, 5, 24, 48 and 72 hours post-inoculation. Cultures for Extraction of Cellular Components Mycobacterium ngig, 510-2, was grown in diphtheria toxin bottles on the surface of 500 ml or 1 liter of modified Proskauer Beck (PB) medium (224). The medium was prepared in 26 liter lots as follows: Reagent Amount Final Concentration Lasparagine 125.0 gm 0.5% KH2P04 125.0 gm 0.5% K2504 .12-5 gm 0.05% Glycerol 500.0 ml 0.15% Dissolve each ingredient sequentially and completely in the order listed in 17.5 1 of distilled H20. Adjust the pH to 7.0 with 40% of NaOH. Dispense 700 ml in each diphtheria bottle. Sterilize (121 C, 50 min). For each bottle of 700 ml, dissolve 1.5 gm.Mg titrate (final concentaation—-0.15%) in 500 ml of water, and sterilize (121 C, 50 min). Aseptically, add the con- tents of one flask to each of the bottles. The medium was incubated at room temperature several days before seeding with M, bovis. To seed, portions of con— fluent, surface growth was transferred with a small wire J'Parke Davis and Company. 58 screen on an inoculating wire. The protions were deposited on the surface of the medium in the horizontal bottles. The bottles were carefully transferred to the 55°C incubator and incubated for the periods of time indicated in the appro— priate sections. After incubation, the contents of the bottles were re- moved by suction into 250 ml centrifuge bottles, centrifuged at 1500 x g for 50 minutes, and the supernatant fluid and cells removed separately. Extraction of Cells with Ultrasound Four ultrasonic extracts (USE) were made differing in the age of the culture from which cells were derived, cell suspension density and duration of insonation. The different ultrasonic extracts are described in Table 2. Table 2. Ultrasonic extracts obtained from viable cells of Mycobacteriumlbovis. i t _ 4— Amount of Suspension Insonation Cells Age of Density Duration Designation (mg wet wt) Culture Percent W/V (min) USE-A 51 6 mo. 10 20 USE-B 45 6 mo. 7 55 USE-C 47 6 mo. 7 60 USE-D 25 2% mo . 7 20 59 A Biosonikl 20 kilocycle ultrasonic apparatus was used for cell disruption. The apparatus consisted of an air cooled 400 watt generator, transducer housing and a solid magnetostrictive type transducer. The transducer was attached to a stainless steel insonation chamber which could be detached and sterilized separately. The system was "tuned" before use by placing approximately 50 ml of water in the chamber and adjusting the electronic circuit to maxi- mum power output. Insonation was performed in a ventilated bacteriological hood fitted with an ultraviolet light. Sixty ml of a pre-cooled cell suspension in 0.002 M phosphate buffer (PB) pH 7.2 were placed in the insonation chamber. The gasketed top was made secure with four wing bolts and the entire chamber was covered with cheesecloth saturated with 5% phenol solution. The transducer housing, coolant reservoir and circulating pump were covered with plastic bags to prevent chance contamination of internal components. The chamber was cooled by an oscillating pump circulating ice water from an insulated reservoir through a cooling coil within the chamber. Preliminary experiments were performed to determine the maximum temperature reached during different intervals of insonation. The cell sus- pension was allowed to reach thermal equilibrium in the chamber before insonation was begun. lBronwill Scientific Division, Will Corporation, Rochester 1, N. Y. 40 Following insonation, the chamber was unopened for approximately twenty minutes. The cell suspension was transferred aseptically to 250 ml centrifuge bottles and centrifuged at 2°C for 50 minutes at 1,500 x g. The super- natant fluid was decanted and the cell debris washed once with approximately 70 ml of sterile 0.002 M PB at 40C. The original extract and the supernatant fluid from the cell washing were combined and stored in 250 ml centrifuge bottles at 4°C. The cell debris was washed again following the same procedure and saved for further extraction. Ultrasonic extracts were sterilized by filtration (size 6, S-1 Seitz filter Sheets and Millipore filter mem- branes, pore size 0.45 u). The extracts were concentrated approximately ten-fold by pervaporation and stored at -700C. Chemical Extraction of Mycobacterial Cells Cells were harvested from six—month-old cultures and washed three times with either 0.002 M tris-HCl buffer pH 8.6 or 0.002 M PB, pH 7.4. Ten percent suspensions of washed cells were placed in 250 ml plastic centrifuge bottles containing the appropriate concentration of extractant. The extractants were as follows: 1.5% sodium desoxycholate in the tris-HCl buffer; 1:520 dilution of Triton-X-100 in tris- HCl buffer; 4.5 M urea in PB; and 4.5 M quanidine-HCl in PB. The sodium desoxycholate (SDE) and triton extracts (TE) were prepared by continuous agitation of the cell suSpenSion in extractant for 18 hours at 2-60C. The urea (UE) and 41 guanidine-HCl extracts (GE) were prepared by continuous agi- tation of cell suspensions in extractant for 18 hours at room temperature. All extractions were performed in a bacteriological hood. Following extraction, the cell suspensions were centri— fuged at 1500 x g for 45 minutes at 4°C and the supernatant fluids were decanted. The cell debris were washed once with 50 ml portions of the suSpending buffer and supernatant fluids from these washings were combined with the original extracts. Extracts were sterilized by filtration through Seitz and Millipore filter pads and dialyzed for 72 hours at 4°C against daily changes of the suspending buffers. Extracts were concentrated approximately ten-fold by pervapor- ation and frozen. Cellular debris from the ultrasonic disruption of bacterial cells from six-month—old cultures were extracted with PB containing ethyl ether (80). Approximately 14 gm wet weight of cell debris contained in 150 ml of 0.002 M PB pH 7.2 were placed in a 250 ml plastic centrifuge bottle. Fifty ml of ethyl ether at —70°C were added and the mixture was shaken vigorously to achieve a stable emulsion. The mixture was frozen at -70°C and remained frozen for approxi- mately six hours. It was thawed and centrifuged at 4°C to separate the organic and aqueous phases. The aqueous phase was decanted and sterilized, dialyzed, concentrated and stored as described for the other extracts. 42 Acetone, Ethanol and Trichloracetic Acid Extractions of Ultrasonic Extract C Carbohydrates from USE-C were precipitated by slowly adding 40 ml of cold acetone with constant stirring to 20 ml of the extract (90). The mixture was allowed to stand overnight at 4°C and the precipitate was collected by centri- fugation at 4°C for 50 minutes at 2,000 x g. The precipitate was washed three times with a cold solution containing one part of distilled water and two parts of acetone. After washing, the precipitate was dissolved in five ml of 0.002 M PB pH 7.2 and dialyzed for 72 hours at 40C against several changes of the same buffer. Twenty ml of USE-C were treated with 60 ml of 95% ethanol by the method described for acetone. A trichloroacetic acid precipitate of USE—C was prepared following a procedure previously described (4). Ten ml of the extract was slowly mixed with an equal volume of 0.5 M trichloroacetic acid at 0°C. After standing for three hours at 4°C, a slight precipitate formed which was removed by centrifugation at 2,000 x g for 50 minutes at 40C. The supernatant fluid was decanted and dialyzed for 48 hours at 4°C against 0.002 M phosphate buffer pH 7.2. The extract was concentrated approximately two-fold by pervaporation and stored at -70°C. Production of Uigrasonic Extract-Specific Antisera One-half ml of a 10% solution of AlCla was slowly added with continuous stirring to each ten ml portion of USE-A. 45 The pH of the mixture was adjusted to 6.5 with 20% NaOH. The inoculum was stored at 40C and was used within 14 days of preparation. Four adult rabbits were each inoculated on days one and 14 with Six ml of the inoculum containing approximately six mg of protein. Each rabbit was inoculated intraperi- toneally on day 14 with one ml (4.0 mg protein) of USE-A with- out adjuvant. The rabbits were bled by carbiocentesis on days 49 and 51. Serums were decanted from the clotted blood and centrifuged for 50 min at 1000 x 9. Each antiserum was tested for precipitins by Ouchterlony gel diffusion and satisfactory antisera were pooled. Merthiolate was added (1:10,000) and three ml portions of serum stored in brucella tubes at -700C. Zone Electrophoresis in Cellulose Acetate Membranes Zone electrophoresis was performed in a Shandon migration chamber with a Vokam DC power supply. Oxoid 12 x 2 1/2 cm cellulose acetate membrane strips were used as the supporting medium. A 0.007 M barbital buffer pH 8.6 was used (129). The composition of this buffer was as follows: Ingredient Quantity Sodium diethylbarbiturate 5.0 gm Sodium acetate (anhydrous 5.25 gm Hydrochloric acid (0.1N) 54.20 ml Calcium lactate* 0.58 gm Distilled water to 1,000.00 ml * Omitted for electrophoresis of bacillary extracts. 44 Five microliter samples were dispensed from a lambda pipette onto a buffer impregnated cellulose acetate mem- brane strip. Sample application was facilitated by placing a ruler across the cathode reservoir. The sample was applied as an even line using the ruler edge as a guide. Serum samples were not concentrated for electrophoresis. Bacillary extracts were concentrated 20—50 fold. A constant current of one ma per strip was applied for 1 1/2-2 hours at 4°C. Following electrophoresis, the protein was stained with 0.2% Ponceau S in 5.0% aqueous trichloracetic acid. Serum protein distribution was measured by a double beam recording and integrating microdensitometer1 and the serum gamma-globulin concentrations computed. Discfiglectrophoresis Disc electrophoresis was performed using the procedures and apparatus described by Ornstein and Davis (127), and later modified by Davis (47). The composition of stock and working solution are given in Table 5. Samples to be electrophoresed were applied to the top of the Spacer gel by displacement. No sample gel was used. Bacillary extracts were concentrated approximately 20-50 fold to contain approximately 280 micrograms of protein in 0.1-0.2 ml. Electrophoresis was performed at room temperature using a Vokkam DC power supply. A constant current of three ma 1N and L—-Joyce Chromoscan. 45 Table 5. Stock and working solutions used for disc electro- phoresis. STOCK SOLUTIONS Reagent A Reagent E IN HCl 48 ml IN HCl approximately 48.0 ml1 TRIS 86.6 gm TRIS 5.98 gm TEMED 0.25 ml TEMED 0.46 ml DHOH to 100.0 ml DHOH to 100.0 ml pH 8.9 pH 6.7 Reagent C Reagent D Acrylamide 28.0 gm Acrylamide 10.0 gm BIS 0.755 gm BIS 2.5 gm DHOH to 100.0 ml DHOH to 100.0 ml Reagent E Reagent F Riboflavin 4.0 mg Sucrose 40.0 gm DHOH to 100.0 mg DHOH to 100.0 ml 1pH adjusted by titrating with IN HCl TRIS--Tris (hydroxymethyl)aminomethane TEMED-—N,N,N1,N1-tetramethylethylenediamine BIS--N,N'-methylenebis acrylamide DHOH-~distilled water Small pore Solutiongfii 1 part A 2 parts C 1 part DHOH pH 8.9 (8.8—9.0) WORKING SOLUTIONS Stock buffer Small pore Large pore solution for Solution #2 solution electrode reservoir Ammonium 1 part B TRIS 6.0 gm persulfate 2 parts D Glycine 28.8 gm 0.14 gm 1 part E DHOH to 1 liter 4 parts F pH 8.5 pH 6.7 (6.6-6.8) 46 per tube was applied for a length of time sufficient to allow the migration of bromphenol blue to within 5 mm from the anodic end of the gel column. Gel columns were stained for protein by immersion in a 0.5% solution of Amido Swarz 10 B in 5% acetic acid for 50 minutes at room temperature. The unbound stain was removed by electrOphoreSis in 5% acetic acid. Polysaccharides and glycoproteins were stained by the periodic acid-Schiff (PAS) procedure described by Canalco (24). Following electrophore— sis, the gel columns were immersed in 7.5% acetic acid for one hour at room temperature. The gel columns were removed and placed in a 0.5% solution of periodic acid and allowed to stand for one hour at 4°C. Excess periodic acid was removed electrophoretically for one hour in 7.5% acetic acid. Following treatment with periodic acid, the gel columns were immersed and stored in Schiff reagent at 4°C. Schiff reagent was prepared by the method described by Crowle (52). The stained disc columns were observed under indirect fluorescent light and diagrammatic representation of line patterns were drawn as composited from several columns run Simultaneously. Rf valves were compiled as the ratio of the migration distance of individual lines to the most anodic component detected. 47 Immunoelectrophoresis Immunoelectrophoresis was performed using a modifica- tion of the Hirschfeld procedure (85). Semi-purified agar was prepared by washing solidified one inch square cubes of 2% Difco agar in distilled water for several months. A bar- bital buffer system was employed for immunoelectrophoresis: Electrode vessel Constituents buffer Agar buffer Diethylbarbituric acid 1.58 gm 1.66 gm Sodium barbital 8.76 gm 10.51 gm Distilled water to 1,000.00 ml 1,000.00 ml A solution of buffered agar was prepared by mixing two parts of agar buffer with one part of distilled water and heating to approximately 70°C. Three parts of melted two percent agar were then added to the heated buffer solution and two and one-half ml of this mixture containing merthiolate (1:10,000) were layered on each 1 x 5 in thoroughly cleaned microscope slide. The agar slides were incubated three-five hours in a humidified chamber at room temperature before use. An LKB1 gel punch was used to cut two, 2 1/2 mm diameter sample basins in the agar approximately 44 mm from the anodic end of the slide and equidistant between the edges of the slide and a centrally located 1 1/2 mm wide antiserum trough. A Shandon migration chamber was adapted to accommodate slides by inserting a 6 x 8 1/2 inch plexiglass casette across 1LKB Instruments, Inc., Rockville, Maryland 48 the chamber bridge. Electrical connections were established with filter paper wicks impregnated with electrode vessel buffer. Two rows of 4-8 slides each were positioned on the casette connected by a filter paper wick. Slides on the anode Side of the casetts served as "blanks." Antigens were added to the sample basins using 26 gauge needles on one—half ml syringes. Bacillary extracts were concentrated 20-fold or greater for immunoelectrophoresis. Serum samples were used without concentration. A constant current of 1.25 ma per Slide was applied for 90 minutes at 4°C using a Volkam DC power supply. Following electrOphoresis, the antiserum was added. Slides were incu- bated in a humidified chamber at 28 C for 72 hours with one replenishment of a 1:5 dilution of antiserum, 12 hours after the start of incubation. Following incubation, the slides were washed in 0.015 M PB-solution pH 7.2 for 72 hours and in distilled water for 12 hours. The slides were then dried and the protein was stained with triple stain (52). Ingredient Amount Thiazine Red R 0.1 gm Amidoswarz 10 B 0.1 gm Light Green SF 0.1 gm Acetic acid 2.0 gm Mercuric chloride 0.1 gm Distilled water to 100.0 ml 49 Ouchterlony Immunodiffusion Ten ml of a melted, buffered agar solution were layered on thoroughly cleaned 5 1/4 x 4 inch glass microscope slides. The composition of the buffered agar solution is given below: Ingredient Amount Difco Agar 0.5 gm 0.15 M KH2P04 7.5 ml 0.15 M NagHPO4 17.5 ml 0.15 M NaCl 25.0 ml Merthiolate 0.02 % PH 7.2 After solidification of the agar, the slides were "aged" for three to five hours in a humidified chamber. Six mm sample basins were made in the agar using a template placed beneath the slide as a guide. Several patterns were employed using inter-basin diffusion distances of six-seven mm. Reactants were added to the sample basins using dispos- able pipettes and the slides were incubated at 280C in humidi- fied, six in diameter plastic Petri dishes. Preliminary experiments were performed to determine optimum antigen and antiserum concentrations for immunodiffusion. The protein concentration of bacillary extracts and their fractions were adjusted to 1.8-2.0 mg/ml for immunodiffusion analyses. Plates were incubated for four days with daily readditions of antigen and antiserum diluted 1:5 with sterile saline solu— tion. Immunoprecipitate patterns were observed and recorded 50 daily. When incubation was completed, the slides were im- mersed for three days in daily changes of 0.15 M PBS solution, pH 7.2. They were rinsed in distilled water, dried to a thin agar film by evaporation through Whatman filter paper and stained with Crowle's triple stain (52). Immunoprecipitates were observed under indirect fluorescent light against a black background. Chromatography Molecular exclusion chromatography was performed using superfine dextran beads (Sephadex)l and acrylamide beads (BioGel).2 Dry beads were swollen in distilled water for varying lengths of time depending upon the degree of cross linkage of the beads. Unjacketed Sephadex laboratory columns3 1.5 x 50 cm and 2.5 x 45 cm were coated with a 1% solution of dimethyl- dichlorosilane prior to packing. The coating solution was heated to approximately 60°C and poured into a clean, dry column and allowed to stand for several minutes. The column was emptied and dried in a hot air oven. This procedure was repeated. Swollen beads were gradually added to a column bed height of approximately 52 and 57 cm in the two column sizes, respectively. The gel beds were washed under low hydrostatic pressure with ten or more void volumes of eluent. 1Pharmacia Fine Chemicals, Inc., Piscataway, N. J. 2Calbiochem, Los Angeles, California 3Pharmacia Fine Chemicals, Inc., Piscataway, N. J. 51 The gel bed was stabilized and 0.2% Blue Dextran 20001 was used to determine the void volume of each column and the homogeneity of packing. The volume of material to be chromatographed varied with the size of the column and the protein concentration of the material. Samples were carefully added to the top of the gel and allowed to "soak" into the gel bed. The sides of the column and the sample pad were washed with several two-four ml portions of eluent before the eluent reservoir was connected to the column for continuous operation. Chromatography was performed at room temperature with flow rates adjusted to 15-18 ml per hour. Three-five ml portions of eluate were collected on an automatic, rotating drum fraction collector fitted with a time drop unit.2 The eluate was analyzed for adsorption at 280 or 260 mu by 3 The corresponding elution diagram a ultraviolet analyzer. was automatically recorded with a recording unit integrated with the analyzer. Tubes containing fractions corresponding to discrete areas of the effluent diagram were identified by an event marker attachment on the recorder. Tubes containing fractions registered under the same peak in the effluent diagram were pooled, concentrated by lyophilization and re- dissolved to the original sample volume. Rf values were 1Pharmacia Fine Chemicals, Inc., Piscataway, N. J. 2Vanguard. 3Ibid. 52 determined for individual fractions by computing the ratio of the void volume (V0) to the eluant volume of the re- spective fraction (Ve). Ion exchange chromatography was performed using selected Type 20 DEAE cellulose:L capacity 0.62 meg/gm as the adsorbent. vThe adsorbent was prepared for use following procedures described by Peterson and Sober (159). Dry powder was allowm ed to sink into 1 N NaOH and become thoroughly saturated. After mixing, the suspension was filtered through a Buchner funnel using Whatman No. 1 filter paper. The adsorbent was washed with 1 N NaOH until the filtrate was clear. It was suspended in a small volume of 1 N NaOH, acidified with 1 N HCl, immediately washed with distilled water, resuSpended in 1 N NaOH, and washed with distilled water. The adsorbent was suspended in starting buffer and "fines" were removed by decanting the supernatant fluid after the adsorbent had settled. The pH of the adsorbent was adjusted to that of the starting buffer and washed thoroughly before the columns were packed. A suspension of adsorbent in starting buffer was slowly poured into dry, "coated" 1.5 x 50 cm Sephadex laboratory columns to a bed height of approximately 25 cm. The adsorbent bed was washed for several days under low hydrostatic pres- .sure with the starting buffer. Ultrasonic extracts were concentrated approximately 20- fold for ion exchange chromatography. Seven ml of ultrasonic 1Carl Schleicher and Schuell Co., Keene, N. H. 55 extract B were dialyzed against the starting buffer for 24 hours at 4°C before application to a column. A continuous concave gradient of decreasing pH and increasing molarity was used to elute proteins from the adsorbent. The gradient was prepared using a modification of the cone sphere method (159). A 500 ml round-bottomed flask containing 450 ml of the starting buffer was connected by thin rubber tubing to a 250 ml Erlenmeyer flask containing 225 ml of the limit buffer. The composition of the buffers were as follows: Buffer Composition Starting 0.005 M TRIS-phOSphate pH 8.6 Limit 0.5 M TRIS-phosphate in 1.7 N NaCl pH 5.0 The flask of starting buffer contained a magnetic stirrer. The sample was applied to the top of the adsorbent bed, washed into the column with small portions of starting buffer and buffer reservoirs were connected to the column for continuous elution. Five ml portions of eluant were collected, analyzed and recorded. Fractions were pooled, dialyzed for 24 hours at 4°C against 0.015 M PBS solution pH 7.2 and concentrated to the original sample volume. Chemical Analyses Protein determinations were made using the Lowry modi- fication of the Folin-Ciocalteu method (109). The reagents were: 4.9% sodium potassium tartrate, 4.0% sodium carbonate and 2.0% copper sulfate 5H20. Reagent A was prepared by mixing one ml of the copper sulfate solution, one ml of the 54 tartrate solution and 100 ml of the carbonate solution. Reagent B was prepared by the addition of one part of Folin reagent to two parts of distilled water. One ml of the protein-containing sample was added to 10 ml of Reagent A with thorough mixing and the mixture incubated 45 minutes at room temperature. After incubation, one ml of Reagent B was added, mixed immediately and incubated 15 minutes at room temperature. Absorbancy was determined in a one ml cuvette at 660 mu using a Beckman DU Spectrophotometer. A standard protein curve was made with bovine serum albumin. Carbohydrate determinations were made using the thymol sulfuric acid reaction described by Shetlar (175). The following reagents were used. Thymol reagent: 10 gms dissolved in 100 ml of abso- lute ethanol. Sulfuric acid reagent: 77% by volume, Specific gravity 1.84. Add 770 ml of sulfuric acid at 150C to 250 ml dis- tilled water. Seven ml of the sulfuric acid reagent were dispensed into fifteen ml st0ppered glass centrifuge tubes and cooled to 4°C. One ml of the test solution containing between five and 100 micrograms of carbohydrate/ml was layered on the chilled sulfuric acid and incubated for 50 minutes at 40C. The tubes were stoppered and shaken after adding 0.1 ml of the Thymol reagent and 0.9 ml of distilled water, and 55 placed in a boiling water bath for 20 minutes. They were cooled at 4°C for five minutes and at room temperature 25 minutes. Absorbancy was determined in a one ml cuvette at 500 mu using a Beckman DU Spectrophotometer. Nucleic acid concentrations were estimated from absorb- ancy measurements at 280 and 260 mu using a one cm light path in a DU spectrophotometer. The nucleic acid concen- tration was read from a standard nomograph based on the ratios of absorbancy at the two wave lengths. Ultraviolet absorption spectra of ultrasonic extracts and fractions were obtained by absorbancy measurements at wave lengths of 200-520 mu using a one cm light path in a Beckman DB Spectro- photometer. Dialysis Fifty ml portions of ultrasonic extract A were dis- pensed into wetted dialysis tubing (Visking Co.) and di- alyzed Six days against 0.01 M PBS solution pH 7.2. The dialysate and retentate were collected and lyophilized. RESULTS [Aptibody Responses Elicited by Cells of Mycobacterium bovis Antibody titers were low and inconsistent in sera from rabbits inoculated subcutaneously with 0.01 mg wet weight of virulent M, nggg cells, strain 510, without adjuvant (Group I). Tuberculous lesions were present in the rabbits at necropsy 8-12 weeks post-inoculation. Antibodies produced by rabbits in Groups II-III during the first two weeks post-inoculation were exclusively ME- sensitive (Figs. 1-10, Tables 4-7). Thereafter, increasing amounts of ME-resistant antibody were detected which decreased the ratios of ME-Sensitive/Me-resistant antibody (Tables 8 and 9). Mercaptoethanol-resistant bacterial agglutinins were detected in sera from most rabbits by three weeks post-inocu- lation; ME-resistant hemagglutinins were detected by four weeks post-inoculation. Most of the rabbits produced ME- sensitive hemagglutinins and bacterial agglutinins until the sixth week post-inoculation. 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It Tl..- m limes.-. m .. m --.”..l m m z . ‘f'i >vca_bca . . scabm_mocn_0cm;u.opdooto: 1,); I >ton_u:m . o>_u_mcom:_o:a£bcoumn0co: .. Wu eiuom tobwouuc: tx.l in N0 720 H3833 A I |.L:l ' .l i \""r‘; lf“.\ U‘Q-“Ia 8‘ .AH oHBSHV > esoto c_ mu_bbmu >2 poospoLd mc_c_u:_mmo _o_t0uomm .m .s: m¥um3 MN mH w h a m w m N H z p l... p L s .II- a 1 p a p o T\ /\.. 1. O A:4 /////l// I. ; /. < t .\ \ H H x»? t. A _ UCQ . \w) ....o bccbmAmouu_o:t:booummoro: .14-): «V\\\\. <\ N >coe_ucm .z. i\\\\\ o>_b_mcom:_0ccruoobcoocoz Ihcfil /<\_ my m A. carom coumouuc: lfiull \\\\\\\\ no . t . l, t -.w t. .l I. J |.\:... ’.Il.i'u (x / . ..\ / I _/ C\ .m J. I u mu... -. . .Irl l \ ,L c .0 ,N .... .LIIIS H5" 1‘: 05‘! (a... UK)” 6:1 \l‘YK" >302mucm H: Hm_moc;_o:d;u ounces 6 azuom caboocbcs .A_ o_aohv _> dsoco c_ mu_bbmc >b couscocd mc_c_H:_mmmEo: 3353 e m s . . .P N :_.‘D L!) .‘N H ..l Q .0 .m _u 2 ”UH Ca? 3233 L. '\ 33310110 . .1 1“! Calvin“ \‘Haq 6 6 >uoa_S:o Lcmbm_ >vob_Hco o>_u_ m0ci_OCw£uooquoLoZ mcon:_o:o;eoobxncuoz Esncm mouccruca Q1... \. .A. o_bmhv_> ozoLo c. mu_e but >2 couscoud mc_:_S3Hm mm _o_toHocm . 35m: e H ..1 KN \\ C\ o. q d (r;- l NOliGTIGY c.) 52238 ‘ n-3— ; MQIIQ‘ \ 67 m0 0.0 m__co po___x to c_£c_> cum: voucHzcoc_ mumbmmu Eocm m0 >.> 0.0 m.m m0 ..K" ._ m_cmh c_ czocm mm boom—sooC_ .QSOLm cod mu_bbmt mouse 5 Quota N .>_o>_coodmoc 0. ob ..ouo N ._ .o wrongsc co_b3__c Eatom mm cocoo om_m OH ..000 cm .o_ .o muob_h_ >.m m0 m.m o.> mi m; _> > .> o .> o .> m0 o.m o > 0.0 >.m o.> 0.0 0.0 070. 2 m6 m6 o.m m0 0.0 o..— I. >.w m.w >.w >.w m.> o.m __ 9 ..-. .- “...-...- .-.-....-. .- ..w- ..-l -..-3.- -_. .. .5... 3-5.--. .c._...e....H.o. .-.. E .. . ...-he. won-...; .- - ...-5.- -l- -l-.-.l--l. 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N .>.o>_0uommo. 0. op...o.m N ._ .0 .0053: co.u:._v 83.00 mm @0000 0N.m Cu ..U0m 0m .0. .0 m.ou_h_ - - . - 0.0 >.0 m.> 0.. 0.. 0.. m.0 0.N 0.0 m.m 0 0.. 0 m.: 0 m.. .0 0.. mg 0.. m0 m. 0.0 N. m... m.m >.m m.m 50 m.0_ 0 0.0 0 0.m 0 0 > m.. 0.. 0.. 0.0 0.0 0.0 0.m 0.0 0.m m.: m.¢ 0.m 0.: 0 0.0 0 0.0 0 0.N 0. -- .... .-.. 0.. m.~ m0 0.m 0.. m.~ 0.> Md N... m... 0.N 0.0 0 0.0 0 0.. :. 0 0.0 m. m0 m.0 0.0 m0 m0 m.0 >0 >.. >... 0.... >.. 0.> 0 m.> 0 0.m _. 2 I J z z I 1 y a z I ..- C. 1.0-...— r... v 0 v 0 m 0 m 0 m m_ m 0 a 0 u 0 m 00 mm N 0 4 I .. 0‘. Cl)” '. | c 5 I I ... u .. 1.51. in! .lall I .. l...- .. 1". \u'. ..u'. i I 13"... .u (I bl. . ’ll.nll‘lll.'|? villi! ‘I-IIP'|II.II«II. .I. .... Iluill-Dh'. .4... .I..r.lx’ '7...§!II(I. .rt .I.l’i.( .-.ilttil’ i m. m > o m S w m _ . 0_..0w.0._ ”heeuxx mum.m.. .1 I. ..y . - .. . .. 3... .. , . . . . f. . o .. . 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I ‘II‘1. ..L-nl.‘ Pi .UW>CL 5;..c.v-.Cu>: go m__uu vo___x LO ..rrw . 0.00 c. mc_c_03.000505 0:000.mogn.o:acuoc0amu.oe 0: . n. > 50.3 0000—3002. 00.000. 200% - c 0. 0 m an: 0cm..oo..mu-.0 - - . a . . .0 .0 . . .o .000... 0 0...-h 0 O> 7O >000_0:0 0:000.00.a.0:oc0000000.0: H Q: .. mm ._ 0.00h c. 03000 00 0000—3000. .0:0.m .00 00.000. 00.;H : 0:0.0N >000.0c0 0>.0.0c00n.0:0£0000000002 .>.0>.000000. 0 00 ..000 .N .. .0 0.0053: :0_u:._0 E3000 00 00000 00m. 00 ..000 0m .0. .0 0.00.0 . - - -- >.0 0.. 0.0 0.. 0.0 0.. 0.0 0.0 ..N 0.0 0.. 0.. 0 >.. 0 0.. _> .0.0 ~.. >.0 0 >.0 0 ..0 >.0 >.m >.0 >.. >.N 0.0 0.0 0 >.. 0 >.0 > 0.0 0 m... 0.0 N... 0 m... 0.0 >.m 0.. 0.0 .0 5. 0.. 0 \... 0 0.. ... .- - - >.0 0 0.0 ..0 0.0 >.0 >.m 0.. 0.0 0.. >.0 0.0 0 0.. 0 0.0 ... 0.0 0.0 0.0 0 50 0 0.0 0 >0. 0.. 5... 0.0 ...N 5. 0 TN 0 0.. : v . ‘, U. 0 ...- -0 .. -- 030-: 0:- 03.30 - ...-z ----_.....- ...-02...... ...-.....-1: ...-..2 ...... .s_.. . u q . .. ... .V ...l. _ _-V._— — 1.4. .w .\ 2 _..l\\.—~ m—_..\.VU DQ—_._x UCr-w O—Q “Jam... TUQQ—SUOC- Wu. . : :— 0 K o 0.00 :. ac.:_0:_000 .0_.0.u«0 00000.000:.0w .;..00cc0.0E 0cm 0>_0_05 0:.0c0:.. 000-.0008 .:a. 50.0 .0 .0.00_H .m o_om. 71 .0c.:.03.mm0500 0:000.00.u.0:000000a00.0e\0>.0.0:00u.0:000000000.0e .0 00.000 : axmz\0um: ¢ . .0..- A 0 N.0 .. 0.00% c. 03000 00 0000.300c. .030.0 .00 00.000. 00.0% I 030.0 ..0 ..0 N.0 m.0 m.0 0.x m.: m.— N.o m.o m.o 0.0 0.0 m.w O.m O m.o 0.0 0.0 0.. m._ o.w 0.0. o.N m... 0.0 m... m... a... 01m 0... .... 00.0 00.0 00.0 m.0 w.0 ..J m.m 0.m .u‘. - ’A-“I 4‘ II 'III I . - .l-\- 1.1! ' "|IO.I. Il‘l . n ‘.'I' .I‘v . II. I < luf- ..‘- 0' - ~rlo1'. l ' t - ‘ . - v II ‘. I | A. I... I .I- 0 I- -..-:4 A. . s ‘ .4 rt, l-II- 4‘ l 4. W". I a 40"ut- I 4 I I lllu‘l '.Il erltv. v4... .l I. t?) \ y \. u . \ N — \ .d pa w. .. l r ... i.\. In. ' .I-‘l....l' . I. 4“- :.l‘...‘¢l r P VI ‘1’ III . . . i ...V ’I I I II : .Ilritvgal, I (I I I. It i _ w .. . _ 0 _ ...-t. . . -OI;IA’IU.P\.IF-.Il.f" 144:- il...f-l-t.h [5| ....Ifl. t I.» l. Fl: ., .. n t. u! III a. .0 «I..- Iv ..Il ‘- I'ii’l I IL I .14 I I}. I. ‘0‘ P — __ — . _ z. . . .. _ . L a. u . Kw. .k. a\ .l C - A r . o o . in l_. (v . - I u. r I i -vII I ..t I. ll 0 I x. I in « III I l. i 1 l I I: 30.» 0.00 c. 0:.: .w.xm0.$;....ru:.uwu.00 0..00 00...x .0 0.00.> 00.3 0000.3000. 00 ' 0-J :3 C ‘- C' ‘- ('3 E U .C 4.) C (U 9'.) U I U) ‘ O L l O C (J ..C C.) C. 4.4 C- (3 U L. 9 L: \. (J, > 00- L) m C Q) U". I C- C 4.) C) O 4.) C‘- C: U L. (=) E H... 0 if: 0 .— Oun- 4.) (‘5 c: .0 “U... 2U N . 72 .0:.:.03.mmu 0:.00.00.u.0:0000000-u.02\0>.0.0:00:.0:000000mw0.ma .0 0.030 : xzu:\m:uum .. 0.0.0 0. 02000 00 00.0—20.4. .mau.0 .00 00.00:. 00.09 1 1:0.0_ 1: :2 N.0 N.0 m.0 m.0 m.0 0.. m.. 0.. _> ..0 m.0 0 0 «.0 N.0 ~._ 0.m \.. N.0 > 0 0 ..0 0 ..0 m.0 N.0 0.0 m._ 0.. >_ in I: 0 N.0 N.0 m.0 0.0 m.m m._ m.0 ___ m0.0 m0.0 0 0 0 N.0 ..0 0.0 m.~ 0.. __ .... Isa-mm.- 0. 1:10.-- a... .- 0 0.. ... .m N _ - ...:oé ...-.... 2.0.0.0...._................_<-00... 00.0.03.00< Nast\m:wz 0.00 c. ls... 0‘ .0.>00 E3..c0wc0oc>: mo 0..00 00...x .0 0.00.> 00.3 0000.300:. 00.000. #3.. 00.0.03.0m0 .0..00000 00000.00.:.0:000000000.05\0>.0.0:00|.o:000000g00.0s mo 00.00a .m 0.009 73 ME-resistant hemagglutinins than rabbits in Groups IV and V. Rabbits in Group II produced significantly more ME- resistant antibody by the third week than rabbits in Groups II and VI. Hemagglutinins were not detected in sera from any of the rabbits in Group V at one week post-inoculation. The ratio of ME- sensitiv’e /ME-resistant hemagglutinins decreased to less than one by four weeks post-inoculation in all groups except Group III. The ME-sensitive/ME- resistant bacterial agglutinin ratio decreased to less than one in all Groups by the fifth week. Mercaptoethanol- sensitive hemagglutinins were detected in sera from some rabbits in all Groups for at least eight weeks post-inocu- lation and in sera from rabbits in Groups IV and V until the 23rd week. Sera from rabbits in Groups III and VI were not tested past the eighth week. Mercaptoethanol-resistant hemagglutinins were detected in sera from rabbits in all Groups tested at 25 weeks post-inoculation. ME-sensitive bacterial agglutinins were detected in serum from rabbits in all Groups five weeks post-inoculation but only in sera from rabbits in Groups IV and VI at the eighth week. Mercaptoethanol-resistant bacterial agglutinins were detected in sera from rabbits in all Groups tested at 25 'weeks post-inoculation. Skin testing of rabbits in Groups II, IV and V with 0.1 ml of second strength PPD-S 14 weeks post-inoculation simu— lated antibody production (Figs. 1-2 and 5-8). Mean hemagr glutinin titers were elevated at the fifteenth week in 74 Groups II and V but remained unchanged in Group IV. Mean bacterial agglutinin titers were higher at the fifteenth week in Group II but remained unchanged in Groups IV and V. Ratios of ME-sensitive/ME-resistant hemagglutinins were correspondingly increased at the fifteenth week in Groups II and V but decreased in Group IV. The ME-sensitive/ME- resistant bacterial agglutinin ratio was higher at the fif- teenth week than at the eighth week in Groups II and V. No reactions were detectable in rabbits in Groups II, IV and V, five hours after skin tests. Delayed reactions occurred which reached their maximum diameters 24 hours post- inoculation. The mean diameters of palpable induration at the skin test sites were greatest in rabbits of Group II and smallest in rabbits of Group V (Fig. 11). Precipitins Specific for culture filtrate antigens were detected in sera from most of the rabbits in Groups II-VI by five weeks post-inoculation. One immunoprecipitate was usually detected in the Ouchterlony double diffusion test, occasionally, two lines occurred. Serum from normal and immunized rabbits was separated into six to eight fractions by zone electrophoresis in cellulose acetate membranes (Fig. 12). Gamma-globulin concentrations increased above normal levels one week post-inoculation (Table 10). A decrease frequently occurred at the second week, and subsequently, a progressive increase. 75 .com.0c0 mo 00.00.3ooc.u0000 0x00: 0. 0-000 .0 co.0oomc. .0E.000.0c. :0 >0 A. 0.00Hv > 0:0 >_ ... nu > 030.0 Ilmwa >_ 030.0 .IMHF ._ 030.0 Ilnvl 0030 .0 00 00.000. 0. 000.0..0 000.0000. 0000 0.00 mZ_H 0.00.... 0.00.0 0.00 0.0000 0 a — , .- ...-1- . c (ww) aaiawvxo NOIIOVBH' ... .0. 0 76 £5.90 0 .355. .05.0: .0 0.00.000o.000.0 0000000 000.3..00 .o 00.3.000. 0..00500.0:00 0>.0000000.000 .N. .0.0 L ofi o c._300.0208300 ._ . to!!!” I \avl' (1.5.. III!- III‘I'I {a 1 I |l. A . . . . _. . 0 m . . . , . ‘ . . . , _ . , _ ._. fl _ m 0. . _ '. u . . 0 . ._ . . . _ . .. '10!" ‘III. II. I. .llllll'p-II -! (III! I- I‘ll! I’ll 1| lllll‘l'll‘tl) ] 77 .comuummcm com_ucm ou Lo_La Umcwmuno mu_aamg .mELOC 50g» EsLom I zN ._ m_amh c_ Czecm mm vmum_300c_ .onLm gma mumnnmg mogzh I anoLu_ o.o_ w.o~ m._N m.m_ o.m_ m.m_ J... m.m_ m.m_ _.:_ m.© > m.m_ w.w_ m.m_ _.m_ m.m_ 0.3— m.~_ o.m N.w m.__ o.m >_ o.mm o._m N.m_ N.m_ m.m_ m.m_ o.N_ ___ I I 5 | \O :3 N Q .d‘ N o.m_ w._m 0.0. m.o_ ..m. m.m_ o.m_ 0.0. 0.0. m... m.m __ :11- i-I.D.I|l’ 3:11.: 1 1 11‘ .I..I.O...Ll’|n, I," |I. ... r. ,'..n.| . (. N3151- |rnl . Ilil‘II-II'Io I. {I .i Illl.l.- c. .1 If ill! ...!!7 \1 ..| I , I.‘|ll!.‘l ‘.§|Jl§fi , "’,-I‘ \ mm m. m o m s m N _ N; .c:oLo 23.99ma2_ :uegq ”gnu“ .cwgypopuruaqufuacwxuum:sagzss:s. ’ - Iv“. Il‘uo.’I-.'U1ulllo1ln. 5.. .i. . .l';\., n . I II 4 u r c.’l'l4.ii||0..“r I’ll. will. 'I. ‘1‘.— .\ ‘ a ‘Orf -. a . ..f ‘ I ... > 0 (F I. o\. . v F ».-A ‘I.:.f' r... 1.. . .( bl? - ... O .\. I... . ‘.l.., . . 1 )‘ll :1" . i.l. u).’r.il€'" I011. Grit-J'lud :1- 4. 1w 1'~‘1. .-.. .,vl lit! IIL”.J' .o ‘rll‘llr‘l.7.l..|'l". .l‘ [I‘l'i ‘1'!!! ... I. .1? D'\\¢.1v I 1.x“. ‘ n I ~v.‘ ‘ .t l‘ ,. f. \, I , ‘ar. III-Ii. a)! cvn. ‘ I . i4 iv'..~ t .,v y . ‘\ ‘ln .. . . ...I. LIA.‘... l|l.4!ila. cbll.y’§\n.t ... .|.1 .nllll I vl"t\l11II-nlll‘l|llnclbvar‘t .l'l "‘IIII"I.V.LI.{| I!!!“ (....i- ‘V‘l .u. w..».cL. 83pow._.uu..:.,.\u>...~ u_o m. 30 U3 _ E Lo o_am_> cu_3 woum_:uoc_ mu_nnmL EQL$ mLom :_ c__:;o~mszEmm $0 ommucouLoq m>muw_om .o. o_amh 78 Chemical Analyses of Bacillary Extracts Prepared by Ultrasound Ratios of absorbancy at 280 and 260 mu and the amounts of protein, carbohydrate and nucleic acid in ultrasonic extracts (USE) are listed in Table 11. Ultrasonic extracts A and B were very similar in chemical composition. There was approximately 4 mg of Folin-reactive material (protein), 0.18 mg of carbohydrate and 0.42 mg of nucleic acid per ml of concentrated extracts. Ratios of absorbancy at 280 and 260 mu were between 0.55 and 0.6. Ultrasonic extract C con- tained significantly more Folin-reactive material, carbohydrate and nucleic acid than USE A or B; 0.5 mg of carbohydrate, 7.8 mg Folin-reactive material and 0.68 mg of nucleic acid were detected/ml. Ultrasonic extract D contained more protein, approximately the same amount of carbohydrate and a somewhat higher concentration of nucleic acid than USE-A or B. Ultra- violet absorbancy ratios at 280 and 260 mu of USE—C and D were similar, and higher than USE-A or B. Bacillary extracts were clear and slightly yellow in color. Some precipitation occurred when frozen extracts were thawed. Chromatography of Ultrasonic Extracts Molecular exclusion chromatography of ultrasonic extracts in Sephadex G-25 separated three major fractions on the basis of absorption at 280 mu or 260 mu (Figs. 15-16). Fraction I was eluted in the external volume of the column and consisted 79 'Irn l!°|.1[l|.l ..r‘ .:E oom pcm owm um >ocmnc0mnm wo omumm : oom\omm¢ p_om o_o_osz u goacmo ozum Ac_ouotav _m_L0umE o>_uoootuc__om Zam— mo.o mm.o ww.o mo.o om.o ¢.o mm.o :.o oom\omm maz agwmxmflmv, «N.o o.m o «.0 0.: m o_.o N.¢ < (- .AI ...-lo . ... ...l . , .lI."..V ‘1!‘IU.A‘ lltlutll .‘. Ill."il 11.1.1, tillltzlllk .i .D.ll(')l' «0 .fi ...“ . ill" ..... ....- .'. a! Q" P, .\QEV comreflm cwrfiwucu_: :mm A e «to w. _ ,,I ..l-_.r;\ a . Ii p .... .14.!) ..II\ ..lvl .. ..-}.afxfi. I. ,(...l. )l.,.ll.llfs ..1 xi: . it \ . .. til}... i I .3, his... II,.(41.rh.I)I.vl.v\lI-| .| v .r. fife... .9 . p: .lvc, s . 1 iv 3} \. .... . ..Ir. .17.... Ibii . ... 1“".I7...,.nzvfilluv)l\’ ’xt.....lt . 9...... {I ...1\.I.l Vllxiv.‘ . ‘ n 3!.I‘.\ffl..(!. {$2.531 .....' ..O-lultluli .’\. ‘K.. Til... .w~>c; SLwctrocaooxm.wo m__oo o_no_> 50;» poc_cuno muomcuxo o_commcu_3.c_ p_om Qua—oz: paw oumtp>aoacoo .c_ou0cc mo muczoa< ... o_coh 80 .mmuu xOpwzgom c_ AN o_pohv < pomtuxo u_commtu_: mo >Ldmmmoumeotso co_m:_oxo cm_:oo_o: mm om mm In... Pi. 4 .533... {if ..I'lnlr‘-. 1.. ‘Ill-I r .1 Ill mumzaz mash mH 3. Kill . a. ti} - m p I I 7...]- I. ' a ..I. lillmfl". III-.1: o :3 (m” 092) Aanvauossv OM— .mi 81 .mmnu xopoggom c_ ammzsz mash om me cc mm cm mm aivlétllnbsliiiangllli w flymweus , : '39—...— --uyy .. . _‘.', -. - . aw H-- no ..u ... O .— 2 ‘5'. c: (.3 AN c.28hv m boacuxo o_:ommtu_: do >zcmcmouceotco co_m:_oxo Lm_:oo_o: VGEOSBV ad ‘ n\ J; (nw 082) A 0.3" .m.m 82 .mmuo xonwgaom c_ Am o_awbv o uumtpxo o_comotu_: co >camtmoum50cgo comm:_oxo L23302 .m. .m_u - u‘_ ‘9 o ”IVSEOSQV a.“ ‘ I (mu 082) @ A3 ._,__ ._ C-..- E“ ‘t H 85 .mN-o xovmzaom c_ AN o_aohv o Homtoxo omcommcu_: mo >rcmrm0umsotso co_m:_oxo Lm_:oo_o: .m— .m_m awmzzz mosh om me av mm om mm om mH ofi m o ll... .. 5...! . . 5L _ . . . . . l, i . \r 4 Z w. . Cu -. o a m . _ Nu m _ WU l. N ”W mm :.m. mm 5.9 . C . ) a I m e .C_H .U g mm a ._ l w a < um :2 _ n 0 Q .-u V H LO.H 84 of a single peak except in USE-C, where fraction one con- sisted of at least two subfractions contained in three dis- crete peaks (Fig. 15). Fractions II and III were eluted within the internal volumes of the columns. In USE-B, fraction II consisted of a single peak. Fraction II from USE-A and C was composed of at least two subfractions (Figs. 13 and 15). Fraction II from USE-D consisted of at least five subfractions consisting of one major peak with four closely associated sholders (Fig. 16). Fraction III usually con- sisted of a single, rather broad 280 mu—absorbing peak. In USE—D, fraction III had two subfractions with a very small peak associated with the major fraction. Fractions I and III were white and readily dissolved. Fraction II was yellow- brown, granular and difficult to dissolve. The distribution of carbohydrate, nucleic acid, Folin- reactive material and 280 mu-absorbing material in chromato- graphic fractions from different ultrasonic extracts are listed in Tables 12-15. Ultrasonic extracts differed in the relative distribution of 280 mu-absorbing material in the three major chromatographic fractions from Sephadex G-25. Fractions I, II and III from USE-A contained 65.4, 22.4 and 14.2% of the 280 mu—absorbing material respectively (Table 12). Although similar in chemical composition, the distribution of 280 mu absorbing material in USE-A and B differed. Ultrasonic extract B contained less fraction I and II and more of fraction III than USE—C (Table 15). Ultrasonic 855 _mmtobcE mcanOmnmnse 0mm u zsoncmu A:_ouoLav _o_uouoe o>_uomotxc__0u ll ozum .mmuo xopmscmm got» pou:_o mco_uomcw o_;cmumoumeoL:o Lofim: 1 Logan: co_uomcu_ m.o N.¢_ o_ o m._ ___ ¢.o $.NN mm o m.mm __ o.— J.mo .m 00— m.No _ - I -3;,-? {Siflflnflafifais§§:;:?i,ésiis.Eéiiiziiiézztifliiiléiiliifi%wbflAbfiflXflM NC mu..< CCN j/tfi MCLU “15m — . o N .N N N .mmxo Xcvugaom Fete sob:_o < bomcpxo owcommtu_: mo mcomuomcm omcgmtmouoaocso Lemme are c_ p_wm omo_u:c pcm .oumtp>£occmo .cmouoLQ mo comuzautwm_e .N~ o~amh 86 _m_coumE mcw320mnouze 0mm u E£oacmu Ac_ouoLQV _mmtoumE o>_uoo0csc__0u Imam mmuo xovmzcom sot» pups—o mco_uoocw o_cdmtmoumE0c£o Lofimz n Loassc comuomtm_ II I. . '21 1"]. (iv all"... .. ii ) bbfll.*. ..r IIIIPI' I) (I. iwkn L‘E‘i'g‘Ob‘ Nm.o _.:m N.@ o w.m ___ oq.o 0.“. m.oo . o m.m¢ __ o._ . m.ms m._m oo. w.ms _ I! pi i .IaQ.Iv-Il\ Al'ihi’.’ a) .v ( .. . , \ ...!» v .v {IA}... 1.1!? .I .I .I. AI lull-viii..- (It...) ...I'I . .I|. : II.- I. In I ..l.l 11,1) v 1100‘-l.ll" II | \ n .Ilialalllrlllvllsll‘ vi, I. o. .\ li’l If! at.‘\l|‘r...rllv, I‘.. O . x. .. «...-II ...a my. ca. :51 m9; Ni...» Ti... 0 O O O _ H1 .J H..— \o Ru xv Ru —.. O m - Lr L n- . . t.||.. .v... .. ... . v .. ,\.l) . . . . o . .~ 8.... .. . . .1 I .\ J L ( .. ..‘Iv .l VIN...|( I! .wv ... LI .. ... n 3.. . \b..( I ...\O.Illl it I V | .' .l .l I .0 b 1 t . I ... .4 ... )r 1 lg! . I I... 7‘3 y i. f .. t I. I K»? L‘P'. ..l I,Pt|4 IFNI... . l ..'n . :1 (If!!! . . ,-.I..uv .(1. I. I a... .O“ a O \ um:c xoncaom sot; poospo m bowcuxo o_commtu_: so mcombootg owccmtmcpmsoLso LOmmE 0;“ :0 when o_o_o:c pcm .obctp>;oncmo .CMOQOLQ mo co_u3nmtum_o .m. 0.2mh 87 III.” III! (..-. mco_momuw o_£motm0umsoLco LOamE 0;“ c_ prom o_o_o:c paw oumtp>goncmo .cmoboLc wo co_u:n_tum _m_coumE mc_aLOmnm|:E 0mm u zconcmo Ac_0u0cdv .mmLOHmE o>_uomoch__ou :zum .mmzo xopmcdom scum pops—o mcomuomtw o_:cotmoumE0cco coho: n Longs: c0_uootu_ o.mm N.om o O ___ m.om m.mm m._ N.mm __ ..o_ #.o. m.mm m.m¢ _ - .. 3.3.314..- H; O. ...... . r - t - -..... .. r ., - is...) .--... . .0.-- if... ... (at. (MAM!!! -1..- 2....qu ..-..w... I... ..-. r..0._:.....w.m.u_..| mlc MC“ .Sogl— MCHUIL 0:10;”— , _ .L L . m a a a a .mmxo xmmmgagm sot; pubs—o u powruxo o_commcu_: mo mo .:_ o_nmh 88 _m_c0ume mc_nc0mnmnne owm u z_uomocuc__om 2am N mmno Xowmgdom E0c$ pups—o m:o_uomcm o_£aoum0um20czo LOmw: n LonE:c co_uoocm_ m.o ..NN 0.: o m.~ ___ m.o 3.0m mm _ _.om __ o._ N: mm mm _¢ _ ‘ v .n ‘01.. it ‘n‘. Infill, . . ll.’ . ... .0: a . .o III. I I '..'C. II .‘I .- -.. l. . .I I. I 4. ..lu (Pi. . I.I. ll. ’Iylla.- ‘, . . . . ...-r- .1 ... '1'... .l.)‘lll..‘bola .. '\ 1'. . I 'D.I'.vl"..n\(( o‘ .1 10“}.7 :tlnll"I.U.O-'.l.l. . Ii-" .2 .........-c..cm S... . 95 ....E a» _ . .2 m J& m N N X —comuuwuu I. .p 4 ‘y ...l V . II. II. .- I|<.. v . .. . In. . I; .l. .I. c ll. I . ‘[.u' I..I$.|I~“1.I.ll Il)lr..llbj.;..u|k\ll.t’..‘ ’.Il.~l‘I-!)l. |--'v ,14Al’|‘t"\.‘£ - .. .. ) ...rltul .1» .II. . 21...}! . ... i..)v.,.f .II- to T ... I. - 1.3.97......)Piull..fl|'bl.l-11. .v o. i. .Il‘u..ll, (-..-.9 .-..V on-l|:||||.-.l‘ .mm:u x.pc:com Soto mops—o o womcoxo o_:0mmcu_: so mco_bomc% omgcmtmcbosoLgo Lemma or“ c_ pxom omo~osc pcm oboup>zoncmo .c_ou0cd mo co_b:n_tumwo .m. o_awh 89 extract C contained substantially less fraction I and more of fraction II and fraction III than USE-A or B. Only approximately 10% of the total amount of 280 mu-absorbing material detected in USE-C was in fraction I. Fraction I, II and III from USE-D contained 42.0, 56.4 and 22.1% respectively of the total amount of 280 mu—absorbing material detected. Ultrasonic extracts differed in the distribution of nucleic acid in the three major chromatographic fractions eluted from Sephadex G-25. In general, elution profiles based upon absorbancy at 260 mu overlayed corresponding pro- files based upon absorbancy at 280 mu although some differ- ences in peak amplitude and width were observed. Nucleic acid concentrations in fraction I from USE-A and B were similar and somewhat lower than in USE-D (Tables 12, 15 and 15). Considerably less nucleic acid (10.1%) was detected in fraction I from USE-C (Table 14). The percent of nucleic acid detected in fraction II was similar in different ultra— sonic extracts and varied between 57 and 60.3%. More vari— ation was observed in the relative distribution of nucleic acid in fraction III from different ultrasonic extracts. Fraction III from USE-D contained 4% of the total nucleic acid. Eight and 10% of the total nucleic acid were detected in fraction III from USE—B and A respectively. Almost one- third of the nucleic acid detected in USE-C was found in fraction III. 90 Carbohydrate was detected in both fractions I and II from USE-C and D and only in fraction I from USE-A and B. The majority of the carbohydrate detected in ultrasonic extracts was present in chromatographic fraction I. The distribution of protein (Folin—reactive material) in the three major chromatographic fractions from Sephadex G—25 varied among the different ultrasonic extracts. Fraction I from USE-A contained 62.5% of the total amount of Folin-reactive material detected in the sample (Table 12). Somewhat lower values were detected in fraction I from USE-B, C and D; 48.8, 45.8 and 41.0% reSpectively were de- tected. Fraction II contained the majority of the remain- ing Folin-reactive material. From zero to 4% of the Folin— reactive material was detected in fraction III. Good cor- relation was observed between the relative percentage of 280-absorbing material and Folin-reactive material in fraction I from USE-A, B and D. No correlation existed between these values in fractions II and III. Rf values were computed for each of the three major 280 mu-absorbing fractions separated from ultrasonic extracts by chromatography in Sephadex G-25 (Tables 12-15). Fraction I had an arbitrary Rf value of 1.0. Fraction II from dif- ferent ultrasonic extracts had Rf values ranging from 0.42— 0.50. Rf values for fraction III varied from 0.29-0.32. Representative ultraviolet absorption Spectra from un— fractionated ultrasonic extract (USE-B) and major chroma- tographic fractions I, II and III eluted from Sephadex G—25 91 are shown in Figures 17 and 18. Absorption spectra of the unfractionated extract and fraction I were virtually identi- cal (Fig. 17). Two broad absorption peaks were observed, one between 280 and 260 mu and a second between 240 mu and 280 mu. Chromatographic fraction II had a single continuous peak with maximum absorption beginning at approximately 260 mu (Fig. 18). Fraction III had two absorption maxima, approximately 250 and 200 mu (Fig. 18). Representative chromatograms of fraction I from ultra- sonic extract B obtained by chromatography in Sephadex G-25 and rechromatographed in BioGels P-100, P-150 and P-200 are shown in Fig.19. Fraction I (G-25) from USE-A was separated into two subfractions in BioGel media on the basis of absorp— tion at 280 mu. Each subfraction consisted of a single peak. In each case, the first subfraction was eluted within the external volume of the column and the second was eluted with- in the internal volume of the column. The percent distribu- tion of 280 mu—absorbing material in each of these sub- fractions from the different BioGels is given in Table 16. Eighty-four percentcxffraction I G-25 from USE-B was eluted in the first subfraction from BioGel P-100. The remaining 16% was eluted in the second subfraction. .Approximately 82% and 18% of fraction I G-25 from this extract was eluted in the first and second subfractions from BioGel P-150 reSpectively. Fifty-nine percent of the 280 mu-absorbing material contained in fraction I G-25 was eluted within the 92’ .meo xovwzaom Soto poc_muao _ comuootm o_:dotmou050c;o pcm Am o_bmpv m boothQ o_:Omocu_: mo weavedm co_uc20mgw uo_o_>ocu_: Asevi:m2ul maul no- . ..N N mNN mum mum m iv .Illyai . ‘14. All 1.! W . null. ..P..1).I ‘I — ill. mNuo _ :o_uomcm I... .l C‘. EDNVLINSNVUL % . Aasv Isuzu; w>gg amw .awm nun and 9-4 omm m uomcuxo o_commcu_: oN— ommh‘ mwm .sou -8 3cm saw 0. I JVIIHSHVHI % 33 93 . .m~-o xosmgaom eotl soc_muso AN o_nmpv m somruxo o_commcu_: socw ___ pcm __ co_uomtm o_;amtmouweot;o mo mtuooam co_uat0mam uo_o_>mcu_: .m. .m_u Asev :sozwn m><3 Anew :pozmn m><3 3N smw 2mm cmw 0mm 0mm .omm 92 3“ 2m 8m 3m 8m Rm l II. t u; . Go." a i (Ila 9. 3 vi.) : OOH ram now can .uw .oh yes row 70 .90 u. m pm m :8 w my mm as mm-o ___ co_lomri .mm mm-o __ co_uomri .mm 0N -QN -1 .s~ | .. ..u 0 {EU FEE. lrlufhuhvurw! .. .. Q SSHVLIHSNVEi % .on oom-m ego Amv om_-a AALcmtmouosotcooa aumxsz MED... aumzsz mash ammZDz was... cw mm om m 3.. cm ma cu m c OH .. inf/iv- .Ifl. . ..Lb... ...... .... .ninb nllr. L; (\i o n . vii. .. nlill,r..-iusnll .. Inn-Ill: afiwsnlnll O .3 -. . wu w. l I” s M... I M“ .1 .. mw “W I . 0 0 mg 0.0 we W. _ w. .v . , _ ..N ....u L _. n4 3 .... ,.\ IA 'A . .J. \) I. ) _ . .. ... a,» _. .... c 0 NW C 0 .n/wu _ C O .x m m g . - n. n L H /.\ , L H ( m\__—; -"'—V--\ ..., o. . I .m.u {‘5 O _ .3... C 7V U nw cez) A“nv3308v r \ 95 m_oo o_m ucogmmmmn 50L. w0u3_o mLan:c co_uomgw ou Logo; __ vcm _m mmsw xouosaom so.» ways—o muomguxo u_commgu_: eoLm _ co_uumgk o_:amLmomeoLgo n mmxu_mm mg_E__ co_m:_uxm Lm_:oo_oE “cmgwwk_v ;u_3 m_mo o_m mLm oomum ncm om_xm .oo_um u E:_v®E >camgm0umEOL;u_ 13': . N. . III ~ «IIV',§ .V III! l. i fit VIII-l Iii? III-«II.YI.LL f ow ON mm Nu w. «m o m mm _. mm mm .0 . Q _: mm m.w_ m._w o. :w m 'I,-\ I ..IIII- ul‘ 1 . ‘.‘i‘-‘ In, ‘. ... U . I o ‘o'IN. .L-|I.!!‘§.f‘l 1‘ . ‘ ‘I' 7‘ ‘- ll: "1 Id .‘.’ .3.DI‘I.A,E III. I ’l-lil I'll “’»‘II".I-‘.E.\I|I‘ .i‘. ‘l' . "flt.l’!‘l ll.‘ 'I'nlll ~ ‘nu??‘|' I ’1‘..I:l ‘ I-OII i- I. .I! ' ’i __ _ __ _ __ m. Lccgpxu owccqu._: . A.“ '0 . ... . n. I. . L .. : 3.! Ilrt! .‘uAtIiI .Iol’ .0. on} .....IIIIIIInuIlIIhIII .. IIIIIIIIOIILTII. I. VIII!) I.I|'. FFOI— * N IL — u n . ..x. .1 .1 .. c n; T .-. w 5L ....-. m ...; II}. .I L .. It . .II’ It. I .0 ...!L a. . n I l v u 4 ... .I u II.‘II I . v 35.. \ 'a-b‘ll u v’. | ..I. . u n . I). . 1 u . ..l a Z. I ...Ir.‘.fi£..‘1IIIII IQNI‘I...|52|I\J.II-i . . . . . . .. . . .I . rczupr: >£CQLCC+GY. .MIU .I‘ 0 r1 .1 x .. .u .I .L I .11 I. Olin. \. I h n...l|ll...l nl III-.IALuiIK. y A: ,A.... ... v,... .Ii III...!¢I-II( a.» . KIN v‘I .. 4...-.I.I.C ls. III... I211 .‘I‘ IIIl u . 1 II: ...IILI .'|.Il h Ian 1 . A1,:Vruéul‘nll- .0 a ....l Idli‘. I. .I .4 V 0..!Ilu n1 ‘ ll.. 4.l ..4 Ini‘I‘I.t.I S.V..I :0rl..I .I i If. a .6! 1 rs I10\I\.5...II LII‘ .muumLux. u020mogw~s <0.» mm o. :c_uomgg mo >52aLmQum¢oLzooL mcm30__0m m_voE .oo own 20;; m:o_uum;» u_;amgmcumE0L:u c_ _m_LoumE mc_nL0macx:E 0mm #0 comu:n_gum_o .o_ o_amh 96 external volume of columns of BioGel P-ZOO. The remaining (41%) was eluted as the second subfraction. Twenty-two percent of fraction I G-25 from USE-C was eluted in the ex- ternal volume of BioGel P—ZOO columns. Twenty percent of the 280 mu-absorbing material in fraction I G-ZS from USE-D was eluted within the external volume of BioGel P-ZOO columns. Representative chromatograms of USE—B, C and D in Bio- Gels P-lOO, P-lSO and P-ZOO are presented in Figs. 20-22. Chromatography in BioGel yielded two major fractions. Both fractions consisted of single 280 mu-absorbing peaks. Fraction I was eluted within the external volume of the columns in every case. Fraction II from BioGel columns was excluded within the columns internal volume. The percent distribution of 280 mu-absorbing material in chromatographic fractions from ultrasonic extracts is listed in Table 17. The percent distribution of fraction I from chromatography of USE-A. C and D on Sephadex G-25 and BioGel P-lOO were considerably different. No significant differences were noted in concentrations of fraction I from BioGel P-lOO and BioGel P-150. Differences were noted in the concentration of fraction I from BioGel P-lSO and BioGel P-ZOO. Ultrasonic extract A was separated into dialyzable and nondialyzable components. Representative elution profiles of these two components from Sephadex G-25 columns are shown in Fig. 25. No dialyzable components were found in fraction I 97 .AUV oom-m saw Ame om.-. .A£dmtm0umEoLgo comma—oxo Lo_:oo_o: mezzz weak awmzzz mmDH xmmZDZ meH cw Mm om m \vo ON mm cm m c mm cm a. .. ... .II; 1.1.... [I I, I ..IF 15.0.. X... c 1 ,. .. III .....- I..I.I ..ullerr o If- In, .Nd sfic uu 0C 8 .n.U _ \L we may WW rid 2A .. m ..u MV c w n m...— ..NA 0 .H 0 ..9H d .oN A3NVCEOSCV nw 032) / l \ 98 ammisz mmsh am ....52 ”.83... r em -..Emmnr PH ...... m ~.. --.. m... . .l IC... 6 0. land—B; .av-‘QI"-' ' ...—— .040 x V I c. m C. . O .. Vt ._.. awe WW w v” . .w l/ ..I o ) afi Md nu w . n I l ”\ m.~ . n . I... \H . .AUV .Asdmtm0umEoLco L\' H oow-¢ was Amv om_-a co_m:_oxo Lm_3oo_o: +nauosev if“, [\UI\I 32) (nm 0 ..N .m_u new ‘\ uyr-\-A(“—‘z JuquQ J [\UA. I'-;’ I ' -‘. Ln“ 0.3) 99 .Auv oom-m new Ame om_-a .Armwtmoumeotxo :o_m3_oxo tm_:oo_o: .Nm .m_u MEGS: MEI; :mQZDz MESH aumzzz was... mm om ma oH m c mm OH m c mH ca m c .. . . :1... .I I Ebb- -....-II ... .. - .H c . ... .... .15. .Inmifini.--fi2. ....n...) o as... -n . rn a 4 -. .. U 0 \t/ . a x , I x . . ,__ . _ __ (3; <3- O .....W0 _ . I . I .Avo uu I .Awo ”V H m S ... S I. c. c. .0 no no . C .. .3. to .3. MW 0 W .. _ .Mw .\ a” C ..-u I . .. u ... A _ .-.. . . .. ., I b F “by .C. H M/W . .U C i — w w . _ n n - . NEH xx N.H /\ _ a.” K H -. _; ......‘uu' * Ian. . k I .. . ...: («J H L. H ‘2 C. H V \u . l ’H‘U‘S Ill ,1" '-“"\‘ nu; 003) [\vnnbunu I k 1100 m_voa >gaagmu¢azoggn figgg find “0 n:o«u\ g“ u:pgnq._w OJ gwwoL ~__ fin, __ ..N O. mfii: :oMWm_o..,.a L443. A..._u1....._).__._U Lko A . .. \..w 1' I . A \. .\‘.. . .. .. I \ I . ..I. ... \, .\ .3 h._>c; aficu owe 0L0 ocng écu om_ym o;_IL .um.o v .,; 0 OJ wg.h;; mayo z :J_t 5 >3 ; . “gru— l\ !\ m C\ at. N F\ \O C \. 4" Y\ \O (\ C3 LIN N o-‘q N D :N @N Km m: o J: .o.om $.mo . < i I Ell-‘l § [0‘ lldt‘l‘i‘tl It'll! ...... tall. 1 I . .--.q l‘li‘ I} ...Il- I o 1...! . I . ' ..IO 0 I IIo|II. I __ _ __ I __ _ ___ d __ _ uomLuxu c_ccuc;;_: it Ci 0 I- .'-| Il'liln' . qI‘lI’liI . 'i \ ....... ii . . . _ . . . . ocp-o oL_.c oo_:a war; - ...III'1 I .III 4;. .D'Il\u b .I'v. |. . .J'IIIDIII—I; «I 11.41.. H . ..- . i {.Il'.‘ . ll; 5 . III}: III! _E;_vo; >ccaLCCchoLLU ‘1- .lli ‘‘‘‘‘ I!!! ‘llI I. It. I III-‘13.» .AII . \IIrlchI—V I'IIICIO’I'II It...“ I‘l.’ .I‘II‘ II. I tilt... gratin-I" I . 7. 11-10.} :‘iillfi Illirll i< . a fit. I II ‘ii‘lt, . \ IF. -3 II, *‘II1 It]- I ain't; II... {I’ll .b'ili . . I}! I .I'I’.I\I I. . .. 41.-II... 'I I. I'll ll‘tllI ‘Illl.vlu.|-'I|.Il .m_>cc E:_Louccgmvxxwwo m__oo o_nm_> EoLm vo:_muno muomguxo o_c0mogu_: mo >zgmLmouoEogso co_m:_oxm Lo_:oo_0E >2 toc_wugo mcomuomgm c_ _m_LoumE mCNQLOmamxae 0mm go :o_u33_gummo .m~ o_amh 101 .mmao Xeumzaom c_ Am o_nohv < uumguxo o_:ommgu_: 50;» m:o_uomgm o_amm>_m_vcoc vcm o_amN>_m_v mo >£qum0umEo;gu co_m:_uxo Lc_:oo_oz .mm .m_m mmmwSZ mash . Mm mm mm am ma oH m o 1.111 ._....11..1|1a 111.1. a. 11.1.1.1. 4.. 1.....1111...,....1.1... a. .. ......11.....a..1£.... ...11 . O I /..~ I/ . If; .. K I ~... If I \I I , ...Nd I s I \ .... ~ p x J a .c I \ q uv .5... m. J 0. .. D. . U H. “...: m c0_uumLu m_nmN>_m_o :1 11.11 .w .@ co_uumgu o_nmN>_m_Ucoz 11111111 c flw scan mw w n 102 but considerable amounts of fractions II and III were de- tected. Small amounts of fractions II and III were detected in the nondialyzable component but it consisted primarily of fraction I. A representative elution profile from ion exchange chromatography of USE-B on DEAE-cellulose is presented in Fig. 24. Seven protein-containing peaks were eluted from the adsorbent using a continuous concave gradient with a phosphate-NaCl buffer system. Immunodiffusion Analyses of Ultrasonic Extracts Between 1.8 and 2.0 mg of protein/ml was optimal for the development of immunoprecipitates using ultrasonic extracts and reference antisera in Ouchterlony immunodiffusion. One percent agar in phosPhate buffered 0.15 M NaCl solutions pH 7.2 was a suitable medium for immunodiffusion analyses of Inycobacterial antigens. Immunoprecipitate formation was not enhanced by incubation at 4 or 37°C, or by the use of poly~ ‘valent sheep anti-rabbit serum. Incubation for four days eat 28°C with daily replenishments of antigen and antiserum cfliluted 1:5 produced the maximum number of immuno-precipitates i_n the reference system. The number of precipitinogens detected by Ouchterlony ixnmunodiffusion in the different ultrasonic extracts is listed in Table 18. Diagrammatic immunograms showing com- parative analyses of ultrasonic extracts and chromatographic 103 .omo_:__oo.mcuo c_ «N o_n cw n oomtuxo o_:ommtu_: to >sdmcm0uoeotzo omcmzox :o_ .sm ea .c3 c3 .Ttn I,” h .c: 1*:- -..- .m_m mm mm mN 9N mg oH . ...11..- -... .11. 11. ..., . N n .I N.o uvgu rad lw.° no." INA _IQH HOSQV n0 VJ ‘11 (nwogz) av) A3 104 m_moLo;doLuoo_oOCJSE_ co_m3u$_po::EE_ >co_cou;o:o mucocoqeoo o>_o_m0anm_;om p_oo o_po_tom u n_u_moauxoo_b 0U_E< u m:o_nou;ozc .m mito:chuoo_o om_p >3 mboccbxo o_comwtb_: c_ tobocbop mococomgco ~m_nnoomnooxz go t33332 .3_ cinch C 105 fractions from Sephadex G-25 are presented in Figs. 25-28. The number of antigen-antibody systems detected in the ultra- sonic extracts were as follows: 15 in USE-A and B (Figs. 25 and 26); 15 in USE-C (Fig. 27); and 16-17 in USE-D (Fig. 28). Several of the immunoprecipitates were faint. Immuno— precipitates frequently occurred in clusters and individual lines could only be reliably distinguished as the different immunoprecipitates developed. No immunoprecipitation occurred when either normal rabbit serum or saline solution was sub- stituted for antiserum. No antigens were detected in any of the unfractionated extracts which were absent in fraction I from chromatography in Sephadex G-25 although the same line in different preparations frequently varied in density and displacement. ‘Immunoprecipitates were not detected in either chromatographic fractions II or III from ultrasonic extracts. The number of immunoprecipitates detected in chroma- tographic fractions from ultrasonic extracts in common identity with the original extract is listed in Table 19. Fewer immunoprecipitates were detected in fraction I from different BiOGels asthe molecular exclusion limit of the chromatography medium increased. . Unheated filtrate from two-month-old cultures of Mycobacterium bovis and USE—D were antigenically undis: tinguishable using anti-USE serum (Fig. 29). However, using anti-filtrate serum prepared against antigens in filtrate 1106 .mmuo XOpwcqom Each L u_:docm0oszocco pco AN o_nwkv A3 Q t we mucoccarco _m_tcbh no _ .i.li‘|i ;no§l}l! ti I..II ‘ ...; I . ..11'. ...s..‘ monocoxo E $0 -..rZ........_.._.,~ .m_ o_boh 111 III-5|- ‘114 It I. IV- lll.‘. .— . 144-It’ll .mm>ob :;_Lonom:pc>: mo moc:u_:o Aouuv p_o:go:orzx_m pco Ammov m_m::o Eon» monocu__w cusp—3o pcw AN o_bohv onumav o boocuxo o_comoLu_: co mamtm0csse co was 03“ o_uo3o;om .mN .oE 112 from six-month-old cultures, only ten antigens were detected in USE-D (Fig. 291. Eleven immunoprecipitates were detected in immunograms of ethanol or acetone precipitates of USE-C (Fig. 50). These preparations were antigenically indistinguishable. Three immunoprecipitates were detected in immunograms of an antigen preparation obtained by TCA precipitation of USE-C (Fig. 51). Electrophoretic Analyses of Ultrasonic Extracts Optimal protein concentration for disc electrophoresis of ultrasonic extracts was between 250 and 500 ug in a sample volume of 0.1-0.2 ml. Schematic disc electrophorograms of different extract preparations are presented in Figs. 52-35. The number of protein-containing components and carbohydrate- containing components in the different ultrasonic extracts are listed in Table 18. Disc electrophorograms of USE-A and B both contained 10 amido black-positive components (Figs. 32 and 55). They differed in only one component having slightly different Rf values. Sixteen and 24 protein components were detected in disc electrophorograms of USE-C and D respectively (Figs. 35 and 54). Sixteen components were common in the four ultrasonic extracts. A nonstained, yellow component in ultrasonic extracts migrated with or preceded the most anodic component. No protein bands were detected in the spacer gel. Rf values for amido-black positive components in disc electro- phorograms of ultrasonic extracts are listed in Table 20. 115 .u Homcuxo o_c0mmtu_: go Ammv mouob_a_ old .o:a:oo Ucm Aa_m:m o_:om_bcm o>_uotsm:oQ.cm OCmH— 114 .u bootoxo omcomoco_: sole voc_mugo Amn_mcm o_com_o:m o>_uwtmas u .— m .m. u 115 .ooo;c>:Ogtmo to; c_mbm ec_;om U_oo omto_tom u mgoacmo LOe :_mbm ww_£om p_om o c_ouoLQ to; c_obm xom EmLmOLOLm0cuoo_o o -11: 51.-E \1 v Q m a... . T _ 1; I 1 ' v ‘I .5. I, m . I4 .I ‘ t t‘v 1.0 i I - . Nu.‘ l\ I 1“ c ‘ Iv‘llnl ED 1 .lrFll' 11X? oomtv>50btmo Low c_wum m%_som p_om o_:o_cum u m<¢ c_ou0nd to; :_obm xon_2 Op_E< n m< .AN o_bmkv u boatuxo omcomwtu_: mo EQLQOLOLmotboo_o cm_t o_uosu;om mcm m: Hill. I. ...1 ill .4!) E '- 1!": _ - - u _ _ _ . 1. CRIB _ - . l— u _ h m _ II ... 4 I l I 4. J .. ... ‘ .uwif ...-0: ..O ...; .1 \t v . H u . Q '1. l ‘o . A 1: .5 I ‘. f 1 g o .l< .I .h 1 ‘En. m r 1-. .1... .. Q. . v .. ta ‘0. .. . x . A p. 1. It ft 1 .. 1 1| .5 L .. ..H. 1. u 1- u . x . . . ,. .. . . ll! . A i. In I It a. o . n . .| ‘. .11- ’.o! n c . a‘ . .. .. . . .9 a‘ . ... '| .11 I In..I.\u. .‘Juti . .. 4‘ i... . m _ fi.‘ It 1. . w w ... _ 4 1 .1 v p- I... vs 1 . 0 l I III. Ei 1118 1111111111! 111 .. oumtp>coacmo to; :_mum mw_com v_om o_po_tom n mx mJo ?:_50m x mm _u to :or 1. AC _ b ”V-o _ no. ..Jl.:.l _ u. .to_m _J_C_loor:J:2__ \Llo_-:Jo.wozo .. mpco. coccoo o>_._m01-_C. com U_oo o_.co_-_:o n mi. mpcocooaoo o>_b. mogzxoo_b Ou_2< u 7;- :_ J_o;ro_BJ _o om .2 z u .1 C: LL] '.’..' v7 :— 3 V . —-L_‘..1 ...Q— (“\Q' S 1 '_! f". \ud 1 u . (lili’I‘ .1 pl 1111’. 5.‘PI.. .. .. . .. lil‘".b'llr.... 1.! .l..‘lll'.l. O’a’ilvk’.)\ 1‘..'Ilt l 1. ’(‘l’rlb‘l’l’i’tli x. ..m m o ......o C M0J. .c::;s__.c:w Fto_m:CC_wxo:3:::_ mp:o_tob_fio:o .mwmchw:x:otuomeTo on_mo -rw_~moms_ _aJm;:v_t :_ woJcpsuxo mocn;gyb;oo _.o_;.n-:uzto%:.uno I(.:.: : .mm ~J_LJC 150 .Am o_cubv A..mm:v a bomtbxo omcommtu_: tam Amsv boo 5rd 20:: Co my h%_ocm o_com_ncm m>_uoccesoo E .m.m 151 < , _ . n .u ..r . Reflux»; .-... .m. .AN o_3obv Am-um:v m bootu>o omccmocu_3 pcm Away bomcu:o Locbo Co m_.>_mcw omroo .:J o>m. _ . «N .1 152 boocuxo o_:omwtb_: Ugo Amomv somcu_ocs o_com_uzm o>_bchmxcu iMmewvxu\ 155 .Am 0.;sCV “n-1mzv 3 L boocb_mco o_:.m_oco o> \ 154 \I coc_bcw o>_umncmiou .m: .m_m o_ncev Ag-mm:v m Dostoxo o_:omcto_3 can Auov .o;tuxo o:_t_:w:m U- 0 (I) U". >\ ,._ (J C: (.3 U 7,, \0J 155 A>vo_3m_> sotC oo:_osno Ambv Doctoxo coo_cb bl ‘ I .-IQIIOIJI vcm Away out: Co .Jn>_ccm o_fi LW>CL ~;._c.s9 n.6o..M Co m__oo Agv Uo___ _c:o 2Jc_bco o>mumcoafiou .3 .\ . \\ \\ \\.. . I..\‘4. \\ \\\\ .\\. I \\\\ Q 156 .-fiiul“ \ ....ull.I)I. .I .157 .w@.;sm w:+flwwwqmw tongs _ 0.1.x--. . . .m_m Co m__oo o_nm_> Co Doolux0mop Es_vtm Co EwLmOLOcaoLuoo_ cases 158 2.2.5.2 Co 3.00 o_._,._> Co some? not: .Co cacaotozgoioo30E...:...___ o_.J.m.:...w-_mE in: .mi 159 .m.>ob E:.tobo -..]. mfiruux...‘ Co m__oo o_bm.> Co Domtbxm oc.v.cm:m Co Emtmo.o£aotuoo_00czcs_ omumEmme_o fJHIJh: ..u-..ll..lll.\\.1ll.. \IV Iii-Ill,“ II”.II.III|I\ .om ...... 140 Schematic disc electrophorograms of the different chemi- cal bacillary extracts are presented in Figs. 51-54. The number of detectable amido black-positive and PAS-positive components in chemical bacillary extracts were six amido- black and four PAS-positive components in TE (Fig. 51); 13 amido black and six PAS-positive components in SDE (Fig. 52); four amido black and three PAS-positive components in UE (Fig. 55); and six amido black and three PAS-positive com— ponents in GE (Fig. 54). Rf values for the disc components detected in chemical extracts are listed in Tables 25 and 24. Zone electrophoresis in cellulose acetate membrane separated each of the chemical bacillary extracts into at least three protein-stained zones (Fig. 55). The relative amounts in each of these fractions differed among different extracts. Schematic immunograms showing comparative analyses of chemical bacillary extracts are presented in Figs. 56-59, common antigens were detected among many of the chemical extracts. Comparative immunodiffusion analyses of chemical bacillary extracts and filtrate from six—month—old cultures demonstrated the presence of common antigens: six in SDE (Fig. 60) and seven in TE (Fig. 60). Skin Testing with Ultrasonic and Chemical Bacillary Extracts Unfractionated ultrasonic extracts and chromatographic fraction I G-25 elicited dermal reactions in normal rabbits 141 .mm>ob Es_touomnoomm mo m__mu m_nm_> mo bumtuxo cou_tu mo EmLmOLOxdotuoo_m om_p o_umEmtmmma ._m .mwm mqm ..c i11- IIH.’P [I III] [III 1142 .mm>ob E:_Louumnou>2 mo m__mu o_nm_> mo uumtuxm mum_o£o>xommp E:_p0m mo EmtmoLOzdotuum_o om_p umumsmtmmmo .Nm .m_m m5. l | I“ ma llHl IHHI l .143 .m_>ob s:_tmuumnoo>z mo m__mu o_nm_> mo uumtuxm mot: mo EmLmot0cdotuum_o om_u u_umsmtmm_a .mm .m_m ac.— . ac . .. VIIIL _ m m g144 .m_>Ob s:_tmuumnoo>z mo m__ou m_nm_> mo uumtuxo oc_p_cmam mo EmLmot0cdotu00_m om_p u_umsmtm_a .:m .m_m I l mg.— filllll|.|||||u 4 1 P- Illrl'i 1145 Table 23. Distribution of amido black-positive components in disc electro- phor09rams of chemical bacillary extracts obtained from viable cells of Mycobacterium bovis. Chemical Baciliarx Extract Rf TE' $052 UE3 GE“ 0.15 x x x 0.17 x x 0.2i x 0.31 x x 0.33 x x x x 0.08 x x x 0.60 x x 0.65 x x x 0.75 x x 0.77 x 0.8} x 0.87 x 1.0 x x x x 1 TE = Triton extract 2SDE - Sodium desoxycholate extract 3UE = Urea extract 4GE = Guanidine extract 1146 Table 20. Distribution of PAS-positive components in disc electrOphorograms of chemical bacillary extracts obtained from viable cells of Mycobacterium bovis. Chemical Bacillary Extract .RF 1:] $052 UE3 GELl 0 4 x x 0.l5 x 0.l7 x x x x 0.28 x x 0.3l I x 0.33 x x l.0 - x x x x TE - Triton extract 2SDE = Sodium desoxycholate extract 3UE B Urea extract 46E - Guanidine extract C .uuuhuxm :0u_cu u up «buncuxo o:_v_cm:m u mo muumcuxu oum_o£u>x0mop E:_vom u mom uuumcuxo wot: u m: .m_>on amicouumnou>z mo m__ou o_nm_> we muumcuxo >cm___umn .mu_so;u mo msmcm0c05a0cuuo_o oumuoum omo_:__mu u... no man u: 147 .mm .m: 1148 .Escom_ucm I m< ”bumcuxo not: a m: muoucuxo cum—Onuxx0mou s:_u0m a mom «yuccuxu c0u_ch u uh .muoocuxo >co___umn .uu_socu mo mom>_mcm u~c0m~uco o>_umLmasou //(6 @ 1 o////.\& / 9 .mm a: 1149 .sacum_uc< u m< uuumcuxo coo—ch u up uuumcuxu o:_v_cmac a mo unoccuxo cocuu a mu .muuucuxo >ca___uma .mu_socu mo mom>_mcm u_:om_ucm o>_umcmdsoo .mm .m_u . 1 b « 6/ @ e«\\\e. 1150 n\. .sscommuc< a m< “numcuxo cocum u mm "yuccuxo uc_v_cm:o a mu muumcuxo cum—ozu>xomou e:_v0m a mom .muumcuxo >cm__~umn .mu_sozo mo mom>_mcm o_com_ucm o>mumcmaeou Kréx . é .w/Mll 97/ .mm .m_m .151 .Escom_uc< u m< muuccuxu Locum a mu “pumcuxo c0u_ch n uh muuocuxo out: a u: muomcuxo cum—o:o>x0mou s:_u0m a mom .muuucuxo >cm___umn .mu_so:u mo m_m>_mcu u_com_ucm o>_umcmasou .mm .m_u 1152 .Amuv m_>0n amwcouumaou>z mo moc:u_:u v.0nzucosux_m sot» voc_muno oumcu__w new Amomv muumcuxo mum—o;u>x0mov E:_v0m new why c0u_cu mo mum>_mcm u_com_ucm o>_umcmasou .om .m_u 79b \ 9 Ao\ _ (a 155 detectable three hours post-inoculation. The erythermatous reactions usually subsided within 24-48 hours. Unfraction- ated ultrasonic extracts and chromatographic fractions I and II G-25 elicited immediate reactions in rabbits sensi— tized with heat-killed fl, bovis, strain 510. Slight re— actions were detected with chromatographic fraction III in sensitized but not in normal rabbits. Reactions elicited in sensitized rabbits were greater and persisted longer than in normal rabbits. Slight reactions were detected in normal rabbits skin tested with the chemical bacillary extracts. All of the chemical bacillary extracts elicited immediate reactions in sensitized rabbits which were greater than in normal rabbits. DISCUSSION Antibody production was studied in experimental models analogous to (a) active tuberculosis, (b) limited (closed) tuberculosis with tuberculoimmunity and tuberculin sensi- tivity, (c) no tuberculosis with tuberculoimmunity and tuberculin sensitivity, and (d) no tuberculosis with tuberculo- immunity and reduced sensitivity. Mycobacterium bovis of different strains or prepared by different methods were in- jected into rabbits to simulate these conditions as follows: (a) active tuberculosis: Group I; viable, virulent g, bovis (strain 510) (b) limited tuberculosis with tuberculoimmunity and tuberculin sensitivity: Group II-—attenuated La. bovis, BCG (c) no tuberculosis with tuberculoimmunity and tubercu— lin sensitivity: Group III heat inactivated.fl, bovis (strain 510) Group IV—BPL inactivated g, bovis (strain 510) Group VI-acetone inactivated g, bovis (strain 510) (d) no tuberculosis with tuberculoimmunity and reduced sensitivity: Group V-betaprone inactivated g, bovis (strain 10) treated repeatedly with acetone and methanol Antibody production in infected individuals is generally considered to be of little diagnostic or prognostic signifi- cance in tuberculosis or tuberculoimmunity. The role of delayed hypersensitivity in either is not known. Virulent g, bovis (strain 510) infects and produces tuberculosis in rabbits. Therefore, a small inoculum 154 155 administered subcutaneously was used to avoid a rapidly progressive, fulminating and fatal disease in rabbits of Group I. It was hOped that the antibody response would vary during the prolonged period of disease. Antibody production by these rabbits was negligible. Antibody was detected in very low amounts and was irregular in occurrence. The irregular occurrence of antibody in diseased individuals is a general characteristic of tuberculosis (159). Sufficient experimental precedent has established the use ME-sensitivity to differentiate IgM from IgG. Myco- bacterial cells elicited first IgM and then IgG in the experimental rabbits. This general pattern existed in all the groups tested, although variations occurred in the temporal and quantitative relationships of hemagglutinins and bacterial agglutinins. Antibody produced during the first two weeks was exclusively IgM. Thereafter IgG was produced in increasing amounts. The IgM persisted through- out the duration of the experimental period (8-25 weeks) in all groups tested except Group I. This is consistent with other reports of prolonged IgM synthesis in rabbits immun— ized with inactivated mycobacteria. This is undoubtedly associated with the relatively slow degradation of myco- bacteria in the host thus providing a prolonged exposure to the antigen. This is in contrast to the reaction elicited by a more rapidly metabolized antigen in that the IgM is produced for a relatively short period of time. 156 The antibody response to infection with BCG (Group II) was comparable to that induced by any of the killed cell preparations. Mercaptoethanol—sensitive hemagglutinin titers were significantly higher at the first and second week post- inoculation in sera from rabbits in Groups II, III and IV than in sera from rabbits in Groups V and VI. Thereafter, rabbits in Groups II, IV, and VI produced significantly more ME-resistant hemagglutinins than rabbits in Groups IV and V. Bacterial agglutinins were produced by rabbits in Group II in significantly greater amounts than by rabbits which received inactivated cells. Because there was multiplica- tion of BCG in the rabbits in Group II, the actual number of cells to which the rabbits were exposed is not known. It would probably be less than the number of virulent fl, boyig cells (Group I) and more than the number of killed cells (Groups III—VI). The significantly better antibody response to BCG than to inactivated cells may be due to differences in antigen dose or to the differences in processing anti— gens in viable, multiplying cells and killed cells. It is interesting that sera from rabbits in Group II, inoculated with BCG, consistently yielded higher titers of bacterial agglutinins using BPL-killed cells than sera from rabbits in Groups IV and V which were inoculated with BPL-killed cells. This indicates the close antigenic relatedness of somatic surface antigens between E, bovis (510) and BCG. A more important point can be suggested from the related- ness of BCG and BPL-killed cells. Betapropriolactone 157 effectively inactivates g, bovis and BPL—killed cells have been shown to elicit tuberculoimmunity in mice and guinea pigs and tuberculin sensitivity in the latter. Betaproprio— lactone reportedly does not denature proteins and therefore provides a means of inactivation without loss of antigenicity. The reaction of the BPL-killed cells with the sera from rabbits inoculated with BCG may indicate promise of the use of inactivated cells in agglutination tests. Rabbits in Group IV and V which received BPL-killed cells produced significantly less ME-resistant antibody than rabbits in the other groups. Moreover, rabbits in Group V failed to produde detectable hemagglutinins during the first week-post-inoculation. These results contraindicate the use of BPL-killed mycobacteria as antigens for antibody produc- tion in rabbits. Results from recent investigations have indicated that simultaneous rather than sequential production of IgM and IgG may occur with different antigens, and that the two antibody responses are independent of each other (210). An explanation of the earlier detection of IgM antibody is thought to be the greater sensitivity of agglutination re- actions for the detection of IgM than for IgG (16). Our results of antibody titrations using agglutination reactions should be interpreted with the understanding that IgM is more efficient in agglutination on a molar basis than IgG and that both immunoglobulin species may be produced simul— taneously. To conclusively determine the temporal sequence 158 of the IgM and 196, a serologic test is needed which is equally sensitive for the detection of both. Hemagglutination is more sensitive than bacterial agglu- tination for antibody determinations. Hemagglutinin titers were consistently higher than agglutinin titers in the same sera with few exceptions. However, the temporal pat- tern of synthesis of ME-sensitive and ME-resistant antibodies was similar. It is not known whether the two tests measured the same or different antigen—antibodies systems. If the two test measured the same antigen-antibody systems, hemag— glutinins would be detected earlier and in greater amounts. Hemagglutinins are reportedly Specific for tuberculOpoly- saccharides. The nature of the agglutinogens are not known other than they are apparently on the surface of the cell. They may also be carbohydrate since the surface antigens of tubercle bacilli are reportedly carbohydrate (116). Because g, bovis cords, that is, forms chains of cells which tightly adhere to other chains of cells, it is diffi— cult to disperse them evenly to standardize inocula. Therefore, there were undoubtedly some differences in the antigen concentration given to each rabbit which could influ- ence antibody responses. However, it is not believed that the slight differences in the cell concentrations would cause significant differences in the serum titers. Due to widespread distribution of mycobacteria in the environment, it is possible that some degree of heterologous antigenic stimulation occurred prior to inoculations. 159 The rabbits were not skin tested prior to inoculation but were obtained from a colony in which the rabbits are and remain tuberculin negative. None of the serum collected prior to inoculation contained detectable antibodies. Skin testing with PPD-S 14 weeks post-inoculation stimulated antibody production in approximately 50% of the rabbits tested. An anamestic-like response following skin testing has been reported to occur in cattle and swine (111). Mean hemagglutinin titers were elevated in Groups II and V but not in Group IV, following skin tests. Bacterial agglutinin titers were elevated only in the serum from rabbits in Group II. Corresponding elevations of the ratios of ME-sensitive/ME-resistant antibodies indicate that skin testing may have preferentially stimulated the production of Ig-M. Whether this is a specific, homologous anamestic response is not known. It is significant, h0wever, that tuberculin testing apparently changed the absolute and relative amounts of Ig-M and Ig-G synthesized. Perhaps more significant changes in the absolute and relative amounts of ME-sensitive and Me-resistant antibody may have been detected if the rabbits were bled at various intervals after skin tests. It is probable that tuberculin testing alters other immunologic processes in still undetermined ways. For this reason, the effect of tuberculin testing should be carefully evaluated when conducting in yiyg, in vitro or serologic tests. 160 Inoculation of rabbits with viable or killed M. bovis induced sensitivity to tuberculin. Rabbits injected with BCG developed greater tuberculin sensitivity than rabbits which were inoculated with BPL-killed.M, bovis or BPL-killed methanol and acetone extracted M, bovis. The ability to induce delayed sensitivity was reduced considerably by the treatment by BPL-killed cells with methanol and acetone. The kinetics of the antibody response to individual mycobacterial antigens cannot be adequately studied until purified antigens are obtained. It is significant that mycobacterial antigen preparations currently employed for serodiagnosis of tuberculosis are chemically and antigenically heterologous. It is only logical that more reliable results can more probably be obtained with a less heterogenous mix- ture. If antigens can be isolated there is a greater prob- ability of obtaining one which is specific. Disruption of viable cells by ultrasound is a safe, effective means of obtaining mycobacterial antigens. The relative amounts of protein, carbohydrate and nucleic acid in extracts differs with the age of the cultures from which the cells are obtained and the duration of insonation to which the cells are exposed. After centrifugation and prior to filtration, ultra— sonic extracts were opalescent and probably contained relatively large amounts of lipoprotein in the form of particulate micells. Membrane filtration clarified the 161 extracts and probably removed some of the soluble protein as well as lipoprotein material. The qualitative and quantitative loss of chemical and antigenic constituents of ultrasonic extracts due to filtration was not determined. It was considered to be the method of sterilization which would most probably reduce the constituents the least. Because of the nature of the organism some method of sterili- zation was necessary. The amount of nucleic acid in ultrasonic extracts was indicative of the extent of cell disruption and was corre- lated with the intensity of insonation. Ultrasonic extract-C which received the longest period of sonication, contained substantially more nucleic acid than USE-A or B, all three extracts were obtained from cells from six-month-old cultures. When the duration of insonation was the same but of cells from two-month-old cultures (USE-D), more nucleic acid was present than in either the USE-A or B. Cells from younger cultures appear to be more fragile than those from older cultures and shorter periods of insonation should be used with the younger cells to achieve the same extent of cell disruption. This may be due to the fact that cells from older cultures are undergoing extensive autolysis and that a considerable percentage of cells are nonviable. Another indication of the extent of cell disruption is found in the carbohydrate and protein concentrations in ultrasonic extracts. Again, USE-C which was irradiated for 162 the longer period, contained more than twice the amount of carbohydrate and significantly more protein than was present in other extracts of cells of the same age but which received a shorter period of insonation. Ultrasound causes varying degrees of depolymerization of biomacromolecules. The distribution of nucleic acid and protein in the three major chromatographic fractions from Sephadex G-25 are illustrative. Molecular exclusion chromatography is an effective method of comparing the rela- tive sizes of molecules. Major fraction I from Sephadex G-25 contains molecules whose average molecular weight is greater than 5000 since they were eluted within the external volume of the columns. Fraction II and III were eluted within the internal volume of the column and contained mole— cules whose average molecular weights are less than 5000. Nucleic acids as they occur in viable cells exist as high molecular weight polymerized biomacromolecules. Approxi- mately 50% of the nucleic acid detected in USE-A and B was present in fraction I whereas only 10% was detected in frac- tion I from USE-C. This indicates that rather extensive depolymerization of the nucleic acid had occurred in USE-C presumably as a result of insonation. Protein was estimated in ultrasonic extracts using two methods; a chemical determination (Folin-phenol reaction) and absorption at 280 mu. Values obtained by these two methods for the relative protein concentrations in chroma— tographic fraction Ig_25 from USE-A, B and D were in close 165 agreement. This was not true of fraction I from USE-C. The reason for this discrepancy is not known. No corre— lation was observed between values obtained for proteins in fractions II and III using these two methods. Protein concentration, at least in bacillary extracts, cannot always be accurately determined using the Folin- phenol reaction. This is illustrated by the observation that of 7.8 mg of protein detected by the Folin reaction per ml of USE—C only 44% or approximately 5.4 mg/ml actually represented material of molecular weight greater than 5000. Ten percent, or 10.34 mg/ml, was detected by absorption at 280 mu. Recognition of this limitation of the Folin- reaction for protein determination is important in standard- izing antigen-containing solutions for serologic and ana— lytical purposes. A correction factor must be introduced when protein solutions contain low molecular weight Folin— reactive material. Protein was apparently "denatured‘l by prolonged ex- posure to ultrasonic vibrations. This can be seen by the rather high relative concentrations of 280 mu-absorbing material in fraction II of the chromatogram of USE-C as compared to the relative concentration in the other ultra- sonic extracts. Chemical analyses of chromatographic fractions I, II and III from Sephadex G—25 indicated that fraction I con- tained proteins of molecular weight greater than 5000, 164 the majority of carbohydrate and a considerable amount of highly polymerized nucleic acid. The presence of nucleic acid in fraction II was detected by the strong 280 mu absorption both in chromatograms and ultraviolet spectra. Fraction II contained a substantial amount of Folin-reactive, 280 mu-absorbing material. It was presumably peptide. Very little carbohydrate and a considerable amount of 260 mu-absorbing material, presumably oligoneucleotides were detected. This fraction was free of detectable antigens but provoked skin reactions in sensitized rabbits. It may be a promising source of sensitins for detecting tuberculo- sis. Fraction III consisted of 280 mu-absorbing material, presumably small peptides, no detectable carbohydrate and some 260 mu-absorbing material, presumably neucleotides. Fraction III from USE—C contained substantial amounts of the low molecular weight nucleotides and peptides, presum~ ably as the result of depolymerization of the respective biomacromolecules. Disc electrophoresis effectively separated many of the constituents of ultrasonic extracts of mycobacteria. The separation achieved by zone electrophoresis in cellu- lose acetate membranes was less satisfactory. Disc electro- phoresis is a means for effective reproducible separation and is economical from the standpoint of time, equipment and sample size required for analyses. 165 The chemical heterogenity of ultrasonic extracts is evident in disc electrophorograms. Between 16 and 24 amido black staining components were detected in differ- ent extracts. It should be noted that although disc electrophoresis is reproducible, variations do occur in gel concentrations, the degree of polymerization and in the relative concentrations of individual components in differ- ent samples. Thus, variations in line intensity and dis— placements do occur and not all electrophorograms contain each component consistently each and every time. Optimal results are obtained by running samples in duplicate, preferably in triplicate, and comparing the disc electro- phorograms following completion of such sets. It may be possible to increase the resolution of the technique by the alteration of gel concentration since disc bands which appear homogenous may contain more than one antigenic com— ponent when separated on a different pore size gel (156). As many as 18 PAS-stained components were detected in disc electrophorograms of ultrasonic extracts. The PAS reaction results in staining of both carbohydrates and glyco~ proteins. No amido—black-positive components were detected in the Spacer gels indicating that the PAS stained compon- ents in this area are carbohydrate. Five to six distinct PAS-stained bands were frequently detected in the Spacer gels of electrophorograms of the ultrasonic extracts. PAS-stained components in the lower gel could be either 166 carbohydrate or glyc0protein. Many of the PAS-positive bands had Rf values identical with amido-black-positive bands indicating that they are probably glycoproteins. The presence in electrophorograms of numerous PAS-positive components from ultrasonic extracts suggests the native state of these molecules since carbohydrate and protein probably exist in the cell wall as complexes containing both carbohydrate and protein. The most anodic amido black and PAS—positive band was quite prominent in disc electrophorograms of all of the ultrasonic extracts. A yellow, nonstaining component was frequently associated with this band and was also seen in disc electrophorograms of chromatographic fraction II from Sephadex G-25. Mycobacterium bovis is generally described as producing no pigment, however, this is in comparison to other strains of mycobacteria and the component detected is undoubtedly pigment. Disc electrophorograms regularly con~ tained discrete, nonstaining opalescent bands. The sig- nificance or chemical nature of these are not known but it is probable that they are of a lipid nature. Analyses by Ouchterlony immunodiffusion tests was the first method used to approximate the number of antigens in ultrasonic extracts of M, boyig. It had been determined in preliminary tests that the maximum number of immuno~ precipitates with the reference system was obtained when the .protein concentration was approximately 2 mg/ml. Since 167 multiple antigen-antibody systems are involved, however, optimal ratios of all of the reactants could not Simul- taneously exist. Thus, it is practically impossible to achieve maximum resolution of the antigenic composition of complex mixtures using this method. Between 15 and 17 separate immunoprecipitates were detected in immunograms of various ultrasonic extracts. While antigenic and chemical analyses of purified fractions obtained from ultrasonic ex- tracts may eventually yield some classification of myco— bacterial antigens, the complexity of the mixtures at this point would make such a classification premature. Maximum resolution was achieved by making daily readditions of dilutions of both reactants. This procedure may, if not properly performed and analyzed, cause splitting of lines and lead to erroneous assumptions. The number of separate antigen-antibody systems enumerated were less than were detected using immunoelectrophoresis. The number of anti- gens detected are undoubtedly less than the number which actually exist. Many of the immunoprecipitates detected in immunograms of ultrasonic extracts were lightly stained and difficult to detect consistently unless individual plates were care- fully analyzed under optimal conditions. The reference system (USE-A with anti-USE-A) yielded 15 immunoprecipitates. This represents the minimum number of antigens in the ex- tract. Seven distinct antigen-antibody systems have been 168 reported in extracts of mycobacteria obtained by mechanical disruption in a pressure cell. Culture filtrate, the same culture filtrate from which cells were used for the prepara— tion of USE-D, and ultrasonic extract D were antigenically indistinguishable using anti-USE-A serum. These results are in general agreement with an earlier report (25) and indicate that bacillary extracts and culture filtrates con- tain the same antigens if filtrates of the appropriate ages are used. The antigenic composition of bacillary extracts is consistent although variations may occur in the relative concentrations of the components. Antigens with Similar diffusion constants but different electrokinetic properties were separated by immunoelectro- phoresis. As many as 20 to 22 immunoprecipitates were detected in USE-D, three to five more than detected by Ouchterlony diffusion. It is perhaps Significant that a maximum of 18 antigen-antibody systems were detected in immunoelectrophorograms with the reference system. Apparently USE-A contained at least 22 distinct antigens in a concen— tration sufficient to stimulate antibody production but only 18 of these antigens existed in sufficient concentration to be detected in immunoelectrophoresis. A region of diffuse immunoprecipitation was frequently observed in immunoelectro- phorograms of ultrasonic extracts. This was perhaps due to the presence of several antigens which do not differ sig- nificantly in electrokinetic properties or diffusion 169 constants and which precipitate together. A comparison of the number of components detected indicates that disc electrophoresis, immunoelectrophoresis and Ouchterlony im- munodiffusion demonstrated decreasing numbers of myco— bacterial constituents. Fractionation of ultrasonic extracts by molecular exclusion chromatography in Sephadex or Bio Gels having different molecular exclusion limits did not yield pure antigen preparations. Fraction I from Sephadex G-25 and unfractionated ultrasonic extract were virtually indis— tinguishable by Ouchterlony immunodiffusion, immunoelectro- phoresis and disc electrophoresis. Thus, fraction IG-gs seems to be an ideal preparation to be used for subsequent fractionation for antigen purification since it is free of much of the low molecular weight material present in the crude ultrasonic extract. Dialysis for extended periods with repeated changes of buffer also yields a preparation rich in chromatographic fraction IG-25 and contains only small amounts of fractions II and III. Chromatography in Sephadex G-25 indicated that approxi- mately 49% of the 280 mu—absorbing material in USE-B had an average molecular weight of greater than 5000. . Of this, 84%, 82% and 59%, had molecular weights of 100,000, 150,000 and 200,000 or greater, respectively. It is apparent that a substantial percentage of the antigenic material in ultrasonic extracts is present in a high 170 molecular weight fraction, greater than 200,000. Ouchterlony immunodiffusion analyses of the fractions eluted from dif— ferent Bio Gels confirmed that although some degree of purification had been achieved, each fraction remained anti- genically complex. Molecular exclusion chromatography alone is not sufficient to yield purified mycobacterial antigens. Hopefully, molecular exclusion chromatography can be allied with other fractionation procedures such as ion exchange chromatography and preparative disc electrophoresis to ob— tain purified antigens from mycobacteria. Ultrasonic ex— tracts provide a rich source of undenatured antigens from which these purified antigens can be obtained. Antigens were obtained by chemical extractions of viable mycobacterial cells but no rigorous chemical or anti- genic analyses were made of these extracts. The amount of cells of M, bovis by its nature were limited and ultrasonic extraction appeared more promising in preliminary experiw ments. The chemical extracts were made by procedures which have been employed for other bacterial species and therefore, optimal conditions for extraction of mycobacterial cells may not have been used. The analyses of these extracts may not represent the maximum number of antigens obtainable by the various methods. Many of the chemical bacillary ex- tracts had antigens in common which indicate that different procedures can remove the same antigens. However, the chemical or antigenic nature of these extracts have not been 171 analyzed sufficiently to classify or identify individual components. They were examined with antiserum Specific for ultrasonic extracts, thus, antigens unique to the chemical extracts if they exist would not be detected. Urea and guanidine denature proteins by the disruption of secondary bonds which disorganizes the tertiary protein structure. Five antigen—antibody systems were detected by immunodiffusion analyses of the urea and guanidine extracts. Differences were detected by immunoelectrophoresis and disc electrophoresis. Because of the denaturative properties of urea and guanidine, the Significance of these differences is uncertain. The mechanisms of action by which tritron X-100 and sodium desoxychobate release components from bacterial cells is speculative. These compounds probably interfere with hydrophobic bondinganflithereby disorganize the three dimen- sional structure of lipoprotein complexes. Ouchterlony immunodiffusion analyses demonstrated 11 antigen-antibody systems in each of these extracts. Immunoelectrophoresis detected 15 and 11 antigens in the triton X- and desoxy- cholate extracts, respectively. Both compounds appear to be effective chemical extractants for preparing mycobacterial antigens. Disc electrophorograms of the triton extract, however, yielded only six amido-black staining bands. Extensive areas of opalescence were present in the gel col- umns after electrophoresis of the triton extract which 172 probably either prevented adequate electrophoretic migration of individual components or interfered with proper staining. It is possible that trace amounts of the triton remain firmly bound to some constituents of the extract and interfere with migration. When the cellular debris remaining after ultrasonic disruption were extracted with phosphate buffer containing ethyl ether, the extract contained only three detectable antigens. There is no doubt that the need exists for specific mycobacterial antigens to be used for the detection of tuberculosis. The need exists equally, if not more so, to study fundamental differences among the mycobacteria and to study the reactions of hosts to separate antigens and various combinations of antigens. Mycobacterial antigen preparations available today are nonSpecific because of their complexity. These preparations cannot be used to differentiate between infection and disease, infection and sensitization, or the species or strain of Mycobacterium causing the infection, disease and/or sensitization. Little is known of the antigenic composition of myco— bacteria despite years of investigation. Neither is the nature of antibody responses to different mycobacterial anti— gens understood nor the complex interplay of the various constituents. Crucial areas of ignorance exist regarding the relationship between delayed hypersensitivity, susceptibility 175 and tuberculoimmunity. In order for these questions to be satisfactorily answered,more detailed information must be obtained regarding the chemical and immunologic properties of the mycobacteria. Purified antigen preparations must be made available and used to investigate fundamental proper- ties of antibody production and delayed hypersensitivity in tuberculosis. Recently developed ig_yiyg_and in_vitro procedures are amenable to this type of study if more puri- fied mycobacterial constituents are available. Extracts obtained by mechanical disruption of M, boyis (510) by ultrasound contain a minimum of 22 antigens detect- able by specific antibodies. This undoubtedly represents the minimum number present in these extracts. Moreover, each of these antigens may well contain multiple antigenic deter— minant sites. Which of these antigens is of potential Sig— nificance in tuberculoimmunity or tuberculin hypersensitivity remains to be determined. Whether any of these antigens are specific is not yet known. SUMMARY Antibody production was studied in rabbits inoculated with different preparations of M, bgyig. Experimental models were designed to simulate (a) active tuberculosis, (b) limited (closed) tuberculosis with tuberculoimmunity and tuberculin sensitivity,(c) no tuberculosis with tuberculoimmunity and tuberculin sensitivity,and (d) no tuberculosis with tuberculo- immunity and no tuberculin sensitivity. Passive-hemagglutina— tion and bacterial agglutination tests were used for antibody determinations. Rabbits (a) injected with virulent M, bovis had gross lesions and produced negligible amounts of antibody and rabbits,(b) injected with attenuated M, bgyig (strain BCG) prOduced significant amounts of antibody comparable to anti- body produced by rabbits,(c) inoculated with killed prepara- tions of,M.lMQyi§. Antibody elicited by heat or acetone-killed cells was somewhat greater than elicited by cells which were killed with betapropriolactone, with or without subsequent extraction with methanol and acetone. A sequential production of first mercaptoethanol-sensitive followed by mercaptoethanol-resistant antibody was observed in all rabbits regardless of the antigen preparation or the serological test used to detect antibody. Antibody detected during the first two weeks post-inoculation was exclusively 174 175 mercaptoethanol-sensitive. Thereafter, mercaptoethanol— resistant antibody was produced in increasing amounts. In most cases, mercaptoethanol-sensitive and resistant antibody was detected for the duration of the experimental period, up to 25 weeks post-inoculation. Skin testing with purified protein derivative 14 weeks post-inoculation stimulated antibody production in approxi- mately 50% of the rabbits tested. Both the absolute and relative amounts of the different types of antibody produced was altered by skin testing. All rabbits inoculated with viable or killed.M, bovis preparations became hypersensitive to tuberculin although extraction of betapropiolactone-killed cells with methanol and acetone diminished their ability to sensitize rabbits to tuberculin. The antigenic and chemical composition of ultrasonic extracts of M, bovis varied with the age of the culture from which the cells were obtained and the intensity of insonation. Ultrasonic extracts of cells from two-month-old cultures con- tained more antigens than were detected in extracts from six- month-old cultures. The number of antigens in ultrasonic extracts was lessened by exposure of cells to long durations of insonation. Disc electrophoresis detected the highest number of chemical constituents in ultrasonic extracts. Between 16 and 24 amido black-stained components and 15-18 PAS—stained components were detected in disc electrophorograms of dif— ferent ultrasonic extracts. 176 Ouchterlony immunodiffusion detected 15—17 distinct antigen-antibody systems in different ultrasonic extracts. As many as 20—22 antigens were detected in ultrasonic ex— tracts by immunoelectrophoresis. A substantial porportion of the antigens in ultrasonic extracts have average molecular weights of greater than 200,000. Molecular exclusion chromatography and ion exchange chromatography provide a method of preparative fractionation for antigen purification. Viable cells of M, bovis were extracted with Triton X-100, sodium desoxycholate, urea, and guanidine, and cellu- lar debris was extracted with phOSphate buffer containing ethyl ether. 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