“(HE NEfiRCUWMMUWERW mm M AND WNW: M68 as MYCQQAWEW MW! ifiACTWA'WD th BETA- memomamm CQMtiAREfi 1‘0 WAT OF 3123. WMQL EHAC‘HVATED W Add!) YEWW same? Thesis £0: flux Beam a? M. S. Méflflfififl STATE WWW Ci‘akpe 42 3E await-View 1% '2 THESIS LIBRARY Michigan State Unive rsity I “3.; (31’3\ ABSTRACT Onyekwere, Okpo, 0. (Michigan State University, East Lan- sing, Michigan). The tuberculoimmunogenicity for mice and guinea pigs of Mycobacterium bgzig_inactivated with beta- propiolactone as compared to that of ECG, phenol inactivated cells, and Youmans' extract. 1962.- Studies were made of the inactivation of Mycobacterium 221;g_with beta-propiolac- tone (BPL) for the preparation of a bacterin. A final con- centration of 0.1% BPL was required to inactivate 3 x 107 cells per ml distilled water if incubated 2 hr at 37 C and the pH maintained at 7.6 t 0.4. A safe effective bacterin was prepared with 0.4% BPL under the same conditions. The greater concentration provided a satisfactory safety factor. Washing the inactivated cells 4 times with distilled water removed toxic products due to the BPL treatment. The tuber- culoimmunogenicity was measured by the difference in the percentage of the vaccinated and unvaccinated experimental animals surviving after challenge. When two-thirds of un- vaccinated mice had died, 72%, 69% and 58% of BCG, BPL-bac- terin and phenol-bacterin vaccinated mice survived, respec- tively.. When two-thirds of unvaccinated guinea pigs had died, 100%, 90% and 80% of BCG, BPL-bacterin and phenol-bacterin vaccinated guinea pigs survived, respectively. Washing the phenol and BPL bacterins with acetone did not improve their efficacy. The BPL-bacterin induced tuberculin sensitivity in guinea pigs. An extract was prepared from g, 221i§_which conferred tuberculoimmunity to guinea pigs assumed to be 512:1 9—” .__.. - Ig—o similar to that conferred to mice by Youmans' extract of g. tuberculosis. THE TUBERCULOIMMUNOGENICITY FOR MICE AND GUINEA PIGS OF MYCOBACTERIUM §QEL§ INACTIVATED WITH BETA-PROPIOLACTONE COMPARED TO THAT OF BCG, PHENOL INACTIVATED CELLS, AND YOUMANS' EXTRACT. BY Okpo 031 Onyekwere A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Microbiology and Public Health 1962 a.) a .- r, I . V. f u ; ' . k m U‘ I - I , x ’ .0 3 "? I.- I n . I f; (- f ‘ ' a. 1 ACKNOWLEDGEMENTS The author wishes to express his sincere thanks to Doctor Virginia H. Mallmann for her sympathetic and able guidance during the progress of the research and prepara- tion of the manuscript and to Doctor w. L. Mallmann and Doctor J. J. Stockton for their counsel. Thanks is eXpressed to everyone in the Tuberculosis Research Project for their friendliness and cooperation. In addition, the author acknowledges the assistance of his wife in typing the manuscript. TABLE OF CONTENTS page INTRODUCTION.......................................... 1 HISTORICAL REVIEW..................................... 3 Tuberculoimmunity afforded by BCG................. 3 Tuberculoimmunity afforded by bacterins prepared With phen010000000000000000.00000000000000000.00.. 12 Extracts of mycobacteria.......................... 15 Specificity of tuberculoimmunity.................. 21 Evaluation of tuberculoimmunity................... 22 Beta-propiolactone................................ 24 MATERIALS AND METHODS Glassware, media, cultures, animals............... 28 Preliminary studies of the inactivation of g. bovis (Ravenel) by beta-propiolactone............. 28 Preparation of bacterins.......................... 30 Standardization of bacterins...................... 31 Extract of g, 221$§°°'-°-°--°°°°'*-°--'°°-°--°°°°- 31 BCG vaccine....................................... 33 Determination of the amount of M.‘29yis to be used to challenge the tuberculoimmunity of mice and guinea p188................................... 33 Vaccination of experimental animals............... 34 Challenge of experimental animals with virulent £0 boyisOOOOOOOOOOOOOO.I...OOOOOOOOOOOOOOOOOOOOOO. 34 Hypersensitivity.................................. 34 RESULTS Preliminary studies of the inactivation of‘fl. bovis With BPLOOOOOOOO0000......000.00.000.00....0 35 Determination of the amount of g. bovis required to cause death of mice and guinea pigs............ 37 page Survival of vaccinated and unvaccinated mice after inoculation with M, bovis................... 40 Survival of vaccinated and unvaccinated guinea pigs after inoculation with‘fl, bovis.............. 40 Hypersensitivity of guinea pigs vaccinated with BPL-bacterin and phenol-bacterin.................. 43 DISCUSSION Preliminary studies of the inactivation of g, b0v18 With BPLOOOOOOOOOOOOOOOOOOOO0.0.000.000.0000 47 Evaluation of the tuberculoimmunity afforded mice and guinea pigs.............................. 48 SWYOQOOOOOOOOOOOOOOOOOOOOOOOOO0.000000000000000000 56 LITER-ATURE CITEDOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO0.0.0... 58 TABLE TABLE TABLE TABLE TABLE TABLE 2. 5. LIST OF TABLES The number of days eight-week-old mice sur- vived after intraperitoneal injections of Micgbactezlum bov1800000000000OOOOOOOOOO0.0. The number of days guinea pigs survived after intraperitoneal injections of M c - bacger;fl m!18000000000000OOOOOOOOOOOOOOOOO The days on which vaccinated and unvaccinated mice died after intraperitoneal injection of 32.0 mg wet weight of Mycgbactgrigg bovis.... The mortality ratio and percentage survival of vaccinated and unvaccinated mice inocu- lated intraperitoneally with 32'mg wet weight of Mycobacterium bovis............... The days on which vaccinated and unvac- cinated guinea pigs died after intra- peritoneal injections of 2.0 mg wet weight of Mycobactegium bovis............... The mortality ratio and percentage survival of vaccinated and unvaccinated guinea pigs inoculated with 2 mg wet weight of Mycgbagteziug bovis............... page 38 39 41 42 44 45 INTRODUCTION Tuberculoimmunity is that refractoriness to infection with the tubercle bacilli which can be induced in man and animals with viable or killed organisms (Crowle, 1958a). It is regarded as an immunologic phenomenon but it is dif- ferentiated from the classical immunity associated with circulating antibodies. Specific antibodies are elicited in various hosts by the tubercle bacilli but no immunity is conferred by the antibodies. Since the discovery of the causal agent of tuberculosis, the immunogenicity of living and killed tubercle bacilli has been investigated. Pertinent literature is abundant and contradictory. It is generally accepted that an attenuated living strain of Mycobacteriumqbgzig, the Bacillus of Cal- mette and Guerin, confers the greatest specific protection. Owing to the inherent dangers in the use of any bacterial vaccine, an equally effective bacterin is more desirable. Beta-propiolactone has been used for the sterilization of serum and plasma fractions and the inactivation of viruses and bacteria other than the mycobacteria. It is non-toxic for man and animals in low concentrations which inactivate microorganisms. 1t reportedly does not denature proteins and therefore, the antigenicity should be retained in a non-toxic bacterin. To determine if the tuberculo- immunogenicity of M, bovis is retained after the inactivatial by beta-propiolactone, a comparison has been made of the specific protection afforded mice and guinea pigs by the administration of the following: (a) g, 291;; inactivated with beta-propiolactone, (b) g, 223;; inactivated with phenol, (c) BCG vaccine, and (d) an extract of g, 221;; prepared by the method of Ioumans (1960). HISTORICAL REVIEW Soon after the discovery of the tubercle bacillus (Koch, 1882), the concentrated culture filtrate, 01d Tuber- culin,was used prophylactically and therapeutically (Koch, 1890). The product proved to be ineffective and, in fact, so hazardous that it momentarily jeOpardized Koch's inter- national reputation. Subsequently, it was found to be of diagnostic value. Tuberculoimmunitz afforded by ECG. The first vaccine investigated for immunization against tuberculosis was the Bacillus of Calmette and Guerin (BOG). The history of this strain of Mycgbacteriggpbgzig_has been reviewed by Irvine (1949) and Rosenthal(l957). The organism was isolated by Nocard in 1902 from the udder of a tuberculous cow. Calmette had established a laboratory for tuberculosis research at Lille in 1894 as a branch of the Pasteur Insti- tute. Guerin joined the laboratory in 1897. While re- peatedly culturing the Nocard strain of4g.‘221;§ on gly- cerine-bile-potato medium, Calmette and Guérin found the bacillus had lost its virulence, first for calves , then for monkeys, guinea pigs, rabbits, horses and cattle. This attenuated strain of M,‘bggig_has been known as BOG since 1921 (Rosenthal, 1957). The first attempt to use BCG prophylactically in man was undertaken by Weill-Kalle (1921). It was administered orally at three, five and seven days after birth to an in- fant born of a tuberculous mother and nursed by a tuberculous grandmother. The child did not develop tuberculosis. As a result, routine vaccination with BCG was initiated at the Charite Hospital in Paris. In 1928, at a technical conference to evaluate vaccina- tion against tuberculosis with BOG, it was reported that BCG was a harmless product and an effective vaccine if ad- ministered orally to 10-day-old infants (League of Nations, 1928). f The tragedy at Lfibeck in 1930 is well known (Rosenthal, 1957). The vaccine was administered orally to 240 children. Seventy-two died of tuberculosis. It was later established that the vaccine was contaminated with the Kiel strain, a more virulent organism. Since then, there has been no uni- versal agreement on the innocuousness or efficacy of BCG. The route of inoculation has also been open to question. Calmette 2;. 2;. (1933) detected BCGin an infant's blood three to five hours after the oral administration of a single dose of 30 mg. Similar results were obtained from two chimpanzees which had received one gram of the vaccine. Subsequently, the oral route was used in South America, Canada, Rumania and Scandinavia, and later in New York by Kereszturi and Park (1936) and Levine, gt,al, (1948). The. general fear was overcome after de Assis (1945) successfully administered a dose as large as 60 mg to infants without production of disease. Encouraged by his initial success, de Assis (1947), within four months, used 100 mg as a single dose for each of 15,000 infants. Over 85 per cent of them became tuberculin positive. The reaction to tuberculin was used as a measure of tuberculoimmunity. Martins and Sampaio (1952) adopted de Assis'_method and within a year vaccinated 152,640 persons which included 81,607 infants. Rosemberg (1952) adopted the "concurrent or concomittant" method by which infants who lived in tuber- culosis contaminated environments were frequently revac- cinated. He recommended this method for tuberculin positive as well as tuberculin negative subjects. The first study of the persistence of tuberculin sensi- tivity after intradermal injections of BCG was undertaken by Anderson and Belfrage (1939). At the Gothenburg tuber- culosis dispensary, 905 BCG vaccinated children and adults were tuberculin tested during an 11 year period. Three hundred and ninety seven of them resided with tuberculin positive individuals. Three hundred and one had not been exposed to tuberculosis while the remaining 207 had probably been in contact with virulent bacilli. Tuberculin sensitiv- ity was about the same in each group. Wasz-Hockert (1948) performed tuberculin tests on 1702 children from six months to seven years after the administration of BCG. Between six and twelve months, 4.1 per cent were negative, 7.5 per cent after one year, 9 per cent after two years, 9.3 per cent after three years, 11.3 per cent after four years, 14.9 per cent after five years, 17.5 per cent after six years and 9.7 per cent in the seventh year. EneIL(1952) found that approximately 90 per cent of 4,000 vaccinated school children remained sensitive to tuberculin for seven to eight years. . Rosenthal developed a multiple puncture modification of the subcutaneous method of administration which provided an appreciable amount of protection and was reportedly safer than the other methods (Rosenthal, 1939; Birkhaug, 1944, 1949; Frappier and Denis, 1945; Frappier, gtfl21., 1952; Manda and Huet, 1952; Hall and wylie, 1952). The greatest sensitivity to tuberculin was produced by a vaccination using 40 punctures (Birkhaug,.l949). There is no signifi- cant difference, however, in the immunity afforded by the various methods (Federal Security Agency, U. S. Public Health Service, 1949; Edwards, gt_a;., 1953; British Medical Research Council, 1959). As a result of a single or multiple-puncture intradermal vaccination with BCG, primary tuberculosis in all age groups and tuberculous meningitis in children under a year has been reduced by 70 to 80 per cent (Dahlstram, 1933; Hyge, 1947; Aronson, 1948; Heimbeck, 1948; Ferguson and Since, 1949; Dahlstrom and Difs, 1951). For example, the study of Dahlstrom and Difs from 1941 to 1947 involved 20-year-old Swedish soldiers of comparable race, housing and diet. The vaccinated group of 36,235 soldiers and the unvaccinated control group of 25,239 were examined periodically by X-ray. Tuberculosis developed in the vaccinated group and in the control group at a 1:3.5 ratio. Vaccination with BCG affords some protection (Shaw and Palmer, 1955; Rosenthal, 1955. 1957; Rosenthal and Leppmann, 1953; British Medical Research Council, 1956, 1959). The British Medical Research Council study of 14-year-old urban school children in England demonstrated that BOG vaccination reduced tuberculosis by 82 per cent in a five year period. They stated in conclusion: "According to the present results, if none of the tuberculin-negative entrants had been vac- cinated, 165 cases of tuberculosis would have been expected among them within thirty months of entry. If all of them had received BOG vaccine, 30 cases would have been expected. The difference of 135 cases represents a reduction of 82 per cent in the incidence of tuberculosis in the tuberculin- negative group." A voledbacillus.(g, micrgti) vaccine was used in the study also and compared favorably with 803. Numerous workers have reported the dissociation of the tubercle bacilli, including BOG, but the use of S and R to denote the colony forms has been controversial (Petroff, 1927; Petroff, 22.31., 1929; Petroff and Steenken, 1930; Steenken, gt,§1,, 1934). Steenken (1935) suggested a new terminology which is accepted as standard by the Trudeau Laboratory. The standard 3 and R are modified by sub let- ters; for example, RV = rough virulent, Ra = rough avirulent, etc. The relatively avirulent strain of BOG R1 has been used in experimental animals over fifty years. It can die- sociate into Rlv which produces progressive disease in guinea pigs with silicosis and R13_which is avirulent. (Steenken and Gardner, 1946). The strain of BOGa used for vaccination of manvandiis less virulent than 31, may die- sociate to R1 . Because of this, and because the administrap tion of BCG may give rise to an accelerated type of local reaction in individuals sensitive to tuberculin, the Ameri- can Trudeau Society (1948) recommended that only tuberculin negative individuals be vaccinated. When one mg BCG is injected intradermally. a local ab- scess accompanied by fever results after three weeks. A dose of 0.05 mg is said to be the optimum amount for a single dose vaccine (Wallgren, 1928). Tubercles are pro- duced in many tissues if BCG is injected intravenously. If intradermal or subcutaneous inoculations are made, the le- sions are localized at the site of injection. Bacilli may be widely disseminated but no microscopic or macroscopic lesions are found in internal organs. The untoward reaction in man after vaccination with BOG, the possibility of reversal to a virulent organism, death of organisms during storage, the difficulty of stan- dardization, and the possibility of contamination with more virulent bacilli, are the basis for the contention that the risks outweigh the usefulness of BCG as a vaccine (Weiss, 1959). In spite of the lack of agreement, Denmark, Norway, France, Brazil and Japan legally enforce vaccination with BCG. It is estimated that approximately 17 million people have been vaccinated (Rosenthal, 1957). Oalmette and Guerin (1927) reported that cattle were afforded protection by the annual intravenous or subcutaneous injections of BOG. Watson.gtflgl., (1928) vaccinated l7 calves four to seven days old with 75 to 100 mg BOG. The calves were fed milk from tuberculin positive herds until six months of age and subsequently pastured and stabled with tuberculin positive cattle. No signs of tuberculosis were detectable during the following two years. When the ani- mals were slaughtered, tubercles were found in many of the organs by gross examination. It was concluded that an im- proper dose of vaccine had been administered. Larson and Evans (1929) inoculated cattle with living and heat inactivated BOG. Six months after vaccination, the animals were housed with tuberculin positive cattle. When the animals were slaughtered, 88.8 per cent inoculated with living BOG, 44.4 per cent inoculated with killed BCG, and 87 per cent of the controls were tuberculous. Vaccination with living BOG had afforded no protection. Buxton and Griffith (1931) and Buxton, etal.(1939) substantiated the findings of Oalmette and Guerin (1927). Cattle which had previously received BOG intravenously survived eight months to three years after.inoculations with virulent organisms. Unvaccinated animals survived inocula- tions with virulent organisms less than 60 days. Swine, at 5 and 20 days of age, were vaccinated sub- cutaneously with 10 to 40 mg BOG (Jundell and Magnusson, 1935). The tuberculoimmunity of the animals was challenged with virulent bacilli 13 weeks after the last vaccination. There was no evidence of disease in the pigs vaccinated lo subcutaneously five to nine weeks after the challenge inocu- lation. Repeated subcutaneous vaccination with BOG increased porcine resistance to tuberculosis. Guinea pigs are highly susceptible to virulent g. bgzig and M, tuberculosis. Increased resistance in guinea pigs by BOG has been demonstrated by many workers (Jensen, 1930; Bogen and Loomis, 1935; Schwabacher and Wilson, 1938; Liebow, g§,al,, 1940). Bogen and Loomis vaccinated more than 325 guinea pigs during a five year period. They found a marked reduction in the number of animals that were tuber- culous as compared to control animals. In a study by Seagle, Karlson and Feldman (1953), 54.5 per cent of control animals.died and none of the vaccinated guinea pigs had died 166 days after the inoculation of virulent bacilli. Schwabacher and Wilson (1937) noted that there was a statistically significant difference between vaccinated and control mice which survived inoculation of Virulent organisms when the vaccinated animals had received 2x107 living BOG organisms. Vaccinated animals survived 125 days. The un- ‘vaecinated animals survived for 72 days. Swedberg (1951) found that the unvaccinated mice died within 19 days and ‘vaccinated mice died 44 days after the inoculation of viru- lent organisms. Different strains of mice vary markedly in their natural resistance to experimental infection (Youmans, 2:... 21., 1959). Lurie (1936) demonstrated that MIEQEEQESEABEHEQZLE was inhibited or destroyed in rabbits vaccinated with.BCG. ll Bacilli suspended in India ink or trypan blue solution were mixed with 6 per cent melted agar and injected subcutaneously into normal and BOG-vaccinated rabbits. At various subse- quent intervals, there were fewer bacilli in BOG-vaccinated rabbits than in the unvaccinated rabbits as determined by plate counts of the bacilli from the agar. Lurie (1942) collected monocytes from normal and BOG- vaccinated rabbits. After allowing them to phagocytize virulent tubercle bacilli igyzitgg in the presence of serum obtained from normal rabbits and rabbits vaccinated with BCG, these mixtures were introduced into the anterior shame ber of the eye of the normal rabbits. .The proliferation of the virulent bacilli was determined.by plate counts and microscopic technique. Fewer virulent bacilli were detected in the monocytes of vaccinated rabbits than in monocytes of unvaccinated rabbits. This was attributed to the increased ability of mononuclear phagocytes of BOG-vaccinated rabbits to ingest the bacilli. The increased phagocytosis was non- specific. Staphylococci, carbon particles and collodion were also phagocytized more readily. Virulent bacilli are not immediately destroyed in BOG- vaccinated animals. The protection arises from the inhibi- tion of basillary multiplication (Levy,‘gt'a;., 1961). Elberg (1960) postulates that the tubercle bacillus resides within the monocytes of BOG-vaccinated rabbits as an "ele- mentary particle". The monocytes prevent the proliferation of virulent bacilli within them. The site of the 12 tuberculoimmunity is cellular. Elberg has compared the cel- lular tuberculoimmunity to lysogeny in those bacteria render- ed immune to infection by homologous viral particles. Cellular resistance has been transferred passively by histiocytes and lymphocytes (Fong, 25.31,, 1962). Cells were extracted from the peritoneal exudates of BOG-vaccin- ated rabbits and whole cells or cell lysates were injected intradermally into unvaccinated rabbits. Cells were re- moved 13 days later from the peritoneal cavities of the recipient rabbits and exposed to virulent‘g, tubercu is 12:1;t22, The cells survived 25 to 42 days. Approximately 47 per cent less of the control cells were living on compar- able days. WW phenol. Bacterins have been prepared from the various spe- cies of mycobacteria with physical and chemical agents. Heat, ultra-violet irradiation, sunlight, urea, oleic acid, iodine, fluorides, chlorine, toluene, petroleum, ether, leci- thin, phenol, formalin, glycerine, hydrochloric acid, sodium hydroxide, nitrous acid, ethylene oxide and prolonged storage have been used (Weiss, 1959). Bacterins prepared with phenol have been widely investi- gated. The degree of tuberculoimmunity conferred is contro- versial. Trudeau (1894) reported that rabbits vaccinated with phenol-inactivated M, gyium_were not afforded tuberculo- immunity. In 1936, Ferguson and Cannon administered 0.1 mg 13 of 5 per cent phenol-inactivated g, tuberculosis to 137 guinea pigs intratracheally. The tuberculoimmunity of the animals was challenged four weeks later with virulent bacil- li by the same route. The average survival time was twice as long for the vaccinated animals as for the control group. When all the animals were killed and examined macroscopical- ly, tubercles were less extensive in the vaccinated guinea pigs than in the controls. Bloch and Segal (1955) and Meyer (1956), in a similar experiment, compared the protection afforded mice and guinea pigs by living BCG and virulent tubercle bacilli killed with phenol. They concluded that living BOG was consistently a superior immunizing agent. Vahlne, 25,31. (1955) administered three subcutaneous injections of phenolized, hexane-extracted tubercle bacilli to guinea pigs at weekly intervals. The resistance of the animals was challenged 36 days later with virulent bacilli. A degree of protection was obtained but the experiment was discontinued after 72 days. The draining tuberculous ab- scesses at the site of injection of the virulent bacilli were considered too hazardous to the handlers. Resistance of mice was increased with phenol-inacti- vated, acetone-washed avirulent‘fl, tuberculosis (Weiss and Dubos, 1955). Weiss (1958) reported that phenol-inactivated bacilli were as immunogenic for guinea pigs as BOG. Acharaya, gtugl. (1958) determined the relative im- munity afforded mice by phenolized and living virulant bacilli as measured by the mouse corneal assay method of 14 Race and Robson (1950) and Robson and Sullivan (1957). (The degree of resistance was estimated by the relative number of microscopic lesions in the infected eye. Phenolized prepara- tions, in adequately large amounts, produced immunity com- parable with that elicited by BOG. Weiss and Wells (1960) treated 1g. ph_l_e_i and virulent M. tuberculosis with 2 per cent phenol for 24 hr . The cells were washed repeatedly with distilled water and acetone. Groups of guinea pigs were inoculated intraperitoneally, subcutaneously or intramuscularly with 1.5 mg to 24.0 mg dry weight cells suspended in saline or Freund's adjuvant. Another group of guinea pigs was vaccinated with 3 x 106 cells of BCG. One group of guinea pigs was inoculated with saline as control animals. The tuberculoimmunity of the animals was challenged at 6, 12 or 36 weeks intramuscularly or by aerosol with virulent‘g, tuberculosis. A total of 330 guinea pigs was used in the study. The criterion was the mean.surviva1 time. Mycobacterium‘pglgi,was devoid of any detectable immunogenic activity, only E, tuberculosis induced an increased resistance. The optimum dose of phenolized cells in saline for mice and guinea pigs was 1 to 1.5 mg administered intraperitoneally. If the killed bacilli were suspended in Freund's adjuvant, 1.0 to 3.0 mg injected sub- cutaneously proved most protective. The tuberculoimmunity Was equal to that induced by BOG. Weiss and Wells made several succinct criticisms of other studies of experimental tuberculoimmunity. If killed 15 organisms or extracts could be injected at a level which would be equal to the total number of organisms resulting from ig;zizg_multiplication of BOG, bacterins might be as immunogenic as BOG. Moreover, the route of administration, the length of time between vaccination and challenge, the species of animal used and the nutritional level, are vari- ables which are frequently disregarded. Extracts of mycobacteria. Various substances obtained from mycobacterial cells are antigenic but there is no evi- dence that a single substance constitutes that component of the tubercle bacilli which elicits tuberculoimmunity. Koch (1897) Produced a water soluble fraction of ground tubercle bacilli. It did not immunize guinea pigs. The residue, "Tuberculin R", was both non-toxic and immuno- genie. Martin and Vsndremer (1906) injected guinea pigs with defatted tubercle bacilli which protected them against experimental infections. Dreyer (1923a, 1923b) removed the lipids from tubercle bacilli to obtain a product he named "diaplyte". Some thera- peutic value was credited to diaplyte and large doses did not produce ulceration at the site of injection. Kettle (1924), Douglas, gt,al., (1925) and Grasset (1925) reported that "diaplyte" possessed no therapeutic value. Brofen- brenner and Straub (1925) found that "diaplyte" hastened the death of tuberculous guinea pigs. Dreyer, Vollum and Amitszll (1934) administered l6 "diaplyte" prophylactically for nine years in a herd of cat- tle. At the beginning of the experiment approximately 90 per cent of the cattle were tuberculin positive. Calves 'were given one intraperitoneal or intravenous inoculation with.175 m8 dry weight of "diaplyte" and at the end of the ninth.year, only 11 per cent of the animals were tuberculin positive. Aqueous bacillary extracts were more immunogenic than lipoidal fractions (Widstron, 1941). Carbohydrate-lipid fractions of a virulent strain of M, tuberculosis possessed prephylactic and therapeutic values if injected subcutane- ously into guinea pigs (Kropp and Floyd, 1947). Seibert (1950) reported that purified tubercle protein and poly- saccharide fractions were non-immunizing. Anderson's (1927) phosphatide fraction of tubercle bacilli did not immunize rabbits (Kubo, gtfl§1., 1951). Boquet and Negre (1923), Négre and Boquet (1930) and Negre (1952, 1956) prepared a heat-killed, acetone washed, methanol-soluble extract of tubercle bacilli, Antigens Methyligue. It contained phos- phatides primarily and possessed some therapeutic and pro- phylactic value for guinea pigs and rabbits. Choucroun (1943, 1947, 1949, 1956) reported that a fraction of tubercle bacillus extracted with paraffin oil was immunogenic for guinea pigs. This was composed prin- cipally of one of the waxes, Wax D (Crowle, 1958a). The most promising of the various tubercle bacillary extracts are those of Ioumans, 23.51. (1957). Youmans,‘gt l7 a_l_. (1959), Kanai and Youmans (1960), Kanai, gt 9;. (1960). The fraction induced tuberculoimmunity in guinea pigs and mice. It was extracted from avirulent M, tuberculosis sus- pended in phosphate buffer-isotonic sucrose solution and disrupted in a ball mill with powdered glass at 4 C. Any intact cells were removed by centrifugation at low speed. The supernatant fluid was centrifuged at increasing speeds ( in a Spinco Model L ultracentrifuge. All sediments ob- tained by centrifuging at less than 144,700 X g were dis- carded. The final product was the sediment from three hrs centrifugation at 144,700 X g. It was enzymatically active, mitochondria-like, tuberculoimmunogenic for mice, and reddish in color. Hence, the extract was named "red fraction". In 1960, these investigators disrupted the cells with the Pressure Cell Method of Ribi, gt_§l. (1958). The immunogenic fraction was not red, and therefore, the name "red fraction" was no longer applicable (Kanai, 23.31., 1960). The particles were large enough to be studied by electron microscopy. They were composed of ribonucleopro- tein and fragments of cytoplasmic membrane. They were not mitochondria-like. An injection of 10 mg of the extract intraperitoneally into CF-l and Strong A strains of mice was the optimum dose to afford protection four weeks later from virulent bacilli injected intravenously. The criterion was the difference in the per cent of vaccinated and unvac- cinated mice which survived 30 days after challenge. No tuberculin sensitivity was induced by the extract. The “Lt-fem y l 18 immunogenicity of the intracellular particles was superior to that of cell wall fraction and intact avirulent M. 5222;: culosis. The cell wall fraction had no immunogenic activity but induced tuberculin hypersensitivity. The component re- sponsible for the induction of hypersensitivity was not destroyed by trypsin, pepsin or ribonuclease but was re- moved by treatment with alkaline alcohol. ( The mechanism by which the particles elicit tuberculo- immunity has not been determined. The particles are rich in enzymes and are principally ribonucleoproteins. Since ribonucleoproteins occur in the ribosomes and are associated with protein synthesis (Allfrey and Mirsky, 1961), the par- ticles may enter into the production of proteins, enzymes or others. They may be directly or indirectly responsible for the production or action of some constituent which is adverse to mycobacterial survival and growth. The immunogenicity of the intracellular particles was destroyed by dilution with distilled water, exposure to 60 O for 30 minutes, by extraction with acetone or ether, by electron bombardment and sonic vibration. The suspending medium during extraction of the particles is critical. Cells are suspended in buffered isotonic sucrose, 0.25 mg, although 0.88 M sucrose is equally effective in preserving the integrity of the mitochondria. There have been no substantiating reports of the im- munogenicity of the extract prepared by Ioumans' method for mice, or its activity in other animals. 19 An extensive review of the studies of the various frac- tions of the tubercle bacillus and their immunogenicity was made by Crowle (1958a). Crude tuberculo-polysaccharides as well as a purified high molecular weight polysaccharide as prepared by Siebert (1950) are antigenic but not immunogenic. Tuberculoproteins from bacillary culture medium or from tubercle bacilli, alone or bound to polysaccharide elicit -=—*-=|a. production of specific_antibodies which are responsible for an immediate type of hypersensitivity. A mixture of-the proteins and waxes inducssthe delayed type of hypersensi- tivity. Lipids are responsible for the majority of the physical and chemical properties of the mycobacteria. For example, tuberculolipids are required to produce delayed hypersensi- tivity, are associated with the acid-fastness, hydrophobic nature and virulence of the tubercle bacillus, and with antibody production and tuberculoimmunity. The lipids may be readily extracted from the cells and are classified as acetone-soluble fats, phosphatides, waxes and "firmly bound” lipids. With the exception of the "firmly bound" lipids, the lipids are extracted from intact mycobacteria with neu- tral organic solvents. Acetone soluble lipids do not immu- nize guinea pigs, rabbits or mice. They are toxic and in- crease the susceptibility of these animals to the tubercle bacillus. It is recommended that bacterins be washed with acetone before they are injected into experimental animals (Crowle. 19583). Phosphatides may be extracted from bacilli With ether- alcohol, some of which are toxic for experimental animals. Antigéne méthyligue, a methanol extracted phosphatide which contains a small amount of fats, waxes and complex nitrogen- ous compounds, possesses some prophylactic and therapeutic properties (Crowle, 1958a). It elicits the production of antibodies specific for the phosphatides. However, if the { phosphatide is carefully purified, it is an incomplete anti- U gen, a complex haptene. The tuberculoimmunity conferred by Antigéne methyligue to experimental animals differs from that by vaccines or bacterins. The acquired resistance is . -w3 present within two weeks after inoculations, no tuberculin sensitivity develops, and the resistance is relatively short-lived. The therapeutic value of the extract and its ability to enhance BOG vaccinations differentiates its ac- tivities from the bacterins and vaccines. Natural or increased resistance to tuberculosis in animals is correlated with a high phosphatidase activity of the tissues. Vaccination with phosphatide extracts or a synthetic phosphatide stimulates enzyme production which is correlated with the increased resistance. This is strong evidence that tuberculoimmunity is not entirely, if at all, associated with the presence of a circulating specific anti- body. The waxes are separated into A, B, C, and D by differ— ences in their solubilities. Wax A contains esters of phthiocerol with fatty acids, and a free fatty acid, mycollc 21 acid. Wax A sensitizes guinea pigs but does not immunize them against virulent M, 293;§,and M, tuberculosis. Wax B has been studied very little. Wax C and Wax D confer no tuberculoimmunity. Wax D is a polysaccharide ester of my- I_colic acid and contains nitrogen and phosphorus. Another wax, PMKO, is extracted from intact tubercle bacilli with warm paraffin oil. It is probably identical to Wax D and f like it, possesses no immunogenic properties (Crowle, 1958a). Specificity of tuberculoimmunity. The specificity of classical tuberculoimmunity is less than that of many well known humoral antibody immunities. For example, the immuni- ty produced by a given type of Diplococcus pneumoniae does not immunize against another type. This is not true of the mycobacteria since cross immunity and sensitivity may occur with M, bovis, M, tuberculosis,lM. balnei,‘M. ulcerans and M. legl (Crowle, 1958a) and taxonomically unrelated bac- teria induce some degree of tuberculoimmunity. Nukada and, Ryu (1936)_demonstrated that a mixture of Salmonella typhosa and Neisseria gonorrhoea injected into pigs or mice in- duced some increased resistance to tuberculosis. A vaccine of Bordetella pertussis also protected mice when subsequent- ly inoculated with‘M..tuberculosig (Dubos and Schaedler, 1956). If mice were previously infected with virulent or a- virulent Brucella abortus and subsequently inoculated with M. tuberculosis they were as resistant as other mice vac- cinated with BCG (Nyka, 1956). This resistance differed 22 from that elicited by BOG in that BOG immunized equally well by the intravenous, intraperitoneal or subcutaneous routes. Brucense were effective only when injected by the intravenous route. Nyka (1956) proposed that the brucellae induced antitubercular resistance because of the common route of infection. The brucellae conditioned the cells to resist a subsequent exposure to virulent mycobacteria. Evaluation of tuberculoimmunity. The evaluation of the tuberculoimmunity afforded animals by vaccination may be influenced by diet (Dubos and Pierce, 1948; Dubos, 1955), the strain of animal (Youmans, 1957), the dose and kind of vaccine or bacterin, the amount and route of administration of the challenge dose (Raffel, 1960), and the criterion chosen for the demonstration of tuberculoimmunity (Ioumans, 1957). These problems have been analyzed effectively by Orowle (1958a) who has stated: "Unfortunately, the word 'immunity' often provokes mental images of vaccinated animals completely re- fractory to infection with the microorganism against which they have been vaccinated, but a standing rule in immunology has been that protection derived from vaccination against any disease can be defeated by severe enough infection. When this happens, whether or not immunity is present can be measured only by comparing the severity of disease in vaccinated and unvaccinated animals. Many kinds of immunity (e.g., against diphtheria) are difficult to overcome. Ac- quired immunity to tuberculosis, on the other hand, seems to be somewhat more easily overcome, but this is no reason to deny its existence. For example, the guinea pig, the favorite animal for tuberculosis research, can be fatally infected with as few as 10 virulent tubercle bacilli (1c). Yet, rarely do ex- perimenters use less than 1000 times\this dose to challenge their immunized animals, and usually this figure is closer to 100,000. It should not be sur- prising, then, that immunized guinea pigs which have been infected with 100,000 LDlOO numbers of tubercle 23 bacilli should seem to be only Presistant' to this disease and not 'immune'. The chronicity of tuber- culosis makes challenge infections approximating those that occur naturally in man rather impractical in the laboratory. But, statistical evidence in man himself has proved beyond reasonable doubt that vac- cination with attenuated or killed tubercle bacilli does induce immunity in the broad classic sense as something which is not infallible but is specifically protective.” In experimental tuberculoimmunity)laboratory animals are vaccinated with BOG or bacterins prepared from the vari- ous mycobacteria (Ioumans, 1957). At some subsequent time varying from three weeks to three months (Bloch and Segal, 1955). their resistance is challenged intravenously, intra- peritoneally or by aerosol with virulent M. tuberculos s or M, 991;; (Weiss and Wells, 1960). Some time after challenge, vaccinated and control animals may be killed and the rela- tive number of microscopic and macroscopic lesions in the organs and tissues recorded (Feldman, 1943; Youmans, 1949; Larson and Wicht, 1962). It is presumed that immunized ani- mals should have less number of tubercles than unvaccinated controls. This method is very subjective and is not reli- able (Youmans, 1957). Moreover, previously sensitized ani- mals have more extensive lesions initially than the con- trols. These lesions tend to diminish with time (Opie and Freund, 1937). The comparative number of acid fast bacilli which are isolated from the organs and tissues of vaccinated and con- trol animals has been used as another index of immunity (Crowle, 1958a; Panisset and Benoit, 1959; Dubos and Oonge, 1960). Its validity is questionable because the reduction 24 in the number of virulent mycobacteria in the organs of vac- cinated animals requires a considerable period of time (Youmans, l957)o The most frequently used method of evaluation of im- munity is the difference in the mean or median days control and vaccinated animals survive following challenge with virulent bacilli. It may be reliable if the deaths of vac- cinated and control animals are normally distributed but Youmans (1957) has demonstrated that the distribution of death of vaccinated mice following challenge with virulent M, tuberculosis was not normal whereas that of unvaccinated mice was normal. The only method of evaluation recommended by Youmans (1960) was the comparison of the number of vac- cinated and unvaccinated animals surviving at a specific time interval after the inoculation of virulent bacilli. The 30th day was the end point most frequently chosen. The results obtained may be equivocal. Bloch and Segal (1955) found that BOG-vaccinated mice did not survive as ‘ long as unvaccinated control mice. This was interpreted as due to the more extensive lesions usually associated with previous sensitization in vaccinated animals. Beta-progiolactone. Beta-propiolactone (BPL) in the concentrated state is a colorless liquid, stable at low temperatures, but quickly hydrolyzed in aqueous solutions into an inactive, non-toxic acid, hydracrylic acid. This acid is removed more quickly in plasma than in water (Testagar and 00.). The formulas are as follows: 25 GHQ-GHQ Q-fzo +~ H20-——90H-CH2-CH2-COOH + 0H-(OH2-OH2-O00Ld; Beta-propiolactone hydracrylic acid hydracrylic acid polymer ~H20 OH2=CH-O00H acrylic acid Beta-propiolactone polymerizes at high temperatures to hy- dracrylic acid polymers. It has a boiling point of 51 O at 100 mm Hg, a specific gravity of 1.1490, a refractive index of 1.4131, a melting point of -33.4 C, a flash point (open cup) at 74 O and a half life hydrolysis in water of three to four hours (Wil- mot, 1959). At ordinary temperatures, BPL is miscible with most or- ganic solvents and reacts readily with hydroxyl, amino, carboxyl, sulfhydryl and phenolic groups (Gresham, g£_§l., 1948). Beta-propiolactone is irritating to the eyes and mucous membranes of the respiratory tract. Beta-prOpiolactone has been used to sterilize plasma (LoGrippo, 1960), inactivate viruses (LoGrippo and Hartman, 1955; Mack and Chtisen, 1956), certain bacteria, bacterial spores and fungi (wilmot, 1959). Huddleson (1955) found that the concentrations required for sterilization varied with different organisms, the time of exposure, and the medium in which the cells were suspended. Ierian and Teodoso (1961) investigated the nature of the antibacterial action of BPL. They studied the cyto- logical changes, viability and respiratory activities of Escherichia coli. No correlation was found to exist between 26 the loss of viability and any morphologic alteration. They demonstrated by the Warburg technique that BPL acted as an inhibitor of respiration. As little as 0.05 per cent BPL inactivated M, gg;;,(1erian and Teodoso, 1961) and 0.2 per cent inactivated viruses (LoGrippo, 1960) but at least 5 per cent BPL was required to detoxify staphylococcal entero- toxin (Hopper and DeValois, 1961). Bruch (1961) used BPL vapor to decontaminate certain enclosed spaces in which spore samples on paper strips were placed. The length of time required to inactivate the dry bacterial spores with the BPL vapor varied with temperature, relative humidity and concentration of the vapor. Beta- propiolactone vapor possessed poor penetrating power. Curran and Evans (1956) killed the spores of Bacillus subtilis, M, stearothermophilus, M, cereus and Clostridium spgrogenes in two hours with 0.3 per cent BPL. Bgcillus globigii spores required 0.75 per cent BPL for one hour or 0.5 per cent for two hours (Testagar and Co.). Continued incubations beyond two hours did not increase the inactiva- tion.(Curran and Evans, 1956). LoGrippo (1960) used BPL to sterilize plasma and tissue grafts and treat viral suspensions for vaccines. The pH of the viral suspension during the period of inactivation was maintained at 6.8 to 7.4. Complete inactivation of virus in 50 ml amounts occurred with 0.2 per cent BPL. However, if seven liter suspensions were treated with a final concentra- tion.of 0.3 per cent ot 0.4 per cent of BPL, all viral \ 27 particles were not inactivated. A desirable inactivating agent for the preparation of bacterins should possess the following properties: 1) lethal for microorganisms, 2) low toxicity for other living cells, 3) a good preservative, 4) no allergenic properties, and 5)_a margin of safety between the killing dose and the toxic dose. Beta-prOpiolactone possesses all these properties (LoGrippo, 1960). Moreover, in the concentrations used for inactivation, BPL does not denature proteins and preserves the antigenic integrity of the microorganisms. For these reasons, the ability of BPL to inactivate M, 221$§ for use as a bacterin appeann worthy of investigation. 28 MATERIALS AND METHODS Glassware. A11 glassware was soaked for 30 min in an alkaline detergent (Wyandotte Chemical Corp.1) containing no wetting agent. After washing in the detergent, glassware was rinsed Eitimes in tap water and 3 times in distilled water. Media. Dubos Agar and Dubos broth with enrichment (Difco2) were the media used for cultivation of the myco- bacteria. Cgltures. A laboratory strain of Myggbacterium bovis (Raveneli5and BOG3 were used as the source of organisms to . - ‘4 prepare the bacterins and vaccine. . Animal . Experimental animals were four-week-old male and female albino Swiss mice4 and female and male albino guinea pigs (450 g to 820 g)5. ,Preliminggy studies on the inactivatign g; M. bovis (ngenel) by beta-prepiolactone. Beta-propiolactone (BPL)6 ‘ was stored in a polyethylene container in the refrigerator at 4 C. It was diluted immediately prior to use. For determination of the inactivation of‘M,‘Q21;ngy BPL, cultures were grown 14 days in broth. They were centri- fuged 20 min at 1680 X g and the supernatant fluid removed. -_1, Wyandotte Chemical Corp., Wyandotte, Michigan. 2. Difco Laboratory, Detroit, Michigan. 3. Tuberculosis Unit, Communicable Disease Center, Atlanta, Ga. (original cultures). 4. Rockland Farms, New York City, N. Y. 5. Breeding stock obtained from Biol. Lab., Camp Detrick, Maryland. 6. Wilmot Castle Company, Rochester, New York. 29 The sediments were pooled, resuspended in 6 ml sterile triple glass distilled water to approximately 1 mg‘wet weight per m1 and redistributed in equal volumes intosnec centrifuge tubes. The tubes were placed in an ice bath and the pH of the contents adjusted to approximately 8.4 with 0.5 M NagHP04-l2 H20. Nine ml each of 0.00001, 0.0001, 0.001, 0.01 and 0.1 per cent BPL were prepared in ice-cold sterile p triple distilled water and added to five tubes of the bac- terial suspensions. Sterile triple glass distilled water was added to the sixth tube for the control. The contents were mixed by agitating the tubes by hand and then were placed in a water bath at 37 C. Ten minutes were allowed for the contents to reach thermal equilibrium. The suspen- sions were incubated for 2 hr during which time the.pH‘ was maintained at 7.6 i 0.4. After incubation, the suspen- sions were centrifuged at 1680 X g for 20 min in an Inter- national centrifuge, Universal Model UV. The supernatant fluid was removed and the sediments were suspended in 1 m1 of sterile distilled water. ‘Ten-fold dilutions and drop plates (Mallmann and Peabody, 1962) were made of the suspen- sions. After incubation, the number of colonies was counted and recorded. To determine the effect of pH and the time of treatment on the inactivation of M, 223;§,by BPL, approximately 10 mg wet weight per ml of M, 221;§_were suspended in 0.4 per cent BPL in three separate tubes. The first tube was incubated for 2 hr at 37 C and the pH adjusted at 15 min intervals to 30 pH 7.6 i 0.4. The second tube was maintained at the same pH but incubated for 1 hr and the third tube was incubated for two hr without adjustment of the pH. A control tube con- tained a comparable number of cells suspended.iml2 m1 of ster- ile glass distilled water and incubated at 37 C for 2 hr at pH 7.6 i 0.4. Drop plates were made and incubated at 35 C. To determine if the efficiency of BPL vere influenced by (51 cell concentration, approximately 15 mg wet weight of cells ; per ml of distilled water were dispensed in each of seven centrifuge tubes, centrifuged, and the supernatant fluid removed. After adjusting the pH to 8.4, BPL was added to the cells in respective tubes to yield a final concentration of 0.001, 0.01, 0.1, 0.2, 0.3, and 0.4 per cent. Triple glass distilled water was added to the cells in the seventh tube as a control. The tubes were incubated 2 hr at 37 C. Pre aration o bacter s.. BPL-inactivated M, nglg: Three-week-old broth cultures were centrifuged 20 min at 1680 X g and the cells resuspended in triple distilled water to approximately 10 mg wet weight per ml. The tubes were placed in ice-cold water and the contents were adjusted to pH 8.4. With constant agitation, BPL was added to make a final concentration of 0.4 per cent. After thermal equili- bration in a water bath, tubes were incubated at 37 C for 2 hr during which time the tubes were shaken and the contents adjusted to pH 7.6 t 0.4 at 15 min intervals. After incubation, the cells were centrifuged at 1680 X g for 20 min and washed 4 times with sterile distilled 31 water. The cells were standardized as indicated below and used as BPL-bacterin. Phenol-inactivated M. 2211.2: Viable M. 29.11% cells were washed as above and resuspended in a 2 per cent phenol solution. The suspension was incubated at room temperature for 24 hr. It was shaken intermittently during incubation. The treated cells were washed 4 times with triple dis- tilled water, standardized as noted below and used as phenol-bacterin. Acetone washed bacterins: Portions of BPL-bacterin and phenol-bacterin were washed once with sterile distilled water and 35 times with cold acetone. The cells were standardized as noted below and used as BPL-acetone-bacterin and phenol-acetone-bacterin. Standardization of bacterins. The bacterins were standardized on the basis of mg wet weight per m1, using the HOpkins Tube Method (Kubica, 1960). Extragt 9f M, m. A modification of the method of . Kanai and Youmans (1960) was used to extract intracellular particles from M, Qgggg, Cultures were grown in Dubos broth for 28 days at 35 C. The cells were centrifuged and washed once with sterile phosphate buffer, pH 7.0. The ' harvested cells were suspended in 150 ml sterile sucrose buffer (0.25 M sucrose dissolved in phosphate buffer) and transferred to a Waring blendor containing approximately 120 g sterile glass beads. The blendor had previously been thoroughly polished with steel wool, washed 3 times, 32 rinsed repeatedly in tapwater mm: 4 times in distilled water, and sterilized at 121 C for 30 min. To maintain a low temperature during blending, a plas- tic bag was inverted over the cylinder to form a pouch into which was poured a mixture of dry ice and 15 per cent NaCl. An outside temperature of -11 C maintained an inside cylinder temperature of less than 5 C. 'The tOp of the cylinder was tightly wrapped with cheese cloth saturated with a disin- fectantl. The Waring blendor was turned to the highest speed for 30 min, allowed to stand 40 min, and the contents transferred to 200 ml centrifuge tubes. The tubes were centrifuged in an International Centrifuge, then in a Serval Superspeed Angle Centrifuge at 4 C and'the sediment discarded. The supernatant was centrifuged in a Spinco Ultracentrifuge, Model L, with Rotor Number 40 for three hr at 100,000 X g (144,700 X g at the base of the tube). ‘The process was as follows: Eggken cells from WaringBlendor I Centrifuged in International Centrifuge, Universal Model UV, 1500 rpm (427 X g) for 10 min. Increased to 3150 rpm (1885 X g) for 60 min. IT I Sediment Supernatant fluid (discarded) I Transferred to Serval Superspeed Angle Centrifuge at 4 C. I Rheostat at 50 (11.730 X g) for 15 min I Increased to 75 (20,360 X g) for 10 mml I Increased to 100 (36,000 X g) for 5 mhl —=1. Septisol (ET 383), Dow Chemical 00., Midland, Michigan.- 33 _h . -1* Sediment Superna'tant fluid (d1 scarded) refrigerated overnight Transferred to Spinco Ultra-Centrifuge Model L, Rotor 40. l 39,000 rpm (1.0 x 105 x g) for 3 hr. Sediment Supernatant fluid (discarded) The final sediment was weighed, suspended in phosphate buf- fer, 100 mg per ml, and injected into experimental animals within 24 hr after preparation. BCG vaccine. Three-week-old broth cultures of BCG were standardized on the basis of mg wet weight per ml using the Hopkin's Tube Method (Kubica, 1960). Determination of the amount of Wm be used to challegge the tubercglgimmunity of mice and guinea pigs. To determine the approximate amount of wet weight orig.-991;§ lethal for mice, intraperitoneal injec- tions of 2.5, 3.5, 5.0, 6.0 and 7.2 mg of two-week-old cul- tures were made, two mice per each amount of inoculum. .The mice were (8 weeks old, the age at which the vaccinated and unvaccinated mice were to be inoculated. 'The number of days after inoculation on which each of the mice died was re- corded. Guinea pigs were inoculated intraperitoneally with 1.0, 2.0 and 5.0 mg wet weight of cells, four guinea pigs per each amount of inoculum. The number of days- after inocula- tion.on which each of the guinea pigs died was recorded. Mice and guinea pigs were examined for macroscOpic 34 lesions after death. Vaccination of experimental animals. A total of 80 mice were inoculated intraperitoneally. Group I (20 mice) received 1 mg BOG. Group II (20 mice) received 15 mg wet weight of BPL-bacterin. Group III (20 mice) received 15 mg wet weight of phenol-bacterin. Group IV (20 mice) received 8 mg extract. Group V (11 mice) were uninoculated and served as controls. Ninety guinea pigs were divided equally into nine groups and inoculated intraperitoneally. Each group re- ceived oneof the preparations as follows:0.5 mg 800, 10 mg wet weight BPL-bacterin, 10 mg wet weight BPL-acetone-bac- terin, 10 mg wet weight phenol-bacterin, 10 mg wet weight phenol-acetone-bacterin, 5 mg extract, 10 mg extract and 25 mg extract.' One group of Mme guinea pigs was. uninoculated. Challenge of experimental animals with virulent M. mg. Four weeks after vaccination, all animals were inoculated intraperitoneally with viable M. 22.1%.; ' cells, 32 mg and 2 mg wet weight for the mice and guinea pigs,respectively. Animals were observed daily for 28.days and the number of deaths recorded. .' Hypersensitivity. Two groups of four guinea pigs each were inoculated intraperitoneally with 5 mg BPL-bacterin and phenol—bacterin. Five weeks later each animal received intradermally 0.1 ml mammalian Old Tuberculinl diluted 1:20 in.saline. The diameter of induration at the site of ~ ‘1. Supplied by Animal Disease Eradication Division, USDA. injection was observed at 48 hr and recorded. 36 RESULTS Preliminary studies of the inactivation of M. p333; with BPL. When approximately 10 mg wet weight of cells per ml were treated with BPL at pH. 7.6 t 0.4 for 2 hr, 0.01 per cent concentration or less of BPL was insufficient to inactivate all cells. All cells were inactivated by 0.1 per cent or more BPL. The number of viable cells per ml as de- termined by the Drop Plate Method (Mallmann and Peabody?” 1962) was 2.7 x 108 prior to the inactivation process, 3.4 x 107 in the control culture which underwent incubation, cen- trifugation and washings withom BPL, approximately 3.0 x 107 when a final concentration of 0.01 per cent or less BPL wasv used, and zero if 0.1 per cent or more was used. A greater concentration of the cells required a greater_ concentration of BPL to inactivate all cells. As the pre- vious results indicated, 0.1 per cent BPL inactivated all of 3 x 107 organisms per m1 when incubated 2 hr at pH 7.6 1 0.4. However, if 1.3 x 1012 organisms per ml were treated with a final concentration of 0.1 per cent BPL, 3.1 x 103 cells per ml were not inactivated. It required 0.2 per cent BPL to inactivate all cells if there were originally 1.3 x 1012 cells per ml. Complete inactivation of all cells was not accomplished with 0.4 per cent BPL if 0.5 M disodium phosphate solution was not added to the suspensions to maintain the pH at 7.6 I 0.4 during the 2 hr incubation at 37 C. The viable cell counts as determined by the Drop Plate Method were 1.8 x 1011 37 per ml in the control culture, zero in the culture in which _the pH was maintained at 7.6 t 0.4, and 6.0 x 102 in the culture in which the pH was not adjusted. In the latter, the pH was approximately 5. All organisms were inactivated when 5.6 x 1010 organisms per ml were treated with a final concentration of 0.4 per cent BPL at 37 C at pH 7.6 i 0.4 for 2 hr. If the period of incubation was reduced to 1 hr, 2.7 x 103 organisms per ml remained viable. I If BPL-inactivated organisms were washed only once with distilled water, sufficient BPL or degradation products were present in 15 mg wet weight of cells to elicit signs of toxicity in mice inoculated intraperitoneally. If the cells _ were washed 4 or more times all detectable toxicity was removed. Determination of the amount of M,ng;i§grequired to cause death of mice and guinea pigs. The average number of days that mice survived after intraperitoneal injection of 2.5, 3.5, 5.0, 6.0, and 7.2 mg wet weight of M, bgzig was 75.5, 27.5, 14.5, 12.5 and 5.5, respectively (Table 1). Gross lesions in the liver, spleen or kidney were present in . only those mice which lived 22 days or more after inocula- tion. The average number of days that guinea pigs survived tafter intraperitoneal injections of 1.0, 2.0 and 5.0 mg wet ‘weight M, 221;§,was 31, 19 and 8.5 respectively (Table 2). Gross lesions in the liver, spleen or lung were present in 38 TABLE 1. The number of days eight-week-old mice survived, after intraperitoneal injections of nggngggggigm m- Number of‘days each Mg inoculum mouse* survived Mean of days per mouse _post-inoculation survived 76m!» 205 75.5 75 22** 3.5 27.5 33** 5.0 14.5 10 15 6.0 12.5 10 7.2 5.5 4 * 2 mice per inoculum ** Gross lesions observed in liver, spleen and/or kidney. 39 TABLE 2. The number of daze guinea pigs survived after intraperitoneal injections of fi¥£§§é§é§£§¥§a§EEL§‘ Mg wet weight Number of days each Average inoculum per guinea pig** survived number of guinea pigg _post-inoculation days survived 1.0 25** 28-3“!- 31 32** 39-11"! ‘ 2.0 15 16 19 19 26** 5.0 6 8 8.5 10 10 * 4 guinea pigs per inoculum ** Gross lesions observed in liver, spleen and lungs 40 only those guinea pigs which lived more than 19 days after inoculations. Survival of vaccinated and unvaccinated mice after inoculation with g. nggs. When unvaccinated control mice and mice vaccinated 28 days previously were inoculated intraperitoneally with 32 mg wet weight of M5 bgzig, less than one half the unvaccinated (control) mice were living on the 6th day after inoculation (Table 3). At the same time interval after inoculation, 81 per cent of the mice which had received BPL-bacterin, 72 per cent which had re- ceived BCG, and 58 percent which had received phenol-bacterin h were living (Table 4). ' 0n the 7th day post-inoculation, 34 per cent of the control mice, 72 per cent or the ECG vaccinated mice, 69 per cent of the BPL-bacterin vaccinated mice and 58 per cent of the phenol-bacterin vaccinated mice were living. 0n the 10th day post-inoculation, 34 per cent of the control mice, 72 per cent of the ECG vaccinated mice, 62 per cent of the BPL-bacterin vaccinated mice and 50 per cent of the phenol-bacterin vaccinated mice were living. At the 24th day post-inoculatibn, 18 per cent of the Control mice, 45 per cent of the ECG vaccinated mice, 42 Per cent of the phenol-bacterin vaccinated mice, and 19 per cen of the BPL-bacterin vaccinated mice survived. WW Biter inoculation witW. The number ‘of days was re- Corded at which vaccinated and unvaccinated guinea pigs 41 TABLE 3. The days on which vaccinated and unvaccinated mice died after intraperitoneal injection of 32.0 mg wet weifiht °W° Unvaccinated Vaccinatedl controls ECG sgpfii Phenol 4012 4e1 4e1 l@2 2a2 2e2 1@5 104 104 1@7 2a7 leg 1e9 2 6 13 1814 4am l@17 ‘1@l7 leis leis 16321 1021 L124 glue lo/ll3 10/18 13/16 8/14 Vaccinations were made with one mg BCG, 15 mg BPL-bacterin 2 (BPL) and 15 mg phenol-bacterin (phenol) The first number indicates the number of mice which died and the second number indicates the day of death post- 3 inoculation with.virulent bacilli. The numerator indicates the number of animals which died and the denominator indicates the total number of animals inoculated. 42 TABLE 4. The mortality ratio and percentgge survival of vaccinated and unvaccinated mice inoculated intra- peritoneally with 32 :15 wet weight of W 2.911% Days after Vaccinated Unvaccinated inoculation BCG BPL Phenol Control 6 death 5/18 3/16 6/14 6/11 survival 72% 81% 58% 46% 7 death 5/18 5/16 6/14 7/11 survival 72% A 69%. 58% 34% 10 death 5/18 6/16 7/14 7/14 survival 72% 62% 50% 34% 24 death 10/18 '13/16 8/14 10/11 survival 45% 19%~ 42% V 9% 1 Intraperitoneal inoculations were made four weeks after vaccinations. Vaccinations were made with 1 mg BCG, 15 mg BPL-bacterin (BPL), and 15 mg phenol-bacterin (phenol). 1 The mortality ratio is eXpressed as a fraction. 1he numerator indicates the number of animals which died and the denominator indicates the total number of animals inoculated. 43 died after intraperitoneal inoculation of two mg wet weight g, ppgip_(Tab1e 5). 0n the 16th day post-inoculation, 40' per cent of the control and 80 to 100 per cent of the vac- cinated guinea pigs were living (Table 6). The groups in which 100 per cent of the guinea pigs survived had been vac- cinated with BCG, phenol-acetone-bacterin, 5 mg extract and 25 mg extract. The groups in which 90 per cent of the gui- nea pigs survived had been vaccinated with BPL-bacterin, BPL-acetone-bacterin, and 10 mg extract. The one group in which 80 per cent of the guinea pigs survived had been vac- cinated with phenol-bacterin. L The only changes in the percentage of animals surviving . I) on the 18th day post-inoculation were that there was 10 per cent less in the 5 mg extract, 10 mg extract and phenol- acetone-bacterin vaccinated guinea pigs and control guinea pigs. All control guinea pigs had died 27 days after inocula- tion. The vaccinmed guinea pigs survived as follows: 90 per cent with 25 mg extract, 89 per cent with BCG, 80 per cent with 5 mg extract, phenol-bacterin and BPL-bacterin, 70 per cent with 10 mg extract and BPL-acetone-bacterin and 60 per cent with phenol-acetone-bacterin. Hypersensitivitx o; guinea pigs vaccinated with BPL- bgcterin and phenol-bacterin., All guinea pigs vaccinated 6 weeks previously with BPL-bacterin and phenol bacterin, responded to tuberculin with an area of induration varying from 16 mm to 21 mm in diameter 48 hr after intradermal 44 .vooeHSoomH mHmaHnm mo gonads Hmaou one moueoHuqH pouenHaomme one end veHe noHn: meeHmm mo mopeds on» moumonmH goodness: one .e ..HHHHomn umoHshH>_muH3 aoHueHdoonHuamoa memo mo sensed one moumoHecH sensed nmooem one was eeHw noHn3_men somst mo gonad: emu moumonqH tones: umaHm one .m .numou hHamo 0» one cousHoxm .m .Awa mm .wa 0H .ws my assuage and Ao< uHomonmv mHmcuomnuocouoomuHonemQ we 0H was 04-4mmv nHaouomniomooooeiqmm we 0H .AHononmv mHuouoenuHononm we 0H .Aqmmv mHamuomniAmm 0H .mom we m.o mqu some one: mnOHuemHoom> .H OHVe H m 0H 0H m mum, eoaumH em a H mm a H mm a H mm a H mm @ H mm @ H mm m H mm m H mm 9 H Hm 9 H om @ H mH @ H mH @ H mH © H mH 9 H mH © H mH @ H wH @ n mH e H 4H @ m «H 3 m MH @ H NH @ H MOH @ H mm m H we mm we 0H «me n o coudeoompeD .390 .Emfimz no: we o.m mo maogofldg Hmong IHno mnunH heave mmHm m mmnH: UmumnHoom>hd use umpmmHoomp nOH£3 no m mu 039 .m m4m .m .mnoHummHooep adage mace: anon eves one: mGOHamHsoonH HmonoanommuumH .H Rom mom Row Row Row Row Row mam R0 Hm>H>nmm oH\H 0H\m 0H\m 0H\e oH\u 0H\m 0H\m m\H 0H\oH memos am mooH mom mom mom new mom mom ROOH men. HmeHeasm .OH\o 0H\m 0H\H 0H\H 0H\m .0H\H 0H\H m\o 0H\a spade mH ROOH mom ROOH ROOH mom mom. mom ROOH mos HeeHeasm OH\o 0H\H OH\o oH\o 0H\m 0H\H 0H\H m\o 0H\e masses eH Illlmwdwxmoo HmoHoeHsoocH memamnHoos> . cogemHooe>nD means mmmn m as H03 as: a m nuHx eoamHsoo smsHa eQnuHooden: one douscHood> no Hm>H>nsm a daemon one oHuma uHHmamoe one .o mqmea 46 testing. The mean size of the reaction in the BPL-bacterin vaccinated animals was 19.0 mm. The mean size of the reac- tion in the phenol-bacterin vaccinated animals was 18.5 mm. 47 DISCUSSION Preliminarystudies of the inactivation of M. ppggg wiph BPL. A number of points are to be made on the use of BPL for the inactivation of M, 2212p, Under the proper con- ditions, BPL inactivated the organisms so that growth could not be initiated on an artificial medium. In addition, the guinea pigs which were inoculated with BPL-bacterin for the hypersensitivity study had not died four months after vac- cination and no lesions were found at autopsy. The final method selected for the preparation of the BPL-bacterin was the use of a 0.4 per cent of BPL with 3 x 107 cells per ml of distilled water incubated 2 hr at 37 0, during which time a pH of 7.6 i 0.4 was maintained. A de- crease in the concentration of BPL, an increase in the con- centration of cells, a shorter incubation period, or failure' to adjust the pH during the incubation resulted in the fail- ure to inactivate all cells. Therefore, it is stressed that the use of BPL for inactivation of M, ppggp requires that all factors must be standardized and maintained. In addition, it is known that medium constituents may hasten the degradation of BPL. If the cells were not washed and suspended in dis- tilled water, this factor would also need to be considered. It was established that under the conditions selected for the final method for the preparation of the BPL-bacterin, the concentration of the BPL was in sufficient excess to pro- vide a satisfactory safety factor. Under those conditions, 0.1 per cent had inactivated all cells, and likewise, 0.4 is (is per cent was able to inactivate a heavier suspension of cells. Therefore, a final concentration of 0.4 per cent BPL with the lesser concentration of cells provided an adequate safety factor. The inactivation of bacteria with BPL possesses two advantages over the inactivation of viruses or sterilization of blood components with BPL. First, the bacteria may be washed prior to treatment and any components in the growth medium removed which may interefere with the action of BPL. Second, after the inactivation of the bacteria with BPL, the bacteria may be washed sufficiently to remove residual BPL and its degradation products which are toxic for animal tis- sues. A greater concentration of BPL may be used to provide a greater safety factor of inactivation without increasing the hazards due to toxicity or other adverse effects of BPL. Evaluation of the tuberculoimmunity afforded mice and guinea pigs. The criterion for the demonstration of tuber- culoimmunity was chosen as the difference in the percentage of vaccinated and unvaccinated mice which survived at a given time interval after the inoculation of a given amount of M, ppgpp. The choice of the amount of inoculum to be used 'was based on the mean number of days that unvaccinated ani- mals survived after different amounts of M, 2213p were injec- ted intraperitoneally (Tables 1 and 2). Two factors were considered in the choice. First, an amount was desired which was small so that it would not overwhelm and mask any tuberculoimmunity conferred on the vaccinated animals. 49 Second, an amount was desired which was sufficiently large to insure death of the animals in a relatively short period of time, prior to the formation of gross lesions. The amounts of wet weight of M, pppgp chosen were 4, 8, l6 and 32 mg for mice and 2.0 g for guinea pigs. Owing to laboratory error, the results of the mice inoculated with 4, 8, and 16 mg wet weight of M. m could not be included. The time interval at which the comparison of the per- centage of vaccinated and unvaccinated mice survived was not selected prior to the inoculations. The percentages are noted at different time intervals and confirm the observa- tions of other workers that the reliability of the evalua- tion of tuberculoimmunity is limited by the criterion chosen. For example, the interpretations may be biased by the number of days after inoculation which is chosen as a point of com- parison of the percentage of animals surviving. If the day is chosen at which approximately one half of the control mice have died, the sixth day after inoculation, the tuber- culoimmunity afforded the mice which received the BPL-bac- terin is superior to that afforded by ECG or the phenol-bac- terin. The percentage of mice surviving was 81, 72 and 58. respectively. ' 0n the 10th day after inoculation, 34 per cent of the control mice lived. More of the mice which had received the iBPL-bacterin survived than those vaccinated with the phenol- ‘bacterin but less than those vaccinated with BCG. The per- centage of mice surviving was 62, 50 and 72, respectively. 50 On the 24th day after inoculation, only 10 per cent more of the BPL-bacterin vaccinated mice survived than the control mice, 19 and 9 per cent, respectively. Mice vac- cinated with BCG or‘the phenol-bacterin survived, 45 and 42 per cent, respectively. Both were markedly superior to the BPL-bacterin at this time interval. Bloch and Segal (1955) obtained such equivocal results. They found that control mice survived longer than mice vac- cinated with BCG. The authors believed that this was due to the differences in the distribution of lesions containing virulent bacilli. Animals sensitized by the vaccination § with BCG developed more extensive tubercles than the con- 3 fi trols which had not been previously sensitized. Criteria other than survival have been used to measure tuberculoimmunity, such as differences in lung densities, numbers of lesions, or the number of bacilli culturable from a given organ (Feldman, 1943; Crowle, 1958a). These criteria have been criticized by Youmans (1957) in that individual animal susceptibility varies markedly and greatly influences the results. Survival of a group of animals is the most re- liable but as noted previously, the interpretation of the results may be heavily biased by the time interval selected. The longer the interval, the more complicated the evaluation becomes due to the complexity of the interplay of the de- 'velopment of infection and hypersensitivity after challenge *with.the hypersensitivity previously induced by vaccination. Shorter time intervals are believed to be more reliable. 51 The amount of organisms used to challenge experimental tuber- culoimmunity undoubtedly far exceeds that which occurs nat- urally and by virtue of the smaller number of organisms, would bring into play less untoward reactions during natural infections. . Weiss and Dubos (1954) stressed that all mycobaCterins are toxic to some degree and animals which survive the toxic effects before their immunity is challenged, are comparative- ly resistant. After the vaccination of the mice, some toxic effects of the extract were evident. Approximately 50 per cent of the mice died before their immunity could be challenged and for this reason, the results of mice vaccinated with the extract could not be included. In preference to repeating the experiment in mice, guinea pigs were chosen on the basis that they are more susceptible to infection with.M, pngp, The measurements of the relative tuberculoimmunity were not as markedly altered by the selection of different time intervals for the guinea pigs (Table 5) as those for the mice*(Table 3). It was apparent that all groups of vaccina- ted guinea pigs had been protected if the day at which ap- proximately one half of the control guinea pigs had died, the 16th day post-inoculation were chosen, or the 18th day, *which.was the mean day, or the 27th day when all guinea pigs had died. The vaccinated pigs surviving at the 16th and 18th day varied from 80 to 100 per cent, and at the 27th day, 60 to 90 per cent. 52 In some instances, the immunity afforded by the vaccine, a given bacterin or concentration of extract appeared to be superior to others. Consistently, BCG and 25 mg extract conferred the greatest protection upon guinea pigs. Initi- ally, 5 mg extract and phenol-acetone-bacterin were equally effective but by the 27th day, 5 mg had protected 10 per cent less and phenol-acetone-bacterin was the poorest of the lot. The tuberculoimmunity afforded by the BPL-bacterin was not as great as that afforded by BCG and 25 mg extract but was consistently superior or equal to that of phenol-bac- terin and 10 mg extract. Of interest are the acetone washed BPL-bacterin and ' phenol-bacterin. Acetone soluble fractions, as an extract or part of the cell, reportedly are toxic for experimental animals and are believed to increase the animal's suscepti- bility to M, ppyip (Weiss and Dubos, 1954; Crowle, 1958a). On this premise, it could be anticipated that the acetone washed bacterins should be more effective in inducing tuber- culoimmunity than the unwashed bacterins. This proved to be true of phenol-acetone-bacterin at the 16th and 18th day, but fewer guinea pigs vaccinated with the phenol-acetone-bac- terin.survived at the 27th day than those vaccinated with phenol-bacterin. Likewise, fewer guinea pigs vaccinated with BPL-acetone-bacterin survived at the 27th day than those vaccinated with BPL-bacterin although they were equal- ly effective at the 16th and 18th day. 53 Kanai and Youmans (1960) reported that 10 mg of an ex- tract of avirulent M, tuberculosis afforded mice better pro- tection than 5 mg or 25 mg. It was demonstrated in the present investigation that 5 mg and 25 mg of the extract from virulent M, ppppp were more immunogenic for guinea pigs than 10 mg. The unpredictability of the optimum dose is a distinct disadvantage in the use of the extract. The extract prepared by Kanai and Youmans (1960), which was highly immunizing for mice, was prepared from‘M. tubepculpsis. It has been demonstrated by the present ex- periment that an extract from M. bovis can confer a tuber- culoimmunity on guinea pigs equal to, or better than, the -intact bacilli inactivated with phenol or BPL. It is sug- gested that similar extracts be made from saprophytic myco- bacteria to determine if they possesstuberculoimmunogenic properties. Saprophytic mycobacteria grow more rapidly and would be less hazardous to handle. The ideal immunization procedure would be one which would induce a significant tuberculoimmunity without also inducing tuberculin sensitivity. The value of the tuber- culin test as a diagnostic tool would be retained. The ex- tract reportedly does not induce sensitivity, and as such, is highly desirable. Unfortunately, massive amounts of cells, by a tedious and hazardous procedure, yields minute amounts of extracts. From approximately 200 ml of packed cells, 600 mg extract was obtained. The BPL-bacterin retained the ability to confer a 54 degree of tuberculoimmunity and also hypersensitivity. Ex- tractions to remove the wax fractions, which with the pro- teins reportedly induce the sensitivity, may yield a product capable of immunization without sensitization. This study was undertaken originally to answer two questions, the second contingent upon the results of the first. The questions were 1) will BPL- inactivate M. _bo_y_i_s., and if so, 2) will BPL-inactivated M, pppgp cells be tuber- culoimmunogenic? An affirmative answer has been obtained to both questions. Under the proper conditions, M, bpvis is inactivated by BPL. The organisms so inactivated are tuber- , culoimmunogenic. However, the tuberculoimmunity conferred . is relative and not absolute. The difficulty of interpre- ting data has been noted. It has been measured not only by the differences in the susceptibility to M, ppp;p of mice and guinea pigs unvaccinated and vaccinated with BPL-bac- terin, but also animals vaccinated with BCG, phenol-inacti- vated cells and extracts of cells. The former, BCG, has been used world-wide, reportedly successfully. It is known that the organism resides for long periods of time in the animal tissues after vaccination, and undoubtedly multiplies. Thus, equal numbers of BCG and cells of a bacterin are not, in reality, equal numbers after inoculations. A comparison of the two is unfavorably biased toward BCG. However, no hazard from infection exists in the use of a bacterin. Therefore, both points must be weighed. A second point is to be made in comparing a bacterin to 55 ‘.a.vaccine. The role of circulating antibodies in tuberculo- immunity is not known. Their presence or absence can not be correlated concluSively with a given stage of the disease or serve as a basis for prognosis. It has been suggested that tuberculoimmunity is dependent upon some intracellular ac- tivity of the host cells which retard the growth of the bacillus, and that antigen-antibody reactions are incidental. If this be true, it is possible that by multiplication in the host, BCG elicits a greater response which can never be equalled by a bacterin. If the stimulating substance is re- tained in the bacterins, the bulk of the bacterin may con- tribute very little to induction of tuberculoimmunity. How- ever, the little that is known of the mechanism of infection, immunity or hypersensitivity of tuberculosis is testimony of the complexities involved and an indication that no one sin- gle factor controls one or all conditions. Mgcobacterium pppgp is inactivated by BPL. Such or- ganisms may serve as a source of non-viable cells from which extracts can.be prepared in large volumes without hazards. {The tuberculoimmunity and sensitivity detected in guinea gpigs after vaccinations with BPL-bacterin establish that the antigenicity is retained in the bacterin. Such antigens may pxassess more potential species differentiation, if it exists, than those commonly used in serologic, identification, and sensitivity studies . 56 ISUMMARY Beta-propiolactone (BPL) inactivated Mycobacterium pppgp, A final concentration of 0.1 per cent of BPL was re- quired to inactivate 3.4 x 107 cells per m1 of distilled water when incubated at 37 C for :2 hr at pH 7.6 I 0.4. All cells were not inactivated if the concentration of BPL was decreased, the concentration of the cells increased, the incubation period was shortened to 11 hr or if pH 7.6 1 0.4 was not maintained. A BPL-bacterin prepared from M, bovis was found to be a safe, effective tuberculoimmunogenic agent for mice and guinea pigs. The BPL-bacterin was made by treating 3 x 107 cells with a final concentration of 0.4 per cent BPL. The excess BPL provided a satisfactory safety factor. The inac- tivated cells had to be washed four times to remove any re- sidual BPL or degradation products toxic for the experi- mental animals. The tuberculoimmunity elicited in mice by BPL-bacterin compared favorablyto that elicited by BCG vaccine and was superior to that elicited by phenol-inactivated cells. When approximately two-thirds of unvaccinated mice had died after the inoculation of virulent organisms, 72 per cent of BCG vaccinated mice, 69 per cent of BPL-bacterin vaccinated mice and 58 per cent of phenol-bacterin mice survived. Tuberculoimmunity was elicited in guinea pigs by the vac- bacterins and extract. After challenge with virulent M, cine, bovis, when two thirds of control animals had died, allguinea pigs uskm 57 vaccinated with BCG and 25 mg extract, 90 per cent of BPL- bacterin and 5 mg extract vaccinated guinea pigs, and 80 per ~ cent of phenol-bacterin and 10 mg extract vaccinated guinea pigs survived. Treatment of the BPL-bacterin and phenol- bacterin with acetone before administration did not increase the protection afforded the guinea pigs. An extract was obtained from M, pppip by a method simi- lar to that by which Youmans (1960) prepared an extract from M. tuberculosis. The extract of M, pppgp conferred tuberculo- immunity to guinea pigs which was assumed to be similar to that conferred to mice by the extract of M, tuberculosis. Extracts from saprophytic mycobacteria which grow more readily and are less hazardous would be worthy of investi- gation. Tuberculin sensitivity was induced in guinea pigs by BPL-bacterin. 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