COMPARISON OF THE ANTEGENIC :mopaa'nes OF SEVERAL DiSSOCIATiD BHASES AND ONE SMOOTH PHASE OF THE GENUS BRUCELLg Thesis for flu Degree of M. 5. MECHIGAN STATE COLLEGE Martha Jane Warner $49 This is to certify that the thesis entitled COMPARISON OF THE ANTIGENIC PROPERTIES OF SEVERAL DISSOCIATED PHASES AND ONE SMOOTH PHASE OF THE GENUS BRUC ELLA presented by Juanita Jane Warner has been accepted towards fulfillment of the requirements for Master of Science degree in Bacteriology W141“. V Major professor Date 77LG/E! COMPARISON OF THE ANTIGENIC PROPERTIES OF SEVERAL DISSOCIATED PHASES AND ONE SMOOTH PHASE OF THE GENUS BRUCELLA by Juanita Jane'Warner A THESIS Submitted to the School of Graduate Studies of Michigan State College of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Bacteriology and Public Health 191.9 1'hbi‘"é Comparison of the Antigenic Preperties of Several Dissociated Phases and One Smooth Phase of the Genus Brucella I. INTRODUCTION 00.00000......0.000000000000000000000000000000 1 II. LITERATURE 00000000000000.0000...o00000000000000.00000.0000 2 III. PROCEDURE .oooooeeeooeoooooeoeeoeoeoeeeeeeoooeoeooeoooecsee 8 A. Culture “Odia 00000000000000.0000000000.000000000000000 8 B. Characteristics of Growth Phases of Brucella Used in this StUdy 000000000000000000000000.00000000000000.0000 9 C. Preparation of Bacterial Antigens for Serum.Agglutina- tiOl’l Tests OOOOOOOOOOOO.COCOOOOOCOOOOO0.00000000000000 12 D.» Preparation of Homologous Agglutinating Sera in Rabbits coooeoeeooeoeeeecocooeeoooeeooecoeo.eeeeeoeeee 13 E. Agglutination TOSt Procedure 90000000000000.0000.so... 1h IV. EXPERIMENTS AND RESULTS ceeosooooeooeeoooeeeoseoeooeoeooeo 15 A. Preparation 0f Stable Antigens eeeeeeoeeeeoeeeoceoeeeo 15 B. Comparative Agglutinability of Antigens Made from Each of Several Dissociated Growth Phases and One Smooth Phase 0f Brucella ooosoeeeeo00000000000000.0000 17 v. DISCUSSION oeooooooooooeeeeeceoeoesocceooeoeeoooeeeoeeeeee 22 VI. SWY 0.00.00.00.00.000000000000000000000000000000000000 214» VII. LITERATURE CITED o.cooooeoosee00000000000000.0000...ooeeso 26 2.18009 -1- COMPARISON OF THE ANTIGENIC PROPERTIES OF SEVERAL DISSOCIATED PHASES AND ONE SMOOTH PHASE OF THE GENUS BRUCELLA The members of the genus Brucella dissociate into many growth phases. It is sometimes difficult to differentiate one dissociated phase from another or even to classify them in the genus Brucella on the basis of cultural characteristics. Therefore, it seemed important to investigate the antigenic relationships between the different dissociated growth phases in order to determine if it 'was possible by serological means to distinguish one phase from another or to identify an unknown culture. Thus far, with the exception of the IR (intermediate rough) phase, it has not been possible to compare the agglutinability of all the dissociated growth phases because they agglutinate spontaneously in solutions of inor- ganic salts. The first part of this study deals with the search for a suit- able method for stabilizing cell suspensions of dissociated phases so that they might be used as antigens in comparative agglutination studies. The second part pertains to the results of the comparative agglutination studies that were conducted with several dissociated phases and one smooth phase of Brucella using homologous and heter- ologous antisera produced in rabbits. -2- LITERATURE Burnet (l) was the first to observe a dissociated phase of Brucella and to describe a test for its detection. He found that a saline suspension of the dissociated form agglutinated when incubat— ed in a water bath at 80° C. for two hours while a saline suspension of the smooth (S) strain remained in suspension. Later, Huddlescn, Scales, and Sorenson (7) found that Burnet's test could not be relied upon for the detection of dissociated phases of Brucella. It was observed that heat-instabilityvvas an uncertain means of detecting dissociation. Di Aichelburg (2) described a simple test for detecting antigen- ic variants of Brucella. Brucella cells suspended in a 132,000 dilution of basic fuchsin in distilled water were incubated for two hours at 37’ C. The dissociated strains were agglutinated in vary— ing degrees at the end of the incubation period while S strains remained in suspension. Dissociated phases of Brucella were detected by Hunger and Huddleson (11) by means of an opsonocytophagic test. Bacterial cells of dissociated cultures were phagocytized in large numbers by polymorphonuclear cells in citrated normal whole blood while cells of S phase cultures were phagocytized in small numbers or not at all. Ross (13) separated the S phase of HE! abortus and §£.:melitensis from the dissociated phase by means of agglutinin absorption tests. The dissociated phase of Brucella'was either not agglutinated or slightly agglutinated by rabbit sera prepared from the S phase. The reverse was also true. He also observed definite agglutination of the dissociated phase with both lactic acid and Michaelis' acid agglutination tests. The S phase was not agglutinated. The dissociated phases could not be distinguished from.one another by either method. marshall and Jared (9) reported that the rough (R) phase of Brucella, when grown in liquid medium, agglutinates spontaneously. However, in the agglutination tests performed, they'were unable to obtain agglutination of R suspensions by an S phase antiserum of high titer. They found that results of the agglutination test served as a rapid method in determining the extent of dissociation since the loss of agglutinability was gradual. Agglutinins for the S phase were produced by the majority of guinea pigs receiving S phase cultures while most of those injected with R phase cultures did not produce anti-S agglutinins. Gwatkin (3) studied a dissociated strain °f.§£f abortus which he designated as an R phase. The R phase was distinctly "stringy" and difficult to suspend. Suspensions of the R phase were made in distilled water and in varying concentrations of sodium.chloride solution with and without phenol. The cells remained in suspension fairly well in distilled water and in concentrations of sodium chloride as high as 0.h.percent. Higher concentrations caused spontaneous agglutination. The presence of phenol did not affect the results. There was no agglutination with positive serum, but the R antigen used was not specified. Guinea pigs injected with R phase cultures produced agglutinins for neither the S nor R phase antigens while guinea pigs receiving a smaller inoculum of the S phase produced only anti-S phase agglutinins. A serological study of dissociated phases of Brucella, desig- nated as R types, was made by Mallman and Gallo (8) to determine the antigenic relationship to homologous S types and to each other. Rabbits were injected with killed cultures of R and S phases of each of the three species. Antigens of both phases were suspended in 0.5- percent phenolized sodium chloride solution. The R phase antigens of 31;. melitensis and BE“ suis were agglutinated equally well by .13.!“ melitensis and £5. suis R antisera while the £5. abortus R phase was not agglutinated by either. 'Eg. abortus R antiserum did not aggluti- nate the R phase antigens of_§5. melitensis and BE. suis but did agglutinate its homologous R antigen. None of the R antigens were agglutinated by any S antiserum while the S antigens were agglutinated equally well by all 8 antisera. Henry (L) stated that the R type of brucella cells was difficult to suspend in sodium.chloride solution due to the presence of the slimy secretion of the organisms. The bacterial cells settled quickly in 0.85-percent sodium chloride. A decrease in sodium chloride concentration retarded the rate of sedimentation. Normal rabbit and bovine serum.hastened Spontaneous sedimentation. .The use of various sodium chloride concentrations, buffers, and surface tension depressants did not lead to uniform.cell suspensions which could be agglutinated in the presence of specific antibody. In most cases, guinea pigs inoculated with large doses of the R phase of 2:. abortus produced no agglutinins for the S type. However, agglutinins for £5. abortus S phase were found in the sera of pregnant rabbits previously injected with the R type of 25‘ abortus. Cattle which were inoculated with 25' abortus R phase produced only a trace of agglutinins for the S phase. Henry stated that the results appeared to indicate that R forms of many 25. abortus cultures contained a residual amount of S antigen. Henry prepared antigens from.several phases of a 25. abortus culture designated as R, S, and SR (pseudo S form) by the alcohol and ether extraction method used by”White (l7). Rabbits were inject- ed intravenously with these extracted antigens. Agglutination tests 'were performed with sera from the rabbits in the presence of the extracted antigens and the stock E3“ abortus S phase antigen. Significant agglutination was obtained only with the S-extracted antigen and the S phase antigen in the presence of the S phase serum. Antigenic preperties of the S-extracted antigen were not lost during the extraction procedure as was indicated by the presence of agglu— tinins in serum from.the rabbit injected with the extracted S phase antigen. Guinea pigs inoculated intraperitoneally with living organisms of the R phase produced slight agglutinins for both R and SR phase organisms. Plastridge and McAlpine (12) failed to observe any difference in the appearance of colonies of the mucoid-like phase and the S phase. Upon suspension in 0.85 percent-sodium chloride, the mucoid cells settled out more rapidly than the S phase cells and appeared viscous. The mucoid and 8 phases of the same strains were suspended separately in phenolized saline and utilized as antigens. The antigens made of mucoid-like growth 0f.§£P abortus were practically inagglutinable in serial dilutions of S antisera while S phase antigens from.the same strains were agglutinated by high dilutions. The mucoid £5. melitensis antigen was agglutinated by homologous S antiserum.nearly as well as was 2E3 melitensis S phase antigen. They believed this indicated that 253 melitensis mucoid cells existed in the mucoid fomm, or the antigen was composed principally of S phase cells. ‘When mucoid cells were injected into rabbits, agglutinins qwere produced for both mucoid and nonmucoid phases. They stated that the mucoid cells differed antigenically from.the S phase cells. Mingle and Manthei (lo) conducted serological studies with two dissociated growth phases of £5. abortus. The colonies were char- acterized by wrinkled surfaces and slightly irregular margins. Agglutinating sera were prepared from each 8 and dissociated strains in rabbits. The S phase antigens were agglutinated equally well by homologous and heterologcus S antisera but were not agglutinated by antisera prepared from the dissociated strains. Antigens prepared from.the two dissociated phases were agglutinated by high dilutions of the antisera produced by the dissociated phases and by low dilutions only of one S phase antiserum. Another S antiserum failed to agglutin- ate the two dissociated phase antigens. Stearns and Roepke (16) studied electrophoretic mobility changes in S and dissociated phases of Brucella in liquid medium. Their resume indicate that dissociation may be a gradual process. The dissociated phase (mucoid) had a greater mobility than the S phase. The disso- ciated phase cells appeared in liquid medium.without the complete disappearance of cells having the mobility of the S phase. After guinea pig passage, the dissociated phase cells, while retaining their mucoid character, decreased in mobility to that of the S phase cells. They found that dissociated phase cells were not entirely satis- factory as antigens due to instability. These were agglutinated by homologous and heterologous dissociated phase antisera and by high dilutions of the S phase antiserum. S phase antigens were not agglutinated by dissociated phase antisera. Comparison of the results of the agglutination tests and the electrophoretic investigation indicated that the factor or factors affecting the electrokinetic preperties of the cell surface are not necessarily the same as those responsible for the antigenic properties. The results obtained by Stearn and Roepke (15, 16) indicate that electrophoretic measurements may provide a means for identifying dissociated phases of Brucella. -3- PROCEDURE A. Culture media. Brucella cells for the preparation of antigens were grown either in liquid medium in bottles or on the surface of tryptose agar plates. Tryptose liquid medium is prepared as follows: Bactc-Tryptose (Difco) 30.0 gm. Sodium.chloride 5.0 gm. Glucose 20.0 gm. Distilled water 1,000.0 gm. These ingredients are dissolved and filtered through coarse filter paper. The pH is adjusted to 6.7 with phosphoric acid and 5.0 mg. of thiamine hydrochloride is then added. The medium is distributed in 250 ml. amounts in one liter bottles and sterilized at 120' C. for 20 minutes. The dilution fluid is prepared as follows: Bacto-Tryptose (Difco) 0.5 gm. Sodium chloride 5.0 gm. Distilled water 1,000.0 ml. The fluid is distributed in 100 ml. amounts in bottles and sterilized at 120’ C. for 20 minutes. Bacto-Tryptose Agar (Difco) was used according to the directions for the preparation of plates and slants. The liver agar for the slants was prepared according to Stafseth (lb) and Huddleson (6). B. Characteristics of Growth Phases of Brucella Used in This Study. The dissociated growth phases of the three species of Brucella 'were obtained by inoculating tubes of'tryptcse liquid medium separate- ly‘with S phase cells of each species (5). The cultures were incu- bated at 57’ C. for at least ten days. A small loopful of each culture was streaked over the surface of tryptose agar in Petri plates to obtain isolated colonies. After a four-day incubation period (37' C.) the colonies were observed and studied under a low power (x 12.5) stereoscopic microscope by means of reflected, oblique light. No other method will reveal graded differences in colony morphology as well as this one. Colonies which appeared to differ in their morphology from the S phase were picked and streaked over the surface of tryptose agar plates and re-examined after four days of incubation. This procedure was continued until the growth was pure phase. 1. Br. abortus smooth (S) phase. ‘Widely separated colonies are 1.0 mm. to 2.0 mm. in diameter. They are circular, smooth, convex, entire, glistening, translucent, and of soft consistency. The color varies from light bluish green to a cloudy green; the centers become slightly opaque with age. 2. Br. abortus rough (R)phase. The colonies are circular, raised, opaque, and have a ground-glass appearance when scattered by a wire pointer. The size of the colony varies from 1.0 mm. to 2.0 mm. in diameter. The color is light orange. They are soft in consistency but are slightly drier than the S phase. Daughter colonies do not appear to develop in R phase colonies. 3. Br. abortus mucoid (M)_phase l. The colonies might easily be mistaken for the S phase when they are close together on the surface of agar plates. ‘Widely separated colonies vary from.l.0 mm. to 2.5 mm. in diameter. They are circular and slightly raised in the center; ‘with rounded margins. The marginal border of the colony is bluish green, but the central area is somewhat Opaque with a slight orange tint. When a fine wire pointer is swept through the colony and lifted upward, the growth has a tenacious, mucus-like consistency. This phase shows no tendency to revert to the S phase or to dissoci- ate into other phases. R. Br. abortus mucoid (M) phase 3. The colonies are circular and convex with no raised edges. The size of the isolated colonies is similar to that of phase 1. They are Opaque and of a faint tan color; they become translucent with age. 'Well-isclated large colonies are quite tenacious. They are more wax-like than mucoid and adhere to the surface of the medium. Daughter colonies of a bluish-green color develop in colonies of this phase. They resemble the S phase. Under certain conditions, M 1 phase colonies also develop from.M 3 phase colonies. 5. Br. suis mucoid (M) phase 2. The colonies are convex, opaque, and slightly orange yellow. The colony size varies from 1.0 mm. to 2.5 mm. 'When a colony is swept with a wire pointer, the entire mass clings to the wire and can be lifted from.the surface of the medium as though it were soft wax. The colony becomes increasingly trans- lucent with age and produces S-like daughter colonies. 6. Br. suis mucoid (M)phase h. The colonies of this phase are slightly convex. The size varies from 1.0 mm. to 2.0 mm. in diameter. 'When viewed by reflected, oblique light, the colonies have a decided -11- ground-glass, opaque appearance. The consistency is markedly mucus- like. 'When colonies of this phase are left at room temperature for 30 days, they develop daughter colonies of several different phases, notably the R phase, SL phase, and M phase 5. 7. Br. suis mucoid (M) phase_5. The colonies are convex, circular and slightly opaque. The smaller ones have a cloudy, glistening surface. The size of this phase varies from.0.2 mm. to 0.5 mm. in diameter. 'lhen a wire pointer is swept through the more confluent growth on the plate, the whole mass is tenacious. This phase dissociates into M phase 6, the colonies of which are much larger than those of phase 5. 8. Br. suis mucoid (M) phase 6. The colonies of this phase vary from 0.6 mm. to 1.5 mm. in diameter. Isolated colonies are slightly convex and cpaque with a slight greenish border. The colonies are mucus-like in consistency. 9. Br. suis smooth-like (SL)_phase:_ Colonies of this phase are easily mistaken for the S phase. Their size varies from 1.0 mm. to 2.5 mm. in diameter. They develop as daughter colonies fromugg..§glg M phase 2 or 3. They are dull bluish green in appearance, are soft, and show no mucoid consistency. SL colonies show no tendency to revert to the S or M phase. 10. Br. melitensis mucoid (I) phase 2. The colonies of this phase are peach-colored with a greenish border. The surface growth is mucus- like in consistency. The size of the colonies varies between 1.0 mm. and 1.5 mm. in diameter. This phase produces SL phase daughter colonies. ll. Br. melitensis mucoid~(M)pphase_3. The colonies of this phase are convex and vary from 1.0 mm. to 1.5 mm. in diameter. They are opaque with a faintly orange center and a greenish edge. The growth is more tenacious in consistency than £5. melitensis M phase 2. This phase also produces SL daughter colonies. C. Preparation of Bacterial Antigens for Serum.Agglutination Testg, The majority of the different phases were grown in tryptose liquid medium in bottles, each of which was seeded with the 2h-hour growth of a liver agar slant suspended in tryptose diluting fluid. The inoculated medium was shaken mechanically and exposed to a measured flow of oxygen during the period of incubation. Shaking during incubation prevented the cells from forming large aggregates that were difficult to resuspend. Those phases which dissociate rapidly when grown in liquid medium in the presence of oxygen were grown on tryptose agar plates. These were BE. abortus M phase 3 and .§£.'§gi§ M phase 5. Only typical phase colonies were harvested from the plates and suspended in 0.5-percent phenol solution. The suspensions of organisms were centrifuged at high speed for 15 minutes and the supernatant liquid discarded. The cells were washed twice with 0.5-percent phenol solution, suspended in the same diluent, and shaken mechanically for 15 to h5 minutes to disperse the large aggregates. The remaining aggregates of cells were removed by centrifuging at low speed for five minutes. The suspended cells 'were stored at h“ C. Before use, all antigens were diluted with 0.5- percent phenol solution to a turbidity of 28 as measured by a Libby Photronreflectometer. '25, abortus S phase organisms were grown upon liver agar slants -13- for 2h hours and removed with a solution containing 0.85-percent sodium chloride and 0.5-percent phenol. The cells were washed and prepared as previously described, except that phenolized saline was used for'washing and diluting to the standard turbidity. D. Prepgration of Homologous AgglutinatingpSera in Rabbits. Brucella growth phases used for producing agglutinating sera in rabbits: Br. abortus S phase Br. abortus R phase Br. abortus M phase 1 Br. abortus M phase 3 --.—- Br. suis M phase 2 Br. suis M phase h Br. suis M phase 5 Br. suis M phase 6 Br. suis SL phase II.— B5. melitensis M phase 2 The cells of each growth phase used for injecting the rabbits 'were obtained either from.2h-hour growths on liver agar slants or from tryptose broth suspensions. The growth was suspended in 5 m1. of a 0.85-percent sodium chloride solution. The rabbits were inject- ed in the marginal ear vein with 1 mg. of living cells (approximately 5 x 109 cells) of the dissociated phases and 0.2 mg. of living cells (approximately 1 x 109 cells) of the S phase. The majority of the rabbits received one injection and were bled from the heart after 10 or 11 days. One rabbit received two intravenous injections of £5. abortus M phase 1. The second injection was given three days after the first. This rabbit was bled 15 days after the first injection. An attempt to give a second injection of Br. suis SL phase to a rabbit h days after the first led to its death immediately after injection. Another rabbit was given a second injection °f.§£f.§23i SL phase 3 days after the first. This time the injection was stopped when the rabbit began to show labored breathing. This rabbit was also bled 15 days after the first injection. After the blood samples were allowed to clot, the sera were removed aseptically, centrifuged, and filtered through a #8 Hormann pad in a Seitz filter and stored at h’ C. until ready for use. E. Agglutination Test Procedure. The agglutinability of each growth phase antigen with normal serum, homologous serum, and heterologous sera was determined by preparing serial two-fold dilutions of each serum from a 1:20 to at least a 1:5, 120 dilution directly in each antigen. An antigen con- trol was included with each test. All tests were incubated for LB hours at 37° C. The tests were read without shaking the tubes, and the results recorded as follows: - = no agglutination, T = trace agglutination, P = incomplete agglutination, and + = complete agglutination. -15- EXPERIMENTS AND RESULTS A. Prgparation of Stable Antigens. Approximately 30 experiments were conducted in order to find an agent which could be added to suspensions of dissociated growth phase cells to maintain.them in suspension for more than.h8 hours at 37° C. Preliminary investigation indicated that most of the water- 'washed cells were fairly stable when suspended in distilled water containing 0.5-percent phenol. However, they became unstable upon the addition of sodium chloride in concentrations as low as 0.2 per- cent. In the absence of sodium chloride, the antigens were generally not agglutinated in dilutions higher than lzh0 by normal, homologous, or heterologous rabbit sera. Cell suspensions containing 0.1-percent sodium chloride were agglutinated by normal rabbit serum.to a titer as high as 1:6h0 and by homologous and heterologous rabbit agglutin- ating sera to titers as high as 1:5,120 dilution. The following agents were added in various concentrations to cell suspensions to increase their stability in the presence of sodium chloride: gum arabic, gum ghatti, dextrin, dextrin and gum ghatti, calcium chloride, ammonium sulfate, ferric sulfate, ferrous sulfate, magnesium sulfate, sodium sulfate, sodium.bisulfite, and sodium thiosulfate. These agents, as a whole, either decreased or failed to increase the stability of the suspensions either in distilled water containing 0.5-percent phenol or in the presence of sodium chloride. The effect of various hydrogen ion concentrations on the stability of several cell suspensions was investigated. The pH of the cell suspensions varied from 6.5 to 7.0 before the addition of acid or -16- alkali. ‘Water suspensions of E5. 5331 M phase 2 were adjusted to a pH of h.0 with phosphoric acid and to pH 8.0, 10.0, and 11.0 with sodium hydroxide. At pH h.0 the stability of the suspensions'was decreased. The cell suspensions maintained at pH 8.0 and those adjusted to pH 10,0, immediately washed, and resuspended in distilled water containing 0.5-percent phenol remained stable; however, they 'were unstable in the presence of 0.2-percent sodium chloride. The cell suspension became viscous at pH 11.0. It was finally found that the suspensions remained stable in the presence of a low concentration of sodium chloride when they 'were adjusted to pH 9.0 by means of sodium.carbonate. The final concentration of sodium.carbonate in the cell suspension was 0.05 and 0.07 percent in the dissociated and S phase antigens respectively; Since antigens at pH 9.0 remained stable for an incubation period longer than h8 hours, the effect of adding various concentra- tions of sodium chloride from 0.0 to 0.5 percent to each dissociated phase was studied. All were found to remain stable for longer than h8 hours. The dissociated phase antigens were prepared in distilled water containing 0.5-percent phenol and 0.5-percent sodium chloride and adjusted to pH 9.0 with a sodium.carbonate solution. Two different S phase antigens of E5. abortus'were used in this study. One was prepared in distilled water containingClBS-percent sodium chloride and 0.5-percent phenol without adjusting the pH (6.3) and the other was adjusted to pH 9.0 with sodium carbonate. B. Comparative Agglutinability of Antigens Made from.anh of Several Dissociated Growth Phases and One Smooth Phase of Brucella. The agglutinability of each phase antigen was determined in the presence of homologous serum and in the presence of each serum produced from.the other growth phases previously mentioned. It may be noted from the results in tables 1 through 5 that most of the different growth phase antigens were partially agglutinated by each of the five norna1.rabbit sera in law dilutions or in a zone ranging between 1:160 and 135,120 dilutions. The zones of agglutin- ation occurred with antigens of _B_1_'_. suis; M phase L; and 6, SL phase, and §£.:melitensis M phase 2 and 3. The zone of agglutination was wider with some antigens than others and varied with each normal rabbit serum. The zone phenomenon observed with certain antigens can not be explained at the present time. S phase antigens'were not agglutinated by normal sera. '25. abortus S phase antiserum.agg1utinated homologous antigen and that of the R phase to approximately the same extent (Guns 6), thus indicating a common antigenic component. Other growth phase antigens were agglutinated slightly by S phase antiserum. .25. abortus R phase antiserum agglutinated homologous and heterologous growth phase antigens (table 7). The titer'was higher 'with'the homologous antigen and several of the mucoid antigens than 'with E3. abortus S phase antigen. .25. abortus M phase 3 antigen was agglutinated incompletely by the antiserum. It may be noted (tables 8 and 9) that all of the dissociated growth phases were agglutinated by EE‘ abortus M phase 1 antiserum -18- but in different degrees. in. abortus M phase 3 and £31. 3113 M phase 5 were incompletely agglutinated in the various dilutions. The S phase antigen was not agglutinated by antiserum from either of two different rabbits which had received one injection each of 133;. abortus M phase 1. The results of only one of these sera were included (table 8). Although the dissociated phase antigens were agglutinated in higher dilutions of the antiserum produced by two injections of 133;. abortus M phase 1 (table 9) than with antiserum produced by one injection (table 8), there was no agglutination of the S phase antigen. Tables 10 and 11 show that the antisera produced by 21;. abortus M phase 3 and _B_1;. _s_1_1_i_§_ M phase 2 exhibited a higher agglutination titer with certain heterologous antigens than with homologous antigens. _B_1_'_. 3.11.”. M phase 5 and E5. _s_u_i_s_ SL phase, were only partially agglu- tinated in the serial dilutions of the 25' abortus M phase 3 anti- serum (table 10). 21;. abortus M phase 3 antigen was incompletely agglutinated by the E5. _s;1_i_s_ M phase 2 antiserum (table 11). 21;. 3133 M phase 5 antiserum (table 12) completely agglutinated its homologous antigen in serum dilutions of 13160 and 13320 after showing a prozone of incomplete agglutination. 2:. abortus M phase 3 antigen was incompletely agglutinated. £5. 1121 M phase 2 antigen was completely agglutinated by a 1320 and 13140 dilution of the anti- serum and incompletely agglutinated up to a 132,560 serum dilution. Both _B_1_'_. abortus S phase antigens were agglutinated completely up to 131,280 dilution of the antiserum. The other antigens were completely agglutinated in a l36h0 or 131,280 serum dilution and partially agglutinated in the next one or two serial dilutions. The agglutina- tion titer of the _B_r_. suis M phase 5 antiserum was approximately the same with homologous antigen as with heterologous antigens. 23:. 3311 M phase 6 and 23:. abortus R phase antigens were agglu- tinated completely in a 131,280 dilution of 15' 511i M phase 6 antiserum (table 13). £5. abortus M phase 3 was only incompletely agglutinated in the serum dilutions. 133:. 2131 I phase 5 antigen also showed a prozone and was agglutinated completely in 1380 and 13160 dilutions of antiserum and incompletely in the other dilutions. The other heterologous antigens were agglutinated in various dilutions of the antiserum. A 13160 dilution of 23;. 3113. M phase 1.; antiserum completely agglutinated its homologous antigen while a 135,120 dilution of the serum only incompletely agglutinated the antigen (table 11...). £5. abortus M phase 3 antigen was agglutinated partially in a 1314.0 dilu- tion of 13;. 3313 M phase L; antiserum. 2:. gig M phase 5 antigen was agglutinated incompletely in all antiserum dilutions except 1320 and 13320 dilutions which showed complete agglutination. 21;. abortus S phase, R phase, M phase 1, 25' 212.3. M phase 6, and £5. melitensis M phase 2 and 3 antigens were agglutinated completely in a higher dilution of antiserum than was the homologous antigen. However, the agglutination titer of the antiserum with the antigens varied between 132,560 and 1310,2h0. The agglutination pattern of the antiserum with the antigens of 23;. abortus R phase and 2:. in}: M phase 6 is similar. _B_r:. _s_u_i_§ SL phase antiserum (tables 15 and 16) agglutinated homologous and heterologous antigens with the exception of the £5. abortus S phase antigens. Another rabbit antiserum produced by one injection of a different .1_3_r_:. suis 81.. strain agglutinated the antigens to essentially the same degree as the one recorded in table 15. The titer of the antiserum which was produced by two injections of SL phase organisms (table 16) was higher for most of the antigens than was the titer of the antiserum which was produced by only one injec- tion (table 15). It may be noted from the results in table 15 that 2:. abortus M phase 3 was agglutinated only incompletely in all antiserum dilutions used. 2:. £21 M phase 2 was agglutinated completely in a 1320 antiserum dilution and incompletely in the other dilutions through 135,120. _B_1;. Ellis M phase 5 was incompletely agglutinated in all serum dilutions except the 13320 dilution (table 5). 21;. abortus M phase 3 antigen was incompletely agglutinated in all E’E.’ 3313 SL phase antiserum dilutions used except the 13320 dilution (table 16). 11;. suis M phase 2 antigen was agglutinated by the antiserum in an unusual zone reaction. I_B_1;. 2232 SL phase anti- serum agglutinated £5. a}: M phase 5 antigen and showed the common prozone phenomenon. All antigens used with the exception of 2:. abortus S phase antigens were agglutinated in all antiserum dilutions used. 133:. melitens is phase 2 antiserum agglutinated both homologous and heterologous antigens in various dilutions of the antiserum (table 17) between 131,280 and 135,120 dilution. 21;. abortus M phase 3 antigen was only incompletely agglutinated by the antiserum. The prozone phenomenon was exhibited by the antiserum in the agglutination of E. suis M phase 5 antigen. 21;. suis M phase 6 and 25. melitensis M phase 3 antigens were agglutinated completely in a 13640 dilution of serum while the 21;. abortus S phase, M phase 1, and the homologous antigens were agglutinated completely in a 13320 dilution of the antiserum. A 13110 dilution of the _B_1:. melitensis M phase 2 anti— serum completely agglutinated both _B_1_'_. suis M phase 2 and 21;. suis SL phase antigens while higher dilutions of the antiserum agglutin- ated the antigens incompletely. coavsnwpsawms epeamfioo 3 + nofipsnapsawws Hdwvusm 3 m newpsdfipsawws mouse 3 a o.m nemavns mo mm Amy noapswfipusws oz 3 3 m.w newwpss he mm Aav .l ' Is 8 H m .H - ' .I ' m UHOOdH: "HunQDvWHQE own-m 3 3 3 3 3 3 3 3 3 3 a N ufioess mandopwaefi .mm. 3 3 3 m, 3 m 3 3 3 3 m 323585 See .Hm 3 3 3 a m m a 3 3 3 a e Bees: e2. .Mm 3 3 3 3 3 3 3 3 3 a m m 38.5 ease .mm 3 3 3 a a 3 3 3 3 3 m a Bees: 33 .mm 3 3 3 3 3 3 3 3 3 a. a m Bees: Steps .mm 3 3 3 3 3 3 3 3 3 3 a H paces: coupons .mm. 3 3 3 3 3 3 3 3 3 3 a .swsom weapons .mm. emetwpammmw is 32.3 u 3 T. 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Ho mH A: 3 3 3 a H 3 3 3 3 3 3 n 302.5 mwlnnofiaoa 39m 3 3 3 H H 3 3 3 3 3 3 m 3025 33333 .MM 3 3 3 H H H H 3 3 3 3 333583 .33. .mm. 3 3 3 H 3 3 3 3 3 3 3 w 33:: 3:. .mm. 3 3 3 3 H H H 3 3 H H m 33:: :3 .mm. 3 3 3 H H H 3 3 3 3 3 a 33:: 3H3 .mm. .3 I I I m + + 3. + + + N ““003: uHSI .Ihlm... 3 3 3 H H H H H H H H m 38:: 33.62. .mm 3 3 H H H 3 3 3 3 3 3 H 388. 2.233 .mm. 3 3 H H 3 3 3 3 3 3 3 £93m .3735] 3mm 3 3 a H H 3 3 3 3 3 3 Amy A308» 35.33an .MIW 3 3 3 H H 3 3 3 3 3 3 AS 5086 33.3% .Hm on no 03 wijmmmm: .3 Z .3 w m % H 0 0 I 3 .35333 nomenq w 33H I 35. .MM 3 35:35 «30053 No 33H Avooau one on... nuudnH 3.3300»qu H.325- .Ho szwpdfivaawmd obavdeHfioo 9 32H o.m 833. Ho HH A3 9m 8335 .3 HH HS 4 33H I 33.5» 3 mm. 3 3533.35 3 3 H H H 3 3 3 3 3 m 38:: $33328 .mm. 3 3 H H H H 3 3 3 3 H 33:: $33333 .HH 3 3 H H H H H 3 3 3 ofiTfiooam :3 .mm 3 3 H 3 3 3 3 3 3 3 0 goon: :3 33mm 3 3 H H H H 3 H H 3 m 38:: :8 .mm 3 3 H H H H H 3 3 3 a 33:: :3 .mM 3 3 3 H H H H H 3 3 m 38:: :8 ma. 3 3 3 3 3 3 3 3 H H m 3 8:: 33.3% .Mm 3. 3 3 H H 3 3 3 3 3 H 300:: 35.39.... 33mm. 3 3 H H 3 3 3 3 3 3 Ana—om 25.3896 .Mm. 3 H. a H H H 3 3 3 3 Amy «3.086 25.3an .Hm. 3 3 3 H H H 3 3 3 3 HS £88 3338.: .Mm m. ”m. a...” “a w mu m W3, W W3 % ouSHH «30on H w. H m H W. 0 .H 88333 8333 daaoosum Ho and: H spoon—u one 33 32:3 vovdwoouawv Hang: Ho Hfiaupdnappamwd obaqudHaoo 4H canda 838...“: 25 5 9m 83%.. H0 H.— H8 m6 8&3. Ho mH AS 3 3 H H 3 3 3 3 3 3 H m 33:: 3833...: .mm. 3 3 H H 3 3 3 3 3 3 3 m 33:: 383:3 .mm 3 3 H H H H 3 3 3 3 3 32350an :2. .mm 3 H. 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H0 HHH H8 m3 nowfiand Ho NH AC 3 H H 3 3 3 3 3 3 3 H m 33:: «Humanism .HH 3 H H 3 3 3 3 3 3 3 H m 38:: 33323 .mm. 3 H H 3 3 3 3 3 3 3 H ofiHépooam 3H3 .Mm. 3 H 3 3 3 3 3 3 3 3 3 0 Boos: 33.3 ....H3m3 3 H H 3 3 3 3 H H H H m 38:: 3H3 .mm. 3 H H 3 3 3 3 3 3 3 H 4 302.2 3:5 3M3m3 3 H H 3 3 3 H H H 3 3 H 33:: :3 .Mm 3 H H H H H 3 H H H H m 3.8:: 33.82. .mm. 3 H H 3. 3 3 3 3 3 3 H H 38:: .3883 .mm 3 H H H H H 3 3 3 3 3 nwsom canoe. .mm 3 3 3 3 3 3 3 3 3 3 3 H8 583m Stock .Mm 3 3 3 3 3 3 3 3 3 3 3 AC H3036 3.5.33.0 .mm. 3 no ooH Hmwmwmmmm: 333 I 3 w. o o m 0 O I .H 38333 w 353 Ill “0 AC :23 Am 33.2. 3.8 3 EouHPHHd H 4302er Ho 0993 subs—Ho 0:0 and 0323 0093333 1305» Ho HHHHdenHvHHHde obHvdeHH—Hoo 0H candn. o.m 53.35 Ho HH 98 m6 5339. .3 HH “5 3 3 3 H H 3 3 3 3 3 3 m cHoo:E_ uwufibpfiflofi .HH 3 .. 3 H H H 3 3 3 3 3 m 38:: 333:3. .mm. 3 3 3 H H H H H H 3 3 ofiTfiooam :3 .mm. 3 3 3 H H 3 3 3 3 3 3 0 Boos: 33.5 .mm. 3 3 3 3 H H 3 H H H H m 38:: :2... .Mmu 3 3 3 H H H H 3 3 3 3 a 33:: :3 .mm. 3 3 H H H H H H H 3 3 m 38:: :3 .Hm. 3 3 3 3 H H H H H H H m 38:: 333.3. .mm 3 3 3 H H H 3 3 3 3 3 H 38:: .389. .mm 3 3 3 H H H H 3 3 3 3 nwsom 33.8% .mm. 3 3 H H H H 3 3 3 3 3 “my 588 3.388. .Mm 3 3 3 H H H 3 3 3 3 3 CV 58%... Stan... .mm. M cm. a...” “6 w W Wm M, 000 W3 W oudgH :36on n m w ,H e 0 O 0 0 T. 3 .30333 om m 33H 2 53323 .mm 3 €335 n 33 daaooam Ho oudAH .3093 23 can noudsH @0332;qu H.395» Ho HpHHHpfiHHvsfime oPHvdeHaoo S candy DISCUSSION A.method for the preparation of stable bacterial antigens from . dissociated growth phases of Brucella has been described. The bacterial antigens of the dissociated phases were suspended in 0.5- percent sodium chloride solution and stabilized in suspension by adjusting the pH to 9.0 with sodium carbonate. Homologous and heterologous agglutination tests were performed using the stable suspensions of dissociated phase antigens and the S phase antigen in dilutions of antiserum.produced in rabbits with live suspensions of different growth phases. The agglutination titers of the various sera were not appreciably different with the £5. abortus S phase antigen adjusted to pH 9.0 than they were with the £33 abortus S phase antigen at pH 6.3. The dilutions of the antiserum exhibiting complete agglutination were the same with both S phase antigens. In a few instances, the final titer of the antiserum showing incomplete agglutination varied by one or two dilutions. The agglutination of the R phase antigen with the S antiserum obtained in one experiment in this study is contradictory to results obtained by Marshall and Jared (9). They were unable to obtain agglutination of an R phase antigen suspension by an S phase antiserum of high titer. Gwatkin (3) was unable to produce agglutinins with .EE‘ abortus S phase for R phase antigen. Also, Mallman.and Gallo (8) did not obtain agglutination of their R antigen suspensions with S phase antisera prepared in rabbits. The results of this study -23- suggest that g5, abortus S and R phases posess common antigens (see table 6 and 7). The dissociated phase antisera were agglutinated by all of the antigens with exception of the 2.1;. abortus M phase 1 and 31;. 3313; SL phase. The fact that several of the M growth phases produced agglutinins to a relatively higher titer for heterologous antigens than for homologous antigens can not be eXplained on the basis of this study. The agglutination results observed in table 6 with the 25. abortus R phase antigen and E53 abortus S phase antiserum are not in accord with those of Ross (15). Also, Ea. abortus R phase culture used in this experiment (table 7) produced agglutinins for the 2:. abortus S phase antigen while the R phase antisera used by Ross (13) either failed to agglutinate his S phase antigens or they were agglutinated only in low titers. Gwatkin (3) and Henrle) obtained results similar to those of Ross (13), but they used guinea pig sera which can not be compared to rabbit sera. The results obtained with the SL phase antiserum (tables 15 and 16) and the S phase antiserum (table 6) indicate that the SL phase can be distinguished from.the S phase by serological means using the S and SL phase antisera with the S phase antigen. The colonies of the two phases appear morphologically identical. This would require further investigation using antigens and antisera prepared from S and SL phases that occur in each species. fig, abortus M phase 1 might be distinguished from the E5. abortus S phase, but the question remains as to whether it could be distinguished from E5. abortus SL phase. -23- suggest that g5, abortus S and R phases posess common antigens (see table 6 and 7). The dissociated phase antisera were agglutinated by all of the antigens with exception of the E5. abortus M phase 1 and E5..ggig SL phase. The fact that several of the M growth phases produced agglutinins to a relatively higher titer for heterologous antigens than for homologous antigens can not be eXplained on the basis of this study. The agglutination results observed in table 6 with th°.§£f abortus R phase antigen and E5. abortus S phase antiserum are not in accord with those of Ross (13). Also, 2:. abortus R phase culture used in this experiment (table 7) produced agglutinins for the fig. abortus S phase antigen while the R phase antisera used by Ross (13) either failed to agglutinate his 8 phase antigens or they were agglutinated only in low titers. Gwatkin (3) and Henry (1;) obtained results similar to those of Ross (13), but they used guinea pig sera which can not be compared to rabbit sera. The results obtained with the SL phase antiserum (tables 15 and 16) and the S phase antiserum (table 6) indicate that the SL phase can be distinguished from the S phase by serological means using the S and SL phase antisera with the S phase antigen. The colonies of the two phases appear morphologically identical. This would require further investigation using antigens and antisera prepared from S and SL phases that occur in each Species. 23, abortus M phase 1 might be distinguished from the £33 abortus S phase, but the question remains as to whether it could be distinguished from Br. abortus SL phase. SUMMARY Bacterial antigens stabilized at pH 9.0 and containing 0.5- percent sodium chloride were prepared from several dissociated phases of Brucella. Agglutination studies show that the growth phases produce agglutinins to a certain degree in rabbits for both homologous and heterologous antigens. However, 21:. abortus M phase 1 and _I_3_1_'_. 3935'. SL phase failed to produce agglutinins for 25' abortus S phase antigen. The results of this study as a whole do not indicate that the different growth phases of Brucella can be distinguished one from another by serological (agglutination) means. ACKNOWLEDGEMENT I wish to thank Dr. I. Forest Huddleson for his patient help and guidance in this study. Also, I wish to thank Dr. R. Evelyn Sanders and Miss Marvis A. Richardson for their helpful suggestions. 1. 3. 9. 10. -26- LITERATURE CITED Burnet, Et. La thermo-agglutination et l'evolution de l'eSpece Brucella. Arch. Inst. Pasteur de Tunis. 17: 128-1h6. marl Di Aichelburg, U. Agglutination aspecifique avec la fuchsine basique dans les microbes due groupe Brucella. Boll. d. sez. ital. 6: 30-52. 193k. Gwatkin, R. Antigenic qualities of a dissociated strain of Brucella abortus. Canad. Pub. Health J. 23: hesquge. 1932. I Henry, B. S. Dissociation in the genus Brucella. J. Inf. Dis. 52: 37h-L02. 1933. Huddleson, I. F. The mucoid phases of the genus Brucella. Am. J. Vet. Res. 8: 5-10. 19u6. Huddleson, I. F. Brucellosis in man and animals. Revised edition. New York. The Commonwealth Fund. 19h}. Huddleson, I. F., J. W. Scales, and 0. J. Sorenson. Non-specific agglutination in the Brucella group. Mich. Agr. Exp. Sta. Tech. Bul. 1&9: 5-20. 1936. Mallman,‘N. L. and F. Gallo. Studies on the dissociation of the Brucella group. J. Agr. Res. b6: 267-279. 1933. marshall, M. S. and D. Jared. Microbic dissociation in the Brucella group. J. Inf. Dis. h9: ‘318-536. 1931. Mingle, C. K. and C. A. Manthei. Bacterial dissociation in Brucella abortus. Am. J. Vet. Res. 2: 181-189. l9hl. 3330?.“ one any 0.0 Swaps. co mm A8 m6 .338. do me CV I? I a m + + + + + + + m m 3853 Segovia: .um I m m + + + + + + + m N Ufloog nHDQOPHHa Hula-m I m a + + + + + + + m 33-583 3:. .M. I m + + + + + + + + + w 3003— 3.43 .Mm. . a m + + + + m m a m m 38:: .3. .mM .. m m + + + + + + + a 4 3.8:: .3. .mm I m m + + + m m m + + m 332. :3 .Mm .. m m a a a + m m a a m 38:: Stop. .mm. I m m + + + + + + + m a 38:: 25.88. .wm. .. a a m a a + + + + + sauce Stops .mm I I I I I I I I I I I Amy £9085 astond nun-ml I I I I I I I I I I I as 5.086 unions .Muml no com mmwmmmmmm: a... I 4 IE 0 o m 0 o I S £3333 nouns: AC :33 Am 35» .mm. I Eamon—«pad «5395 Mo 3.9.3 spoon; one use 2593 00930033 Hench: Mo hpafindfipsamws ofivsuemfioo 0..” 04.0.69 0.0 nowfipus mo mm “NV m6 33%. no mm as m 092% 3 3305.36 .wlmu I 5.3»wa I I I a m I I I I I I m 33% massages :6 I I I a m m a m a I I .fiTfioosm £3 .Mm I I I m m + + + + + + 0 309.5 neon .M I I I I a m I a a m m m 38% :3 .mm I I I a m m m + + + + J 30052 3.5m .Mm. I I a a a m a m. m I I m 38:: 33 .mm I I I I a a a a a a a m 38:2 Stops .mm I I I a m m I I I I I H 3352 mangoes .mm I I I a m a a I I I I ewsom «ease. .mm. I I a a m m I I I I I “my 535m «58°... .mm I I I a a a I I I I I A: 585 "3.3.... .mm. my ”me. u...” he cu m We. M, We W: ”W madam newcomm 1. IE 3 MW 8 . J O 0 0 0 O T... .H 68333 $393 «3005.5 mo @923 4.3005» 93 was 3923 povdwoomufiv Hduobou mo bvfiflfipdnwpsawms obfipdasmfioo S cannon. DISCUSSION A method for the preparation of stable bacterial antigens from dissociated growth phases of Brucella has been described. The bacterial antigens of the dissociated phases were suSpended in 0.5- percent sodium chloride solution and stabilized in suspension by adjusting the pH to 9.0 with sodium carbonate. Homologous and heterologous agglutination tests were performed using the stable suspensions of dissociated phase antigens and the S phase antigen in dilutions of antiserum.produced in rabbits with live suspensions of different growth phases. The agglutination titers of the various sera were not appreciably different with the £5, abortus S phase antigen adjusted to pH 9.0 than they were with the 2:, abortus S phase antigen at pH 6.3. The dilutions of the antiserum exhibiting complete agglutination were the same with both S phase antigens. In a few instances, the final titer of tne antiserum showing incomplete agglutination varied by one or two dilutions. The agglutination of the R phase antigen with the S antiserum obtained in one experiment in this study is contradictory to results obtained by Marshall and Jared (9). They were unable to obtain agglutination of an R phase antigen suspension by an S phase antiserum of high titer. Gwatkin (3) was unable to produce agglutinins with .EEI abortus S phase for R phase antigen. Also, Mallman.and Gallo (8) did not obtain agglutination of their R antigen suspensions with S phase antisera prepared in rabbits. The results of this study -23- suggest that £3, abortus S and R phases posess common antigens (see table 6 and 7). The dissociated phase antisera were agglutinated by all of the antigens with exception of the E5. abortus M phase 1 and 25?.EEEE SL phase. The fact that several of the M growth phases produced agglutinins to a relatively higher titer for heterologous antigens than for homologous antigens can not be explained on the basis of this study. The agglutination results observed in table 6 with the g5: abortus R phase antigen and E5. abortus S phase antiserum are not in accord with those of Ross (13). Also, Ea. abortus R phase culture used in ‘ this experiment (table 7) produced agglutinins for the fig. abortus S phase antigen while the R phase antisera used by Ross (13) either failed to agglutinate his S phase antigens or they were agglutinated only in low titers. Gwatkin (3) and Henry (L) obtained results similar to those of Ross (13), but they used guinea pig sera which can not be compared to rabbit sera. The results obtained with the SL phase antiserum (tables 15 and 16) and the S phase antiserum (table 6) indicate that the SL phase can be distinguished from the S phase by serological means using the S and SL phase antisera with the S phase antigen. The colonies of the two phases appear morphologically identical. This would require further investigation using antigens and antisera prepared from S and SL phases that occur in each Species. E3, abortus M phase 1 might be distinguished from.the 25, abortus S phase, but the question remains as to whether it could be distinguished from Br. abortus SL phase. SUMMARY Bacterial antigens stabilized at pH 9.0 and containing 0.5- percent sodium chloride were prepared from several dissociated phases of Brucella. Agglutination studies show that the growth phases produce agglutinins to a certain degree in rabbits for both homologous and heterologous antigens. However, 21;. abortus M phase 1 and 135. Ellis SL phase failed to produce agglutinins for 25. abortus S phase antigen. The results of this study as a whole do not indicate that the different growth phases of Brucella can be distinguished one from another by serological (agglutination) means. ACKNOWLEDGEMENT I wish to thank Dr. I. Forest Huddleson for his patient help and guidance in this study. Also, I wish to thank Dr. R. Evelyn Sanders and Miss Marvis A. Richardson for their helpful suggestions. 1. 3. 9. 10. LITERATURE CITED Burnet, Et. La thermo-agglutination et l'evolution de l'eSche Brucella. Arch. Inst. Pasteur de Tunis. 17: 128-1h6. ngi Di Aichelburg, U. Agglutination aspecifique avec la fUchsine basique dans les microbes due groupe Brucella. Boll. d. sez. ital. 6: 30-32. 193h. Gwatkin, R. Antigenic qualities of a dissociated strain of Brucella abortus. Canad. Pub. Health J. 23: h85eh92. 1932. Henry, B. S. Dissociation in the genus Brucella. J. Inf. Dis. 52: 37h-h02. 1935. Huddleson, I. F. The mucoid phases of the genus Brucella. Am. J. Vet. Res. 8: 5-10. 19u6. Huddleson, I. F. Brucellosis in man and animals. Revised edition. New York. The Commonwealth Fund. 19h}. Huddleson, I. F., J.'W. Scales, and 0. J. Sorenson. Non-specific agglutination in the Brucella group. Mich. Agr. Exp. Sta. Tech. Bul. 1h9I 5-20. 1936. mailman,'fl. L. and F. Gallo. Studies on the dissociation of the Brucella group. J. Agr. Res. b6: 267-279. 1933. marshall, M. S. and D. Jared. Microbio dissociation in the Brucella group. J. Inf. Dis. u9I 1318-336. 1931. Mingle, C. K. and C. A. Manthei. Bacterial dissociation in Brucella abortus. Am. J. Vet. Res. 2: 181-189. l9hl. 11. 12. 13. 17. Munger, M. and I. F. Huddleson. The detection of antigenic variants of Brucella by means of an opsonocytophagic test. J. Bact. 35: 255-259. 1938. Plastridge, W} W} and J. G. McAlpine. Microbe dissociation in the abortus-melitensis group. J. Inf. Dis. £6: 315-323. 1930. Ross, G. R. The value of non-specific agglutination in the differentiation of the genus Brucella. J. Hyg. 26: 279-283. 1927. Stafseth, H. J. Studies in infectious abortion. Part II. Mich. Agr. Exp. Sta. Tech. Bul. b9: 7-11. 1920. Stearns, T.'W. and M. H. Roepke. Electrophoresis studies on Brucella. J. Bact. L2: h11~h30. 19hl. Stearns, T. W. and M. H. Roepke. The effect of dissociation on the electrophoretic mobility of Brucella. J. Bact. hZI 7h5-755o 19h1. White, P. B. On the relations of the alcohol—soluble constituents of bacteria to their spontaneous agglut- ination. J. Path. and Bact. 30: 113—132. 1927. uluum:willwillmlmuiwuuu1mm“‘ 723