0N THE CHEMECAL NATURE C)? THE FAC'YOR RESPQ'NSIBLE FOR ZONE REACTBCHS {N THE TUBE AGGLUTKNATEGN TEST FOR PULLORUM DiSEASE EN T‘JRKEYS Thais‘zs, fer rhea Emma 0% M. S. M‘smfififéfié my mums ‘Wéhcr E. Hfir‘é‘fi-‘fiz 3"- ‘ f'hrf- 139 2:; ’33 THESIS This is to certilg that the thesis entitled "0n the Chemical Nature of the Factor Reaponsible for Zone Reactions in the Tube Agglut ination Test for Pullorum Disease in Turkeys" presented b1] Walter E. Hewes, Jr. has been accepted towards fulfillment of the requirements for M . S . Bacteriology degree in W/ QM A ._ 7 I Iawr professor Date MID/7 2J1: /fJ_fl 0-169 \ A _.-.‘__. _ ON THE CHEMICAL NATURE OF THE FACTOR RESPONSIBLE FOR ZONE REACTIONS IN THE TUBE AGGLUTINATION . TEST FOR PULLORUM DISEASE IN TURKEYS * By Walter E. genes, Jr. 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*fihe degree of MASTER OF SCIENCE Department of Bacteriology 1950 THESIS Acknowledgemm t s the author wishes to acknowledge, with sincerest appreciation, fine technical advise and criticisms offered by Dr. Henrik J. Stafseth, Head of the Department of Bacteriology and Public Health. He also wishes to express gratification to Dr. Walter N. Mack, Assistant Professor of Bacteriology and Public Health, for his technical advise and interest. The work on this thesis was supported, in part, by a gqant from Swift and Company, Chicago, Illinois. Table of Contents Introduction Materials and Methods Experimen tal Procedure Chemical and Physical Treatment of Anti-sera 1. 2. 3. 4. 5. 6. Effect of Phenol Effect of Filtration Effect of Freezing Effect of Using 20% Bovine Albumin as Di luent for the Bacterial Antigens Effect of Absorption Effect of Heat Chemical Fractionation of Antisera Specificity of Zone Phenomenon Discussion and Results Summary Bibliography Page COCOCDG) 10 10 11 11 12 14 57 59 II III IV VI VII VIII IX-A . IX-B IX-C Tables Preliminary Serum Titrations Effect of Phenolation and Centrifugation Effect of Filtration Effect of Freezing Effect of Using 20% Bovine Albumin as Diluent for Antigens in Tube Agglutination Tests Retitration of Antisera Following Absorption with Homologous Antigen Effect of Heat Reaction of Antisera with Heterolo- gous Antigen to Show Specificity of the Zone Reaction Chemical Fractionation of Antisera, Showing No Zone Reaction, Before and After Absorption With Homologous Antigen Chemical Fractionation of Antisera, Showing Strong Zone Reaction, Before and After Absorption With Homologous Antigen Chemical Fractionation of Antisera, Showing No Agglutination, Before and After Absorption with Homolgous Antigen Page 15 17 19 21 24 26 29 31 34 55 36 INTRODUCTION This work has been done in an attempt to form at least a basis from which further studies on the nature of the factor responsible for zone reactions in tube agglutination tests can be carried out. Aside from giving some insight into the Chemical nature of this factor, it is also»desired to show some possible methods of removing the factor when conducting routine tube agglutination tests. The zone, or prozone, phenomenon in various types of serological reactions has been reported on numerous occasions. It was generally assumed by the workers concerned that fine zone phenomenon was due to some in- hibitive substance which combined with the antigen and thus prevented the formation of’the specific antigen- antibody complex. For example, Benton and Bailey (1926), in their studies on the protective action of pneumococcus antiserum.in mice infected with pneumococci, observed zone reactions. They fbund.that in some in- stances larger doses of antiserum were less effective than smaller doses. They attributed this zone pheno- menon to a substance, differing from.the protective antibody, that was capable of paralyzing the defensive mechanisms of the host. The exact nature of the inhibitive factor re- sponsible for zone reactions has been the subject of (1) much speculation. Eisenberg and Vblk (1902) attempted to explain.the appearance of the zone phenomenon in agglutination reactions on the basis of Ehrlich's conception of agglutinins in which.the agglutinin consisted of a haptophore and a zymophore group. The haptophore group was considered to be responsible for the specific union between the agglutinin and the antigen, and the zymophore group was considered to be responsible for agglutination. These workers assumed that in cases there zone reactions were encountered, the zymophore group was destroyed, while the haptophore group was not affected. They called this modified agglutinin an agglutinoid, and assumed further that the agglutinoid had a greater affinity for’the antigen than the agglutinin and could inhibit agglutination. Zinsser (1923) suggested that the zone phenomenon is due to the presence of a non-specific protein colloid substance which coats the antigenparticle, and thus prevents the formation of the antigen-antibody complex. Another hypothesis has been.advanced by Iandsteiner (1945) in which he states that the zone phenomenon in agglutination tests are due simply to the physico- chemical laws which govern the absorption of all colloidal substances and that optimum prepcrtions of two colloids are necessary for complete agglutination (2) in all dilutions. If antigen or antibody is present in excess of these optimum.proportions there will be an incomplete agglutination. In recent studies on the agglutination of red blood cells by anti-Rh sera, Wiener (1944), (1945), Race (1944), Levine and Waller (1945), Hattersby and Fawcett (1947), and.Diamond and Denton (1945) have shown the presence of both "complete“ and “incomplete" antibodies. The "incomplete" antibodies or "blocking" antibodies, according to Wiener (1945) are univalent and are capable of combining with receptors or individual erythrocytes but will not link receptors of two or more erythrocytes, thus preventing agglutination of the red blood cells. Diamond and Denton (1945) observed that the zone phenomenon was present only in the serum.of individuals who had received repeated antigenic stimuli prior to being tested for Rh agglutination. Individuals who had not received a severe antigenic stimulation.before the agglutination tests were made showed no zone reac- tion. These authors state, "After repeated stimulation of experimental subjects or following multiple pregnancies in some women, inhibitor or "blocking“ antibody appears. This may be called the late, nature or complete anti- body." ..... "The term 'early, or immature, antibody' would seem to apply to that causing agglutination in saline suspensions, whereas 'late, or mature, antibody' (3) is more descriptive of the 'blocking' antibody which is recognizable only in albumin or plasma media and is as— sociated with more severe or prolonged sensitizati on." This concept is in direct opposition to that proposed by Wiener (1945). Wai and Stafseth (1950) reported that zone reactions in tube agglutination tests are due to the presence of an inhibitive colloidal substance which is adsorbed by the bacterial cell and prevents reaction with agglutinins. These authors state that the colloidal substance is non-specific in its protective action. MATERIALS AND METHODS Twelve, four month-old, White Holland turkey hens were obtained from a "pullorum-free" flock. The birds were kept in an indoor- pen previously cleaned with hot lye solution. They were kept in this same pen through- out the experiment ard were thus maintained under similar conditions of management. The antigens used in the experimental stimulation of antibody production in the turkeys, and in the tube agglutination test, consisted of three standard strains of Salmonella pullorum obtained from the U. S. Bureau of Animal Industry. These strains, designated as strains 4, 10, and 11, were classified in the Poultry Clinic as _S_,_ mllorum on the basis of morphology, motility, and biochemical reactions. They were then identified (4) serologically by the author according to the method of Edwards and Bruner (1942). The antigens for the tube agglutination tests were prepared by seeding a layer of nutrient agar (Difco) in 52 ounce, screw-cap bottles with a 24 hour culture of the respective strain followed by incubation at 37°C. fbr 48 hours. The culture was washed off the agar with a small amount of physiological saline (0.85 per cent NaCl). This suspension was then diluted with physiological saline to an optical density corresponding to tube 2 of a McFarland Nephelometer (McFarland 1907). Phenol was used as a preservative and was added to the suspension to a final concentra- tion of 0.5 per cent. The resulting antigens were adjusted with brompthymol-blue pH 7.8 with 0.1 N NaOH. The three antigens were designated as 4, 10, and 11 (corresponding to the three strains of §,_ pullorum used in their preparation. The antigens fer experimental stimulation of anti- body production in the turkeys were prepared by seeding nutrialt agar (Difco) slants with the three respective strains of S; pullorum. After 24 hours incubation at 57°C. the cultures were washed off the slants with a small amount of physiological saline. The suspensions were diluted with physiological saline to an optical density corresponding to tube 1 of'a McFarland Nephelo- meter. (5) In the experimental work described below, the standard tube agglutination test, using constant amounts of antigen (1 ml.) and two-fold serial dilu- tions of antiserum, was used in all cases, The serum- antigen mixtures were incubated at 57°C. in all cases unless otherwise designated. The results of the tests are expressed in terms of degree of agglutination as follows: Complete agglutination-- +-++-+ Marked agglutination---- +~+-+ Moderate agglutinationp- +-+ Slight agglutination---- + No agglutination -------- - EXPERIMENTAL PROCEDURE The turkeys were divided into four groups of three birds each, designated Group 4, Group 10, Group 11, and Control Group, the numbers corresponding to the strain of organism used in immunizing the birds. Group 4 Group 11 Turkey 5702 Turkey 5708 Turkey 5705 Thrkey 5709 Turkey 5704 Tur key 5710 (6) Group 10 Control Group Turkey 5705 Turkey 5711 Tur key 5706 Tur key 5712 Turkey 5707 Turkey 5713 On September 21, 1949 the turkeys in each group were inoculated intravenously with 1.0 ml. of a heat killed (60°C. for 1 hour) saline suspension of tie strain of §_._ pgllorum corresponding to the number of the group. The control group was not inoculated. The turkeys sub- secpent 1y were inoculated intravenously according to the following schedule: Volume of Days after Initial Inoculation Inoculation 1 10 2 l5 3 20 5 25 The turkeys were tested, by the mole blood plate agglutination method, (Stafseth 1958), using Redigen "Turkey“ Antigen and Lederle Polyvalent K Antigen, at 5-day intervals after the final inoculation. At the 15th day there was only a slight agglutination and daily tests were made from this time until 19 days after the final inoculation. At this time, all turkeys showed complete agglutination by the whole blood method. The turkeys were then bled from the brachial vien. (7) The blood was collected in sterile bottles and allowed to clot at room temperature. After complete clotting, the blood was placed in a refrigerator at 4°C. and the sera was allowed to separate. The sera were decanted into sterile test tubes ami stored at 4°C. until used. The sera from the turkeys in Group 4 thus contained anti-strain 4 §L_pullorum.antibodies, those from.Group 10 contained Anti-strain 10 S; pullorum antibodies, those from.Group 11 contained anti-strain 11 S; pullorum antibodies, and the sera from the birds in the Control Group contained no S; pullorum antibodies as confirmed by tube agglutination tests. Since the sera from turkeys in each group showed similar titers, in respect to sera from the three birds in each group, they were pooled. This was done so that a larger number of tests might be condlcted on each group of sera. Chemical and Physical Treatment of Sara. 1. Effect of Phenol. (a) The antisera were diluted with an eqzal volume of phenolated saline (2 per cent phenol by volume in 0.85 per cent NaCl solution) giving a final concentra- tion of l per cent phenol by volume. (b) The antisera were diluted with an equal volume of 4 per cent phenolated saline, giving a final concentration of 2 per cent phenol by volume. (8) These treated sera were placed at 4°C. for 24 hours, and the precipitate that formed was sedimalted by centrifugation at 2000 rpm. far 50 minutes. The supernatant fluids were decanted and were used as antisera in the tube agglutination tests. The serump antigen mixtures were incubated at 57°C. and results were read at 24 and 50 hours. 2. Effect of filtration. (a) Samples of each group of sera were passed through a No. 5 Seitz EK filter. The filtrates were tested by the tube agglutination test at 57°C. Results were read at 24 and 50 hours. ‘ 5. Effect of Freezing (-57°C.) (a) Samples of antisera were stored at -57°C. for periods of 24, 48, and 96 hours. At the end of the storage period the sera were thawed at room temperature and one smnple from each group was tested for agglutin- ability. (b) Another sample from each group was centri- fuged at 2000 rpm. for 50 minutes. (c) A.third sample from each group was passed through a No. 5 Seitz EK filter. All treated sera were than tested by the agglutina- tion test at 57°C. Results were read at 24 and 50 hours. 4. Effect of using Bovine Albumin as the Diluent for Bacterial Antigens in the Tube Agglutination Test. (9) (a) In this experiment the phenolated antigens used for the tube agglutination tests were centrifuged at 2000 rpm. for 1 hour. The alpernatant fluids were decanted and the sediments were allowed to drain. The sedimented organisms were resuspended in a 20% bovine albumin solution in distilled water to original volume. The tube agglutination tests were conducted on all groups of sera using the bovine albumin suspen- sions as antigen. Incubation was at 57°C., room temperature (approximately 22°C.), and 4°C. with different smnples of sera. Results were read at 18, 24, and 50 hours. 5. Effect of .Absorption. (a) Tube agglutination tests were con- ducted on several sanples of each group of sera. Incubation was at 57°C. and at 4°C. with different samples of sera. At his end of 24 hours results were read, and all tubes were centrifuged at 2000 rpm. for 50 minutes. The supernatant fluids were decanted.and retested using equal volumes of fresh antigen. These mixtures were all incubated at 57°C. and readings were made at 24 and 50 hours. (10) 6. Effect of Heat (a) Sanples of each group of antisera were heated in a water bath at 51°C. for l.hour, and other samples were heated at 60°C. for 1 hour. After heating, all sera were centrifuged at 2000 rpm. for 45 minutes, and standard tube agglutination tests were incubated at 57°C. and readings were made at 24 and 50 hours. Chemical Fractionation of Antisera. 1. In this experiment, representative antisera, one showing a strong zone reaction, one showing no zone reaction, uni one showing no agglutination (Control serum), were fractionated by the nethod of Greenburg (1929). The serum constituents (albumin, globulin, and total protein) were determined photome- trically on a Cenco No. 41000 photelometer, using a green filter. This method of determination has been proved to be accurate to within 1 to 5 per cent of the results obtained by the Micro-Ejeldahl method. 2. The above experiment was repeated following absorption of the antisera with.homologous antigen at 4°C. and 57°C. The absorption was carried out by mixing equal volumes of antisera and.homologous antigen. After the incubation period the serum-antigen mixtures were centrifuged at 2000 rpm. for 1 hour. The super- natant fluids were then fractionated and the serum proteins determined as above. (11) The method of Greenberg for the determination of serum proteins is as follows: "1. To prepare the albumin solution place 9.5 ml. of 22.2% Negso4 solution in a centrifuge tube. Add 0.5 ml. of serum and mix well. Add 5.0 ml. of ethyl ether, stopper the tube and shake vigorously for a few seconds. Stopper the tube loosely and centrifuge at moderate speed for 10 minutes. Pipette 5.0 m1. of the lower albumin solution into a 50 m1. volumetric flask. 2. To determine total protein, dilute 1 m1. of serum with 9.0 m1. of 0.85% NaCl solution and mix. Pipette 2.0 m1. of the di luted serum into another 50 ml. volumetric flask. 5. To each of the flasks add about 55 ml. of distilled water, exactly 4.0 m1. of 10% NaOH solution and 5.0 ml. of phenol reagent (Folin-Ciocalteu, 1929). Dilute to volume with water an! mix well. After 5 minutes read in the photelometer using a “green filter (about 520 milli-microns).u Specificity of the Zone Phenomenon 1. To determine the "strain-specificity" of the zone phenomenon, samples of each group of antisera were used in the tube agglutination test with heterolo- gous antigen. Serum from Group 4 was tested with strain 10 antigen, Group 10 serum was tested with strain 11 antigen, and Group 11 serum was tested with (12) strain 4 antigen. 2. To determine "species-specificity”, samples of sera showing zone reactions were tested with Salmonella enteritidis (I, IX, XII); Salmonella gallinarum, (IX, XII); and Salmonella typhimurium var. copenhagen (IV, XII). (13) RESULTS AND DISCUSSION The data presented in Tables I -- IX-C represent a summation of fine results obtained from.several tests conducted with each serum. These data are considered to be representative for each serum. The preliminary serum titrations, Table I, present an interesting picture in that sera from only Group 11 show a zone of inhibition. A11 birds in.this group present an almost identical agglutination pattern. The zone of inhibition is in practically the same position in the pattern and the en titers are about the same in all three individual sera. The sera from Group 4 and Group 10 show no zones of inhibition and have similar and titers. There is an indication, therefore, that the antigen used to stimulate antibody production.may play some role in the formation of an inhibiting factor. (14) Ache. ceaeemv I I I I I I I I I I I Ha.oae: macho flamenco, I I + ++ + + ++ +++ +++ ++ + an oafim hexasn I + +++ ++ + + ++ +++ ++ + + an mafia hexasa I I + + +++ +++ ,+ ++ + + I AH movm banana I I + + ++ ++ +++ +++ ++++ ++++ ++++ oa Aueaoomv ca macaw I I + + ++ ++ +++ +++ ++++ ++++ ++++ OH . behm heunsa I I I I + + ++ +++ ++++ ++++ ++++ 0a worm hexane I I + + ++ ++ +++ +++ +++ ++++ ++++ 0H mahm heuhjfli I I + + ++ +++ +++. ++++ ++++ ++++ ++++ .4 Aueaeemv.: macho I I + + ++ +++ +++ ++++ ++++ ++++ ++++ .J adhm heunsa I I I + ++ ++ +++. +++ ++++ ++++ ++++ _4 mahm hexhaa I I + ++ ++ +++ ++++ ++++ ++++ ++++ ++e+i .: Noum hexane acetone names comm omen one can ecu em as em as accessed. - ummwrstn sBaem managed we seesaw- euoaelhvap some. maendsaaoam .H canoe (15) Effect of Phenolation When pehnol was added to the sera, in a final concentration of l per cent, and stored at 4°C. fir 24 hours, a precipitate formed. In some cases the pre- cipitate formed immediately, while in others no pre- cipitate was visible by macrosCOpic observation after several hours. Upon removal of the precipitate by centrifugation, the supernatant fluids revealed, through agglutination tests, a shift in the position of the zone of inhibition and the end titers totthe left, Table II. There was a tendency to remove the zone of inhibition, while at the same time there was a reduction in the agglutinin.content of he sera. It is generally'agreed, by most workers, that antibodies are present in the various globulin frac- tions of serum. Since the agglutinin content and the zone of inhibition are both reduced by treatment with 1 per cent phenol, there is an indication that the agglutinins and the inhibitive factor are both related in molecular structure. (16) I I I I I I I I I I I m 2.2.: Theo I I I I I I I I I I I m a S 295 I I I I I I I I I I I m S. 2 hate I I I I I I I I I I I u 4 a 96.5 I I I I I I I I I I I H 2.3.: donate I I I + ... 1. 1. +1. +1. ... I H .3 .3 95am I I I I ... ... 1. 1.... +1. +1.... 1...... .n S o." 30.6 I I I I ... 1. 1. 1.... 1.... +1. 1.1. a 4 A 99.6 .tao 8sz come 83 So can 02 8 e: on 3 Sq . douche Cheese 93.35. $3.33 Show no .88 Sandstone... es. 83.15% no nodes .2 .2: (17) IL Effect of Filtration It has been reported by Wai and Stafseth (1950) that zones of inhibition, in agglutination tests with §;_pullorum, have been removed by filtration of the sera through a bacterial filter prior to agglu- tination. AsIan readily be seen by comparing Tables III and I, this work could not be duplicated in the present studies. Filtration.of'the antisera through a Seitz No. 5 RE filter before agglutination tests resulted in practically no Change in the agglutinability of the sera. Similar results were Obtained by filtering sera that had been previously heated to 56°C. for :50 min- utes. If, as has been proposed by Zinsser (1925), the zones of inhibition were due to a.non-specific colloid coating on the antigen, it might be expected that filtration of the serum would remove this substance and permit complete agglutination. However, since neither the inhibitive factor nor the agglutinin content were affected by filtration, it is further indicative that the two substances are similar. (18) I I + ... 1.... 1.... 1...... 1.1. + + 1.+ 1. .3 H." 9.5.6 I I I I + 1. 1. 1.... 1.1. 1.1. 14+ 1.1. 0H 3.. 95.6 I I I ... 1. .1. +1. 1.... 1.... 11. +1.... 1.1. d 4 93.5 accrued demos 8% 8mm 82 one can o3 on 3 on 3 83...: «.8.er .8333 5...... $.33 Show me 839.32 «c vacuum .HHH .2: (19) Effect of Freezing Freezing at ~57°C. for as long as 96 hours seems to have very little effect on either the end titers or zone of inhibition of treated sera, Table IV. Filtra- tion and centrifugation of these treated sera did not alter the results. This experiment proved little except that the in- hibitive factor is stable and persists after freezing and thawing. The experiment was done on the assumption that file inhibitive factor might be a non-specific colloidal protein of large molecular size which might be inactivated or denatured by freezing and thawing. Effect of Using 20% Bovine Albumin as Diluent for the Antigens in Tube Agglutination Tests It was reported by Diamond and Denton (1947) that certain Rh Antisera, showing no agglutination of human erythrocytes suspended in saline, did show high agglutination titers when the erythrocytes were suspend- ed in human plasma. They further demonstrated that other substances could be used in place of plasma. Bovine albumin in concentrations of 20 and 50 per cent seemed to give the best results and there were no non-specific effects observed. From their observations they concluded that the inhibition of agglutination in saline suspensions of erythrocytes was due to the presence of "complete“ antibodies. The "complete" (20) I. .i .‘(Iflm ....._ .' . I I I I I I I I I I I Q¢ HHaOHeJ Hahvfléo I I I I I I I I I I I 04 Hecoaq4 Heapnoo I I I I I I I I I I I in 3.2.: H.381. I I + + ++ ++ +++ +++ ++ + + no Ha AH @9099 I I + + ++ ++ ++++ ++ ++ + ++ m4 HH HH mocha I I I + ++ ++ ++ +++ +++ + + 4N HA AH macaw I I + ++ ++ +++ ++++ ++++ ++++ ++++ ++++ mm OH OH myehw I I I + +++ ++++ ++++ ++++ ++++ ++++ ++++ m4 OH OH macho I I + ++ +++ ++++ ++++ ++++ ++++ ++++ ++++ 4m OH OH muons I I I + ++ ++ +++ ++++ ++++ +++ ++++ em .4 .4 macaw I I I I + ++ +++ +++ ++++ ++++ ++++ m4 .4 .4 niche ovHMD ommm comm OQNH +++ +++ ++++ ++++ +++ +++ .4Nr I.4 .4 niche Aw®IIIAfldfillddfllldflfiIIAfifillda, ,- . I 3.333 .5...” a ., cu mmocMMmV». £38. 98.35 I 8% moanemsuaavnee one unawoeum no roommm .bH canes (21) antibodies had the ability to react with the erythrocytes but lacked the ability to cause agglutination. The results obtained by these workers would seem to indi- cate support for the zymophore-haptophore concept. The "complete"\ antibodies apparently have a greater affinity for the antigen and thus prevent reaction of the "incomplete" antibodies with the antigen. The rather unusual conclusions expressed by these workers were discussed in the Introduction of this thesis. The author wishes to re-emphasize the fact that these conclusions are contrary to the more widely regarded concept in which the "incomple to“ antibody is univalent and acts to inhibit agglutination. These workers also observed.that the ”incomplete” antibodies were only present in serum from individuals who had received the antigenic stimulation a relatively short time prior to being tested. Serum from individu- als receiving the antigenic stimulus years before being tested were found to show a high incidence of ”complete" antibodies and showed inhibition of agglu- tination. They concluded from this observation that during the process of formation, antibodies pass through various phases. After they are completely formed, the antibodies are capable of reacting with the antigen but are incapable of causing agglutination. These workers did not attempt to explain why the presence of human blood plasma or bovine albumin prevented the (22) l: Ill IlIllIAI. Ill-Ill)! I‘ll/Ill: . ll)llll ll I’lll lllll/ |)||II "complete" antibodies from inhibiting the action of the "incomplete" antibodies. 1 Table V shows that the use of 20% bovine albumin as a diluent for the bacterial antigens produced re- sults similar to those of Diamond and Denton. The zone of inhibition was removed completely and the end titers were increased slightly. Due to the lack of a sufficient supply of turkey L. blood plasma, this experiment does not include the use I of bacterial antigens suspended in turkey blood plasma. ; The results obtained in this experiment show only that the presence of a relatively high concentration of a colloidal protein solution, such as bovine albumin, prevents the inhibition of agglutination. An explanation of'this phenomenon is not offered. The fact that zones of inhibition of agglutina- tion can‘be removed by using bovine albumin in place of 0.85% NaCl as diluent for bacterial antigens affords a practical solution to the problem presented by the occurenCe of false negatives in routine tube agglutina- tion tests employing only a few serum dilutions. (25) Ha.oaq4 I I I I I I I . m4 ”chance I I I I I I I .ecem HH.eH.a enhance ++ +++ ++++ ++++ ++++ ++++ ++++ .om4 Ha AH macaw +++ +++ +++ ++++ ++++ ++++ ++++ cochm Ha AH macho +++ +++ ++++ ++++ ++++ ++++ ++++, .om4 OH OH macho +++ +++ +++ ++++ ++++ ++++ ++++ .oehm 0H 0H macaw ++ ++ ++ +++ +++ ++++ ++++ .om4 .4 .4 macho ++ ++ +++ +++ ++++ ++++ ++++ onehm .4 .4 macaw one, ohm, or .1mm ow om . nehacnnenH homered Incaaae< . uneavsfian spasm no .9309 «are» neapenavsamwe can» ma Issuance can can encodes as can: .559 “mow m5»: we gonna .b canes (2h) The Effect of Absorption with Homologous Antigen In 1948, Jamil.and Stafseth demonstrated the removal of zones by retitration of absorbed sera after removal of bacterial cells by centrifugation. The data in Table VI show that similar results were obtained in this experiment. The temperature at which absorption was carried out had no effect on the results. The inhibitive factor was complete- ly removed by absorption to the bacterial cells. In the tubes originally Showing zone reactions, a high agglutinin content was demonstrated by retitration with fresh antigen. . The inhibitive factor apparently has a greater affinity fer'the antigen than do the agglutinins. This experimalt also proves that the prozones are not due to the absence of optimum.proportions of antigen and antibody. The removal of prozones by absorption supports several oi'the theories presented in the Introduction of this work, and no definite conclusions may be offered as to the nature of the inhibitive factor. (25) Tang I I I I I I I I I I . 04 3.3.4 "0.3.30 I I I I I I I I I I .ecem 3.3... H.380 I I + + ++ +++ +++ 1.++ 1.++ ++++ .004 d." H." A45.8 I I ++ ++ ++ +++ 1.++ 1.++ 1.++ ++1. .00bm .5 .2” A50.5 I I I I I + + +1. +1.... 1.++ .004 0H 0H $6.5 I I I I I + .1. +1. +++... ++++ .005m 3 0a 90.6 I I I I + 1. ++ +++ 1.++ 1.++ .004 4 4 95.5 I I I + + 1. +++ ++++ ++++ 1.1... .0esm 4 4 990.5 8% 8mm Sela Sc 8M on. on 3 8 3 83.82: 83......3 some»... 98.35. . .8333 8.8 u. .539 guano-be want—0:00 0.30.3.5 00 3393.35 0.30.. 4m .80 Jenn use .004 no 000300 0500303.. A»: .Hb 0.3.09 (26) .lllllljl IIII Effect of Heat The effect of heat on prozones in sera was one of the first phenomena reported having a bearing on this subject. Dreyer-and Jex-Blake (1906) demonstrated that by heating "normal" antisera at 70°-7500. fer a few minutes, an intermediate zone of inhibition was produced. They attributed this to the fact that the heat probably I..-IJ-r:. produced a substance which was capable of impeding agglutination. Shibley (1929) made further studies on the production of zones by heat treatment of anti- sera. He was unable to demonstrate zones of inhibi- tion in unheated sera. Sera heated at 60°-70°C. showed zones of inhibition in the lower dilutions while agglutination occured at the higher dilutions. He also demonstrated that heating the sera at 76°C. produced no zones but did lower the end titer of the sera tested. Spencer (1930) was able to demonstrate that, with sera heated at 56°C. for periods varying frmm 10 to 60 . minutes, the zone of inhibition widened in proportion to the time of heating. Corpron, Bivens and.Stafseth (1947) made similar observations. The end titers were decreased in the heated sera. The following experiment was performed to sub- stantiate these data, since the determination of the heat on zones of inhibition is a necessary and help- (27) ful step in determining the chemical nature of the inhibitive substance. The results shown in Table VII correlate with those Obtained by other workers. Sera showing no zones when unheated, show a def- inite zone of inhibition when heated at 51°C. for 1 hour. Sera showing zones When unheated, show a definite widening of fine zones when heated at 51°C. for 1 hour. Heating at 60°C. for 1 hour completely inactivated the agglutinins,in all sera tested. There are several possible explanations for this phenomenon. The most reasonable one seems to be_that some of'the agglutinins are partially denatured by heat. These altered agglutinins seem to have the ability to react with.flae antigen but do not cause agglutination. The zones produced by heat seem to occur in the higher serum dilutions. That they do not come pletely inhibit agglutination.can be explained by the dilution factor in that the numbers of unaltered agglu- tinins exceed.the numbers of altered agglutinins. Since some sera show zones of inhibition prior to any heat treatment, it is reasonable to assume that the inhibitive factor is similar to that produced by heating these sera. This seems to indicate, therefdre, that the inhibitive factor is a partially formed agglu- tinin, as in the case where zones are found without treating the sera, or a partially denatured agglutinin (28) I I I I I I I I I I I . .00 ”H.0H94 Hogonuo I I I I I I I I I I I .0.Hm 44.0”.4 Hoapnoo I I I I I I I I I I I .003 .5 .2" @0000 I ... +++ +++ ... I I ... I I I . 0.3 a." .2” @0000 I I I I I I I I I I I .ooow 0H 0H usage I I I ... ...... +++ ...... +++... ... ... I .001.“ 0..” 0." @9000 I I I I I I I I I I I .0.00 .4 .4 00.00 I I I I , ... ...... +++ 1.... ... ... I .003 4 4 @0000 .»0000. omam come can” 040 own 00H em 0: cm 0H ..4 a you . use»..m n.0apsq ....aasq . anuapnflan_usuom 0. ....u 1|:0 soapsmsuaupn.. en. a..n a. 0..eum .HH>..Hnus (29) as in the case where heat treatment is required to produce the zones. On the Specificity of the Zone Reaction It has been reported by Zinsser (1923), Iandsteiner (1942) and Wai and Stafseth (1950) that prozones are due to a non-specific agent. The following experiment was perfbrmed in an E effort to determine the specificity with respect to 5 antigenic structure. As shown in Table VIII, the inp hibitive factor was specific for the antigen used. Al- 2 though some inhibition is shown when Group 11 serum 1: reacts with Sngallinarum, the relationship between g; gallinarum and S; pullorum is so close that inhibi- tion is to be expected. However, when S; enteritidis was used as antigen, there was no inhibition. The same result was obtained using S; typhimurium.var. copenhagen as antigen, although the agglutination was much lower. This is to be expected since only the XII somatic antigen is present as fine common factor. It may be concluded from this that the inhibitive factor is specific for the antigen used to stimulate antibody production. This is further indication that the inhibitive factor is similar in molecular structure to the agglutinins. (50) 2.3. .fi 0. :4 5000000 35.5.3 I I I + + ++ ++ +++ +++ ++++ ++++ udddndudnuulqu. 55 @0000 3:35 Hun—slang .3 00000 . 3:33 .. .I + ++ +++ +++ ++ +++ +++ ++++. +++ ++ .luududdflduldu 55 @0050 I ... I. +++ ++++ ...... ... ... ...... ... ... A HHH.HH V4 .5 @0000 AHHN.NH.HV I I I I I + ++ ++ +++ +++ +++ .4 @0000 I I I I I I + + + ++ ++ .4 @0000. I I I + + ++ +++ ++++ ++++ ++++ ++++ .4 @0000 I I + + ++ +++ +++ ++++ ++++ ++++ ++++ A HHM.NH v OH .4 @0000 .080 83 8mm 83 20 8m 3.: 8 3 on S L .8035 ...-35 1‘ 000500550.I0000 00500000 0000 on» 00 hyaeauaee@u :00. 0» 0005000 000005000004 0050.00005000 «0 noavesem .HHH>.05nUa (31) I I V ' ll ' l ’ l ' t ' ' I I I. .I . ... .. ' . . I I II II I. '- l . 'I .II .II Chemical Fractionation of Antisera The most significant differences in Tables IX-A, IX-B, and IX-C are found in.the globulin fractions of ,the sera. The sera showing no zone reaction (Group 4) had a relatively high serum globulin content, and after absorption with homologous antigen this Serum.globulin was greatly reduced. However, even after absorption, Y'FIIH the concentration.of'the globulin fraction.was §ufficient 3. to give an inverted albumin-globulin ratio. The a normal A/G ratio is about 1. 5. i In Table IX-B the antisera showing a strong zone reaction (Group 11) had low globulin content. Ab- sorption with homologous antigen lowered the globulin content to about the same extent (0.4 grams%) as in the Group 4 sera. The albumin content was somewhat higher in the Group 11 sera than in the Grogpl4 and Control sera, but it is not lowered after the ab- sorption process. The albumin fraction.was not, therefore, responsible fer the zone reactions since the zone was removed by absorption,but the albumin fraction was not altered. This indicated that the agglutinins found in the globulin fraction are responsible for 31 increase in serum globulins. The occurence of zone reactions seems to indicate the pre- sence of partial or incomplete agglutinins as evidenced (52) by the low globulin content of such sera. Summation of the results of the several experi- ments indicate that zones of inhibition in tube agglutination tests for pullorum disease in turkeys were found to be caused by the presence of partial agglutinins. The partial agglutinins were specific with regard to serological activity and had a greater affinity for bacterial antigens than did "normal" agglutinins. The partial agglutinins did not have the ability to cause agglutination but finey did re- act with the antigens. It is realized that the evidence presented here is not complete, and further work should be undertaken along this line. It is suggested that electrophoretic analysis of zone antisera and further chemical frac- tionation studies be carried out. (53) 00.0 00.0 00... 00.4 .03 m 6.4 0.0 00000000 4 @0000 ...a 40 6.0m 00.0 $0 8.0 $4 , 0. 0.0.83. 4 08.5 two 85 00.0 3.0 0.00.53. 4 08.6 3“ 0.0003 3“ 00003 G“ 00003 . 30.0 a} 505.3 5.502 .5330 03.0 - 000000009 0000304 000300005 flovo0@ 0.0000 .0... m 6.4 I I I I I + ++ ++ +++ ++++ +++. 0. 0.0.3.0. 4 4 0300 ..a 40 .900 I I I I ... ... ++ +++ +++... +++... +++... 0.0 00900000 4 4 @0000 I I ... ... .I. +++ +++ +++... 1.++ +++... +++... 0000000000 4 4 @0000 .008 83 8mm 83 040 can 000 8 04 cm 3 0.3.3.00 . 8300.34 0.035 .0335 00 .900.“ - .8333 .50.... . 000.300 00000505: .30? 0000300000 00000 000 000000 50.3.0000 0000 00 00.2.2.0 .0000300 00 00.300030000 50000040 .dINH 05000 (311) no.0 04.0 00.0 omI4 0000 m .004 00 00000000 as 00000 00.0 mm.0 00.0 mNIa ..00.00 .0 00.00000u00 Ad 00000 .00.0 00.0 00.0 no.4 , 0.0.0.0.00 00 00.00 .000.0 0\¢ 00000000 0000000 00.0000 0.000 . . 000000000 00000004 3“ 000000, 000000000 500000 00000 900.0.004 I I I I I + ++ +++ ++++ ++++ ++++ 00 00000000 an HH @0000 .000 :0 .005m I I I I + ++ +++ +++ ++++ ++++ ++++ 00 00000000 Ha HH 00000 I + + +++ +++ + + + + I 0.000.000: 00 00 00000 .080 0000 0000 0000 30 000 000 00 2 00 00 83.0080 0000000004 0000000 00000004 000a00nun.lu0om 00 .mlba 0000000 0:000H0000 0000.0000000000 00000 0000000000..00000000 0000 000000 0000000 .00000000 00 0000000000000 H0000000 .mIMH 0000a (35) SA ou.m 8.m 8.m .3. m .o 4 pi conuouma H9330 Oahu a to NM om; om.N om.m 0.7m v: nonhuman .3."ng mm.” 2.m . 8.m .8.m @3835 3380 ,. # c“ a: v § saw“. § 29% Spam 3} 5.1.33 . £53 £225 7%.... pus—Bucks 30.35 30303..“ 332m 850m .hiumm.oaa ...5 fl .ooum ' ' ' | ' ' | a! ' ' ' c.ph°.p‘§ HHIOHJ thvuoo .paoo omam comm coma cam omm ow” on a: on o” nuflp¢naoua gapmnound nova»: ago-.35 \mmmmmmflqa canon no .gaua Humans pgachhaom and! gavmuoupu you: and ououon 503.3353»: » 2.. warns. .3335 «o nodpdnogoé 70.328 39.5” ode-H (36) SUMMARX An attempt has been made to determine the chemical nature of the factor responsible for zone reactions in tube agglutination tests for pullorum disease in turkeys. g; pullorum antisera were prepared by immunizing three groups of'turkeys with saline suspensions of 3:, different strains of g; pullorum. These antisera were treated by various chemical and physical methods and fractionated by chemical means. The results obtained were as follows: 1. Addition of phenol, 0.1 per cent final ' concentration, to samples of antisera had a tendency to remove zones of inhibition and to reduce the end titers of all sera tested. 2. Filtration of antisera, before and after heating, using a No. 3 Seitz EK Filter, did not alter the zones. 3. Freezing the antisera at -37°C. fer varying periods of time and thawing at room.temperature had no effect on the zones. 4. Using 20% bovine albumin as the diluent for bacterial antigens in tube agglutination tests resulted in the removal of’zones of inhibition. 5. Absorption of antisera with homologous anti- gens resulted in fihe removal of zones. 6. Beating the sera at 51°C. for 1 hour pro- duced an increase in the width of the zones and pro- (37) duced zones in sera that showed no zones before heating. Heating the sera at 60°C. for 1 hour in- activated all agglutinins. 7. All chemical fractionation of antisera showed that sera giving no zone reactions had a higher globulin content than did sera eXhibiting strong zone reactions. E- 79 These results indicate that the inhibitive factor is a "partial" agglutinin with a greater affinity for the bacterial antigen than the affinity of the normal agglutinins. (39) BIBLIOGRAPHY Corpron, R. Bivins, J. A., and Stafseth, H. J. 1947. Pullorum disease studies in.turkeys. I. Poultry Sc. gg; 540-551. Diamond, L. K., and.Denton, R. L. 1945. Rh agglutination in various media with particular reference to the value of albumin. J. lab. and Clin. Med. 50: 821-850. Dreyer, G. and Jex-Blake, A. J. 1906. On the agglutina- tion of bacteria. J. Path. Bact. 11:1. Edwards, P. R. and Bruner, D. W. 1942. Serological i identification of salmonella cultures. Uni- ‘ versity of Kentucky Agriculture Experiment Station, Circular 54. Eisenberg, P. and Volk, R. Quoted by Zinsser, H. 1925. |l Infection and resistance. 5rd. ed. The Mac- 5 Millen 00., New York. Felton, L. D. and Bailey, G. H. 1926. Biologic signifi- cance of the soluble specific substance of pneumococci. J. Infec. Dis. 58: 151. Greenburg, 1989. A.method for the determination of serum proteins. J. Biol. Chem. 82: 545. Hattersby, P. C. and Fawcett, M. L. 1947. The prozone - phenomenon in Rh blocking serums. Am. J. Clin. Path. 17: 695. Jamil, M. and Stafseth, H. J. 1948. Pullorum disease studies in turkeys. II. Zone reactions in the tube agglutination test. Poultry Sc. 28: 571- 576. Iandsteiner, M. 1945. The specificity of serological reactions. Harvard University Press, Boston, M8880 Levine, P. and Waller, R. K. 1946. On the blocking antibody and the zone phenomenon in human anti-Rh sera. Science 105: 589. McFarland, J. 1907. The nephelometer. J. Amer. Med. Assn. 49: 1176. Race, R. R. 1944. An "incomplete" antibody in human serum. Nature 155: 771. _ (39) Shibley, G. S. 1929. Studies in agglutination. IV. The agglutination inhibit on zone. J. Expt. Med. 50: 825. _ Stafseth, H. J. 1958. The whole blood agglutination test for pullorum disease. Vet. Med. 55:1. Wai, W. Y. and.Stafseth, H. J. 1950. Pullorum disease studies in turkeys, III. Studies on the bactericidal and agglutination properties of serum and plasma of normal and pullorum infected turkeys. In press. Wiener, A. s. 1944. A new test (blocking test) for Rh sensitization. Proc. Soc. Exptl. Biol. Med. 56: 175. Wiener, A. S. 1945. The Rh blood.types and some of their applications. Amer. J. Clin. Path. 15: 106. Zinsser, H. 1925. Infection and resistance. 5rd ed. The MacMillen Co., New York. (40) UNIVERSITY LIBRARIES 1 AN STAT E II 3 1293 03085 2 19 MICHIG