M " Hl' W l ' ’ll. M WI H' tl’ ? 1"! l i HI A :8: ll MOON I ! ZONE REAC‘HGNS N THE AGGLUWNAHON ’E‘ES? FOR PULLORUM DiSEASE N ”fURKEYS Thesis for the 009m of M. S‘ MICHEGAN STATE COLLEGE Mohammaé Jamil 1948 IHESIS This is to certify that the thesis entitled Zone Reactions in the Agglutination Test for Pullorum Disease in Turkeys presented by Mohammed J mail has been accepted towards fulfillment of the requirements for Master' 3 degree in Bacteriology Mk Major professor Date May 27, 1948 ~ “-795 ZONE REACTIONS IN THE AGGLUTIIIATION TEST FOR PULIORUM DISEASE IN WYS by MOHAMMAD JAMIL A THESIS Submitted to the Graduate School of Michigan State College of Agriculture and Applied Science in partial mlfillment of the Requirements for the degree of MAS'I'ER OF SCIENCE Departmen t of Bac te riology 19% It is characteristic of Science and Progress that they continually Open new fields to our vision. Pasteur THhC'F TABLE OF CONTENTS Page IntmductionOooooooooo0.0000000000000000. 1 materials and. LlethOdSOOOooeooooo00000000. 3 Experiments and results.................. 5 1. Influence of temperature of incu- bation on agglutination tests....... 5 II. Effect of dilution of the antigen... 7 III. Effect of heated serum..... ...... ... 9 IV. The shift of zones in the serum Of turl‘my 3950000.00.000000000000000 11 V. Agglutinins absorption in zone 81.88.8000. ooooo coo... ...... 00000.00. 11 ma.ry.000000000000.0.0.0000... ooooooo o. 11 Bibliographyocoooooooo0000000000000oooooo 1% IKLRODUCTIQE Pullorum disease in turkeys was first recorded by Hewitt in 1928. Since then a great deal of work has accumulated on means of preper diaga nosis of diseased and carrier birds. The official tests prescribed by the National Turkey Improvement Plan (19h6) include: (1) Standard tube agglutination test (U. S. Live Stock Sanitary Association Meeting. 1932), (2) The rapid serum test (Runnells. Coon. Farley and Thorp. 1927), and (3) The rapid whole-blood test. In the case of the standard tube agglup tination test. it is recommended that either of the two serum dilutions 1:25 or 1:50 may be employed. Corpron (l9h5), while studying nullorum disease in turkeys found that in standard tube agglutination taste some birds which gave the max» imum agglutination reaction in 1:100, 1:200, 1zhoo and even in 1:800 serum dilutions showed only slight reactions in 1:25 and 1:50 serum dilu- tions. This phenomenon, referred to in the literature as zone reaction. prozones, prezones, zones of inhibition and pro-agglutinoid zones is not uncommon in antigen-antibody reactions. Thus Coventry (1930). while studying the trynanocidal effects of immune serum in rats infected with Trypanosoma lewisi. found that 1.1 m1 of the immune serum had no trynan- ocidal effect, 1.5 ml was markedly trypanocidal. 1.9 m1 had no such efl. feet. 213 ml was only slightly trypanocidal and 2.7 ml was markedly try- panocidal. She also found that these zone phenomena were independent of the inactivation of the serum. Taliafferro and Johnson (1926) observed similar zones of inhibition in mice infected with 23 ecuinum. reiton and Bailey (1926) observed lone reactions in their studies on the protective action of pneumococcus anti—serum in mice infected with pneumococci. They found higher doses of the antiserum less effective as compared to certain smaller doses in some mice. They attributed this absence of protection when higher doses of antiserum were used to a substance different from the protective antibody. but which para— lyzed the defence mechanisms of the mice. Lorgan and Nigg (1928) observed that in some positive Wassermann reactions for syphilis in leprous individuals. there was more comple— ment fixation in tubes containing smaller amounts of patients serum than in the ones containing the maximum amounts. They found the incidence of such reactions to be as high as 50% of the positive Wassermann reac- tions. Priestley (1931) observed zones of inhibition in standard tube I agglutination tests in sera from animals infected with the Brucella group of organisms. He, also. found that. provided a standard tech- nique was adhered to. the zone generally occurred in the same posi— tion. Seddon (1915) made a similar observation. White (1931) has drawn attention to the occurrence of prozone inhibition phenomenon in tests performed with freshly drawn sera of typhus sufferers and conval- escents. Shibley (1929) has made an extensive study on genes produced artifically by heating immune ears to various temperatures for differ- ent periods of time. The present study was undertaken to gain more information on the subject of zone reactions as they occur in standard tube agglutin- ation tests for pullorum disease in turkeys. -3- Materials and Methods Bacterial strains: The organisms used in this study were: 1. Salmonella pullorum strain...........h 20 Salmonella 1311110111”) Strain-00000000010 3. Salmonella pullorum strain..........11 All antigens discussed in this report were prepared from the orb ganisms listed above and are referred to by their numbers hereafter. '.All organisms were plated on tryptose agar (Difco). and incubated for 2% hours at 370 0. Single. smooth colonies as indicated by reflected light were picked up and all antigens were prepared from cultures orig- inating from such colonies. The morphological. cultural and biochemical reactions of all the three strains correspond to those listed in Bergey's Manual (1948). ?reparation of standard antigens: Kalle flasks containing tryp- tose agar (Difco) were seeded with about 2 ml of 2h hours tryptose broth (Difco) cultures. Simultaneously a loopful of the same broth culture was streaked on a tryptose agar plate. and after 2% hours incubation at 37° 0., the plates were examined by reflected light for the presence of any rough or dissociated forms. Kolle flasks corresponding to plates showing only smooth colonies were selected for antigen preparation. The Kblle flasks. after EM—MS hours of incubation, were washed with phenolized (0.5%) physiological sodium chloride (0.85%) solution (referred to hereafter as 0.5% carbol—saline solution) to form thick suspensions. These suspensions were filtered through sterilized cotton pads, and the filtrates stored in the refrigerator as stock antigens. JI— To prepare standard antigens, each stock antigen, just before use. was diluted with enough 0.5% carbol-saline solution to correspond in turs bidity to tube three in McFarland's nephelometer (McFarland: 1907). To prepare polyvalent standard antigen. equal quantities of each of the stock antigens h. 10 and 11 were mixed in a test tube and the mixture then diluted with 0.5% carbol-seline solution to match in tura bidity of tube three in McFarland's (1907) nepholometer. Sera used: - Three turkeys (previously tested and found negative to the agglutination test for pullorum disease) were inoculated intra- venously with 0.5 m1 of 2h hours tryptose broth culture, as follows: Turkey 395.. ....... ...S. pullorum strain...... M Turkey 615............s. pullorum strain......10 Turkey 11921..........S. pullorum strain......10 The turkeys were bled at various intervals of time. and the sera were separated by centrifugation of the coagulated blood. All sera were stored in the refrigerator without added preservatives. The time of the post-infection bleeding was indicated by labelling the serum containers as follows: ll-day serum or 16-day serum, etc. :Mgthgggz The standard.macrosc0pic tube agglutination test was used. All serum—antigen mixtures were incubated in a.water bath at 56° C. for 18 hours unless otherwise stated. In view of the considerable import- ance attributed to convection currents in effecting an increase in the speed of clumping in agglutination tests(Topley and Platte: 1918, Fleming 1928) the tubes were immersed in the water bath up to three-fourths of the depth of the contained fluid. ~5- The degrees of agglutination have been indicated on the following +++ --- Complete agglutination.- supernatant fluid clear .++ --- Markedly granular with flocculation + --- Granular without flocculation : -- Dbubtful - Negative Experiments and Results I. Influence of temperature of incubation on agglutination tests Shibley (1929) observed a serum (pneumococcus Type I, horse) in which an inhibition zone appeared only when the incubation was at 56° C. but disappeared when incubation was at 37° C. Heidelberger and Kendall (1929). while studying precipitation reaction, observed flocculations at icebox temperatures in mixtures which showed no precipitation at room temperature. Spencer (1930) on the other hand. found that the zone was destroyed if the serum-suspension mixtures were incubated at 56° C. Priestley's (1931) results indicated a definite narrowing of the zone when the incubation temperature was 56° C. The following experiments were planned to study the comparative influence of the incubation temperatures of 37° C. and 56° C. on agglu— tination tests in pullorum positive sera. 7Agglutination tests in duplicate were performed with the ll. 16 and 21—day sera of turkeys 615 and 11921. One set of tubes of each serum sample was incubated at 37° C. and the other at 56° C. '. Table I shows the results of these experiments. -6- t .. ... H ...... .++ ++ ++ ... H... o omm Hm HmmHH u I t H + ++ ++ ++ +++ +++ u can Hm HmmHH I I H 1. ...... +1. +4.... ....I. ++ .1. .0 0mm ma HmmHH ... .. H + ++ .1 ....I. 1.... +++._ .I. 0 0R mH HmmHH .. I ..... ... .1. .1. ....I. ....I. ....I. ...... 0 0mm .3 8m: .. .. .. H ..... ... +1 +1. .11 +1. 0 can HH HmmHH .. .. - t H i. .1 ++ ... H u 0mm Hm m6 _ .. u .. u u u ... . .1 +1. 1... 0 can Hm mHm .. n u ... .1 +1. 1.... ...I. .I. t... 0 0mm mH mHm .. .... t + + .I. W ++ a +++ +1. 1. 0 0R ma mam u .. .. + .1 .I +1.7 1+ 1.... ... o omm HH mHm ... a .. ..... t... 1.... 1+ .1... ....I. .1... 0 0R HH t mHm package mm balm mmm louvre mm. mm— mml bum lgfigoga named H5 .Hopgn H H H H H H H H H 53283 3:3er 332:3 222a. f . _ :1 .2 :1 Jean .3 Hamel L anoHEEHop pudendum: poms gauged deHMtdeHoHHa no zaagoqao» Mo oonoHHCHHH H 0.309 II ' I II i'x'nl -7- It will be seen from this table that there were no zones of inhibi- tion in 11 and 21-day sera when the incubation temperature was 37° C. but definite though weak zones appeared in 1:10 serum dilution of the 11—day serum and well marked zones appeared in 1:10 and 1:20 serum dilutions of 21-day sera. when tests were incubated at 56° C. In the case de161-day sera. the already existing weak 2 ones in 1:10 serum dilutions widened to 1:20 serum dilutions at 56° 0. incubation. This observation is very significant since in any pullorum eradi- cation pregmem in turkeys. where a single dilution method is employed and the tests are incubated at 56° 0. some positive birds are liable to be missed and will be potential sources of infection for the healthy stocks II. Effect of dilution of the antigen Heur (1922) and da Costa Cruz (1929) showed that zone reactions in agglutination tests could always be demonstrated when very light bac- terial suspensions were titrated in constant amount, against increasing dilutions of antiserum. Seddon (1915) feund that by reducing the density of the bacterial suspension. the dilutions in which zone occurred were raised. Priestley (1931) by halving the density of the bacterial suspen- sion raised the zone to the next serum dilution. Spencer (1930) made a similar observation. Gorpron (l9h5) did not find much difference in the zone reactions by changing the density of the antigen. In the following experiments the results obtained by the use of standard antigens and the standard antigens diluted 1:1 with 0.5% carbol-saline solution are shown in Table II. H8.- oH ncwHaas eoaaHHn In oH aumHaaa essence» an I I I I _u ++ ++ +++ + I .sHoa snap» coasHHn m I .aHom I I I I ... +1. .1... +++ .I. I .Hcsaam m I I I I I _n ++ +++ I I n Hm I I I I I ..... 1.... .1 I I n mH I I I I I + ++ +++ + I m mH I I I I + _+ ++ +++. ++ + I a HH I I I I IN .I. ....I. ....I. .I. ... H m HH I I I I H + + +++ +++ I I a m I I I I I ..... ++ ....I. 1.... ... I m m .228 ml mum man. as mm. mm. a... 8 mm. B. ...; as a muses H H H H H H .H H H H noprgq aoHpommuH Ipubmrhnruzanrua mono» no 33.28 on» mo 583.33 no ooaoHHCnH lane. on» nH 2.. 0.369 -9- By studying the date in Table II it will be observed that by re- ducing the density of the antigens by one half the zones of inhibition became more pronounced and in most cases these are raised to 1:20 and then to 1:h0 serum dilutions. It thus seems that the position of the zone¢jf inhibition in the agglutination test depends on the ratio between the amount of serum and the number of organisms. From this it will be seen possible that further dilutions of the antigen might extend the po- sition of the zones to 1:80 serum dilutions or even higher. These observations have important bearings on the central program of pullorum disease. The use of one serum dilution of 1:25 or of two serum dilutions of 1:25 and 1:50 with a light bacterial suspension as recommended by the National Turkey Improvement Plan (19h6) and the sub— sequent incubation of the serum-suspension mixtures at 560 C. might fail to detect some diseased and carrier birds. 2 In view of the above observations. a better and more definite way to detect the diseased and carrier birds would probably be either to re- place the use of two serum dilutions of 1:25 and 1:50 as recommended by the National Turkey Improvement Plan (19h6) by four serum dilutions to include 1:100 and 1:200 serum dilutions or if only two serum dilutions must be used, a serum dilution of 1:25 combined with 1:100 or 1:200 serum dilutions to cross over the area of occurrence of zone. III. Effect of heated serum_ Detre (1927) stated that the '. hone was removed and agglutination occurred when zone sera were inactivated by heat at 53° 0. Spencer (1930) showed that if the serum was inactivated at 56° C. for periods varying from 10 to 60 minutes. the zone widened in prOportion to the length of time of inactivation. In some of the sera used by him. heat treatment introduced new zones of inhibition. Priestley (1931) and Corpron (1935) also made similar observations. Corpron (19h5) also notiéed a fall in the end-titres of the heated sera. Dreyer and Jen. Blake (1906) studied the production of "Zone ef- fects” with sera heated at 70-75° 0. They noticed intermediate zones of inhibition in such sera and attributed them to the production by heat of a substance which impeded agglutination. Shibley (1929) also investigated the production of zones by heat treatment of immune sera. Bis emperiments showed three states of immune serum induced by heat. (a) The unheated serum gave agglutination in all concentrations to the limit of the titer: (b) After heating at about 60 to 70° C. a zone of inhibition occurred in the lowest serum dilution, but agglutination took place in the higher dilutions to the limit of the titer: (c) After further heating at about 76° 0. there was no zone, but the titer was lowered. and with serum heated to 18° C. no agglutination appeared. The following experiments were conducted to determine the effect of heat treatment on 11-day sera of turkeys 615 and 11991. Sera used in this study were heated at 56° C. and 65° C. for % hour, the ones which were heated at 65° c. were diluted 1:5 with 0.85% sodium chlor- ide solution before heat treatment to avoid their coagulation. Table III shows the results of this experiment. It will be seen from the results thet heating the sera at 56° c. for % hour did not cause any material change in the position of the L11- \ .. .. .. .. u .. .. .. .. .. ... oum HmmHH ... .. J ... .. + ... ...... ...... 1+ 1. 0mm HmmHH u t a t u ++ ++ ++ +++ +++ ++ eopoonep HmmHH .. .. .. .. .. .. .. n m u .. o8 _ me i .. .. ... 1. ....I. ....I. +1. +1. .I. .. 0mm mHm t u t .H ++ ++ +++ +++ +++ r+++ ++ eoaoognp mHm. Hats mm mew he. as em am e. 8 mm. a. :2 133 H H H H H H H H H H Java. esoeoKHop Eooaoem I coma someqd [wwom mo madamcg hm oopoommm om mnOHaomoa anon HHH oHpoa -12- zone reaction but when the sera were heated at 65° C. no agglutination appeared. Apparently all the agglutinhms had been destroyed at that temperature. IV. The shift of zone in the serum of Turkey_39§ The agglutination reactions of turkey 395 are shown in Table IV. It will be seen from that table that the position of the zone shifts from 1:320 serum dilution in the 16-day serum to 1:80 serum dilution in the 20-day serum, to 1:20 in the 25—day serum and 1:10 and 1:20 serum dilutions in the 30—day serum. This shift in the position of the zone coincides with the fall in the end-titers of the sera. which shifts from 1:6h0 in the 16-day serum to 1:320 in the 20-day serum and 1:80 in the 25 and 30-day sera. A similar reaction was observed by Spencer (1930) who noted in his studies that in the later samples of the serum from the same patient. the zone of inhibition had shifted from the in- termediate position to the tubes of maximum serum concentration. This observation is in line with that obtained in experiment II (Influence of dilutions of antigen: Table II). There is apparently a correlation between the prOportion of serum and antigen and the occur— rence of zone. It was noticed in experiment II that if the antigen was diluted. the zone shifted to include a correspondingly higher di- lution of the serum. In this experiment when the amount of antibodies is reduced the zone shifts to higher concentrations of the serum. V. Agglutinin absorption in zone areas The following experiment was planned to see what happens to the agglutinins present in the tubes exhibiting zone reactions. For such -1}. a study, it was considered necessary to produce wider and definite zones and so a lighter bacterial suspension (Standard antigen 10 diluted 1:1 with 0.85% sodium chloride solution) was employed as an antigen. Eleven. sixteen and twenty-one-day sera of turkey 615 were titrated against the diluted antigen and the tubes incubated at 56° C. for 18 hours. Table V shows that there were definite zones present in all the three sera(reac- tions A, B and C). All the tubes showing reactions A. B and C were cen- trifuged for one-half hour at 3,000 r.p.m. and the supernatant fluid from each tube was again titrated against the diluted antigen. The re- sults obtained are detailed in Table V (reactions A', B' and C‘). and show that the supernatant fluids from A, B and 0 contain fairly high concentration of agglutinins in the zones of inhibition. as indicated by ++ agglutination reactions. This indicates that in the original ag- glutination tests (Aw B and C), few, if any, antibodies combined with the antigen . The absence of agglutination in the presence of antibodies reveals the presence of an inhibitory factor. The exact nature of such a.factor has been the subject of much speculation and dehgte;. Two hypotheses have been put forward to explain the appearance of zone phenomenon in agglutination reactions. Eisénberg and Volk (1902) explained the phenomenon on the lines of Ehrlich's conception of the ag- glutinin as consisting of a "haptOphore" and a ”Zymophore" group. the haptophore group bringing about the specific union of the agglutinin with the bacteria and zymophore group causing agglutination. They further as- sume that by heating or aging, some of the agglutinin is so modified -1M- I I I I I I H + +++ + I On I I .. I I I u A + ++ + +1 . mm _ I I I I I + ++ I +++ +++ +++ ON I I I H ++ + +++ +++ +++ +++ +++ mH 4828 cmHW ohmn m 08H 8m 8m omH mm. B mm mm was 5 H H H H H H H H H H meHemsHp I weaposmnalawop MO 055%! mmm*1woaasp mo sauce aw msoduoawa snowing» mo wnwumflnm >H manna -15. .8325 does fin. .noHasmshHapnoo.wnHkOHHom .0 was .m .4.aopnp spam ucHde andpenaoasu monHa scam mcHaHamou uaoHposoa n .o ..m ..4 .unoHuotha onstHa quHpnuIESHom kneadam aH noHpoeoa n o .m .4 . o I I I I I I + + e! Hm . m I I I I I I ... .I. .I. mH m I I I I H ++ ++ u. I mH . 4. I I I I I I _H ++ ++ HH .3333 H828 ommH mum 0% EM mm .9 mm .8. name 5 m5 InQuHucu ashow H H H H H H H H IpooHp uoHpoownH Ianop mo oaHm mHm_hthna ashcm I :OHpaHom ccHaOHgo aaHpom mow.o 39H; HnH copaHHp 0H soprsa caspnsme coma smmeg4 > OHQGB ~16- (agglutinoid) that the clumping component is destroyed without, however, affecting the binding portion. To explain the inhibition of reaction in lower serum dilutions it is assumed that the agglutinoid in these concentrations has a greater affinity for the bacteria and is. there- fore bound to them to the exclusion of effective agglutination. The second hypothesis was put forward by Zinssar (1923) and has been widely accepted. It explains the inhibition of clumping in a zone of stronger serum concentration. due to the presence of a nonnspecific protein colloid which coats the bacterial bodies in the antigen and thus prevents their agglomeration. Recently'W1ener (l9hh) and Race (IShM) have shown the presence of both "incomplete" and complete antibodies in the Anti-Rh serum of the in- dividuals showing zone reactions in lower serum dilutions. Incomplete- antibodies. according to Wiener (19MB) are univalent and are unable to serve as a link between red cells that effectively combine with the re- ceptors on the red cells thus preventing agglutination by bivalent or complete antibodies - a trend back to Ehrlich's hypothesis. -17.. SUMMARY Weak, if any, zone reactions were produced when the serum-antigen mixtures were incubated at 37° C. but definite and wide zones were no- ticed when the mixtures were incubated at 56° C. Dilution of the antigen raised the zone to as high as 1:30 serum dilution. It is suggested that in any pullorum eradication program, either of four serum dilutions of 1:25. 1:50, 1:100 and.l:200 should be employed or if only two dilutions must be used. serum dilutions of 1:25 and 1:100 or 1:200 should be preferred. Heating the sera at 56° C. for fi'hour did not produce any change in the position of the zone reactions. but heating the sera to 650 G. destroyed all agglutinins in them. The shift of zones in one turkey correlated to shifts in its end titer. Supernatant fluids from tubes showing zone reactions revealed the presence of high titre of agglutinins when titrated with fresh antigens. , Literature on the subject is reviewed. -18— BIBLIOGRAPHY 1.Corpron, R..A. 19h5. Pullorum disease studies in turkeys. Thesis for 2. 6. 7. 8. 9. 10. M.S. degree at Michigan State College, East Lansing. Michigan. Costa Cruz, J. da. 1929. Quoted by Topley; W.W.C., and Wilson. G. S. 1936. The principles of bacteriology and immunity. 2nd. ed. The Williams and Wilkins Company, Baltimore. Coventry, F..L., 1930. The trypanocidal action of specific antiserum on Trypanosoma 1ewi§i_“in vivo". Amer. J. Hyg., 1g, 366. Detre. L. 1927. Quoted by Priestley, F. W. 1931. The intermediate zone phenomenon encountered in certain Brucella abortus age glutinating sera. Jr. Path. Bact., 29. 81. Prayer. G. and Jex—Blake,.A. J. 1906. On the agglutination of bacteria. J} Path. Bacto. 11’ lo Eisenberg. P.. and Yolk. R. 1902. Quoted by Zuisser. H. 1923. Infec— tion and resistance. 3rd. ed., The MacMillan Co.. New York. Felton. L. D.. and Bailey, G. H. 1926. Biologic significance of the soluble specific substances of pneumococci. J. Infec. Dis. 18.. 131- Fleming; A. 1928. On the influence of temperature on the rate of agg1u~ tmation or bacteria. Brit. J. Int. PPthOg 2, 2250 Heidelberger; Mo. and Kendall. F. E. 1929. A quantitative study of the precipitive reaction between type III Pneumococcus polysacchar- ide and purified homologous anitbody. J. Expt. Med..‘52. 809. Heuer. G» 1922. Quoted by TOpley, W. W. 0.. and Wilson. G. S. 1936. The principles of bacteriology and immunity. 2nd. ed. Williams and Wilkins Co. Baltimore. Larsen. N. P. and Higg, C. 1928. Some observations on the Wassermann and Kalm reaction. J. Lab. Clin. Med. 1}. SN}. Mciarlend. J. 1907. The nephelometer. J. Amer. Med. Ass.,‘E9, 1176. National Turkey Improvement Plan. 19h6. United States Department ovagri- culture. Washington. D. 0.. Miscellaneous Pubdications No. 555. Priestley. F. W. 1931. The intermediate zone phenomenon encountered in certain Brucella abortus agglutinating sera. J. Path. Bect. 33, 81. 15. 16. 17. 18. 19. 23. 2h. 25. 26. ~19- Race, R. R., 19hh. An 'incomnlete' antibody in human serum. Nature. 153. 771. Runnells, R..A., Coon. C. 3., Farley, H., and Thorn. F. 1927. An application of the rapid-method agglutination test to the diagnosis of bacillary white diarrhea infection. J. Amer. Vet. Med. is... 10 (N.S. v 23). 660. Seddon. F. R. 1915. Some observations on the methods using the agglutination test in the diagnosis of the disease in bo- vines caused by the bacillus of contagious abortion. J. Camp. Path. Therm fl. 29‘ Shibley. G. S. 1929. Studies in agglutination. IV The agglu- tination inhibition zone. J. Expt. Med..‘§Q. 825. Soencer. R. R. 1930. The zone nhenomenon in agglutination tests. ‘1' InfGCto D180, 31g, 138. Taliaferro. W. H.. and Johnson. T. L., 1926. Z one phenomenon in "in vivo" trynanolysis and the therapeutic value of trypano- lytic sera. r. Prev. Med...l. 85. Tepley, W.W.C.. and Platte. S. G., 1918. The effect of convection currents on agglutination. Lancet 1. 800. United States Live Stock Sanitary Association. Proceedings. 1932. h87. White. P. 3., 1931. Observations on Salmonella agglutination and related phenomena. J. Path. Bact. 13. 23. Wiener,.A. 8.. l9hh. A new test (Blocking Test) for Rh sensitiza— tion. PTOC. SOC. Expte 31010 Med. 5, 173. Wiener. A. 5. l9h5. The Rh blood types and some of their annlica— tions. Amer. J. Clin. Path.. 15. 106. Z insser. H. 1923. Infection and resistance. 3rd. ed., The Macmillan Company. New York. figmmmmemir My sincere thanks are due to Dr. H. J. Stafseth for his guidance in the prosecution of this work and helpful suggestions in the preparation of this manu- script.