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A new vaccine for use in treating diphtheria carriers Weld, Charles Beecher 1924

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1 U.B.C. LIBRARY CAT. W.Ubkdty*J&2£!i&Jil4t*& hcc. no. J££3jLL, I A NEW DIPHTHERIA VACCINE, by C.B. Weld, B.A. April 25th, 1924. Sfiye $nttn>ratty at Irttrah, (Unlmtttrta OF BACTERIOLOGY VANCOUVER, C A N A D A May 2, 19 24. John Ridington, Esq., Librarian, University of B. C., Dear Sir: I beg to a d v i s e that the a t t a c h e d t h e s i s , e n t i t l e d "A New D i p h t h e r i a Vaccine" by C. Beecher Weld (No. 949 ) has been approved. i Yours t r u l y , Head of Department. A HEW VACOIHE FOR USE IN TREATING DIPHTHERIA CARRIERS, by Charles Beecher Weld. A Thesis submitted for the Degree of MASTER OF ARTS in the Department of BACTERIOLOGY. THE UNIVERSITY OF BRITISH COLUMBIA. APRIL, 1984. i . A HEW DIPHTHERIA YACCIHB. COSTEHTS. Page 1 The D i p h t h e r i a C a r r i e r . As an Epldemeolog lca l Problem, n e c e s s i t y for new methods of t rea tment . D r e y e r ' s new method f o r p r e p a r i n g v a c c i n e s . 2 D r e y e r ' s Season ing and o u t l i n e s of h i s work. 3 Immunology of D i p h t h e r i a . A brief discussion with special reference to the disappearance of the Diphtheria bacilli from the tissues, the role of phagocytosis in the disappearance, and the possible advantage to be gained by an eff-ective vaccine. 8 Preparation of the Vaccine. Method of cultivating the bacilli. Method of defatting the bacilli. Method of Standardizing the vaccine thus obtained. 11 Animal Experimentation. Selection of, use of, and inoculations of Guinea Pigs and Rabbits. 14 Examination of Sgra Produced. Technic of Agglutination Tests. Technic of Complement fixation Tests. o ii. CQggBNTS (continued). Page 16 Observations on the results of Sera examinations, 19 Conclusions. APPENDIX I to 711. Experimental Data. " VIII. Bibliography. -1-A HEW DIPHTHERIA VACCINE. The Diphtheria Carrier. It is veil known that many Diphtheria patients, who have recovered from the disease, carry virulent Diphtheria bacilli in their throats for weeks and even months after they have recovered. There are also other normal people apparently immune to the disease, who har-bor, without symptoms, in their throats and nasal cavit-ies virulent organisms. Both these types of carriers are capable of conveying these organisms to other and less immune persons, who may promptly contract Diphtheria, and they constitute a serious problem among Public Health workers, and in Spidemeology. She only way, which is sure of success, of treating the problem is to rigidly isolate and quarantine all carriers, until they have been shown to be free of bacilli. If at the same time all susceptible people are immunised against Diphtheria, the disease will be completely wiped out once and for all. Shis however is Impossible at the present time, even though the methods for testing one's susceptibility are simple and accurate, and the immunising process effec-tual and not as a rule uncomfortable. Even the possibll* ity of isolating all carriers is remote, as it is impos--2-sible to tell how long the quarantine will have to last. The carrier would have to be segregated in an institut-ion, and not only would this be a great expense to the state, but the personal inconvenience to the carrier would be so great that it is improbable that all would submit to the isolation. However if some method could be devised which should ensure the "clearing up" of the carrier within a reasonable time, some definite policy could be arrived at. At present there is no method of treatment whereby a persistent carrier can be freed of the baoilli with any degree of certainty. Dreyer (1] has recently developed a new method of preparing vaccines which he claims is of general applic-ation. His work has been chiefly with the Tubercle Bac-illus, and the early results are encouraging, though as yet they have not been thoroughly confirmed. is it pos-sible to use Dreyer's method, or some modifioation of it, and produoe a vaccine which will aid in the olearing up of carriers ? Dreyer's Reasoning and outlines of Method and Wort. In brief, Dreyer states that successful vaccines are made from Gram Negative Organisms, while in general Gram Positive organisms do not make good vaccines. He -3-allows many exceptions to this rule but claims it to hold in general. Gram negative bacteriae differ chiefly from gram positive ones in that they contain less of the lipoidal class of substances. "Acid Fast" organisms contain even more of these lipoids. is there then some connection between the lipoids and the antigenic valve of the bacterial suspension ? To answer this question he developed a method of extracting these fatty substan-ces, which consists of treating a mass of baoteriae with formalin and then extracting with acetone. After this treatment, Gram positive, and even acid fast organisms are rendered Gram Negative. Then, using "Defatted" tubercle bacilli he showed by animal inoculation that the antigenic qualities of the bacterial suspension were not destroyed. Indeed, judging from the antibody res-ponse they seemed rather to be increased. Human cases, treated with this new tuberculin have given very encour-aging results, but of course as it usual in dealing with Tuberculosis, considerable time will have to elapse be-fore early indications can be confirmed. Immunology of Diphtheria. The clinical symptoms of Diphtheria are due for the most part to the production, during the growth of the , organism, of a soluble toxin which profoundly affects the 4-looal tissue. This toxin diffuses through to the blood stream, whence it is carried throughout the body, and a general toxemia produced. In addition to this however it stimulates the body to produce an antitoxin which neutralises the toxin free in the blood, and when present in adequate quantity it has neutralized all the free toxin in the body, and is combining with all fresh toxin as it is liberated. Thus the disease is checked, though the damage whieh the toxin has already done is not right-ed. After this necessary amount of antitoxin is obtained, either by natural production, or by artificial adminis-tration, the Diphtheria bacilli usually but not always disappear from the throat. Why does1the organism disappear, when this antitoxin is obtained ? The three following explanations suggest themselves: (1) That the bacillus is unable to grow in the absence of toxin or in the presence of an excess of antitoxin. (2) That in the presence of an excess of antitoxin it is more readily overgrown and smothered out by other organisms. (3} That when its toxin is neutralized it is more sus-oeptible to the body defences than before. The first two possibilities do not seem reasonable -5-when one considers that Diphtheroids, suoh as d. hoffmanni, fl.xerosis and a virulent but otherwise typi-cal a. Diphtheria are frequently found in the nose and throat. 7drulent diphtheria baoilli too are quite often found present eren when the patient has in his blood more than 1/30 unit antitoxin per oo. which is the amount considered necessary for a person to be immune. The car-riers referred to belong to this latter class. In any •Tent, It has been shown (xj that virulent Diphtheria baoilli grow readily in vitro in a glucose broth medium containing 100 units of antitoxin per CO., and that in such a medium the baoilli will not be outgrown by contam-inants any more readily than in the same medium contain-ing no antitoxin, The third possibility however opens up a question whioh is far too large to be discussed completely here, though a few details may be oonsldered. In the first plaoe, what are the body defences, and in the seoond plaoe how do they aot in the removing of the baoilli from the nose and throat T If the Diphtheria organisms are present in the throat or nasal oavlties, some will be on the surface of the (x) - See appendix I for details of experiments. -6-membranes, and others will be more or less embedded in the tissues. Those on the surface, are out of reach of the phagocytes, and antibodies in the circul-ation and the tissues, but are seadily reached by the mouth fluids, These, and the nasal secretions do not seem to have any harmful effect on the bacteriae nor-mally. However the possibility of specific antibodies being present in these secretions must be thought of, and probably many organisms are washed away to the stomach and intestines, whose secretions may exert a bactericidal effect on the foreign organisms. Those bacilli, which are embedded in the tissues however are in a position to stimulate the production of antibac-terial antibodies, and are also within reach of these substances. Phagocytosis must play an important part in the removal of these organisms from the body. Keque (2) has shown that the Diphtheria bacillus is very sus-ceptible to phagocytosis; Turnicliff (3) states that the phagocytic power of the blood of Diphtheria carriers is increased, but that at the same time the nose and throat may contain large numbers of leucocytes not engaged in phagocytosis. These findings indicate that while Fhago-cytosis does play an important part in the removal of the bacilli, other factors must be involved. Some antibodies 7-or substances are probably present in the convalescing case which is not produced in the carrier, and which either exert a bactericidal action themselves or aid the Phagocytes in their task. If some means could be found to stimulate the production of these antibody substances, the time required for the clearing up of carriers would be very materially shortened. Park i4) mentioned that the ordinary immunisation procedure, using a 'I'oxin-Antitoxin mixture has no appar-ent effect on clearing up carriers. Park and Zingher (5) attempted to remedy this by adding 1000 million killed Diphtheria bacilli to each oc. of 'I'oxin-Anti toxin mixture, but at the time of writing of their article, sufficient time had not elapsed to determine its efficacy. aeebohm (6) discusses the use of a Diphtheria vaccine to clear up carriers, and reports that in two weeks, 53.5% are clear; that in three weeks, 77%; and that in four weeks, 89.5% are clear. His results however are exceptional and the general opinion is that Diphtheria vaccines are not suc-cessful. This, according to Dreyer's (1) hypothesis is only what is to be expected, as the bacilli are Gram positive 9 organisms; but if they are "defatted" by his process, they might prove capable of producing the necessary antibodies. 8-An attempt was made to investigate this possibility in practice. Preparation of the Yacolne, She Diphtheria bacilli are grown on Loeffler's serum medium for forty-eight hours. In order to obtain as large a surface for growth as possible, the serum is solidified on the flat side of eight ounce medicine bot-tles. Five or six of these will produce sufficient growth for convenient handling. The mass of bacilli is washed off with sterile physiological salt solution con-taining 0.9% sodium chloride, into sterile centrifuge tubes. So free the suspension of traces of liquid serum, whioh are present in the condensation liquid, and which will give an undesired precipitate when formalin is added, it is washed three times with saline and then once with formalin. She washed bacterial mass is ground up in a sterile agate mortar with pure fomalin, (40% formaldehyde) using 20-30 ocs. of formalin to every gram of bacilli weighed wet; and the resulting suspension is poured into a sterile flask, connected to a "retlux-condenser** and heated in a water bath at 100° Oentigrade for four hours. At this stage Dreyer filters the suspension through calcium free filter paper and washes it with acetone. Using ordinary filter paper it was found very difficult even when using a Buchner funnel and suction to force the -9 solution through the paper, and that when the filtering was finally complete, it was impossible to scrape the precipitate from the paper. So avoid this difficulty, the suspension is oentrifuged and the sediment washed three times with acetone. It is then suspended in ace-tone, poured into a coarse alundum thimble and placed in a Soxhlet extractor with ground glass joints. Acetone is the extracting fluid used, and the whole apparatus is immersed in a water bath to the level of the top of the thimble and kept at 65-70°C. for 24 hours. Ihe thimble is now removed from the apparatus, and the extracted bac-terial mass dried, if the bacilli on staining prove to be completely Gram negative, the extraction has been effec-tive, and they need only to be emulsified in saline and the suspension standardized. If however they are not def-initely negative to Gram, the whole process from grinding in formalin to extracting in acetone must be repeated. Dreyer's method of standardizing is to weigh out exactly 0.1 gram of the dried extracted mass, and grind it up thoroughly in an agate mortar with 10 cc». saline. He then centrifuges down the coarser particles and deter-mines the weight of baoteriae left in suspension by sub-tractlng from the original 0,1 gram, the weight of the oentrifuged sediment, not forgetting to allow for the -10-welght of sodium chloride which will be dried with it. The chances of error with such a method are great, and working with Diphtheria it was found impossible to ob-tain an accurate standardisation. For this reason, and also because grinding in a mortar is a tedious process and one where proper aseptic precautions are difficult to maintain, the following procedure was adopted. The dried extracted mass is weighed out carefully into a sterile raeeine shaker bottle containing a dosen or so glass beads, and 10 oos. sterile saline added for every 0.1 gram of the defatted bacilli. The whole is now shaken In a meohanical shaker for six or seven hours and Immediately pipetted into a weighed sterile centri-fuge tube and centfcifuged at 3000 revolutions per minute for about fire minutes. The supernatant fluid, which is somewhat turbid but must contain no granulation risible to a 6X lens, is the vaccine and is diluted as desired. From this point, there are three convenient methods of standardising the vaccine. (1) That involving the use of the weighing described above. The shaken emulsion contains exactly 0.01 grams defatted bacilli per oc. The weight of the centrifuge tube is known, and the weight of the residue in the tube after oentrifuging can easily be determined by weighing li-the tube with its contents after dessicating, and sub-tracting the former from the latter. The weight of salt (0.9jb of the Tolume in cos, of solution left in the tube after centrifaging and decanting) must be calculated and allowed for. She weight of solid still in suspension will be then the difference between the original 0.1 gram per co. and the corrected weight of residue. This method however gives as the concentration of the vaccine by weight, and the normal method is to speak of the num-ber of organisms present. (2) She individual baoteriae not haring been broken up by the shaking, it is possible to count the number pres-ent. A "Levy" counting chamber, ruled In squares 1/20 mm. square, and with a chamber 1/50 mm. deep is satisfac-tory, if a reinforced eoverslip, thin enough to allow the use of the oil immersion lens, is used. (3) A standard having once been prepared by either of the above methods, future preparations may be matched to it by the opacity method. This is a rery simple pro-cedure and is sufficiently accurate. Animal Experiments. It is impossible to give a vaccine prepared in such a radically new way to human patients without first proving its harmlessnes8 to animals, and without obtain-12 ing at least suggestive evidence that the giving of the vaccine would be beneficial to the patient. In this ser-ies of experiments, Guinea Pigs were first used. Six healthy guinea pigs were selected, weighing between 350 and 400 grams. Two were given a polyvalent Diphtheria vaccine, consisting of equal parts of three known viru-lent type strains, "Park," "Durand," and "American" and three looal avirulent strains from the Vancouver General Hospital, of unknown agglutinative type. The polyvalent vaccine was used in order to ensure as far as possible the production of antibodies against the particular type conoerned in the particular case. For use in animals however it has no advantage, and was later dropped in favor of the monotype vaccine, in order that results could be more closely compared. Two guinea pigs were in-oculated with Diphtheria strain Park and two with Dur-and strain. The suspensions given these four animals were of living virulent organisms washed repeatedly with saline in the manner suggested by Witz (ierald (7) who found that by using washed living organisms a serum could be obtained with an agglutinating titre of from 1/1200 to /4000 in three or four weeks. Bell (6), using a sus-pension of bacilli killed by heat succeeded in obtaining a titre of only 1/500 to 1/400 after many weeks of -13-immunising. After a month or more of treatment (see Appendix ), no evidence of the presence of antibodies could be shown, judging by examinations for agglutinins. On this aocount it was decided that Guinea Pigs were of no use for Diphtheria bacterial antibody production* and Rabbits were selected as being the most likely to prove useful. Three young rabbits were chosen* not full grown at the beginning but after the course of immunisation they had attained normal weight. It was thought that young animals might react more strongly to the injections than old ones. One was given strain number 100 from the Gen-eral Hospital, which was known to be very virulent. One was given strain "Park*" a standard laboratory strain of known virulenoe. Both these suspensions consisted of liv-ing organisms* washed 10 x with saline. The third was given Park strain, defatted suspension* prepared aooording to the teohnio described above. The two rabbits given Park, were twins* identical in weight* and were given precisely the same dosage throughout. All three were inooulated on three successive days, weekly* as is advised by Gay and Fits Gerald (9). At no time did any of the animals become sick, nor did they appear to suffer any ill effects from the vaccination. For full details as to inoculations and 14-and dosage, refer to the individual animal charts in the appendix* From time to tine blood was taken from the ani-mals and the titre of agglutinins determined. This is a comparatively simple test and, it was thought that if the antigen was stimulating the production of agglutinins the production of other, perhaps more important, and possibly unknown, antibodies would be stimulated simul-taneously. Examination of Sera. feohnic of Agglutination lasts. Blood is taken from animals, and the serum separat-ed from the cells. This serum keeps well in the ice-box and is diluted just before use with sterile saline. As there is no way of foretelling the titre of the serum to be tested, a series of dilutions must be used. In an animal in the early stages of immunisation a series of dilutions ranging from 1 part in 10 to 1 in 200 will prob-ably indioate the titre present, but animals well immun-ised may give a serum which will agglutinate in a dilution anywhere between 1 in 1000 to 1 in 10,000. In detail the test Is as follows; A suspension of the strain un-der consideration is made by washing off the growth of one or two Loefflar's slopes in saline. This is shaken -15-free of clumps and is standardised by the opacity method to about 500 million organisms per oc. A series of small, clear glass test tubes is placed in a rack and 0.1 oo. of suspension put in each. To each tube is then added 0.9 ccs. of a serum dilution already prepar-ed, A control tube is set up which contains suspension and saline but no serum. She rack is then shaken, incu-bated at 56°c. for two hours, and placed in the ice-box overnight, The results are read in the morning, and the tube containing the highest serum dilution which shows agglutination indicates the titre of the serum. As a rule there is no difficulty in reading the tests, those tubes in which complete agglutination has taken place will be perfectly clear, while the others, and the con-trol tube remain turbid. If there is only partial agg-lutination the sediment must be shaken up, and the coarse-ness of the granulation as compared to that of the con-trol will determine the result. The tubes must not be shaken vigorously as the flocculi that are formed are easily broken up, especially the small ones that are hard to see. One or two light flicks of the tube will suffice to throw up the sediment and allow the granulation to be seen. " 16 Tochnlc of Complement Fixation Teats. The teehnic is that described in detail by Jreyer (1). He titrates the complement against sensitised sheep's red blood corpuscles, and dilutes it so that two minimal haemolytic doses are contained in five drops. He then titrates the antigen against the complement and sheep cells, (sensitised cells are used throughout -0.26 oos. of a 5jfa suspension) and dilutes it so that the largest doe* which is not anticomplementary is contained in 15 drops. The test then oonsists in adding graded amounts of the unknown serum to fixed amounts of comple-ment (5 drops) and antigen (15 drops) and incubating for one hour at S7£°C. and then adding 0.25 oos. of sensi-tised sheep cells to each tube and Incubating another hour* The smallest quantity of serum being tested which will gire complete fixation of the complement can thus be readily determined. ObserTations on Results of Sera Examinations. For all details refer to the animal records in the Appendix. Ho evidence of the presence of antibodies was ob-tained from any of the Guinea Pigs. The Babbits however, all sho? good response to the -17-antigenic stimuli. That one receiving # 100 had in twenty days developed a serum showing an agglutinating tltre of more than 1/720 and in four weeks a titre of about 1/1800. The one receiving live Park baoilli had in fifteen days, a titre of more than 1/240; in twenty four days, a titre of about 1/2000; and in thirty days, using glyoerinated serum, a titre of about 1/6000. The Babbit receiving the "Defatted" Park baoilli seemed to have developed no agglutinins in 15 days but in 24 days the titre was 1/2000, the same as that given by the other Park rabbit. This serum also showed the presence of Complement fixation antibodies in considerable quantities. An attempt was made to preserve these sera with glycerine, but unexpected results occurred. On adding the glycerine, the agglutinating power of serum # 100 and Defatted Park serum seemed to be immediately des-troyed, and that of the live Park serum while not affec-ted at first was completely lost after a few weeks. The glycerine was added as a preservative and its action is rather puzzling, unless one assumes the presence of cer-tain unknown impurities in the glycerine. The agglutinating titre of sera from all three rab-bits is high and was rapidly formed. The Park serum and the Defatted Park serum in 24 days both showed the same -18-titre. Unfortunately, no agglutinations were done with the 3 0 day serum from the Defatted Park animal, before glycerine was added. However the differences of the sera are so small that they might readily be explained as due to individual animal idiocyncrasies. It would appear then that Defatted Diphtheria bac-illi have antigenic powers equal to that of live organ-isms. Shis is noteworthy because Fits Gerald 17 J has shown that live Diphtheria bacilli make a better antigen-ic stimulus for the production of agglutinins than dead organisms. Judging from this then it would seem that De-fatted bacilli make a better antibody produoing antigen than do Diphtheria bacilli killed by heat as is done in making ordinary vaccines. -19-Oonoluslons. (1) The question of the carrier of virulent Diphtheria bacilli is becoming very important, and new •ethods of treatment are necessary to hasten the removal of the bacilli from these carriers. (2) A vaccine which would effectually produce anti-bodies either bactericidal in themselves or with power to aid the Phagocytes would be useful. (3) A Diphtheria vaocine has been made by defatting the bacilli as suggested by Dreyer. (4) She vaccine has proved harmless to Guinea Pigs and to Babbits. (5) 'i'he antibodies produced by rabbits, as eviden-ced by agglutinin*, are stimulated by the Defatted bacilli equally well as by washed living organisms, and therefore better than by dead organisms. (6) As the vaccine has proved in animals to be an efficient and harmless antigen, it would be safe and ad-visable to try its efficacy in human cases. • B i ^ M - ^ ^ _ i ^ » — APPENDIX!. Comparison of Growth of B. Diphtheria in media containing Antitoxin with that in media contain-ing no Antitoxin. 16/4/24. To two of four similar tubes of Glucose broth containing 5 oos. each, 500 units of Diphtheria antitoxin were added to each. Then all four tubes were inoculated with one loopful of a suspension of Diphtheria bacilli, strain # 77 from the General Hospital. 17/4/24. The growth in each of the four tubes, judging by the opacity, is equal to that of any other. 18/4/24. A tube of glucose broth • 500 units Diphtheria Antitoxin was sown with a field culture containing a few Diphtheria baoilll. A normal tube of glucose broth was similarly sown. 22/4/24. The opacity of the growth in both tubes is similar. In a stained smear the proportion of Diphtheria bacilli to the other organisms is about 50% in each culture. APPEHDIX II. Animal # 1. Guinea Pig. Curly Hair, Black, White and San. Inoculated with living "Park" organisms, washed toxin free by 14 washings. 1 cc. = 250 million. On Jfov. 26 with 0.25 ess. su'ocu taneously. » i t n i t w i t i t • t i t i t i t Boo. tt i t I t I t t l n 27 28 S 7 11 15 18 2 1 26 H I I N I t I t I t I t I t I t 0 .75 0 .75 1 . 0 1 . 0 1 . 0 1 . 0 1 . 0 1 . 0 1 . 0 u t f • n H r i f t t t M f t f t I I I t I t I t I I I t I t Bled Sec. 18. igglutinations against live Park strain were all negative. Bled Deo. 27. Agglutinations - negative. Animal f 2. Guinea Pig. Ourly Hair, Black, White and Tan. Inoculated with living "Park" strain, washed 14 X. 1 oo m 250 Billion. Inoculations. On Nov. SO with 0.25 ccs. intraoardially N N f t I t I t I t f t tt Deo. » N tt •1 rt i t t t 1 2 5 7 1 1 18 21 26 n N N f t I t H I t tt 0 .25 0 .25 0 . 4 0 . 5 1 . 0 1 . 0 1 . 0 1 . 0 i t n n H M I t I t W It I t I t I t subcutaneously. » N tt Bled Dec. 18. Agglutinations against Park strain, in dilutions from 1/50 up - negative. Bled Dec. 27. Agglutinations - negative. APPENDIX III. Animal #3, Guinea Pig. Grey, Light Brown. Animal § 4. Guinea Pig. Black, White and Brown, Both inoculated with Defatted Diphtheria Bacilli, Polyvalent mixture containing equal parts of "Park","Durand" and "American" strains, and of three local avlrulent strains, # 5, Inoculations. 1 cc. s 250 million. 14 H It On Nov. 26 w i t h 0 , 5 c o s . s u b c u t a n e o u s l y . H if 27 " 0 . 7 5 " » n 2 Q » 0 # 7 g « " D i e . 3 " 1 . 0 " It M 7 « 1 # 0 tl tt H H n " 1 . 0 " ' " It M l g * 1 , 0 " M M M ! g " 1 . 0 ? " H N 21 " 1 . 0 " M " " 26 " 1 . 0 " " Bled Sec. 21, and 27. With live Park bacilli as antigen all agglutinations were negative in dilutions from 1/50 up. • 4. Inoculations. 1 cc, « 250 million. On Dec, 5 with 0.25 ccs. intracardially. ii tt g ii Q 4 ii it n i t 7 " 0 5 " " •I It H tt Q[ 7 g » It " " 15 " 1 . 0 ? " it it ! g " 1 . 0 " " " " 21 • 1 . 0 " " H it 26 " 1 . 0 " " Bled Deo. 27. Using live Park suspension as antigen .-agglutinations were negative in dilutions as low as 1/50. APPEHDIX 17 . Animal # 5 . guinea P i g . Brown and Fawn. Animal # 6. Guinea P i g . White , Tan and Xel low. Both are i n o c u l a t e d w i t h a suspens ion of l i v e Surand S t r a i n D i p h t h e r i a b a c i l l i , washed 10 X. # 6. I n o c u l a t i o n s . 1 eo z 250 m i l l i o n . On Nov. 30 w i th 0 .25 co . i n t r a c a r i d a l l y . " Dec. 1 " 0 . 2 5 w " n « 2 " 0 . 2 5 " H M M 5 H Q # ^ .i n M M 7 ft Q # 5 M H • H 11 * 1 .0 n s u b c u t a n e o u s l y . " " 16 " 1 .0 " H " n 18 ** 1 .0 " " H tt 2 1 » 1 # 0 It ft tt tt 26 •* 1 . 0 n " Bled Dec. 27. Using live "Durand" strain bacilli as antigen, there were no agglutinations in any dilution from 1/50 up. # 6. Inoculations. 1 cc. • 250 million. on Hov. " Dec. it tt It M tt ft tt ft tt ft N It tt tt It tt 30 1 2 5 7 11 15 18 21 26 with tt w n it it •t tt •t rl 0 .5 0 .5 0 . 5 0 .5 1 .0 1 .0 1 .0 1 .0 1.0 1.0 0 0 8 . i t ft ft fi ft i i it N n subcutaneously. tt tt it it n tt it M ft Bled Deo. 27. Using lire NDurandN strain bacilli as antigen, there were no agglutinations in any dilution from 1/50 up. APPENDIX V, Animal $ 7 . Rabbit. Young, grey, white ring around neck. Injected with a living suspension of strain B.C. 100 washed 10 X and proved toxin free by intracutaneous in-jection into a normal Guinea Pig. Inoculations. On Dec. 22 with 0.25 cos. intraveinously. •1 I I I I I t tt I I I t I t It I I tt It t t t t tt I I I I It Jan. tt H t t i i tt •t H i t it i t tt 23 24 26 3 4 5 8 9 10 15 16 17 22 23 24 n it n i t it •t tt It It I I I I I t It It I I 0 . 4 0 . 5 1 . 0 1 . 0 1 . 0 1 . 0 1 . 0 1 . 0 1 . 0 1 . 0 1 . 0 1 . 0 1 . 0 1 . 0 1 . 0 It tt I I I t I I H tt I t It I I I I I I I I I t I t It I I I I I t It It I I It I t It I I It II I t I I Bled Jan. 10. With strain # 100 as antigen, agglutinations were + in all dilutions made up to 1/720. Bled Jan. 18. With strain # 100 as antigen agglutinations were strongly + up to 1/1200 and weakly + in 1/1800. Using strains Park, Durand and American as antigens there was no agglutination. The animal was bled out and killed Jan. 29. The serum was separated and an equal volume of glycerine added. On Feb.7 with #100 as antigen, agglutinations were negative in dilutions 1/2000-1/6000 On Feb. 12 " 11 « tt tt were •£ up to l/l600 and - from 1/2400-1/3200. On April 16 " n " " M " - in dilutions from 1/100 - 1/900. APPEHDIX VI. Animal f 8. Babbit. Young, grey with white left front foot. Injected with a living suspension of "Park" strain washed 10 X and proved toxin free by inoculation intracutaneously into a normal Guinea Pig. 1 cc. * 250 million. Inoculations-On Sec. 26 with 0.25 cos. intraveinously. H If It N M M It M n it »t ft It It It It It It Jan. it N It tt it •t H H •t ft It 27 28 SI 3 4 5 6 9 10 15 16 17 22 22 24 n M N It It » It It M It It It it N It 0.5 fi.O 1.0 0.5 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 It It N It It tt N tt It It it It It n It M H II M It M It It tt It It tl It It It Bled Jan. 10. Against live Park,agglutinations were * in all dilutions made, up to 1/240. Bled Jan. 18. Against live Park, agglutinations were + up to 1/19 20 and - in 1/2160. The animal was bled out and killed Jan. 31 and an equal volume of glycerine added to the serum. On Feb.4 against live Park, there was • agglutination in all dilutions made up to 1/4000. On Feb. 7 " " " * was strong + agglutination up to 1/5000 and a weak + agglutination in 1/6000. On Apr. 10 " live Park there was J agglutination up to 1/200 and - agglutination in 1/400. " APPENDIX 711. Animal # 9. Rabbit. Young, grey, white hind feet. Injected with a suspension of Defatted wParkH bacilli. 1 cc. • 250 million. Inoculations. On Dec. 26 with 0.25 cos. intraveinoualy. I I If If It If N " Jan M M it If If W » It it II n w ii « M If II it I I I I ft If 27 28 31 3 4 5 8 9 10 15 16 17 22 23 24 •t it it t i i t it t i M I I If n it It I I If 0 .5 1.0 110 1 .0 1 .0 1 .0 1.0 1 .0 1 .0 1 .0 1 .0 1 .0 1.0 1 .0 1 .0 If It It I I I I I I If I I I I It I I 11 I I H If I I If I I I I I t I I It I I It •I I I •1 H I I ft Bled Jan. 10. Agglutinations against live Park and Defatted Park were negative in dilutions from 1/60 up. died Jan. 18. Against live Park agglutinations were + up to 1/1800 and' - in 1/2510. The animal was bled and killed Jan.31, and an equal volume of glycerine added to the serum. On Feb. 4 and 7 against live Park there was no agglutination in dilutions 1/200 - 1/4000. On Feb. 12 there was no agglutination against live or Defatted Park In dilutions 1/200 - 1/3200. On Feb. 7. Using Dreyers Technic a oomplement fixation test showed complement to be fixed in tube # 4 containing 10 drops of a l/20 dilution of serum. The Antigen consisted of a heavy suspension of live Park bacilli and was not anticomplementary in any concentration. On April 16 against Defatted Park bacilli agglutination was A in 1/200 and 1/400 and r in 1/600. APPENDIX 71II. BIBLIOGRAPHY. (1) Georges Dreyer. "Some Hew Principles in Bacterial Immunity, their Experimental foundation, and their Application to the treatment of Refractory Conditions." - British Journal of Experimental Pathology. Vol. 4. (2) H.A. Reque. "Some Observations of the Phagocytosis of Diphtheria Bacilli". Journal of Infectious Diseases - Vol. 3. Ho. 3. (3) Ruth Turnioliff. "Some observations on Phagooytosis and Leukocytic Activity in Diphtheria Carriers." Journal of infectious Diseases. Vol. 19. Ho. 1. (4) W.H, Park. "Toxin Antitoxin Immunisation Against Diphtheria". Jour, of the American Medical Association. Vol. 79 P. 1584. (5) W.H. Park and A. Zingher. "Active Immunisation with Diphtheria Toxin-Antitoxin Mixture". Department of Health, City of Hew York, Reprint Series, #39. Jan. 1916. (6) Hans Seebohm. "Abstracts of Bacteriology." Vol.5. Ho. 1022. (7) J.G. Pits'Gerald and D.G. Doyle. "Methods of Produoing Agglutinating Sera for B. Diphtheria." Transactions of the Royal Society of Canada. Section V. 1923. (8) Major A.S.G. Bell. "Serological Differentiation of some strains of B. Diphtheriae." Journal of the Royal Army Medical Corps. Jan. 1922. 19 J Gay and Pit* Gerald. University of California Public-ations. Pathology 1912. 

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