LIBRARY OF THE UNIVERSITY OF CALIFORNIA. RECEIVED BY EXCHANGE Class SIlj? Htutt?r0tig of OUttrago THE MECHANISM OF NATURAL AND ACQUIRED STREPTOCOCCUS IMMUNITY A DISSERTATION SUBMITTED TO THE FACULTY OF OGDEN GRADUATE SCHOOL OF SCIENCE IV ('AXIHDACY FOR THE DEGREE OI' 1 DOCTOR OF PHILOSOPHY (DEPARTMENT OF PATHOLOGY AND BACTERIOLOGY GUSTAV F. RUEDIGER THni\>ersits of Cbtcago FOUNDED BV JOHN D. ROCKEFELLER THE MECHANISM OF NATURAL AND ACQUIRED STREPTOCOCCUS IMMUNITY A DISSERTATION SUBMITTED TO THE FACULTY OF THE OGDEN GRADUATE SCHOOL OF SCIENCE IN CANDIDACY FOR THE DEGREE OF DOCTOR OF PHILOSOPHY (DEPARTMENT OF PATHOLOGY AND BACTERIOLOGY) BY GUSTAV F. RUEDIGER h ' CHICAGO 1907 (From the Memorial Institute for Infectious Diseases, Chicago.) THE MECHANISM OF NATURAL AND ACQUIRED STREPTOCOCCUS IMMUNITY. EY GUSTAV F. RUEDIGFR. PROFESSOR OF BACTERIOLOGY AND PATHOLOGY, UNIVERSITY OF NORTH DAKOTA, GRAND FORKS, N. D. INTRODUCTION. A great deal of work has been done on the mechanism of strepto- coccus immunity both with normal and with immunized animals, but many points are still left entirely in the dark. In the experiments de- scribed in this paper, an attempt is made to throw additional light on some of these points, but on account of the many inherent difficulties,, the results which I have achieved by no means exhaust the problem. Shortly after the publication of his classical paper on phagocytosis in daphnia, MetchnikofP made a study of phagocytosis of streptococci : in the human body, in cases of erysipelas. He concluded from his ob- servations that streptococci gain entrance through abrasions of the skin,, multiply and set up an inflammation. At the same time there is a gath- ering of microcytes about the streptococci, and these take up the latter and destroy them. Macrocytes are also found in considerable numbers, but they do not take up the streptococci, but have, nevertheless, a phago- cyte action in that they take up and remove dead and disabled micro- cytes. In 1895 Denys and Leclef* studied the mechanism of immunity in rabbits which had been repeatedly injected with small but gradually in- creased doses of virulent streptococci. Their conclusions may be briefly stated as follows : Immune rabbit serum is not so good a culture medium for streptococci as normal serum but it does not possess any marked, streptococcidal powers. The cell-free fluid from a leucocytic exudate *Virdiow's Archiv. 1887, evil., 209. **La Cellule, 1805, XI, 177. 183473 retards multiplication of virulent streptococci and sometimes kills them. Normal leucocytes in normal serum, and leucocytes from an immune rabbit suspended in normal serum, do not greatly retard multiplication of virulent streptococci. Leucocytes from a normal rabbit, or those from an immune rabbit, suspended in immune rabbit serum rapidly take up and destroy virulent streptococci. The serum has acquired something in the process of immunization \vhich neutralized something in the cocci by virtue of which they were protected against phagocytosis. Denys and Marchand* showed that there is better phagocytosis ot virulent streptococci when inoculated into a suspension of rabbit's leu- cocytes in normal rabbit serum to which had been added I per cent, of immune horse serum, than when they were inoculated into the same mixture of leucocytes and rabbit serum with the addition of I per cent, of normal horse serum. Bordet* was able to find no protection against streptococci in normal and immunized rabbits and guinea-pigs except that due to phagocy- tosis. If a rabbit was treated with antistreptococcic serum, and later injected with several times the minimum fatal dose of streptococci, the organisms were all taken up by phogacytes and destroyed. In untreated rabbits he also found phagocytosis, but the organisms soon got the upper hand and the rabbit died of streptococcus infection. Marchand* studied phagocytosis of virulent and non-virulent strep- tococci, and came to the conclusion that the failure on the part of the leucocytes to take up virulent streptococci depends on a physical property of the organisms and not on a secretion. Tchistovitch** injected rab- bits intravenously with fatal doses of a highly virulent streptococcus, killed the animals in one-fourth to six hours and examined the organs for evidence of phagocytosis. He was always able to find some phagocy- tosis in the lungs and in the livers of these animals, and concluded that this phagocytosis of virulent streptococci may be due to the fact that every culture contained some cocci which are less resistant than others, and that those are the only ones which are taken up by the phagocytes. Simon* found very little evidence of phagocytosis, in vitro, when rabbit's leucocytic exudate was inoculated with a non-virulent strepto- coccus. The washed leucocytes suspended in salt solution, and also the cell-free exudate, killed non-virulent streptococci, but not the virulent organisms. Leucocytic exudate mixed with rabbit serum had no effect Quoted from Donys, (Vntralbl. f. Bact, 1898, XXIV, 685. **Aun. tie I'lhst. J'Mstour. ISiMi. X, 104, and 18!)7, XI, 177. Archiv. do M('<]. K.\><>r.. ISDN. x. r>r>:j. **An. de 1'Inst. Pasteur. IfXiO. XIV, 802. *Ontnilbl. f. liMkt. V.xn. XXIX. SI and li:',. on non -virulent streptococci. He .was unable to confirm the view ad- vanced by Bordet and others that a rabbit which is injected with strep- tococci into a pleural cavity containing leucocytic exudate can stand a larger dose than a rabbit of the same size which is injected in the normal pleura. The pleural exudate was produced by the injection of aleuronat suspension forty-eight hours previously. These results are quite contradictory to those of other investigators, and it was, therefore, thought desirable to repeat these experiments with leucocytes. Neufeld and Rimpatt* have shown that the leucocytes play an im- portant role in combating streptococcus infections in immunized rabbits. They have shown also that the addition of antistreptococcus serum to a suspension of leucocytes and streptococci aids phagocytosis and that this is not due to a stimulation of the phagocytes but to an effect of the serum on the cocci. The specific substance in the immune serum they found to be fairly resistant to heat, it being unchanged by heating at 59 C. for ^2 hour. TEST TUBE EXPERIMENTS WITH LEUCOCYTIC EXUDATE FROM NORMAL ANI- MALS. The leucocytic exudate used in these experiments was obtained by injecting a 6-8% suspension of aleuronat in Na Cl solution into the right pleural cavity of a rabbit or large guinea-pig and bleeding the animal to death eight to ten hours later. The aleuronat suspension is best prepared by sterilizing the aleuronat in dry heat at 150 to 160 C. and suspending it in sterile physiological salt solution. No sodium oxalate was added to the exudate, as this salt is strongly streptococcidal, but the coagulation of the exudate was prevented by occasional agitation of the tubes during the first hour. The contents of each tube were 2 c. c., which were inoculated with three loopfuls of a twenty-four-hour broth streptococcus culture and three loopfuls were plated at intervals. Tube 7 differs from Tube 6, in that it contains the washed leucocytes which had been centrifugated out of 2 c. c. of the exudate, suspended in rabbit serum, whereas Tube 6 contained a mixture of I c. c. of the entire exu- date -f- i c. c. of serum. The fluid of the exudate in Tube 7 was removed to show that the effect of these mixtures is not due entirely to the liquid portion of the exudate, but in a large measure to the leucocytes. The Tables show that non-virulent streptococci are destroyed by the leuco- cytic exudate, by mixtures of leucocytes and serum and by leucocytes in clefibrinated blood. Defibrinated blood and serum alone, the heated exti- **D. Med. Wool]., 11)04, XXX, 1458. date and a suspension of leucocytes in salt solution or in heated serum- have little or no effect on these organisms. The virulent streptococcus is not destroyed by the leucocytic exudate nor by a suspension of leuco- cytes in serum or blood. Smears made in one to two hours from tubes in which there is destruction of cocci show evidence of phagocytosis. Table 2 shows the same experiment as that shown in Table I ; except that guinea-pig's leucocytes and blood were used. With these leucocytes the TABLE 1. SHOWING THE EFFECT OF RABBIT'S LEUCOCYTIC EX L- DATE AND MIXTURES OF EXl DATE AXD BODY FLUIDS OX STUEI'TOCOCCI. Strepto- Immecli- eocci. ately. 3 hrs. 18 hrs. 300 Leucocytic exudate 4GOO 10000 300 Exudate 58 for half hour 3250 8000 Many 300 Defibrinated blood 4800 4700 Many 300 Blood 1 c. c. + exudate 1 c. c 4000 750 1 500 300 Rabbit serum 4200 4200 Many 300 Serum 1 c. c. -f exudate 1 c. e 5200 1100 Many 300 Leucocytes -f serum 5000 800 Many 300 Leucocytes in heated serum 5500 3560 Many 300 Leucocytes in NaCl sol 4900 5000 Many 300 Leucocytes in NaCl sol. -f Sensitized streptococci 570 32 .",000 324 Leucocyte exudate 420 11 Many 324 Defibrinated blood 430 700O Many 324 Blood 1 c. c. -f- exudate 1 c. c 750 540 Many 324 Rabbit serum 552 0500 14000 324 Serum 1 c. c. + exudate 1 c. c 320 1 Many B104 Leucocytic exudate 3500 3600 Many 270 Leucocytic exudate 1700 2 Many Sources of streptococci: Nos. 300 and 324 from the heart's blood of scarlet fever patients, postmortem. No. B104 from an abscess in a guinea-pig which had been injected with a fungus. This organism has been passed through 78 rabbits and is very virulent for both rabbits and guinea-pigs. eighteen-hour plates are often sterile, or nearly so, while it is only occa- sionally that they are sterile when rabbit's leucocytes are used. Special attention must be called to the fact that the suspension of leucocytes in salt solution destroys many of the sensitized a virulent strep- tococci. These results are therefore in harmony with the work of Wright and Douglas/-' who have shown that phagocytosis takes place only after the bacteria have been sensitized, that is, have been acted on by the opsonin of the serum. There is no phagocytosis in a suspension of washed leucocytes in salt solution or in heated serum, when untreated *Proc. of Royal 'Soc., 1903, LXXJI, 357, and J904, LXXIII, 128. 5 Bacteria are, added, but when sensitized bacteria are added to such a suspension of leucocytes there is good phagocytosis. This work was confirmed and extended by Hektoen & Ruediger** and by Bulloch & Atkin.*** In consideration of these facts we would expect that the TABLE 2. -SHOWING THE EFFECT OF GUINEA-PIG*S I.EUCOCYTIC EXUDATE AND MIXTURE OF EXU- DATi: AND BODY FLUIDS' ON STREPTOCOCCI. Strepto- Immedi- cocci. ately. 3 hrs. 18 hrs. 300 Leucocytic exndate 6500 70 170 A Leucocytic exndate 650 600 Many B104 Leucocytic exudate 8000 10000 Many 300 Cell-free exudate 5500 800 GOflO 7H04 Cell-free exudate 8000 10000 Many 300 Defibrinated blood .6000 5000 Many 300 Defibrinated blood + leucocytes 6800 240 72 300 Serum 6000 6000 Many 300 Serum + leucocytes 6000 2000 Many Streptococcus Xo. 300 is not virulent for guinea-pigs. Nos. B104 and A are virulent. No. A was isolated from a peritonsillar abscess following an attack of follicular tonsillitis and was used in this experiment in the first generation on artificial media. TABLE 3. PHAGOCYTOSIS OF STREPTOCOCCI BY RABBIT LEUCOCYTES AND GUINEA-PIG LEUCOCYTES. Strepto- Phagocy- cocci. tosis. 300 Rabbit leucocytes in blood 20 B104 Rabbit leucocyte? in blood : . 300 Guinea-pig leucocytes in blood 30 13104 Guinea-pig leucocytes in blood 1 300 Washed rabbit leucocytes in NaCl solution 1 300 Washed rabbit leucocytes in heated rabbit serum 1.5 300 Washed rabbit leucocytes -f sensitized streptococci 12 Each tube contained 2 c. c. of suspension of leucocytes -(- 2 c. c. of suspen- sion of streptococci. Tubes were incubated 1 hour, smears were made and stained and the degree of phagocytosis determined by counts. extent of destruction of streptococci in the test tubes should run parallel with the degree of phagocytosis of these organisms and Table 3 shows that this is the case. * ' If we read the three tables together we notice that the virulent **Jour. of Infect. Dis., 1905, II, 128. ***Proc. of. Royal Soc., 1905, LXXIV, 379. organism (6104) is not taken up by the rabbit leucocytes and guinea-pig^ leucocytes in blood, neither is this organism destroyed or killed by the leucocytic exudate and suspensions of leucocytes from these animals in blood or fresh serum. The avirulent organism (300) is ingested by the leucocytes in blood and in serum but not by washed leucocytes in Na Q solution or in heated serum unless the cocci have been treated before- hand with fresh unheated serum (sensitized). Similarly this organism is destroyed by suspensions of leucocytes in blood or serum but not by suspension of washed leucocytes in NaCl solution or heated serum. The- washed leucocytes do however destroy the sensitized avirulent organisms. EXPERIMENTS WITH ORGAN CELLS. It seems quite reasonable to suppose that the organ cells might play an important part in the protection of the body against invasion by streptococci ; and with this point in mind the following experiment was carried out: Experiment. Two guinea-pigs, A and B, were bled to death and' the blood collected under aseptic precautions. The organs were now care- fully removed from the body, placed in sterile glass dishes and rubbed up: (A) with guinea-pig's serum, and (B) with defibrinated guinea- pig's blood. The organs and blood were not allowed to become cold. TABLE 4. SHOWING THE EFFECT OF THF SUSPENSIONS OF ORGAN CELLS OF GriNEA-PK.S ON STREPTOCOCCI. Strepto- Iinmedi- cocci. ately. ' lirs. IN hrs. B104 -+- Defibrinated blood 1800 HHM 1 500- B104 Blood + muscle 2000 8000 Many B104 Blood -I- spleen 1900 Many Many B104 Blood + liver 1800 Many Many B104 Blood -f- kidney 1900 Many Many B104 Blood +Hdrena1 1500 Many Many B104 Blood + lymph gland 1500 0000 Ma ny 300 Blood -f bone marrow 2500 lOoo Many 300 Serum 3SOO :;75o (5000 300 Serum -j- leucocytes 2700 1800 1500 300 Serum + spleen 2800 50OO Many 300 Serum -j- liver 2000 12000 Many 300 Serum + kidney 2400 12000 Many Control liver + serum " !> Control ppleeu + serum 0- The strain of streptococcus B104 used in this experiment has not been passed through rabbits and has lost its virulence. The suspensions of organ cells were now introduced into small test tubc.% inoculated with two loopfuls of a 24-hour broth culture of a non-virulent streptococcus, and two loopfuls were plated at intervals. The colonies that developed in each plate were carefully estimated and the results are shown in Table 4. In no instance did the suspension of organ cells have an inhibitive effect on the multiplication of the streptococci, but on the contrary the organisms multiplied more rapidly in these suspen- sions than in either the serum or the defibrinated blood alone. If, how- ever, bone marrow was added to the defibrinated blood or serum, the number of streptococci in these tubes was greatly diminished during the first three to five hours. These results agree with those found by Bail and Pettersson* in their study of the mechanism of anthrax immunity in chickens, and show that suspensions of organ cells from the guinea-pig possess no strepto- coccidal power, except the suspensions of bone marrow. Suspensions of marrow cells in defibrinated blood or serum kill large numbers of avirulent streptococci, but that is not the case with suspensions in salt solution. These results have also been obtained with suspensions of rabbit's bone marrow. PHAGOCYTOSIS OF LIVING STREPTOCOCCI IN VIVO. It having been found that leucocytes ingest and destroy streptococci in test tube experiments, the following experiment was made to show that there is phagocytosis of living streptococci in the animal body: A guinea-pig was injected in the right pleura with 7 c. c. of aleuronat suspension. Twelve hours later he was injected in tin's pleura with 2 c. c. of an 1 8-hour glucose broth culture of a non- virulent streptococcus, and two hours after this injection he was bled to death. Several cubic centimeters of leucocytic exudate were now taken from the right pleura, under aseptic precautions. Smears made with this exudate contained no free streptococci, but many of the leucocytes contained cocci in large numbers. Figure i is a photomicrograph of one of these smears showing streptococci in the body of a leucocyte. Preparation of Slides. By means of a sterile, soft cotton swab a small quantity of the exudate was now gently smeared on each of eight agar-covered slides ; the slides were incubated in a moist chamber for four to six hours, stained and examined under the microscope. The prep- aration of these slides requires the following special technic. Three ordinary clean glass slides and a heavy watch glass are placed in a large *Centnilbl. f. Bakt, 1903, XXXV, 102. 8 Petri dish and sterilized in dry heat. When the Petri dish has cooled .sufficiently to be handled the slides are taken out with sterile forceps and hot glucose agar is poured on each to form a uniform thin film. Each .slide is immediately replaced in the Petri dish and the agar allowed to solidify. The exudate which contains many streptococci in the bodies of the leucocytes is now gently smeared on these agar films, the watch .glass is half filled with sterile water, and the large dish is placed in the incubator for four to six hours. The water is used to prevent dry- ing of the agar. Staining. The slides are now taken out of the Petri dish and dried Fig. 1. Photomicrograph x 1300. Streptococci in guinea-pig's leucocytes, taken from the pleura t\vo hours after injection of streptococci. in a thermostat at 55 C. for fifteen to twenty minutes, or longer if nec- essary; fixed in 40 per cent, formalin for four or five minutes, washed in water, stained for only a few seconds with Loeffler's methylene blue solution, again washed in water and dried in the thermostat. The best stains were obtained when the methylene blue solution was poured over the wet slide, the slide being held so that the stain ran off readily. The slide is then immediately placed in a dish of cold water and gently rinsed in several changes of water. Violent agitation in the water pulls off the film of agar. When the agar is perfectly dry (which requires only about half an hour), a cover-glass is placed on it, either 9 in glycerin or Canada balsam. The slides are now ready to be examined with the oil immersion lens. Examination. Examination in various stages shows that the strep- tococci in the leucocytes multiply, burst open the cell and form a small colony surrounding the cell which contained them. Figures 2 and 3 are photomicrographs of such preparations at different stages. In Figure 2 the leucocyte has just been burst open and the streptococci are beginning to grow out of it; in Figure 3 a considerable colony has already formed around the ruptured leucocyte. These colonies may be found in all stages, by varying the time of incubation. Fig. 2. Photomicrograph x^W- Multiplication of streptococci after their ingestion (in vivo) by a guinea-pig's leucocyte. The leucocytes do, however, kill streptococci if the conditions are right, as is shown by the following two facts: i. Organism No. 300 is not virulent for guinea-pigs, and we have seen that the leucocytes take' up the cocci alive. The guinea-pig must destroy these organisms some- where, and we can find no substance in its body that is capable of doing this except the leucocytes and the bone marrow. We have seen in Table 2 that the leucocytes destroy streptococci in vitro, and the inference is- that they also destroy them in vivo. 2. Plates made with three loopfuls of the exudate when taken from the guinea-pig contained 2,540 colonies of streptococci after twenty-four hours' incubation, while plates made 10 with three loopfuls, after the exudate had stood in a test tube in the incubator for six hours, contained only 480 colonies. Evidently some of the streptococci were killed in this tube, but the smears show that all of the organisms are in the leucocytes, hence they must have been killed in the leucocytes. EXPERIMENTS WITH LEUCOCYTIC EXUDATE IN VIVO. In order to determine whether a rabbit is more resistant when in- jected in a pleura containing leucocytic exudate than when injected in the normal pleura, the following experiments were carried out : Fig. 3. Photomicrograph x ]50 . Multiplication of streptococci after their ingestion (in vivo) by a guinea-pig's leucocyte, in slightly later stage than Fig- ure 2. Experiment A. A gray rabbit weighing 1,450 grams was injected into the right pleura with 7 c. c. of aleuronat suspension. Fifteen hours later 2, c. c. of a 4<>hour culture of a virulent streptococcus were injected into this pleura and also into the right pleura of a normal rabbit weigh- ing 1,500 grams. The normal rabbit was found dead in 18 hours, and the one which had been previously injected with aleuronat died 45 hours later. Experiment B. A gray rabbit weighing 2,100 grams was injected in the right pleura with 6 c. c. of aleuronat suspension. Eighteen hours later i c. c. of a 24-hour culture of a virulent streptococcus was injected 11 into the pleura and also into the right pleura of a normal gray rabbit weighing 2,150 grams. The normal rabbit died in about 18 hours, while the aleuronat rabbit died only after 45 hours 21 hours after the death of the control. The experiment was now varied so that the conditions were the same as in Simon's experiments. Experiment C. A rabbit weighing 1,700 grams was injected in the right pleura with 8 c. c. of aleuronat suspension and 48 hours later this rabbit and a normal rabbit weighing 1,700 grams were each injected in the right pleura with I c. c. of a 3O-hour streptococcus culture. In this experiment the rabbit treated with aleuronat died in 24 hours, while the control lived for five days. Experiment D. A rabbit weighing 1,500 grams was injected in the right pleura with 9 c. c. of aleuronat suspension. Forty-eight hours later this rabbit and a normal rabbit weighing 1,800 grams were each injected in the right pleura with 1.5 c. c. of a 24-hour streptococcus cul- ture. The normal rabbit died in 37 hours, while the rabbit treated with aleuronat died in 45 hours eight hours after the death of the control. The results of the last two experiments agree fairly well with those obtained by Simon, and we may now ask why they are different from the results of Experiments A and B. Apparently this difference is due to the fact that in Experiments C and D the inoculation was made some thirty hours later than in Experiments A and B. That this is the correct explanation is indicated by the following ex- periment : Experiment E. A rabbit weighing 2,300 grams was injected in the right pleura with 9 c. c. of aleuronat suspension. Forty-eight hours after this injection the rabbit was bled to death, and on opening the pleural cavity it was found to contain no leucocytic exudate. Had this rabbit been inoculated in the right pleura forty-eight hours after the injection of the aleuronat suspension, the inoculation would not have been made into leucocytic exudate. That being the case we would not expect that this rabbit could stand a larger dose of streptococcus than a normal rabbit. In Simon's experiments, however, the inoculations into the pleural cavity were made forty-eight hours after the injection of aleuronat, and this is probably the reason w'hy he did not find these rabbits more resistant than normal rabbits. Had he made his inocula- tions within twenty-four hours after the injection of aleuronat his re- sults would very probably have been different*. At the suggestion of Professor Hektoen I tested filtrates of virulent streptococci to determine whether they are toxic for leucocytes, as has 12 been shown by Van de Velde,* and by Neisser and Wechsberg,** to be the case with staphylococcus filtrates. The organism used in these experi- ments was BiO4, which has been passed through 78 rabbits, and is very virulent for rabbits and for guinea-pigs, and produces an active hemoly- sin when grown in heated rabbit serum or in a mixture of equal parts of rabbit serum and ascites fluid. The filtrates used were from iwenty- four to thirty-six-hour cultures in a mixture of rabbit serum and ascites fluid, which has been heated to 560. for one-half hour before inoculation. Neisser and Wechsberg have shown that living leucocytes reduce methylene blue solution when the supply of oxygen is shut off, while dead leucocytes no longer possess this property. In my experiments this fact was made use of to determine whether or not the leucocytes had been killed by the filtrate. 0.3 c. c. to 0.4 c. c. of leucocytic exudate from a rabbit was added to 1.5 c. c. of ~the filtrate, incubated for two hours, then two drops of methylene blue solution added and the contents of the tube shaken up and covered with a layer of sterile olive oil. If there was reduction in two hours the result was considered negative, but if there was no reduction in that time this fact was taken as evidence that the leucocytes were dead. Control tubes with a mixture of equal parts of heated rabbit serum and ascites fluid were always made, and the results recorded only when there was marked reduction in the control in two hours. A large number of these experiments were carried out, but it is not necessary to tabulate more than one of them here. Experiment. .3 c.- c. exudate + 1.5 c. c. 24-hour filtrate; no re- duction in 2 hours, but a trace of reduction in 3 hours. .3 c. c. exudate -|- 1.5 c. c. 36-hour filtrate; no reduction in 3 hours. .3 c. c. exudate 4- 1.5 c. c ascites-serum ;* nearly complete reduction in 2 hours. The leucocytic exudate was obtained from the pleura of a rabbit which had been previously injected with aleuronat suspension The methylene blue solution was made according to the formula given by Neisser and Wechsberg, which is given below : Solution A. Methylene blue, i; absolute alcohol, 20; distilled water, 29. Solution B. i c. c. of solution A ; 49 c. c. of physiologic salt solu- tion. Two drops of solution B, which must be sterile, are added to each tube in the experiments. *An. de 1'Inst. Pasteur, 389G, X, 580. **Zcitschr. f. Hyg., 1001. XXX VI, 209. *The term "asdles-serum" as used here refers to a mixture of equal parts of ascites fluid and rabbit serum, heated to 55 . for one-half hour. 13 The fact that the filtrates have a decided effect on leucocytes has also been shown in a second experiment. Experiment. Two cubic centimeters of a rich leucocyitc exudate were placed in each of five small test tubes and centrifugated. The clear, supernatant' fluid was now decanted from each tube and replaced by 2 c. c. of bouillon, heated ascites-serum or filtrate from a 24-hour ascites- serum streptococcus culture. The leucocytes in each tube were evenly suspended in the fluid, the tubes placed in a water bath at 37 C. and kept there for I to i l /2 hours. Now .3 c. c. or normal rabbit serum (guinea- pig serum when guinea-pig's leucocytes were used) was added to each tube, the tubes inoculated with a loopful of a non-virulent streptococcus broth culture and plates made at intervals, the tubes being kept in the incubator. The following table shows the results of an experiment: Immed. 4-5 hrs. 20 hrs. Leucocytes -f 2 c. c. filtrate B104 + .3 c. c. normal serum. .2000 3200 Many Leucocytes -f 2 c. e. filtrate 10 A + .3 c. c. normal serum . .1200 2000 Many Leucocytes + 2 c. c. filtrate 300 + .3 c. c. normal serum ..1100 24 Many Leucocytes + 2 c. c. ascites serum -j- .3 c. c. normal serum . .1000 140 Many Leucocytes -f- 2 c. c. bouillon -f- .3 c. c. normal serum 1800 GO Many The numbers B10-1, 10A and 300 refer to different strains of streptococci. B104 has a high virulence and 10A a medium virulence; both these strains pro- duce liemolysin in heated serum, although 10A has never been passed through animals. It was recently isolated from suppurating axillary glands. 300 is non-virulent, and does not produce hemolysin. The suspensions of leucocytes in filtrates from serum cultures of virulent streptococci have no effect on streptococci while the suspension in filtrates from non-virulent organisms, in heated ascites-serum, and in bouillon destroy large numbers of cocci. These experiments do not always give positive results, but there is a small percentage of tubes in which the filtrate has apparently no effect on the leucocytes. This is not surprising, however, when we consider the fact that in about .TOO filtrates which I have tested for hemolysis, there were nearly 10 per cent, that had no hemolytic properties, whereas some of them were so active that .005 c. c. completely laked i c. c. of a 2]/2 per cent, suspension of rabbit's washed corpuscles in two hours. TEST TUBE EXPERIMENTS WITH HUMAN LEUCOCYTES. The foregoing experiments show that the leucocytes and opsonin are the most important, if not the only, factors concerned in the destruction of streptococci in the body of infected rabbits and guinea-pigs. In strepto- 14 coccus infections in man the tissues are invaded by virulent streptococci but in a majority of these cases the cocci disappear sooner or later and the patients make a complete recovery. Freshly drawn human serum has no streptococcidal powers in vitro and it is reasonable to suppose that it has none in the body during life. We must look therefore for some other agent than the serum alone to account for the disappearance of the cocci from the tissues during convalescence. According to Metchnikoff and his followers this agent is found in the phagocytes and this view seems to explain the facts better than any other theory that has been advanced. I next undertook a series of experiments to determine more accu- rately what factors are concerned in the destruction of the invading cocci in cases of human streptococcus infections, and to analyze more fully this phenomenon. In all instances where blood was used it was drawn from the vein at the elbow by means of a Luer syringe and de- fibrinated by gently whipping with a sterile wire. If the defibrinating is carefully performed not a very large proportion of the leucocytes are destroyed. In the experiments each tube contained from 0.8 to i.o c. c. of blood or serum which was inoculated with one loopful of streptococcus culture and two loopfuls from each tube were plated in glucose agar at intervals. The tubes were always kept in the incubator at 36 C. When highly virulent organisms were used 0.3 to 0.4 c. c. of defibrinated rabbit TABLE 6. THE EFFECT OF DEF1B1IINATED NORMAL HUMAN HLOOD AND HUMAN SERUM ON STREP- TOCOCCI. Strepto- cocci. 300 300 300 300 300 300 300 298 298 298 B104 B104 B104 B104 381 300 298 Defibrinated Blood. I II I la III IV V VI VII VII VIII I la III IV V VI Serum IV Semin VII Colonies in Agar Plates. limned. 2 to 3 hours. 1100 .... 3000 .... 680 675 2600 2700 3000 1050 540 360 1100 500 1600 600 1600 600 600 80 76 56 1800 2800 1600 3000 500 600 2000 2300 1400 2000 1700 2500 5 hours. 160 1060 1300 3500 2600 240 390 315 315 16 510 Many Many 900 Many Many 10000 15 blood was added to each tube of melted agar to facilitate the counting of colonies, which often are very small if no blood has been added. Table 6 shows that, although human serum in vitro is a good culture medium for streptococci, normal defibrinated blood has a slight strepto- coccidal power. Occasionally we may find a sample of normal blood which destroys many non-virulent streptococci, but the virulent organ- isms usually multiply in this blood. Table / shows that defibrinated blood from patients suffering from an acute infection has a much greater destructive effect on these organisms than has normal blood. (The non- virulent cocci are ingested very freely by the leucocytes while the viru- lent strain is not taken up so freely, as shown by counts in stained smears.) TABLE 7. THE EFFECT OF DEFIBRIX.VTKH PLOOD FROM CASES OF SCARLATINA, ERYSIPELAS AND PNEUMONIA ON STREPTOCOCCI. Strepto- cocci. 300 ?>00 300 300 300 300 300 300 300 300 300 300 300 300 381 B104 B104 381 381 381 381 B104 381 B104 381 381 381 Colonies Defibri nated Leucocyte Blood. count. Immed. Scarlatina I 10500 1100 II 10600 2000 III 15000 690 IV 550 V 1500 VI 1000 VII 500 VIII 13000 1380 IV 1200 X 13400 1250 XI 1950 XII 10800 1800 XIII 1800 XIV 12300 1500 III 15000 1200 IV 600 V 1800 VI 220 VII 130 VIII 13000 360 IX 13000 2200 IX 330 X 13400 *640 X 400 X 320 XII 10800 200 XIII 540 in Agar 2 to 3 hours. 270 18 300 350 32 102 125 245 150 700 300 31 1500 462 1800 51 41 1180 1950 190 290 160 650 2000 900 Plates. 5 hours. 6 140 116 3 150 43 12 9 14 120 19 130 45 2 3500 415 5000 1100 Many Many Many 1500 2500 165 Many Many Many 16 300 Erysipelas I ..... G30 64 300 II 15000 800 14 4 300 H;i 1100 41 8 300 III 740 120 160 300 IV 1650 480 150 298 V 10400 3100 420 02 I 1200 1080 960 B104 II 96 30 13 B104 IV 133 110 480 300 Tonsillitis I 16000 620 34 2 JB104 I 300 59 70 300 II 1400 74 6 300 Pneumonia 1 2600 3 500 " II 600 5 Serum 300 Scarlatina VII 1500 3500 10000 300 " XIV 1200 1400 1300 300 Erysipelas II 670 760 7.".0 300 III 700 1800 6 298 Conv. washed corp. -f Neph. ser 725 GOO 01 20 nearly as great a streptococcidal power as the defibrinated blood from the patients without nephritis. The corpuscles were washed twice in a large amount of NaCl solution which is usually sufficient to prevent phagocytosis in a suspension of corpuscles in NaCl solution. It is not likely, therefore, that the washing had not been carried far enough. But the results of these experiments seem to indicate rather that the leuco- cytes as well as the serum from these nephritis patients have undergone some change which renders them less efficient in the destruction of bac- teria. In fact, it would seem that the leucocytes have suffered more than the serum. Whether or not these facts may serve to throw light on the cause of some of the terminal infections can not be determined at this time. It is an interesting question whether the opsonin is increased or not during the acute infections. Normal leucocytes in normal serum take up large numbers of cocci ; hence, it is difficult to determine if leucocytes in erysipelas serum, for instance, take up more cocci than those in normal serum. This question had, therefore, to be approached in a different way. Experiment 4. Two sets of tubes were made and 0.2 c. c. of washed corpuscles introduced into each. To one set of tubes were added falling quantities of normal serum and to the other set falling quantities of erysipelas serum. The contents of each tube were made up to 0.4 c. c. with NaCl solution, and to each tube was added 0.4 c. c. of a suspension of streptococci. The tubes were incubated for one hour at 36 C., smears were made and the average number of cocci in each leucocyte determined "by counting those in 30 leucocytes. The results are shown in Table 12. Table 12. Normal serum. Phagocytosis. Erysipelas serum. Phagocytosis. 0.2 c. c 13.5 0.2 c. c 11.5 0.1 c. c 0.4 0.1 c. c 11.2 0.05 c. c 0.7 0.05 c. c 8.2 0.025 c. c 4.7 0.025 c. c 0.0 0.012 c. c 2.2 0.012 c. c 4.3 This experiment indicates that there is a slight increase of opsonin in the erysipelas serum as compared with normal serum. A similar in- crease of opsonin has been noted by Wright and Douglas* after treat- ing with their staphylococcus vaccine a person afflicted with furunculosis. Hektoen** also observed a rise in opsonin after injecting a person with **Proc. Royal Sue., 1904. **Journ. A. M. A., 1006, XLVI, 1400. 21 heated streptococci. A diminution of opsonin in persons subject to at- tacks of furunculosis, sycosis, etc., has also been observed by Wright and Dong-las. During the course of this work I had an opportunity to study a case of acute purulent rhinitis, in the discharge of which streptococci were found in pure culture. Patient. A man 27 years of age. During the first two days the discharge was clear and watery and contained very many streptococci in short chains and in diplococci. Figure 4 shows a photomicrograph of a smear make on the second day. There were only a few leucocytes, and those did not contain any streptococci. On the fourth day the discharge Fig. 4. Photomicrograph X 1500. Streptococci hi nasal discharge on sec- ond day of sickness. had become thick and purulent, and smears made at that time presented an entirely different picture. There were no free streptococci, but very many leucocytes, some of which were loaded with streptococci. Figure 5 shows a photomicrograph of such a preparation made on the fourth day. The purulent discharge continued for about a week, and during this time it was easy to find leucocytes in the smears which contained large numbers of streptococci. The streptococci finally disappeared, and the patient made a complete recovery. In this case of streptococcus infection phagocytosis of the organ- isms by the leucocytes seemed to be an important factor in combating 22 the infection. In the smears made on the fourth day no free streptococci could be found, although diligent search was made for them. Plates made with a small quantity of discharge, which was obtained high up in the nares by means of a sterile cotton swab, contained, however, many colonies of streptococci after twenty- four hours' incubation. The fact that many colonies developed in the plates, although no free streptococci could be found in the smears, seems to indicate that the organisms were not dead when they were taken up by the phagocytes, but were taken tip alive. It is possible, however, that there were free streptococci in this purulent discharge, even though they were not found in the smears. Fig. 5. Photomicrograph X 1500. Streptococci in a leucocyte from nasal discharge on fourth day of sickness. IMMUNIZATION OF RABBITS WITH STREPTOCOCCI. There can no longer be any doubt about the statements of many in- vestigators that laboratory animals acquire a fair degree of immunity after repeated injections of small doses of virulent streptococci. I have injected a large number of rabbits first with several doses of heated cul- tures (65 C. for 20 minutes) and then with living cultures of medium virulence, and have found that some animals acquire enough resistance after 4 or 5 injections to withstand injections of several times the mini- mum fatal dose of the streptococcus. Thus, for instance, a full grown rabbit weighing 2000 G. had received 2 large injections of heated culture 23 and 4 small injections of the living culture "381" at intervals of 8 to 10 days. This rabbit was now injected under the skin of the back with 4 c. c. of a suspension of streptococcus "381" and at the same time a nor- mal rabbit weighing 2100 G. was injected in the corresponding locality with 2 c. c. of the same streptococcus suspension. The normal rabbit died on the 3rd day while the immune rabbit showed no signs of sickness except that it lost 150 G. in weight, which was regained at the end of one week. Experiments with rabbit leucocytes in immune serum in vitro. Ex- periments were now undertaken to make a further study of the mechan- ism of this immunity than has already been made by.Neufeld and Rimpau and others. An immune rabbit and a normal rabbit were each bled 5 c. c., the bloods defibrinated, divided into two equal quantities and leucocytes added to each blood. One portion of each blood received leucocytes from the normal rabbit and the other portion received washed leucocytes from the immune rabbit. The whole blood instead of the serum alone was used because rabbit blood contains only a very small number of polymorphonuclear leucocytes which may be neglected, and it has been found that smears from tubes containing leucocytes in blood are more TABLE 13. PHAGOCYTOSIS OF STREPTOCOCCI BY NORMAL AND BY IMMUNE RABBIT LEUCOCYTES JN NORMAL AND IN IMMUNE RABBIT BLOOD. Strepto- Phagocytosis by normal leucocytes in : cocci. Normal Blood. Immune Blood. Laura* G.2 14. 381 2.2 4.8 Joe R. 4.4 7.G Stuart 1. 4.0 Puerperal 1. 1.6 Phagocytosis by normal leucocytes in : Normal Blood. Immune Blood. Laura 5.5 15.2 381 1.7 5. Joe R. 5.3 7.2 Stuart 1. 4.2 Puerperal 5. 2.5 **Sources of Streptococci: "Laura" from a* scarlatinal otitis media, and used to immunize the rabbit, the serum of which was used hi this experiment; "Joe R." from a scarlatinal sore throat ; "381" from pericardial fluid of a scarla- tinal body ; "Stuart" from a case of erysipelas and "Puerperal" from a blood cul- ture of a case of puerperal sepsis. All were only of medium virulence. 24 satisfactory than those made from suspensions of leucocytes in serum. Four-tenths of a cubic centimeter of each suspension of leucocytes in blood was put into a number of small test tubes arid .2 c. c. of a fairly thick suspension of virulent streptococci added to each tube. The tubes were incubated at 36 C. for i hour, smears were made and the average number of cocci ingested by each leucocyte was determined by counting the cocci in 30 leucocytes. The results of such an experiment are shown in Table 13. The Table shows that there is a larger number of cocci taken up by the leucocytes in immune blood than by those in normal blood; that the immune leucocyfes do not ingest appreciably more cocci than the normal leucocytes and that the increase is most marked with that strain of streptococcus which was used in immunizing the rabbit. Four other experiments with the bloods of different immune rabbits gave practically the same results. The increased phagocytosis always was most pronounced with that strain of streptococcus which had been used in immunizing the rabbit whose defibrinated blood or serum was used in the experiment. Several strains of streptococcus were used in the immunization and all that were of medium virulence gave an im- munity that could be demonstrated in the test tube. Rabbits were also treated with a highly virulent streptococcus, but this organism was scarcely taken up at all by rabbit leucocytes suspended in this or in other immune sera. An experiment was now carried out to show how the immune serum promoted phagocytosis of the virulent cocci. For this purpose normal rabbit leucocytes were suspended in normal and some in immune rabbit serum for */?, hour, then centrifugated out of the sera, washed twice in NaCl solution and suspended in normal serum. To each tube was now added the same amount of suspension of a virulent streptococcus, the tubes were incubated for i hour, smears were made from each and the degree of phagocytosis determined by counts. In neither tube was there any phagocytosis, showing that the immune serum is not capable of changing the leucocytes so that they will ingest virulent streptococci which have not been treated with immune serum. A second pair of tubes was carried through at the same time, using the same leucocytes, streptococcus suspension and sera. In this instance virulent streptococci were treated J/ hour with immune rabbit serum and some with normal rabbit serum, then washed once in a large amount of NaCl solution, sus- pended in NaCl solution and each lot added separately to a suspension of normal rabbit leucocytes in NaCl solution. The tubes were in- cubated i hour, smears were made as above and the degree of phagocy- tosis determined by counts. In this pair of tubes the degree of phago- 25 cytosis was strikingly different. The cocci which had been sensitized in normal serum were not ingested by the leucocytes, while those that had been sensitized in immune serum were taken up freely. That is, the serum has acquired something in the process of immunization by virtue of which it is capable of so changing the virulent streptococci that they become susceptible to phagocytosis. The results of the count are shown in Table 14. TABLE 14. PHAGOCYTOSIS OF VIRULENT STREPTOCOCCI SENSITIZED IN IMMUNE RABBIT SERUM. 1. Washed rabbit leucocytes + normal rabbit serum + streptococcus "Laura" 0.4 2. Rabbit leucocytes suspended in immune rabbit serum y 2 hour, washed in NaCl solution, resuspended in normal serum + streptococcus "Laura" 0.0 3. Washed rabbit leucocytes in NaCl solution + streptococcus "Laura" sensitized in normal rabbit serum 0.6 4. Washed rabbit leucocytes in NaCl solution + streptococcus "Laura" sensitized in immune rabbit serum 8.0 The destruction of virulent streptococci by rabbit leucocytes in im- mune serum can also be shown by means of plate cultures. For this purpose 2 tubes were prepared containing (a) I c. c. defibrinated nor- mal blood + .2 c. c. of a thick suspension of rabbit leucocytes, and (b) i c. c. defibrinated immune blood -(- .2 c. c. of the same suspension of leucocytes. Each tube was inoculated with one loopful of a virulent streptococcus culture, incubated at 36 C. and plates made at intervals with 2 loopfuls of blood from each tube. The plates were incubated for 24 hours and the colonies carefully estimated with the following result: Strepto- Colonies in Agar plates, cocci. Immeil. 2 hours. 5 hours. 381 Leucocytes in normal blood 480 1200 SOOO 381 Leucocytes in immune blood 460 300 5000 In the tube containing immune blood + leucocytes the number of organisms decreased during the first two hours but then began to in- crease rapidly, while in the other tube they increased rapidly from the beginning of incubation. The immune serum alone has no destructive action on these organisms but acts as a culture medium for them. The following experiment was now made to determine whether the opsonin of the immune serum can withstand a higher degree of heat than that of normal serum. Experiment. .2 c. c. of normal serum and .2 c. c. of immune serum in two small test tubes were heated to 60 C. for y 2 hour. To each 26 tube were now added .2 c. c. suspension of washed rabbit corpuscles containing leucocytes from a pleural exudate and .2 c. c. suspension of streptococci of medium virulence. Two additional tubes with unheatecl normal and immune serum were prepared in the same manner and all tubes incubated ]/ 2 hour. Smears were now made from all tubes and stained and the degree of phagocytosis in each tube determined by count- ing the cocci in each of 50 leucocytes, with the following result : Phagocytosis. Heated normal serum ~j- washed corpuscles + streptococci . ! 1.1 Heated immune serum + washed corpuscles + streptococci 4.5 Normal serum -f washed corpuscles + streptococci 4.3 Immune serum + washed corpuscles -f- streptococci 7.3 Washed corpuscles in NaCl solution + streptococci 1.1 The experiment shows that the opsonin of the immune serum is only slightly disturbed by heating at 60 C. for y 2 hour, whereas that of nor- mal serum is entirely destroyed at that temperature. CONCLUSIONS. The normal sera of man, rabbits and guinea-pigs have no strep- tococcidal power and do not acquire such a property in the course of a streptococcus infection. Suspensions of organ cells of guinea-pigs have no streptococcidal powers. Defibrinated human blood is distinctly streptococcidal and this prop- erty is roughly proportional to the number of leucocytes the blood con- tains per cubic millimeter. Normal leucocytes of rabbit, guinea-pig and man, suspended in normal serum or blood, freely ingest non-virulent streptococci and de- stroy them. The washed leucocytes in NaCl solution or in heated serum do not ingest these organisms, but the latter multiply in these suspen- sions. If, however, the streptococci are treated with normal serum, washed and then added to a suspension of washed leucocytes they are ingested and destroyed by the leucocytes. In the phagocytosis of strepto- cocci, therefore, it is essential that the organisms should first be sensi- tized, that is, acted upon by the opsonin of the serum. The leucocytes of guinea-pigs and of man take up living strepto- cocci in vivo, and in all probability destroy them. It seems evident, therefore, that the phagocytes, acting in conjunction with the opsonin of the serum, are the most important (if not the only) factors concerned 27 in combating streptococcus infections in man and in the lower animals. Hektoen and Ruediger* have shown that m-8 solutions of many salts and other substances inhibit phagocytosis, presumably because these sub- stances bind the opsonin. I have carried out a number of experiments with rabbits for the purpose of determining whether or not intravenous injections of weak solutions of antiseptics can prolong the animal's life when inoculated with a fatal dose of streptococcus, but have found that the animals thus treated invariably died earlier than the controls. We must conclude, therefore, that great harm can be done by the indiscrimi- nate use of drugs, or antiseptics, for the purpose of combating strepto- coccus infections. The remedy used may act on the opsonin, for in- stance, so as to hinder phagocytosis and thus do harm rather than good. Virulent streptococci are not freely, or scarcely at all, ingested by normal leucocytes suspended in normal serum or blood, and hence these organisms multiply in suspensions of leucocytes. Human leucocytes take up virulent streptococci somewhat more freely than do the leucocytes of rabbits and guinea-pigs. Rabbits can be successfully immunized with streptococci of mediunr virulence, and this immunity is clearly dependent upon phagocytosis. The immune rabbit serum has no streptococcidal power, but the leucocytes suspended in the immune blood or serum readily take up and destroy that strain of streptococcus which was used in the immunization. Other strains of virulent streptococci are not taken up so readily by leucocytes suspended in the immune serum. This is an important fact to be recog- nized in the serum therapy of streptococcus infections. The action of the immune serum is not that of a stimulus for the leu- cocytes but the effect is on the cocci. The immune serum has acquired the power to change the cocci so that the leucocytes will ingest them, this power being possessed only slightly by the normal serum. The opsonin of human serum is increased during the course of a streptococcus infection, and according to Hektoen also after a subcuta- neous injection of heated streptococci. The immune opsonin is more resistant to heat than normal opsonin. *Jonr. of Infect. Dis., 1905, II, 128. 183473 m? THE UNIVERSITY OF CALIFORNIA LIBRARY