ill THE EISENHOWFF 3 1151 02716 9089 AN EXPERII.IENTAL STUDY OF PHAC-OCYTOSIS IN RELATION TO TERMINAL INFECTIONS Dissertation Submitted to the Board of University Studies of the Johns Hopkins University in Conformity with the Requirements for the Degree of Doctor of Philosophy by Howard B. Cross Baltimore 1921 n^.i -ir TABLE OF CONTENTS Introduction 1 Historical Sketch 2 Plan of Investigation 5 Technique 6 Subject I.'atter 9 Pneuraococcus Infections 9 Dog #11 9 Dog #12 14 Guinea Pig #31 16 Staphylococcus Infections 19 Dog #14 19 Dog #15 22 Guinea Pig #16 23 Guinea Pig #17 25 Typhoid Infections 26 Dog #13 26 Dog #19 31 Streptococcus Infections 33 Guinea Pig. #33 33 Cat #33 36 Cat #35 36 Cat #37 36 Cat #38 39 Miscellaneous Infections 41 Rabbit #41 41 Guinea Pig #20 42 Dog #47 44 Infections In Man 45 Patient ^m 46 Patient #23 47 Patient #13 48 Patient #11 49 Patient #5 50 Functional Variations of Leucocytes 52 Inadequacy of the Vvright Teclinique 55 Discussion 60 Conclusions 63 Biography 64 Bibliography 65 AN EXPERIlffiNTAL STUDY OF PliAGOCYTOSIS IN RELATION TO TEF1!INAL INFECTIONS INTRODUCTION The circumstances which originally suggested this in- vestigation were the instances of energetic phagocytosis fre- quently observed at autopsy. The surprising number of bacteria occasionally found within the leucocytes in individuals dying of fatal infection or intoxication suggests the possibility that the diminution of phagocytic activity sometimes associated with destructive infections may not be as nearly universal as is cominonly supposed. It farther seems probable that in mori- bund animals, "in which the resistance fails" (1), the phago- cytic defense may remain unimpaired, and, occasionally at least, function at a level considerably above the normal. These observations also indicate that terminal infections can not be accounted for by assuming a collapse or a decrease in the ac- tivity of the phagocytic functions of the animal. Fig. 1 illustrates the extent of phagocytosis which may occur in animals dying of fatal infections. # 1' ^ J> Fig. 1. — Illustrating the extent of phagocytosis which may occur during the late stages of terminal infection. A. and B. Cells from the peritoneal cavity of a guinea pig dying of B. coli peritonitis. C. Phagocyte from lung of patient dying of influenza pneumonia. D. Leucocyte from peritoneal cavity of a guinea pig dying of staphjrlococcus peritonitis. E. Leuco- cyte from blood stream of a guinea pig dying of generalized staphylococcus infection. F. Phagocyte containing diphtheria bacilli from lung of child with terminal pneumonia following diphtheria. '^G. Cell containing mixed flora from lung of pa- tient dying of terminal pneumonia. H. Phagocyte from pleural exudate of a dog dying of generalized pneumococcus infection. HISTORICAL SKETCH The widespread occurrence of terminal infections was strikingly expressed by Osier when he wrote that, 'Persons rarely die of the diseases with which they suffer." (2) However, the nature of the rupture in the defensive mechanism of the individual which permits the rapid invasion of the body before death by even the feebly aggressive bacteria, has never been satisfactorily explained. Flexner (3) undertook a study of this condition as early as 1896. He, however, limited his investigations to an examination of the hvimoral defense with- out any attempt at ascertaining whether cellular modifications might not be a contributing cause in terminal infections. He thought that he discovered, at least in a few cases, a slight decrease in the bactericidal actions of human serum against staphylococci » The correctness of these findings, however, was soon placed in doubt by V/right, (4) who together with 'Windsor demonstrated in 1902 that hanan blood exhibits an almost total absence of any bactericidal influence against staphylococci. Bordet C5) was one of the first to suggest that the phagocytic activity of the body might be a relatively stable function and not easily influenced by conditions which pro- foundly effect other vital activities. He demonstrated that deep chloroform anesthesia which "completely deadens" (8) the central nervous system has no disturbing influence upon phago- cytosis. This investogator also observed that diptheria ' (2) toxin exerts little, if any, effect on phagocytic activity (6). Tunnicliff (7), in an investigation of the opsonic in- dex during the leucopenia resulting from measles, found that there was a slight decrease for streptococci, staphylococci, and tubercle bacilli. She suggested that the decrease in phagocytic activity against these organisms might "account for the secondary infections" commonly associated with measles. Bartlett and Ozaki (8) in 1917 injected a dog with a massive dose of B. coli and five hours later, when the animal was in a moribund condition, inoculated it witn a quantity of staphylococci. They observed no decrease in the opsonic index for staphylococcus. This experiment was not repeated and the authors themselves were not entirely convinced of the adequacy of their controls. There is, of course, and extensive literature (luetchni- koff (9); Fevre (11); Wright (10); Bull (12); Hektoen (13); Opie (14), etc.) indicating a sharp and very considerable de- cline, in some instances at least, in the phagocytic activity of txhe body against the specific infecting organism in fatal infections. There seems to have been no organized inquiry, however, concerning the opsonic index for those bacteria which take no part in the original infection but which later invade the weakened body giving rise to the destructive phenomenon known as terminal infection. Bacterial invasion of the wasted body immediately pre- ceeding death is sometimes so complete and sudden that it has (3) been assu.iied the process is unopposed. The collapse of the defensive mechanism against bacteria in terminal infections seems to be thorou["h and complete. Zinsser (15) expressed this idea in a discussion of fatal infections when he wrote: "The infectious process becomes rapidly generalized, the bacteria enter the blood stream and lymphatics, and the defensive powers are overwhelmed." The possibility of a distince part of this protective adaptation of the body remaining intact with unim- paired function. has not been believed and awaits confirmation. It is proposed, therefore, in this research to undertake an investigation of the phagocytic activity in animals after kataphylaxis* has occurred, and to determine what change, if any, occurs in the opsonic index for those organisms which most frequently overrun the bod3r late in fatal infections and intoxications . *The word kataphylaxis was introduced by Bullock and Cramer (Proc. of the Royal Society, Series B, vol. 90, p. 515) and was defined by them as a rupture in the local defensive mechanism against bacteria. As used in the present paper the word will be understood to indicate a break-down in the gen- eral defensive mechanism sufficiently complete to permit the infection to lead uninterruptedly to the death of the animal. (4) PLAN OF INVEST I Cr AT ION. The investigation of this problem has been done almost entirely in vitro because there is no thoroughly dependable method of controlling results when the experiments are under- taken in vivo. Wright's (16) work has established that even slight changes in the phagocytic activity of the animal can be demonstrated by in vitro methods, so that if there is a decline in opsonic power sufficient to account, even in part, for the bacterial invasion occurring in terminal infections, there could be no difficulty in demonstrating that decrease in effectiveness by the methods employed in this investigation. The bacteria used were those most commonly associated with terminal infections — a pyogenic coccus, Staphylococcus , aureus . recently isolated from a case of human f urunculosis, and a gram negative bacillus, B. coli communis. In addition, two other organisms were commonly used, 3, bronchi gepticus was selected because it is readily taken up in large numbers, making possible dependable counts in weakly pliagocytic cells, while 3. typhosus resisting engulfment to considerable extent, permits reliable enumeration when phagocytosis with other bac- teria is so vigorous that even a depressed phagocytic capacity is quite sufficient to fill the cells beyond counting. 7;hen- ever possible the specific infecting organism was included in each series of experiments. (5) TECHNIQUE The opsonic teciinique used in this research -was essen- tially the same as that originally employed by Wrirht and Douglas (17). Certain modifications and refinements, however, were introduced which seemed to insure ,c;reater uniformity in the results, and to make this generally confusing technique more dependable and satisfactory. The blood providing the cells was received into a centri- fuge tube containing several volumes of citrated calt solution. Before sedimentation the cells were uniformly suspended by re- peated suction and ejection with a pipette. This process was repeated before each centrifugation and is a most effective means of preventing the aggregation of the platelets .around the leucocytes. Tne centrifugation was accomplished at a speed which permit Led the sediu.entation of the white cells in four to is five minutes without throwing down the platelets. This^most important for it eliminates the platelets from tne leucocytic layer and prevents packing of the white cells. The washings were repeated three times and the leucocytes suspended in a volume of physiological salt solution equal to one third t.:e the original volume of blood. After standing for one hour, cells A were uniformly suspended by a gentle continued agitation of the tube and all samples of any one experimental series were taken immediately. it was early observed that the ingesting capacity of phagocytes was not always as reliable and vigorous if they were used immediately after washing as when they were (6) allowed to suand for an hour. This is probably occasioned by a disturbance in the osmotic pressure resulting from repeated washings and centrifugation. Confusing and contradictory results invariably accom- panied a careless or hurried preparation of the bacterial sus- pension. The or.-anisms used in this work were grown, v/henever possible, on moist plain agar for fifteen hours. The tube was then washed out with saline to remove lint and debris. Five cubic centimeters of salt solution were next added and the tube gently agitated until the resulting turbidity was equal to or slightly in excess of that desired. This was then transferred to a second clean tube and vigorously shaken to break up pos- sible clumps. Finally the tube was centrifuged at a speed sufficient to remove from suspension all but the single bac- teria. Two and five-tenths cubic centimeters were tnen drawn from the upper portion and reserved for the bacterial suspen- sion. All the samples for any experimental series were regu- larly taken from Lhe same level in order to insure a more near- ly uniform number of organisms, dacteria which do not grow upon plain agar or which could not be uniform.ly suspended from solid medium were grown in meat infusion broth. For growing massive cultures of pneumococci, whole blood was added to the broth. The bacteria v;ere sedimented, washed and suspended in salt solution. A turbidity was selected which presented a delicate opalescence in indirect light. Sera were taken in the usual manner except that those (7) containing an excess amount of fat were, -wiienever possible, avoided.' Those containing agglutinins for the experimental cells, although thorou^^hly annoying, can be used without in- validating the results. '-Vhile a few hours difference in the age of the sera results in no demonstrable difference in their opsonic reactions, all sera for an experimental series were collected as nearly as possible at the same time. The phagocytic mixtures were prepared according to the Wright technique, and incubated at 37° for twentj'- minutes. The smears and stains were made in the manner described by Gross (18). All smears showing gross differences in the num- ber or distribution of the leucocytes were discarded and the preparations repeated. The organisms in fifty polymorpho- nuclear leucocytes were counted and these cells were always enumerated from corresponding areas on the control and experi- mental slides. Cells containing an excessively large number of bacteria show a tendency to collect in portions of the smear which can be predicted, and the most confusing and contradic- tory deviations present themselves if the selection of corres- ponding areas for enumeration is not rigorously observed. All preparations exhibiting marked variations from the normal or expected were repeated throughout. Whenever the phagocytes revealed unusual inequalities in the number of ingested organ- isms, parallel preparations were made and the average enumera- tion ta^cen as the true count. Polymorphonuclear leucocytes alone were considered, and no attempt was made at enumeration in cells containing m.ore than twenty-five organisms. In all (8) 3HNS HOPKINS HOSPITAL BULLETIN, FEBRUARY, 1921 PLATE VI Fig. 2 4 < ^F^SwK'iT^g ;?-Q Si t\veilt\ Ihi' lITi'-; ;itli'i- llir iii;;i'-l 11,11 i.l I'. |iMil,n-, Mip I,:hIi|1:i ;iri' ~il vacuoles. One bacillus, partially iligcstcil, Ikis Inst its staining cliaractcrisi is associated with erythrocytes. (C) Smear from a luno; abscess containing and spirochetes. (D)" Polymorphonuclear leucocyte containing a colon bacill digestive vacuole. counts the percentage, of ingesting leucocytes as well as the total number of intx'acellular bacteria were determined. Fig. 2, taken from the Johns Hopkins Hospital Bulletin (18) illustrates the appearance of the cells from which the enumera- tions recorded in this paper were made. SUBJECT MATTER The data presented in this paper are selected from, a study of 85 cases of infection and intoxication which resulted in death. An effort was made to secure the widest possible variety, including both spontaneous and induced infections in animals, supplemented by a series of human cases. The period elapsing between the primary inoculation and death ranged from a few hours to several weeks, and even much longer in some of the human cases. The arrangement of subject matter and condi- tion of each experiment are clearly set forth in the various protocols . EXPERIMENTAL DATA PART 1 A. Pneumococcus Infections Experiment 1. — The dog - No. 11 - used in this experi- ment was an adult female weighing 15^ pounds. It was inoculated intravenously on November 27, 1920, with a sublethal dose of virulent Pneumococcus, Type I. The sublethal doses were con- tinued on dates indicated in the protocol until the blood ex- hibited a high bacteriotropic action. The intravenous inocu- (9) lations were then gradually increased until a sufficient dose was administered to bring about the death of the animal. This result was hastened -toward the end of the experiment by injec- tion of pneuraococci into the left pleural cavity. A sample of blood was taken before each injection and allowed to clot. Two hours later the serum was withdrawn and the opsonic index determined in the manner described above. The normal dogs pro- viding the leucocytes and control serum were always bled at tae same time as the experimental animal. The results of all op- sonic determinations are included in the followinp; tables: Date Remarks Bacteria Ani- mal Ko . of Cells Ko. Ceils \vith Bacteria K 0 , Cells Total Ko.of Fha;-o- cytic 11/27 20 Dog #11 Before injection wt. 15 i- lbs Inj. 25cc Pn.I S .aureus ,^11 Gnn 50 50 39 42 11 8 145 121 2.9 2.4 Pn. I #11 Con 50 50 1 4 49 46 2 7 0.0 0.1 B. coli #11 Con 50 50 41 40 9 10 86 93 1.7 1.8 B. typho- sus • #11 Con 50 50 43 45 7 5 95 104 1.9 2.0 11/29 20 Dog #11 wt. 14*lbs Inj. 50cc Fn.I S. aureus #11 P.nn 50 42 43 8 7 175 171 3.5 3.4 Pn. I ,fll Con 50 50 0 0 50 50 0 0 0.0 0.0 B. coli ;fll Con 50 50 43 46 7 96 104 1.7 2.0 B. typho- sus ,t11 Con 50 50 35 38 15 12 66 70 1.3 1.4 12/1 20 Dog ,7-11 wt. 13-^ lbs Inj. lOOcc Pn.I S. aureus #11 Con 50 50 41 44 9 6 220 212 4.4 4.2 Pn. I #11 Con 50 50 26 5 2- 45 101 6 2.0 0.1 B. coli .yii Con 5C 50 19 25 31 2u 50 55 1.0 1.1 B. typho- sus #11 Ccn 50 50 13 15 37 35 21 20 0.4 0.4 (10) [■"' Dat€ Remarks Bacteria Ani- mal No . of Cells Count. l!o .Cells with Bacteria Ko. Cells Empty Total No. of Bact. Phago- cytic Index 12/3 20 Dog i/^11 v.'t. 14 lb *Inj. 150CC Pn. I S. aureus Con 50 50 24 27 26 23 57 60 1.1 1.2 Pn.I #11 Con 50 50 31 2 19 48 100 4 2.0 0.1 B. coli #11 Con 50 50 23 24 27 26 28 55 0.5 0.7 B. typho- sus Trll Con 50 50 39 42 11 S 82 1.6 1 .6 12/lC Dog ;,-ll wt. 13 lb Inj. 200CC Pn.I S. aureus /rll Con 50 50 4; 9 12 166 145 2. '9 Pn.I #11 Con 50 50 38 c 12 42 162 22 3.2 0.4 B. typho- sus #11 Con 50 50 21 20 29 30 30 45 0.6 0.9 12/17 Dog ,fll IVt. 13 lb InJ. 250CC Pn.I S .aureus ,rll Con 50 50 40 43 10 7 269 231 3.3 4.6 Pn. I ,rll Con 50 50 25 10 25 40 75 21 1.5 0.4 B. coli .rll Con 50 50 32 33 18 17 77 80 1.5 1.6 B. typho- sus #11 Con 50 50 32 38 18 12 70 86 1.4 1.7 12/22 Dog #11 wt. 13 lb Inj. 250CC Pn.I S. aureus #11 Con 50 50 33 25 17 25 121 104 2.4 2.0 Pn. I itll Con 50 50 25 0 25 50 161 0 3.2 e.o B. coli .fll Con 50 43 38 7 12 113 105 ' 2.2 2.1 B . typho- sus ,rll Con 50 30 31 20 19 50 68 1.0 1.3 12/26 Dog ,-11 wt.l3 lb Inj. 250CC Pn.I *P fusio ood. re se :ated in 2 neumoc n brot The b diment in th 5 cc. occi wer^ h contair acteria f ed from t e table, of broth 5 growl 1 in meat 12/30 Dog ;^11 wt.l3 lb Inj. 2o0cc Pn.I in bl = we di ed ling 1% whole or each injection 1/10 21 Dog #11 wt.l3 lb inj. 25Ccc Pn. I he quantity in- and then suspend- before inoculation (11) Date Re.Tiarks Bacteria Ani- laal Uo . of Cells Count. i:o .Cells with Bacteria Ko. Cells Empty Total Kg. of Bact. Phago- cytic Index 1/10 21 Dog #11 wt. 13 lb In J. 250CC Pn. I S. aureus Con 50 50 37 33 13 17 199 leo 3.9 3.6 Pn. I ,rll Con 50 50 22 8 28 42 62 16 1.3 0.3 B. coli Con 50 50 41 45 9 5 103 140 2.0 2.8 . B. typho- sus Con 50 50 26 23 24 27 27 38 0.5 0.7 1/20 21 Dog ,7^11 wt. 13 lb *Inj. 245CC Pn. I 3. aureus Con 50 50 36 35 14 15 145 171 2.9 3.4 Pn. I #11 Con 50 50 0 0 50 50 0 0 0.0 0.0 B. coli #11 Con 50 50 42 41 8 9 93 95 1.9 1.9 B. bron- chicep. ffll Con 50 50 27 42 23 8 65 73 1.3 1.4 1/22 *Inj. 50cc Pn. I 1/24 Dog #11 v;t. 11 lb No inj. 3. aureus vr^ll Con 50 50 34 31 15 19 128 115 2.5 2.3 Pn. I #11 Con 50 50 0 0 50 50 0 0 0.0 0.0 B. coli .fll Con 50 50 41 41 9 9 78 71 1.5 1.4 B. bron- chicep. ,/ll Con 50 50 37 38 13 12 136 137 2.7 2.7 1/25 Dog #11 wt. 11 lb This read- ing made 10 hrs be- fore death Ko.inj. S. aureus ,vll Con 50 50 35 32 15 IS 235 199 4.7 3.9 Pn. I -/ll Con 50 50 4 0 46 50 8 0 0.1 0.0 B. coli #11 Con 50 50 15 14 56 45 50 0.9 1.0 B. bron- chicep. ;/ll Con 50 50 39 37 11 13 212 196 4.2 3.9 B. typho- sus #11 Con 50 50 7 8 43 42 15 19 0.3 0.3 The bacteria from 25 cc. of this inoculation were in- jected into the left pleural cavity. The remaining quantity was given intracardially . "- (12) J»ig. 3. Dog No . 1 1 --Pneuinv^c .ccua T:i:'. ? tion Opsonic ^ndex Duration 59 ^'ays Death /? ~"7 Ti 7j To Is" H aP v? «? ?5 ^^ 7f Day Opsonic Ividex C'arve for Staph, aure'is . i I o J' -> .r zo ts Jo as- ^ Opsonic Iri(:ex Ourvj for '^ . c ili Mf ja rs- jr^ '■z o » /e /s JO 3S Jo J-^ ^ ?ps-:iic Inde;-. : .rv : -2 yj Date Remarks Bacteria Ani- .-aal I-o.of Cells County Ko. Cells with Bacteria ICo. Cells Emptv Total No. of Bact. Phago- cytic Index 1/25 21 Dog #11 wt. 11 lb This read- ing made at the time of death. 3 .aureus Con 50 50 34 33 15 17 251 199 5.0 3.9 Pn. I Con 50 50 7 0 4S 15 0 0.3 0.0 B. coli /fll Con 50 50 14 14 36 36 47 50 0.9 1.0 B. bron- chicep. #11 Con 50 50 33 37 17 13 226 195 4.5 3.9 B .typho- sus #11 Con 50 50 6 8 44 42 16 19 0.3 0.3 Anatomical Dia^'^nosis: Slight emaciation, bronchopneumonia, purulent exudate in both pleural cavities, generalized edema. Pneumococcus , '-^ype I, cultivated in pure culture from pleural exudate and from heart blood. Kataphylaxis is this animal probably occurred on Jan- uary 20, five days before its death, and was first indicated by the appearance of pneumococci in the blood sufficient quan- tity to be recovered in the culture. By referring to the curves in Fig. 3 it will be observed that there was no post- kataphy lactic decline in the opsonic index for any except the specific infecting organism. Samples of blood taken a few moments before death, when the animal was in a state of com- plete collapse, revealed a phagocytic activity in all respects equal to the normal and for staphj'-lococci it was even somev.hat increased. The opsonic index for the infecting or^^anism behaved throughout in accordance with the findings published by Keu- feld and TBpfeT (19) and Rosenow (20). Virulent pneumococci (13) are not phagocyted at all by the normal animal, but after one or two inoculations of this or^-anism the opsonins for pneumc- cocci increase and can be still further increased by subsec^uent injections, provided a lethal dose is not administered. Once this quantity is given, however, there is a rapid decline in phagocytic activity, the zero mark ordinarily being reached some time before the death of the animal. The variation in the opsonic curve for*S. aureus re- quires some explanation. Up to December 3 the curve foi' this organism had remained normal, but at this time a culture of Pneumococcus I, contaminated with*S. aureus was injected and five days later the dog's blood showed an increase in opsonins for staphylococci. This organism was almost immediately over- come, for it W3 s never obtained in subsequent blood cultures. The increased opsonic activity for staphylococci continued a few days, but gradually decreased until at the end of a month the index w&s again normal. However, on January 20 pneumo- cocci appeared in the blood and the reaction against this homologus antigen resulted in a non-specific increase in the residue of antibodies remaining from the previous staphylo- coccus inoculation. This w& s immediately reflected by a rise in the opsonic curve for staphylococcus. These observations are in agreement with the findings of Beiling (21). Experiment 2. — This experiment is presented as a parallel to the preceeding experiment and was performed through- out in a similar manner, except that it was found possible to (14) ♦Staph, aureus. produce death by increasing intravenous injections without the supplementary pleural inoculations. Only B. coli and 3. aureus were used in addition to the infecting organism, Eneu;nococcus , Type I. Date Remarks Bacteria Ani- mal No. of Cells Count. No. Cells with Bacteria No. Cells Ernotv Total No . of Bact, Phago- cytic Inde?: 8/1/20 Dog #12 *Inj. 5co Pn.I S. aureus /rl2 Con 50 50 49 48 1 2 190 167 3.8 3.3 Pn. I B. coli #12 Con 50 50 0 0 50 50 0 0 0.0 0.0 ,rl2 Con 50 50 46 45 4 5 90 83 1.8 1.6 8/3 Dog ;r'12 Inj. 20cc FN. I S. aureus #12 Con 50 50 50 49 0 1 162 150 3.2 ■. 3.0 Pn. I .12 Con 50 50 43 6 7 44 132 12 2.6 0.2 B. coli #12 Con 50 50 47 42 o 8 100 105 2.0 2.1 S/5 Dog ,v'l2 No inj. S. aureus #12 Con 50 50 48 48 2 2 184 167 3.6 3.3 Pd. I /rl2 Con 50 50 36 2 14 48 190 4 3.8 0.0 3. coli vfl2 Con 50 50 43 44 1 6 76 86 1.5 1.7 8/9 Dog #12 Inj. 200CC Pn. I S. aureus #12 Con 50 50 45 49 5 1 384 348 7.6 6.9 Pn. I ;fl2 Con 50 50 48 6 44 265 11 5.3 0.2 B. coli #12 Con 50 50 _ _ ~ S/12 Dog #12 Inj. 250CC ?n. I S. aureus #12 Con 50 50 49 47 1 3 290 280 5.8 5.6 Pn. I ,rl£ Con 50 50 44 0 6 50 415 0 8.3 0.0 B. coli /rl2 Con 50 50 48 47 2 3 195 214 3.9 4.2 8/16 Dog ,fl2 This read- ing was made a few minutes be- fore death S. aureus #12 Con 50 50 45 45 5 5 450 308 9.1 6.1 Pn. I #12 Con 50 50 46 2 4 48 100 2 2.0 0.0 B. coli ,rl2 Con 50 50 46 47 2 3 203 191 4.0 3.8 B.proteus #12 Con 50 50 50 47 0 3 553 445 11.0 8.9 (15) i?Tr n — r— 1 — I — I — \ — r 'action. _ — I 1 — ^ — I — _^_ ^ Dui-'a:! ra 16 day.. . 5: ' o .-:: 11 o . 1 2 - - Pn e urn o c o .: ; 'j u f O.os on 1 c T .-. , 1 .- ^ • Ji^th iyi> o t u i t /i /i- '* Cpsoni: 7 .dex ^.nrve for .;-taph. a-i.reup. t 0 i W I 1 7i 7i 77 7Z 1^ 0,^3 >nic T.-.C1., ^tiHtea;;:i:;tei.:-:[:: ' ■"■■ B. coli . Anatomical Diagnosis: Slight emaciation; no gross lesions; no exudates. Pneumococcus , Type I, was recovered in pure culture from the heart's blood. The results of this experiment coincide with tiie find- ings in the previous experiment so far as the heterologous organisms were concerned. There was no decrease after kata- phylaxis in the phagocytic activity against any bacteria except the one responsible for the primary infection. The opsonic curve for pneumococcus, however, is not what we vi/ould expect from the work of Rosenow (22). While there was a decrease in the opsonins for this bacterium after kataphylaxis this de- crease never carried the curve down to the normal level. Here, then, was an animal dying of an infection with its phagocytic defense against the specific organism not only intact but functioning with an efficiency distinctly above tne normal. Whatever the explanation for the defeat of this animal in its struggle against the infection, it does not seem possible to account for it by a rupture in the phagocytic defense. Experiment 3. — The experimental animal used in this test was an adult guinea pig - No. 31 - weighing 375 grams. It was injected intravenously with gradually increasing amounts of Pneumococcus, Type I^ and died 13 days later in a state of extreme cachexia. Full details of the experiment, together with the re- sults of the opsonic determinations, are fully set forth in the accompanying tables. (15) Date Remarks Bacteria Ani- mal No. of Cells Count. No. Cells with Bacteria No. Cells Empty Total No. of Bact. Phago- cytic Index 11/12 Pig#31 wt .before injection 375 grams Inj. 1.5CC Pn. I S. aureus /f31 Con 50 50 35 41 15 9 165 215 3/3 4.3 B. coli /f31 50 50 47 45 3 5 180 200 3.6 4.0 Pn. I #31 Con 50 50 0 0 50 50 0 0 0,0 0.0 11/21 Pig .^31 wt. 355 0-. Inj. 2.5CC Pn. I S. aureus #31 Con 50 50 40 36 10 14 75 60 1.5 1.2 Pn. I #31 Con 50 50 3 47 48 3 4 0.0 0.0 B. coli #31 Con 50 50 41 38 9 12 125 95 2.5 1.9 11/24 9:00 A. M. Pig ,f31 S. aureus #31 Con 50 50 42 46 8 4 250 290 5.0 5.8 Pn. I #31 Con 50 50 47 46 3 4 4 4 0.0 0.0 B. coli ;r31 Con 50 50 36 38 14 12 70^ 80 1.4 1.6 11/24 6:00 P.M. Pig jr^l wt. 302 g. No inj S. aureus #31 Con 50 50 36 47 14 3 245 290 4.9 5.8 Pn. I #31 Con 50 50 4 3 46 47 4 4 0.0 0.0 B. coll #31 Con 50 50 41 38 9 12 67 80 1.3 1.6 11/24 10:50 P. iv:. Pig #31 wt. 302 g. No'inj. S. aureus -/31 Con 50 50 45 46 5 4 303 290 6.0 5.8 Pn. I #31 Con 50 50 4 2 46 48 6 4 0.1 0.0 B. coli #31 Con 50 50 35 38 15 12 90 £3 l.S 1.5 11/25 Pig #31 Dead S. aureus #31 Con 50 50 50 46 0 4 299 290 5.9 5.S Pn. I #31 Con 50 50 2 3 48 47 3 6 0.0 0.1 B. coli #31 Con 50 50 41 36 9 14 80 70 1.6 1.4 Anatomical Diagnosis; Extreme emaciation, no macroscopic lesions. Pneumococcus , Type I^ recovered in pure culture from heart's blood. (i7) ^i S-r -I- 30 ?. US Infecitl on Opsonic Inde:-: O-irve f r "^u,, . r '0 /* // ••'•.v.yfj j'.^p Pneurnoc 5c::u3, Type I, i : I : n r-'-T:.!:::lj:J::.Tln i T:.:l.j;:;ateteiiiliaitei Throu 'hout this experiment there was no discoverable increase in the opsonic activity of the blood against pneumo- cocci. This is probably due to the fact that the initial in- jection was so large as to closely approximate the lethal dose. The reaction to the initial inoculation was so violent that the animal lost 15,^ of its body weight in eight days. The sub- sequent injections resulted in equally violent disturbances so that the animal was practically in a kataphylactic state during the whole period of the experiment. Yet in this weakened con- dition, a state commonly characterized as "defenseless", the opsonic blood revealed no diminution whatever in its^ activity against the bacteria commonly associated with terminal infections. In these experiments we have reviewed three types of fatal pneurnococcus infections — one in vjhich the phagocytic index for pnexmococcus was very high during the early stages of the disease, but fell rapidly after the infection began to gain on the bod^/-, and reached zero sometime before tne death of the animal; in the second case there was a corresponding increase in opsonins during the early stages of the infection but the decrease following the break was gradual and the index never at any time fell to the level of the normal; the third type was one in which the character of the infection from the start was so severe that there was no increase whatever in the phagocytic activity above that observed in the normal. These three types, however, are alike in that none shows a decrease in the opsonic index for either Staph, aureus or B. coli. during the period immediately preceeding death. (18) B . Staphylococcus Infections Experiment 4. — An adult female dog - No. 14 - weighing 15 pounds was selected as the experimental animal in this test. She was ^iven an initial intravenous injection of Ice. of a broth culture of Staph, aureus on November 27, 1920. Gradually increasing amounts of this organism were given until the dog, in an emaciated and helpless condition, died 23 days later. The opsonic determinations, together with other data concerning this experiment are recorded in the appended table. Date Remarks Bacteria Ani- mal ro.of Cells Count. No. Cells with Bacteria Ko . Cells Emptv Total ro.of Bact . Phago- cytic Index 11/27 20 Dog #14 wt. before injection 15 lbs Inj. Ice 3. aureus S.auraus Con 50 50 41 42 9 8 121 120 2.4 2.4 Pn. I #}4 Con 50 50 5 4 45 46 10 8 0.2 0.1 B. typho- sus .A 4 Con 50 50 42 45 8 5 106 104 2.1 2.0 11/29 Dog #14 wt. 13g-lbs Inj. 2 cc. 3. aureus S .aureus #14 Con 50 50 44 44 6 6 297 170 5.9 3.4 ?n. I #14 Con 50 50 0 0 50 50 0 0 0.0 0.0 B. coli #14 Con 50 50 38 46 12 4 96 104 1.$ 2.0 B. typho- sus #14 Con 50 50 35 38 15 12 100 70 2.0 1.4 12/1 Dog #14 wt. 13-;jlbs IIo inj. S. aureus #14 Con 50 50 50 44 0 6 309 212 6.2 4/2 Pn. I #14 Con 50 50 4 5 46 45 10 6 0.2 0.1 B. coli Con 50 50 34 35 16 15 56 55 1.1 1.1 B. typho- sus #14 Con 50 50 14 15 36 35 IS 20 0.3 0.4 (19) Date Remarks Bacteria Ani- mal No. of Cells Count. Nc. Cells with Bacteria No . Cells Total No. of 3a ct. rhago- cytic Index 12/3 20 Dog #14 wt. 13 lbs Inj. 5 cc S. aureus S. aureus -M4 Con 50 50 28 27 23 66 61 1,3 1.2 Pn. I #14 Con 50 50 3 47 48 7 4 0.1 0.0 B. coli ,rl4 Con 50 50 31 34 19 16 50 35 1.0 0.7 B .typho- sus #14 Con 50 50 36 42 14 8 76 85 1.5 1.6 12/7 Dog -14 wt. 12ilbs "o inj. S. aureus 7rl4 Con 50 50 35 34 15 16 133 ' 150 2.6 3.0 Pn. I #14 50 B. coli #14 Con 50 50 49 39 1 11 156 131 3.1 2.6 B. typho- sus #14 : 12/10 Dog #14 wt. 11 lbs No inj. 3. aureus #14 Con 50 50 41 39 9 11 76 115 1.5 2/3 Pn. I ^4 Con 50 50 - B. coli #14 Con 50 50 31 33 19 17 73 75 1.5 1 -5 B. typho- sus #14 Con 50 50 19 23 31 27 42 33 0.8 0.6 12/15 Dog #14 wt. 11 lbs No inj. S. aureus #14 Con 50 50 29 25 21 25 105 171 2.1 3/4 Pn. I #14 Con 50 50 _ B. coli #14 Con 50 50 38 42 12 8 57 73 1.7 1.5 B. typho- sus #14 Con 50 50 27 28 24 22 55 46 1.1 0.9 12/17 Dog #14 No Inj. 3 .aureus .fl4 Con 50 50 44 43 6 7 554 231 7.0 4.6 Pn. I #14 Con 50 50 9 10 41 40 22 21 0.4 0.4 B. coli #14 Con 50 50 35 33 15 17 80 1.4 1.6 B. typho- sus #14 Con 50 50 40 38 10 12 92 86 1.8 1.7 (20) 1 1 : I t -I Duraui ci Zeath 1 d J V ^ jr 7i 7> /? ^^ ^7 Iff Ji. Opsonic Inde:-: Curve for Sta^h. 'tLir-ua. ^'' i J i » 75 7* 7* 7Z 77 7^ 5a 0;X£.onic Index Jurve f^," B. -ill. ff i V /i /v /^ // ia Ji EHEEB::.B^l^fd,-FJff^--T-^-''rr-rvjq;::F:i:^^ Date Remarks Bacteria Ani- mal No. of Cells Count. No. Cells with Bacteria No. Cells Empty Total No. of Bact. Phago- cytic Index 12/19 20 9:00 ii . -. . Dog #14 wt. 11 lbs Reading made 14 hrs before death S .aureus #14 Con- 50 50 40 38 10 12 121 2C0 2.4 4.4 Pn. I #14 Con 50 50 9 5 41 45 22 }2 C.4 0.2 B. coli #14 Con 50 50 38 43 12 17 114 91 2.2 1.8 B. typho- sus #14 Con 50 50 34 30 16 20 70 49 1.4 1.0 12/19 11:00 P.!-.:. Do- #14 Reading made at time of death. S. aureus #14 Con 50 50 36 3c 14 12 121 219 2.4 4.4 Pn. I #14 Con 50 50 4 5 46 45 18 12 0.3 0.2 B. Coli ;^14 Con 50 50 41 42 9 8 111 91 2.2 1.8 B. typho- sus #14 Con 50 50 29 30 21 20 72 49 1.4 1.0 Anatomical Diagnosis: I'arked emaciation, Staphylococcus bronchopneumonia, multiple abscess in bodj'' wall, heart, kidneys and spleen* acute nephritis, staphylococcus cystitis. Staph. aureus in pure culture obtained from heart blood, kidneys, spleen and urine. The variations in the opsonic index for Staph, aureus in this experiment are in agreement with the results published by Wright (23) and Neufeld (24). Under the stimulation of sub- lethal inoculation there is an increase in phagocytic activity but as soon as the lethal dose is given there is a sharp de- cline wnich often forces the index below the normal. One es- pecially interesting feature of the opsonic index for staphylS- coccus in this experiment is that on December 7th the index was somewhat lower than it was at the time the aniraal died. Although phagocytosis is regarded as the chief defensive mechan- (21) ism imposed against staphylococci, this animal Vi/ith an uncom- plicated infection, survived the period of its lowest phago- cytic activitjr and died v.'hen its opsonic index was only slight- ly lower than the normal. Experiment 5. — The dog - No. 15 - used in this experi- ment was anesthetized and an incision made in the abdominal wall. The right kidney was exposed and one-third of an agar slant of Staphylococcus aureus injected into the renal pelvis. The dog died in 52 hours with a generalized staphjrlococcus peritonitis and septicemia in addition to the violent reaction in the right kidney. A sample of blood was taken and the following opsonic determinations secured. Date Remarks Bacteria ani- mal IJo.of Cells Count. No. Cells wi th Bacteria No. Cells Empty Total No . of Bact. Phago- cytic Index 3/11 21 Dog ,fl5 2 hours after death S. aureus #15 Con 50 50 48 50 2 0 319 303 6.4 6.1 B. coli #15 Con 50 50 34 37 16 13 61 57 1.2 1.1 B. bron- chisep. .fl5 Con 50 50 37 40 13 7 77 69 1.5 1.4 Anatomical Diap-nosis: *7ell nourished, generalized staphylococcus peritonitis, septicemia. Staphylococcus aureus in pure culture cultivated from the blood, right kidney and peritoneum. Although staphylococci had already appeared in the blood there was no decrease in the opsonic index for this organism. The shock associated with deep and prolonged anesthesia and (22) openins the peritoneal cavity did not occasion any modifica- tion in the normal phagocytic activity a,2;ainst any of the three bacteria tested in this experiment. Experiment 6. — For this experiment a large adult guinea pig - No. 16 - weighing 540 grams was inoculated intracardially with 0.5 cc. of a 24 hour broth culture of Staph, aureus on Kovember 30, 1920. The first two days the animal exhibited no symptoms of infection but on the third day staphylococci ap- peared in the blood and during the next 72 hours the guinea pig suffered a phenomenal loss in weight. It died on December 4th, in a state of the most extreme emaciation and weakness. Full details of the experiment, together with the routine opsonic readings appear in the subjoined table. Date Remarks Bacteria Ani- mal Ko.of Cells Count. Ko. Cells with Bacteria Ko. Cells Emptv Total Ko.of Bact. Pha.-^o- cytic Index 11/30 20 Pig #16 wt. before inj. 540 g. Inj. 0.5CC S. aureus S. aureus #16 Con 50 50 43 42 7 8 270 276 5.4 5.4 Strep, hemo. #16 Con 50 50 43 46 7 4 144 140 2.8 2.8 B. coli Jrl6 Con 50 50 43 44 7 6 75 73 1.5 1.5 12/4 Pig #16 wt. 475 g. Ko inj. S. aureus #16 Con 50 50 38 36 12 14 95 2.4 1.9 Strep. hemo. /rl6 Con 50 50 40 40 10 10 106 125 2.1 2.5 B. coli ,/16 Con 50 50 40 39 IC 11 45 40 0.9 0.8 12/5 Pig ;fl6 wt. 375 g. 12 hours before death. No inj. S .aureus ;fl6 Con 50 50 41 42 o 8 195 170 3.9 3.4 Strep, hemo . #16 Con 50 50 20 25 30 25 103 100 2.0 2.0 E. coli ,fl6 Con 50 50 46 45 4 5 225 220 4.5 4.4 (23) ^1 3 N ^ . 1 j--.3tapiiiiQii.bj UB I Ti-ifieotl on. Duration 6 days Opsor.ic Index J ijeath. ^psonl;": Index ']urv9 for ~t',;^;i. airous. <3 / 5 3 ■«' rf Opsonic Ind3X Curve Tor 3t-ep, enolyti Ipsoaio Index ■JTr^ve :'or '3, coii ms 1 "In I 1 ! Date Remarks Bacteria Ani- mal No. of Cells Count. ro.. Cells with Bacteria No. Cells Empty Total No. of Bact . Phago- cytic Index 12/5 20 Pig #16 Death S. aureus ,rl6 Con 50 50 45 42 5 8 160 170 3.2 3.4 Strep. henio . #16 Con 50 50 17 25 33 25 104 100 2.0 2.0 B. coli ;/l6 Con 50 50 50 49 0 1 273 220 5.4 4.4 Anatomical Diagnosis: r.iarked emaciation, purulent exudate in pleural cavities; kidneys studded with small abscesses. Pelvis of each kidney and bladder contained pus. Staph, aureus in pure culture cultivated from pleural cavities, kidneys, bladder and heart's blood. This case represents an infection of the most violent and destructive character. The animal lost 28/j of its body weight in five days. The pleural cavities and urinary system were filled with pus containinf^ abundant organisms, and the staphylococci were so numerous in the blood stream at death that 2000 colonies were cultivated from a single loop-full of blood. Yet in spite of this tremendously overwhelming infec- tion the phagocytic system suffered practically no impairment. Only the opsonic index for the specific organism was below the normal, and this by a difference so small that it mijht easily be regarded as an experimental error. That the test affords an accurate estimate of conditions in the body is attested by the fact tnat many of the polymorphonuclear leucocytes of the blood were activeljr phagocytic, some containing as many as 35 cocci. Likewise, almost every polymorphonuclear leucocyte examined from the pleural exudate contained organisms. This (24) case is perhaps somewhat exceptional, especially the phagocy- tosis in the exudates, for Opie (25) has shown that exudates are often deficient in opsonins. It does, however, indicate that phagocytic collapse does not always accompany fatal infection. Experiment 7. — The guinea pig -No. 17 - selected for this experiment weighed 360 grams. It was injected intraven- ously with 2 CO. of a 24 hour broth culture of Staph, aureus. Death occurred at the end of eighteen hours. The experimental details are sumrnerized in the follow- ing table. Date Reraarks Bacteria Ani- mal Ko.of Cells Count. Ko. Cells ;^ath Bacteria Ko. Cells Empty Total No. of Bact. Phago- cytic Index 11/12 20 Pig #17 wt. 360 g. Inj. 2 cc. S. aureus S. aureus #17 Con 50 50 41 35 9 15 185 165 3.7 3.3 B. coli #17 Con 50 50 45 47 5 3 200 ISO 4.0 3.6 11/13 Pig ,;-17 Dead 3. aureus B. coli -717 Con 50 50 36 40 14 10 1 101 133 2.0 2.6 -rl7 Con 50 50 43 42 7 8 125 115 2.5 2.3 Anatomical Diagnosis: Ko macroscopic lesions. Heart blood yielded a pure culture of Staph, aureus. A review of the preceeding staphylococcus infections reveals that^ overwhelming of an animal by Staph, aureus may b( accompanied by a sharp and subnormal decline in the opsonic index for that organism, but that this is not invariabljr the condition. It would seem that especially in rapidly fatal (25) infections the phagocytic activity may not be disturbed. In these cases, no matter how violent and destructive the cause of the infection, no matter how exhausted and wasted the animal, there V;as not one instance of a decrease in phago- cytic activity, even in the hours immediately preceeding death, against any organism not concerned in the primary infection. C. Typhoid Infections Experiment 8. — In this experiment an adult male dog - No. 13 - weighing 13-^ pounds was injected intravenously on November 27, 1920, vsith 2 cc. of an IS hour broth culture of B. typhosus. Sublethal inoculations v;ere continued until the blood revealed a high opsonic content and then the injections were gradually increased until the dog died on December 2S , 1920. Samples of blood were taken before each injection and the usual opsonic determinations made under the conditions de- scribed in the followinn- table. Date Remarks Bacteria Ani- mal :"o.of Cells Count. Ko. Cells v;ith Bacteria No . Cells Empty Total Kio . of Bact. Phago- cytic 11/27 20 Dog ,rl3 wt. before infection 13|lbs Inj. 2 cc. B . typho . S. aureus #13 Con 50 50 48 42 2 8 133 121 2,6 2.4 Pn. I Con 50 50 3 4 47 46 4 8 0.0 0.1 B. coli ,rl3 Con 50 50 - - - B . typho- sus #13 Con 50 50 42 45 8 5 97 104 1.9 2.0 (26) Date Remarks Bacteria .-ani- mal Ko. Ox"" Cells Count. NoCells with Bacteria No. Cells Enpty Total Ko.of Bact. Pnago- cytic Index 11/29 20 Dog #13 wt. 13ilbs No inj. S. aureus #13 Con 50 50 47 44 3 6 150 170 3 .0 o . 4 ?n. I /fl3 Con 50 50 0 0 50 50 0 0 0.0 0.0 B. coli //-■I 3 Con 50 50 41 4 c 9 4 83 102 1.6 2.0 B. typho- sus :/^13 Con 50 50 43 39 7 11 95 71 1.9 1.4 12/1 Dog yrl3 wt. l^ijlbs inj. 1 cc . B. typho- sus 3. aureus ,rl3 Con 50 50 43 45 7 5 218 212 4.3 4.2 ?n. I #13 Con 50 50 4 4 46 46 8- 6 0.1 0.1 B. coli ,rl3 Con 50 50 32 2o IS 25 53 55 1.0 1.1 B . typho- sus ,fl3 Con 50 50 17 15 33 35 45 20 0.9 0.4 12/3 Dog ,fl3 v.t. 13 lbs Inj. 4 oc. B. typho- sus S. aureus ,,-13 Con 5© 50 23 27 27 23 70 63 1.4 1.2 Pn. I ^13 Con 50 50 4 46 48 8 4 0.1 0.0 B. coli ,fl3 Con 50 24 24 26 26 37 35 0.7 0.7 B. typho- sus /fl3 Con 50 50 49 42 1 8 300 £5 6.0 1.7 12/17 Dog #13 wt. 11 lbs Inj. 5 00. B. typho- sus S .aureus ,rl3 50 50 45 43 4 7 232 231 4.6 4.6 Pn. I ,fl3 Con 50 50 7 10 43 40 25 21 0.5 0.4 B. coli ,rl3 Con 50 50 40 33 10 17 84 80 1.6 1.6 B. typho- sus ;rl3 Con 50 50 42 38 8 12 211 86 4.2 1.7 l.V'2^ Dog #13 wt. 11 jibs Inj. 5 cc. B. typho- sus 3 .aureus /fl3 Con 50 50 26 25 24 25 119 104 2.5 2.1 Pn. I -rl3 Con 50 50 0 0 50 50 0 0 0.0 0.0 li. coli ;rl3 Con 50 50 41 38 9 12 130 105 2.6 2.1 B. typho- sus ,ri3 Con 50 50 40 31 10 19 211 6 c 4.2 1.3 (27) i^_Dos Jo. 13--T,;p^i3id. Inrejtion, Oosonic r^-" "! I Death 3 i f ,'x /^- // ji 3» J/ J'/ Jay 3 jpsoaic Ind:;-x Gurvj for- Ztapli. .areas. o 3 I 9 ~ ~ 77 ^/ J* 77 7/ . Onsonic Ind^. ''or Pn e uni o c v:;;u.- , T.-.-pe I. J 1 f /I /^ /r -»/ Jf a/- ^z- 0p3o-rl3 Ind3::: ":u;;-vo for '3. coll. Date Remarks Bacteria ani- mal ..o.of :ells :ount. Ko. Cells with Bacteria Ko. Cells Linptv Total Ko.of Bact . Phago- cytic Index 12/26 20 9:00 Do^ ,7-13 wt. 9-i^lbs ::o inj. 3 .aureus ,rl3 Con 50 50 45 41 5 9 256 250 5.1 5.0 Pn. I /fl3 Con 50 50 0 0 50 50 0 0 0.0 0.0 B. coli ;fl3 Con 50 50 25 37 25 13 43 47 0.8 0.9 B.t.-pho- sus /1 3 Con 50 50 27 23 46 77 10 1.5 0.2 12/26 5:00 PJ'. Dog ,t13 wt. 9 lbs No inj. 3 .aureus ,rlo Con 1 50 50 35 41 15 9 227 £50 4.5 5.0 Pn. I ,t13 Con 50 50 0 50 50 0 0 0.0 0.0 B. coli ;rl3 Con 50 50 21 29 13 42 47 0.8 0.9 B. typho- sus #13 Con 50 50 36 4 14 46 78 10 1.5 0.2 12/27 Dog #13 No inj. S .aureus ,rl3 Con 50 50 25 37 25 13 149 180 3.0 3.6 Pn. I ;rl3 Con 50 50 0 0 50 50 0 0.0 0.0 3. Coli #13 Con 50 50 28 36 22 54 49 1.0 0.9 B. typho- sus ,rl 3 Con 50 50 35 19 15 31 56 26 1.1 0.5 12/28 Dog //--IS 3 hours before death. 3. aureus ,fl3 Con 50 50 23 37 27 13 146 160 2.9 3.6 . X^Yi. I ,fl3 Con 50 50 2 0 48 50 2 0 0.0 0.0 B. coli /rl3 Con 50 50 36 36 14 14 62 49 1.2 1.0 B . t^/pho- sus if 1 5 Con 50 50 32 19 18 31 50 26 1.0 0.5 12/2E Dog 'rlo wt. Silbs Death S. aureus w-13 Con 50 50 27 37 23 13 143 180 2.8 3.6 Pn. I -fl3 Con 50 50 0 0 60 50 0 0 0.0 0.0 3. coli Con 50 50 24 36 26 16 58 49 1.1 1.0 B. typho- sus ■// 1 3 Con 50 50 38 ]9 12 31 70 1.4 0.5 (28) Anatomical Diagnosis: Emaciated; no characteristic typhoid lesions except acute splenic tumor. B. typho- sus obtained from heart's blood. B. enterid- itis cultivated from heart's blood and gall bladder. -t-ataphylaxis in this animal probably occurred on Decem- bei- 26th when there was a marked decline in the opsonic index for the t^rphoid bacilli. The decrease, however, was not suf- ficient to bring the index down to the normal. The phagocytic activity of the blood against B. typhosus was at the time of death more than twice as great as that of a normal control. It is possible, too, that the test gives an inadequate measure of the real opsonic strength of the experimental animal's blood, for Harrison (26) has shown that the lytic action of anti- typhoid serum is so great against the typhoid bacilli as to lower the observed index by dissolving the bacteria before they can be taken up by the leucocytes. That this condition actually existed in this case was demonstrated when the inactivated im- mune serun gave a higher phagocytic index than when the serum was used unheated. Date Remarks Bacteria Ani- mal No. of Cells Count. No. Cells with Bacteria No . Cells Empty Total No. of 3act. Pnago- cytic Index 12/18 20 Unhtd serum B . tyjpho . ,fl3 50 38 12 70 1,4 inactvd " B.. typlio . ,rl3 50 42 8 124 2.4 That the phagocytic cells in this dog, dying of a typhoid infection, rare not functionally impaired is indicated by the following tests: (29) A quantity of blood was taken in citrate from dog Ko. 13 and from a control animal. Each lot was washed twice, using 175 volumes of salt solution. Samples from the resulting cells were then separately incubated with Staph, aureus and B. typho- sus. The results are civen in the table. Date rvemarks Bacteria ani- mal No . of Cells Count. No. Cells with Bacteria No. eells ^mpty Total No . of Bact. Phago- cytic index 12/28 20 oOmin. incu- bation. S. aureus ;fl3 Con 50 50 1 2 40 48 4 2 0.0 0.0 ^Onin .incu- bation. B. typho- sus Con 50 50 32 0 ■ 18 50 57 0 1.1 C> . 0 Whether the energetic phagocj'-tosis of B. typhosus by the washed cells of the immune animal, in the absence of serum can be interpreted as the action of an immune cell, or whether there was left upon the cell enough highly potent i.amune serum to op- sonize only the specific bacteria is a matter to be decided by more detailed observations. That the last explanation, however, is probably the true one is suggested by Illein's (27) work on the dilution of sera. A quantity of polymorjihonuclear leucocytes were recovered from the urine of dog No. 13 and when incubated with sensitized bacteria were actively phagocytic, as indicated in the follow- in-' results. Bate Remarks Bacteria ^\ni- m.al No. of Cells Count No. Cells with Bacteria No. Cells Empty Total No. of Bact. Phago- cytic Index 12/2S Cells from urine dogl3 B. typho- sus ,/8 Con 50 50 33 23 17 27 128 37 2.6 0.7 inimal providing serum. (30) These findings, then reveal an animal dyinn- of an infec- tion with its phagocytic mechanism, so far at least as experi- mental standards can determine, functioning with an efficiency considerably above the normal ar;ainst the specific or.^anism^ and without any demonstrable decrease in opsonic activity against the oiher bacteria used in the test. Experiment 9. — This experiment v/as intended as a dupli- cate of the preceeding experiment. The conditions of the test, paralleling as nearly as possible those of No. 8, are given in the accompanying table. Although this infection resulted in death it ran a much m.ilddr course than the typhoid infection recorded above. There was only slight emaciation at death, and there was a complete absence of macroscopic lesions. Date Remarks Bacteria r.ni- mal No. of Cells Count. No. Cells with Bacteria No . Ceils Empty Total Nc.of Bact. Phan-o- cytic index 1/18 21 Dog rr^9 wt. before injection 12llbs. Inj. 3cc. 3 . typho- sus S. aureus ^19 Cop 50 50 33 34 17 16 252 159 5.0 3.2 3. coli ,-19 Con 50 50 43 40 7 10 98 95 1.9 1.9 3. typho- sus ,fl9 Con 50 50 10 7 40 43 15 20 0.3 0.4 B. bron- chisep . ,fl9 Con 50 50 42 27 23 51 65 i.e 1.3 1/24 Dog #19 wt. llilbs Extreme weakness To inj. 3. aureus ,;15 Con 50 50 31 34 19 16 115 128 2.3 2.3 B. coli ,rl9 Con 50 50 41 41 9 9 78 71 1.6 1.4 B. typho- sus ,fl9 Con 50 50 8 6 42 44 19 16 0.3 0.3 B. bron- chisep. ^19 Con 50 50 38 38 12 12 137 136 2.7 1^ . 7 (31) iElS.9 0 -)5->ni( Ind. Jn -■ :-it i :>n 14 .;a" o z Opaonio "■; 1 73 73 7i Day s urve for otapri.. -^.ureus /i /v 0.1 ic Ind'^x :jurve f jns-)nl: Inde:-: Curve for :?. coll, Op£:'^nic Index G irve Tor 3. bronL^diaopLicus. fci'P i p t. ::: :tefe3ifei5fej^;:fa:1-^ iilPis:^: r P^i P^-hidi±dgd;g Date Renarks Bacteria Ani- mal No. of Cells Count. No;Cells with Bacteria No. Cells Empty Total No. of Bact. Phago- cytic Ir.dex 1/25 21 Dog /rl9 Wt. 11 lbs To inj. S. aureus ./■I 9 Con 50 50 ■ : - : 3. coli Con 50 50 14 15 36 35 50 48 ■ 1.0 0.9 B.tjrpho- sus #19 Con 50 50 8 7 4^^ 45 19 15 0.3 0.3 B. bron- chi eep. #19 Con 50 50 39 37 13 19c 212 3.9 4.2 1/29 Dog !fl9 V.'t. 11 lbs No inj. S. aureus #19 Con 50 50 42 45 5 226 244 4.5 4.8 B. coli |19 (^on 50 50 44 45 6 5 183 182 3.7 3.6 B. typho- sus #19 Con 50 50 36 30 12 20 70 54 1.4 1.0 B. bron- chi sep. #19 Con 50 50 34 31 16 19 182 101 3.6 2.0 2/1 Inj. 5 cc. B. typhosus 2/2 Dog #19 Dead S. aureus -fl9 Con 50 50 24 27 26 23 c9 81 1.7 1.6 B. coli .■19 Con 50 50 38 3^; 12 16 81 70 1.6 1.4 B. typho- sus #19 Con 50 50 44 13 6 37 157 18 3.1 0.3 B. bron- chi 6=^ p . -rl9 Con 50 50 50 50 C 0 408 310 8.1 6.2 Anatomical Diagnosis: Slight emaciation; no macroscopic lesions. B. typhosus in pure culture cultivated from heart's blood. The findings in this experiment are for the most part in agreement with the results obtained in No. 8. The character- istically high opsonic index for the specific organism suffered no decrease after the infection had overcome the general body (32) resistance. This may perhaps be due to the short pei'iod which elapsed between the fatal inoculation and death. Also there was, as in all preceeding tests, no discoverable change in the indices for any of the non-specific bacteria, either during the course of the infection, or within the agonal period, the time when terminal infections commonly appear. The results of these experiments with typhoid infections challenge the validity of Bordet's (28^^ conception that "yield- ing to an infection is primarily due to an inability of the phagocytes to take up the infectin_- agent." D. Streptococcus Infections Experiment 10. — The experimental animal in this test was a large adult guinea pig - Ko. 23. It was given an initial intravenous injection of 1.5 cc. of a 24 hour broth culture of hemolytic streptococci on November 24, 1920. The inocula- tions were repeated every second day, but were discontinued as soon as it was apparent that a fatal quantity had been given. The guinea pig died sixteen daj'^s after the first injection. During the last six days the infection assumed the most destruc- tive character. Over this period the animal lost 29;^ of its body weight, and throughout the last 24 hours of its life was in a state of astonishing emaciation and weakness. The opsonic determinations, together with other data concerning the experiment are recorded in the appended table. (33) Date Reraarks Bacteria Ani- mal No. of Cells Count. No. Cells with Bacteria ^^o. Cells Empty Total ICo.of Bact. Pnago- cytic Index 11/24 20 Pig -f23 wt. before injection 555 g. Inj. 1.5CC Strep.hemo. S. aureus ,f23 Con 50 50 40 38 10 12 160 131 3/.^ 2.6 B. coli #23 Con 50 SO- 45 47 5 3 176 150 3.5 3.0 Strep . heme . Con SO 50 42 46 8 4 142 150 £.8 3.0 11/26 Inj. 1.5CC Strep.hemo. 11/28 Inj. 1.5CC Strep .henio . 11/30 Pis #23 wt. 550 g. Inj. 1.5CC Strep.hemo. S. aureus #23 Con 50 50 41 42 9 8 263 276 5.2 5.5 B. coli #23 Con 50 50 41 45 9 5 80 75 1.6 1.5 Strep, hemo . .f23 Con 50 50 50 46 0 4 223 140 4.4 2.6 12/4 Pig ,t23 wt. 540 g. Ho. inj. S. aureus #2S Con 50 50 34 37 16 13 110 113 2,2 2.2 B. coli #23 Con 50 50 40 39 10 11 45 50 0.9 l.C Strep. nemo . ,r23 Con 50 42 25 25 520 100 6.4 2.0 l£/6 Inj. 1.5CC Strep.hemo. 12/S Inj. 1.5CC Strep.hemo 12/9 Inj. 1.5cc Strep.hemo 12/10 Pig #23 wt. 375 g. 4 hours be- fore death To inj. S .aureus ,r23 Con 50 50 40 45 10 5 244 204 4.8 4.1 B. coli ;;-23 Con 50 50 18 29 32 21 60 48 1.2 0.9 Strep. hemo . #23 Con 50 50 10 17 40 3" 44 93 0.8 1.8 12/10 Pig #23 wt. 375 g. Death S. aureus ,-23 Con 50 50 43 45 7 5 215 204 4.3 4.0 B. coli ,r23 C(?n 50 50 26 20 22 30 50 48 1.0 0.9 Strep . iiemo . ;V-23 Con 50 50 13 17 37 35 44 92 0.9 1.8 (34) 'i-. V Streptococcus Infedtlorj. :;u:'it:on 16 iays '^oaonii^ Inde ^■j3-)nl 1 '/ i t /d /I 7i Days O 2 ^ t Z Jo /^i /■^ /{, ■'vo for B, coll. 5 :It_-: : ::i ANDRUS&CHUR Anatomical Diagnosis: Extren.e emaciation; generalized hemorrhage; purulent exudate in pleural cavities. Strepto- coccus hemoljrticus in pure culture from pleural exudate. Streptococcus hemolyticus and B . pyo- cyaneus cultivated from heart's blood. The variations in the opsonic activity against the in- fecting agent in this experiment coincide with the observations of Denj'-s and Leclef (29) in their investigation of streptococcus immunity in rabbits. It is interesting in the present experi- ment that the marked decline in the phagocytic activity against streptococci, after the infection had overcome the general body resistance, parallels almost exactly the curve for loss in weight. There was at no time throughout the experiment any change in opsonic index for any of the non-specific bacteria examined. Experiment 11. — In this experiment are presented the results of a study of four cases of fatal spontaneous hemolytic streptococcus infection in cats. The epidemic, of which these cases were a part, appeared in a room containing twenty-five cats, and ran a course so severe that of the whole number only two survived. The onset of the attack was characterized by a nasal discharge and violent sneezing. The cats lost rapidly in weight and died, generally after four or five days. The last 24 hours of the infection was especially severe. The animal was in a state of extreme emaciation and exhaustion, (35) being all the time unable to stand ?nd in the last hours of life responding only to such violent stimulation as a cardiac puncture . Samples of blood were taken from the heart at intervals indicated in the tables and the usual opsonic determinations made . 1 — Ani- fJo.of No. Cells No. Total Pha,n;o- Date Remarks Bacteria mal :ells with Cells No. of cytic :;ount. Bacteria Empty Bact. Index 2/13 Cat jf-5-5 S. aureus #33 50 43 7 210 4.2 21 at time of Con 50 46 4 18£ 3.7 B. coli #33 50 29 £1 51 1.0 death Con 50 27 23 40 0.8 B. bron- #33 50 50 0 380 7.6 chisep. Con 50 46 4 285 5.7 Strep. //■33 50 25 25 101 2.1 hemo . Con 50 32 18 266 5.3 1 2/14 Cat ,r35 S. aureus ~w--35"^ 50 38 12 161 3.2 Con 50 38 12 137 2.7 B. coli ,;35 50 27 23 55 1.1 1 hour be- fore death Con 50 25 25 62 1.2 B. bron- -7-35 50 37 13 166 3.3 chisap. Con 50 34 16 172 3.4 B. typho- //35 50 36 14 71 1.4 sus Con 50 33 17 66 1.7 2/14 Cat #36 3. aureus #36 50 37 13 2.7 at time of Con 50 38 12 137 2.7 B. coli ;f36 23 27 58 1.1 death Con 50 25 25 62 1.2 B. bron- #36 50 43 7 216 4.3 chicep. B. typho- Con 50 34 16 171 3.4 -36 50 25 25 55 1.1 sus Con 50 33 12 86 1.7 2/15 Cat ,f?.7 S. aureus #37 50 37 13 101 2.0 Con 50 50 0 co6 5.1 B. coli #37 50 48 2 225 4.5 Con 50 48 2 216 4.3 3. br on- ;r37 50 50 0 294 5.8 chic: ep. Con 50 42 8 250 5.0 Strep. #37 50 25 25 146 2.9 nenio . Con 50 42 8 213 4.3 (36) 1': "I"!""!' Dur-xtion '' Indo Op£. jnic Invlex Curvj Cot Itn^'n. aurouj OTs;>nic ladex Curve Tor B. coli. )nio Index Curve for B. bronchis '^ptioun . i,^^:,^.A^iA.j,A^j^^i^i^i..^,^^ •NDRUS I, CHURCH, Date Remarks Bacteria ani- mal Ko.of Cells Count. No ..Cells with Bacteria No. Cells Empty Total No. of Bact. Phago- cytic Index 2/17 21 Cat if 37 48 hours uef ore death. S. aureus 7f37 Con 50 50 39 46 11 4 145 168 2.9 3.7 B. coli #37 Con 50 50 25 18 25 32 45 42 0.9 0.5 B. bron- chisep. #37 Con 50 50 46 48 4 2 263 284 5.2 5.6 Strep. nemo . #37 Con 50 50 34 36 16 14 176 266 3.5 5.3 2/19 Gat #37 at time of death S.auraus #37 Con 50 50 41 46 9 4 305 340 6/.1 6.8 B. coli #37 Con 50 50 42 29 8 21 216 159 4.3 3.2 B. bron- chi Sep. /f37 Con 50 50 50 50 0 0 720 793 14.4 15.9 Strep. heme . #37 Con 50 50 32 41 le 9 160 201 3.2 4.0 2/19 Sat ,f37 1 hour after death 3. aureus #37 Con 50 50 46 48 4 2 335 327 6.7 6.5 B. coli ,r37 Con 5^ 50 50 45 0 5 150 120 3.0 2.4 B. brcn- chisep . #37 Con 50 50 50 50 0 0 304 297 6.0 5.9 Strep . hemo . ;f37 Con 50 50 50 50 0 0 201 386 4.0 7.7 Anatomical Diagnosis: All the cats in this experiment presented the same appearance; Marked emaciation; greenish purulent exudate filling the nasal sinuses and extending over the surfaces of the naso-pharynx and down into the trachea as far as the bifurcation. There was a complete ab- sence of any lesions in any other organs. No. haemorrhages. Streptococcus hemolyticus in pure culture obtained from heart's blood of all four animals. This was also the predomin- ating organism in the exudate of the upper respiratory tract. There is, in general, agreement in the results obtained from these four cases. In all the opsonic index for the spe- (37) cific organism was below normal at the time of death. The opsonic indices for the heter;logous bacteria, in each instance, differed from the normal by less than v;hat can properly be regarded as experimental error. There was in the study of cat No. 37 one feature of very special significance and interest. Tnis animal when first examined on February 15th already revealed advanced emaciation and weakness. The blood culture on this date showed an abund- ance of streptococci and a few colonies of Staph, aureus. Two days later this latter organism waB found abundantly in the blood, but by February 19th it had considerably decreased and had completely disappeared before the cf.t died on February 20th. Ten cubic centimeters of blood cultured at the time of autopsy failed to develop a single colony of Staphylococci. Strepto- cocci were sufficiently numerous in the blood at all times to be recovered hy plating a single loop of blood. Vvhen this cat was placed under observation it was the subject of a double infection and showed evident indications of being rapidly overcome. That the streptococcus was the dominant etiological factor' in the infection seems evident from the fact that the symptoms revealed by this cat were in every way similar to those in animals suffering with a pure strept- coccus infection. Cat No. 37, then, was being overcome by a rapidly fatal infection. It had in some way become secondarily infected with Staph, aureus, and this latter organism had become so abundant (38) in the blood stream that there resulted a marked decrease in the opsonins for staphylococci. Yet this enfeebled animal in its losing struggle against a streptococcus infection was able to react against a secondary infection of staphylococcus in a manner so vigorous as to completely banish these bacteria from the body. During this time there was an unmistakable increase in the opsonic index for stapnylc coccus, and at the time of death this index was only slightly below the normal. The op- sonic increase is ,^iven in the following table: Animal :-.'ate Feb. 17 Feb. 19 Tot „-1V C>p.j.nd 0 . 40 0.7- L' . c -z. It would s-^em tnat the body reacted against the secon- dary infection in -n independent manner, and that the recovery from one infection and death from cmother occured, by chance, at the same time. Experiment 12. — In this experiment an attempt was made to reproduce experimentally the spontaneous infections described in Experiment 11. An adult female cat. No. 38, was injected intravenously with 3cc of an 18 hour broth culture made directly from the heart's blood of a cat dying of the epidemic strep- tococcus infection. I'he cat came down with the cnaracteristic sjmiptoms and died after 72 nou. s. The extent of emaciation and collapse Vi/as not as marked as in the .receeding cases. The routine determinations are recorded in the accom- panying table C39) Date Remarks 1 Bacteria Ani- mal llo . of Cells lio. Cells with Ko. Cells Total Ko.of Phago- cytic 2/18 21 Cat #38 Before Injection Inj. 3cc Strep. hemo . S. aureus #38 Count 50 Bacteria 40 iiimpty 10 Bact. 167 Index 3.3 B. coli Con ,7^38 Con 50 50 50 46 29 IS 4 21 32 103 50 42 3.7 1.0 0.8 B. bron- chisep. ,f36 Con 50 50 47 48 3 2 301 284 6.1 5.6 Strep, hemo . 7^38 Con 50 50 35 36 15 14 306 266 6.1 5.3 2/20 Cat #38 ICo inj. S. aureus #38 Con 50 50 39 46 11 4 400 341 8.0 6.8 B. coli #38 Con 50 50 45 28 5 22 155 159 3.1 3.2 B. bron- nhiRep. #38 Con 50 50 50 50 0 0 350 790 17.0 16.0 Strep, hemo . ;r-38 Con 50 50 29 42 21 8 191 202 3.8 4.0 2/21 Cat 7f38 Death S. aureus #38 Con 50 50 40 47 10 3 313 328 6.2 6.6 B. coli #38 Con 50 50 39 45 11 5 59 120 1.2 2.4 B. bron- chieep. ,r38 Con 50 50 50 50 0 0 320 297 6.4 5.9 Strep, hemo . ,r38 Con 50 50 33 20 17 30 2ii4 286 4.5 7.7 Anatomical Diagnosis: Slight emaciation; Streptococcus hemo- lyticus in pure culture cultivated from heart's blood. The findings in this experiment are throu3hout similar to the results obtained in the spontaneous streptococcus infec- tions . (40) E. Miscellaneous Infections jJxperi.'nent 13. — This experiment was conducted on an adult rabbit - No. 41. It was injected intravenously viith in- fluenza bacilli washed from a slant of cooked-blood ajjar. The rabbit died 18 hours later without any macroscopic lesions, but with a tremendous number of influenza bacilli in the blood. The opsonic results are given below. Date Remarks Bacteria Ani- mal No. of cells No .Cells with Bacteria No. Cells •^mpty Total No. of Bact. Phago- cytic Index 2/4/2] Rabbit if^l wt .before injection 2500 s. 3 .aureus r-41 Con 50 50 41 37 9 13 97 93 1.9 1.9 B, coli ;f41 Con 50 50 40 37 10 13 79 74 1.6 1.5 B.influ. m Con 50 50 17 10 35 40 22 25 0.4 0.5 2/5 Rabbit jfAl 3. aureus ^41 Con 50 50 36 40 14 10 94 105 1.9 2.1 B. coli ,v-41 Con 50 50 41 40 9 10 lOB 95 2.1 ■ l.S B.influ. ,r41 Con 50 50 13 17 37 33 13 29 0.3 0.6 Although this animal was overcome by a rapidly fatal in- fection, aided perhaps by the intoxication resulting from a n. ^ large initial infection, its opsonic defense, except for the specific infecting bacteria, remained intact up to the very hour of its death. 3xp^riment 14. — The two cases presented below are cats which died of a spontaneous infection of B. bronchigepticus. the infection in each case had lasted for three days and both animals presented moderate emaciation. During the last 24 (41) hours of life the cats were in a state of extreme weakness, being unable to stand and responding only to violent stimu- lation. Samples of blood were taken from the heart shortly before death. The routine determinations are given in the appended table. Date Remarks Bacteria Ani- mal No . of Cells No. Cells with Bactei^ia No. Cells Empty Total Mo. of Bact. pha?-o- cytic Index 1/9/21 Cat ,f27 at tiT.e of death S. aureus rr27 Con 50 50 ^3 35 17 15 125 116 2.5 2.3 B. coli r^27 Con 50 50 50 48 0 2 305 264 6.1 5.2 B. bron- chisep. #23 Con 50 50 50 50 0 0 337 363 6.7 7.2 Pn. I ,/27 Con 50 50 14 8 36 42 42 30 0.8 0.6 B. typho- sus /r27 Con 50 50 44 45 6 5 96 94 1.9 1.9 1/9/21 Cat ;r'28 S. aureus 7,^28 Ccn 50 50 38 35 15 136 116 2.8 2.3 B. coli ,,-28 Con 50 50 48 48 2 2 271 264 5.4 5.2 B. bron- chiBsp. #28 Con 50 50 50 50 0 0 254 363 5.1 7.2 Pn. I #28 Con 50 50 lo 8 4C 42 'd6 30 0.4 0.6 B. typho- sus ^8 Con 50 50 44 45 6 5 90 94 1.3 1.9 Anatomical Diagnosis Moderate emaciation. Marked lobular pneumonia. B. bronchiaepticus from blood and lungs in both cases. Experiment 15. — The guinea pig - No. 20 - of this ex- periment was inoculated on November 8, 1920, with the sediment (42) of 10 cc. of spinal fluid from a child dead of tubercular meningitis. By December 3 the pig had reached an advanced stage of generalized infection and was extremely weak and emaciated. The pig was sacrificed and a sample of blood ob- tained from the heart for opsonic determinations. Date Remarks Baci-eria Ani- mal I3o.of Cells Count. No. Cells with Bacteria Ko. Gelx,. ^mpty Total I.o.of Bact. Phago- cytic Index 12/3 20 Pig #30 at ti;:ie of death 3 .aureus #30 Con 50 50 45 41 5 9 305 273 6.1 5.5 3. coli #30 Con 50 50 36 41 14 9 70 72 1.4 1.4 Pn. I #30 Con 50 50 0 0 50 50 0 0 0.0 0.0 Strep, he mo . #30 Con 50 50 48 46 2 4 600 625 12.0 12.5 Anatomical Diagnosis: Advanced generalized tuberculosis. Inguinal glands easilj?- palpable; extensive infiltration, haemorrhage, necrosis at site of inoculation. Omentum, studded with tubercles, was rolled into a characteristic- ally snarled mass. There was an extensive distribution of tubercles over the diaphragii and peritoneal wall. Smears from the en- larged glands and omentum revealed the pres- ence of a larp-e number of tubercle bacilli. Notwithstanding the destructive changes which the in- fection had produced in this animal, there was no lowered opsonic efficiency for any of the non-specific organisms examined. Experiment 16. — The dog studied in this experiment had, when first seen on December 27, already developed charac- (43) teristic symptoms of "distemper." There .was a continual nasal discharge associated with frequent sneezing and dyspnea. The animal was emaciated and staggered about when forced to its feet. A small grara-negative bacillus, probably Pasteurella canis . v/as present in enormous numbers in both the nasal secre- tion and blood stream. However, on December 30 the dog began to improve and continued to gain in strength until January 12. On this date it suffered an overwhelming and paralysing col- lapse. After this, the dog was unable to move and lay in a continual stupor. It died o'anuary 14, in a state of wealaiess so profound as at once to set it apart from any other animal observed during this investigation. It does not seem possible thao an animal could attain a more defenseless condition than this dog presented during the last 36 hours of its life, and yet throughout this period of extreme physiological depression there was no discoverable rupture in its phagocytic defense. Full details of the experiment are set forth in the accompanying table. Date Remarks Bacteria Ani- mal No. of Cells Count. No. Cells with Bacteria No. Cells mpty Total No. of ^act. rhago- cytic Index 12/27 20 Dog #47 wt. 5.7 Kg. S. aureus #47 Con 50 50 33 37 17 13 ISO 199 3.6 3.9 B. coli #47 Con 50 50 45 Ac 5 8 140 2.8 102 , 2.0 B. bron- chisap. #47 Con 50 50 23 26 27 24 38 0.7 27 1 0.5 (44) :le: ■ on ( jnr-'jrv 0 i * i r /a /2 /¥ u /fD'i J / ) " I.Kiex CiirvG for Stip:;. -.Mrouy. 5 / Op-jnio :n:.o. :-r^vo for 3. coll. « ^ -C ^ 7 /« /-^ "f /i ■e for B, i^roncliis _oti2u3 « /e /a /M /i /g 'or 3. typ:iO£;U3. JI4Jari:;ai#mj?-i44:^i:g;it^4?;T.4'^ Date Re:narks Bacteria Ani- mal i:o.of Cells Count. No. Cells with Bacteria i;o. Cells Em;:tv To'tal No. of Bact. Phago- cytic Index 1/12 21 Dog ;f47 wt . 5 Kg . S. aureus ,f47 Con 50 50 42 46 8 4 205 202 4.1 4.0 B. coli ,r47 Con 50 50 40 47 10 3 132 156 2.6 3.1 B. bron- chisap. #47 Con 50 50 3S 44 12 6 94 62 1.9 1.2 B. typho- sus #47 Con 50 50 5 45 40 6 10 0.1 0.2 1/13 Dog '7-47 wt. 4.8Kg. S. aureus #47 Con 50 50 36 44 14 6 162 202 3.2 4.0 B. coli #47 Con 50 50 46 47 4 3 146 156 2.9 3.1 B. bron- chieep. ,-M7 Con 50 50 4c 44 2 6 110 62 2.2 1.2 B. typho- sus #47 Con 50 50 11 10 39 40 12 10 0.2 0.2 1/14 Dog #47 wt. 4.6Kg Death S. aureus #47 Con 50 50 41 46 9 4 163 202 3.2 4.1 B. coli #47 Con 50 50 47 47 3 3 163 156 3.S 3.1 B. bron- chisep. #47 Con 50 50 46 44 4 6 112 62 2.2 1.2 B . typho- sus #47 Con 50 50 5 10 45 40 6 10 0.1 0.2 Anatomical Diagnosis I.'arked emaciation; purulent exudate in nasal cavity and pharynx; extensive broncho- pneumonia. Pasteurella canis cultivated from blood and from naso-pharyngeal exudate. F. Infections in Man It is desired to submit a study of the following human cases as a supplement to the animal experiments presented earlier in this investigation. 'i/Vhile the number presented is small, the (45) exact agreement throughout with the results previously obtained endows these findings with considerable significance. The method of procedure was the same as that emploj'-ed in previous experiments. A sample of blood was obtained from the patient, or if from autopsy, as soon after death as possible, and after two hours the serum was separated and the usual op- sonic determinations made. The control serum and leucocytes were obtained from a normal individual at the same time the experimental sample was taken. In some instances leucocytes were also obtained from the patient and here the "cytophagic" as well as ihe opsonic index v/as determined. These results are given in a subsequent table. The experimental conditions and results of the study of human cases are tabulated below. Case No. 1. Clinical History: '.'.'hite female; age 69 years Duration of present illness: July 1920 until March 8, 1921. Entered hospital January 22, 1921; died March 8, 1921. Diagnosis: Arteriosclerosis, hypertension, chronic nephritis, hemiplegia, acute bronchopneumonia.. Bacteriology: Blood culture repeatedly negative. *The clinical and autopsy data of the following human cases were secured from the Johns Hopkins Hospital records. (46) The complete opsonic data in this., as in the remaining cases, are condensed into the tables following the clinical histories . Date Remarks Bacteria Per-, ^'0. of son Cells :;;ount. Jo. Cells with Bacteria Ko . Cells £mpty Total No. of Bact. Phago- cytic Index 3/2/2] Patient #1^ S.aureusti^l? Con. 50 50 45 47 5 3 335 297 6.7 5.9 B. coli #17 Con. 50 50 32 36 18 14 77 81 1.5 1.6 B, bron- chisep #17 Con. 50 50 50 50 0 0 423 361 8.5 7.2 Pn. 1 #1^ Con 50 50 9 5 41 45 16 8 0.3 0.2 3/3 Patientj/^IV S. aureus #17 Con 50 50 50 47 0 3 432 381 8.6 7.6 B. coli #17 Con 50 50 32 35 18 15 51 SO 1.0 1.6 B. bron- chisep #17 Con 50 50 50 48 0 2 356 345 7.1 6.9 Fn. I ^17 Con 50 50 19 10 31 40 63 56 1.2 1.1 3/5 Patient#17 S. aureus #17 Con 50 50 50 50 0 0 436 408 8,7 S.l B. coli /rl7 Con 50 50 25 28 25 2L 45 46 0.9 0.9 B. bron- chisep #17 Con 50 50 50 49 0 1 236 221 4.7 4.4 Pn. I r/r? Con 50 50 0 0 50 50 0 0 0.0 0.0 Anatomical Diagnosis: Autopsy not permitted. Case No. 2. (Autopsy Ko . 6473) Clinical History: White male; age 50 j'-ears . Duration of present illness: December 1920 to Jan- uary 31, 1921. (47) Entered hospital January 7, 1921; died January 31, 1921. Diagnosis: carcinoma of bladder; hydroureters; pyo- nephrosis; arteriosclerosis; with occlusion of coronary arteries; chronic myocarditis; jaundice. Date Remarks Bacteria Per- son i: 0 . 0 f Cells Count. To. Cells with Bacteria Cells Empty Total Mo. of Bact. Phap;o- cytic Index 1/30 21 Patient/f23 2 hours after death S. aureus #23 Con 50 50 50 42 0 8 390 248 7.8 4.9 B. coli #23 Con 50 50 43 50 7 0 159 116 3.2 2.3 B. bron- chisep #23 Con 50 50 50 50 0 0 600 297 12.0 5.9 B. typho- sus ,;^23 Con 50 50 22 25 28 25 38 46 0.8 0.9 Anatomical Dia^^nosis: "Carcinoma of bladder with extension to perivesical structures. Acute cystitis. Hydroureters and pyonephrosis. Arteriosclero- sis with marked thickening and occlusion of coronary arteries. Chronic fibrous myocard- itis. Jaundice. Anisocoria. Bilateral hydroceles. Healed tuberculosis of lung and bronchial nodes." Case No. 3. (Autopsy No. 6486) Clinical Historir; Colored female; age 32 years. Duration of present illness: May 1920 to January 10, 1921. Entered hospital November S, 1920; died jc'ebruary 10, 1921. Diagnosis: Carcinoma of cervix, with metastasis to brain; pyelitis; bronchopneumonia; siphylis. Panh^rsterectomy , Januarj/- 10, 1921. Died 8 hour':^ later. (48) 1 ' Date Records Bacteria Per- son No . of Cells Count. No. Cells with Bacteria No. Cells Empty Total No. of Bact. Phago- cytic Index 2/11 21 Patient/fl3 S. aureus ,fl3 Con 50 50 42 4o 8 2 298 316 6.0 6.3 B. coll ^13 Con 50 50 11 12 39 38 14 17 0.2 0.3 . B . bron- chisep . #13 Con 50 50 48 46 2 4 250 196 5.0 4.0 B. typho- sus ^13 Con 50 50 10 10 40 40 IS 15 0.3 0.3 Anatomical Diagnosis : "Unhealed operative wounds of abdominal wall. Adhesions about spleen, liver and in- testine. Retroperitoneal abscess involving righu iliopsoas muscle; abscesses in ieft kidney; dense adhesions (bilateral); acute bronchitis; mitral endocarditis; acute splenic tumor. Thrombosis of longitudinal sinus and cerebral veins, thrombosis of pelvic veins." Case No. 4. (Autopsy No. 6428) Clinical History: Colored male baby; age 10 days. Diagnosis: Congenital sj^-philis; jaundice; osteochondritis. 1 Date Remarks Bacteria r-er- son No. of Cells Count. No. Cells with Bacteria No. Cells Empty 'iutal No. of Bact. rna^c- cytic Index 12/12 20 Patient #11 32 hours after death 3 .aureus /rll Con 50 50 41 40 o 10 213 204 4 .2 4.1 B. coli #11 Con 50 50 48 49 2 1 167 160 3.3 3.2 B. typho- sus /rll Con 50 50 Ic 13 32 37 20 17 0.4 0.3 B. pyoc. #11 Con 50 50 50 48 0 0 298 224 5.9 4.5 Strep. heme. ,/ll Con 50 50 7 1£ 43 38 12 32 0.2 0.6 Strep. yiri. #11 Cpn 50 50 7 11 43 39 13 21 0.2 0.4 (49) Anatomical Diagnosis: dice: 'Congenital sypiiilis; hepatitis: osteochondritis . " Case No. 5. (Autopsy No. 6468) Clinical History: V/hite male; age 39 years. Duration of present illness January 27, 1921. April 1920 to Entered hospital January 13, 1921; died Jan- uary 28, 1921. Operated January 21 for removal of carcinoma of bladder . Streptococcus hemolyticus septi- cemia developed January 26. Diagnosis: Carcinoma of bladder; streptococcus hemolyticus septicemia; uremia; chronic urethritis; acute prostatitis. Date Kemarks Bacteria Per- son i:o .of Cells Count. Ko.uells vath Bacteria r.o . Cells Empty Total No. of iiact. Fha.ro- cytic index 1/28 21 Patient #5 3. aureus #5 Con 50 50 41 38 9 12 271 294 5.4 5.9 B. coli /r5 Con 50 50 44 38 6 12 87 105 1.9 2.1 B. bron- chisep. #5 Con 50 50 50 50 0 0 300 228 6.0 4.5 Strep. hemo . /f5 Con 50 50 3 16 47 34 9 SI 0.2 1.6 Anatomical Diagnosis: "Squamous cell carcinoma of bladder; gonorrhoeal urethritis; cystitis; ureteritis; pyonephrosis; pyelonephritis; prostatitis; surgical drainage of prostate; purulent peri- tonitis; bronchopneumonia; Adenoma of thyroid.* Bacteriological findings: Streptococus hemolyticus in pure culture from heart's blood. (50) The results obtan^ed here are in strict agre'Tmt-nt with .he findin.^s ^;re^'iousl ■ revieu'ed. There i.-. sometimes, tiiough by no means in eveiy case, a depressed opsonic activity against the si^ecific infeotin • organism. Nowhere, however, even in the most chronic oases, is there any discoverable decrease in the phagocytic effectiveness against an^r bacteria not concerned in the primary infection. The downward variations in the opsonic index for these organisms are always within the possible range of experimental error. (51; PART II FUNCTIONAL VARIATIONS OF PHAGOCYTES While it is unquestionably true "that the essential regulating influence affecting phagocytosis rests upon the ac- tion of the serura upon the bacteria" , (30) this should be weighed against the possibility that there may be variations in the functional capacities of the leucocytes themselves, independent of the action of the serum. Park and Biggs (31) pointed out that there vms often a demonstrable difference in the phagocytic power of the leucocytes of normal persons, and in 1910 Glynn and Cox (32) obtained a similar result in persons suffering with staphylococcus and tuberculous infections. They introduced a new factor in opsonic determinations which they termed the "opsono-cytophagic-index, " and which was obtained by determining tne relative phagocytic power of the leucocytes and serum of one person compared with phagocytic activity of the leucocytes and serum of a control individual. Tunnicliff (33) demonstrated the difference in opsonic capacity exhibited by the leucocytes of a baby and those of an adult, emphasizing the necessity of obtaining cells and serum from children for opsonic determinations on babies. Jhat there is considerable technical difficultjr in com.paring the phagocytic powers of two suspensions of leucocytes was demonstrated by Fleming (34) in an experiment in which he showed that given two unequal suspensions of leuco- cytes there 'was always less phagocytosis in the sample contain- ing the greater number of leucoc^-tes. This result was probably due to two factors, the relative number of bacteria in the sec- ond suspension was less, and there was also a greater amount of non-specific absurption of opsonins in the suspension con- taining the larger number of cells. Tihe following tabulated results, selected from cases presented earlier in this paper, reveal the difficulty of ob- taining an accurate and dependable measure of the relative phagocytic powers of leucocytic suspension obtained from dif- ferent animals. As in the preceeding experiments the opsonic determinations are based upon a count of the bacteria taken up by 50 neutrophile leucocjrtes . The phagocytic m.ixtures were made by incubating the normal and experimental leucocytes separately with both the control and experimental sera. The kind of bacteria used and other conditions of the experiment are explained in the following table: Date Remarks * Bacteria * Ani- mal Normal Leuco . ^Exioerimen. Leuco. ^Total Ko.of Bact. Phago- cytic Index Total No. of Bact. Phago- cytic Index 9/18 20 Dog #19 B . muc . capsu. #19 Con 1 3 0 0 120 to 2.4 1.6 12/7 Pig #23 S. aureus ,r23 Con 133 150 2.6 3.0 150 190 3.0 3.8 B. coli #23 Con 259 255 5.1 5.1 156 132 3.2 2.7 Strep, hemo . Con 300 398 6.0 7.9 198 196 3.9 3.9 12/10 Pig #23 4 hours before death S. aureus #23 Con 244 204 4.S 4.1 110 104 2.2 2.1 B. coli #23 Con 60 48 1.2 1.0 45 30 0.9 0.6 Strep. hemo. 7f23 Con 44 93 0.8 l.£ 2.0 17 0.4 0.3 *Nc. of bacteria in 50 neutrophile leucocytes, ♦♦Animal providing serum. (53) Date Remarks **Ani- Bacteria mal 1: or mal Leuco. Excerimen .Leuco . *Total No. of Bact. Phago- cytic Index *Total No. of Bact. Phago- cytic Index 12/10 20 Pig ;/^23 Death S. aureus ^23 Con 215 204 4.3 4.1 102 104 2.0 2.1 B. coli ;f23 Con 50 46 1.0 1.0 42 30 0.8 0.6 Strep. /r23 Con 44 92 0.9 1.8 22 17 0.4 0-3 -37373IT Pat; -nt 7rl7 S. aureus .17 Con 38 1 b.6 247 252 4.9 5.1 B. coli ttIV Con 51 80 1.0 1.6 44 16 0.9 0.3 Pn. I #17 Con 63 56 1.2 ] .1 21 25 0.4 0.5 B. bron. ..^17 Cnn 326 6.6 6.Q 382 440 7.6 8.5 12/26 20 Dog ,fl3 B. typho- sus .fl3 Con 78 10 1.5 0.2 128 37 2.6 0.7 *No . of bacteria in 50 neutrophile leucocytes. **Animal providing serum. While this table reveals a considerable variation in the "cytophagic" indices of the normal and infected animals, the nature of this variation is by no means constant. Sometimes the index for the treated animal is lower for the non-specific bacteria than in the control animal but quite as often it is higher. Altogether the variations observed are no greater than the v;ork of Fleming (34) would lead us to expect for two lots of normal leucocytes. V/hile experiments of this kind are exceed- ingly difficult to control and dangerous to interpret, it is contended that the results set forth in the above table are sufficiently definite to indicate that there is no invariable decline in the function of the phagocytes during the last stages of fatal infection. (54) PART III INADEQUACY OF THE V^RIGHT TEGHI^tIQUE The remaining experimental portion of this paper will be devoted to an analysis of the adequacy of the usual opsonic methods for extimatinn- the phagocytic capacity of an animal. During the course of this investigation there have been several occasions when the opsonic index, determined in the accepted manner, has iven not only an inaccurate gauge of the animal's phagocytic defense but has indicated a result the exact opposite of what was subsequently demonstrated to be the true condition. For these reasons it seems altogether worth while to examine the additional factors necessary to make vJrijht's method a de- pendable technique for determining, so far as it is possible to determine, the true phagocytic strength of an animal. Wright (35) has t'emonstrated by a series of experiments which have been repeatedly confirmed that the stimulating ef- fect of serum on phagocytosis is directed almost entirely against the bacteria. And it is reasonable to suppose from Bordet's (36) work tnat whenever one bacterium in a medium has been sufficient- ly opsonized to permit its being taken up by a leucocyte, all similar bacteria oT comparable virulence in that environment will likewise be sufficiently sensitized to effect their engulf- ment. Neglecting, then, the possibility of non-specific absorp- tion of opsonins by the lecocytes, the extent of the subsequent phagocytosis would be determined entirely by the number of phago- cytic cells present. Accordingly, it would be impossible to (55) estimate the comparative phagocytic capacity of tv/o animals without- taking into consideration not only the white count of the blood but also a differential determination in order to ob- tain the number of neutrophile leucocytes, the cells most com- monly concerned in the phagocytosis of bacteria. Even by this method of determining a more exact measure of phagocytic capacity, no account is, or can be, taken of the tremendous phagocytic powers of the "fixed" cells, j.ietchnikof f (37) first pointed out that these cells pla^r an important role in resistance, and in a more recent investigation Bartlett and Ozaki (8) demonstrated that the phagocytic capacity of the "fixed" cells, of ten reveals a comoensatorjr increase whenever there is an exhaustion of tne circulating phagocytes. it wouM therefore seem necessary to balance any demonstrable decrease in phagocytic activity of the blood against the possibility tnat this loss in effectiveness might be compensated for by in- creased activity of the infinitely more numerous "fixed" cells. There remains one other factor that should be considered before concluding that a lowered opsonic index, secured accord- ing to the method of '.Vright, indicates an absolute depression in phagocytic defense. It is reasonable to suppose that in con- sequence of the rapid destruction and replacement of neurtophile leucocytes daring infection tiiat these cellular elements are younger in infected than in normal animals. Hektoen (38) sug- gests that it is possible to account for the increased phago- cytic activity which Tunnicliff (39) demonstrated in exudates (56) and in recovering pneumonia cases^ by the fact that the cell obtained under these circumstances are youngei' than the leuco- cytes in the blood stream of norrual individuals. This suggests, tlisn, that it would not be possible to demonstrate conclu- sively a depression in phagocytic effectiveness without first eliminating the possibility of an increased "cytophagic" index which might even over compensate for the decline in opsonins. The results of this investigation would seem to warrant the conclusion that with the possible exception of cases with extreme leucopenia the relative phagocytic power of an infected animal is never lov;er than the opsonic index indicates, but that it is sometimes, indeed it is commonly, very much higher. The opsonic index, with the exception noted above, never does more than express the minimal limitations of the animal's phagocytic defense. VvTiile it is manifestly impossible , by any experimental means whatever, to estimate accurately the phagocytic capacity of an animal, it is quite useless to employ the Wright technique unless a correction is made for the observed variations in the number of neutrophile leucocytes present in the experimental animals. The factor by which this correction is made in the following table vjas obtained in each case by dividing the number of neutrophilic leucocytes present in 1 cmmi. of experimental blood by the corresponding count in the control. The vVright . opsonic index is tnen multiplied by tae above factor. This new measure of rela.tive phagocytic capacity may for convenience (57) be designated as the differential opsonic index. The following data are intended to show the results of opsonic determinations made by taking into consideration the white count of the infected animals. Both the Wright and differential opsonic indices are given. Date Remarks Normal Experimen. Bacteria Factor Wrighl Cpson Index Dif .' Opson Index Av. White Count ^Keu- trcph. Leuco. Av. white Count %Neu- troph. Leuco. 1/26 21 Dog #51 Death Bronchi. Infect. 10600 *75 27000 *85 S. aureus 2.0 1.3 2.6 B. bron- chisep. 2.0 1.3 2.6 B. coli 2.0 1.1 2.2 2/2 Dog #19 Death %phoid Infect, 10600 75 3500 35 S. aureus 0.16 1.1 0.17 B. bron. 0.16 1.2 0.19 B. coli 0.16 1.1 0.17 B. typho 0.16 9.0 1.4 1/24 Dog ffll 36hr. be- fore deati Pn.I Infec 10600 , 75 3000 75 S. aureus 0.2o 1.0 0.28 B. bron. 0.£c 1.0 C.28 B. coli 0.28 1.1 0.30 Pn. I 0.28 1.0 0.28 2/21 Cat #38 Death Strepto Infect . 14800 69 50000 73 S. aureus 3.5 C.9 3. 1 B. bron. 3..U 0.9 3.1 Str.hemo o.o 0.5 1.7 2/20 Cat #38 39 hrs. before death 14800 69 100000 90 3 .aureus 8 . S 0 . 9 8.0 B. bron. 8.8 c.y 8.0 B. coli 8.8 1.3 il.4 3. hemo. 8.8 0.8 7.0 2/18 Cat #37 15 hrs before death Strepto Infect. 14800 69 12000 SS S. aureus 1.0 1.0 1.0 B. bron. 1.0 1.0 1.0 B. coli 1.0 1.2 1.2 S. heme. 1.0 0.5 0.5 ** Differential Opsonic Index. ^Average counts compiled from re- sults obtained in this laboratory, supplemented by the counts of Klieneberger and Carl (42). Date Remarks Kor::,al Experi:!ien Bacteria i^actor Wright Opson Index Dif. Opson Index Av. IVhite Count /JNeu- troph. Leuco. Av. v'ihite Count ,;oKeu-- troph. Leuco. 3/2 21 Fat. ,fl7 5500 69 11400 74 3 .aureus 2.2 1.0 2.2 B. coli '^.2 1.0 2.2 B. bron. Cj . C. 1.0 2.2 Pn. 1 2 .2 l.C 2.2 1/3 Pat. ,/--23 5500 69 19300 73 S. aureus 5 - l.o 5.7 B. coli 3.8 1.3 4.9 B. bron. 3.8 1.2 4.5 B. typho. 3.C 0.9 3.4 1/28 Pat. ,o 5500 69 32840 89 S .aureus 7 . •/- 0.9 6.9 B. coli 7.7 0.9 6.9 :o . oi'on. 7.7 1.3 10 .0 3. ie- c , 7 . 7 C.12 0.9 2/11 Pat. #13 5500 69 24000 75 S. aureus 4.7 0.9 4.2 B. coli 4.7 0.8 3. 7 B. brcn. 4.7 1.2 5.6 B. tj-nho. 4.5 1.0 4.5 A study of the above table reveals that an actual depres- sion in the phagocytic activity associated with infectious dis- ease is not common, and indeed is exceedingly rare, except in individuals v^ith a lovif leucocytic count. In most cases the in- crease in neutrophile leucocytes over compensates for the ob- served decrease in opsonic efficiency revealed by the Wright technique. It seems reasonable, then, to suppose that infections may triumph, not because of a rupture in phagocytic activity of the animal but in spite of a very considerable increase in the effectiveness of that defensive mechanism. (59) DISCUSSION Throughout this investigation it has been the custon in each enumeration to determine not only the total number of bacteria taken up by 50 neutrophile leucocytes, but also to record the number of these cells actually taking part in the en,c;ulfment . A survey of these records reveals in general a parallelism between the variations in the phagocytic indices and fluctuations in the percentage of phagocyting cells. Com- monly a high phagocytic index is associated with a large per- centage of phagocyting leucocytes, while a low index generally is obtained in preparations with few ingesting cells. However, this is not uniformly true and of the two methods of determining the degree of phagocytic activity it would appear from this research tiiat the technique of counting the number of or^:anisms ingested is the more delicate and reliable. It has frequently happened that the experimental animal revealed the same per- centage of phagocyting cells as the control but yet exh.ibited the utm.ost variation in the total number of bacteria ingested. There is always a quest-ion whether the opsonic results obtained by incubating samples of cells and serum with in- different suspensions of bacteria can be accepted as indicating even approximately the conditions whicn prevail in the animal body. It is, of course, at oncn recognized that a culture of organisms grown on artificial medium may differ fundamentally in their reaction to living cells, from bacteria which develop (60) within the body. Bordet (40) has shown,' for example, that even v^/hen tne most virulent organisms are injected into a susceptible animal, there is an initial phagocytosis. This he explains as a selective process. Supposing that any culture, no matter how virulent, contains numbers of feeble and weakly aggressive individuals and these the phagocytes at once select and devour. The remaining hi^'^hly selected bacteria then, in some cases, extend the infection without any phagocytic interference. Zinsser (41) likev^ise has pointed out that it is possible to obtain agglutination of "culture" pallida but quite impossible to do so when the spirochetes are taken directly from a lesion. A certain reasonableness therefore attaches itself to tne objection that with phagocytic determin- ations in vitro one m.ay be dealing largely/- with the enfeebled non-pathogenic fraction of the cultures examined and that the findings are v;orthless as an index of vital phagocytic defense. It cannot be denied, hov^^ever, that opsonic determinations possess some significance and diagnostic value. It is possible that such observations in a specific case may be of uncertain value, but when extended over a large series, representing a wide variety in species and conditions, a uniform result gives ample justifi- cation for conservative deductions. Perhaps the most striking feature of this investigation has been the unfailinr-- effectiveness of phagocytic activity against the non-specific bacteria. In not one instance has there been any appreciable decline in the opsonic index for any microorganisms not concerned in the primary infection. -^his (6}) has been uniformly true even in cases of • extended infection, associated during the last dajT-s with the most destructive emacia- tion and with what appeared to be complete collapse of the ani- mal's vital defensive mechanism. It has also been developed in this paper that the Wright opsonic index is useful for determining the lower limits of phagocytic capacity but may ^ive no adequate measure of the actual extent of an animal's opsonic defense. It appears cer- tain that the percent of infected animals dying because of a collapse in the pha^^ocytic defense is very much smaller than is commonly supposed. Indeed, it is quite often as inaccurate and meaningless to say that an infected animal is overcome on ac- count of a rupture in its phagocytic defense as it would be to contend that a man was run down by a train because of a break in his running technique, although at the time of the disaster he was exhibiting a speed beyond anything he had ever developed before in his life time. (62) GONGLUSIONS The results obtained in this investigation would seem to warrant the following conclusions: 1 . Although thsre is sometimes a decrease in the onaonic in.iex for the specific infecting organism during the 1-ite stages of fatal infections, there is no decrea.-.e in the phagocytic activ- ity against any bacteria not concerned in the primary infection. 2. Neutrophilic leucocytes from an animal in the late stages of a fatal infection are as actively phagocytic as normal cells when placed in a medium containing suitable opsonins. This in- dicates that there is no decrease in the phagocytic function of these cells. 3. The "differential" opsonic index is a more reliable measure of the animal's phagocytic capacity than the '7right opsonic index. 4. Absolute depression in phagocytic effectiveness against the infecting organism is not an Invariable phenomenon in fatal infections. In closing this paper I v/ish to thank Dr. MacCallum for his courtesy and assistance, and I desire to -icknowledge a very especial debt of gratitude to Dr. Bayne- Jones for the substan- tial contributions he has made to the development of this problem. (63) Biography Howard B. Cross was born in Conway Springs, Kansas, July 31, 1889. He graduated from the public schools of Okla- homa in 1905 and after completing three years of hi^h school work taught for two years in a rural school. Then graduating from the Oklahoma Preparatory School, he entered the University of Oklahoma in the fall of 1911. The following year he was ap- pointed Student Assistant in the Department of Zoology, and in September 1913 was selected as Acting-Head of the Department of Biology in the Southwestern State Normal School. After a year at this institution, Mr. Cross returned to the University of Oklahoma as a student and Assistant in Botany, and graduated in 1915. He then studied at the Marine Biological Laboratory tliroughout the summer of 1915, and in the fall returned to the University of Oklahoma to accept the position of Instructor in Zoology. He continued in this position until 1917. The summer of 1916 Llr. Cross spent in the University of Chicago and in 1917 he returned to that University as a graduate student and Fellow in the Department of Zoology. After serving in the Army Neuro- surgical Laboratory, Baltimore, during 1918, I.'r. Cross was ap- pointed Assistant in the Department of Pathology and Bacteriology Johns Hopkins University. He enrolled in the graduate school of this University in 1920 and completed the work necessary for his den-ree under Professor iiacCallum. (64) Bibliography 1. Ivlac Call urn, '/» . i}., A Text, Book of Pathology, Kew York, 1920, 528. 2. Osier, Wm., Practice of L'edicine, Mev; York, 1901, 165. 3. Flexner, S., J. Exper. M., N. Y., 1896, I,. 559. 4. Vv'right, A. S. and Windsor, F, V. . , J. 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Dis., Chicago, 1910, VII, 698. 34. Fleming, A.^ Practioner, Lond., 1908, LXXX, 607. 35. V/right, A. E. and Douglas, S. r. . , Proc. Roy. Soc, Lond., 1904, LXXIII, 128. 36. 3ordet, J., Studies in Immunity, New York, 262. 37. Metchnikoff, E., Ann. de I'Inst. Pasteur, P;ir., 1901, XV, 665. 38. Hektoen, L. , J. Am. M. Ass., Chicago., 1911, LVII , 1579. 39. Tunnicxiif ,^ R. , J. Infect., Chicago, 1911, VIII, 302. (65) 40. Bordet, J,, Studies in Immunity, New York, 1909, 13. 41. Zinsser, H. , Hopkins, J., and KcBurney, r.. , J. Expex'. Med., N. Y. , 1916, XXIII, 341. ■ 42. Klieneberger, G. and Carl, W., vie Blut-Morphologie der ■Laboratoriums-Tiere , Leipzig, 1912. (66)