.. yap. ‘- fit.“ 3r, ‘ Onyx} ”a.“ mi In... new. nun . G ? Aiiéfi C 3Q RU {1' E {"7 l ‘t 9" ‘ 605 E .17 to. 551’: a: i». W "i V 2 h a I an ”Mn .1. 3? id C .1 :x.' 1 r ‘i r". .r-W q {'1 ‘w ‘ pd “got: u ‘3 3 f I UN "" L a 3353’ . 5A R} 'E .3. ('1 I? 1" L mw- aka-.82“: \, 3 «,5 AV S W C a: ¢ 4 . mu.» 8 mm W a s \. .mu 1.. «U .‘gf:2:=__,__‘__:_3_____;__:5;_‘__.__ mmm M-795 Date -,+~._-v.~.. .— on -g-¢ n‘JI-M”- This is to certify that the thesis entitled presented by has been accepted towards fulfillment of the requirements for AJ—Q: _ _degree in _/;_‘:‘:(Z:z" ”‘4' 77 - “”‘-_"‘—__—V*'—_ a?" "OBSERVATIONS ON THE EFFECT OF CHICKEN BLOOD PIASEA.AND SERUM ON THE GROWTH OF $322014th PULLORD“-Ii." BY WONG Y . ml A.TEESIS Submitted to the School of Graduate Studies of Michigan State College of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Bacteriology and Public Health Year December 1946 TABLE OF CCLTELTS Page Introduction 1 Review of literature . 2 Materials and Kethods 7 Table No. 1 10 Table K0. 2 11 Table NO. 3 12 Table N0. 4 13 Table No. 5 14 Table N0. 6 15 Table No. 7 16 Table No. 8 17 Table No. 9 18 Table No. 10 19 Table No. 11 20 Table to. 12 21 Table No. 13 ( 22 Discussion and Results 23 Summary. 27 References 28 Acknowledgement 29 .1 37885 1. INTRODUCTION The object of this experiment was to observe the bactericidal action of chicken serum and plasma. First, observations were made on the killing power of normal chicken serum and plasma and then on the killing power of infected and hyperimmune serum and plasma. For comparison, human serum and plasma were used. It was hoped that with the collective re- sults, thus obtained, a definite contribution toward a better understanding of Salmonella_pullorum disease in chickens would result. Much work has been done by others on.normal animal serum using various species of organisms. The bactericidal action of each serum varies with each species and strain of organisms. Since the bactericidal action varies toward each Species of organisms, it is necessary to test the Specific serum and plasma on Specific organisms. The first work concerning the bactericidal action of chicken serum on S. pullorum was done by Bahler, Hodes and Hartsell in 1941. They experi- mented on the bactericidal action of normal chicken serum on g. pullorum and found that the bactericidal property of fowl serum increased with the age of the birds. This work had emphasis on the bactericidal action of infected and hyperimmune serum and plasma as well as the normal serum. . e , . ‘ l r s - \ t‘ s I ~ ' f 4 , Q o 1‘ . J a . \ ‘ x. I x- - , u ' . I , t .. _. . v i O I - ~ 0 . T , »-v0' «3 lo REVIEW'OF LITERATURE A revieW'of the literature shows that much work on the reactions of various serums was done by Nuttall. The classical observations of of Nuttall1 (1888) and many others first contributed to our knowledge of serum reactions. It has been recognized that the bactericidal property of serum is a variable one, differing according to the animal species and the type of organism. There has been uncertainty regarding the Specificity or non-specificity of natural bactericidal effects. Muir and Browning2 (1908) reviewed the literature on this subject and studied the Specificity of these reactions by absorption methods. They found that treatment of a normal serum with increasing amounts of bacterial suspension produced first a diminution of the bactericidal action towards the homologous bacterium, and also a decrease in the effect of natural complement-fixing and agglutinating antibodies. This sug- gested the likelihood that the bactericidal effects of normal serum may be due to multiple Specific antibodies sensitizing bacteria to the lytic action of complement. Thjotta:5 (1919) has Shown that during immunization there is produced along with the antibodies, a complement-inhibiting substance which he believes to be separate and distinct from agglutinins, precipitins and bactericidal amboceptor. If sufficient dilution and if extra complement is added, the serum will show bactericidal action, while undiluted, fresh immune serum, mixed with the homologous organisms, exhibits little if any bactericidal effect. Gordon and Wormall4 (1928) have shown how bacteriolysis of Shigella _dysenteriae (Flexner) by normal guinea pig serum depends on the combined ,. I35 action of complement and a thermostable factor removed from the serum by absorption with particular organisms. The question is further compli- cated by the fact that different mechanisms may be concerned in the bactericidal action of normal sera and that the facters involved may vary with different organisms. The killing of anthrax bacilli by normal serum was attributed by Gruber (1908) to a product of blood platelets (plakanthrakocidine) which is independent of complement. Leucocytic extracts (leukins) have been shown to possess marked bactericidal proper- ties for certain bacteria (Ledingham 1922). Pettersson5 (1928) has classified the bactericidal agents of serum into alpha lysine and beta lysine. The former apparently represent the complement acting along with a sensitizing agent analogous to an immune body. The latter, according to Pettersson, consist of a stable "activating”agent (resisting a temperature of 63° C. for 1/2 hour) and an activable principle which unites with the bacteria in the presence of the activing agent. Finkelstein6 (1931) summarized his results as follows: 1. An analytical study has been.made of the mechanism of natural bactericidal action by the serum of various animals towards certain organisms exhibiting the maximum reactivity to this effect. 2. The serum-complement has no bactericidal action by itself and an antibody-like agent invariably acts as an intermedi- ary agent, "sensitizing" the particular organism to the action of the complement and capable of being "absorbed" by it from s erum at 0° C. 8. This sensitizing agent is stable at 55° C. but labile at GOO-65° C. In this respect it resembles natural hemolysins 4. and agglutinins, but contrasts vdth the more stable immune antibodies and the more labile nature comple- ment-fixing antibodies. 4. The absorption tests demonstrate the high degree of specificity of these natural bactericidal antibodies forparticular bacteria. 5. .A.non-sPecific extracellular substance occurs in bac- terial cultures which may neutralize or inhibit these antibodies, and interfere with their sensitizing action even at 00 C. This substance is liberated in large amounts in cultures heated to 1200 C. In 1932. Finkelstein7 demonstrated that the bactericidal property of normal serum towards gram-negative bacteria is labile at 55° C. for 30 minutes; the factors reaponsible for the corresponding effect on gram-positive bacteria are stabile at this temperature. Thus the gram- negative and gramrpositive organisms are acted on by separate mechanisms, the "thermolabile" and ”thermostable" bactericidins reapectively. Bactericidal effects are more frequent and pronounced towards the gram- negative than the gram-positive bacteria. The "thermolabile" bactericidin consists of complement and a sensitizing antibody. The lability of the bactericidin is due to the lability of the complement. The antibody is stable at 60° C. and specific for the particular organism acted on. The "ihermostable" bactericidin in undiluted serum withstands a temperature of 57.50 C. though labile at 60° 0.; its lability is considerably increased in diluted serum and in slightly alkalmized serum though unaltered by slight acidity. The work 0f Gordon8 (1933) demonstrated that the absorption of both normal and heated sera by dead bacteria fails to yield any evidence of the existence of a series of Specific antibodies in serum. The loss of bactericidal power consequent upon absorption is never Specific for the absorbing organism but is always general. Liudd9 (1933) showed that sensitization by serum renders various dissimilar bacteria similar with respect to their surface properties. This convergence of surface properties is carried further by homologous immune than by heterologoun or normal sera, and the homologous immune sera are effective in higher dilutions. The work of Gordon and Johnstone10 (l940)also Showed that the absorption of a normal serum with a series of strains of one organism causes a general diminution in bactericidal poweri?or all the strains, but there is a more striking diminution forihe strain with which the serum was absorbed. Three strains of Micrococcg§.3atarrhalis was used to absorb the guinea pig serum. They also demonstrated that the complement titer of guinea pig serum was high, of human serum low and of rabbit serum still lower. The results shows low bactericidal action of human serum on the gonococcus, whereas guinea pig serum with a higher and rabbit serum with a lower complement titer were both markedly bactericidal. In this experiment one human serum had no bactericidal action on Vibrio cholerae but another human serum killed Vibrio cholerae in one hour. The rabbit serum, which had a lower complement titer than the guinea pig serum, was again.the more bactericidal, and inactivation of complement completely destroyed the bactericidal action of both sera. Gordon and Johnstone showed that many species Specific antibodies can be individually absorbed or that there is a general bactericidal anti- body which can betn modified by contact with.a large excess of any particular organism or strain as to render it specifically inactive 6. for that organism or strain. Bahler, Hodes and Hartsellll (1941) Studied the normal bacterici- dine of the domestic fowl. They found that bactericidins, active against a certain strain of §, pullorum, vary with the age of the bird, appearing first at 37 days of age, increasing to a maximum of 143 days and showing fluctuating activity for 164 days. The presence of sodium citrate in the plasma did not cause any reduction in the bactericidal action of most samples. LATBRILLS AhD RETJODS Preparation of culture. A smooth strain of Salmonella ppllorum (p. 19) was used in this experiment. The organism was grown on nutrient agar slants for 24 hours at 37° C. The growth was removed by means of a sterile‘wire loop and suspended in sterile diluting fluid, which consisted of 0.05% tryptose peptone at 0.5% sodium chloride in distilled water. Ten ml. of this diluting fluid was usually used for each agar slant. The bacterial suspension was transferred into a sterile test tube then thoroughly mixed and then diluted to a scale of 40 on the photolometer. 010 The standard suSpension contained from 8 x 101 to 8 x l of live §, nullorum organisms per ml. Serial dilutions ranging from 8 x 10"2 to 8 x 10"6 were made from this suSpension in the same diluting fluid. The number of organisms present was determined by plating 0.5 ml. of 10"9 and 10"8 dilutions. The pour plate method was used. The nutrient agar was melted, cooled to 45° C. and poured into the Petri dish.which was rotated to mix the content well before the agar solidified. When the agar was solidified the plates were incubated for 3 to 4 days at 37° C. and then the colonies were counted. The initial number of bacteria added to the blood plasma or serum from differentciilutions can be calculated by multiplying the number counted by the dilution factor. Preparation of blood. Each bird was tested for S. oullorum infection by using the stained antigen rapid whole blood test. Blood for the bactericidal test was drawn aseptically from the heart of the bird and placed in a sterile bottle. For the tests requiring 8. plasma 0.1 ml. of sterile saturated sodium citrate solution for each 10 ml. of blood was placed in the bottles. 'When heparin was used as a substitute for the sodium citrate, 0.1 ml. of heparin.was used for every 10 ml. of blood. The plasma was separated from the whole blood by centrifuging at 2,500 R. P. M. for 25 minutes. The supernatant was poured off aseptically into another sterile tube. Serum was obtained by allowing the blood to clot after which it was centrifuged if necessary. All tests were madewithin 24 hours after collection of blood. During this period the blood specimens were kept in an ice box (4° 0.). Infected chickens were hyperimmunized by injecting 1. ml. of live .§. pullorum suSpended in saline (8 x 1010) intravenously. One week later 1 m1. of dead 8. pullorum suSpension (susPension was boiled to kill the organisms) was injected into the same chicken intravenously. Blood was drawn a week after the last injection. The purpose of using the live and dead organisms was to raise the antibody titer to the mAximum. - The bactericidal test. In order to Show the maximum bactericidal activity of serum or plasma on S. pullorum, two methods of setting up the tests were used. In one the volume (1 ml.) of undiluted serum or plasma was kept con- stant in a series of tubes to which.were added the same volume of diluting fluid containing live organisms in varying numbers. In the second, serial dilutions of serum or plasma, in 1 ml. amounts, were placed in sterile tubes to which were added the same volume of diluting fluid containing a constant number of live S, pullorum. The tubes were shaken and incubated at 37° 0.; the length of time varied with the experiment. At the end of the period of incubation 0.25 ml. of the mixture was taken from each tube and placed in a sterile Petri dish. halted nutrient agar was cooled to 45° 0. Ten ml. amounts were then poured into each Petri dish. The contents were mixed by rotation and'then allowed to harden after which the plates were incubated at 370 C. for 3 days. Colony counts were made and compared with hose of the control tubes. The same procedure was repeated at the end of 4 hours, 8 hours, 24 hours and 48 hours. Table No. 1 shows how the dilutions are being prepared and the amounts of §, pullorum suSpension used. TABLE NO. 1 DILUTICNS OF SAHZCNLLIA PULLORDM SUSPVZSION USES IN THIS EXPERIMENT Tubes 1 2 5 4 5 6 7 8 9 10 Saline 9. m1. 9. m1. 9. ml. 9. ml. 9. mi. 9. m1. 9. m1. 9. m1. 9. ml. 90 m1. Added Pullorum 1. ml. 1. ml. 1. ml. 10 ml. 10 ml. 1. m1. 1. ml. 10 ml. 1. m1. 1. ml. Suspension Added -—1-‘? -—r-> “———l -->- ——J+ —-—-~+ -—-—«r+ ~-r~> "V” “1+ Dilutions l-lO l-lOO l-T l-lOT l-lOOT l-M l-lOM l-lOOM l-B l-lOB line milliliters of saline solution (containing 0.05% tryptose peptone and 0.5% sodium chloride) were placed in.all test tubes, then pipetted 1. m1. of SUSpenP si1>n into the first tube. The content was well mixed, then pipetted 1. ml. of tube no. 1 mixture into tube no. 2 and mixed. The following tubes were carried out likewise. #3 " Thousand E: n Million U1 " Billion TABLE KO. 2 AGGLUTILATION TEST I ! ZMmber 0f Dilutions Chicken. 1 1/20 1/40 1180 1/160 112120 1Z640 1/1280 i/zsso 1/5120 Jbrmal No. 533 ‘++ NO. 530 4'4' NO. 546 ’4' Infected No. 45 ++++ ++++ ++++ +++ ++ No. 563 ++++ ++++ ++++ +++ +4 + No. 591 ++++ ¢+++ ++4+ +++ +4 + Hyperimmune No. 508 ++++ ++++ ++++ ++¢+ +4++ +++4 9+4 *4 + 30. 555 ++++ ++++ $+++ ++++ ++++ ++++ ++4 4++ + No. 588 ++++ ++++ ++++ +4++ ++++ 444+ +44 +4 + ruman E0. 1. ++++ +++ + The Salmonella pullorum antigen was adjusted to pH 8.2, then 1.9 m1. of the above antigen was placed in tube no. 1 and 1. ml. in the following tubes; pipetted 0.1 m1. of the testing serum into tube no. 1 and mixed well, then transferred 1. ml. into the second tube. The following tubes were carried out likewise. ”a. ._._. . V. __ .— —,. .. -—. _-.. . gem—w...— ___7-_.._. »- wv< ___.__. .4 n ”—4 . ._.._.__.._. ,_, .y—. ._.,..n,... , .._. .,_ ._~ - .o. _, H TABLE NO. 3 SODIUM CITRATE PLASMA Rate of Bactericidal Action of Normal Plasma acting Number of Colony Count Substance Bacteria Period of Incubation in Hours Added 4, 8 24 48 Undiluted 7 x 108 677 29 2 o blasma 7 x 107 29 3 o o 7 x lo6 2 1 o o 7 x 105 o o o o ' 7 x 104 o o o o [Biluted 1: 4 7 x 105 707 62 o o : 8 7 x 105 N 161 1 o 1:16 7 x 105 N N N N 1:32 7 x 105 N N N N 1:64 'Lx 105 N N N N Rate of Bactericidal Action of Infected Plasma Undiluted 7 x 108 N N N N Plasma 7 x 107 ‘ N N N N 7 x 106 46 35 N N 7 x 105 3 8 5 0 j x 104 o o o o Diluted ; - l: A a 7 x 105 N N N N 1: 8 7 x 105 N N N N 7. 1:16 7 x 105 N N N N ' 1:32 7 x 105 i N N N N 1:64 7 x 105 N N N N icontrol l 7 x 104 J N N L N N fii The results on this table show that the normal chicken plasma has a better bacteriéidal action than that of the infected dhicken plasma in vitro. N = Too numerous to count 0 = No growth TABLE NO. A SODIUM CITRATE PLASMA Rate of Bactericidal Action of Normal Plasma Reacting Number of Colony Count Substance Bacteria Period of Incubation in Hours Added :4 ' 8 24, 48 Undiluted 6 x 108 400 203 0 0 Plasma 6 x 107 15 6 O O 6 x 106 0 0 0 0 6 x 105 0 0 0 0 6 x 104 0 0 0 0 Diluted 1: 4 6 x 105 400 159 0 0 1: 8 6 x 105 N 967 501 105 1:16 6 x 105 N N‘ N N 1:32 6 x 105 N N N N , 1:64__ [.6 x 105 l N 4. N J_‘ N N Rate of Bactericidal Action of Infected Plasma Undiluted 6 x 108 N N N N Plasma 6 x 107 N N 500 84 6 x 106 126 121 14 7 6 x 105 22 23 1 0 6 x 104 _Ji #5 O 0 Diluted 1: 4 6 x 105 N N N N 1: 8 6 x 105 N N N N 1:16 6 x 105 N N N N 1:32 6 x 105 N N N N 1:64, 6 x 105 N N N N Lgontrol 6 x 104 I N J N N N The results on this table show that the normal chicken plasma has a better bactericidal action than that of the infected chicken plasma in.vitro also. TABLE NO. 5 SODIUM CITRATE PLASMA Rate of Bactericidal Action of Infected Plasma Reacting Number*of Colony Count Substance Bacteria Period of Incubation in Hours Added 4_ 8 24. 48 Undiluted 8 x 108 N N N N Plasma 8 x 10; N N N 1016 8 x 10 N 964 N 358 8 x 105 963 551 181 302 8 x 104 333 254 161 200 Diluted : 4 8 x 104 N 102 977 N : 8 8 x 104 N N N N 1:16 8 x 104 N N N N 1:32 8 x 104 N N N N 1:64. 8§_l04 N N N N Rate of Bactericidal Action of Infected Serum ’Undiluted 8 x 108* N N N N Serum 8 x 107 N N 967 N 8 x 106 N 988 664 N 8 x 105 699 232 433 N 8 x 104 201 199. ‘_J_ 349 N Diluted 1: 4 8 x 104 N N N N 1: 8 8 x 104 N N N N 1:16 8 x 102 N N N N 1:32 8 x 10 N N N N _lé§fii 8 x 104 N N N N [Contrqlp [ 8 x 104 I] N l N I N I‘— Ii The results on the above table indicate that the infected chicken plasma has a better bactericidal action than the infected chicken serum in prolonged incubation in vitro. TABLE NO. 6 SODIUM CITRATE PLASMA Rate of Bactericidal Action of Normal Plasma Reacting Number of Colony Count 1 Substance Bacteria Period of Incubation in Hours Added A A? 8 24, 48 Undiluted 8 x 103 1.121 987 13 11 Plasma 8 x 10; 452 46 10 4 8 x 10 42 5 4 O 8 x 105 15 0 0 o 8 x 104 2 0 0 0 D i In t e d 1: 4 8 x 104 16 1 0 0 1‘ 8 8 x 10"+ 411 62 3 o 1:16 8 x 104 N N 671 0 1:32 8 x 10" N N N N _1:64 @3104 N N N N Rate of Bactericidal Action of Normal Serum Undiluted 8 x 108 997 195 6 4 ' Serum 8 x 107 343 36 3 1 8 x 105 48 7 1 0 8 x 105 11 4 1 0 8 x l 4 3 l Ofifi Diluted ' l ' 1: 4 8 x 104 12 8 18 61 1: 8 8 x 104 992 48 254 N 1:16 8 x 10’+ N 123 N N 1:32 8 x 104 N N N N 1:64 8 5:104 N N N N [Control I 8 x 10" N N N N The results on this table indicate that the normal chicken plasma and the normal chicken serum have a slight variation in bactericidal action. TABLE NO. 7 SODIUM CITRATE PLASMA Rate of Bactericidal Action of Normal Plasma RaaCting I Number ofT Colony Count Substance Bacteria Period of Incubation in Hours Added A: 8 #17 24, 48 Undiluted 8 x 108 976 644 22 r 10 Plasma 8 x 102 368 87 5 7 8 x 10 73 17 3 6 8 x 105 15 7 1 0 8 x 104 6 2 1 2 Diluted ' 1: 4 8 x 104 48 4 2 2 1: 8 8 x 104 300 160 5 6 1:16 8 x 104 601 989 N N 1:32 8 x 104 N N N N 1 1:64 8 x 104 N N N N Rate of Bactericidal Action of Infected Plasma Undiluted 8 x 108 N N N N Plasma 8 x 107 N N 89 12 8 x 106 1099 877 19 8 8 x 105 382 249 7 5 8 x 104 210 90 5 L Diluted I 1: 4 8 x 104 485 273 27 17 1: 8 8 x 104 1110 988 N N 1:16 8 x 104 N N N N 1:32 8 x 104 N N N N 1:64 8 x 104 N N N N Control 8 x 104 N J N N N The above results again indicate that the normal chicken plasma has a more effective bactericidal action thap'that of the infected chicken plasma in vitro. TABLE NO. 8 HEPARIN PLASMA Rate of Bactericidal Action of Normal Plasma Reacting Number of Colony Count' Substance Bacteria Period of Incubation in Hours Added 4. 8 ' 24 y 48 Undiluted 8 x 108 N : 1126 g N z N Plasma 8 x IOZ 951 438 f N i N 8 x 10 139 ' 109 i N N 8 x 105 33 e 24 i N I N 8.5 104 10 1 20 ’ N . N biluted T E E T :1: 4 8 x 104 68 3 486 i N i N 1: 8 8 x 104 411 i N f N i N 1:16 8 x 104 ' N 3 N g N i N 1 1:32 8 x 10? N i N i N i N ' 1:64. 8 x 10“ . N 3 N ._E N ;_ N Rate of Bactericidal Action of Infected Plasma hndi1uted 8 x 108 g N g N N N {Plasma 8 x 10 g N N N N i 8 x 106 g 1000 3 1115 N ; N g 8 x 105 g 486 g 586 561 534 g__ 8 x 104 E 261;_ 3 .230 171 160 Diluted i 5 , ‘ g 1: 4 * 8 x 104 g 966 3 1153 N N 5 1: 8 l 8 x 104 g N § N N N E 1:16 8 x 104 i N t N N N 1:32 8 x 104 f N g N N N .41:64_ i 8 x 104 .f N * N' N N 3 'r j T i‘ m mmmmi i &le i it i N “ N i N The above results indicate that the heparin plasma has less bactericidal power than that of the sodium citrate plasma in pro- longed incubation periods. The sodium citrate may have some influence on bactericidal action. TABLE NO. 9 HEPARIN PLASMA Rate of Bactericidal Action of Normal Plasma Reacting Number of Colony Count Substance Bacteria Period of Incubation in Hours ' Added 4 8 24 48 Undiluted 9 x 108 N 1109 N N Plasma 9 x 10; 1188 179 N N 9 x 10 204 53 N N 9 x 105 58 28 N N 9 x 104 _39 5 ._911 N ‘Diluted 1: 4 9 x 104 12 5 N N 1: 8 9 x 104 82 408 N N 1:16 9 x 104 238 1112 N N 1:32 9 x 104 286 N N N 1:64 9 x 104 339_ N N N Rate of Bactericidal Action of Infected Plasma Undiluted 9 x 108 N N N N Plasma 9 x 102 N N 979 1010 9 x 10 1109 486 165 251 9 x 105 408 237 65 '76 9 x 104 188 96 _35 20 Dilutcd‘ 1: 4 9 x 104 66 118 126 300 1: 8 9 x 104 68 154 214 N 1:16 9 x 104 75 193 998 N 1:32 9 x 104 79 360 1121 N w_l:64; 9 x 104 87 394. N N l_90ntrol 1 9 x 104 N N_] N I N The above results indicate that the heparin plasma of the infected chicken has a better bactericidal action'thmz that of the normal chicken plasma in prOlonged incubation periods. TABLE NO. 10 HEPARIN PLASMA Rate of Bactericidal Action of Human Plasma Reacting Number of Colony Count Substance Bacteria Period of Incubation in Hours Adds; 4 8 24 18 Undiluted 9 x 108 N 346 746 N Plasma 9 x 107 1201 63 8 28 9 x l 246 9 4 l9 9 x 105 89 3 4 16 9 £2104 37 l 1 2 Diluted 1: 4 9 x 101,» 49 5 3 18 l: 8 9 x 104 96 9 6 28 1:16 9 x 104 182 66 53 63 1:32 9 x 104 468 445 980 N . 1:64k 9 x 104 551 886 N N Rate of Bactericidal Action of Human Serum Undiluted' 9 x 108 N ' N 142 i 55 Serum 9 x 102 1221 262 97 2 9 x 105 236 27 12 0 9 x 10 47 7 3 0 9 x 10" 9 _ l 9 0 Diluted 1: 4 9 x 104 1 0 0 0 : 8 9 x 104 6 l 0 0 1: 9 x 104 6 1 l 0 1:32 9 x 1 , 87 13 N N , 1:6é 9 x 104 119 82 N N Control 9 x 104 I N I N J N r N 1 fiw The above results show that the human plasma and serumare more bactericidal in prolonged incubation periods. TABLE NO. 11 HEPARIN -PIJ‘:SI‘:’IA OF THE HYPERIIMUNIZED CHICKEN Rate of Bactericidal Action.of'Infected Plasma Reacting Number of Colony Count Substance Bacteria Period of Incubation in Hours Added L r 8 24 48 Undiluted 12 x 108 N N N N Plasma 12 x 10'7 N N N N 12 x 106 N N 910 822 12 x 105 1211 1112 263 301 12 x 104 270 221 196 99 Diluted 1: 4 12 x 104 154 156 526 600 1: 8 12 x 104 161 334 710 889 1:16 12 x 104 170 412 762 N 1: 32 12 x 104 174 616 .1110 N 1:64 12 x 104 216 248 N N . 3 1 I Undlluted 12 x 10 N N N N Plasma 12 x 107 N N N N 12 x 106 N N 1088 418 12 x 105 1190 1144 892 408 912 x 104 245 225 222 78 TBiluted 1: 4 12 x 104 144 187 449 586 1: 8 12 x 1 155 257 460 578 1:16 12 x 104 176 286 650 N 1:32 12 x 104 191 408 780 N 1:84 12 x l 219 pg448 971 N 61 I 12 x 104 4 I N N I N I The above results indicate that the initial bactericidal action of the hyperimmune chicken plasma is about the same as the normal plasma, but as the incubation period prolonged, the hyperimmune chicken plasma is more effective in vitro. TABLE N0. 12 HEPARIN PLASMA Rate of Bactericidal Action of Normal Plasma Reacting Number of T Colony count Substance Bacteria Period of Incubation in Hours Added 4L_A 8 39: 48 Undiluted 9 x 1010 N N N N Plasma 9 x 103 N N N N 9 x 10.7 1431 492 N N 9 x 106 422 178 1250 N 9 x 105 116 32 0 226 9 x 10 14 6 O O 9 x 104 6 l 1 0 9 x 103 1 0 o 0 9 x 10% o O 9 0 _9 x 10 o a 0 0 Rate of Bactericidal Action of Infected Plasma I Undiluted 9 x 1010 N N N N Plasma 9 x 109 N N N N 9 x 108 N 1124 506 216 9 x 102 1074 750 207 52 9 x 10 275 234 101 8 9 x 105 57 69 48 25 9 x 104 8 11 5 o 9 x 103 1 2 0 o 9 x 102 1 0 0 o 3 x o 0 0 0 1 I ngtrgl I 9x105 T j 11L QN F NI trol 1 x 101 I 22 36 I N I N I The hyperimmune chicken plasma indicates a better bactericidal action than that of the normal chicken plasma in vitro. TABLE N0. 13 HEPARIN PLASMA Rate of Bactericidal Action of Mixed Plasma heating Number of Colony Count substance Bacteria Period of Incubation in Hours Added 4 8 3.4 L8. [ ndiluted 9 x 1010 N N N N d 9 x 109 N N N N Plasma 9 x 108 N 918 217 66 9 x 107 1024 317 123 35 9 x 106 211 146 66 33 9 x 105 51 31 25 1 9 x 104 3 6 3 O 9 x 103 2 4 1 0 9 x 102 0 0 o 0 9 x 101 0 0 0 9—4 The above table shows the results of mixed plasma. 2:2 ...._.1 The mixed plasma has 50% hyperiImnunized chicken plasma and 50% normal chicken plasma. than anyone plasma acting alone. It shows a better bactericidal action RESULTS.AND DISCUSSION Table No. 2 shows the results obtained where normal, infected, hyperimmune chicken sera and human serum are used on S. pullorum agglutinating antigen. The purpose of the agglutination test is to find out if the agglutinating titer has any relationship to the bactericidal action of serum. According to the results of this experiment, as shown in.Table No. 3, when.a limited number of S. pullorum was mixed with a definite quantity of normal chicken plasma none of the plates showed any colonies after 4 hours of incubation. The plasma either inhibited the growth or killed the organisms. Further eXperiments are necessary in order to prove whether the organisms were killed or merely inhibited. 'When the number of organisms was increased from 7 x 104 to 7 x 106 in 1 m1. of plasma it required about 8 hours of incuba- tion before no growth occurred on the plates. As the number of organisms increased, the period of incubation required for bactericidal action lengthened. 'When an excess number of organisms (8 x 108 to 1 ml. of plasma) was mixed with a constant quantity of plasma many colonies were present on the plates even after 48 hours of incubation, however, the number of colonies was always declining as the incubation period lengthened. Table No. 4 shows the results of normal and infected chicken plasma bactericidal action on g. pullorum also. The number of colonies are varied in.eech corresponding plate which indicates thelrariability of plasma in different chickens. In Table No. 5 the results indicate the number of organisms surviving in the infected chicken plasma and serum. It indicates that the infected chicken plasma has a better bactericidal action than the infected chicken serum. In Table N0. 6 the results show that the normal chicken plasma has a slight variation in bactericidal action when comparing it with the normal serum. In some cases the normal chicken plasma seems to possess a better bactericidal action than the normal serum. The sodium citrate added to the blood may have had some influence on the bactericidal effect. For this reason heparin.was used for comparison in the latter part of this experiment. Table Nos. 7 and 8 show the results of 2 sets of tests. Table No. 7 shows that sodium citrate was used and Table No. 8 shows that heparin was used. The same number of organisms were added to each set and were treated under identical conditions. The plasma which had theesodium citrate showed a continuous decrease of organisms while the plasma in.which heparin.was usedhad about the same number of organisms after the first 4 hours of incu- bation, but the decrease of organisms was less than that in the tubes containing szbdium citrate in the second 4 hours of incubation, however, showing a definite increase in number in the 24 and 48 hours' incubation period. This condition occurred when a large number of organisms were added to the heparin plasma tubes. By in vitro tests it was shown that plasma of the infected chickens is less effective than plasma of normal chickens. Table No. 9 indicates that the tubes of the infected chicken plasma showed a greater number of organisms surviving. Human plasma showed the same potency of bactericidal action as normal chicken plasma in the first44 hours of incubation, but it was more effective in the longer incubation period (this indivi- dual immunized against typhoid fever) as shown in Table No. 10. The plasma from hyperimmunized chickens seems to have a better bactericidal action than the normal chicken plasma. It had a more effective and prolonged bacteriostatic action somewhat like the normal sodium citrate plasma. (The results are shown in Table No. 11.) In Table No. 12 the results indicated that the hyperimmune chicken plasma had about the same degree of bactericidal action in the first 4 hours of incubation. As'the incubation period proceeded the tubes with an excess number of organisms (9 x 108) showed a de- crease in number of colonies, while those of normal chicken plasma tubes showed increased in number of colonies. Table No. 13 shows the results when mixed plasma is used. The mixture consisted of 50% hyperimmunized plasma and 50% normal plasma. It indicates an even better bactericidal action than when either normal or hyperimmune plasma alone was used. Chicken plasma and serum showed a noticeable bactericidalsiction in dilutions up to 1:8. When the dilutions were higher, the bactericidal effect diminished rapidly. Human plasma and serum showed a noticeable bactericidal action in dilutions up to 1:16. The agglutination test showed that the normal chicken serum agglutinated E; pullorum at 1:20 dilution. The infected chicken serwmlagglutinated E3 pullorum at 1:520 dilution. 26. The hyperimmune chicken serum agglutinated §, pullcrum at lflfifio dilution. The human serum agglutinated é, pullorum at 1:40 dilution. (Individuals had been vaccinated against typhoid paratyphoid.) Serum with the increase of agglutinating titer generally shows the increase of bactericidal action also, but this does not hold true with the infected chicken serum or plasma. The failure of plasma of infected chickens to exert a more effective bactericidal action may be due to any one of the following factors: (1) its low complement content, (2) the absence of complement- Binding groups on a portion of the bactericidal antibodies, or (3) the presence of anticomplement. Although the infected chicken plasma may not kill many bacteria per volume concentration in vitro, the agglutination factor alone may aid the prevention of bacteria from Spreading in vivo, which could be a protective factor or mechanism. SUMLARY The normal chicken serum and plasma have a better bacteri- cidal action than the infected chicken serum and plasma. The human serum and plasma have a more effective bactericidal action than the normal chicken serum and plasma only in prolonged incubation periods. The hyperimmune chicken serum and plasma possess a greater bactericidal action than the normal chickentserum and plasma. The mixed chicken plasma (50% hyperimmune chicken plasma and 50% normal plasma) has the most effective bactericidal action. 1. 2. 3. 4. 5. 5. 7. 9. 10. ll. 12. 28, REFERENCES Iuttall, G. F. H., 1888 - 1904; Blood Immunity and Blood Relationship and Zeitschr F. Imnuitats 4, 353 huir, R. and Browning, C. H., 1908; on the Bactericidal Action of Normal Serum, Jour. Pathology and Bacteriology 13, 76 Thjotta, 1919; on the so-oalled Neisser - hechsberg Inhibiting Phenomenon in Bactericidal Immune Sea Jour. Immunology 5, 1 Gordon, J. and'Wormall,.A., 1928; The Relationship Between the Bactericidal Power of Normal Guinea Pig Serum and Complement Activity, Jour. Pathology and Bacteriology 31, 758-768 Pettersson, Am, 1926 - 8; Zeitschr F. Immunitats 48, 233 Mackie, T. J. and Finkelstein, M. H., 1931; Natural Bactericidal Antibodies: Observations of the Bactericidal mechanism of Normal Serum, Jour. Hygiene 31, 35-55 Mackie, T. J. and Finkelstein, M. H., 1932; The Bactericidins of hormal Serums; Their Character, Occurrence in Various Animals, Jour. Hygiene 32, 1-24 Gordon, J., 1933; The Bactericidal Power of Normal Serum, Jour. Pathology and Bacteriology 37, 367-386 hudd, S., 1933; Sensitization of Bacteria'With hormal and Immune Human Serum, Jour. Immunology 26, 447-454 Gordon, J. and Johnstone, K. 1., 1940;’ The Bactericidal Action of Normal Sera, Jour. Pathology and Bacteriology 50, 483-490 Bahler, Hodes and Hartsell,,l94l; The Studies on the Normal Bactericidins of the Domestic Fowl, Jour. Bacteriology 41, 102-103 Huddleson, I. F., 1945; The Bactericidal.Action<>f Bovine Blood for Brucella and Its Possible Significance, Jour. Bacteriology 50, 261-277 ACKL‘IO‘LED GLIJJLT The writer wished to acknowledge his apprecia- tion to Dr. H. J. Stafseth, Dr. J. Bivins and hrs. V. Bleil for their assistance and helpful suggestions during the course of these studies. will {11111118111311 fillijlfllflhllflifli fills