THE USE OF $YSLEESYROL LYSOZYME AND POLYVALENT VACCINES IN THE CONTROL OF EXPERIMENTAL STAPHYLGCDCCAL {INFECTIONS H4 LABORATORY ANIMALS Wait fear flue W cf {511. O. MICHIGAN STATE UNiVERSITY Chesfier A. Hamback 3962* THIS” This is to certify that the thesis entitled The Use of Stllbestrol, Libozyme, and Polyvalent Vaccines in the Control of Experimental Staphylococcal Infections in Laboratory Animals presented by CHESTER A. HORNBECK has been accepted towards fulfillment of the requirements for Microbiology and Public Health L/:/Z‘ Uf/ per cm width of strip produced separations of 6 to 8 cm after eighty min- utes. After the separation was finished, the strips were immersed in fresh 3% aqueous trichloroacetic acid (TCA) for 5 minutes, then stained with Ponceau S stain (2% in 3% TCA) for 5 minutes. Clearing of the strip background was accom- plished by washing the strips in three successive trays of 5% acetic acid (Korotozer et al., 1961). The strips were dried under pressure to prevent curling. The stained frac— tions were excised and eluted with 0.1 N sodium hydroxide. The optical densities at 560 mu were found, and subsequently the percent protein determined from the curve (Figure 3). 6Consolidated Laboratories, Inc., Chicago, Illinois. 32 Serum transferases Three enzymes, serum glutamic oxalacetic transaminase (SGOT), serum glutamic pyruvic transaminase (SGPT), and ornithine carbamyl transferase (OCT) were included in this study (Molander et al., 1953; and Wroblewski and La Duce, 1956). The enzyme SGOT catalyzes the following general reaction: a Ketoglutarate + L-Aspartate-—a-Glutamate + Oxalacetate The enzyme SGPT catalyzes the following general reaction: a Ketoglutarate + L-Alanine-—o-Glutamate + Pyruvate In the method used for this study (Reitman and Frankel, 1956, modifying the Karmen, 1955 procedure) the amount of oxalacetate or pyruvate which forms a highly colored hydra- zone with 2,4-dinitrophenyl hydrazine hydrochloride in sixty minutes is determined colorimetrically. The calibration curve, Figure 4, for this analytical procedure plots optical density (505 mu) against arbitrary Sigma-Frankel units per m1 as well as certain ratios of pyruvate to o—ketoglutarate. The enzyme OCT catalyzes the following special reaction: Arsenate‘_ Citrulline Ornithine + Carbon Dioxide + Ammonia This reaction forms the basis for determining OCT activity by measuring the rate of ammonia formation when '71 0.4 0.2 Change in Optical Density (505 mp) igure 33 Sigma-Frankel units 20 4O 60 80 IOO l l l l _J SGPT b SGOT )— 1 1 1 J g 0.4 0.6 0.8 |.8 I .6 I4 I 1.1M Pyruvate pM m-keloglutorote 4 Relation between the activity of transaminase and change in optical density, expressed both in Sigma- Frarkel units and as concentration of pyruvate and corresponding decrease in(1~ketog1utarate (total 2 HM), as measured by their 2,4-dinitrophenyl hydrazones. 34 Citrulline is incubated in the presence of an arsenate. This method (Reichard, 1957) determines the amount of am- monia absorbed by 0.01 N hydrochloric acid in a Conway microdiffusion unit by nesslerization. Optical density at 410 muwes compared to the standard curve (Figure 5), with results stated in uM ammonia per 0.5 ml serum. Serum samples from each animal were measured prior to treatment, post—immunization, and post-challenge. Optical Density (4/0 my) 0 .0 m on .0 .n 0.2 Figure 5. Lu 1 l 1 L 0.5 I.O l.5 2.0 .uM of NH3 per 0.5 ml Serum Reference calibration curve measuring ornithine carbamyl transferase expressed as HM of KH3 per 0.5 m1 of serum (determined by.nesslerization) plotted against optical density (410 mu). 2.5 RESULTS Untreated and Experimentally Infected Mice The data in Figure 6 show the disappearance of staphy- lococci from the blood as well as the uptake by certain tissues at several intervals following a caudal intravenous challenge dose of l x 107 organisms. The intravenous route of inoculation was selected as the most desirable since preliminary experiments showed survival time was the longest when compared to either the intracerebral or intraperitoneal route. It was assumed also that this route of injection produced an infection which most closely resembled the staphylococcal septicemia occurring in clinical cases. The greatest number of viable organisms was noted in the blood sample where there was a thousand-fold reduction in viable organisms in fifteen minutes; and after thirty min- utes there remained only 0.1% of the original number injec- ted. The liver retained 90% of the infecting dose at thirty minutes while the lungs accounted for 1%. After two hours, the kidneys showed an increase over the initial number of staphylococci injected which indicated an early establishment 36 Viable Count (log) LL) \1 oKidney _. ALiver ALung OBlood 8 l- l 6 _. 4 _ \ 1 l l [#3\ 4 fi 30 60 90 l20 I2 hrs. Time (minutes) Figure 6. Viable counts per whole tissue taken from mice after intravenous injection with the Smith strain of Staphylococcus aureus. 38 and reproduction in this tissue. Assays beyond twelve hours showed incomplete removal of bacteria from blood, liver, and lungs; although levels were very low in these tissues, the kidneys consistently harbored a much higher population. Effect of Stilbestrol upon Experimental Infection Table 1 lists the viable counts of staphylococci per liver at two intervals of time after challenge of the mice undergoing treatment with stilbestrol via various routes of administration. In all groups of animals with and without treatment and regardless of route of administration, the liver captured from 7 to 20% of the challenge. However, the number of viable organisms measured at the second interval was considerably smaller than the first. The result of the intravenous injection of the challenge dose of staphylococci as measured in the bloodstream is shown in Table 2. In both the control group and those re— ceiving the oral and subcutaneous stilbestrol, there was found a marked reduction in numbers of bacteria. However, the group receiving stilbestrol intravenously showed a bacterial reduction of only approximately one-quarter as much as the others. 39 TABLE 1. Viable cell counts per liver of mice treated with stilbestrol via various routes after challenge with Staphylococcus aureus. Liver Counts Route Dosage After Challenge Period 20 min 70 min X 105 X 105 1 Oral 6 days 7.5 3.2 12 days 15.0 11 0 2 Subcutaneous Daily for 5 days 20.0 5.2 3 Intravenous Single injection 13.0 9.0 4 Control ---------------- 11.0 5.0 1Mice were fed orally ad libitum with prepared Rockland mouse diet containing 100 ug of stilbestrol per gram of feed. 2Mice were given a daily injection of 500 ug of stil- bestrol in 0.1 ml of corn oil. 3Mice were injected with 250 ug of stilbestrol contained in 0.05 ml of 50% ethanol. 4Control mice were fed plain Rockland mouse diet during entire experimental period. 5Challenge dose of 10 million organisms was adminis- tered IV. 40 TABLE 2. Viable cell counts per m1 of blood of mice treated with stilbestrol via various routes* after chal- lenge with Staphylococcus aureus. Liver Counts Route Dosage After Challenge Period 20 min 70 min 3 3 X 10 X 10 Oral 6 days 12.1 5.3 12 days 9.3 4.3 Subcutaneous Daily for 5 days 12.6 4.7 Intravenous Single injection 42.0 18.2 Control ---------------- 10.0 2.6 * Experimental conditions same as in Table l. 41 Table 3 shows the survival time after experimental in- fection of all groups of mice regardless of treatment. Of the untreated controls only 5% failed to survive at least thirteen days. With the exception of 20% of the group which were treated with stilbestrol subcutaneously, all other stilbestrol treated animals died after the experimental infection. Under the conditions of this experiment stil- bestrol exerted no prophylactic action against the staphy- lococcal infection. In comparison with the oral control group which received no medication, the control groups receiving plain corn oil and plain 50% ethanol showed some decreased resistance to the challenge dose. Effects of Lysozyme upon Staphylococcus aureus Ih_vitro studies Typical results of the effects of lysozyme and coagulase upon each other are shown in Table 4. In concentrations of 100 1%? per m1, lysozyme exerted no demonstrable antagonis- tic or synergistic effect upon coagulase. None of the coague lases from the seven strains had any effect upon the lysozyme. It was observed also that incubation for eighteen to twenty hours at 37 C had little effect upon lysozyme activity. 42 TABLE 3. Survival of mice challenged with Staphylococcus aureus after treatment with stilbestrol via var- ious routes.* Adminis- Survi— Mor- Maximum tration Dosage Period vors tality Days of Route Total Survival 96 Oral 6 days 0/15 100 12 12 days 0/15 100 4 None (control) 19/20 5 13 Subcutaneous Daily (5 days) 4/20 80 6 None (control) 17/20 15 5 Intravenous One injection 0/15 100 2 None (control) 8/10 20 5 * Experimental conditions as in Table 1. 43 TABLE 4. The inactivity of lysozyme and coagulase upon each other during active growth of Staphylococcus aureus. Actual Tests Activity Index** Strain Supernatant Supernatant Supernatant Fluid Plus Supernatant Fluid Plus Fluid Lysozyme* Fluid Lysozyme Reciprocal Coagulase Titer 3C Negl Neg 0.93 4.70 6 Neg Neg 1.00 4.30 70 1280 1280 0.98 5.05 44A 640 640 1.00 4.90 Smith 640 320 0.98 4.00 7 320 320 0.95 5.20 80/81 160 320 0.95 4.80 Test Controls Medium (uninoculated) 1.00 Supernatant fluid after growth 0.98 Supernatant fluid and lysozyme after growth 5.20 Fresh medium and lysozyme 5.15 *100 pg per ml, diluted 1:10 for testing. substrate control 540 test *‘kOD lNegative at 1:20 dilution. 44 Table 5 summarizes the effect of lysozyme on total bacterial growth. In the case of three strains there was a very slight reduction in optical density, while there was an opposite effect shown by the other four. Apparently lysozyme had no appreciable effect at the concentration of 100 ug per ml of medium. Results of lysozyme inhibition experiments are sum- marized in Table 6. Dilutions of 1:5 and 1:10 both serum and globulin, showed complete inhibition of lysozyme with respect to the controls containing substrate only. There seemed to be little if any difference between the antiserum or globulin in potency. Since the data shown in Table 4 gave little indication of lysozyme inactivation by growing cells, it was decided to expose lower concentrations of lysozyme to increasing numbers of various strains of staphylococci. The organisms were harvested from BHI broth, and washed three times prior to mixing with the lysozyme solution. After the mixture was allowed to stand approximately eighteen hours at 37 C, it was centrifuged, and the supernatant examined for enzyme activity. The sedimented organisms were washed four times with phosphate buffer, and resuspended in buffered 45 TABLE 5. The relative inactivity of lysozyme upon growth of various strains of Staphylococcus aureus* in vitro. Strains Medium Only Medium Containing Lysozyme X 108 X 108 3C 9.5 8.5 6 12.1 14 5 70 20-0 21.0 44A 15.5 14.0 Smith 11.1 13.2 7 34.7 31.2 80/81 23.5 24.5 * Number of organisms after 18 hours incubation. 46 TABLE 6. Lysozyme inhibition by antilysozyme serum and antilysozyme globulin. Test Dilution of Inhibition Material Antibody Material Index* 1 5 1.00 1 10 0.98 Pooled rabbit sera 1:20 0.77 1 40 0 71 l 80 0.34 Control** 1.00 l 5 1.00 l 10 1.00 1:20 0.83 Globulin 1:40 0.67 1 80 0 33 Control** 1.00 Lysozyme controll 5.90 Control * Rec1proca1 of OD540 Test ** Serum (or globulin) and substrate only. 110 pg per m1. 47 physiological saline for agglutination tests. Figure 7 reflects the adsorptive capacity of four strains, 6, 80/81, 7, and Smith, previously mentioned in Table 5. It can be seen that all strains caused decrease in the lytic activity of the lysozyme solution. No one strain appeared to display superior adsorptive ability. Further, the most adsorptive strain, PS 6, removed less than 50% of the initial lysozyme activity present before absorption with the cells. lh_vivo studies The effectiveness of lysozyme as a therapeutic agent in experimental infections of mice was examined. The pre- sumed prophylactic dose of 1000 ug in 0.1 ml was given simultaneously or just prior to the challenge infection. The observed averaged results are shown in Table 7. Re- gardless of treatment route, groups receiving lysozyme all showed increased survival rates over the untreated controls. The highest survival rate was found among those receiving lysozyme immediately after challenge. The control animals receiving lysozyme only showed no untoward effects. The effect of in vivo administration of antilysozyme globulin upon subsequent sublethal challenge infection in Optical Density ( 5 4 0 my I 0.8 r /$Ub$lrate Control (no lysozyme) l. 1 1 Of) 3 6 9 I2 Number of Cells )thI0 Figure 7. Decrease in lytic activity of lysozyme solution (10 ug per ml) after absorption by each of four strains of washed staphylococcal cells. 49 TABLE 7. Effect of lysozyme upon experimental staphylo- coccal infection in mice. Treatment with Challenge Survivors . Surv1vors Lysozyme Dose Total % Intravenous Simultaneous 18/30 60 Intracutaneous Preceding treatment 21/32 66 Intracutaneous Following treatment 23/31 74 None Yes 0/15 0 Intracutaneous None 15/15 100 Intravenous None 15/15 100 50 mice was investigated. Typical results are given in Table 8. There appears to be some interference with the defense mechanism of the host by the antilysozyme globulin as in- dicated by a decrease in survival percent. Additional experiments where treatment was given several hours before challenge showed survival rates of more than 80%. Treatment several hours after challenge gave results slightly more comparable to the control group which all survived. Effect of Various Vaccines A series of rabbits immunized with partially purified coagulase was challenged by intracutaneous injection using thirteen strains of staphylococci. The degree of resulting erythema and pyogenesis was measured. The results are presented in Table 9. It is noted that the coagulase- treated animals showed an exaggerated response to the challenge dose over the controls. With respect to degree of erythema strains 77 and 83 produced the greatest contrast over the controls. Polyvalent vaccine, inactivated by one of three methods, was administered to another series of rabbits. The response to intracutaneous infection is summarized in Table 10. 51 TABLE 8. Effect of antilysozyme globulin upon sublethal experimental staphylococcal infection of mice. Route of Treatment Challenge Survivors Survi; Treatment Dose Total vors % Intracutaneous Yes Preceding treatment 4/10 40 Intracutaneous Yes None 10/10 100 None Yes 10/10 100* * Two mice ill, but survived experimental period. 52 TABLE 9. Responses to intracutaneous infection of rabbits immunized with partially purified coagulase. Immunized Rabbits Controls Phage Strains Erythema Pyogenesis Erythema Pyogenesis Avg cm Avg cm 6, 55, 73 0.9 — 1.0 0.1 0.8 0.1 187, 3A, 42D*, 53, 70, 81 1.1 - 1.2 0.3 1.0 0.1 29, 80* 1.3 - 1.4 0.4 1.1 0.2 77, 83* 1.5 - 1.6 0.5 1.0 0.3 * Homologous strains. 53 TABLE 10. Responses to intracutaneous infection of rabbits immunized with polyvalent vaccine. Vaccine and Immunized Rabbits Controls Administration Erythema Pyogen— Erythema pyogen_ esis esis Avg cm Avg cm , 1 Multiple I.C. A-H * 0.8 0.05 A_F ** 0.4 < 0.05 0 9 0.1 A_Z*** 0.1 < 0.05 2 I.M. and I.C. A-H 0.6 0.05 A-F 0.5 0.05 l 0 0-1 A-Z 0.2 < 0.05 *A-H is heat inactivated **A—F is formalin inactivated ***A-Z is Zephiran inactivated lMultiple intracutaneous injections 2 . . . Intramuscular injection (1), intracutaneous injection (1). (8 - 10) followed immediately by Repeated twice. 54 The data presented in this table indicate that the heat- inactivated vaccine was the least effective of those tested. Little protection was evident when the intracutaneous route of immunization was used. Formalin inactivated vaccine in both cases was of intermediate effectiveness. Again, in both methods of immunization, Zephiran seemed to be the most efficacious inactivating agent when administered by multiple intracutaneous injection. Rabbits, previously immunized with coagulases from S, aureus strains of phage groups I and II, were injected with the Zephiran-inactivated polyvalent vaccine intradermally in order to determine if any increased protective effect could be demonstrated upon challenge. It can be seen from Table 11 that the control animals responded to the challenge dose with a smaller degree of erythema and pyogenesis than those injected previously with coagulase(s) and polyvalent vaccine. Pyogenesis in all cases was not critically different. Using the data accumulated during these studies, the relative infective index was calculated from degree of erythema and pyogenesis of the immunized groups over the control groups. In Table 12 an infective index of less 55 TABLE 11. Responses to intracutaneous infection of rabbits immunized with various coagulases and subse- quently with polyvalent vaccine. Coagulases Strains Group I Group II :22;::: Ery- Pyogen- Ery- Pyogen- Ery- Pyogen- thema esis thema esis thema esis Avg cm Avg cm Avg cm 3A 1.6 0.2 1.2 0.3 1.2 0.3 55 1.1 0.1 1.1 0.1 0.8 0.1 29 1.2 0.1 1.3 0.1 1.1 0.1 80 1.5 0.2 1.1 0.2 1.0 0.1 81 1.3 0.0 1.1 0.1 0.5 0.0 83 1.5 0.2 1.2 0.1 0.6 0.1 42D 2.0 0.1 1.3 0.2 1.0 0.1 56 TABLE 12. Infective index of immunized rabbits. V ' d Strain Vaccine aCClne an Coagulase Coagulase Only 6, 55, 73 0.5* 1.28 1.25 187, 3A, 42D, 53, 70, 81 0.47 1.51 1.30 29, 80 0.39 1.15 1.35 77, 83 0.41 2.15 1.57 Controls 1.00 1.00 1.00 * Calculation of infective index: Ratio of average diameter of erythema plus average diameter of pyogenesis of the immunized animal to the average diameter of ery- thema plus average diameter of pyogenesis of the control animal. Infective index = OU'I 0.1 0 2 — 0.5 + + 57 than unity signifies protection against the infective dose while a value greater than unity signifies decreased pro- tection when compared to the untreated controls. From the results it is seen that coagulase alone or in combination with polyvalent vaccine exacerbated the challenge infection whereas the vaccine alone reduced the response to infection by 50% or more. It is common knowledge that continued cultivation on the usual laboratory media tends to lower virulence of microorganisms. Data showing the protection offered by a polyvalent vaccine prepared from the stock phage propa- gating strains against a challenge dose of freshly isolated staphylococci are found in Table 13. In comparison with results from control rabbits observed in preceding tables, the erythematous response to the recent isolates was, in most cases, more than two-fold greater while the pyogenesis was three to ten times greater. Protection against the challenge dose of recently isolated strains was not as marked as that against old stock strains. Another series of paired rabbits was immunized with a polyvalent vaccine prepared from six recent isolates. In this series, Carmine Red was administered subcutaneously 58 TABLE 13. Responses to intracutaneous infection of rabbits immunized with recent isolates from human lesions. Immunized Rabbits Controls Vaccine and Administration Ery- Pyogen— Ery- Pyogen— thema esis thema esis Avg cm Avg cm 1 . A—Z Multiple I.C. 1.1 0 5 B-22 Multiple I.C. 1 8 0.5 2.0 1 2 A-Z I.M. and I.C. 1.5 0 7 A-Z I.M. and I.C. 1.3 0.5 Vaccine A—Z: 2 . VaCCine A—Z: 25% propagating strain (PS) 42D, 20% PS6, 25% ps 3A, 25% PS 29, and 5% PS 187. 25% PS 42D, 20% PS 77, 25% PS 55, 25% PS 80, and 5% PS 187. Both vaccines were Zephiran inactivated. 59 TABLE 14. Responses to intracutaneous infection of rabbits immunized with recent isolates from human lesions. . . . Immunized* Rabbits Controls Administration ' 1 Adjunct Ery- Pyogen- Ery- Pyogen— thema esis thema esis Avg cm Avg cm Multiple I.C. 0.9 < 0.05 Multiple I.C. Carmine Red 0.1 0.00 1.4 0.7 I.M. and I.C. 0.3 < 0.05 I.M. and I.C. Carmine Red 0.4 < 0.05 lCarmine Red (carminic acid) was administered subcu- taneously just prior to the vaccine. * A polyvalent vaccine made from 6 strains of the recent isolates, inactivated with Zephiran. 60 as a saturant of the RES just before vaccine injection. The average figures for erythema and pyogenesis are given in Table 14. These data indicate a good degree of protection; the infective index ranged from less than 0.1 to a maximum of 0.43. Carmine Red seemed to cause an increase in pro- tective effect when used in conjunction with the intracu- taneous route of vaccination. There was no similar effect when the intramuscular-intracutaneous route was utilized. It is of interest to note that the virulence as indicated in the controls decreased from that shown in Table 13, but it was still greater than that of the old stock strains. Serum protein studies In addition to dermal response, a series of hemato- logical parameters were selected for quantitative measure- ment which included electrophoretic analysis of the serum proteins and determination of a group of serum transferases. Table 15 gives the results obtained from determination of total serum protein of rabbits immunized with polyvalent vaccine. There was no particular change resulting from either immunization or subsequent infection. Table 16 shows the results of the electrophoretic anal- ysis of various protein components averaged from the sera TABLE 15. The effect upon total serum protein of rabbits by polyvalent vaccine and subsequent challenge dose of Staphylococcus aureus. Percent Total Protein Rabbit Post- Post- Normal Immunization Challenge (48 hr) 1 5.6 6.1 6,5 2 6.35 6.35 6.1 3 5.9 5.5 5.7 4 5.5 5,0 4.85 5 5.5 6.1 6.1 6 5.9 6.35 6.35 7 6.1 6.1 5.7 8 6.1 6.2 5.9 9* 5.9 6.0 6.3 10* 6.0 6.5 6.4 Avg 5.88 6.02 5.99 * Non-immunized controls. 62 TABLE 16. The effect upon the electrophoretic pattern of rabbit serum proteins by polyvalent vaccine and subsequent challenge by Staphylococcus aureus. Average* Percent of Fraction Found Fraction Post- Post- Normal I , t' Challenge mmuniza ion (48 hr) Albumin 61.3 59.6 55.8 Globulins 8.1 8 8 d1 7 5 o 7 8 6.9 8 6 2 - - 4.9 0‘3 81 9 l 8.3 9 l 82 6 4 8.5 6 4 k 7 9 8.6 7 4 -k Average of 10 rabbits. 63 of ten rabbits. Two days after the challenge dose theci— globulins showed a small increase while the B-globulins remained relatively unchanged. After an initial rise of about 9% following immunization, the y—globulin decreased to a value slightly below normal in 48 hours. Serum transferases Serum glutamic oxalacetic transaminase values are shown in Table 17. The only rabbit (#10) showing an ele- vated SGOT value had a focal subacute granulomatous myo- carditis which was probably caused by several cardiac punctures. The other rabbits gave values within normal limits. Serum glutamic pyruvic transaminase activities are given in Table 18. All values were normal, falling mostly in the lower half of the range. Abnormally elevated ornithine carbamyl transferase values, Table 19, were found in all post—challenge serum samples. In the majority of cases little difference be- tween immunized rabbits and controls was noted. Patholo— gical examination of several livers indicated glycogen infiltration. In all cases, there was a slight increase of OCT activity in all sera taken six days after immunization. 64 TABLE 17. The effect upon rabbit serum glutamic oxalacetic transaminase values by polyvalent vaccine and subsequent challenge dose of Staphylococcus aureus. Post- Post- Rabbit Normal I unized Challenge (48 hr) Sigma-Frankel Units* l 16 19 16 2 16 38 27 3 14 22 21 4 11 17 22 5 22 53 27 6 30 19 38 7 22 30 3O 8 21 45 32 9** 22 22 26 10** 21 97 53 * Value in Sigma-Frankel (S-F) units; 1 unit will form 4.82 x 10’4 uM of glutamate per minute (pH 7.5 @ 25 C). Normal values 8 - 40, post infarction 40 — 200, and liver necrosis to 2000. ** Non-immunized rabbits. 65 TABLE 18. The effect upon serum glutamic pyruvic transam- inase values of rabbit serum by polyvalent vaccine and subsequent challenge with Staphyloc— cus aureus. Post- Post- Rabbit Normal . . Challenge Immunization (48 hr) 1 l4 16 ll 2 24 ll 14 3 11 14 16 4 10 21 22 5 18 18 16 6 19 11 16 7 16 12 14 8 20 27 28 9* 19 19 21 10* 16 19 24 lS—F Units; tentative normal values, 5 - 35, post infarction 40 - 100, liver necrosis over 100. * Non-immunized controls. 66 TABLE 19. The effect upon rabbit ornithine carbamyl trans- ferase values1 by polyvalent vaccine and subse- quent challenge dose of Staphylococcus aureus. Sera Examined Rabbit Post— Post- Normal Immunization Challenge (48 hr) 1 0 07 0.12 0 53 2 0 12 0.14 0 62 3 0 15 0.21 0 31 4 0.15 0.33 0.67 5 0.13 0.23 0.27 6 0 15 - 0 42 7 0 02 - 0 53 9* 0.30 - 0.53 10* 0.30 - 0.57 1Values of uM NH per 0.5 ml of serum, normal range 0 —-0.25, cirrhosis 6f liver 0.5 - 1.2, acute cholecysti- tis 1.0 - 8.0, and infectious hepatitis 0.5 - 25. * Non-immunized controls. DISCUSSION Stilbestrol From results in Tables 1 and 2, it is clear that admin- istration of stilbestrol, regardless of route, failed to increase materially the disappearance of viable organisms from the blood and tissues. From smears of peripheral blood, the number of macrophages was shown to remain rela- tively constant. Heller et a1. (1957) believed that in the absence of increased numbers of cells the explanation must lie in the probability that increased disappearance of carbon was caused by an enhancement of the activity of each cell. While this explanation may well be correct, one must consider the great difference between an inert carbon par- ticle and the dynamic surface of a bacterial cell. Evidence from the present studies did not indicate increased phago— cytic activity under the conditions used. Had there been increased phagocytic activity, a longer survival period and a lower mortality rate would have been expected. Neither alternative was seen. The groups receiving the stilbestrol over the longest period of time showed the lowest capacity to remove the 67 68 microorganisms from the bloodstream. Cuppage and Block— worth (1960) reported that administration of stilbestrol for 50 to 100 days caused distinctly abnormal bile duct proliferation in rabbits. In fact 80% of the liver was replaced by bile duct cells in one rabbit. The injection intravenously of mice with ethanol-stilbestrol solution caused a reduction of about 75% in the ability of the liver to remove staphylococci. Since the administered amount of this solution was approximately 2% of the total blood volume, this treatment was probably the most physiologi- cally disturbing since death occurred rapidly within 48 hours. Perhaps the longest dosage period used in this study may also have caused subclinical liver damage which reduced the trapping of bacteria. Frank and Pounden (1961) found that mastitis-producing staphylococci were inhibited by 3.5 pg per ml of stilbestrol in vitro. On the basis of the regimen in this study blood levels of more than 3.5 pg per ml were possibly obtained; yet, compared to the con— trols, no marked reduction of viable staphylococci occurred. Again the difficulty of comparison between the test tube and the animal system is underscored. Heller et a1. (1957) found significant hepatomegaly 69 following stilbestrol administration in mice, and further, that based on radiocolloid studies the activity per gram of liver was decreased. It is suggested therefore that stilbestrol administration may adversely effect the liver causing interference with normal organism trapping effi- ciency. This in turn could hamper the normal defense response resulting in a subsequent fatal outcome. It was concluded that stilbestrol administered under the conditions given here was not valuable as a thera- peutic agent in experimental staphylococcal infection of mice. Lysozyme According to Elek (1959) the known production of proteinases by staphylococci may lead to auto-destruction of coagulase (Birch—Herschfield, 1940). Since egg white inhibits the proteinase action against coagulase (Lominske, Morrison, and Smith, 1955), it was desirable to study possible interaction between lysozyme and coagulase. More- over, because Ekstedt and Nungester (1955) and Yotis (1962) have shown that coagulase exerts an inhibiting effect on an active protective factor in normal serum, possible 7O interaction of these two enzymes could create a unique effect upon the host-staphylococcus relationship. As previously indicated, data presented in Table 4 reveal no antagonism between coagulase and lysozyme. The combination gave no evidence of any synergistic relationship. Although there appeared to be no definite effect upon total bacter— ial population as measured by optical density, slight decreases in lysozyme activity suggested the possibility of bacterial adsorption of lysozyme. This supposition was subsequently borne out when after repeated washing, the cells were consistently agglutinated by both antilysozyme serum and its globulin fraction. Since the titer never exceeded 1:20, perhaps a limited number of active sites ‘were available for lattice formation. According to Raffel's discussion of the lattice hypothesis (1961), it is probable that the lysozyme-antiserum specific aggregate has the lnaximum spatial freedom for lattice formation for saturating the available active sites. The agglutination reaction, on 'the other hand, suffers first from the fact that the basic liroperties of lysozyme would be attracted by negatively dharged groups on the bacterial surface. Secondly, as a ;result of the adsorption of the lysozyme, the residual 71 sites available on the exposed facets may be inadequate to provide the minimum necessary lattice for strong agglutination. The results of tests in mice with lysozyme (Table 7) indicated a moderate degree of protection. Both the intra- venous and subcutaneous routes gave similar results. Time of administration of lysozyme and the challenge dose was immaterial provided the interval between the two was short. If the interval extended beyond two hours, the detectable life of the enzyme, protection dropped off sharply. This protective action seemed to be related to the degree and recency of bacterial adsorption of additional lysozyme. Savini and Mercurelli (1947) reported that the adsorption of lysozyme did render the staphylococci more susceptible to phagocytosis. Also Melsom and Weisser (1958) reported that in lysozyme-treated mice there was a greater tendency for pneumococci to adhere to phagocytes. The position that the lysozyme was a factor in the protective action was strengthened by the observation that the administration of antilysozyme globulin decreased by 60% the survival of mice challenged with a normally sub- lethal number of staphylococci (Table 8). 72 Recently, Flannagan and Lionette (1955) showed the lysozyme content of blood was due to its liberation from damaged polymorphonuclear cells since plasma cells, ery- throcytes, lymphocytes, and platelets contain none. This concentration suggests a relatively constant source of lysozyme to participate in phagocytosis. Thus, the injec- tion of antilysozyme globulin could appreciably reduce phagocytic action by neutralizing naturally occurring lysozyme. Even though it is probable that the passive specific antibody would be quickly destroyed, the amount of globulin solution was the equivalent of approximately 16% of the total blood volume of a normal adult mouse. It seems quite reasonable to assume that this rather massive dose of antilysozyme globulin could effect, at least tem- porarily, neutralization of normally present lysozyme. This action could, in turn, allow the infecting organisms to overwhelm the host as a result of the altered defense mechanism. It has been reported by Lepow et a1. (1959) that par- tially purified properdin contains a small amount of lyso- zynmn It is interesting to contemplate the possible role of lysozyme in the known inhibitory effect of properdin on some bacteria and viruses. 73 Vaccines The polyvalent vaccines used in this study excluded treatment with Dornase. However, the results of Greenberg and Cooper (1960) with polyvalent vaccine subjected to Dornase indicated that in every instance their preparation protected against a greater variety of strains than one without the Dornase treatment. McCoy and Kennedy (1960) found good therapeutic results using an autogenous whole cell vaccine in antibiotic- resistant staphylococcal infections. In agreement with our findings (Table 10) these authors also preferred the use of a minimum quantity of a mild antiseptic over the drastic denaturation caused by heat. They also believed that only essential subculturing be done to lessen changes in bacterial cells due to artificial culture medium. None of the above workers considered the possible use of coagulase as potentially valuable in either a vaccine system or alone. As previously mentioned, the fact that coagulase appeared to promote virulence in coagulase- negative staphylococci suggested several experiments to determine whether immunization with this substance would reduce the virulence of staphylococci injected into rabbits. 74 The experimental results (Tables 9 and 11) using semi- purified coagulase were not encouraging when used alone or combined with a polyvalent vaccine. The heightened ery- thematous and pyogenic response when coagulase had been used suggested at first a hypersensitivity phenomenon. If this were the case, one would expect the greatest reaction with the most active coagulase producing strains; however, this was not borne out. The strain (PS 70) producing the greatest amount of coagulase ranked in the middle so far as infective response is concerned (Table 9). In January, 1962, Yotis presented evidence that coagu- lase appears to neutralize some serum factor with anti- bacterial activity located in the supernatant portion ob- tained following 65% (NH4)ZSO4 saturation of the water- soluble globulin portion and precipitation by ethanol. Since the rabbits were injected with coagulase and Freund's adjuvant, the protracted release of coagulase from the sterile abscess formed from inoculation may well have served to keep the antibacterial activity in the rabbit serum at a constant and prolonged low level, allowing a given infective dose to produce a greater reaction than in the control. It seems, also, that this effect could perhaps 75 be interfering with antibody production after vaccination. Derbyshire and Helliwell (1962) reported that their @- 1ysin—coagulase-leucocidin vaccine enhanced resistance to a challenge of staphylococci via the mammary gland. The severe mastitis found in one—third of the experimental group may have been due to continued release of coagulase from the AlPO4 used as the adsorbant for the vaccine. Also in the matter of inactivation, Zephiran was found to be the least disruptive of the cationic agents tested against staphylococci as demonstrated by a lower percent of phosphorus and nitrogen released into the medium (Hotchkiss, 1946). This leakage has been employed as a measure of permeability of the bacterial cell. The study of total serum proteins (Table 15) was not rewarding. The experimental infection had no measurable effect upon the total protein. The variation for each period assayed was approximately 3%. When the various serum fractions were quantitated after elution from the acetate paper strips (Table 16), there appeared to be a slight decrease in the albumin fraction, the most marked reduction occurring 48 hours after intra- venous staphylococcal injection. The y-globulin fraction 76 was the only one to show a decrease below normal values 48 hours after challenge. Presumably this would be expected since antibody is concentrated in this fraction. Caution needs to be exercised in assessing these changes in the separate reactions because failure to properly relate the changes to the total protein may give a false impression of the degree of change. The enzymic response in serum of rabbits to both immu- nization and experimental infection was considered poten- tially valuable in understanding systemic and tissue effects. White (1960) suggests that enzymes showing activity in serum are normally there as the natural products of cellular wear and tear; therefore, increased metabolism of any major tissue resulting in greater cell replacement should be reflected in increased levels of its constituent enzymes in the blood at a given time. Three enzymes were selected which are involved in hepatic disorders, serum glutamic oxalacetic transaminase (SGOT), serum glutamic pyruvic transaminase (SGPT), and ornithine carbamyl transferase (OCT). The first two are not solely restricted to liver, but are fairly widely distributed in mammalian tissue. OCT is highly localized in liver to the extent that abnormal 77 activity is found only in patients with hepatitis (Reich- ard, 1957). The major detoxicating organ is the liver; thus, if immunization provokes a systemic response by the intro- duction of foreign material requiring inactivation, an increase in liver activity should produce an increase in the enzymes listed above. No general increase in either SGOT or SGPT was found either after immunization or after the challenge dose. A subacute granulomatous myocarditis determined after necropsy of a rabbit with an abnormally high SGOT may have been caused by cardiac punctures (Table 17). The OCT values were found to be increased moderately after immunization, but the high values attained after challenge indicate liver disturbance. Rabbits displaying the highest values were necropsied and glycoqen infiltra- tion of the liver was found by pathological examination. These findings intimate that rabbits undergoing immuniza- tion may show detectable liver involvement in terms of elevated OCT levels. SUMMARY The effect of stilbestrol, lysozyme, and various vac- cines upon the experimental infection of laboratory animals with Staphylococcus aureus has been studied. Tissue localization and blood clearance of staphylo— cocci were determined in mice experimentally infected by intravenous inoculation. Mice were variously treated with stilbestrol in food, or dissolved in corn oil and injected intracutaneously, or dissolved in 50% (v/v) alcohol-water solution and adminis- tered intravenously. The results suggested that in vivo use of stilbestrol exacerbated a subsequent infection with staphylococci. Concentrations of 100 ug per ml of lysozyme had no apparent effect upon growth of staphylococci in vitro. Lysozyme activity was completely inhibited by both anti- lysozyme serum and its globulin fraction in vitro. No apparent interaction occurred between lysozyme and coagu— lase in the in vitro studies. Washed staphylococci were found to adsorb lysozyme from solution and were agglutinated by both antilysozyme serum and its globulin fraction. 78 79 In vivo tests in mice indicated that lysozyme exerted a therapeutic effect based upon increased rate of survival against staphylococcal infection. On the other hand, anti- lysozyme globulin administered by subcutaneous injection increased the mortality rate with normally sublethal in— fecting doses. Semi—purified coagulase used as an immunizing agent alone or followed with polyvalent bacterial vaccines failed to promote resistance in rabbits after intracutaneous in- fection. The superiority of the methods for inactivating the vaccines was in the order of Zephiran, formalin, and heat. Polyvalent vaccines made from recent isolates of human lesions showed a better immunizing ability and a greater virulence than the laboratory strains. In the use of Carmine Red as an adjuvant during the immuniza- tion schedule with polyvalent vaccine, results found after the challenge dose indicated that there was some beneficial action if multiple intracutaneous administration had been used. The results were less favorable if immunization had been accomplished by the intramuscular route. 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