OBSERVATIONS ON THE HEMOLYTIC PROPERTIES OF TYPHUSRICKETTSIAE1

JOHN C. SNYDER, MARIANNA R. BOVARNICK, JUDITH C. MILLER, ANDR. SHIH-MAN CHANG

Department of Microbiology, Harvard School of Public Health, Boston, Massachusetts,Veterans Administration Hospital, Brooklyn, New York, and Department of Medicine,

College of Medicine, State University of New York at New York City

Received for publication December 4, 1953

In 1948 Clarke and Fox reported that crudesuspensions of typhus infected yolk sacs causedthe lysis of erythrocytes of several animal speciesin vitro. When it was learned that typhus rickett-siae could be purified by differential centrifuga-tion and treatment with celite in such a way asto reveal their independent metabolic activity(Bovamick and Snyder, 1949) and to preservetheir infectivity for cotton rats (Bovarnick et al.,1950), it seemed of interest to determine whetherthe purified suspensions would still retain thehemolytic properties of the crude tissue supen-sions. Preliminary tests showed that this was thecase, and further experiments with purifiedrickettsial suspensions have made it posible todetect factors affecting the reaction and to de-velop a rapid, sensitive hemolysin test. Themethod as evolved during several years in twodifferent laboratories is based on a 2.5 hour in-cubation of rickettsiae and red blood cells, fol-lowed by colorimetric determination of the hemo-globin released. For convenience this method isreferred to subsequently as the "short hemolysintest" to distinguish it from the overnight pro-cedure of Clarke and Fox in which the end pointwas determined by inspection.

This paper describes our standard procedurefor the short hemolysin test and its applicationto quantitation of rickettsial suspensions undervarious conditions. Experiments on factors af-fecting the hemolytic activity of typhus rickett-siae are reported.

1 The work was supported in part by researchgrants from the Lederle Laboratories Division ofthe American Cyanamid Co., the Research Labo-ratories of Chas. Pfizer and Co., the Division ofResearch Grants of the National Institutes ofHealth, U. S. Public Health Service (E29C4 andC5, E167C).

MATERIAS AND METHODS

Diluents. The diluent for the rickettsiae andred cells in the standard short hemolysin test wassucrose PG (sucrose, 0.218 x; KH2PO4, 0.00376m; KHIP04, 0.0071 m; potasium glutamate,0.0049 m; pH, 7.0; sterilized by autoclaving)(Bovarnick et al., 1950) to which was added,after autoclaving, 10 ml of 0.1 M MgCIg per 100ml. This was designated as sucrose PG + Mg.

In certain experiments solution NK 7 G wasused for washing and resuspending the red bloodcells. It contained KCl, 0.042 m; NaCl, 0.089 m;Na,lHPO4, 0.0079 m; KH2PO4, 0.004 m; potas-siurm glutamate, 0.005 M; pH 7.0. After auto-claving this solution, the following supplementswere added per 100 ml: 0.4 ml of 50 per centglucose and 8 ml of a solution composed ofMgC12,0.095 m; CaCl2, 0.0025 m; and MnCl2, 0.0025 m.

Rickett8iae. The Breinl strain of epidemictyphus, the Wilmington strain of murine typhus,and the human avirulent Madrid E strain(Gallardo and Fox, 1948) were maintained bypropagation in embryonated egg (Cox, 1941)for the various experiments as indicated below.The infected yolk sac were homogenized in aWaring blendor, in the proportion, 50 g yolksac for each 50 ml sucrose PG and shell frozenin a dry ice-alcohol bath for stoage at -72 C.Suspensions of "once washed" rickettsiae (Bovar-nick et al., 1950) were stored in the same manner.

Toxicity for mice. The toxicity of variousrickettsial suspensions was determined by inocu-lation of 0.25 ml of each dilution into the tailveins of 12 to 18 g mice, using 4 mice for eachdilution. The LD60 was calculated by the methodof Reed and Muench (1938) on the basis ofsurvivors at 18 hours.

Erythrocytes. Sheep blood was drawn asepticallyinto an equal volume of sterile modified Alsevers

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solution (sodium citrate, 0.027 M; sodium chlo-ride, 0.072 m; glucose, 0.114 M; and citric acid,0.0038 M) and stored in small screw cap bottlesat +2 to 4 C. Sheep blood prepared in this waycan be used for at least 4 weeks. On the day ofthe hemolysin test, the blood was centrifuged,and the supernatant plasma and buffy coat werediscarded; the red cells were washed by centrifu-gation at low speed first with several volumes ofsucrose PG, then twice more with sucrose PG +Mg, and finally suspended to a concentration of25 per cent in the latter diluent.

Procedure of the short hemolysin test. Serialhalf-log dilutions of the rickettsial suspensionswere made in sucrose PG + Mg; 0.2 ml portionsof each dilution were placed in Kahn tubes, and0.4 ml portions of the 25 per cent suspension ofsheep erythrocytes were added to each tube. Acontrol containing diluent in place of rickettsiaewas set up at the same time. If the rickettsialsuspension was turbid, as was the case with crudeyolk sac suspensions, the hemolytic property ofone set of dilutions was destroyed by heatingat 56 C for 30 minutes before addition of the redcells to allow correction for the turbidity in thesubsequent colorimetric measurements. Rickett-sial dilutions and mixtures of red cells and rickett-siae were kept at 0 C until all suspensions to betested at any one time had been prepared. Thetubes were incubated then at 34 C for 2.5 hours.At the end of this time the hemolytic reactionwas arrested by the addition to each tube of 2.0ml of 0.85 per cent NaCl solution containing 0.2ml formalin per 100 ml The contents of eachtube were well mixed, and the tubes were cen-trifuged at 2,000 rpm for 15 minutes. The opticaldensity due to the hemoglobin released by theaction of the rickettsiae was determined at wave-length 545 m,u in a Coleman Junior spectro-photometer. The sedimented intact red cells atthe bottom of each tube were well below the lightbeam and thus did not interfere with the reading.For nonturbid suspensions, the tube containingdiluent and red cells was used as the blank; forturbid suspensions, the corresponding heateddilution was used.The end point was determined by plotting the

optical density readings against the appropriateconcentration of the rickettsiae. The concentra-tion of the initial suspension was considered to be1.0. The concentration of rickettsiae required toproduce an optical density of 0.3 was read from

the curve. (This was arbitrarily chosen as theend point because in this range a direct pro-portionality had been observed between concen-tration of rickettsiae and optical density.) Thevalues thus obtained were of the same order ofmagnitude as the toxic LD50 for mice.

Example. Serial half-log dilutions of once-washed Breinl strain typhus rickettsiae, pool A,were tested as above. The readings were:

Dilution

1/101/31.61/100

Rickettsialconcentraties

0.1000.0320.010

Opticaldensity

0.970.370.14

These values are plotted in figure 1 from whichthe concentration of rickettsiae required to givean optical density of 0.3 is found to be 0.024 or adilution of the initial suspension of 1/42. Thisvalue is referred to hereafter as the hemolysinend point (HE). Pool A was shown to have atoxic LDIo for white mice at a dilution of 1/35.

Because of the short time required for the as-say (2.5 hours at 34 C) it was not necessary toobserve sterile precautions. The high concentra-tion of red cells used in the above procedure wasfound essential to insure reasonable sensitivitysince with lower concentrations the amount ofhemolysis is very low. With the concentration of

1.0r

0.9

02s

Fnz 0oLUJ-i

(.) 0.4

a-o 0.

02

0.1

0

0

.7O .01 .02 .03 .04 .05 .06 .07 .08 .09

RICKETT SIAL CONCENTRATION(INITIAL SUSPENSION * 1.0)

Figure 1. Example to illustrate determinationof hemolysin end point of once-washed epidemictyphus rickettsiae, pool A.

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rickettsiae normally used, the degree of hemolysiswas small although it was possible to get com-

plete hemolysis if a sufficiently high concentra-tion of rickettsiae was present (approximatelytwo hundred times that required for the chosenend point).

RESULTS

Reproducibility of hemolysin end point. Thehemolysin end points for 5 different ampules of asingle pool of once-washed Breinl rickettsiae were

determined in separate standard short hemolysintests performed on different days. The hemolysinend point values were 0.037, 0.040, 0.036, 0.032,and 0.036; the mean value was 0.036; and thestandard deviation, 0.0029. A similar comparisonwas made with a pool of once-washed Madrid Estrain; the hemolysin end point values were0.048, 0.050, 0.050, and 0.056; the mean was0.051; and the standard deviation was 0.003.These tests were run with erythrocytes from thesame sheep, and the chance variations under thesecircumstances amount to less than 10 per cent.When erythrocytes from different animals were

used, there was somewhat more variability.Sensitivity. With the Madrid E strain the

rickettsial end point was two to threefold higherwith rabbit than with sheep red cells. It wasusually more convenient to use sheep red cells

TABLE 1Effect of various metals on the extent of lysis of

sheep erythrocytes by typhus rickettsiae*

SUBSTANCZS ADDED

SOLIUTION

None Mgt Mg, Cat M ant yNokrmal

NK 7 G 11§ 68 76 84 125Sucrose PG 28 65 57 69

* The rickettsiae in the above experiment wereMadrid E strain and had been purified as describedby Bovarnick and Miller (1950).

t The final concentration of metals when pres-ent was: Mg, 4.1 X 10-' M; Ca, 1.1 X 104 m;Mn, 1.1 X 10-4 M.

$ For the NK 7 G solution, the normal yolksac was added to the medium containing Mg,Ca, and Mn; for the sucrose solution, it wasadded to the mixture containing only Mg.

§ The figures represent the denominator of thedilution required to produce an optical densityof 0.3.

because they are available in larger quantitiesin most laboratories.The sensitivity with sheep erythrocytes can be

increased by substitution of solution NK 7 G forsucrose PG. In this case the red cells were washedand suspended in NK 7 G containing magnesium,manganese, calcium, and glucose, and the rickett-siae are diluted in NK 7 G without metals. Forassaying purified rickettsiae it was advisable toadd normal yolk sac that had been heated forthirty minutes at 56 C in a final concentrationof 0.5 per cent to the NK 7 G solution. This ap-proximately doubles the titer with the MadridE strain but makes a more cumbersome pro-cedure than the standard short hemolysin test.The effects of various metals and of heated nor-mal yolk sac on the hemolysin end points areshown in table 1.

Comparison of hemolysin end points and toxicLDso. Titration of concentrated suspensions oftyphus rickettsiae by intravenous inoculation ofwhite mice is a very reliable and reproducible

TABLE 2Comparison of the toxic LDso for white mice and

the hemolysin end point (HE) of various poolsof typhus rickettsiae

RICEETTSI4L NO.r0 Ratio: LD#/HESTRAIN PREPARLATION COMPAX-______

isoNS Mean S.D.

Breinl epi- crude yolk 40 1.03 0.51demic sac

Breinl epi- once washed 4 1.26 0.28demic

Madrid E crude yolk 5 0.96 0.22sac

Madrid E once washedt 3 0.56 0.20Madrid E once washed 12 1.68 0.42

Wilmington crude yolk 16 0.51 0.45murine sac

Wilmington crude yolk 3 0.27 0.11murine sac

* The LDso and the HE values used in calcu-lating the ratios were expressed as concentrationof rickettsiae referred to the initial suspension as1.0. If, as in other tables, denominators of endpoint dilutions are used in the calculations, thevalues of ratios are the reciprocals of those indi-cated in this column.

t This pool was frozen and thawed twice afterwashing.

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procedure with sharp end points (referencescited by Neva and Snyder, 1952). Comparisons ofthe hemolysin end points and the toxic LD&ovalues were carried out by performing the twotests simultaneously each time an ampule ofrickettsiae was thawed out for use. Table 2 showsthe results of 83 comparisons with seven differ-ent rickettsial preparations.

Since a difference of one mouse in the toxicitytitrations, as performed in these experiments,involves an appreciable change in the LDIovalue, the variability recorded in table 2 is ap-proximately what would be anticipated. Theratios vary somewhat from strain to strain andfrom one pool to.another of the same strain. Thedifferences observed between crude yolk sacpreparations, washed preparations, and frozenwashed preparations may reflect partial inijury,resulting in selective destruction of one or theother property (compare table 3). The physio-logic state of the rickettsiae at the moment ofharvest of the yolk sacs, e.g., the proportion offully infective to inactive eells, may be anotheraspect of importance. Finally, the strains them-selves may differ in relative toxic and hemolyticactivities.

TABLE 3Comparison between loss of toxicity for mice and

hemolytic activity of rickettsiae on incubationfor 16 hours at 84 C

HEXOLYTIC ACTIVITYt TOxICITY FOR HICEtEXPERI- SOLU-_MENT TION Initial Final Per cent initial Final Per cent

titer titer change titer titer change

1 A 5.8 3.3 -44 5.5 <0.7 >-88B 5.5 4.4 -20 5.5 1.4 -74

2 A 6.4 6.4 0 5.5 1.7 -69B 6.4 6.8 +6 5.5 2.8 -49

* In these experiments washed rickettsiae weresuspended in a concentration equivalent to 10per cent original infected yolk sac, and the sus-pensions were assayed before and after incubation.The solutions used were: A, the sucrose mediumdescribed by Bovarnick, Allen, and Pagan (1953)or B, the same medium with the addition of di-phosphopyridine nucleotide.

t The figures represent the denominators ofthe dilutions required to produce an optical den-sity of 0.3 in the hemolysin assay, or death of 50per cent of the mice in the mouse toxin asay.

In view of the degree of correlation found be-tween these two quite different phenomena, it isevident that the in vitro test can be used underordinary circumstances in place of the animaltest for the assay of crude or once washed typhusrickettsiae.The parallelism between toxicity for mice and

hemolytic activity did not always hold whenrickettsiae were subjected to conditions leadingto partial inactivation. When rickettsiae wereincubated for 16 hours at 34 C, the loss in toxicityfor mice appeared to be appreciably more rapidthan the loss in hemolytic activity, as can be seenin table 3.

Factors affecting the hemolysis by typhus rickett-siae. In working out the procedure for the stand-ard short hemolysin test, the influence of variousconditions was studied. It was found that theoptimum pH was about 7, and that changes inthe time of incubation from 2 to 6 hours had verylittle effect on the end point. Glucose had no ef-fect on the end point but was included in the NK7 G medium to decrease the spontaneous hemoly-sis of the red cells.When purified rickettsiae were used, it was

possible to show that both magnesium (see table1) and glutamate (see table 4) were requlired forthe lysis of red cells by rickettsiae. When thesalt medium was used, calcium and manganese

TABLE 4Effect of glutamate on the lysis of red cells by

typhus rickettsiae*

NO GLUTAXATEtTYPE 0F TIME OF NOUIA Y ADDED ATRED CELLS INCUB3ATION GLUSZTATE________PSET 0 time 2 hours

hr

Sheep 2 71 184 20 83 71

Rabbit 3 10 133

The figures represent the denominator of thedilution required to produce an optical densityof 0.3.

* The above experiments were carried out asusual, except that glutamate was omitted fromthe sucrose PG+Mg solution used in washingthe red cells and in diluting the rickettsiae.

t Glutamate, 0.02 ml of 0.15 M, was added to thered cell-rickettsiae mixtures either before incuba-tion or after two hours' incubation at 34 C, asindicated.

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had a small additional effect in increasing the de-gree of hemolysis. The magnitude of the effect ofthe added metals and glutamate varied with thepurity of the rickettsiae, the greater the concen-tration of yolk sac, the less apparent the require-ment for any of these factors, presumably due totheir presence in the yolk sac tissue.Using rabbit erythrocytes it was noticed that

one other substance, namely adenosine triphos-phate (ATP), had a marked effect on the degreeof hemolysis. A concentration of washed rickett-siae, sufficient to produce a low degree of hemoly-sis corresponding to the end point chosen in theabove described procedure under the usual con-

TABLE SInfluence of adenosine triphosphate (ATP) on the

lysie of rabbit erythrocytes

CONENTRTION0 ATn

RICKETTSIAE OR HZMOLYSATEI ADDZD 0 0.0004m

Optical densityreading

None 0.0 0.0Rickettsiae* 1/2,000 0.04 0.05Rickettsiae* 1/600 0.13 0.8Rickettsiae* 1/200 0.28 1.3Rickettsiae* 1/60 0.78 1.3Rabbit cellt hemolysate 1/8 0.08t 0.08Rabbit cellt hemolysate 1/4 0. 17t 1.4Rabbit cellt hemolysate 1/2 0.34t 1.4

In the above experiment, each tube contained0.3 ml 8 per cent rabbit cells. The other con-stituents were added in the following amounts,where indicated: rickettsiae, 0.2 ml; ATP, 0.0024m, 0.1 ml; hemolysate, 0.2 ml; sucrose PG+Mgto bring the total volume to 0.6 ml. The diluentused throughout was sucrose PG+Mg. The re-mainder of the experiment was carried out asusual.A lower concentration than usual of rabbit

cells was used in these experiments so that opticaldensity readings on complete hemolysis could beobtained. An optical density reading of 1.4 cor-responds to complete hemolysis in this test.

* The rickettsiae were a washed preparation ofthe Madrid E strain.

t The hemolysate was prepared by addition of15 ml water to 1 ml packed, washed rabbit cells.After 3 to 4 minutes, 3.7 ml 42.5 per cent sucrosewas added to restore isotonicity.

+ These readings represent the color due tothe added hemolysate.

ditions, brought about complete hemolysis of therabbit cells when adenosine triphosphate (0.0004M to 0.005 m) was added to the reaction mixture.This phenomenon has been found to be relatedonly indirectly to the rickettsiae. Rabbit erythro-cytes alone were unaffected by the presence ofadenosine triphosphate. When a small amount ofa water hemolysate of rabbit erythrocytes as wellas adenosine triphosphate was added to un-damaged rabbit cells, complete hemolysis tookplace after one to two hours at 34 C. The amountof hemolysate required was of the same order ofmagnitude as that produced by rickettsiae in theabsence of adenosine triphosphate (see table 5).Sheep cell hemolysate and adenosine triphos-

phate did not have a similar action on sheep cells,nor was the degree of hemolysis of sheep cells byrickettsiae altered by the presence of adenosinetriphosphate. Crude yolk sac suspensions ofrickettsiae did not show enhanced hemolysis ofrabbit cells on addition of adenosine triphosphate,presumably because of the high concentration ofadenosine triphosphatase in such preparations.

Meabolic inhibitors. Several metabolic in-hibitors were tested for their effect on the lysis ofsheep red cells by typhus rickettsiae. Table 6shows that cyanide, azide, fluoride, and iodo-acetate reduce the hemolysis under the condi-tions of the test.

TABLE 6Effect of inhibitors on the lysis of sheep erythroeytes

by typhus rickett.ae

SOLUTION

rritot NK 7 G j Sucrose PGt

Expt I ExPt2 Expt jI Expt 2

Per cenm InhibileuNaCN, 1 X 10 M 94 93 64 65NaNs, 1 X 1l' M 61 55 24

2 X 10'Mm 57NaF, 4 X 10-3M 89 89 93 93Iodoacetate, 10-3 M 68 62 41

2 X 10-8M 73Malonate, 10' m 7 0

* The NK 7 G solution contained also Mg, Ca,and Mn, and heated normal yolk sac in the con-centration described in the test.

t Mg was added to the sucrose PG solution.The inhibitors were added to the mixture of

red cells and rickettsiae at 0 C immediately beforetransferring the tubes to 34 C.

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Effects of antibiotics on the hemolysin end point.Several tests were carried out to determine theeffect of aureomycin, chloramphenicol, and terra-mycin on the hemolysin end points of representa-tive pools of the Breinl, Madrid E, and Wilmingtonstrains. In each test the rickettsiae were washedonce before the antibiotics were added. Thegeneral plan was exposure of the rickettsial sus-pension at 34 C for varying intervals up to 4hours, to different concentrations of antibioticsup to 300 ug per ml in sucrose PG solution, withsuitable controls. The red blood cells were addedthen, and the tests were run as described above.In two experiments the attempt was made toeliminate the antibiotic from the rickettsial sus-pension by two cycles of high speed centrifuga-tion at 2 to 4 C before addition of the red cells.The results of a typical experiment are shown intable 7.The hemolysin end points were reduced sharply

by exposure of rickettsiae to aureomycin andterramycin; chloramphenicol had no effect underthe conditions of these tests. Furthermore, thehemolysin end point values were unchanged by

TABLE 7In vitro effect of antibiotics on hemolysin end points

(HE) of epidemic typhus rickettsiae

HEKOLYSIN END POINTS AmRSPECITIED TIME OF CONTACT

AT 34 CATIBIOTIC RATION-

4 hr thenZero 1 hr 4 hr washed

twice

pg/miNone 0 49 59 51; 51t 38t

Aureomycin lOOt 36 32 25316 22 <3 <3316 <2t <2t

Terramycin lOOt 46 48 45316 56 45 <3316 20( 8t

Chloramphenicol 100 44 50 38316 36 44 44

* Figures are denominators of dilutions re-quired for optical density of 0.3.

t Concentrations less than 100 ug/ml had noappreciable effect on the HE values.

t Madrid E strain; all other values shown referto the Breinl epidemic strain.

two cycles of washing of the rickettsiae followingtheir contact with the antibiotics.

DISCUSSION

The short hemolysin test which has been de-scribed in this paper is a convenient assay pro-cedure for typhus rickettsiae in relatively con-centrated suspensions. During its application intwo different laboratories over several years, ithas been shown to have the same order of sensi-tivity and reproducibility as the titration oftoxicity by the intravenous inoculation of whitemice. If a laboratory has an abundant supply ofsheep or rabbit red blood cells, the test is lessexpensive to perform than the toxicity titrationin mice. The short procedure herein described hasobvious advantages over the original procedureused by Clarke and Fox (1948). It is apparent,however, from the variety of factors which affectthe degree of hemolysis brought about by a givenconcentration of rickettsiae that the hemolysintest is much more subject to influence by othersubstances present in the rickettsial suspensionthan is the mouse toxin assay.The phenomenon of in vitro lysis appears to be

a process which requires magnesium and glu-tamate and possibly calcium and manganese aswell. The effect of normal yolk sac in increasingthe hemolysis by washed rickettsiae even in thepresence of glutamate and magnesium presum-ably is due to its stabilizing effect on the rickett-siae. It is of interest to note that glutamate canstill promote hemolysis after the red cells andrickettsiae have been incubated together for twohours alone (see table 4), and thus it would ap-pear that glutamate must be required for somereactions which precede or take part in thehemolysis. It may be pertinent in this respectthat glutamate is the only known substrate whichis metabolized rapidly by rickettsiae.The inhibition of hemolysis by cyanide, azide,

and iodoacetate parallels closely their inhibitionof the oxidation of glutamate by typhus rickett-siae (Wisseman et al., 1951; also, unpublishedobservations of the authors). On the other hand,the hemolysin reaction is very much more sensi-tive to fluoride than is the oxidation of glutamate.The best known example of a reaction highlysensitive to fluoride is that of the enolase ofglycolysis. Rickettsiae show no evidence of gly-colysis, but red cells do. Whether this indicatesthat some reaction of the red cells also contributes

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to their own lysis remains to be determined.In agreement with this hypothesis, however, isthe fact that the lysis of rabbit cells brought aboutby rabbit hemolysate and adenosine triphosphatealso is inhibited strongly by fluoride, though notaffected by cyanide (unpublished experiments).In any case, the significant inhibition of therickettsial hemolysis by a variety of metabolicinhibitors indicates that at least one and prob-ably several enzymatic reactions are involved inthis hemolysis. A similar conclusion was reachedby Gardner and Morgan (1952) in a study of themumps hemolysin although the effective in-hibitors differ markedly in the two reactions.The effects of aureomycin, terramycin, and

choloramphenicol on the hemolysin are quitesimilar to their effects on the oxidation of glu-tamate by rickettsiae (Karp and Snyder, 1952).The unsuccessful attempts to restore hemolyticactivity of rickettsiae after exposure to terra-mycin and aureomycin by centrifugation and re-suspension suggest that these antibiotics becomefirmly attached to the rickettsial cells, or thatthey exert an irreversible effect on the enzymesystems involved in the hemolysin reaction.

SUMMARY

A rapid test for the demonstration of hemolyticproperties of typhus rickettsiae is described.The test is of the same order of sensitivity and re-producibility as the titration of toxicity by intra-venous inoculation of white mice. The hemolysinreaction appears to require glutamate and mag-nesium and possibly calcium and manganese aswell. Metabolic inhibitors reduce the hemolyticactivity of rickettsiae. Aureomycin and terra-mycin reduce the hemolytic activity of rickett-siae, whereas chloramphenicol does not.

REFERENCES

BOVARNICK, M. R., AND MILLER, J. C. 1950Oxidation and transamination of glutamate

by typhus rickettsiae. J. Biol. Chem., 184,661-676.

BOVARNICK, M. R., AND SNYDER, J. C. 1949Respiration of typhus rickettsiae. J. Exptl.Med., 89, 561-565.

BOVARNICK, M. R., MILLER, J. C., AND SNYDER,J. C. 1950 The influence of certain salts,amino acids, sugars and proteins on thestability of typhus rickettsiae. J. Bact.,59, 509-522.

BOVARNICK, M. R., ALLEN, E. G., AND PAGAN, G.1953 The influence of diphosphopyridinenucleotide on the stability of typhus rickett-siae. J. Bact., 66, 671-675.

CLARKIE, D. H., AND Fox, J. P. 1948 The phe-nomenon of in vitro hemolysis produced bythe rickettsiae of typhus fever, with a noteon the mechanism of rickettsial toxicity inmice. J. Exptl. Med., 88, 25-41.

Cox, H. R. 1941 Cultivation of rickettsiae ofRocky Mountain spotted fever, typhus, andQ fever groups in embryonic tissues of de-veloping chicks. Science, 94, 399 403.

GALLARDO, F. P., AND Fox, J. P. 1948 Infectionand immunization of laboratory animals withRickettsia prowazekii of reduced patho-genicity, strain E. Am. J. Hyg., 48, 6-21.

GARDNER, B. J., AND MORGAN, H. R. 1952 Ef-fect of certain enzyme inhibitors on hemo-lytic and hemagglutinating activity of mumps.Proc. Soc. Exptl. Biol. Med., 79, 133-135.

KARP, A., AND SNYDER, J. C. 1952 In vitroeffect of aureomycin, terramycin, and chlor-amphenicol on typhus rickettsiae. Proc.Soc. Exptl. Biol. Med., 79, 216-219.

NEVA, F. A., AND SNYDER, J. C. 1952 Studieson the toxicity of typhus rickettsiae. J.Infectious Diseases, 91, 72-78.

REED, L. J., AND MUENCH, H. 1938 A simplemethod of estimating fifty percent endpoints.Am. J. Hyg., 27, 493497.

WISsEMAN, C. L., JACKSON, E. B., HAHN, F. E.,LEY, A. C., AND SMADEL, J. E. 1951 Meta-bolic studies of rickettsiae. I. The effects ofantimicrobial substances and enzyme inhibi-tors on the oxidation of glutamate by purifiedrickettsiae. J. Immunol., 67, 123-136.

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