THE VIRULENCE AND IMMUNOGENICITY OF SALMONELLA TYPHOSAGROWN IN CONTINUOUS CULTURE

SAMUEL B. FORMAL, L. S. BARON, AND WALTER SPILMANDivision of Immunology, Walter Reed Army Institute of Research, Walter Reed Army Medical Center,

Washington 12, D. C.

Received for publication December 27, 1955

In recent years the technique for the continuouscultivation of microorganisms has been adaptedfor a variety of uses. Gerhart (1946) and Barnesand Dewey (1947) have reported on the advan-tage of continuous cultivation over conventionalmethods for obtaining increased yields of bac-terial cells, and von Hofsten et al. (1953) haveextended the method to the cultivation of fungi.

Continuous cultivation techniques offer severaladvantages. In some instances, the formation ofa given enzyme may be inhibited by a constit-uent of the growth medium. By employingcontinuous culture, it is possible to grow largeamounts of bacterial cells in a medium containingsuch low concentrations of the inhibiting con-stituent that enzyme formation is unaffected(Cohn and Torriani, 1953). In addition, metabolicproducts which may block the formation of someenzymes do not accumulate under conditions ofcontinuous growth (Monod, 1950).Workers in the field of microbial genetics have

also employed continuous culture as a tool inthe study of mutation (Novick and Szilard, 1950,1951), while others (Bryson and Szybalski,1952) have used a modification of the techniqueas a means of selecting bacterial mutants whichare highly resistant to such substances as anti-biotics.

Continuous cultivation techniques may alsobe applicable to the production of bacterialvaccines where the advantage of obtaining largequantities of cells in a relatively short period isobvious. On the other hand, a shift in the bac-terial population during continuous growth fromone of high protective potency to one of reducedimmunogenicity would restrict the usefulness ofthis method in the large-scale production oforganisms for vaccines. It cannot be denied thatthe opportunity eists for mutation and selec-tion during continuous growth. Therefore astudy of the effects of growth in continuousculture on the stability of the virulence and

immunogenicity of SalmoneUa typhosa wasundertaken.

MATERIALS AND METHODS

Continuous culture apparatus. A diagram ofthe continuous culture apparatus used in thiswork is given in figure 1. Several methods ofinjecting fresh medium into the growth tube havebeen used by other workers. In the present study,a Brewer automatic pipetting machine provedto be satisfactory, since the amount of mediintroduced into the growth tube and the fre-quency of injection can be easily and accuratelycontrolled with this apparatus. When less than10 injections per min were required, it was foundnecessary to activate the pipetting machine bymeans of an automatic timer.

In pilot experiments, the culture in the growthtube was agitated by bubbling air through it;however this invariably resulted in excessivefoaming. This difficulty was eliminated byemploying a magnetic stirrer. Clumps of cellstended to accumulate on the sides of the growthtube after several days, but this difficulty wasobviated by coating the vessel with silicone.The component parts of the apparatus were

sterilized separately and reassembled underaseptic conditions. This assembly process wasgreatly simplified by employing male and female"luer-lok" adapters to join the rubber tubing ofthe components.

Cultures. Two strains of S. typhosa wereemployed: a mutant of strain Ty2 which did notrequire tryptophan, and strain V58. Strain Ty2has been utilized extensively by workers forexperimental typhoid studies and for vaccineproduction. It exhibits maximum virulence formice, and is agglutinated by anti-Vi but not byanti-O serum. The Panama Carrier strain (V58)is employed by most investigators in the U. S.for vaccine production. It is less virulent for micethan strain Ty2 when administered as an 18-hr

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G

Figure 1. Diagram of contiA = Source of sterile mediumB = Brewer automatic pipetting machineC Automatic timer

D = Magnetic stirrer37 C water bath

culture in a saline suspension, but scarcely anydifference is observed with 6-hr suspensions.Unlike Ty2, strain V58 is agglutinated by 0 aswell as Vi antiserum.

Culture media. The basic ingredients for all

culture media used in these experiments were:dibasic potassium phosphate, 7 g per L; mono-

basic potassium phosphate, 3 g per L; andmagnesium sulfate, 0.1 g per L. Glucose andammonium sulfate in various concentrations wereadded to this basal medium, and in some experi-ments, cystine and tryptophan were also in-corporated.Mouse virulence tests. Suspensions of celLs for

challenge experiments were prepared from theTy2 strain of S. typhosa collected directly fromthe continuous culture apparatus in a flaskmaintained at 0 to 3 C. When a sufficient volumehad been obtained, the bacteria were centri-fuged and resuspended in saline to a densityof approximately 5 X 108 cells per ml. Whitemice (Bagg strain) weighing 14 to 16 g were

then injected intraperitoneally with 0.5-mlquantities of dilutions of the cell suspensions.Groups of 20 mice containing equal numbers of

inuous culture apparatusF - Growth tubeG = SiphonH = 3-way stopcockI = Outlet to collection tankJ = Outlet to sampling tube

males and females were used to test each dilu-tion. Deaths were recorded after 72 hr and theLD5io and Standard Error (S. E.) were estimatedby the method of Miller and Tainter (1944).Whenever the mouse virulence titrations were

performed on samples from the continuous cul-ture apparatus, parallel determinations on themouse virulence of strain Ty2 grown on agaralso were conducted. For this portion of thework, lyophilized ampules of Ty2 prepared 10years previously were employed. A new ampulewas opened for each virulence determination,and the mouse virulence of these cells grown onnutrient agar was asumed to be relativelystable. Thus this additional test was includedto provide some indication of the variation insusceptibility of the mice to experimentaltyphoid infection over the course of the experi-ment.

Preparation of vaccines. The organisms usedfor the preparation of vaccines were collectedfrom the continuous culture apparatus in a flaskmaintained at 0 to 3 C. As soon as a sufficientvolume had been obtained, the cells were har-vested by centrifugation.

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Dehydrated vaccines were prepared from cellsof strain Ty2 by washing the harvest withacetone, collecting the precipitated bacterialcells on a Buchner-type fritted glass filter, anddrying the organisms by air suction through thebacterial mass. Final traces of acetone andmoisture were removed by desiccation in vacuo.When the cells were to be assayed for their mouseprotective potency, they were suspended insaline to make a concentration of 24 mg cellsper 100 ml. The total nitrogen content of eachvaccine was determined on 5-ml aliquots by themicro-Kjeldahl method.

Heat-killed vaccines were prepared from cellsof strain V58 by suspending the harvest in salineto make a concentration of 109 cells per ml, andthen heating the suspension for 1 hr at 56 C.Phenol was then added to give a final concentra-tion of 0.5 per cent. Finally the total nitrogencontent of the vaccine was determined on 5-mlaliquots by the micro-Kjeldahl method.Mouse protective potency tests. Vaccines pre-

pared from cells grown in the continuous cultureapparatus were compared with a standard vac-cine consisting of cells cultivated by conventionalmethods. Groups of mice containing equal num-bers of males and females were immunized bysingle intraperitoneal injections of 0.5 ml-volumes of 5- to 10-fold dilutions of the vaccine.Six days later the mice were challenged. Miceimmunized with the acetone-killed and driedorganisms were challenged with 100 million celLsof an 18-hr culture of strain Ty2 suspended insaline, while mice receiving the heat-killedphenol-preserved organisms were challengedwith one to two thousand cells of an 18-hrculture of strain V58 suspended in 5 per centhog gastric mucin. These challenge suspensionssufficed to fatally infect at least 85 per cent ofthe control unvaccinated mice.The EDso and Standard Error (S.E.) of these

protection tests were estimated by the methodof Miller and Tainter (1944).

Bacterial counts. Viable bacterial counts weredetermined by the usual pour plate technique,averaging the number of colonies on 4 to 6 plates.Total bacterial counts were determined in aPetroff-Hauser counting chamber with eachrecorded count representing the average of fourseparate determinations.

Serological tests. The phenomenon of 0 inag-glutinability exhibited by S. typhosa is taken bymost workers to be indicative of a high Vi antigen

content. Strains not agglutinated by the 0 anti-serum are considered to have more Vi antigenthan cultures which are agglutinated by 0 anti-body.The ability of strain Ty2 grown in continuous

culture to resist agglutination by typhoid 0antiserum was examined daily. Cells were col-lected from the apparatus, centrifuged, andresuspended in saline. One-half ml of the suspen-sion was added to 0.5-ml volumes of dilutions of atyphoid 0 antiserum (titer 1: 2560). The testswere incubated for 2 hr at 37 C and then over-night at 4 C before reading.The same bacterial suspension was also tested

for its ability to react with Vi antibody. Theidentical procedure was employed as describedabove except that a serum prepared against aVi-containing strain Paracolobactrum ballerup(Vi titer 1:1280) was used.

RESULTS

S. typhosa strain Ty2 was grown in continuousculture for 24 days in the basal medium whichhad been supplemented with an excess of glucose(2 g per L) and a suboptimal amount of am-monium sulfate (52 mg per L). Fresh culturemedium was added to the growth tube (volume- 320 ml) at a rate of 2 ml per min without anychange in the density of the growing culture.With the culture in the "steady state," this isequivalent to 0.53 divisions per hour (Monod,1950).The density in terms of per cent light transmis-

sion, the viable count, and the total count fromthe third to the eighth day of the experiment isshown in figure 2. Although no further countswere done after the eighth day, the turbidity ofthe culture during the remaining 15 days of theexperiment remained constant.

Virulence tests were conducted on the effluentculture of strain Ty2 taken from the growth tubeon the third, eighth, and twenty-third day andalso on 16-hr agar-grown suspensions of the samestrain. The results of these tests are given intable 1. The data show that over the course ofthe experiment the mice were equally susceptibleto typhoid challenge, and that bacteria takenfrom the apparatus after 23 days of contin-ous growth were as virulent for mice as theywere when continuously cultured for only 3 days.The immunogenicity of acetone-killed and

dried vaccines prepared from Ty2 celLs collectedafter 6 and 24 days of continuous culture was

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4

x

0

2

A A

* Percent Tronsmission* Viable CountA Totol Count

3 4 5 6 7 a 9DAYS AFTER INOCULATION OF GROWTH TUBE

Figure S. Density, viable count, and totalcount of Salmonella typhosa strain Ty2 grown incontinuous culture in a nitrogen deficient medium.

compared with that of a standard acetone-driedpreparation. The results of these tests presentedin table 2 show that a vaccine prepared fromcelLs taken after 24 days of continuous growthprotected mice as well as the one made from cells

taken early in the experiment, and both experi-mental products were comparable to the standardvaccine in their mouse immunogenicity.

The continuously cultured cells were examinedat intervals during the experiment for theirresistance to agglutination by typhoid 0 anti-serum titer (1:2560). Cells collected during thefirst 15 days were not agglutinated by the anti-serum in a dilution of 1:80, but cells collectedsubsequently were agglutinated by progressivelyhigher serum dilutions and finally reached thetiter of the serum on the nineteenth day ofcontinuous culture (table 3). These results were

obtained in several experiments and varied onlyin the time the celLs became 0-agglutinable.However, it is interesting to note that althoughthese organi became 0-agglutinable, no

concomitant decrease in mouse virulence or

immunogenicity was observed.Experiments were also conducted in which

S. typhosa strain Ty2 was cultured continuouslyin the basal medium supplemented with am-

monium sulfate, 2 g per L and glucose, 390 mgper L. Here the nitrogen was in excess while theglucose was in suboptimal concentration. Freishculture medium was added to the growth tube(volume - 320 ml) at a rate of 2 ml per mi.The results insofar as the virulence and im-

TABLE 1The mouse virulence of Salmonella typhosa strain TyS grown in continuos culture*

Dar Strain Ty2 Grown in Continuous Culture Strain Ty2 Grown on Nutrient Mar (16-hr Suspension)tCon- _ _ _ _ _6_ _ _

tin-

ture ~ ~ , 0 94Z "0 0 U

27 X 106 17/20 41.5 X 10* 18/203 6.9 X 10*1:1280 <180 6/20 11 X 10 2.5 X 10 10.4 X 1061:1280 <1:80 10/20 10 X 10 2.4 X 10*

1.7 X 106 1/20 2.5 X 106 2/20

32.7 X 10' 19/20 56.2 X 10* 19/208 8.2 X 10 1:1280 <1:8010/208.2 X 10' 2.6 X 10 14 X 10*1:1280 <1:80 7/2018 X 10'6.1 X 10'

2 X 10 1/20 3.5 X 10' 1/20

43.2 X 10' 18/20 51.7 X 10' 20/2024 8.8 X 10' 1:1280 1:2560 13/20 6 X 10 1.9 X 10' 12.9 X 10' 1:1280 <1:80 13/20 5.8X106 3 X 10'

2.2 X 106 4/20 3.2 X 106 8/200.5 X 106 1/201 0.8 X 106 2/20

Medium deficient in nitrogen.t The mouse virulence of strain Ty2 grown on nutrient agar is included to give an indication of

variation in mouse susceptibility to experimental typhoid infection over the course of experiment.t Titer to which challenge suspension reacted in an antiserum prepared against Paracolobactrum

ballerup.I Titer to which the challenge suspension reacted in an antiserum prepared against Salmonella ty-

phosa strain 0-901.

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FORMAL, BARON, AND SPILMAN

TABLE 2

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Comparison of the immunogenicity for mice of acetone-killed and dried suspensions of Salmonella typhosastrain Ty2 grown in continuous culture with a standard acetone-killed and dried preparation

DescriptionoeNitrogenVaccine* Dose in MIDescription of Vaccine Content oED S.E.

0.025 0.005 0.001 0.0002 0.00004

mg/S ml

Salmonella typhosa Ty2 grown in con-tinuous culture for 6 days. 0.10 36/40 30/40 17/40 15/40 5/40 0.0008 0.0005

Salmonella typhosa Ty2 grown in con-tinuous culture for 24 days. 0.10 36/40 33/40 26/40 13/40 11/40 0.0005 0.0004

Standard acetone-killed and driedvaccine.0.15 33/40 30/40 19/40 13/40 8/40 0.0007 0.0006

* 12 mg of dried bacilli suspended in 50 ml saline.t Challenge dose: 100,000,000 S. typhosa Ty2 suspended in saline and injected intraperitoneally.$ Numerator denotes number of survivors; denominator denotes total number vaccinated.

TABLE 3Reactions with Vi* and Ot antibodies of Salmonella

typhosa strain TyS grown in continuous culture(medium deficient in nitrogen)

Days ofContinuous Vi-Titert O-TitertCultivation

1-15 1:1280 <1:8016 1:1280 1:32017 1:1280 1:16018 1:1280 1:64019-24 1:1280 1:2560

* Antiserum prepared against a Vi-containingstrain of Paracolobactrum ballerup.

t Anti-serum prepared against S. typhosastrain 0-901.

t Tests incubated for 2 hr at 37 C and over-night at 4 C.

munogenicity tests are concerned were the sameas those described previously. The cells growncontinuously for 8 days were as virulent andimmunogenic for mice as conventionally culturedpreparations. However, in contrast to theprevious experiment, the cells became 0-ag-glutinable by the eighth day. The total nitrogencontent of the experimental vaccine in this ex-periment was slightly greater than that of theprevious experiment (0.12 mg per 5 ml as com-pared with 0.10 mg per 5 ml). While this mighthave resulted from errors in experimental tech-nique, it could also have been due to the differencein nitrogen content of the two media in whichthe bacteria were grown. The latter explanationwould agree with the findings of Virtanen and De

Ley (1948) who found that cells of Escherichiacoli contained less nitrogen when cultured on a

low nitrogen medium than on a medium withlarger concentrations of nitrogen.The results so far described were obtained with

strain Ty2 as the test culture. This strain hadbeen selected for this study, since on the basis oflaboratory criteria, acetone-killed suspensionsof these cells have been demonstrated to besuperior vaccines (Henderson et al., 1951, Landy,1953). Nevertheless, heat-killed, phenol-pre-served vaccines prepared from S. typhosa strainV58 are widely used for immunization. It was ofinterest therefore to determine the effect ofcontinuous cultivation on the immunogenicity ofstrain V58. In these experiments, heat-killed,phenol-preserved preparations of the cell harvestwere used as immunizing agents. The bacteria

6r

x 3

_i

2

a,- u

* Percent Tronsuisuien* Viable CountA Total Count

100

so~_

80 vI

_60 z

40

20.0.z

w

ERX(

3 4 5 6 7 8 9DAYS AFTER INOCULATION OF GROWTH TUBE

Figure 8. Density, viable count, and total countof Salmonella typhosa strain 58 grown in a mediumdeficient in cystine and tryptophan.

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TABLE 4Comparison of the immunogenicity for mice of heat-killed, phenol-preserved suspensions of Salmonellatyphosa strain V58 grotn in continuous culture with a standard heat-killed, phenol-preserved preparation

Niroe Vaccine* Dose in 1K1Description of Vaccine Nitrogen c set S.E.

0.15 0.015 0.0015 0.00015

Salmonella typhosa strain V58 grown incontinuous culture for 7 days ........ 0.12 36/40 29/40 12/40 7/40 0.0042 0.0018

Standard heat-killed phenol-preservedvaccine ............................ 0O.15 25/40 6/40 3/40 0.085 0.088

* One billion cells per ml.t Challenge dose: 1-2 thousand S. typhosa strain V58 suspended in 5 per cent hog gastric mucin.t Numerator denotes number of survivors; denominator denotes total number vaccinated.

were cultured for 7 days in the basal mediumcontaining ammonium sulfate, 1.8 g per L; glu-cose, 0.65 g per L; tryptophan, 0.2 mg per L;and cystine, 0.2 mg per L. In this medium bothamino acids were in suboptimal concentrations.Fresh medium was added to the growth tube(volume 360 ml) at a rate of 1.4 ml per minute

with no change in the density of the culture.The density, viable count, and total count ofthe culture effluent from the growth tube overthe course of the experiment are given in figure 3,while the results comparing the immunogenicityof the vaccine prepared from the effluent cellscollected on the seventh day with a standardheat-killed, phenol-preserved vaccine are givenin table 4. It is apparent that the vaccine pre-pared from cells grown in continuous cultureequalled and possibly exceeded the standard inmouse protective potency.

DISCUSSION

The studies outlined were designed to deter-mine the effect of continuous cultivation on thevirulence and antigenic properties of two widelyused and studied strains of S. typhosa. Althoughdirected primarily at determining the suitabilityof continuous cultivation techniques for theproduction of acceptable vaccines, the experi-ments described led to certain findings of more

general interest.In the present instance, the organisms were

first grown in a medium deficient in nitrogen,but with an adequate source of glucose. Cells ofE. coli grown continuously in a nitrogen-deficientmedium are said to contain an increased carbo-hydrate fraction. Since the Vi antigen, establishedas being of major importance in the mouse viru-

lence of typhoid strains, is known to be a poly-saccharide, it appeared likely that a nitrogendeficient medium would favor the growth of Vicells. Data have been presented which show thatstrain Ty2 can be continuously cultured for atleast 3 weeks in this medium without loss ofmouse virulence or immunogenicity. However,after 2 weeks, a measurable change occurred inthe culture, i. e., cells previously able to resistagglutination by typhoid 0 antiserum were nolonger able to do so.When the same strain was grown continuously

in a glucose deficient medium, a more rapiddevelopment of 0-agglutinable cells was noted.However, as in the previous experiment, thiswas not reflected in a detectable loss in mousevirulence or immunogenicity. This experimentlasted only 8 days, and it is possible that if thecells were cultured continuously for a longerperiod of time under similar conditions, a reduc-tion of mouse virulence and immunogenicitymight have been observed. The relatively rapidloss in ability to resist agglutination by 0 anti-serum can probably be attributed to the limitedglucose concentration in the growth medium, anobservation which is in agreement with the find-ings of Gladstone (1937).These results would appear to differ from the

observations of Felix and Pitt (1934), who foundthat an 0-inagglutinable strain of S. typhosawas more virulent and immunogenic for micethan another 0-agglutinable culture. However,in the case reported here the same strain wasstudied both before and after it became 0-ag-glutinable. Thus, several explanations may beoffered. The change which an 0-inagglutinableculture undergoes when it becomes 0-agglutinable

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may not be accompanied by a decreased contentof Vi antigen, or perhaps some factor in the cellother than the Vi antigen is also of importance indetermining mouse immunogenicity. Althoughthese may be logical hypotheses, we know of nodata to support them. A more likely explanationin our opinion is that in some cases when an0-inagglutinable culture becomes 0-agglutinable,the accompanying decrease in Vi antigen contentmay be too small to measure by mouse virulenceor protection tests.

It is also interesting to note that the totalnitrogen content of the vaccines prepared fromorganisms grown in continuous culture was lowerthan those made from cells cultured by conven-tional methods. Since it was necessary to con-centrate the bacteria grown in continuous culturein order to obtain the required vaccine density,the bacteria were centrifuged, and the se nteither acetone-killed and dried or resuspendedin saline and heat-killed. Thus a total nitrogencontent lower than that of the unwashed standardheat-killed vaccine could have resulted from this"washing" procedure. However, in the case ofthe acetone-killed and dried vaccines, both thestandard and the experimental agents werewashed. Thus in this instance at least, washingcannot account for the difference in nitrogenvalues which were noted. It does seem likely thatculturing the bacteria which made up all theexperimental vaccines in simple synthetic mediacould have resulted in immunizing agents oflowered nitrogen content (Virtanen and De Ley,1948). However, this point remains to be eluci-dated.From the results reported, cells of S. typhosa

grown by continuous cultivation techniqueswould meet the presently accepted criteria foruse in a vaccine for immunization of humans.Organisms cultivated by this method are asimmunogenic as conventionaRly grown prepara-tions when tested by the mouse protectivepotency tests presently employed as the standardmethod of assay.Some investigators have been critical of this

test and are of the opinion that it is not justifiableto correlate mouse protective potency withhulman inmmunogenicity since the typhoid in-fection produced in the mouse does not resemblethe disease in man. While this point is welltaken, no substitute for this test has yet beenwidely considered or officially accepted. Recently,carefully controlled field and laboratory studies

on pertussis vaccine have indicated that, in thiscase at least, a positive correlation between mouseprotective potency (as determined by intra-cerebral challenge) and human immunogenicitymay occur (Evans and Cockburn, 1951). It ishoped that work now in progress in this labora-tory (Edsall et al., 1954) and in a controlledfield trial will determine whether a similar cor-relation exists with typhoid vaccine. Untilfurther results along these lines are available,however, it would appear that there is no alterna-tive but to continue to employ the mouse pro-tective potency test for assaying typhoid vac-cines, and to be guided by the results of suchtests in selecting procedures for vaccine prepara-tion.

ACKNOWLEDGMENTS

The authors wish to express their appreciationto Dr. Geoffrey Edsall and Dr. Maurice Landyfor their interest in this work, and to Mr. AlbertGroffinger and Mr. Elmer Martino for technicalassistance.

SUMMARY

The effect of continuous cultivation on themouse virulence and immunogenicity of strainsof SalmoneUa typhosa was studied. Two strainswere employed.

Strain Ty2 was continuously cultured for 24days in a medium deficient in nitrogen with nodiscernible loss in mouse virulence. Dehydratedvaccines prepared from the cultures taken earlyin the experiment and on the twenty-fourth daywere of comparable immunogenicity for mice,and both experimental vaccines were of thesame order of potency as a standard prepara-tion. However, a measurable change in theculture was observed after the fifteenth day ofcontinuous growth since organisms previouslyable to resist agglutination by typhoid 0 anti-serum became 0-agglutinable. When strain Ty2was cultured continuously in a medium deficientin glucose, the cells became 0-agglutinable bythe eighth day. At this time no loss in mousevirulence or immunogenicity was noted.The second culture, strain V58, was grown

continuously for 7 days. A heat-killed phenol-preserved vaccine was prepared from organismson the seventh day. This preparation proved tobe equal or possibly superior to a standard heat-killed vaccine in mouse protective potency. Thesignificance of these results is discussed.

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GERHART, P. 1946 Brucella sui8 in aerated brothculture. III. Continuous culture techniques.J. Bacteriol., 62, 283-292.

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