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....... SlRICKETTSIAL GROWTHSIN SUSPENSION CELL CULTURES

IN SERUM-FREE AND.DEFINED MEDIA

AUGUST 1964

UNITED STATES ARMYBIOLOGICAL LABORATORIES

FORT DETRICK

U.S. ARMY BIOLOGICAL LABORATORIESFort Detrick, Frederick, Maryland

TECHNICAL MANUSCRIPT 155

RICKETTSIAL GROWTH IN SUSPENSION CELL CULTURESIN SERUM-FREE AND DEFINED MEDIA

Henry R. Tribble, J).

Horace B. Rees, Jr.

Henry J. Hearn, Jr.

Medical Bacteriology Divisionand

Virus and Rickettsia DivisionDIRECTOR OF BIOLOGICAL RESEARCH

Project IC522301A05901 August 1964

1

This publication or any portion thereof may notbe reproduced without specific authorization fromthe Commanding Officer, U. S. Army Biological

Laboratories, ATTN: Technical Releases Branch,Technical Information Division, Fort Detrick,Frederick, Maryland. 21701. However, DDC isauthorized to reproduce the publication for U. S.Government purposes.

The information in this publication has not beencleared for release to the public.

DDC AVAILABILITY NOTICE

Qualified requestors may obtain copies of thispublication directly from DDC.

Foreign announcement and dissemination of thispublication by DDC Ts limited.

AB8TRACI

Rickettsia t and Rickettsia rickettail were propagated incultures of cat kidney and L cefl lines that were grown in a serum-free suspension system. One growth medium (LAE) consisted of lact-albumin hydrolyzate and glutamine as nitrogen sources, glucose andpyruvate as carbon sources, vitamins, salts, and additives such asMethocel, insulin, and phenol red. A second and third medium con-sisted of essentibi amino acids (EAA) with or without nonessentialaino acids (NZAA), respectively as nitrogen sources, salts, carbonsources, vitamins, and the additives, Methocel, insulin and phenolred. LAN supplemented with 10 per cent calf serum was used as acontrol.

The two cell lines vere carried as stock cultures in serum-free suspensions at least a month prior to the experiments. Thestudies were conducted with 100-al serum bottles (30 ml mediumper bottle) and wroe agitated on a Neo Brunswick shaker at 130 rpmat 3°C. A mximal yield of 1063 yolk sac (Y") LD% of R. typhioccurred at 7 days in cat kidney cells grown in EAA; titers wereslightly less in LAN and considerably less in IWAA cell cultures.

In contrast, maximal yields of 106'8 YSLD6 of R. rickettsiioccurred at 4 days in L cqlls grown in NEAA; slightly-lover titerswere found in BAA. In LAN only minimal amounts of rickettsiae werefound. Titers obtained with cultures infected with R. typhi wsg-gested cyclic rickettsial growth.

These data, obtained in serum-free and chemically defined media,demonstrate the importance of nutritional environments for maximumsurvival, penetration, and proliferation of rickettsial speciesand suggest a new approach to studying rickettsial metabolism.

I. INTRODUCTION

Studies that define the nutritional requirements for rickettsial multi-plication in animal cell cultures have received increasing attention duringthe last two decades. A limiting factor in these studies has been the needfor a serum for the growth of animal cell cultures. A system in which ex-periments could be conducted with serum-frer media or with chemically de-fined media would be desirable. Several suck investigations have been re-portedWat meetings and in the literature in recent years. This paperpresents the results of preliminary studies on the growth of Rickettsiarickettsii and Rickettsia typhi in suspended cell cultures employing serum-P and heicallydef~te37m-•ia.

II. MATEPJALS AND MET•DS

The media employed were as follows: (a) A serum-free lactalbumin hy-drolyzate medium designated as LAH (Table I) which contained lactalbuminhydrolyza~e and glutamine as nitrogen sources, glucose and pyruvate .as carbonsources, vitamins, salts, plus such additives as Methocel and phenol red;(b) a defined medium designated as EAA (Table II), which contained 13 essen-tial amino acids, carbon sources, salts, vitamins and Methocel and phenolred; and (c) the defined medium plus six amino acids found nonessential forcell growth, and hereafter referred to as NBAA. In studies with R. typhi, afourth type of medium, LAB supplemented with 10 per cent calf serum, was em-ployed. The medium with serum was added to serum-free cat kidney cellsgrown Just prior to infection. This combination served as an initial posi-tive control for the growth of rickettsiae.

Animal cell cultures, suspended in 30 ml of media contained in 100-mlserum bottles were incubated at 34 0C in a New Brunswick gyrotory shakerrotating at 130 rpm. Both cat kidney and L cell lines were derived fromstocks maintained in either the LAB medium or the defined medium for atleast a month before infection with rickettsiae.

The Bitterroot strain of R. rickettaii and the Wilmington strain of R.typhi (R. mooseri) were prepared as inocula from infected yolk sacs of em-bryonated eggosby a modified, molar potassium chloride method of Ormtbee. 4

After the final centrifugation, rickettsiae were suspended in Bovarnick's 5

sucrose-phosphate-glutamate containing 0.6 per cent bovine albumin (Armour'sFraction V) and stored at -65 0C until the mixtures were thawed and employedas inocula.

TABLE I. LACTALBUMIN HYDROLYZATE MEDIUM FOR SUSPEINDED CELL GROWTH

C Concentra- ooncentra-Component tion, mg/l Component tion, mg/i

Nitrogen Sources: Salts:

Lactalbumin hydrolyzate 2500 NaCI 7400L-Glutamine 300 KCI 400

NaH2 PO4 .H20 100Vitamins: NaHCO3 300

CaCl 2 "-2 20 265D-biotin 1.0 MgCI1•6H20 275Choline.Cl 1.0Folic Acid 1.0 Carbon Sources:Niacinamide 1.0Ca pantothenate 2.0 Glucose 1000Pyridoxal.HC1 1.0 Sodium pyruvate 110Thiamine •HCI 1.0i-inositol 1.0 Antibiotics, etc.:Riboflavin 0.1B 0.002 Methocel 15 cps 1000

SPhenol red 10 mg/i

II

I

7

TABLE II. CHEMICALLY DEFINED M4EDIUM4 FOR SUSPENED CELL GROWT~H

_-cntA COoncentra~-component titpýgi Component on m/

Amino Acids (.uuiential): Saits:

L-Arginine*ECl - 1.00 NaCl 74&00L-Cysteine-BCl 75 XCI. 400L-Hiuti-dine*HC1 60 IN8310)4-320 100L-Isoleucinb 1.50 NaRC03 500L-Leucine 300 CaC1:2*9g0 265L-Lysizie 300 MgCla 6Hgs) 275L41etbionine 60L-Pbenyialanine 120 Carbon Sources:L-Tbreonine 135L-Tryptophan 60 Glucose 10ooL-Tyrosine 120 Sodium pyruvate no0L-Valine 150L-Giutsidne Ji,0 Vitamins:

Amino Acids. (aioneumtial): D-biotin 1.0Choline*Cl 1.0

Glycine 60 Folic Acid 1.0L-Alanine 120 liacinamide 1.0L-Serine 150 Ca pantothenate 2.0L-Cystine*HC1 75 Pyridoxal*E1 1.0L-Aspartic Acid 270 Thimaim.3C1 1.0L-Giutamic Acid 31.5 i-inositol 1.0L-Proline 115 Riboflavin. 0.1

B12 0.002Antibiotics, etc.:

Methocel 15 cps 1000

Phenol red 10 mg/i

Cell cultures with populations from 5 x l10 to 1.5 x 108 cells per mlwere inoculated with these partially purified rickettsiae. The mixture wasincubated for 45 minutes on the rotary shaker-incubator and centrifuged tosediment the cells. Supernatant medium and unadsorbed rickettsiae were re-moved by decanting, and the remaining cells resuspended in fresh medium.Theme resuspended cells were then sampled for a rickettsial assay and a cellcount. The cultures were separated from spent medium by centrifuging everyother day and resuspended in fresh medium. Additional culture samples wereobtained daily, diluted 1:1 with normal calf serum, sealed in glass ampoules,and stored at -65WC until 'essayed for rickettsial titer in embryonated eggs.Viable tissue cell counts were made using the 0.5 per cent trypan bluemethod.

III. RESULTS AND DISCUSSION

To assay rickettsial infectivity, tenfold dilutions of each sample wereinoculated into the yolk sac of 7-day embryonated eggs. After incubatingthe eggs at 350 C in a commercial incubator for 11 days, embryo deaths weredetermined daily by candling. Embryo deaths occurring in the typical patternfor rickeltsial infections were employed to calculate the LDSOtiter per mlby tho method of Reed and Muench.

The stabilities of rickettsial infectivity at 340 C in a shaker-incubatorwere determined first in LAN and defined media without tissue cells. Tenper cent skimed milk was included also for comparison. R. t hi and R.rickettsii showed similar losses of infectivity (Figure 1T. - lostoneor more logaritbms of infectivity after four hours at 340 C. Other data in-dicated little or no infectivity remained after 24 hours of incubation.These findings suggested that rickettsial infectivity found after 24 hoursmust have been a result of cell-rickettsial association.

When cells of the cat kidney line were infected with R. typhi (Figure 21,cultures grovn either in LAH or LAH plus 10 per cent calf serum appeared toproduce cyclic yields of rickettsiae. The highest yields of rickettsiae-were about 10 YSLDH per ml and occurred in the LAE medium on the 4th, 14th.,and 20th day after inoculation, as shown by the open circles in the figure.The highest yield found for the cells grown in the medium with serum was 10.5infectious units and occurred on the ninth day of the infection. Neitherrickettsial yields nor the cell proliferation appeared to be enhanced by theaddition of 10 per cent calf serum to the LAH.

The number of cat kidney cells remained static for the first six days ofrickettsial infection, then, a precipitous drop in the number occurred onthe Day 7. This was followed by a slow decline in cell numbers until com-plete cell lysis occurred on Day 15 in the medium with serum, or on Day 20

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in the LAH medium. Increases in the rates of cell lysis were generallyassociated with increased rickettsial yields.

When groiith curves of R. typhi were determined in cultures of the catkidney line propagated in 1A a NEAA media (Figure 3), the tissue cellsgrown in MAA yielded greater amounts of rickettsiae than did those cellsgrown in the same medium supplemented with nonessential amino acids. Thisis shown by the closed circles in the figure, which depict maximal rickett-sial yields of 1CO-8 YSLW5 per ml in the defined medium, compared to 1Ci-2YSL•0 per ml for cells in the defined plus NZAA medium (open circles). Aculture life of 8 days was not sufficient to confirm the cyclic yields ofrickettsiae observed in the previous experiment. However, a comparison ofthe rickettsial yields with the number of cells in cultures again suggestedthat maximal infectivity was closely associated with cell lysis.

With R. rickttaii in L cell cultures the beneficial effect of incorpo-rating the nonessential acids was observed (Figure 4). Cells infected withspotted fever rickettsiae and grown in NZAA produced a maximal yield of 104JYSLDU per-ul on the sixth day after infection, shown as the solid circles inthe figure. By comparison, cells in the defined 3AA medium produced only1016 Y8Ltý per al. In marked contrast, infected cells suspended in LANmedium grow well but failed to support proliferation of the rickettsiae.

In sumary, a neow approach to studying rickettsial metabolima has beendemonstrated in cells propagated in serum-free and chemically defined envi-ronments. Preliminary data have suggested rather striking differences amongmedia and rickettsial species for maximal survival, penetration, and prolif-eration.

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LITERATURE CITED

1. Tribble, H.R., Jr., and Higuchi, K. "Studies on the nutrition andmetabolism of animal cells in serum-free media: II. Cultivation ofcells in suspension," J. Infect. Diseases 112:221-225, 1963.

2. Nagle, S.C., Jr.; Tribble, H.R., Jr.; Anderson, R.E.; and Gary, N.D."A chemically defined medium for growth of animal cells in suspension,"Proc. Soc. Exptl. Biol. Med. 112:340-344, 1963.

3. Bozeman, F.M.; Hopps, H.E.; Danauskas, J.X.; Jackson, E.B.; and Smadel,J.E. "Study on the growth of rickettsiae: I. A tissue culture sys-tem for quantitative estimates of Rickettsia tsutsugamushi," J. Immunol.76:475, 1956.

4. Ormsbee, R.A. "A method of purifying Coxiella burneti and other patho-genic rickettsiae," J. Immunol. 88:100-108, 1962.

5. Bovarnick, M.R.; Miller, J.C.; and Snyder, J.C. "The influence of cer-tain salts, amino acids, sugars, and proteins on the stability ofrickettsiae," J. Bacteriol. 59:509-522, 1950.