Vol. 44, No. 3INFECTION AND IMMUNITY, June 1984, p. 559-5640019-9567/84/060559-06$02.00/0Copyright © 1984, American Society for Microbiology

Biochemical and Immunochemical Analysis of Rickettsia rickettsiiStrains of Various Degrees of Virulence

ROBERT L. ANACKER,l* ROBERT N. PHILIP,2 JIM C. WILLIAMS,'t ROBERT H. LIST,' AND RAYMOND E.MANN'

Laboratory of Microbial Structure and Function' and Epidemiology Branch,2 Rocky Mountain Laboratories, Hamilton,Montana 59840

Received 27 December 1983/Accepted 27 February 1984

Six strains of Rickettsia rickettsii from Montana and North Carolina were examined in an effort to identifyrickettsial constituents associated with virulence. Fever responses, scrotal reactions, and mortalities ofmale guinea pigs inoculated intraperitoneally with 1,000 PFU of rickettsial strains revealed that the twoMontana patient strains (Sheila Smith and Norgaard) and one Montana strain (Sawtooth Y 2) from the woodtick, Dermacentor andersoni, could be placed in the group of highest virulence, the two North Carolinastrains (Morgan and Simpson) in the group of lesser virulence, and the Montana strain (HLP) from the rabbittick, Haemaphysalis leporispalustris, in the group of lowest virulence. The HLP strain was differentiatedfrom the other strains by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by stainingwith Coomassie brilliant blue or with silver. The patient strains could not be differentiated from each otherby these procedures. All of the strains apparently had three heat-modifiable proteins. Analysis of proteinaseK-digested rickettsial lysates by sodium dodecyl sulfate-polyacrylamide gel electrophoresis suggested thatthe strains had a complex mixture of polysaccharides. These putative polysaccharides probably were notrelated to the differences in virulence of the strains, since the patterns for all of the strains were identical. Atleast five antigens (molecular weights of 128,000, 105,000, 84,000, 30,500, and 20,500) were demonstrated byradioimmune precipitation tests employing extracts from radioiodine-labeled rickettsiae and antibodiesfrom infected guinea pigs. With these same sera a minimum of 14 antigens was detected in these strains byan immunoblotting procedure. The apparent molecular weights of several of the HLP antigens differed fromthose of the presumed corresponding antigens of the other strains. The electrophoretic techniques utilized inthis study were not sufficiently sensitive to demonstrate compositional differences in the patient strainswhich differed in their virulence for guinea pigs.

A variety of evidence suggests that strains of the etiologi-cal agent of Rocky Mountain spotted fever, Rickettsiarickettsii, differ in virulence for humans and experimentalanimals. Before the advent of effective antibiotics, onlyabout 5% of individuals afflicted with Rocky Mountainspotted fever in south-central Idaho succumbed, whereasthe majority of unvaccinated patients in certain areas, suchas the Bitter Root Valley of western Montana and Thermo-polis, Wyo., died (11). Similarly, strains isolated from theAmerican dog tick, Dermacentor variabilis, collected in theeastern United States were less virulent for guinea pigs thanwere some of the strains isolated from the western woodtick, Dermacentor andersoni (15). Also, strains isolatedfrom the rabbit tick, Haemaphysalis leporispalustris, werefound to be less virulent for guinea pigs than were the mostvirulent strains from D. andersoni (12). Recently, it wasfound that one strain (HLP), although serologically closelyrelated to the D. andersoni strains of R. rickettsii and stillconsidered to belong to the R. rickettsii species, could bedistinguished from the wood tick strains by the microimmu-nofluorescence test (13). Thus, it seems clear that R. rickett-sii can exist in forms which vary with respect to the severityof illness induced in their hosts.The factors responsible for the virulence of R. rickettsii

are unknown. In an effort to identify those componentsassociated with virulence, eastern and western strains of R.rickettsii from patients and ticks were compared by a variety

* Corresponding author.t Present address: U.S. Army Medical Institute of Infectious

Diseases, Fort Detrick, Frederick, MD 21701.

of methods. The studies, outlined in this report, demonstrat-ed that the strains selected differed in their virulence forguinea pigs and that differences in the composition of one ofthese strains could be revealed by the experimental methodsemployed. It has not yet been established whether thesestructural differences are indeed related to differences invirulence.

MATERIALS AND METHODSRickettsial strains. The six strains used in this study are

described in Table 1. Four of the strains (Sheila Smith,Norgaard, Morgan, and Simpson) have been cloned. TheNorgaard strain was isolated from a nymph obtained from apatient 3 days after the onset of Rocky Mountain spottedfever. Since the nymphal D. andersoni was the only tick onthe patient, this isolate is presumed to be the one whichcaused the illness in this patient. In all cases, rickettsiaefrom the last one or two egg passages were used in theseexperiments.

Cultivation of rickettsiae. The rickettsiae were grown inthe yolk sacs of chicken embryos or in L cells as previouslydescribed (16).Guinea pigs. Male Hartley strain guinea pigs raised at

Rocky Mountain Laboratories, Hamilton, Mont., were usedin the virulence test.

Purification of rickettsiae. Rickettsiae were purified bycentrifugation in Renografin density gradients (17). Thepurified rickettsiae were suspended in 10 mM potassiumphosphate buffer (pH 7.0) to a concentration of about 5 to 10mg of protein per ml and held in 0.1- to 0.2-ml amounts at-75°C until used.

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TABLE 1. Strains of R. rickettsii studied

Strain (reference) Host Geographical source Year No. of passagesa

Sheila Smith (4) Human Western Montana 1946 8 EP/1 TC/1 GP/4 TC/2-4 EPNorgaard (13) D. andersoni Western Montana 1975 1 M/4 EPI4 TC/2-3 EP

nymphSawtooth Y 2 (5) D. andersoni adult Western Montana 1961 23 EPMorgan (13) Human North Carolina 1975 1 M/3 EP/S TC/2-3 EPSimpson (13) Human North Carolina 1975 1 M/3 EP/5 TC/2-3 EPHLP (12) H. leporispalustris Western Montana 1948 50 EP/9 TC/3-4 EP

adulta EP, Egg passage; M, Microtus; TC, tissue culture; GP, guinea pig.

Radioiodination of rickettsiae. Rickettsiae were iodinatedin the presence of 1,3,4,6-tetrachloro-3a,6a-diphenylglyco-luril (Iodogen; Pierce Chemical Co., Rockford, Ill.) (9).Purified rickettsiae were thawed and diluted with K-36 buffer(18) to contain 2 pug of protein per pl. A sample (0.1 ml) of theappropriate suspension was then incubated with 10 pI ofcarrier-free Na125I (50 puCi/pI) for 5 min with occasionalgentle stirring in a small glass vial coated with 10 pg oflodogen. Next, the labeled rickettsiae were washed twicewith 1.0 ml of K-36 buffer (Microfuge B centrifuge; 5 min).The final pellet was resuspended in 0.5 ml of 10 mMpotassium phosphate buffer (pH 7.0).SDS-PAGE. Samples were analyzed by sodium dodecyl

sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) es-sentially by the procedure of Laemmli (8). The rickettsialsuspensions were thawed, mixed with solubilizing buffercontaining 4% SDS and 8% 2-mercaptoethanol, and heatedat the desired temperature. Samples (25 plI) containing 25 pigof rickettsial protein were added to each well and electro-phoresed on gels consisting of a 5% stacking gel and a 12.5%separating gel at a constant current of 40 mA. The gels werefixed in 25% isopropanol-7% acetic acid and stained eitherwith Coomassie brilliant blue R-250 or with silver for poly-saccharide or polysaccharide and protein (6). For someexperiments, the rickettsial samples were solubilized at100°C for 5 min, and then the samples (60 pI) were treatedtwice with proteinase K (20 pu1 containing 2.5 mg of protein-ase K per ml of solubilizing solution) (Boehringer MannheimBiochemicals, Indianapolis, Ind.) at 60°C for 1 h each time todigest proteins (6).

Unlabeled high- and low-molecular-weight protein stan-dards (Bio-Rad Laboratories, Richmond, Calif.) or a 14C_methylated protein mixture (Amersham Corp., ArlingtonHeights, Ill.) were generally run along with the rickettsialpreparations on the polyacrylamide gels.

Virulence of rickettsiae for guinea pigs. Virulence wasassessed basically as described earlier (1). A total of 1,000PFU of each strain grown in chicken embryos and diluted inSnyder I solution (7) was inoculated intraperitoneally intoguinea pigs, which weighed ca. 450 g, in groups of six. Rectaltemperatures were determined daily for 12 days with atelethermometer (Yellow Springs Instrument Co., YellowSprings, Ohio). Areas under the fever curves were deter-mined with a model 9100A Hewlett-Packard calculator cou-pled to a 9125B Hewlett-Packard calculator plotter andcompared by the student t test (2). Scrotal reactions ofinfected guinea pigs were assigned a score of 0 to 4+ on thebasis of grossly observable responses, from undetectable (0)to severe with necrotic changes (4+).Radioimmune precipitation test. Radioiodinated rickettsiae

(200 p.g of protein) were extracted for 30 min at 37°C withlysing buffer (0.1% SDS, 0.5% sodium deoxycholate, 0.5%Triton X-100, 0.85% NaCl, 0.02% NaN3 in 10 mM Tris [pH

7.4]) and then centrifuged at 39,000 x g and 20°C for 30 min.A sample (50 RI) of the supernatant was incubated with 50 ,u1of pooled, inactivated guinea pig sera for 30 min at 37°C inmicrofuge tubes (1.5 ml). Then 100 1LI of 5% protein A-Sepharose CL-4B (Pharmacia Fine Chemicals, Inc., Pis-cataway, N.J.) in 67 mM phosphate buffer plus 0.85% NaCI(pH 7.4) was added, and incubation was continued foranother 30 min at 37°C. Afterwards, the preparations werecentrifuged at 9,600 x g for 3 min, and the pellets werewashed three times with 1 ml of lysing buffer, with centrifu-gation as described above. The pellet obtained after the lastwash was heated in 100 R1 of sample solubilizing solution at100°C for 5 min. Samples (25 pI) were loaded onto polyacryl-amide gels and electrophoresed in the standard way. Thegels were fixed in 25% isopropanol-7% acetic acid, dried,and incubated with Kodak X-Omat AR film.

Immunoblotting. Rickettsial constituents separated bySDS-PAGE were transferred to nitrocellulose paper(HAHY; Millipore Corp., Bedford, Mass.) in a Trans-Blotcell (Bio-Rad) by the procedure of Batteiger et al. (3).Unoccupied protein-binding sites on the nitrocellulose paperwere blocked with 0.05% Tween 20 in 50 mM phosphatebuffer (pH 7.4) containing 150 mM NaCl and 0.02% sodiumazide (PBS-T), and the paper was then incubated for 2 h with

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DAYS POST CHALLENGEFIG. 1. Fever curves for male Hartley strain guinea pigs inocu-

lated intraperitoneally with 1,000 PFU of the appropriate strain of R.rickettsii. Each point represents the mean temperature of six ani-mals. V, Sawtooth 9 2; 0, Norgaard; *, Sheila Smith; A, Simpson;O, Morgan; Q, HLP; 0, diluent control. Points where two or morecurves interact are noted by asterisks.

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ANALYSIS OF RICKETTSIA RICKETTSII STRAINS 561

1:100 dilutions of inactivated guinea pig immune sera or anti-yolk sac serum in PBS-T. After the removal of unboundserum proteins by washing in phosphate-buffered saline, thenitrocellulose paper was incubated with 125I-labeled proteinA in PBS-T, washed again in phosphate-buffered saline,dried, and incubated with Kodak X-Omat AR film.

RESULTSVirulence of strains for guinea pigs. Average temperatures

of guinea pigs inoculated intraperitoneally with 1,000 PFU ofthe apropriate strain are presented in Fig. 1. Analyses of theareas under the fever curves showed that the two strainsfrom Montana patients and the Sawtooth 9 2 strain from D.andersoni caused significantly more fever than did thestrains from the North Carolina patients (P < 0.01). TheNorth Carolina strains induced more fever than did the strainisolated from the rabbit tick (P < 0.001). Differences in theareas under the fever curves for guinea pigs within the abovegroups were not significant (P > 0.2). The identical assign-ment of these strains into groups was made on the basis ofthe severity of the scrotal reactions. There was no grosslyobservable scrotal reaction in animals infected with the HLPstrain. Mean maximum scores for the other Montana strainsranged from 2.1 (Sheila Smith) to 3.0 (Norgaard), whereasscores for the North Carolina strains were 0.5 (Morgan) and1.3 (Simpson). During the 21 days the animals were heldafter infection, two animals inoculated with the Norgaardstrain and one animal inoculated with the Sheila Smith straindied. Based on these criteria, Montana strains Sheila Smith,Norgaard, and Sawtooth 9 2 were most virulent, NorthCarolina strains Morgan and Simpson were less virulent, andthe HLP strain was least virulent. All but the Sawtooth 9 2strain were selected for further study.

1 2 3 4 5 6 7 8 9 10MW(K)

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FIG. 2. Coomassie brilliant blue-stained SDS-PAGE profiles of

rickettsial strains solubilized at either 37C for 30 min (lanes 1 to 5)

or at 1000C for 5 mmn (lanes 6 to 10). All of the strains had three heat-

modifiable proteins (noted by asterisks). Major differences in pat-

terns between HLP and other strains are marked with arrows. Lanes

1 and 6, Sheila Smith; lanes 2 and 7, Morgan; lanes 3 and 8,

Norgaard; lanes 4 and 9, Simpson; lanes 5 and 10, HLP. MW,

Molecular weight.

1 2 3 4 5 6 7 8 9 10MW(K) ^*

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FIG. 3. Silver-stained SDS-PAGE profiles of rickettsial strainssolubilized at either 37°C for 30 min (lanes 1 to 5) or at 100°C for 5min (lanes 6 to 10). Major differences in patterns between HLP andother strains are noted with arrows. Lanes 1 and 6, Sheila Smith;lanes 2 and 7, Morgan; lanes 3 and 8, Norgaard; lanes 4 and 9,Simpson; lanes 5 and 10, HLP. MW, Molecular weight.

Comparisons of strains by SDS-PAGE. Whole-cell prepara-tions 'of strains solubilized at either 37 or 100°C wereexamined by SDS-PAGE, followed by staining with Coo-massie brilliant blue (Fig. 2). All of the strains had threemajor heat-modifiable proteins (noted by asterisks in Fig. 2).Two of the heat-modifiable proteins appeared to be identicalin all strains, but the intermediate heat-modifiable proteinfrom the HLP strain migrated slightly faster than did thecomparable proteins from the other strains. The SDS-PAGEprofile of the HLP strain differed from that of the otherstrains. Some differences between HLP and the other strainsare noted by arrows in Fig. 2.

Next, whole-cell lysates of the test strains were subjectedto SDS-PAGE and then stained with silver to determinewhether this stain might reveal differences (Fig. 3). The heat-modifiable proteins and the proteins differentiating the HLPstrain from the patient strains were observed as in theCoomassie brillant blue-stained gel, but silver staining didnot indicate compositional differences in the Montana andNorth Carolina patient strains.When rickettsial lysates were treated with proteinase K,

electrophoresed, and then stained with the silver stain forpolysaccharides, the patterns presented in Fig. 4 wereobtained. A nearly continuous series of seemingly identicalbands was observed in the lysates of all of the strains of R.rickettsii. Particularly obvious were the relatively intensebands occurring in the middle and upper portions of thelanes. When this gel was reoxidized to clear the silver stainand then restained with Coomassie brilliant blue, only thetwo lowest bands stained (very slightly), suggesting thatthese substances which stained with silver but not withCoomassie brilliant blue were polysaccharide in nature.Radioimmune precipitation of R. rickettsii antigens. Pooled

21-day sera from guinea pigs inoculated intraperitoneally

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562 ANACKER ET AL.

3 5MW(K)

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FIG. 4. Silver-stained SDS-PAGE profiles of rickettsial strainssolubilized at 100°C for 5 min (100 p.g of rickettsial protein in 60 ,ul)and then incubated consecutively at 60°C for 1 h with two additionsof 20 ,ul of solubilizing solution containing 50 ,ug of proteinase K.Lane 1, Sheila Smith; lane 2, Morgan; lane 3, Norgaard; lane 4,Simpson; and lane 5, HLP. MW, Molecular weight.

with 1,000 PFU of the patient strains (Fig. 1) were tested fortheir ability to precipitate rickettsial antigens in the radioim-mune precipitation test (Fig. 5). Qualitatively, antibodies toat least five antigens (molecular weights of 128,000, 105,000,84,000, 30,500, and 20,500) not observed in significantamounts in the control lanes were detected in all sera,although there appeared to be some quantitative differencesin the antibodies in some sera as judged by the amount oflabeled antigen bound.

Immunoblotting of R. rickettsii antigens. Immunoblottingresults obtained with the five strains tested against pooledsera from guinea pigs inoculated intraperitoneally with 1,000PFU of the Sheila Smith strain or anti-yolk sac serum arepresented in Fig. 6. (Results obtained with antisera to theother strains were comparable.) Antigens detected in thelysates of the rickettsial strains had apparent molecularweights ranging from ca. 15,000 to 165,000. The HLP strainhad several antigens, noted by asterisks in Fig. 6, whichdiffered slightly in mobility from the presumed correspond-ing antigens present in the patient strains.

DISCUSSIONOur results from limited virulence assays of various

strains of R. rickettsii in guinea pigs corroborate the conclu-sion of Price that some western strains are more virulentthan some eastern strains (14, 15). In our tests the twopatient strains and the D. andersoni strain from western

Montana produced higher and longer-lasting fever and moresevere scrotal reactions than did the two patient strains fromNorth Carolina. In addition, only guinea pigs from thosegroups inoculated with the western strains died during theperiod of observation. On the other hand, the HLP strain ofR. rickettsii obtained from a rabbit tick in western Montanawas the least virulent of the strains tested; it produced inguinea pigs on the average only 1 day of fever and no visiblescrotal reaction. This result also is consistent with an earlierreport (12).Our attempts to differentiate our selected strains of R.

rickettsii were only partially successful. The HLP straincould be distinguished from the Montana and North Carolinapatient strains by several of the procedures we tried. Withthe SDS-PAGE technique and Coomassie brilliant blue orsilver staining, bands with apparent molecular weights of ca.14,000, 18,500, and 27,000 were demonstrated in lysates ofthe strain of least virulence, HLP; lysates of the morevirulent strains from patients did not exhibit bands withthese molecular weights. In addition, several antigenic dif-ferences between the HLP strain and the patient strains wererevealed by immunoblotting experiments. Possibly some ofthe differences in composition of these strains shown bythese techniques are responsible for the slight but demon-strable differences of these strains in microimmunofluores-cence tests (13). Although our methods showed differencesbetween the HLP strain and the patient strains, none of theelectrophoretic techniques was satisfactory for differentiat-ing the western patient strains of highest virulence from theeastern strains of lesser virulence.The reason for our inability to differentiate by electropho-

retic methods patient strains which differ in their virulencefor experimental animals is not clear. Perhaps the differencesin the virulence factors for the two groups are relativelyminor, so minor that they are not reflected in differences inelectrophoretic properties. Alternatively, quantitative, rath-er than qualitative, differences may be responsible for thevirulence difference, or the virulence factors may not bedemonstrable by the techniques utilized for this study.At present the significance of the differences observed by

SDS-PAGE and immunoblotting between HLP and the otherstrains of R. rickettsii is unknown. Perhaps the "modified"constituents present in the patient strains are responsible fortheir greater virulence, but we cannot exclude the possibilitythat some or all of these differences may be completelyunrelated to the kinds of interaction between parasite andhost usually associated with virulence. Instead, the observeddifferences may represent evolutionary changes in the rick-ettsiae, permitting enhanced survival in their particular tickand mammalian hosts.

It is unclear why disparate results were obtained inradioimmune precipitation and immunoblotting experi-ments, but current experiments with monoclonal antibodiesto rickettsial antigens may provide at least a partial explana-tion (R. L. Anacker, R. H. List, R. E. Mann, S. F. Hayes,and H. C. Caldwell, manuscript in preparation). Some of ourmonoclonal antibodies are able to reveal carbohydrate anti-gens by immunoblotting but not by radioimmune precipita-tion tests, in which only proteins are labeled with radioio-dine. Conversely, other monoclonal antibodies function inthe radioimmune precipitation test but not in immunoblot-ting tests. Possibly the later antibodies are specific for"conformational" antigens which are destroyed by the SDSand 2-mercaptoethanol solubilization treatment required be-fore electrophoresis.The presence of multiple silver-staining components in

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ANALYSIS OF RICKETTSIA RICKETTSII STRAINS 563

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FIG. 5. Precipitation of antigens labeled extrinsically with 1251 and extracted from rickettsiae with a mixture of SDS, sodium deoxycholate,and Triton X-100 by pooled sera from guinea pigs infected with strains of R. rickettsii. Antigens precipitated by sera from infected guinea pigsbut not by the control guinea pig anti-yolk sac serum are noted by asterisks. Source of extracts: panel A, Sheila Smith, lanes 1 to 4 and 9; Mor-gan, lanes 5 to 8 and 10; panel B, Norgaard, lanes 1 to 4 and 9; Simpson, lanes 5 to 8 and 10. Sera (both panels): anti-Sheila Smith, lanes 1 and5; anti-Morgan, lanes 2 and 6; anti-Norgaard, lanes 3 and 7; anti-Simpson, lanes 4 and 8; anti-yolk sac, lanes 9 and 10. The soluble extractswere run in panel A, lane 11 (Sheila Smith), lane 12 (Morgan); panel B, lane 11 (Norgaard); lane 12 (Simpson). MW, Molecular weight.

proteinase K-digested lysates of rickettsiae suggest thatrickettsiae have a complex mixture of polysaccharides. Inaddition, the patterns of these silver-staining rickettsialconstituents are reminiscent of the ladder-like patterns char-acteristic of lipopolysaccharides extracted from wild-typeenteric bacteria (6). Although polysaccharides have been

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6 7 8 9 10

implicated in virulence of gram-negative bacteria (10), thefact that the rickettsial strains examined in this study havepresumed identical polysaccharide compositions arguesagainst the concept of a major role for polysaccharides as adifferential determiner of virulence of R. rickettsii.

ACKNOWLEDGMENTSThe authors are grateful to Alan Barbour, Harlan Caldwell, John

Swanson, and Penny Hitchcock for their helpful advice and to SusanSmaus for her excellent secretarial assistance.

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immunoblotting with pooled guinea pig sera from animals infectedwith the Sheila Smith strain of R. rickettsii. Immune serum, lanes 1to 5; control anti-yolk sac serum, lanes 6 to 10. Lysate in lanes 1 and6, Sheila Smith; lanes 2 and 7, Morgan; lanes 3 and 8, Norgaard;lanes 4 and 9, Simpson; lanes 5 and 10, HLP. The bands in lane 5identified by asterisks are the antigens which differentiate the HLPstrain from the patient strains. MW, Molecular weight.

LITERATURE CITED1. Anacker, R. L., T. F. McCaul, W. Burgdorfer, and R. K.

Gerloff. 1980. Properties of selected rickettsiae of the spottedfever group. Infect. Immun. 27:468-474.

2. Anacker, R. L., R. F. Smith, R. E. Mann, and M. A. Hamilton.1976. New assay of protective activity of Rocky Mountainspotted fever vaccines. J. Clin. Microbiol. 4:309-311.

3. Batteiger, B., W. J. Newhall V, and R. B. Jones. 1982. The use ofTween 20 as a blocking agent in the immunological detection ofproteins transferred to nitrocellulose membranes. J. Immunol.Methods 55:297-307.

4. Bell, E. J., and E. G. Pickens. 1953. A toxic substance associat-ed with the rickettsias of the spotted fever group. J. Immunol.70:461-472.

5. Burgdorfer, W. 1963. Investigation of "transovarial transmis-sion" of Rickettsia rickettsii in the wood tick, Dermacentorandersoni. Exp. Parasitol. 14:152-159.

6. Hitchcock, P. J., and T. M. Brown. 1983. Morphological hetero-geneity among Salmonella lipopolysaccharide chemotypes insilver-stained polyacrylamide gels. J. Bacteriol. 154:269-277.

7. Jackson, E. B., and J. E. Smadel. 1951. Immunization againstscrub typhus. II. Preparation of lyophilized living vaccine. Am.J. Hyg. 53:326-331.

8. Laemmli, U. K. 1970. Cleavage of structural proteins during theassembly of the head of bacteriophage T4. Nature (London)227:680-685.

9. Markwell, M. A. K., and C. F. Fox. 1978. Surface-specific

ii

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iodination of membrane proteins of viruses and eucaryotic cellsusing 1,3,4,6-tetrachloro-3a,6ot-diphenylglycoluril. Biochemis-try 17:4807-4817.

10. 0rskov, F. 1978. Virulence factors of the bacterial cell surface.J. Infect. Dis. 137:630-633.

11. Parker, R. R. 1935. Rocky Mountain spotted fever: results often years' prophylactic vaccination. J. Infect. Dis. 57:78-98.

12. Parker, R. R., E. G. Pickens, D. B. Lackman, E. J. Bell, andF. B. Thrailkill. 1951. Isolation and characterization of RockyMountain spotted fever rickettsiae from the rabbit tick Haema-physalis leporis-palustris Packard. Public Health Rep. 66:455-463.

13. Philip, R. N., E. A. Casper, W. Burgdorfer, R. K. Gerloff, L. E.Hughes, and E. J. Bell. 1978. Serologic typing of rickettsiae ofthe spotted fever group by microimmunofluorescence. J. Im-munol. 121:1961-1968.

14. Price, W. H. 1953. The epidemiology of Rocky Mountain

spotted fever. l. The characterization of strain virulence ofRickettsia rickettsii. Am. J. Hyg. 58:248-268.

15. Price, W. H. 1954. Variation in virulence of Rickettsia rickettsiiunder natural and experimental conditions, p. 164-183. In F. W.Hartman, F. T. Horsfall, Jr., and J. G. Kidd (ed.), Dynamics ofvirus and rickettsial infections. The Blakiston Co., Inc., NewYork.

16. Stoenner, H. G., D. B. Lackman, and E. J. Bell. 1962. Factorsaffecting the growth of rickettsias of the spotted fever group infertile hens' eggs. J. Infect. Dis. 110:121-128.

17. Weiss, E., J. C. Coolbaugh, and J. C. Williams. 1975. Separationof viable Rickettsia typhi from yolk sac and L cell host compo-nents by Renografin density gradient centrifugation. App. Mi-crobiol. 30:456-463.

18. Weiss, E., H. B. Rees, Jr., and J. R. Hayes. 1967. Metabolicactivity of purified suspensions of Rickettsia rickettsi. Nature(London) 213:1020-1022.

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