spotted fever group rickettsiae by protein immunoblotting

Am. J. Trop. Med. Hyg.. 39(5), 1988, pp. 497—501(8-051)

Copyright ©1988 by The American Society of Tropical Medicine and Hygiene

ANTIGENIC

ANALYSIS

OF CHINESE

STRAINS

OF

SPOTTED FEVER GROUP RICKETTSIAE BY PROTEIN IMMUNOBLOTTING MING-YUAN FAN, XUE JIE YU,* ANn DAVID H. WALKER* Department of Rickettsiology, Institute of Epidemiology and Microbiology, Chinese Academy of Preventive Medicine, Beijing, People's Republic of China, and *Department of Pathology, University of Texas Medical Branch, Galveston, Texas 77550 Abstract. Protein immunoblotting demonstrated that 6 Chinese strains of spotted fever group (SFG) nickettsiae from northern China had antigenic polypeptides identical to those of Rickettsia siberica (strains 246 and 232),and dissimilar to otherSFG rickettsiae. The variousspeciesof otherSF0 rickettsiae exhibitedserologically distinct epitopesas wellas many cross-reactive epitopes in protein immunoblotting. All SFG rickettsiae examined in this study had major antigenic polypeptides of 113—160kDa. Wisseman, Jr. (University of Maryland, Balti The spottedfevergroup(SFG) rickettsiae con more, MD). sistofa multitudeof pathogenicand nonpatho were cultivated in specific genic strainsof obligateintracellular bacteria. All rickettsiae pathogen free embryonated hens' eggs.4 Yolk sac Endemic diseases caused by SF0 rickettsiae are stock suspensions of SF0 rickettsiae were diluted transmitted to humans through the bite of in 1/100 in sucrose-phosphate-glutamate (SPO) fected ticks and mites. Six strains of rickettsiae buffer (0.2 18 M sucrose, 0.0038 M KH2PO4, have been isolated in northern China during re 0.0072 M K2HPO4, and 0.0049 M monosodium cent years. ‘@ In this report, we compare the pro L-glutamic acid,pH 7.0)forinoculation intoyolk tein immunoblots of 6 Chinese strains of SFG sacs for production of antigens.5 After death, all rickettsiae and prototype strains of 6 species of infectedeggs were incubatedan additional48 SFG rickettsiae: R. siberica, R. rickettsii, R. cono hours.4Rickettsiae were purified by centrifuga ru, R. parkeri, R. akari, and R. austra/is. tion in a 30%-36%-42% discontinuous Reno grafin d ensity gradient.6 Both thelight a nd heavy MATERIALS AND METHODS bands of rickettsiae were collected and pelleted at 22,500 x g for 20 mm. The pellets were re Rickettsiae suspended in distilled water and stored at —70°C. The Chinesestrains of SF0 rickettsiae inthis study are An-84, Se-85,XJMT-84, XJFT-84, Jinghe-74and IMTO-85.'3 An-84 and Se-85 Antisera strains were isolated from the blood of patients Sera were preparedaccordingto the method intheXinjiangUygur Autonomous Region and of Philipand othersfor serotypingby micro InnerMongolia,respectively. The other4 Chinese imunofluorescence.7 Female Swiss-Webstermice strains ofSF0 rickettsiae were isolated from Der were injected intravenously (iv) via the tail vein macentor nutta//i ticks. R. conorii (Malish 7 with 0.5 ml of heavy bands of Renografin-pun strain), R. akari (Kaplan strain), and R. siberica fled rickettsiae diluted 1/10 in SPO on days 0 (strain 246) were obtainedfrom the American and 7 and were exsanguinated on day 10. The Type Culture Collection. R. siberica (strain 232), sera from each group of mice were pooled, di R. austra/is (strain W58), and R. parkeri were vided, and stored at —70°C. obtained from C. Pretzman (Department of HealthLaboratory, Columbus, OH). R. rickettsii (Sheila Smith strain) was provided by C. L. Protein immunob/otting Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed.@'°

Accepted 7 June 1988. 497

498

FAN,YU, AND WALKER

123456

1234567 .‘

I FIGURE 1.

Protein

immunoblotting

of mouse anti

Se-85strain serumdiluted 1/250reacted withIMTO 85strain (lane 1), Se-85strain (lane 2),XJMT-84 strain (lane 3), XJFT-84 strain (lane 4), An-84 strain (lane 5),and Jinghe-74 strain (lane6).The arrowsatleft designate themolecular weights: 200,116,97,66,and 42 kDa.

Stackinggelsconsisted of4%, and separating gels of 12.5% acrylamide. Gel electrophoresis was performed at 20 mA per gelin a cold room at 4°Cfor 12 hr. Western blot analysis was per formed in a 4°Ccold room.'°'2 The nitrocellu lose sheets were air-dried; nonspecific protein bindingwas blocked with 5% milk in 0.01 M phosphatebufferedsaline(PBS),pH 7.5for1 hr at room temperature with shaking. After 2 wash es with 0.05% Tween-20 in 0.01 M PBS (pH 7.5) for 10 mm each, the nitrocellulose sheets were incubatedfor30 mm withantisera diluted1/100 to 1/250in0.01M PBS containing1% milk and washed as before. The nitrocellulose sheets were incubatedfor 30 mm with an anti-mouseIgG horseradish peroxidase conjugate (Bio-Rad Lab oratories, Richmond, CA) diluted 1/500 in 0.01 M PBS (pH 7.5) containing 1% milk. After a wash with 0.01 M PBS, the nitrocellulose papers were incubated with bis-benzidine. The color was allowed to develop for 5—10mm.

—— FIGURE 2.

Protein

immunoblotting

of mouse anti

IMTO-85 strain seradiluted 1/100reacted withXJFT 84 strain (lane1),An-84 strain (lane2),R. siberica strain 232 (lane 3), R. conorii (lane 4), R. rickettsii (lane 5), R. parkeri (lane 6), and R. australis (lane 7). The arrowsat left designate themolecular weights: 200, 116,97,66,and 42 kDa. RESULTS

All 6 isolates from China were demonstrated by protein immunoblotting with antisera to con tain antigens that were indistinguishable from those of R. siberica. Antisera against each of the isolates reacted with the electrophoretically sep arated proteins of each isolate in protein im munoblots revealing 2 major proteins (130 and 118 kDa) of similar electrophonetic mobility and reactivity. Figure1isrepresentative oftheresults obtained. The Chinese isolates are closely relat ed, if not identical. Comparison ofrepresentative human and tick

ANALYSISOF CHINESESTRAINSOF SFG RICKETTSIAE

1234

499

567891011121314 \

FIGURE 3. Protein immunoblotting of mouse anti-R. akari serum diluted 1/250 reacted with uninfected yolk sac control (lane 1), R. parkeri (lane 2), R. akari (lane 3), R. australis (lane 4), R. rickettsii (lane 5), R. conorii

(lane6),R. siberica strain 246 (lane7),R. siberica strain 232 (lane8),IMTO-85 strain (lane9),Se-85strain (lane10), XJFT-84strain (lane11), XJMT-84 strain (lane12), An-84strain (lane13), andJinghe-74 strain (lane 14). Arrows at left indicate the molecular weight standards: 200, 116, 97, 66, and 42 kDa.

isolates ofSF0 rickettsiae from China withother SF0 rickettsiae by protein immunoblotting demonstrated that the polypeptide antigens of the Chinese rickettsiae, R. siberica, R. conorii, R. rickettsii, and R. parkeri contained shared ep itopes. However, the polypeptides of R. siberica showed thesame electrophonetic mobilityas the ChineseSF0 strains whereasthepolypeptides of R. rickettsii (151 and 133 kDa), R. parkeri (151 and 133 kDa) and R. conorii(136and 113 kDa) showed different electrophoretic mobilities. Fig ure 2 is representative of the results obtained with antiseraagainstthe Chinese SF0 rickett siae,R. siberica, R. conorii, R. rickettsii, and R. parkeri. Figure 2 shows less resolution of the 130 and 118 kDa polypeptidesand highlyvisible lower molecular weight bands in the range oc cupied by lipopolysaccharides, likely a result of a greater quantity of rickettsial antigens in the gels and broader cross-reactivity of this anti serum. Antisera against R. akari and R. austra/is dem onstrated that the polypeptides of R. akari and R. austra/is differ considerably from those of the

ChineseSF0 rickettsiae and theotherSF0 tested. Mouse anti-R. akari sera reacted strongly with a polypeptide of R. akari having the electropho reticmobilityof 110 kDa and weakly with a polypeptide of R. austra/is having a molecular sizeof 115 kDa (Fig.3).Thisserum didnotreact with any othermembers of the SF0 by protein immunoblotting. Mouse anti-R. austra/is serum reactedstronglywith 2 polypeptides of R. aus tra/is with the molecular sizes of 160 kDa and 115 kDa and reactedweakly with the polypep tidesoftheotherSF0 rickettsiae tested(Fig.4). DISCUSSION

Protein immunoblotting using mouse antisera prepared by the method of Philip and others7 documented that 6 strains of SF0 rickettsiae iso lated from northern China contain immuno dominant polypeptides that are antigenically identical withthoseofR. siberica and thatdiffer from the other SF0 rickettsiae tested. These con firm previous data obtained by microimmuno fluorescence serotyping.'3

500

FAN, YU, AND WALKER

1 2 3 4 5 67

8 91O11@13

FIGuRE4. Mouse anti-R. australis serumreacted withuninfected yolksac(lane1),R.parkeri (lane2),R. akari (lane 3), R. australis (lane 4), R. rickeztsii (lane 5), R. conorii (lane 6), R. siberica strain 246 (lane 7), R. siberica strain 232 (lane 8), IMTO-85 strain (lane 9), XJMT-84 strain (lane 10), XJFT-84 strain (lane 11), An 84 strain (lane12), and Jinghe-74 strain (lane13).The arrowsmark themolecular weights: 200,116,97,66, and 42 kDa.

All SFG nckettsiae tested had antigenic poly indicated thatcross-reactivity ofvarious species peptides of113-160kDa molecular weightwhen ofSFG rickettsiae withR.siberica varies. These reacted withhomologousmouse antisera. Itis studies included Westernimmunoblotting ofan difficult to define which bands contain species tigens ofalltheChinesestrains (R.siberica, R. specific

antigens

because

of cross-reactivity

rickeusii,

R. conorii, R. akari, R. austra/is,

and

among thepolypeptides oftheseSF0 rickettsiae. R. parker,) with antisera to each of these rick Nevertheless, species-specific monoclonalanti ettsiae. The apparent antigenic relatedness ofR. bodiesindicate which antigenic bandscontain siberica, R. rickettsii, R. parkeri, and R. conorii species-specific epitopes. The antigenic band of forms a cluster that is relatively distant from R. molecular weight130kDa fromboththeUSSR akariand R. austra/is asshown ina bioassay by and Chinese

strains of R. siberica is recognized

Bell and Stoenner.'4

It is clear that the antigens have distinct differ them. Apparently the cluster of

by a murinemonoclonalantibody thatisspecificof R. akari and R. austra/is for R. siberica (D. H. Walker, personal com munication) and is also recognized by polyclonal

ences between

mouse antisera against other SF0 rickeusiae. This

ettsii, R. parkeri, and R. conorii maintained

SF0 nckettsiae comprising R. siberica, R. rick sub

polypeptide consists ofbothspecies-specific ep stantial stability of antigenic relatedness despite itopesand groupreactive epitopes, possibly in thegreat geographic separation andprobable long addition to some epitopes that cross-react with passage oftimesince their evolution fromacom a limited subsetofSFG rickettsiae. mon progenitor. Although the electrophoretic major polypeptides of most

mobilities of the SFG nckettsial

species aredistinct, R. rickettsii and R. parkeri ACKNOWLEDGMENTS havesimilar patterns ofprotein electrophoretic mobility. Protein immunoblotting withantisera This work was supported by research grant Al Institute ofAllergy and preparedaccordingto the mmcroimmunoflu 22224fromtheNational orescence method fordistinguishing serotypes Infectious Diseases. We wishtothankMarilyn

ANALYSISOF CHINESESTRAINSOF SFG RICKETTSIAE

501

croimmunofluorescence. J Immunol 121: 1961— 1968.UI:79049236 8. Laemmli UK, 1970. Cleavage of structural pro teins during the assembly of the head of bacte riophage T4. Nature (London) 227: 680—685. REFERENCES 9. Dasch GA, Burans JP, Dobson ME, Jaffe RI, Sew dl WG, 1985. Distinctive properties of corn 1.Kong ZN, Lao GY, Zhang YF, Chen GS, 1982. ponents of the cell envelopes of typhus group Isolation and identification of a spotted fever rickettsiae. KazarJ,Ormsbee RA, Tarasevich rickettsia from JingheCounty,Xinjiang.Mi IN,eds.Rickettsiae andrickettsial diseases: pro crobiology (China) 9: 11—13. ceedings of the 2nd International Symposium on 2. Fan MY, Wang JO,JiangYX, Zong DO, LenzB, Rickettsiae and Rickettsial Diseases, Smolenice, Walker DH, 1987. Isolation of a spotted fever June 21—25.1976. Bratislava, Czechoslovakia: groupnickettsia froma patient and related eco VEDA, Publishing House of the Slovak Acad logicinvestigations inXinjiang Uygur Auton emy of Sciences, 55—58.UI:800 1282 omous RegionofChina.J ClinMicrobiol 25: 10.FengHM, WalkerDH, Wang JG, 1987.Analysis 628—632.UI:87195371 of T-cell-dependent and -independent antigens 3. Fan MY, WalkerDH, LiuQH, Han L, BaiHC, ofRickettsia conorii withmonoclonalantibod ZhangJK,LenzB,CaiH, 1987.Rickettsial and ies.Inftct Immun 55:7—15. UI:87082297 serologic evidence for prevalent spotted fever 11.HanffPA, Fehniger TE, MillerJN, LovettMA, rickettsiosis ininnerMongolia.Am J TropMed 1982. Humoral immune responsein human Hyg 36: 615—620.UI:872 10806 syphilis to polypeptides of Treponema palli 4. Stoenner HG, Lackman DB, Bell EJ, 1962. Fac durn. Jlmmunol 129: 1287—1291.UI:82267054 tors affecting the growth of rickettsias of the T,GordonJ,1979.Electro spotted fever groupinfertile hens' eggs.Jlnfect 12.TowbinH, Staehelin phoretic transfer of proteins from polyacryl Dis110:121—128. amide gelsto nitrocellulose sheets: procedure 5. Bovarnick MR, Miller JC, Snyder JC, 1950. The andsome applications. ProcNatlAcadSciUSA influence ofcertain fats, amino acids, sugars, and 76: 4350—4354.UI:80056736 proteins on the stability of rickettsiae. J Bac 13.Wang JO, WalkerDH, 1987. Identification of teriol 59: 509—522. spotted fever group rickettsiae from human and 6. Hanson BA, Wisseman CL Jr, Waddell A, Silver tick sources in the People's Republic of China. man DJ, 1981. Some characteristics of heavy J Infect Dis 156: 665—669.UI:87309920 and light bands of Rickettsia prowazekii on HG, 1960.Immunologicrela Renografin gradients. Infect Immun 34:596— 14.BellEl,Stoenner tionships among the spotted fever group rick 604. UI:82074804 ettsias determined by toxin neutralization tests 7. Philip RN, CasperEA, Burgdorfer W, GerloffRK, inmicewithconvalescent animalserums.JIm Hughes LE, Bell EJ, 1978. Serologic typing of munol 84: 171—182. rickettsiae of thespottedfevergroupby mi

Jones for assistance in the preparationof the manuscript.