JOURNAL OF CLINICAL MICROBIOLOGY, Jan. 1976, p. 51-61Copyright C 1976 American Society for Microbiology

Vol. 3, No. 1Printed in U.SA.

Microimmunofluorescence Test for the Serological Study ofRocky Mountain Spotted Fever and Typhus

R. N. PHILIP,* E. A. CASPER, R. A. ORMSBEE, M. G. PEACOCK, AND W. BURGDORFER

National Institute ofAllergy and Infectious Diseases, Rocky Mountain Laboratory,Hamilton, Montana 59840

Received for publication 12 September 1975

A microimmunofluorescence test was used to study antibody responses tovarious spotted fever group and typhus group rickettsiae during Rocky Moun-tain spotted fever (RMSF) and epidemic typhus (ET). Patients with RMSFreacted most strongly to Rickettsia rickettsii; those with ET reacted predomi-nantly to R. prowazekii. The degree of cross-reaction to other rickettsial strainsvaried from patient to patient, but a particular pattern of cross-reaction was

consistently observed in serial sera from the same patient. Fresh isolates fromthree Montana RMSF cases were indistinguishable from each other and fromstrain R ofR. rickettsii used as a standard antigen in all tests. ImmunoglobulinM (IgM) antibodies were usually present in high titer in early-convalescent-phase sera from RMSF, as well as ET, patients. After RMSF, IgM antibodiespersisted for a few months and, in one instance, for as long as 10 months. IgMresponses to laboratory-acquired infections were infrequent in persons previ-ously vaccinated with antigens related to the infecting strain. Previous antigenicconditioning from infection or vaccination may have accounted partly for theapparent lack of IgM response in a few study participants.

Indirect immunofluorescence (IF) is not newto serological study of rickettsial infections.Goldwasser and Shepard (7) first demonstratedits potential value for the study of typhus, andBozeman and Elisberg (3) found it helpful inthe investigation of scrub typhus, in whichantigenically diverse strains complicate sero-logical responses. Hazard et al. (10) used IF inconjunction with complement fixation (CF)tests to identify cases of Rocky Mountainspotted fever (RMSF) in New England, andGutman et al. (8) reported its use to confirmdiagnoses of tick-borne typhus in Israel. How-ever, IF has not been used extensively in theinvestigation of RMSF, nor has a practicableprocedure been described that is applicable toroutine serodiagnosis of rickettsial infections.

Five years ago, Wang (18) developed a micro-IF method that is widely recognized as a simple,reliable procedure for immunological classifica-tion of trachoma-inclusion conjunctivitis andlymphogranuloma venereum chlamydiae (19).Since then, it has been used increasingly tocharacterize serological responses to trachoma-inclusion conjunctivitis and lymphogranulomavenereum infections in humans (5, 9, 16, 20).The test is sensitive, reproducible, and oftenmeasures antibodies to species-specific antigensof infecting strains.On the supposition that micro-IF might also

be efficacious for the study ofhuman rickettsialinfections, particularly RMSF, a resurginghealth problem in the United States, we evalu-ated the procedure in tests on sera from epi-demic typhus (ET) and RMSF patients. This re-port describes the method and presents generalobservations on IF antibody responses to RMSFand ET. Use of the procedure relative to otherserological tests in serodiagnosis of RMSF andtyphus will be reported elsewhere.

MATERIALS AND METHODSRickettsial strains. Six strains were used for the

preparation of antigens routinely used in all tests(standard antigens). Three were from the spottedfever group and three were from the typhus group.They included: Rickettsia rickettsii, strain R, 53chicken embryo passages (EP), a virulent strainisolated from Dermacentor andersoni ticks in Mon-tana in 1945 (2); R. akari, strain 29 of Americanrickettsialpox, 17 EP; R. conorii, Malish strain ofSouth African tick-bite fever, 12 EP (2); R. prowa-zekii, strain Breinl, 164 EP; R. typhi, strain Wil-mington, 14 EP; R. canada, strain 2678, 11 EP (12).

Other spotted fever group strains were occasion-ally included in a given series of tests. These rick-ettsiae were isolated from ticks from various areasof the United States but have not yet been incrimi-nated as agents of human disease. They includedthe Haemaphysalis leporispalustris strain ofR. rick-ettsii (15), the maculatum strain of R. parkeri (14),the M/5-6B strain of R. montana (1), and a recently

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52 PHILIP ET AL.

described rickettsia isolated from Rhipicephalussanguineus ticks in Mississippi (4).

Antigens. Standard antigens were rickettsiaethat were cultivated in chicken embryo yolk sacs,inactivated with 0.2% formalin, and purified by themethod of Ormsbee and Peacock (submitted forpublication). Stock suspensions of purified rick-ettsiae were standardized in normal saline contain-ing 1:10,000 merthiolate solution (Eli Lilly & Co.,Indianapolis, Ind.) to given 200 gg of rickettsiae/ml.All stock suspensions were prepared between April1971 and December 1972 and held at 4 C. Workingantigens were prepared as needed by adding 0.5 mlof stock suspension to 0.5 ml of 0.01 M phosphate-buffered saline, pH 7.4, containing 5% uninfected(normal) yolk sac and held at 4 C.

Several experiments were performed to deter-mine the effects of inactivation, purification, andstorage on reactivity of antigens. For this purpose,5% crude yolk sac preparations containing viablerickettsiae of the same strains were compared withworking preparations of standard antigens in micro-IF tests of sera from convalescent RMSF cases andantisera prepared against the same suspensions ofactive rickettsiae in mice. Homologous antibodytiters were usually the same, and never more thantwofold higher, against active rickettsiae thanagainst inactive rickettsiae in working preparationsof standard antigens used regularly for as long as4 months. Furthermore, patterns of reactivity ofthe two kinds of antigens with heterologous serawere similar. These observations, plus the fact thatstock suspensions of standard antigens had beenprepared 3 to 4 years earlier, indicated that thesuspensions were remarkably stable when storedunder the conditions described and that formalinhad had little or no effect on reactivity.

Micro-IF tests. The procedure for micro-IF testswas that of Wang (18). The important features of themethod are that small quantities of reagents areused, as many as nine dilutions of one serum (orsingle dilutions of nine sera) can be tested on oneslide, and the same drop of diluted serum is reactedagainst many (usually nine) antigens simultane-ously. Rickettsial antigens were applied by dip-penpoint (C. Howard Hunt Pen Co., Camden, N.J.) tomicroscope slides over premarked templates in ninesets of dots, nine antigens per set. Six were stand-ard antigens; the other three were various prepara-tions described above, introduced into a particularseries of tests for comparison. After application toslides, antigens were dried for 30 min and fixedin acetone at room temperature for 10 min. Serialtwofold dilutions of serum in phosphate-bufferedsaline containing 10% normal yolk sac to reducebackground fluorescence were then placed on anti-gen sets with a 3-mm bacteriological loop. An appro-priate positive or negative control serum was addedto the antigen sets in the upper left corner of eachslide. The slides were incubated in moist chambersfor 30 min at 37 C and washed in two changes ofphosphate-buffered saline for 5 min each. Afterdrying, antigen sets were overlaid with loopfuls offluorescein isothiocyanate-labeled goat anti-humanglobulin (Baltimore Biological Laboratories, Cock-

eysville, Md.) or conjugated rabbit anti-humanimmunoglobulin M (IgM), F, specific (Industrial Bio-logical Laboratories, Inc., Rockville, Md.). Theanti-Ig conjugate detected antibodies in both IgMand IgG classes. This was determined after absorp-tion of the conjugate with purified IgG or IgM intests of typhus antisera with and without IgM anti-bodies. Evidence for specificity of the anti-IgM con-jugate was described elsewhere (16). After con-jugate was added, the slides were incubated for 30min at 37 C, washed for 5 min each in two changesof phosphate-buffered saline, and mounted in buff-ered glycerol.A typical test consisted of 20 slides (20 serum

titrations) set up in the morning and examined inthe afternoon. The slides were read independentlyby two persons who did not know the identity ofa particular serum being examined. End pointsagainst each antigen were the highest serumdilutions conferring definite fluorescence to rick-ettsiae. The lower of the two end point readingswas designated as the antibody titer. Although endpoints were subjectively determined, reproducibleresults were readily obtained with experience.

Sera. About 1,700 sera have been tested since thiswork began in October 1973. Included were severallarge groups of paired sera from Ethiopian patientssuspected to have ET or louse-borne relapsing fever(Ormsbee, Peacock, Plorde, Philip, and Casper,submitted for publication); sera submitted to theRocky Mountain Laboratory in 1973 and 1974 byphysicians in northwestern states from patientswith tick-associated illnesses, many of which wereconsidered to be RMSF or Colorado tick fever; serafrom Mississippi patients with tick-associated ill-nesses in 1973; and all sera referred to the NorthCarolina Division of Health Services in 1974 forrickettsial serology (Philip, Casper, Thomas,Anacker, Burgdorfer, MacCormack, and Sexton,submitted for publication). Small groups of serafrom various sources and clinical entities were alsoexamined primarily to determine specificity of thetest and parameters of reaction. Included amongthese were stored sera from eight laboratory-ac-quired rickettsial infections that occurred between1959 and 1974.

All sera were screened at 1:16 dilutions for rick-ettsial antibodies, and those reacting were titratedto end point. Most sera were also tested for CF anti-bodies by the standard procedure used at RockyMountain Laboratory (21). Many were also testedfor typhus and RMSF antibodies by microagglutina-tion (6) and for spotted fever group antibodies bypassive hemagglutination (R. Anacker, submittedfor publication).The results herein are based on all micro-IF

reactive sera from the above groups. The precisenumber of typhus and RMSF cases represented isunknown, and it is not intended in this report toestablish criteria for definition of a seropositivecase. However, some concept of the numbers ofcases involved may be gained from the fact that59 and 94 patients with paired sera had fourfold orgreater rises in micro-IF antibody titers to R.prowazekii and R. rickettsii, respectively. Most of

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these persons also had other serological evidence ofinfection by these rickettsiae.

RESULTSDescription of micro-IF reactions. Purified

rickettsial suspensions were combined withnormal yolk sac for three reasons. (i) Innatefluorescence of rickettsiae is obscured by homo-geneous, low-background fluorescence of yolksac (Fig. 1A). When specific binding of antibodyoccurs, rickettsiae become visible (Fig. 1B, C).End points based on the disappearance of rick-ettsiae can be sharply determined. (ii) The pres-ence of yolk sac facilitates the location of anti-gen dots. (iii) Rickettsiae plus yolk sac adhereto slides better than rickettsiae alone.

Specific reactions were characterized bysharp, uniform, and intense staining of rick-ettsiae in particular antigen plaques. Theywere particularly evident against R. rickettsiiin sera from RMSF cases (Fig. 1B, E, F) andagainst R. prowazekii in sera from typhuspatients (Fig. 1C). Cross-reactions of varyingdegree sometimes occurred to antigens bothwithin (Fig. 1E, F) and between (Fig. 1D) thespotted fever and typhus groups. Staining char-acteristics of cross-reacting antigens variedwidely. In some instances, rickettsiae stainedspecifically but only with low dilutions ofserum. In others, staining was erratic; i.e.,intensity of fluorescence varied, and organismswere indistinct and appeared granular or fuzzy,as though antibody were bound only to fewantigenic determinants or were loosely bound.Antibody responses in laboratory-confirmed

RMSF. Organisms characterized as fully viru-lent R. rickettsii were isolated from threeclinically typical cases of RMSF from the BitterRoot Valley, Mont. Active rickettsiae in crudeyolk sac preparations of the three isolatedstrains were compared with standard antigensin micro-IF tests of acute and convalescent serafrom the patients. The results are shown inTable 1. Only one patient had diagnostic (-four-fold) rise in CF spotted fever antibodies, but allthree showed pronounced responses to spottedfever group antigens by micro-IF. No differ-

ences were noted in reactivity among isolatesor between the isolates and strain R of R. rick-ettsii.

Heterotypic antibody responses in RMSF.To date we have not found sera from tick-asso-ciated illnesses in the United States that re-acted more specifically to other spotted fevergroup agents or R. canada than to the Rstrain of R. rickettsii. Although all sera fromconvalescent RMSF patients showed at leastsome cross-reaction with other spotted fevergroup antigens, the degree of response wasalways as high or higher against the R strain.However, patterns of reactivity to heterologousantigens of the spotted fever group varied frompatient to patient. Often there were no cross-reacting, typhus group antibodies. When thesedid occur, titers were usually low and patternsof reactivity to particular typhus group rick-ettsiae were variable. Variability in reactionpatterns seemed to be host-associated ratherthan a peculiarity of a given serum. Thus, aparticular pattern of cross-reaction was con-sistently observed in serial sera from the samepatient.Some variations in antibody responses in

RMSF are shown in Tables 1 and 2. In Table 1,patient 1 had rather broad antibody responsesto typhus, as well as spotted fever, groupstrains as compared with the specific responsesto R. rickettsii in patients 2 and 3, even thoughthe three isolates from these patients wereindistinguishable when tested against the con-valescent-phase sera.

In Table 2, patient 1 was unusual for thelate appearance of high antibody titers quitespecific for R. rickettsii. Early-convalescent-phase sera were broadly reactive at low levelsto all spotted fever group strains. None of hissera cross-reacted with typhus group antigens.

Patient 2 developed high antibody levelsearly, particularly directed toward R. rick-ettsii. Although she had modest titers to R.conorii, she developed only low titers to R.akari. She also had transient antibodies in lowtiter to R. canada but none to other strainsof the typhus group. The tendency toward spec-

FIG. 1. Micro-IF standard test antigens overlaid with 1:16 dilutions of serun and stained with fluo-rescein-conjugated goat antihuman globulin. x1,200. (A) Rickettsia prowazekii, left, and R. rickettsii, right,overlaid with serum from a patient with Colorado tick fever. Antibody titers against both antigens were<1:16. (B) R. prowazekii, left, and R. rickettsii, right, overlaid with serum from a patient with RMSF.Antibody titers were <1:16 against R. prowazekii and 1:2,048 against R. rickettsii. (C) R. prowazekii,left, and R. rickettsii, right, overlaid with serum from a patient with ET. Antibody titers were 1:32,768against R. prowazekii and <1:16 against R. rickettsii. (D) R. prowazekii, left, and R. rickettsii, right,overlaid with serum from a patient with indeterminate rickettsial disease. Antibody titers were 1:4,096against R. prowazekii and 1:1,024 against R. rickettsii. (E) R. akari, left, and R. rickettsii, right, overlaidwith serum from a Mississippi patient with RMSF. Antibody titers were 1:2,048 against both antigens.(F) R. akari, left, and R. rickettsii, right, overlaid with serum from a Montana patient with RMSF.Antibody titers were 1:32 against R. akari and 1:4,096 against R. rickettsii.

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FIG. 1A,C,E54

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TABLE 1. Micro-IF serum antibody responses in RMSF patients from whom rickettsiae were isolated

Micro-IF titers tob:

Standard test antigensdPatient Disease

nessCFti- Isolated strainsc

onset day ter' Spotted fever group Typhus group

RS HH MW rick akar cono prow typh cana

1. RS male, 5 yr 7-13-70 4 0 0 0 0 0 0 0 0 0 0old (0) (0) (0) (0) (0) (0) (0) (0) (0)

25 8 8,192 8,192 8,192 4,096 1,024 1,024 16 32 128(8,192) (8,192) (8,192) (4,096) (512) (1,024) (0) (32) (128)

2. HH male, 25 yr 6-2-73 0 0 NT NT NT 0 0 0 0 NT NTold (NT) (NT) (NT) (0) (0) (0) (0) (NT) (NT)

40 12 128 128 128 64 16 0 0 0 0(64) (128) (64) (64) (16) (0) (0) (0) (0)

137 48 8,192 8,192 8,192 8,192 32 128 0 0 0(0) (0) (0) (0) (16) (0) (0) (0) (0)

3. MW male, 6 yr 5-4-74 2 0 0 0 0 0 0 0 0 0 0old (0) (0) (0) (0) (0) (0) (0) (0) (0)

24 6 256 256 256 256 32 32 0 0 0(256) (256) (256) (256) (32) (0) (0) (0) (0)

51 8 16 32 32 16 16 0 0 0 0(0) (0) (0) (0) (0) (0) (0) (0) (0)

a CF antibody titers to R. rickettsii, R strain. 0 = titers < 1:6.Numbers not in parentheses indicate titers of antibodies undifferentiated as to Ig class. Numbers in parentheses

indicate titers of IgM antibody. 0 = titers < 1:16; NT, not tested.c Crude yolk sac preparations (5%) containing viable rickettsiae.d rick, R. rickettsii; akar, R. akari; cono, R. conorii; prow, R. prowazekii; typh, R. typhi; cana, R. canada.

ificity to R. rickettsii was frequently, but notexclusively, encountered in RMSF patientsfrom the Rocky Mountain area.

Patient 3 is notable for his unusually pro-nounced antibody response to several typhus,as well as spotted fever, group antigens. IfIgM antibodies to R. rickettsii had not beenpresent, serodiagnosis of this case would havebeen difficult. Previous ET vaccination isunknown.

Patient 4 had broad reactivity in convales-cent serum to spotted fever and typhus groupstrains. Because of his age and IgM response,it is doubtful that he had received typhus vac-cine. His reactivity probably represents a pri-mary response to antigens shared by bothgroups of organisms.Antibody responses to typhus. All sera from

convalescent Ethiopian typhus patients reactedmost strongly to R. prowazekii. Nevertheless,there was uniformly pronounced cross-reactionto R. typhi (titers averaging fourfold lower thanto R. prowazekii) and modest reactions to R.canada (titers averaging eightfold lower than toR. prowazekii). Although cross-reactions wereoften also noted against spotted fever groupantigens, particularly R. conorii, titers andreaction patterns varied, and some patientswere devoid of spotted fever group antibodies.

Examples of variations in reaction are shown inTable 3.

Patient 85, devoid of antibodies early, subse-quently developed high typhus titers, particu-larly to R. prowazekii, but barely detectablecross-reacting, spotted fever group antibodies.

Patient 225 was also devoid of antibodiesearly, but later he acquired high levels ofbroadly reacting spotted fever group, as well astyphus group, antibodies.

Patient 162 had equally high titers of anti-body to R. prowazekii, R. rickettsij, and R.conorii on day 13 of illness but virtually noreaction to R. akari. By day 27, a fourfold risein titer to R. prowazekii had occurred, but titersremained the same against other antigens. Thismay have been ET in a person who had ex-perienced a spotted fever group infection.

Patient 155 illustrates the extremely highantibody responses to R. prowazekii occasion-ally noted. Homologous antibody responses inET were generally higher t-han they were inRMSF.IgM antibody responses. IgM antibodies

were detected in 85% of persons with diagnosti-cally significant antibody responses to R. rick-ettsii or R. prowazekii. Some IgM responses areshown in Tables 1 to 3. Frequency of homolo-gous IgM antibodies was similar in typhus and

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TABLE 2. Variations in micro-IF serum antibody responses in RMSF patients

Micro-IF titers tob:

PatientDisease on- Illness CF ti-

Spotted fever group Typhus groupset day tera Spotted fever_groupTyphus_grourick akar cono prow typh cana

1. JD male, 12 yr old 5-16-74 5 0 0 0 0 0 0 0(0) (0) (0) (0) (0) (0)

15 24 256 256 256 0 0 0(128) (128) (256) (0) (0) (0)

32 32 512 256 128 0 0 0(128) (64) (64) (0) (0) (0)

111 64 4,096 256 256 0 0 0(0) (0) (0) (0) (0) (0)

2. HC female, 63 yr old 8-19-73 8 8 256 0 16 0 0 0(32) (0) (0) (0) (0) (0)

19 16 8,192 128 1,024 0 0 128(1,024) (32) (256) (0) (0) (16)

59 6 8,192 128 1,024 0 0 16(1,024) (32) (256) (0) (0) (0)

322 8 1,024 0 256 0 0 0(128) (0) (32) (0) (0) (0)

3. RL male, 43 yr old 8-11-63 6 0 0 0 0 0 0 0(0) (0) (0) (0) (0) (0)

52 16 2,048 64 512 4,096 1,024 16(64) (0) (32) (0) (0) (0)

4. BC male, 4 yr old 7-17-73 10 0 32 32 16 32 0 0(64) (64) (32) (64) (16) (16)

22 16 256 128 128 32 tr 16(256) (128) (32) (128) (32) (64)

a CF antibody titers to R. rickettsii, R strain. 0 = titers < 1:6.b See footnotes b and d, Table 1.

TABLE 3. Variations in micro-IF serum antibody responses in Ethiopian ET patients

Micro-IF titers tob:

Patient no. Disease on Illness day CF titere Spotted fever group Typhus group

rick akar cono prow typh cana

85 4-10-70 4 0 0 0 0 0 0 0(0) (0) (0) (0) (0) (0)

17 64 32 32 32 32,768 16,384 4,096(16) (32) (32) (8,192) (2,048) (512)

225 3-26-71 4 0 0 0 0 0 0 0(0) (0) (0) (0) (0) (0)

17 256 1,024 2,048 1,024 16,384 2,048 1,024(0) (0) (0) (1,024) (1,024) (512)

162 3-4-70 13 64 2,048 32 2,048 2,048 512 256(0) (0) (0) (2,048) (512) (256)

20 256 2,048 32 2,048 4,096 512 256(0) (0) (0) (1,024) (128) (64)

27 128 2,048 64 2,048 8,192 256 256(0) (0) (0) (32) (64) (32)

155 8-23-70 15 32 32 64 16 4,096 1,024 256(0) (0) (0) (0) (0) (0)

30 64 64 512 128 65,536 8,192 4,096(0) (0) (0) (0) (0) (0)

a CF antibody titers to R. prowazekii, Breinl strain. 0 = titers < 1:4.b See footnotes b and d, Table 1.

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RMSF patients. In general, IgM responses cor-responded to Ig responses in-time of appearanceof antibody, antibody titers in early-convales-cent-phase sera, and spectra of reactivityagainst heterologous antigens. Although late-convalescent-phase sera were few, there wasevidence that IgM, but not Ig, antibodies hadbegun to decline several months after RMSF(e.g., patient 2, Table 1; patient 1, Table 2).No late-phase sera were obtained from typhuspatients.

There were exceptions to these observations.Occasionally in very early-phase sera, lowlevels of IgM antibody were detected in theabsence of Ig reactivity. The anti-IgM conjugatewas probably more efficient than the anti-Igconjugate in detecting IgM antibodies. Titers ofIgM antibody were sometimes quite low rela-tive to Ig titers in early-, as well as late-, con-valescent-phase sera. Occasionally, fluores-cence with the anti-IgM conjugate was lessintense at low than at high serum dilutions,as though binding of IgM antibody were beinginhibited in the test system, perhaps by moreavid IgG antibody. Some RMSF and typhuspatients with strong heterologous rickettsialgroup Ig responses showed no cross-reactingIgM antibodies (e.g., patient 3, Table 2; pa-tients 225 and 162, Table 3). Some of thesepersons may have had earlier contact withheterologous antigens, either from infection orimmunization. Finally, IgM antibody did notalways disappear within a few months afterinfection (e.g., patient 2, Table 2, still hadhomologous IgM reactivity more than 10months after RMSF).About 15% RMSF, as well as typhus, pa-

tients showed no IgM response. Sometimes thiswas observed in the presence of extremelyhigh levels of Ig antibody. Yet, usually absenceofIgM reactivity seemed to be characteristic fora particular patient rather than a particularserum. Thus, early-convalescent-phase serawith modest Ig titers from such patients weredevoid of IgM reactivity (e.g., patient 155,Table 3).Laboratory-acquired RMSF and typhus in

vaccinated persons. Acute- and convalescent-phase sera, stored frozen, were still availablefrom two laboratory-acquired cases of RMSF,five cases of typhus (presumably murine), andone undifferentiated spotted fever group infec-tion. All patients had received RMSF and/orET vaccine before illness.Table 4 shows micro-IF responses to illness in

five of these patients. Patient 1 acquired RMSFby tick bite in 1959. He had received RMSFvaccine in at least 4 different years; the lastdose was given 7 years before illness. Spotted

fever group antibodies were detected by CF,but not by micro-IF, on day 1 of illness. On day10, a specific micro-IF response of Ig antibodiesto R. rickettsii was demonstrated. IgM reac-tivity was absent.

Patient 2, an RMSF case, was probably in-fected by aerosol in 1962, 4 months after re-ceiving two doses of RMSF vaccine. On day 6,CF and low levels of micro-IF antibody againstR. rickettsii were detected. By day 30, he hadhigh levels of micro-IF antibody quite specificfor R. rickettsii. An IgM response was neverdetected.

Patients 3 and 4 were infected simultane-ously by aerosol with murine typhus rick-ettsiae. Patient 3 had had ET vaccine 3 yearsearlier but no spotted fever vaccine. He de-veloped CF and micro-IF responses specific fortyphus group rickettsiae with highest titersdirected toward R. prowazekii. Typhus anti-bodies persisted for at least several years. Pa-tient 4 had had both RMSF and typhus vac-cines 6 years earlier. He developed high levelsof antibodies against typhus rickettsiae, par-ticularly R. prowazekii, and also low, broadlyreacting antibody titers against spotted fevergroup rickettsiae. After 2 years, modest titersof typhus antibodies were still demonstrable bymicro-IF. IgM antibodies were not detected ineither patient.

Patient 5 acquired typhus, probably murine,by aerosol in 1970. He had been vaccinatedannually for many years against RMSF andET, but the last dose of typhus vaccine wasreceived 18 years before illness. Between day 4and 24, he developed high CF and micro-IF anti-body titers against typhus group and moderatetiters against spotted fever group rickettsiae.Titers were highest against R. prowazekii.Low levels of antibody against both groups oforganisms were still present 2 years later. Ofparticular interest was the fact that this patientalso had a transient IgM antibody responseagainst R. prowazekii and R. canada but notagainst R. typhi.These observations suggested that antibody

response to illness in vaccinated persons wasdirected particularly toward the related vaccinestrain. Heterologous rickettsial group antibodyresponse to illness was sometimes observed inpersons who had received heterologous groupvaccines. IgM responses to infection were infre-quent (two of eight patients) in persons previ-ously sensitized to related antigens.

DISCUSSIONAn advantage of IF over most serological

procedures for study of rickettsiae is that anti-body interaction with surface antigens of indi-

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TABLE 4. Micro-IF serum antibody responses to laboratory-acquired RMSF and typhus in vaccinated persons

Micro-IF titers to":

Patient, disease, vaccination historya Illness CF titerb Spotted fever group Typhus group

rick akar cono prow typh cana

1. GS RMSF 6-1-59 1 48 0 0 0 0 0 0Vac: RMSF-1947, 1948, 1949, 10 128 128 0 0 0 0 01952ET-1942, 1947

2. LP RMSF 9-5-62 6 128 128 0 0 0 0 0Vac: RMSF-May 1962 (2 14 256 1,024 16 32 0 0 32doses)ET-June 1962 (2 doses) 30 256 4,096 32 128 16 0 16

3. JC Typhus 12-26-69 5 0 0 0 0 0 0 0Vac: RMSF-none 18 .64 0 0 0 256 64 32ET-1966 27 .64 0 0 0 256 128 64

1 yr 16 0 0 0 256 32 162 yr 16 0 0 0 128 32 0

4. KB Typhus 12-26-69 8 12 0 0 0 64 16 0Vac: RMSF-1963 13 .256 64 64 16 4,096 1,024 256ET-1963 29 128 16 16 tr 4,096 512 512

1 yr 12 0 0 0 128 64 162 yr 8 0 0 0 128 32 0

5. CR Typhus 8-23-70 4 0 64 0 0 0 0 0Vac: RMSF-28 yr, 42+ doses (0) (0) (0) (0) (0) (0)ET-8 yr, 18+ doses (last, 24 128 512 64 64 8,192 4,096 2,0481952)

(0) (0) (0) (1,024) (0) (512)149 64 64 32 16 256 64 32

(0) (0) (0) (0) (0) (0)2 yr 8 32 0 16 64 16 0

(0) (0) (0) (0) (0) (0)

a Vac, vaccination.I CF antibody titers to R. rickettsii, R strain (RMSF patients) or R. prowazekii, Breinl strain (typhus

patients). 0 = titers < 1:6.r Numbers not in parentheses indicate titers of antibodies undifferentiated as to Ig, and IgM class titers

are indicated in parentheses. IgM antibodies were detected only in patient no. 5.

vidual organisms is visualized. The micro-IFtest permits direct comparison of such inter-action with different populations of organisms.Both qualitative and quantitative charac-

teristics of antigen-antibody interaction indicatethe specificity ofthe IF reaction. Rickettsiae are

complex organisms and probably possess manysurface antigens differing among strains inkind and number. The extent to which antigensare shared determines the degree of related-ness. Theoretically, specificity of antigen-anti-body reactions is determined by the degree towhich the host immunologically responds tovarious antigenic components of the infectingstrain, availability of the kinds of antibodyneeded to combine with all surface antigens ofthe test strain, and avidity of antibody forspecific antigenic determinants. If these as-

sumptions are correct, then rickettsiae in testantigens that stain sharply, uniformly, and in-tensely with convalescent serum are morelikely to resemble infecting strains than rick-ettsiae with poorer staining characteristics.

Convalescent-phase human sera in this studygenerally showed rather broad reactivity to allstrains within either the typhus or spotted fevergroup. Goldwasser and Shepard (7) noted diffi-culty in differentiating epidemic from murinetyphus infections by IF. In their studies, theshared antibody was removed by absorptionwith heterologous strains. In our study, thesera from Ethiopian ET cases cross-reacted ex-tensively with both R. typhi and R. canada.However, in every instance titers were as highor higher against R. prowazekii, and stainingcharacteristics appeared to be more specific

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60 PHILIP ET AL.

against this rickettsia than against R. typhi andR. canada. We have not had an opportunity totest sera from murine typhus patients thatwere known not to have received ET vaccineto determine whether they would react morespecifically against R. typhi than against R.prowazekii.

Although cross-reactions among spotted fe-ver group antigens were usually quite exten-sive, sera from some convalescent RMSF pa-tients reacted rather specifically to R. rickettsii,and antibody titers in all instances were atleast as high against this organism as theywere against other spotted fever group rick-ettsiae. The fact that three isolates from RMSFpatients were indistinguishable in reaction pat-terns from the R strain of R. rickettsii, usedas the standard antigen in all tests, is evidencethat this strain is suitable for serodiagnosis ofRMSF in the Rocky Mountain area. However,specific reactions to the R strain seemed to beless frequent in convalescent-phase sera fromeastern patients. We have not yet had an op-portunity to compare fresh isolates of westernand eastern strains from RMSF patients todetermine whether minor antigenic differencesexist.The remote relationship between typhus and

spotted fever group rickettsiae, as evidenced bycross-reacting antibody responses to human ill-ness, is well recognized. Cross-reactions werealso observed in this study. In some instances,they seemed to be heterologous primary re-sponses shown by presence of IgM antibodies.That others may have been secondary re-sponses in persons who were previously sensi-tized by heterologous vaccination or infectionwas suggested by high Ig antibody titers with-out demonstrable IgM reactivity.

Variations in reaction patterns to heterolo-gous strains within the spotted fever groupwere often noted. The reasons for this havenot been established. Such differences in hostresponse might reflect minor antigenic differ-ences in infecting strains of rickettsiae, but thisis not likely the only explanation. The threeisolates from RMSF patients (Table 1) wereantigenically indistinguishable, yet the breadthof antibody reactivity to other spotted fever andtyphus group antigens in the corresponding pa-tients varied.Antibody response to illness in vaccinated

persons is pertinent to this issue. Specificityseemed to be particularly pronounced in previ-ously sensitized persons and was directed to-ward the antigen contained in the vaccine.Goldwasser and Shepard (7) observed that per-sons vaccinated against ET responded more

vigorously to the vaccine strain than to R.typhi during subsequent infection with murinetyphus rickettsiae. This phenomenon may par-tially explain why western patients with RMSFreacted more specifically to R. rickettsii than didmost eastern patients. RMSF vaccine has beenused quite extensively in the Rocky Mountainstates, and information on previous vaccinationwas not obtained from most study participants.

Aside from antigenic conditioning, it seemsplausible that individuals within a species asgenetically complex as man will respond toantigenic stimuli in varied ways. There is nodoubt that differences in response occur amonghost species. For example, the antibody re-sponse of mice to infection with certain spottedfever group and typhus group rickettsiae ismore specific than that of humans (17; Philipet al., unpublished data). In cross tests ofmouse antisera, R. akari and R. canada arecompletely distinct from other spotted fever andtyphus group strains, whereas sera from RMSFand typhus patients frequently react with theseorganisms.Murray et al. (13) showed that humans re-

spond to primary typhus infection by earlyformation of 19S (IgM) antibodies. Tests withour anti-IgM conjugate indicated that mostRMSF, as well as typhus, patients developedIgM antibodies. However, we could not detectan IgM response in about 15% of patients. SomeEthiopian cases may have been recrudescenttyphus in which IgM antibodies were not re-called (13). We have no firm explanation forlack of IgM response in some RMSF cases.Steric hindrance of IgM reactivity by moreavid IgG antibodies has been observed duringconvalescence from chlamydial infection (11).We did not fractionate sera into Ig classes todetermine whether this sometimes occurs inrickettsial infection.The duration of the IgM response was not

clearly defined in this study. However, it wasevident that the response varied. In one in-stance, IgM antibodies were demonstratednearly 1 year after RMSF infection. Never-theless, IgM antibodies are probably indicativeof primary infection. Their determinationshould help to establish diagnosis of primaryrickettsial infection, provided the conjugate isknown to be specific and the patient has notbeen previously sensitized by vaccination or in-fection to related antigens.

ACKNOWLEDGMENTS

We are grateful to James J. Plorde, Seattle VeteransAdministration Hospital, Daniel Sexton, Duke UniversityMedical Center, and J. Newton MacCormack, North Caro-

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lina Division of Health Services, for providing manysera used in this study, and to Robert Gerloff, Rocky Moun-tain Laboratory, for the antigen preparation of R. san-guineus agent. We also appreciate the technical assistanceofJames Whitlock, Anthony Mavros, and Robert Karstens.

LITERATURE CITED

1. Bell, E. J., G. M. Kohls, H. G. Stoenner, and D. B.Lackman. 1963. Nonpathogenic rickettsias relatedto the spotted fever group isolated from ticks,Dermacentor variabilis and Dermacentor andersonifrom eastern Montana. J. Immunol. 90:770-781.

2. Bell, E. J., and E. G. Pickens. 1953. A toxic substanceassociated with the rickettsias of the spotted fevergroup. J. Immunol. 70:461-472.

3. Bozeman, F. M., and B. L. Elisberg. 1963. Serologicaldiagnosis of scrub typhus by indirect immunofluo-rescence. Proc. Soc. Exp. Biol. Med. 112:568-573.

4. Burgdorfer, W., D. J. Sexton, R. K. Gerloff, R. L.Anacker, R. N. Philip, and L. A. Thomas. 1975.Rhipicephalus sanguineus: vector of a new spottedfever group rickettsia in the United States. Infect.Immun. 12:205-210.

5. Dwyer, R. S. C., J. D. Treharne, B. R. Jones, and J.Herring. 1972. Results of micro-immunofluorescencetests for the detection of type-specific antibody incertain chlamydial infections. Br. J. Vener. Dis.48:452-459.

6. Fiset, P., R. A. Ormsbee, R. Silberman, M. Peacock,and S. H. Spielman. 1969. A microagglutinationtechnique for detection and measurement of rick-ettsial antibodies. Acta Virol. (Engl. Ed.) 13:60-66.

7. Goldwasser, R. A., and C. C. Shepard. 1959. Fluores-cent antibody methods in the differentiation ofmurineand epidemic typhus sera; specificity changes result-ing from previous immunization. J. Immunol. 82:373-380.

8. Gutman, A., H. Schreiber, and R. Taragen. 1973. Anoutbreak of tick typhus in the coastal plain of Israel.Trans. R. Soc. Trop. Med. Hyg. 67:112-121.

9. Hanna, L., E. Jawetz, B. Nabli, I. Hoshiwara, B. Ostler,and C. Dawson. 1972. Titration and typing of serumantibodies in TRIC infections by immunofluorescence.J. Immunol. 108:102-107.

10. Hazard, G. W., R. N. Ganz, R. W. Nevin, A. H. Nauss,E. Curtis, D. W. J. Bell, and E. S. Murray. 1969.Rocky Mountain spotted fever in the eastern UnitedStates. Thirteen cases from Cape Cod area of Massa-

chusetts. N. Engl. J. Med. 280:57-62.11. Juchau, S. V., W. D. Linscott, J. Schachter, and E.

Jawetz. 1972. Inhibition of antichlamydial IgM anti-body by IgG antibody in immunofluorescence tests. J.Immunol. 108:1563-1569.

12. McKiel, J. A., E. J. Bell, and D. B. Lackman. 1967.Rickettsia canada: a new member of the typhus groupof rickettsiae isolated from Haemaphysalis leporispa-lustris ticks in Canada. Can. J. Microbiol. 13:503-510.

13. Murray, E. S., J. M. O'Connor, and M. Mulahasanovic.1965. Serologic studies of primary epidemic typhusand recrudescent typhus (Brill-Zinsser disease). I.Differences in complement-fixing antibodies: highantigen requirement and heat lability. J. Immunol.94:723-733.

14. Parker, R. R. 1939. Observations on an infectiousagent from Amblyomma maculatum. Public HealthRep. 54:1482-1484.

15. Parker, R. R., E. G. Pickens, D. B. Lackman, E. J. Bell,and F. B. Thrailkill. 1951. Isolation and characteriza-tion of Rocky Mountain spotted fever rickettsiae fromthe rabbit tick Haemaphysalis leporis-palustris Pack-ard. Public Health Rep. 66:455-463.

16. Philip, R. N., E. A. Casper, F. B. Gordon, and A. L.Quan. 1974. Fluorescent antibody responses to chla-mydial infection in patients with lymphogranulomavenereum and urethritis. J. Immunol. 112:2126-2134.

17. Pickens, E. G., E. J. Bell, D. B. Lackman, and W.Burgdorfer. 1965. Use of mouse serum in identifica-tion and serologic classification of Rickettsia akariand Rickettsia australis. J. Immunol. 94:883-889.

18. Wang, S.-P. 1971. A micro-immunofluorescence method.Study of antibody response to TRIC organisms inmice, p. 273-288. In R. L. Nichols (ed.), Trachomaand related disorders caused by chlamydial agents.Excerpta Medica, Amsterdam.

19. Wang, S.-P., and J. T. Grayston. 1971. Classification ofTRIC and related strains with micro-immunofluores-cence, p. 305-321. In R. L. Nichols (ed.), Trachomaand related disorders caused by chlamydial agents.Excerpta Medica, Amsterdam.

20. Wang, S.-P., and J. T. Grayston. 1974. Human serologyin Chlamydia trachomatis infection with micro-immu-nofluorescence. J. Infect. Dis. 120:388-397.

21. Welsh, H. H., F. W. Jensen, and E. H. Lennette. 1959.Q fever studies. XX. Comparison of four serologictechniques for the detection and measurement ofantibody to Coxiella burnetii in naturally exposedsheep. Am. J. Hyg. 70:1-13.

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