Copyright X 1976 American Society for Microbiology Printed in U.SA.
Antibody
Response
to
Rocky Mountain Spotted
Fever
R. H. KENYON,* P. G. CANONICO, L. S. SAMMONS, L. R. BAGLEY, AND C. E. PEDERSEN, JR.
U.S.Army Medical Research Institute of Infectious Diseases,Fort Detrick, Frederick, Maryland 21701 Received for publication17 February1976
Various techniqueswerecomparedtodetermine themostsensitivemethod for
detection ofRocky Mountain spotted fever antibody. A radiometabolic technique for detection of Rocky Mountain spotted fever antibody is also described. In
infected monkeys, the fluorescent antibody technique yielded the earliest
evi-dence ofseroconversion; with some monkeys the microagglutination procedure
was equally effective. The fluorescent antibody and microagglutination
meas-urementsshowed higher titers than those for complement fixation, Weil-Felix, orthe radiometabolic techniques.
A variety of immunological techniques for
the detection of humoral antibody in Rocky
Mountain spotted fever (RMSF) has been
de-scribed. These include complement fixation (CF) (10), Weil-Felix (16), microagglutination
(MA) (5), and fluorescent (3) antibody (FA)
re-actions. The present studies were designed to
determinethemost sensitive and reliable
tech-nique for the detection of RMSF antibody. Standard methods for antibody detection are
compared and the temporal course of antibody
response is described after experimental RMSF infection of Macaca mulatta and accidental, laboratory-acquired infections in two humans. The median lethal dose of RMSF rickettsial
preparations has been difficult to determinefor
rhesus monkeys in this laboratory. Monkey
deaths have occurred using inocula with 101 through 107 plaque-forming units (PFU)/ml. Generally, the greater the infection dose, the
shorter the incubation period and the greater
the percentage of deaths. Monkeys that had
survived RMSFinfectionwereselectedtostudy antibody response overaperiod of time.
Anti-body response was also measuredin two
mon-keys with fatal infections. Inaddition, a radi-ometabolic technique for rapid detection of
RMSF opsonizing antibody is described and evaluated.
MATERIALS AND METHODS
CFtest.CFtests wereperformedwith the micro-titermethod ofCasey (2).Astandard rickettsial CF antigenwassupplied bythe Center for Disease
Con-trol, Atlanta, Ga.
MAtest. MA testsfor RMSFantibodywere per-formedas describedbyFiset etal. (5). Antigenfor
thistestwaspreparedfrom the Sheila Smithstrain
of Rickettsia rickettsii grown in duck embryo cell (DEC) cultures: rickettsial suspensions collected after differential centrifugation were inactivated
with 0.1% formalin and extracted twice with ethyl ether to remove egg lipids. The antigen suspensions werestandardized to contain 1 mg of rickettsiae/ml. Antigen-antiserum suspensions were incubated at 20 C for 18 h, atwhich time 25 u1of 0.02% acridine orange wasadded; results were recorded at 24 h.
Weil-Felixantibody tests. Proteus OX2 and OX19 slide antigen,tube antigen, and positive antiserum were purchased from Difco Laboratories, Detroit, Mich. Serum titrations were performed in microtiter plates with 200
Al
ofdiluted serum and 25A1l
of 1:8 saline dilution of slide antigenineach well. Sealed plates were incubated in a 37C water bath, and agglutination was recorded after4h. The tube test wasperformedasdescribed by Weil and Felix (16). IndirectFAtest.WI-38 cells grown on cover slips in Leighton tubeswere infected with R. rickettsii at amultiplicity of infection of5rickettsiae/cell. At72 hpostinfection, cells were fixed and stored in ace-tone at -70C. Human or monkey test sera were diluted serially in 0.015 Mphosphate-buffered sa-line, pH 7.3, and applied to infected cells for 25min in a moist staining chamber. Preparations were washed with phosphate-buffered saline, andconju-gatedrabbit anti-human globulin (Microbiological
Associates, Bethesda, Md.) or rabbit anti-monkey globulin (preparedin ourlaboratory)wasapplied for 30 min. Slides were washed with three changes of phosphate-buffered saline and mounted with phos-phate-buffered glycerol. The lowest dilution of the immune serashowed nopositivestaining on unin-fected WI-38 cells. The conjugate itself showed no positive stainingoninfectedWI-38cellsatthe work-ingdilution forbothconjugates.
Stained cells were observed with a Zeiss micro-scopefitted with BG-38 and BG-12 exciter filters,
10xoculars, anda 40 xPlanapochromat objective.A titrationend pointwasconsideredtobethehighest
dilutionoftest serumwith whichspecificrickettsial fluorescencewasobserved.
Opsonizing antibody test. Serumopsonizing
ac-tivitywasevaluated witharadiometabolic assayas describedby Canonicoetal. for Francisella tularen-sis (1). Rhesus monkey peripheral neutrophils
(PMN) were harvested from heparinized venous 513
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bloodas described by Skoog and Beck (12).
Leuko-cytesseparatedby dextran sedimentation were
sus-pended inglucose-free Hanks balanced salt solution
at 107PMN/ml.
Test antigen was prepared from DEC-grown R.
rickettsii; rickettsiae collected after differential
cen-trifugation were resuspended in Hanks balanced
saltsolution and inactivated by ultraviolet light (45 min, 4 cm from two 15-W germicidal lamps). The antigensuspensionwasstandardizedby directcount (11)atapproximately 2x 109 rickettsiae/ml. For the
radiometabolicassays,0.025 mloftestserum,0.1ml of antigen, and 0.2mlofHanks balancedsalt solu-tionsupplemented with4mMKCN and 0.5 mCi of [1-'4C]glucoseweremixed inanonsiliconized plastic
test tube with a rubber cap from which a plastic
center well (Kontes, Vineland, N.J.) containing filter paper saturated with 0.1 ml ofhyamine hy-droxidewassuspended. Aftera 15-minopsonization
period, 0.1 ml of leukocyte suspension was added
and the reactionwas incubated for 30 minat37C
withvigorousshaking (120 strokes/min). The
reac-tionwasstoppedby injection of 0.5mlof trichloroa-cetic acid. Released '4CO2 wascollected during an
additional 45-min incubation period before the plas-tic centerwellwastransferredto10 mlof Scintolute (Isolab, Akron, Ohio)ina scintillation vial. Radio-activity was counted with a liquid scintillation
counter(Nuclear-Chicago Corp., Austin, Tex.). Op-sonizing activity was equatedto hexose monophos-phate shunt activation and reported as counts of '4CO2 released per minute from theoxidation of
[1-'4C]glucose by 106 PMN/30 min.
Rickettsiae. The Sheila Smith strain of R.
rickett-sii wasused. Seed stockwaspropagatedin embryo-nated chickeneggs(15),and all dilutionsweremade
using sucrose phosphate glutamate buffer, pH 7.2
(7).Viable yolk sac-grownrickettsiaeweretitrated by the plaquing method described byWeinbergetal. (17),and rickettsemia levelsweredetermined bythe
methods of Wike and Burgdorfer (18).
Serumdonors. Sixwell-conditioned, healthy
rhe-sus monkeys, weighing 3 to 4 kg, were purchased
from Prime Laboratories (Farmingdale, N.J.), housed in individual cages, and fed a commercial
ration (Wayne Feed Supply Co., Gaithersburg, Md.). Monkeys selected for use had the following serological profile: MA s 1:2, CF - 1:16, OX2 and
OX19 - 1:4, and FA c 1:4. A fourfold or greater
increase in titer was considered significant.
Mon-keyswereinfected byintravenous (i.v.)or
subcuta-neous (s.c.) inoculation with the indicated dose of
viable yolksac-grown rickettsiae. Priorto
inocula-tionandonindicated days thereafter, monkeyswere
bled from the femoral vein. Samples ofserum for
antibody determinations and of whole blood for rick-ettsiaelevelswerestoredat -70 C.
Adultrhesus monkeyswerevaccinatedwith inac-tivated DEC-grown or commercial yolk sac-grown
RMSFvaccine (8). Group I (four monkeys)was
im-munizedwith three inoculationsat2-weekintervals with 0.5 mlof DECvaccine.GroupII(four monkeys)
wassimilarly immunized withcommercial vaccine.
Monkeys were bled for serum to test for opsonic activity just priorto the second and third
inocula-tions and at 2-week intervals thereafter for 3 months.
Two human patients developed laboratory-ac-quiredinfections with RMSF. Patient histories indi-cated that both individuals wereinvolved in
sepa-rateunreportedlaboratoryaccidents withyolk sac-grownR. rickettsii 6to 7days priortoearly
symp-tomsof illness. In bothcasesthesuspected infection
was by aerosols, which were accidentally created
while working withaneedle andsyringe. Patient 2 had received immunization with a commercially
available RMSF vaccine 4yearspreviously. Within 72hafter the firstsymptomsappeared, eachpatient
wasseenbyaphysician and tetracycline
chemother-apy was initiated. The presumptive diagnosis for
bothwasconfirmedby isolation of R. rickettsii from
guinea pigs inoculated with blood samples obtained prior to chemotherapy; rickettsemia, however, proved too low for successful recovery by plaque
techniques. Serum samples collected at intervals during and after hospitalization were stored at -70C. Preinfectionserumsamples for both
individ-uals wereavailable from aprevious immunization
surveillanceprogram.
RESULTS
Microtiter technique for Weil-Felix
reac-tion.Acomparisonof themicrotiter, slide, and
tube techniques for the Weil-Felix reaction is
shown in Table 1.Although titerswere
compa-rable with the three methods, end point
reac-tionswereachievedmorerapidly andwere
eas-iesttoread with the microtitertechnique.
Con-sequently, in this study, all Weil-Felix
determi-nationswereperformedby microtitration.
Temporalcourseofantibodyresponses.
An-tibody titers and rickettsemia levels were
de-terminedin sequentialblood samples from
indi-vidual monkeys infected by the i.v. route with
50 or 106 PFU of R. rickettsii (Fig. 1). The
monkey inoculated with the 106 dose was
rick-ettsemicfromdays 1 to 7, with maximum
rick-ettsemia on day 4, and the monkey inoculated
with the 50 PFU dose was rickettsemic from
days 3 to 11, with the maximum on day 9.
Antibody titers were detected earlier and
peakedmore rapidly after the 106 dose; by day
4, when rickettsemia was maximum, CF
anti-body, OX19 antianti-body, FA, and MA responses
were detectable. Opsonizing activity appeared
onday7andachievedmaximumvaluesondays
TABLE 1. Comparisonofslide, tube, and microtiter techniquesfordetectionofWeil-Felix antibody'
Technique Reciprocal Antibody titer (0X2) (0X19)
Slide 320 320
Tube 160 320
Microtiter 256 256
" Knownpositive sera.
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ANTIBODY RESPONSE TO RMSF 515
E
CL&
0
w
Ni~
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o000
500: A
DAYS AFTER INOCULATION
FIG. 1. Rickettsemia levels and antibody activity in monkeys that survived i.v. infection with (A) 50 R.
rickettsiior (B) 106 R.rickettsii. Opsonic activity is reported as arithmetic mean'4C counts per minute per106
PMN; eachhorizontal band represents the mean value ± 2standard deviations for preinoculation sera. Other antibody measurements are presented as reciprocallog2 titers.
11to 15. Incontrast, after the 50 PFU dose, FA,
CF, and MAantibodies weredetectable before
maximum rickettsemia, i.e., on day 4 for FA
andonday8for CFandMA;therefore, FAand
MA titers increased concomitantly and more
rapidlythandid CFactivityuntil all achieved peak values ondays 15 to 17. As was observed
after the
10"
dose, opsonizing activity was notdetected during rickettsemia; it appeared on
day11andincreased rapidly to achieve a
maxi-mum value on day 18. Minimal amounts of
OX19 and OX2 agglutinins werepresent from
days 8to 28. Despite the difference ininjected
dose, maximum CF, FA, MA, and opsonic
ac-tivities wereessentiallyidentical for both
mon-keys.
Responses ofmonkeys after s.c. inoculation
with 10:1 or 104 PFU ofR. rickettsii are
pre-sentedin Fig. 2and3, respectively. Both
mon-keys injected with 101 rickettsiae had rickett-sial levels
.10:1
PFU/ml of bloodonday
3 and continuedtoincrease until bothdied of RMSFinfection. Onemonkey thatdiedby day9failed
todevelopdetectable antibody; the second
mon-key died on day 12, soon after an antibody
response, detected by FA, MA, CF, and
Weil-Felixtechniques, was initiated. Serumopsonic
activity, however, failed to increase in either
monkey.
Twomonkeys thatreceived104PFUs.c.
sur-vived infection and had elevated FA and MA
titers by day 6, when maximum rickettsemia
wasobserved (Fig.3). A moderate CF
antibody
response was detected somewhat later (days 9
and 12), and antibodyagainst OX19,
inappar-ent in one monkey, nearly coincided with CF
antibodyin the other. Increased opsonic activ-ity developed more slowly, appearing on days
12and17; inthe first monkey the opsonic
anti-body level was at a maximum on day 21 and
thereafter decreased rapidly, but in the second itcontinuedtoincrease through day 90.
Monkeys vaccinated with commercial or
DEC-grown vaccine failed to show enhanced
opsonic activity at any time aftervaccination.
Although these monkeys were notchallenged,
twoothergroupssimilarly immunized resisted subsequent s.c. challenge with 104 R. rickettsii
30days after the last inoculation.
Antibody response patterns for the two
hu-mans with RMSF were somewhat different
(Fig. 4). At no timedidserafromthese
individ-uals have CF orOX2antibodytiters or
detecta-bleopsonicactivity.Onlyserafromthe
individ-ual who had no prior RMSF vaccination
(pa-tient 1) exhibited MA activity; the titer
in-creasedrapidlyandremained elevated
through
day 90. However, at 2 to 3weeks after initial
signs of illness, both individuals
developed
gradually rising FA titers that continued at
maximumlevelsthrough day90. AweakOX19
antibodyresponsewasinitiated somewhat ear-lier and remained at maximum levels from
days21 through 90.
DISCUSSION
The primary purpose of this investigation
was todetermine the most sensitivemethodfor
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20-
15-10d
5-A B
IOX19
FA
CF,MA
OPSONIC ACTIVITY
0 3 6 9 12 0 3 6 9
DAYS AFTER INOCULATION
FIG. 2. Rickettsemia levels and antibody activity in monkeys that succumbed to infection after s.c.
inoculation with10OR.rickettsii.Opsonicactivityisreportedasarithmeticmean'4Ccountsperminuteper106
PMN; eachhorizontal bandrepresentsthemeanvalue+ 2 standarddeviations for preinoculationsera.Other
antibodymeasurementsarepresentedasreciprocal log2 titers.
500
4 1
40962
0
C-)
z
QC) L~J cr
512-
64---O FA
_ MA
't CF
--25- OPSONIC ACTIVITY
N
20-0
15-I-
10,f
_
5-3 69 12 17 21 26 90
-B
OPSONIC ACTIVITY
-O FA
> MA
< CF
AXI
I --.-I
03 6 9 12 17 21 28 90
DAYS AFTER INOCULATION
FIG. 3. Rickettsemia leuels and antibody activity in monkeys that suruived s.c. infection with 104 R.
rickettsii.Opsonicactivityisreportedasarithmeticmean '4Ccountsperminuteper 106PMN; each horizontal
bandrepresents themean value +2 standarddeuiations for preinoculation sera. Otherantibody
measure-mentsarepresentedasreciprocallog2titers.
-J
LL.
oJ Z
r
.-3O w w
N
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ANTIBODY RESPONSE TO RMSF 517
PATIENT 2
51OPSONICACTIVITY
0 12 21 24 32
MA , FA
-0'
CF
40 90
o_-
-.
--OX19
MA
go . CF
OPSONIC ACTIVITY
0 7 15 21
4,0
90DAYS
FIG. 4. AntibodyactivityinserafromtwohumanpatientswithRMSF(patient1,notvaccinated;patient 2, vaccinated). Opsonic activity is reported as arithmetic mean 14C countsper minuteper 106 PMN. Other
antibodymeasurementsarepresentedasreciprocal
log,
titers.thedetection of RMSF antibodies. Inour
stud-ies with infected monkeys, the FA technique
consistently yielded the earliest evidence of
ser-oconversion, and with some monkeys the MA
procedurewasequally effective. Generally, FA
and MAmeasurementsdemonstrated
parallel-ism of antibody development, and invariably
FA and MA titers were higher than those for
CForWeil-Felixantibodies. Elisberg and
Boze-manreported that rickettsial agglutination isa
moresensitivetestthan CF for the detection of
RMSF antibody and thatrickettsial
agglutin-ins appear earlier and persist longer than CF
antibodies (4). Weil-Felix serology, routinely
used as adiagnostic screening test forhuman
antibodyagainst spotted fever and typhus
rick-ettsiae, was of little value for investigations
with the monkey model. Except for the 10- to
14-dayserafromonemonkey infected with 106
PFUi.v., titerswere l1:64 withOX19antigen
and c 1:4 with OX2 antigen. The microtiter
Weil-Felix technique, however, employingthe
standard stainedpreparation ofslide-test
anti-gen, provedto be as sensitiveas the standard
tubeorslideprocedures.Inaddition to theease
ofperformance and conservationofreagents,it
has the advantage of requiring a relatively
short reaction time and of developing end
pointsthatareeasier to determine thaneither
tubeorslidetests.
Although rickettsiaewererecoveredfrom all
infectedmonkeyswithin 3days ofinoculation,
the timerequiredtoreach maximum titerswas
somewhat relatedtothe numbers of rickettsiae
inoculated. After 106 PFU were administered
i.v., a peak titer of1.2 x 103 rickettsiae/ml of
blood was detected on day 3; after the 10' s.c.
dose, 3 x 102 to 5 x 102 rickettsiae/ml were
foundonday6; and after the 10'.7i.v.dose,5 x
102/ml werefound on day 9. In surviving
ani-mals, antibody titers appeared soon after the
peak response and often before rickettsemia
disappeared. Unlike the time of appearance,
ultimateantibody titerswereessentially
unaf-fected by the size of the infective dose, since the
antigenic stimulus conferred by infection
ap-pearedtobe ofthesameorderofmagnitude for
all surviving monkeys. In fatal infections,
deathusually occurredatthe time of maximum
rickettsemia and before significant antibody
waselicited.
All monkeys that survived RMSF infection
developed significantly increased serum
op-sonicactivity. This activitywas demonstrated
approximately 1weekafterpeak rickettsemia,
5 to 6 days after the detection of FA and MA
activity, and somewhat later than CF antibody. It had been postulated that specific opsonin
activity for R. rickettsii could be usedto
evalu-ate host protection after immunization. The
procedure was rapid, easy to perform, and
seemed especially appropriate in view of the
findingsof Gambrill and Wisseman (6) with R.
mooseri.These investigatorsdemonstrated that
antibody-sensitized rickettsiae were destroyed
by cultured human macrophages, whereas phagocytized nonsensitized rickettsiae
contin-ued to replicate and eventually destroy the
macrophages.Theirfindings indicatedthat the
protective action of humoral antibody in
mu-rine typhus can be associated with
enhance-ment ofphagocytosis and preparation of
rick-ettsiae for intracellular destruction. However,
our inability todemonstrate enhanced opsonic
activity insera from vaccinated monkeys that
were resistant to infection suggests that the
radiometabolic assays with PMN cannot be
used to evaluate protection against RMSF. In
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addition, the absence of opsonizing activity in
serafrom the two humanpatients with RMSF
suggests that the radiometabolic assay for
op-sonizing activity is less sensitive for the
detec-tion ofantibodythan are FA or MAprocedures.
No attempt was made to differentiate the
classes of antibodyresponsibleforthereactions
measured. Sinceserological agglutination
usu-ally involves reaction with immunoglobulin
M(IgM) antibody (9), it seemsreasonableto
as-sociatethe early MAactivity withspecific IgM
antibody. Similarly, since IgG antibody
ap-pearsafterIgM and isprimarily responsiblefor
fixation of complement by antigen-antibody complexes, CFreactions inRMSFareassumed
torepresentspecificIgGantibody activity. The
temporal relationship of FA to MA response
andofopsonic response toCFactivity suggests
association with IgM andIgG, respectively.
Antibody responses in two human patients with laboratory-acquired RMSFinfection were
markedly less than those that followed
experi-mental infection of monkeys. Our inability to
detect enhanced CF and opsonic activity
sug-gests that the IgG response was minimal.
These findings are in accord with the response
observedafter antibiotictherapy inhuman
pa-tientswith scrub typhus (13). Withthe RMSF
patients, rickettsemia never approached the
minimumleveldetected in monkeys;thisresult
of antibiotic therapy instituted in the early
stagesof infectionundoubtedly affectedthe
an-tigenic mass available for stimulation of
anti-body production and modified the detectable
response. Absence of an MA response in the
previously vaccinatedpatient (no. 2) mayimply
that the antigenic stimulus was
sufflcient
foractivation ofmemory cells but not for
expan-sion of the reactivecellpopulation.
These studies on the sequence of humoral antibody events initiated by RMSF infection indicate that the indirect FA test and
rickett-sial MA arethe most sensitive techniques
cur-rently available for the detection of RMSF
anti-body. Bothprocedures are simple to perform in
clinicallaboratories when appropriate reagents
are available. For human infections, the FA
technique would appear to be the method of
choice, particularly for patients with previous exposure to RMSF antigens. In addition, the apparently longer duration of FA reactivity
suggests the superiority of the method for
fol-lowing response to RMSF vaccines. Additional
studies are underway to evaluate the relative importance of humoral and cellular reactions in protective immunity.
ACKNOWLEDGMENTS
We wishtothank JosephMangiafico for the performance ofcomplement fixationtestsand HallSaylor for his techni-cal assistance.
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