• No results found

Antibody response to Rocky Mountain spotted fever

N/A
N/A
Protected

Academic year: 2020

Share "Antibody response to Rocky Mountain spotted fever"

Copied!
6
0
0

Loading.... (view fulltext now)

Full text

(1)

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 25

A1l

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, and

conju-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

on February 7, 2020 by guest

http://jcm.asm.org/

(2)

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.

on February 7, 2020 by guest

http://jcm.asm.org/

(3)

ANTIBODY RESPONSE TO RMSF 515

E

CL&

0

w

Ni~

0c

a.

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 not

detected 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 bloodon

day

3 and continuedtoincrease until bothdied of RMSF

infection. 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

VOL. 3, 1976

on February 7, 2020 by guest

http://jcm.asm.org/

(4)

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

0

x

a-\

on February 7, 2020 by guest

http://jcm.asm.org/

(5)

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

90

DAYS

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

z

w

0 2

0~ cr *i L

C,-0F

tr

w

x

,0

a-VOL. 3, 1976

on February 7, 2020 by guest

http://jcm.asm.org/

(6)

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

for

activation 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.

LITERATURE CITED

1. Canonico, P.G., A. T. McManus, J. A. Mangiafico, L. S.Sammons, V. G. McGann, and H. G.Dangerfield. 1975.Temporal appearance ofopsonizingantibodyto Francisellatularensis: detectionbyaradiometabolic

assay.Infect. Immun. 11:466-469.

2. Casey, H. L. 1965. Standardizeddiagnosticcomplement fixation method and adaptationtomicrotest.II. Ad-aptationof the LBCF method tomicrotechnique, p.

31-34. InPublic Health ServiceMonogr.no.74. U.S. Government PrintingOffice, Washington, D.C.

3. Elisberg,B.L.,and F.M.Bozeman. 1966.Serological diagnosis of rickettsial diseases byindirect immuno-fluorescence. Arch. Inst. PasteurTunis 43:193-204.

4. Elisberg, B. L., andF. M.Bozeman.1969.Rickettsieae

(sic), p.826-868. InE.H.Lennetteand N. J. Schmidt

(ed.), Diagnostic procedures for viral and rickettsial infections, 4thed. AmericanPublic Health Associa-tion,New York.

5. Fiset, P., R.A. Ormsbee, R.Silberman, M.Peacock,

and S. H.Spielman.1969. Amicroagglutination tech-nique for detection and measurementofrickettsial antibodies. Acta Virol. (Engl.Ed.) 13:60-66.

6. Gambrill, M.R., and C. L.Wisseman,Jr. 1973. Mecha-nismsof immunity intyphus infections. III. Influence of human immuneserumandcomplementonthe fate of Rickettsia mooseri within humanmacrophages. In-fect. Immun. 8:631-640.

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

8. Kenyon,R. H.,L.S. Sammons, and C. E.Pedersen, Jr. 1975. Comparison of three Rocky Mountain spotted fever vaccines. J. Clin. Microbiol.2:300-304.

9. Pike, R. M. 1967.Antibodyheterogeneity and serologi-calreactions. Bacteriol. Rev. 31:157-174.

10. Plotz, H., and K. Wertman. 1942.Theuseof the

com-plement fixation test inRocky Mountainspotted fe-ver. Science 95:441-442.

11. Silberman, R., and P. Fiset. 1968. Method for counting rickettsiae and chlamydiae inpurified suspensions. J.Bacteriol. 95:259-261.

12. Skoog, W. A., and W. S. Beck. 1956. Studies onthe fibrinogen, dextran andphytohemagglutinin meth-ods ofisolating leukocytes.Blood 11:436-454.

13. Smadel,J. E.1954.Influence of antibioticson

immuno-logicresponsesinscrubtyphus. Am. J.Med. 17:246-258.

14. Spencer, R. R., and K. F. Maxcy. 1930. The Weil-Felix reactioninendemic typhus fever and in Rocky

Moun-tainspotted fever. Public Health Rep. 45:440-445.

15. Stoenner, H. G., D. B. Lackman, and E. J. Bell. 1962. Factors affecting the growth ofrickettsias of the spot-ted fever group in fertile hens' eggs. J. Infect. Dis. 110:121-128.

16. Weil, E., and A. Felix. 1916. Zur serologischen Diag-nose des Fleckfiebers. Wien. Klin. Wochenschr.

29:33-35.

17. Weinberg, E. H., J. R. Stakebake, and P. J. Gerone.

1969.Plaque assay for Rickettsia rickettsii. J. Bacte-riol. 98:398-402.

18. Wike, D. A., and W. Burgdorfer. 1972. Plaque

forma-tion in tissuecultures by Rickettsia rickettsii isolated directlyfrom whole blood and tick hemolymph. In-fect. Immun. 6:736-738.

on February 7, 2020 by guest

http://jcm.asm.org/

References

Related documents

Figure 3 shows the simulated magnitude, phase response and group delay of the proposed multi- resonator circuit, consisting of 8 resonators of varying W i as shown in Figure 1(a)..

This research adopts the analytical hierarchy process to prioritize the performance indicators which have been determined in the digital divide balance scorecard.. The

Keywords: autonomy, the essence of learner’s autonomy, the types and forms of autonomous work of learners, autonomous learning skills, learning a foreign language, higher

In addition to the lumbar spine, the average t-score and t-score mean difference of femoral neck were calculated for both groups.The densitometry of two groups showed that the

Acute asthma is a common, potentially life- threatening medical emergency and is responsible for an increasing hospital admission rate in several Western countries.’ An increase in

These indicate that there is a positive relationship between prevalence of corruption and financial statement fraud and business failure in Nigeria with the

Signal coordination is a tool available in the hand of traffic engineers by means of which they control, regulate and manage the traffic in the signal system in such a way

Rural Luddism and the makeshift economy of the Nottinghamshire Framework Knitters.. Copyright and