Immunity to Rickettsia mooseri Infection of Guinea Pigs

Vol. 27, No. 3INFECTION AND IMMUNITY, Mar. 1980, p. 730-7380019-9567/80/03-0730/09$02.00/0

Mechanisms of Immunity in Typhus Infection: Analysis ofImmunity to Rickettsia mooseri Infection of Guinea Pigs

JAMES R. MURPHY, CHARLES L. WISSEMAN, JR.,* AND PAUL FISETDepartment ofMicrobiology, University ofMaryland School ofMedicine, Baltimore, Maryland 21201

To study the mechanisms of immunity to Rickettsia mooseri (R. typhi) infec-tion, sera and splenic cells collected from nonimmune and immune guinea pigswere inoculated separately into syngeneic nonimmune recipients which weresubsequently challenged intradermally. Protection was measured by comparingthe course of the challenge infections of recipients with infections initiated withthe same rickettsial inocula in nonimmune animals. Recipients of splenic cellscollected 21 days after donor infection were protected from lesion development atsites of intradermal challenge and showed fewer rickettsiae in their kidneys. Cellsobtained from nominmune donors did not protect against either skin lesiondevelopment at sites of challenge or kidney infection. Antibody-containing seracollected 21 days after donor infection, but not normal sera, reduced levels ofkidney infection, but immune sera did not protect against the development oflesions at sites of intradermal challenge. It was concluded that both immune seraand immune splenic cells possess capacities to effect a partial control of thesystemic phase of R. mooseri infection in guinea pigs, but that immune spleniccells possess a capacity not shared by immune sera, i.e., the capacity to protectfrom infection at local sites of intradermal inoculation.

It has been shown that, over a period of about9 days after intradermal (i.d.) inoculation intononimmune guinea pigs, Rickettsia mooseri (R.typhi) establishes a local infection at the site ofinoculation, subsequently infects the draininglymph nodes, and thereafter achieves systemicdistribution (11). About midway through thisinterval, acquired immunity develops and ismanifested by (i) the elimination of rickettsiaefrom the primary site of i.d. inoculation, (ii) thedevelopment of a capacity to resist a second i.d.challenge delivered at a site distant from that ofthe primary infection, and (iii) the developmentof humoral antibody (10, 11). These studies,therefore, have demonstrated that the eventsleading to systemic infection progress despiterapidly increasing titers of serum antibodiesand, furthermore, have suggested that systemicinfection occurs despite the presence within in-fected animals of a population of lymphoid cellswhich is capable of expressing a cellular immu-nity at i.d. sites of R. mooseri infection (12).These findings suggest that the requirements forthe expression of immunity to R. mooseri indeep organs may differ from those which effectprotection from dermal infection at the inocula-tion site.The studies presented here were designed to

examine further the nature of acquired immu-nity to R. mooseri infection in guinea pigs. They

show that specifically sensitized splenic cellsprotect adoptive recipients from local infectionsat i.d. sites of inoculation and that both humoralfactors and specifically sensitized cells, whentransferred to naive recipients subsequentlychallenged i.d. with R. mooseri, are capable ofproviding a degree of protection from the estab-lishment of infections in deep tissues but cannotprevent such infections.

MATERLALS AND MEHODSRickettsiae. Two seed lots of R. mooseri Wilming-

ton strain were employed. Seed 1 was prepared frommonkey kidney cell culture (12EP/15GP/5EP/lGP/4BSC-1) and was used for immunizing infections (seebelow). Seed 2, which was prepared from embryonatedchicken eggs (12EP/15GP/5EP), was used for chal-lenge infections (see below). Some characteristics ofthese seed preparations and the methods employedfor their production and characterization have beenpublished previously (10, 13). Guinea pigs infectedwith the seed prepared from monkey kidney cell cul-tures did not develop hypersensitivity to normal yolksac components.Animals. Syngeneic strain 13 guinea pigs (weight,

350 to 500 g) were purchased from R. C. Rosecrans,Hamilton, Mont., housed individually, maintained onGuinea Pig Chow (Ralston Purina, Co., Saint Louis,Mo.), and provided with water ad libitum.

Serological procedures. In the complement fixa-tion (CF) test the microtiter adaptation of the Labo-ratory Branch complement fixation procedure was

730

IMMUNITY TO R. MOOSERI 731

used (4). Particulate antigen (7) prepared from R.mooseri grown in the yolk sacs of embryonatedchicken eggs was used at a concentration of 8 U.

In the microagglutination (MA) test of Fiset et al.(6), the particulate antigens were used at concentra-tions of 333 jig/ml.

Diluents. In experiments which involved the inoc-ulation of guinea pigs with spleen cells, the diluent(designated D/2 medium) was half-strength Dulbeccomodification of Eagle minimal essential medium withEarle salts containing 0.1% glucose (GIBCO Labora-tories, Grand Island, N.Y.) (16) without any addedserum or antibiotics. Sera were diluted with 0.15 MNaCl. The diluent for the yolk sac R. mooseri seedwas sucrose-phosphate-glutamate (3). The diluent forthe kidney cell culture seed was 3.7% brain heartinfusion (BBL Microbiology Systems, Cockeysville,Md.).

Hypersensitivity tests. Skin tests were performedby id. inoculations of 0.1-ml amounts of soluble orparticulate R. mooseri antigens. The particulate anti-gen (7) was used at a dose of 100 pg/test site. Thesoluble antigen, which was used undiluted, was pre-pared from homogenates of infected yolk sacs and hada CF titer of 1:64 when tested in block titration againsta reference antiserum. Individual animals were testedwith each antigen at different sites.

Preparation of sera and spleen cells. Blood wascollected by cardiac puncture. The separated sera werestored at -20'C in sterile rubber-stoppered vaccinebottles.

Using sterile technique, spleens were collected,minced with scissors, gently pressed through an 80-mesh stainless steel screen into cold (40C) D/2 me-dium, passed through five thicknesses of sterile surgi-cal gauze, washed by low-speed centrifugation, andsuspended in a 0.85% NH4Cl solution to lyse erythro-cytes. After 1 min the NH4Cl solution was diluted bythe addition of 5 volumes of D/2 medium, and afterlow-speed centrifugation the washed cells were resus-pended to the desired concentration in D/2 medium.More than 80% of the cells prepared by these proce-dures were viable, as measured by trypan blue exclu-sion.

Except for one experiment where serum was admin-istered intraperitoneally, sera and splenic cells weredelivered to the recipients by subcutaneous inocula-tion in the nape.Experimental model. (i) Immunizng infection.

On the basis of previous reports (10, 11), id. inocula-tion of 3.5 x 104 plaque-forming units (PFU) of R.mooseri (kidney cell seed) into the outer aspect of thethigh was used to initiate immunizing infections. Thisinoculum and route were selected because the result-ing infection and aspects of the host immune responseoccur in a reproducible sequence (11).

(ii) Challenge infection. The challenge infectionwas also selected on the basis of previous observations(10, 12) and consisted of i.d. injections of 8.2 x 104PFU of R. mooseri (egg seed) into each of three sitesalong the central portion of the shaved back lateral tothe midline. This challenge produces readily observa-ble and characteristic reactions in the skin of nonim-mune animals at the sites of inoculation (10, 12).

Quantitation of rickettsiae. A modification (13)of the primary chicken embryo tissue plaque assaytechnique of Wike et al. (18, 19) was used. Exceptwhere noted otherwise, tissues collected from a mini-mum of four animals per group were pooled and ho-mogenized. Samples were frozen at -70°C until plaqueassay. At least two plaque assays were performed witheach sample. Although the pooling oftissues precludeda definition of the level of variation within a group,variation appeared to be low, as was shown by anexperiment in which six animals were given the chal-lenge infection (see above) and sacrificed 11 days later.Kidneys, spleens, and whole blood were collected fromeach of the animals, and three pools of each tissue,each containing the tissues from two animals, weretitrated for R. mooseri content. That the infectionsprogressed similarly in each of the groups of animalswas shown by the recovery of 3.01 x 104 PFU/kidney(standard deviation, 0.43 x 104 PFU/kidney), 2.06 x104 PFU/spleen (standard deviation, 1.33 x 104 PFU/spleen), and 1.41 x 103 PFU/ml of whole blood (stan-dard deviation, 0.43 x 103 PFU/ml).Assay of protection. (i) Systemic infection. Ex-

perimentally treated and naive guinea pigs were in-fected i.d. with the same R. mooseri inoculum. Differ-ences in the progress of infections determined directlyby rickettsial titration were used to measure the levelof protection. The results are expressed either as (i)number of PFU per organ or per milliliter of wholeblood or (ii) relative protection, a derived value. Rel-ative protection is the difference between the logo ofthe geometric mean number of rickettsiae recoveredfrom control guinea pigs and the logo of the geometricmean number recovered from experimentally treatedanimals. Positive values denote increased resistance toR. mooseri, whereas negative values signify enhancedR. mooseri infection. In some experiments rickettsiaewere not recovered from recipients of immune sera orimmune splenic cells. To allow calculation of relativeprotection in these instances, a value of 102 PFU/organ, the approximate lower limit of the plaque assay,was assigned to the groups from which no rickettsiaewere recovered.

In most experiments the capacity of R. mooseri todisseminate from i.d. sites of inoculation to deep or-gans was determined by measuring the number ofrickettsiae in kidneys on day 11 after i.d. challenge.The selection of this organ and interval was based onthe previous demonstration (11) that after i.d. inocula-tion, infections in spleens, whole blood, and kidneysdevelop on about day 9 and peak on about day 11.Thereafter, rickettsiae are rapidly cleared from spleensand blood but persist in kidneys through at least day28. Because infections in kidneys develop in parallelwith infections in other deep organs but persist muchlonger, this organ was considered to provide a sensitivemeasure of systemic dissemination of rickettsiae.

(ii) Local infection at sites of i.d. challenge.The local reactions at sites of i.d. inoculation of R.mooseri were observed for 5 days after challenge. Onthe basis of previously published results (10, 12) thedevelopment of large grossly observable lesions wasconsidered indicative of the inability of an animal tocontrol a local i.d. R. mooseri infection. The failure of

VOL. 27, 1980

732 MURPHY, WISSEMAN, AND FISET

animals to develop such large lesions was interpretedas indicative of the existence of immunity to i.d. infec-tion.

RESULTSDemonstration that protection from sys-

temic R. mooseri infection can be trans-ferred with immune sera or with immunespleen cells. Figure 1 presents a summary oftwo experiments which were conducted to de-termine whether transferred sera or splenic cells,which were collected at intervals with respect toinfection of the donors with R. mooseri, werecapable of protecting R. mooseri-naive recipi-ents from homologous challenge. Recipientswere inoculated with either 10 ml of serum or 2x 109 viable splenic cells and 6 h later challengedwith R. mooseri. Recipients of serum were givena second 5-ml inoculation of the same serum onday 6 after infection. At 11 days after challenge,the expected peak of infection in kidneys (11),the animals were sacrificed, and the number of

Serum Recipients Spleen Cell Recipients

30 DONORS_W Noemnel Gui.na Plg

R MOOSER INFECTED GUINEAPIGS *

L SDe 12

INDE2120

z*

~ 0

00

FIG. 1. Demonstration that recipients of immuneserum or immune splenic cells are protected fromsystemic R. mooseri infection. Sera or splenic cellswere collected from the indicated donors and trans-ferred to recipients as described in the text. Subse-quently, recipient and normal animals were chal-lenged with R. mooseri by i.d. inoculation on theback, and 11 days later the animals were sacrificedand the numbers of rickettsiae in their kidneys weredetermined. The histograms show the difference be-tween the log10 geometric mean numbers ofrickettsiaerecovered from normal control and treated animals.Asterisks denote groups of recipient animals fromwhich no rickettsiae were recovered. The sites of i.d.challenges were observed daily through day 5 afterchallenge. The groups of recipient animals uhichdeveloped lesions of approximately the same magni-tude as those of nonimmune controls are denoted bya plus sign, whereas groups of animals which did notdevelop large lesions are signified by a minus sign.

R. mooseri PFU in the kidneys was determined.Figure 1 shows that recipients of serum or

spleen cells collected from nonimmune guineapigs were not protected from systemic R. moos-eri infection; the numbers of rickettsiae in kid-neys were close to the numbers recovered fromuntreated control animals challenged with thesame inoculum. Similarly, recipients of serum orcells collected 12 days after donor infection werenot protected. However, the recipients of spleniccells did not develop lesions at the i.d. sites of R.mooseri challenge, suggesting that the 12-daysplenic cells conferred immunity to local infec-tion in skin in the absence of protection fromsystemic infection. The demonstration thatsplenic cells collected 12 days after donor infec-tion protect recipients from infection at i.d. sitesof challenge is consistent with previously pub-lished data (12).

In contrast to the preceding results, both se-rum and cells collected 21 days after donor in-fection provided recipients with protection fromsystemic R. mooseri infection. The quality of theprotection is illustrated by the fact that no de-tectable rickettsiae (i.e., <102 PFU) were re-covered from the kidneys of recipients of eitherimmune serum or immune cells, whereas thesame inoculum delivered to untreated animalsproduced infections of 4 x 103 PFU/kidney (se-rum recipient controls) and 2 x 104 PFU/kidney(cell recipient controls), respectively. Notably,the recipients of day-21 serum, although pro-tected against systemic infection, developedlarge lesions at the sites of i.d. challenge, whereasthe recipients of day-21 cells did not. Thus, itappears that protection from systemic infectioncan be expressed in the absence of protectionfrom local infection at sites of i.d. challenge.

It should be noted that spleen cells collected21 days after donor infection contain about 10250% infective doses of R. mooseri per 2 x 109splenic cells (12). Thus, cell recipients were in-fected with R. mooseri by both i.d. challengeand injection of a spleen cell preparation.

In all subsequent experiments, recipients ofi109 live day-21 immune spleen cells resisted

lesion formation at sites of i.d. R. mooseri chal-lenge, whereas recipients of immune serum werenot capable of resisting the development of le-sions at sites of i.d. challenge.Requirements for the transfer of protec-

tion. The following experiments employed im-mune serum or immune spleen cells collected 21days after infection of donors with R. mooseri.The protocols for serum and cell transfers wereidentical to those of the preceding experiment.

Figure 2 shows a direct relationship betweenthe amount of immune serum injected or the

INFECT. IMMUN.


2.0

coz

u 1,0w

-0.

-4 -2 -I 0 5.3 73 8.3 9.3DILUTION OF SERUM (logto) NUMBER OF SPLEEN CELLS lsogo1

FIG. 2. Effect of the amount of immune serum ornumber of immune spleen cells transferred on thelevel ofprotection from systemic R. mooseri infection.Points marked with asterisks denote recipient groupsfrom which no rickettsiae were recovered.

number of immune cells transferred and thecapacity to protect recipients from systemic R.mooseri infection. Thus, R. mooseri was notrecovered from the kidneys of recipients of un-diluted serum or serum diluted 1:10, whereaslarge numbers ofrickettsiae were recovered fromthose animals treated with serum which hadbeen diluted 21:100. Similarly, 22 x iOW immunespleen cells protected recipients, whereas s2 x

107 cells did not.The experiment just described showed that a

substantial number of immune splenic cells (.2x 108) was required to protect recipients fromsystemic R. mooseri infection. However, it wasalso shown that serum could be diluted 1:10 andretain its capacity to protect recipients. Thus, itwas possible that a preformed soluble mediatormight have been carried to recipients ofimmunesplenic cells and that this preformed solublefactor might have been the mediator of theobserved, apparently cell-transferred protection.This possibility was investigated in the nextexperiment.Immune cells were collected and divided into

two lots, one of which was treated to kill thecells; the second lot was maintained at 40C.After similar intervals in vitro, live cells or killedcells were delivered at a dose of 109 cells perrecipient. It should be noted that the killed cellswere not washed before transfer and that theprocedures selected for killing cells did not causean appreciable loss of guinea pig antirickettsiaeantibody, as measured by the MA test (Murphy,unpublished data).

Figure 3 shows that killing immune spleencells either by heating at 560C for 30 min or byfreezing and thawing through three cyclesablated their capacity to protect recipients. It isunlikely, therefore, that the protection conferred

upon recipients by infusion of live immune cellsresides in the transfer of a preformed quantityof a molecular mediator. Additional evidencesupporting this view is presented below.Demonstration that immune serum or

immune spleen cells do not prevent sys-temic infection. Figure 3 shows that a smallnumber of rickettsiae were recovered from thekidneys of the recipients of 109 live immunespleen cells, which were the controls for theheat-killed cell experiment. This was the firstrecovery of viable rickettsiae from recipients ofprotective amounts of immune serum or protec-tive numbers of immune splenic cells, and therecovery of these raised the question of whetherthe efficacy of the transfer procedures was lim-ited to delaying the onset of systemic infection.This possibility was tested by taking advantageof the observation that, once systemic infectionoccurs, substantial numbers ofrickettsiae persistin kidneys for an extended interval (11). There-fore, it was predicted that, if the effect of thetransfer procedures was to delay the onset ofsystemic infection in recipient animals, rickett-siae would be recovered from kidneys collectedat intervals ofmore than 11 days after challenge.

Figure 4 shows that kidneys collected either15 or 21 days after R. mooseri challenge ofrecipients of either 15 ml (total volume) of im-mune serum or 2 x 109 immune splenic cellscontained approximately the sane number of R.mooseri PFU as did kidneys collected from un-

treated animals challenged with the same R.mooseri inoculum. The transfer of immune se-rum or immune splenic cells delayed the onsetof measurable levels of systemic infection.

Heated Cells Frozen Cells

RECIPIENTS OF:

inLive Cel Is

go 1.0 Eff -Treated Cells

z 1.0

I0

FIG. 3. Demonstration that killing of immunespleen cells ablates their capacity to convey protec-tion to recipients. The asterisk denotes a group fromwhich no rickettsiae were recovered.

Serum Recipients Spleen Cell Recipients

IJL~~~,," ,o

VOL. 27, 1980


03

<2

Nnrmui Animais m Serum Recipienis Cell Recipients

FIG. 4. Demonstration that systemic R. mooseri

infection occurs at an extended interval after chal-

lenge ofrecipients ofimmune serum or immune spleen

cells. The interval between challenge of recipients

and collection of kidneys was extended to 15 or 21

days.

It was possible that the failure of the transfer

procedures to provide absolute protection was

resident in a technical limitation, such as the

infusion of an inadequate amount of the active

component. Also, because the kidney is a "priv-

ileged" site with respect to the interaction of

host defenses and R. mooseri (11), it was possible

that the transfer procedures might have ablated

the acute phase of systemic infection while being

incapable of preventing the establishment of

persistent infections in privileged sites. These

possibilities were investigated in the next exper-

iment. Because it was not feasible to collect

larger numbers of spleen cells, the questions

were pursued with antibody-containing serum

only.

In an attempt to maintain a high level of

circulating antibody for an extended interval,

guinea pigs were inoculated with 10 ml of im-

mune serum 6 h before R. mooseri challenge and

given additional 10-mi inoculations of the same

serum pool on days 5, 10, 15, and 20. Figure 5

shows the number of R. mooseri PFU recovered

from blood, spleens, and kidneys of normal and

immune serum recipient animals at intervals

after infection. This figure shows that, for nor-

mal untreated guinea pigs, rickettsiae were pres-

ent transiently on day 9 in whole blood and

spleens but that by day 9 they established infec-

tions in kidneys which persisted through at least

day 21; this was the expected pattern of R.

mooseri infection nonimmune guinea pigs

(11). In contrast with this pattern of infection,

recipients of immune serum did not have de-

tectable rickettsiae (i.e., >102 PFU) in their kid-

neys through day 15. However, on day 21 rick-ettsiae were recovered from the kidneys ofguinea pigs which had received a total of 50 mlofimmune serum. The results suggest, therefore,that immune serum is capable of delaying theonset of measurable levels of kidney infection,but is incapable of preventing dissemination tokidneys of i.d. inoculated R. mooseri. Further-more, the demonstration of infections in serumrecipient kidneys but not in blood or spleenssuggests that immune serum may have ablatedthe acute measurable phase of infection in thosetissues from which the parasite is rapidly elimi-nated during the course of a primary infectionbut was incapable of similarly controlling infec-tions in kidneys, an organ within which intactanimals are incapable of rapidly eliminatingrickettsiae (11).That the plaque-forming agent recovered

from serum recipients on day 21 was R. mooseriwas confirmed by 50% infective dose titrationsin guinea pigs, utilizing serological conversion asan indicator of infection.Levels of serum antirickettsial antibody

in recipients of immune serum or immunespleen cells. Systemic R. mooseri infections ofnonimmune guinea pigs develop about 9 daysafter i.d. inoculation of the organism, an intervalgreater than that required for the generation of

2 ,^v' WHOLE BLOOD

Fr *-.*Normal Guinea PigsI,I- s..O- Immune Serum Recipients

<I ,"-V" < I----t~~~-------------.SPLEEN

3

0 <2 X --

1>l~~l 4̂, ^~IDNEY

D ,D3

2 /<2 f

o 9 12 15 18 21

DAY AFTER INFECTION

FIG. 5. Course of R. mooseri infection in normaland immune serum recipient guinea pigs. Serum re-cipients received 10 ml of the immune serum pool ondays 0, 5, 10, 15, and 20.

INFECT. IMMUN.

VOL. 27, 1980

a primary immune response to R. mooseri (11).It was possible, therefore, that the administra-tion of immune serum or immune spleen cells atabout the time of id. infection might protectrecipients either (i) by a direct inhibition ofrickettsial growth or dissemination or, alterna-tively, (ii) by an indirect pathway, such as byaltering the immunological potential of thehosts, thus allowing the de novo development ofa unique class of immunity which, in turn, pro-vides protection from the organism. The follow-ing experiments were conducted to determinethe effects of the transfer procedures on thecapacity of recipients to generate or to maintainhumoral antirickettsial antibody. Serum andspleen cells collected 21 days after the donorinfection were used.

(i) Persistence of transferred antibody.To determine the persistence of antibody afterpassive transfer, guinea pigs were inoculatedintraperitoneally with 10 ml ofan immune serumpool which had pre-inoculation titers of 1:128and 1:512 in the MA and CF tests, respectively.Figure 6 shows significant titers of circulatingantibody at 12 h after intraperitoneal inoculationand also shows that the titer of antibody in-creased through day 1. Subsequently, antibodytiters remained relatively constant through day4 or 6 and then fell slowly. By day 14 no anti-bodies were detected by the CF test, but MAantibody persisted.

(ii) Recipients of immune serum or im-mune spleen cells show altered antibodyresponses to R. mooseri challenge. Guineapigs were inoculated subcutaneously with 10 mlofimmune serum or 2 x 109 immune spleen cellsand 6 h later challenged i.d. with R. mooseri.The recipients of serum received a second 5-mlsubcutaneous dose of the same immune serumpool on day 6 after infection. Blood sampleswere collected from recipient and normal ani-mals immediately before R. mooseri challenge(i.e., 6 h after transfer) and at intervals thereafterthrough day 28.

Sera collected from the nonimmune guineapigs first showed humoral antibody on day 5after infection (Fig. 7). Thereafter, the titer ofantibody increased rapidly through day 12 andremained at high levels through at least day 28.This is the expected pattern of the response ofstrain 13 guinea pigs to R. mooseri infection (11).In contrast to sera collected from nonimmune

animals, sera collected from guinea pigs whichhad been passively immunized with the pooledimmune serum showed significant titers of MAantibody at 6 h after passive transfer, before R.mooseri challenge. Clearly, the antibodies inoc-ulated subcutaneously rapidly entered the blood


512 r

wI

zwX.

I-)w0w0:

128

32 MA

2J I- /C

0 2 3 4 5 6DAY AFTER TREATMENT

7 14

FIG. 6. Persistence of passively transferred anti-body as measured by CF and microagglutinationtests.

I-

w0w

0

A:to

1024

256

64

16 -

4 -

<4t0 5 10 15 20

DAY AFTER INFECTION25 30

FIG. 7. Effect ofpassive immunization with anti-body-containing serum or adoptive immunizationwith immune splenic cells on the level of humoralantirickettsial antibody in animals subsequentlychallenged with R. mooseri.

of the recipients. After infection, however, pas-sively immunized animals did not develop excep-tionally high titers of circulating antibody.Rather, the transferred antibody appeared toinhibit the capacity of the recipients to generateantibody in response to challenge. The immuneserum pool used for this experiment reacted inthe MA test through a dilution of 1:64 and inthe CF test through a dilution of 1:256.The antibody response of recipients of im-

mune spleen cells was similar to that of thecontrols through day 5; i.e., no antibody wasdemonstrable from 6 h after transfer throughday 3, but on day 5 significant titers were ob-served. However, after day 5, the progress of theresponse in the recipients of immune cells wasarrested, whereas that of controls progressed.Evidently, immune cells inhibit the maturationof the antibody response of a recipient. It is clearfrom this experiment that adoptive immuniza-tion with immune spleen cells does not providerecipients with (i) a measurable quantity of pre-formed antibody, (ii) an accelerated capacity toproduce those antibodies demonstrable with theMA test, or (iii) a capacity to produce exception-

.- I 0--j-


ally high levels of these antibodies.Figure 8 shows the titers of MA antibody

recorded on day 11 after R. mooseri challenge ofrecipients of increasing amounts of immune se-rum or increasing numbers of immune spleencells. The data show that the level of the hu-moral MA antibody of the recipients on day 11was inversely related to the dose of transferredserum or cells. A comparison of Fig. 8 and 2shows that protection from kidney infection wasexpressed by the animals which had the lowerMA antibody titers. The data in Fig. 8 wereobtained from sera collected at the time of sac-rifice of the animals used for the experimentshown in Fig. 2.Failure to transfer skin hypersensitivity.

Splenic cells collected 21 days after donor infec-tion were inoculated at a dose of 2 x 109 cellsper recipient. After 6 h the recipients were in-oculated i.d. in the back at different sites withparticulate and soluble R. mooseri antigens, andthe sites of antigen inoculation were observedsubsequently at 12, 24, and 48 h. No immediateor delayed inflammatory reactions were ob-served. That our failure to detect hypersensitiv-ity did not result from an inability of theseantigens to elicit such responses in appropriatelysensitized guinea pigs has been established inother studies (L. Gluck, R. W. I. Kessel, and C.L. Wisseman, Jr., manuscript in preparation).

DISCUSSIONThe present studies show that live splenic

cells collected either 12 or 21 days after donorinfection protect recipients from lesion forma-tion at i.d. sites of R. mooseri inoculation. Be-

cause it has been established (12) that adoptiveprotection from lesions at sites of i.d. challengereflects a capacity to restrict rickettsial replica-tion at these sites, the present studies establishthat an imunizng infection causes the gener-ation of a population of splenic cells, most prob-ably lymphocytes with antirickettsial activity,by day 12 of donor infection, and they also showthat cells with antirickettsial activity persist inthe donors through at least day 21 postinfection.The relatively early development of these cellsafter an immuniing infection is not surprisingbecause the donors resist second homologouschallenge from day 5 of immuniin g infection(11), and a previous study showed adoptivetransfer of immunity to i.d. infection with cellscollected at day 10 (12). These results clearlysuggest the possibility that it is this lymphocyte-mediated, adoptively transferable antirickettsialactivity which, in guinea pigs undergoing pri-mary infection, is responsible for clearance ofrickettsiae between days 4 and 6 (10, 11) fromthe primary sites of id. inoculation and is themediator of the rapidly developing (by day 5)protection from a second i.d. challenge. If thisargument is accepted, however, it becomes clearthat systemic infection, which begins on aboutday 9 and peaks on day 11 or 12, occurs after,and progresses in spite of, the presence of thelymphoid mediator of Wd. resistance. Therefore,this mediator must lack the capacity to restrictthe development of systemic infection. Directevidence in support of this view was provided inthe present study by the demonstration that,recipients of immune day-12 cells were solidlyresistant to i.d. challenge but nevertheless de-

RECIPIENTS OF IMMUNE SERUM

01ob

.9 Untreated- LI.~I1 mu< _-------- Control

I

I-4 -3 -2 -I 0DILUTION OF SERUM (log1o)

RECIPIENTS OF IMMUNE CELLS

10241-

256

64

16

4'

Ic Untreatedi 1 \ Control

1

5.3 6.3 7.3 8.3 9.'NUMBER OF CELLS (logo)

Z 1024wI-

z 2564 o

o. 64_ +1

wa 160L0

4'

FIG. 8. Demonstration that the suppression of recipient antibody titer observed after transfer of immuneserum or immune cells is inversely related to the dose of serum or number of cells transferred. Dilutions oserum were administered 6 h before (10 ml) and 6 days after (5 ml) R. mooseri challenge, and increasingnumbers of cells were administered 6 h before challenge. Recipient and normal animals were challenged wit)the same R. mooseri inoculum, and sera were collected 11 days after infection.

I

INFECT. IMMUN.


veloped by day 11 after challenge the same levelsof infection in their kidneys as identically in-fected, otherwise untreated animals.

In contrast to the failure of day-12 cells andserum to protect against systemic infection, bothimmune cells and immune serum collected 21days after donor infection delayed the onset ofmeasurable levels of systemic infection. Signifi-cantly, the day-21 serum protected from sys-temic infection without protecting from localinfections at i.d. sites, thus suggesting that themechanisms of protection from local id. infec-tion and systemic infection differ. In this regard,the present experiments do not establishwhether day-21 immune cells, which protectagainst both local i.d. infection and systemicinfection, do so by the same or by differentmechanisms at the two sites. The capacity ofday-12 immune cells to protect from local butnot systemic infection might suggest that differ-ent mechanisms are involved. It might also beargued, however, that the difference is merelyquantitative in that more immune cells are re-quired to prevent kidney infection than skininfection.On the other hand, it is not likely that the

protection from systemic infection transferredwith day-21 immune splenic cells resulted fromthe concomitant transfer of effective amounts ofthe same preformed soluble mediator responsi-ble for the protection transferred by the serum.Thus, freezing of the immune cells ablated theprotective capacity, whereas freezing of the pro-tective serum did not. The results neither pre-clude nor support the possibility that live cellsmay cause the production within the recipientof the same mediator of protection which ismeasured by the serum transfer experiments.

In the guinea pig model, R. mooseri infectsthe kidneys along with other organs during thesystemic dissemination phase of the infection,but it can be detected in the kidneys long afterthe organism have been cleared from the site ofinoculation and from other organs, such asspleens (11). The reasons for this are unknown,but it can be postulated that either (i) the kid-neys trap and concentrate small numbers oforganisms originating from other sites and pres-ent in the blood in numbers below the levelsdetectable by our laboratory methods, or (ii) thekidneys represent a privileged site, in whichexpression of immunity is somehow restricted.In any case, persisting kidney infection also ap-pears as a sensitive, cumulative indicator ofwhether systemic dissemination of infection hasoccurred. Thus, in the present study, infectionof the kidneys was used as an indicator ofwhether systemic dissemination of R. mooserihad occurred after i.d. challenge of rickettsia-

naive animals which had received either serumor spleen cells taken from R. mooseri-infectedguinea pigs at different times after infection. Oneconsequence of using this organ to measure dis-semination of infection is that immunity asmeasured in kidneys might not necessarily cor-respond to immunity as expressed in other or-gans from which rickettsiae can be rapidly elim-inated. It is possible, therefore, that a host de-fense incapable of preventing dissemination tokidneys might be fully capable of preventingacute infections of other organs and tissues.

It is clear that the immunological defense torickettsial infection is complex and involves rolesfor both humoral and cellular factors. Althoughantibody-containing sera do not exert a directrickettsiacidal action (2, 22), they have beenshown to reduce the severity of disease in hu-mans (5, 9, 14, 17, 23, 24, 26), to reduce signs ofinfection in animals (9, 25, 26), to opsonize rick-ettsiae for polymorphonuclear cells (20, 21) andmacrophages (1, 8), and to prepare rickettsiaefor macrophage-mediated killing (1, 8). The re-sults of previous studies, therefore, demonstratethat humoral factors can protect against signs ofinfection and suggest that in vivo antibodiesmight cause enhanced clearance of rickettsiae.However, direct evidence that antibodies are theeffectors of the protection transferred with an-tibody-containing sera is not available.Immune lymphoid cells which appear to be T

cells can protect mice from lethal R. tsutsuga-mushi infections (15) and guinea pigs from localR. mooseri infections at i.d. sites (12), and re-cently a soluble, presumably nonantibody factorhas been shown to restrict intracellular rickett-sial growth (Wisseman, manuscript in prepara-tion; C. Nacy-Mahady and J. V. Osterman,Abstr. Annu. Meet. Am. Soc. Microbiol. 1978,D39, p. 38). The relative importance of thesedifferent elements of the immunological arma-mentarium to control of rickettsial infections invivo remains to be determined, as does the se-quence in which they are brought to bear againstinfection. The importance of this latter point isreinforced by the present studies, which showthat the ontogeny of the protective response toR. mooseri is such that a cellular mediator israpidly deployed during the early stages of theresponse and a humoral protective componentdetected only significantly later, when the re-sponse has matured (11).

ACKNOWLEDGMENTSWe thank Lillian Snyder for her excellent assistance.This research was supported by contract DADA 17-71-C-

1007 with the U. S. Army Medical Research and DevelopmentCommand, Office of the Surgeon General, and by PublicHealth Service Training Grant AI 00016 from the NationalInstitute of Allergy and Infectious Diseases. J. R. M. received

VOL. 27, 1980


partial support from the training grant as a predoctoraltrainee.

LITERATURE CITED

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INFECT. IMMUN.

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Immunity to Rickettsia mooseri Infection of Guinea Pigs