JOURNAL OF CLINICAL MICROBIOLOGY, May 1989, p. 1081-10850095-1137/89/051081-05$02.00/0Copyright © 1989, American Society for Microbiology

Characterization of Sicilian Strains of Spotted Fever GroupRickettsiae by Using Monoclonal Antibodies

GIUSTINA VITALE,' ROSA Di STEFANO,2 GUIDO DAMIANI,3 AND SERAFINO MANSUETO1*Cattedra di Patologia Speciale Medica e Metodologia Clinica, Piazza delle Cliniche, 2,1 and Istituto di Microbiologia,2

90127 Palermo, and Istituto di Chimica Biologica, 16132 Genoa,3 ItalyReceived 29 September 1988/Accepted 19 January 1989

Twenty-two hybridomas producing anti-Rickettsia conorii monoclonal antibodies were obtained by ninefusion experiments. The strain chosen for immunization of mice was MAVI, an R. conorii strain isolated froma Sicilian patient with Boutonneuse fever. When tested for immunoglobulin isotype by an indirect Immuno-fluorescence (11F) assay, 46.6% of supernatants from the 22 hybridomas were immunoglobulin M. Thesupernatants were tested in the IIF assay for binding to the MAVI strain and four spotted fever group rickettsiastrains isolated from Sicilian ticks (two virulent and two nonpathogenic when inoculated intraperitoneally inmale guinea pigs). Only five of the supernatants showed a positive IIF result on all tested strains, although theyproduced different titers to the various strains, possibly an indication that they recognized an antigen commonto spotted fever group rickettsiae. Immunodominant epitopes for humans were determined by using patientsera to analyze inhibition of binding to the MAVI strain. Although a limited number of serum samples were

screened, a high percentage of Boutonneuse fever patients produced antibodies recognizing the same epitopesas were recognized by the mouse monoclonal antibodies. A striking heterogeneity was found both in theexpression of mouse-recognized epitopes on the five rickettsial strains and in the serum antibody responses ofBoutonneuse fever patients to these epitopes.

Rickettsia conorii is the causative agent of Boutonneusefever (BF), which causes several cases of illness in Italy eachyear (10, 14). Sicily, Latium, and Sardinia in particular areendemic areas for this disease and have shown a risingincidence in recent years. Rickettsiae are transmitted tohumans through the bite of the tick Rhipicephalus san-

guineus (family Ixodidae), which parasitizes mainly dogs.Rickettsiae are also transmitted by ticks in a transovarianmanner. Therefore ticks represent both a reservoir and avector of the infection (1).

Preliminary studies conducted in Sicily by using a biolog-ical assay for pathogenicity in guinea pigs indicated thatinoculation of different local isolates induced different sero-logical and clinical responses (14; G. Vitale, R. Librizzi, etal., Acta Medit. Patol. Infet. Trop., in press; G. Vitale, A.Virga, et al., Acta Medit. Patol. Infect. Trop., in press). Toextend these observations on the epidemiology of BF, weused a novel approach to analyze the antigenic heterogeneityof spotted fever group (SFG) rickettsiae in a relatively smallgeographic region. A marked heterogeneity would indicatean antigenic shift or drift of the SFG rickettsiae even in asmall geographic area. Such variation could be due either toan intrinsic genic variability of the pathogen or to selectivepressure during the host response to infection. In any case,development of effective vaccines and diagnostic tests mustrely on a clear understanding of the extent and nature of thisphenomenon. To begin to clarify these issues, we used a

panel of monoclonal antibodies raised against the MAVIstrain of Rickettsia conorii, isolated from a BF patient, toinvestigate the antigenic phenotypes of four other isolatesfrom ticks. Our results clearly show that most of the epitopesrecognized by the monoclonal antibodies were expresseddifferently in the five SFG rickettsia isolates. These findingsconfirm the remarkable antigenic variation in this group ofmicroorganisms.

* Corresponding author.

MATERIALS AND METHODSRickettsiae. The antigen used both for immunization of

BALB/c mice and for screenings of hybridoma supernatantswas an isolate of R. conorii MAVI obtained from a 42-year-old male Sicilian patient showing classic signs of theacute phase of BF (fever, characteristic maculopapular rash,tache noire at the site of the tick bite, and high levels ofspecific anti-R. conorii antibodies as determined by anindirect microimmunofluorescence method) (S. Mansuetoand G. Vitale, Acta Medit. Patol. Infet. Trop., in press).Four other isolates of SFG rickettsiae were used: Villaf-

rati, Cammarata, Palermo, and Baida. These were isolatedfrom ticks essentially as described by Vitale, Virga, et al. (inpress). Briefly, ticks were collected from dogs with pincersand monitored individually for the presence of SFG rickett-siae by the hemolymph test (3).Each lot of ticks was washed in succession with the

following solutions: 12 volumes of H202, 90% ethanol, 70%ethanol, penicillin (50 IU/ml) and streptomycin (50 ,ug/ml),and 2.5 ,ug of amphotericin B (Fungizone; Flow Laborato-ries, Milan, Italy) per ml. Each wash lasted 3 min. Tickswere then minced with a mortar in minimum essentialmedium (Flow) without antibiotics, resuspended, and usedto infect VERO cells (see below).For tests of the virulence in guinea pigs of each isolate,

rickettsiae were isolated and passaged in VERO cells (Flowcatalog no. 03-230) as follows. Cells were cultured in mini-mum essential medium containing 5% fetal calf serum (FCS)(Flow), glutamine (10 mg/ml; Flow), and antibiotics (peni-cillin [200 TU/ml] and streptomycin [100 ,ug/ml]; Flow).VERO cells were maintained at 37°C in a humidified incu-bator with a 5% CO2 atmosphere in air. Bacterial andmycoplasmal contamination was checked regularly.To infect VERO cells with rickettsiae, the temperature

was lowered to 33°C. VERO cells in a 75-cm2 flask (Falcon3024; Becton Dickinson and Co., Lincoln Park, N.J.) werewashed, and 15 ml of antibiotic-free complete medium was

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added. When the cellular monolayer was confluent (after 24h), a 5-ml sample of venous heparinized blood from thepatient (MAVI strain) or 5 ml of a suspension of ticks(Villafrati, Cammarata, Palermo, and Baida strains) was

added.Infection and intracellular growth of rickettsiae were

monitored by indirect immunofluorescence (IIF) as de-scribed below by using hyperimmune serum (IIF titer,1:1,280) from a patient with BF (clinically, serologically, andbacteriologically confirmed) and a second anti-human immu-noglobulin fluorescein-conjugated antiserum (Bio Merieux,Charbonnieres-les-Bains, France) (11).

Antigen preparation. In a typical preparation, rickettsia-infected VERO cells from 15 175-cm2 tissue culture flasks(Falcon 3028) were detached with a cell scraper, centrifugedat 8,000 x g for 30 min, suspended in 30 ml of sterile salinesolution (0.9% NaCi), and sonicated on ice for 20 s atmaximum power output in an MSE sonicator (frequency, 22,um). These sonication conditions were found preliminarilyto be optimal. The sonication time of a pellet of infectedVERO cells was increased in 5-s increments from an initial 5s at maximum power output (frequency, 22 ,um). After eachadditional sonication, a sample was removed, and bothVERO cell disruption and rickettsial viability were checkedby IIF.

Undisrupted cells and cell debris were eliminated bycentrifugation at 1,000 x g for 15 min. The supernatant was

centrifuged at 35,000 x g for 1 h. The pellet was suspendedin 6 ml of sterile saline, and rickettsial titers were deter-mined. The pellet was then divided into equal portions,which were kept at -20°C. This preparation was designatedthe antigen preparation.

Titration of rickettsiae was performed by IIF with doubledilutions of the antigen preparation. One drop of eachdilution was allowed to dry on a microscope slide, and an IIFtest was carried out. At a dilution of about 1:25 in phosphate-buffered saline (PBS), the rickettsiae appeared to be wellseparated. The protein content of the 1:25 dilution was 15iig/ml. The same antigen dilution was used for screeninghybridoma supernatants.

Assay of virulence in guinea pigs. Virulence in guinea pigswas determined by intraperitoneal (i.p.) inoculation of amale guinea pig. The degree of virulence was determinedaccording to the presence (alone or in combination) of fever,orchitis, and seroconversion (14).

TIF. The IIF assay was performed as described elsewhere(11) by allowing 5-pi portions of antigen (1:25 dilution inPBS) to dry on microscope glass slides. Antigen was fixedwith cold acetone for 7 min and left to dry. A 10-,ul amountof each supernatant was then added, and the preparation wasincubated at 37°C for 30 min. After two washes of 15 mineach in PBS containing 0.05% Tween 20 and a brief wash indouble-distilled water, 10 ,u of an appropriate dilution of a

fluorescein-conjugated anti-mouse immunoglobulin antise-rum (catalog no. F 261; Dakopatts, Milan, Italy) was addedfor 30 min at 37°C. After two washes in PBS-Tween, slideswere mounted with glycerol and examined with a LeitzDialux microscope equipped for fluorescence.Mice and immunizations. BALB/c mice (Charles River

Breeding Laboratories, Como, Italy), 12 to 18 weeks old,were immunized by repeated i.p. injections of 0.5 ml ofemulsion containing 4.5 tg of rickettsial antigen in completeFreund adjuvant (Difco Laboratories, Detroit, Mich.).

Antirickettsial antibody titers were evaluated regularly by11F, and mice with a positive antibody response were

boosted by an intravenous injection of 0.3 ml of antigen inPBS (total of 4.5 jLg).

Cell fusion and cloning of hybridomas. Three days after thebooster injection, mice were sacrificed and their spleen cellswere fused with P3/NS-1/1-Ag 4-1 (NS-1) mouse myelomacells by using polyethylene glycol (PEG) 1500 (Sigma Chem-ical Co., St. Louis, Mo.). When indicated, PEG 4000 (Sigma)was used, and the fusion conditions were varied accordingly.Table 2 shows the experimental conditions followed in eachfusion experiment. Fusions were performed as described byFazekas de St. Groth and Scheidegger (7), with minormodifications. After fusion, cells were suspended in com-plete selective medium: Dulbecco modified Eagle medium(DMEM) (Flow) containing 20% FCS, glutamine, and anti-biotics in the presence of hypoxanthine, aminopterine, andthymidine (HAT; Flow). The cells were then seeded in24-well tissue culture plates (Falcon 3047) at a density of 2 x106 cells per well in a 0.5-ml volume. Plates were incubatedat 37°C in a humidified incubator with a 5% C02 atmospherein air.

After 7 days, 0.1 ml of fresh HAT-containing completemedium was added. The same amount of fresh medium wasremoved, and 0.1 ml of fresh medium was added at 72-hintervals thereafter. At about 20 days after fusion, vigorouscell growth was observed in most wells.

Supernatants from each well were sampled and monitoredfor the presence of antirickettsia antibodies. The assay wasperformed by IIF on the MAVI strain. Cells giving a positivereaction weré expanded to mass culture, frozen, and clonedby limiting dilution in DMEM containing 20% FCS in 96-wells plates (Falcon 3072).At 24 h before limiting-dilution cloning of hybridomas, a

96-well plate with feeder cells was prepared as follows: aspleen cell suspension was prepared from the spleen of anuntreated BALB/c mouse, suspended in DMEM-20% FCS,and plated at 105 spleen cells per well in a 0.1-ml volume.After incubation at 37°C in 5% C02 for 24 h, and after asterility control, hybridoma cells were added in suitabledilutions to obtain a single hybridoma clone in each well.Monoclonal antibody immunoglobulin class and subclass

were determined by IIF, using fluorescein-coupled anti-mouse immunoglobulin Gi (IgGl)-, IgG2a-, IgG2b-, IgG3-,and IgM-specific antisera (Miles Laboratories, Inc., Milan,Italy).

Ascites production. BALB/c mice were injected i.p. with500 mg of pristane (2,6,10,14-tetramethylpentadecane;Sigma catalog no. T 7640) 2 weeks before i.p. injection of 3x 106 to 4 x 106 hybridoma cells in DMEM without FCS.Mouse ascites fluid (MAF) was collected from days 15 to 20after cell injection, filtered with 0.2-ptm-pore-size membranefilters (Millipore Corp., Bedford, Mass.), divided into equalportions, and frozen at -20°C.

Determination of immunodominant epitopes for humans.Inhibition experiments using IIF were performed to checkfor the existence of immunodominant epitopes for humans inthe MAVI strain. Serum samples from patients with BF weretested for 30 min and washed twice with PBS-Tween;hybridoma supernatants were then added for 30 min. Thecontrol was serum from a healthy blood donor. Inhibition byhuman sera or by a high-titer serum sample from an infectedguinea pig was tested by adding the indicated serum dilu-tions to the dried antigen for 30 min. Washings with PBS-Tween and distilled water and evaluation ofthe reactionwith anti-mouse immunoglobulin fluoresceinated antibody(DAKO) were performed as described above. The tests werecarried out in duplicate. Titers of inhibitor sera were as

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TABLE 1. Characteristics of SFG rickettsiae isolated in Sicily

Pathogenicity for guinea pigs'Strain Origin H/Ta Serocon-

Fever Orchitis version

MAVI Human + + +Villafrati Tick 7/40 - + +Cammarata Tick 8/18 - + +Palermo Tick 7/17 -

Baida Tick 2/12 -

a Hemolymph-positive ticks to total ticks examined.b After culture in VERO cells for 1 month.

follows: serum samples 40471, 33725, 39374, and 39457,1:640; samples 39178 and 38877, 1:320; sample 28470, 1:40;and sample 42296 (guinea pig serum inoculated with theMAVI strain), 1:320.

RESULTS

Isolation and characterization of the strains of SFG rick-ettsiae used in this study are shown in Table 1. The MAVIstrain (from a patient with BF) and the Villafrati, Palermo,and Baida strains (from ticks) were isolated from areas ofrelative proximity, whereas the Cammarata strain was iso-lated about 90 km away.

When tested by a biological assay on guinea pigs, only theMAVI strain produced fever, orchitis, and seroconversion;the other strains were nonpathogenic for guinea pigs.Nine fusion experiments were camied out according to the

protocols summarized in Table 2. Because of its pathogenic-ity in guinea pigs, the MAVI strain was chosen for immuni-zation of mice. All immunized mice responded by serocon-

version, as determined by IIF. Four fusions (C, B, E, and A)yielded positive hybridomas. The fusion that yielded thehighest number of positive hybridomas (C) was obtainedafter immunizating mice three times i.p. and once intrave-nously (booster) at 7-day intervals and fusing cells 3 daysafter the last injection. The IIF titer in mouse serum was

1:1,280 at the time of sacrifice; PEG 1500 was used in thisfusion.

Of 563 supernatants from wells with cells growing inselective HAT medium, 22 were positive for the presence of

TABLE 2. Experimental conditions for fusion experiments

No. of No. No. ofimmuni- M wel anti-

Expt time (days) IIF titer mPolEwtb S/M' positive MAVitmdy)in serum

MIwfor positive

between growth hybri-injectionsg domas

A 3; 7 1:640 1,500 10:1 70 11 3; 15 1:640 4,000 10:1 51 02 3; 15 1:1,280 4,000 10:1 58 03 2; 30 1:640 4,000 5:1 46 04 2; 30 1:1,280 1,500 10:1 67 0B 3; 15 1:1,280 1,500 5:1 70 3C 3; 7 1:1,280 1,500 5:1 72 16D 3; 7 1:1,280 1,500 5:1 66 0E 2; 30 1:1,280 1,500 5:1 63 2

In each case, a booster was given 7 days after the last inoculation.b Fusion conditions varied according to the molecular weight of the PEG

used (see Materials and Methods). PEG 1500 was added to cells for 7 min;PEG 4000 was added for 1 min. Thereafter, serum-free medium was addedgradually.

Spleen cells to myeloma cells.

TABLE 3. Reciprocal IIF titers of supernatants of monoclonalantibodies to SFG rickettsiaea

Class and Reciprocal titer of SFG strain:subclass MAVI Villafrati Cammarata Palermo Baida

CA IgM 32 16 128 2 NegCB NDC 2 2 Neg Neg NegCC IgG3 4 16 Neg 2 NegCD IgM 4 2 Neg Neg 2CF IgG2a 8 Neg 2 Neg NegCG ND 2 2 Neg Neg NegCI ND 2 2 Neg Neg NegCL IgG2a 64 64 8 2 2CM IgM 16 32 16 Neg NegCN IgG2a 8 4 Neg Neg NegCO IgM 16 4 32 32 2CP IgG2b 128 256 Neg Neg NegCQ IgG2a 64 64 2 2 NegCR IgM 16 8 32 32 2CS ND 16 16 8 2 NegCU ND 16 16 Neg Neg NegBA ND 4 Neg Neg 4 NegBB IgG2a 16 2 32 2 NegBD IgG2a 4 Neg 4 2 4E15 IgM 64 4 64 64 64E17 IgM 8 8 64 32 2Ai IgG3 64 128 Neg Neg Neg

a Only fusion experiments yielding positive hybridomas are shown. Testswere carried out in duplicate.

b The MAVI strain was isolated from a human; the others were isolatedfrom ticks. The Villafrati and Cammarata strains were virulent when inocu-lated i.p. in a male guinea pig. Neg, Negative.

ND, Not done.

antirickettsia (MAVI strain) antibodies. Eight of these hy-bridomas were used for production of MAF by i.p. injectioninto BALB/c mice. IIF titers of MAFs to the MAVI strainranged from 1:80 to 1:2,560.

Supernatants of the hybridomas were tested by the IIFassay to determine immunoglobulin isotypes. No superna-tant was ofthe IgGl subclass; 33,3% Were IgG2a, 6.6% wereIgG2b, 13.3% were IgG3, and 46.6% were IgM (Table 3).The 22 supernatants reacting with the MAVI strain were

then tested for binding in the IIF assay to the other SFGrickettsial isolates to investigate the presence of the recog-nized epitopes in these strains (Table 3). Most of themonoclonal antibodies reacting with the MAVI strain did notbind to all of the other strains tested. Only five of thesupernatants, CL, CO, CR, E15, and E17, gave a positiveIIF result, although with different titers, on all strains, anindication that they nmay have recognized epitopes commonto SFG rickettsiae. E15 was the only monoclonal antibodythat recognized the Baida strain at high titer and the Villaf-rati strain at low titer (relative titers). CP, CQ, and Ai werepositive only with the Villafrati and MAVI strains (CQreacted at a 1:2 dilution with the Cammarata and Palermostrains, versus a 1:64 dilution with the MAVI and Villafratistrains). CB, CG, CI, CN, and CU showed the same patternof reaction but bound at low titers. Monoclonal antibodiesthat had low titers of binding to the MAVI strain (arbitrarilydefined as 1:8 or less) were included in the analysis, sinceseveral showed higher titers with one or more of the otherstrains (e.g., E17). Despite the observed heterogeneity,some of the monoclonal antibodies (e.g., CB, CG, and CI)possibly recognized the same epitope, as judged by patternand extent of reactivity.To investigate whether epitopes recognized by mouse

monoclonal antibodies were also recognized by human anti-

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TABLE 4. IIF assay for immunodominant epitopes in humans: inhibition by BF patient sera of binding to the MAVI strain'Inhibition by serum sample":

Hybridoma37164' 40471 33725 39374 39178 38877 39457 28470 42296'

BD + + - - - - + + -

CF + - - - - ND ND ND +CL + + + + + + + + +CQ + - + + + + + + +E17 + + + + + + + +CC + + + + + + + + +E15 + + + + + + + + +BA + - + - + + - + +BB + + + + + ND ND ND +CN + - + + + + - + +Ai + + + + + + + + +CB + + + + + + + + +CR + - + - + + + + +Co + + + + + + + +CM + _ + + + + +

'Hybridoma supernatants were used undiluted; sera were used at a 1:40 dilution. Tests were carried out in duplicate.b +, No inhibition; -, inhibition; ±, partial inhibition; ND, not done.c Negative control (negative titer to the MAVI strain in the IIF assay).d Sample from the patient from whom the MAVI strain was isolated.e Guinea pig serum inoculated with the MAVI strain.

bodies present in serum samples from BF patients, weperformed an inhibition assay as described in Materials andMethods (Table 4). Each test was carried out in duplicateand showed satisfactory reproducibility. All of the superna-tants exhibited variable behavior except possibly BD andCF, which were negative in the IIF assay after absorptionwith most of the patient sera. This result indicated saturationof the antigenic epitopes recognized by the monoclonalantibodies.

Binding of CF, CQ, BA, CN, CR, and CM was totallyinhibited, and binding of six other monoclonal antibodieswas partially inhibited, by patient serum 40471. In contrast,serum 28470 (obtained from the patient from whom theMAVI strain was isolated) partially inhibited only the CNsupernatant. The other five serum samples from patientsshowed intermediate patterns, with preferential inhibition ofBD and CF supernatants; however, these monoclonal anti-bodies showed low IIF titers (1:4 and 1:8, respectively, onthe MAVI strain). Guinea pig serum from an immunizedanimal inhibited BD and CM and partially inhibited CF.Although a limited number of serum samples were screened,a high percentage of BF patients produced antibodies thatrecognized the same epitopes as did mouse monoclonalantibodies BD and CF, whereas human antibodies to theepitopes recognized by the other monoclonal antibodiesseemed less represented in the humoral immune response toBF.

DISCUSSIONWe used different protocols for immunization and for cell

fusion to try to improve the yield of positive hybridomas,since there have been few reports on the production ofmonoclonal antibodies to intracellular parasites and there isno established strategy to obtain high percentages of positivemonoclonal antibodies. Moreover, there is only one previ-ous report of monoclonal antibodies to R. conorii (8).

Results of IIF analysis of binding of a panel of monoclonalantibodies to different local isolates of SFG rickettsiaeclearly showed a strong antigenic heterogeneity among thefive isolates tested. The differences in binding titers amongthe monoclonal antibodies reacting mainly or exclusively

with the MAVI and Villafrati strains, CP, CQ, and Ai (hightiters) and CB, CG, CI, CN, and CU (low -titers) can beexplained on the basis of higher or lower expression of thesame epitope on rickettsial isolates. Alternatively, the dif-ferences may have resulted from differences in the immuno-globulin subclass specificity of the second fluorescent anti-body used or from lower immunoglobulin production by thehybridoma. On the other hand, the latter possibilities cannotexplain differences in binding titers for the same monoclonalantibody (e.g., E15, CP, and Ai). It is therefore likely thatthe E15 epitope is actually expressed at a low level by theVillafrati strain and at high levels by the other four strains.The same conclusion applies to the CP (positive on theMAVI and Villafrati strains only) and Ai (same pattern)monoclonal antibodies.Some of the MAVI-positive monoclonal antibodies re-

acted more strongly with the tick-derived Villafrati andCammarata strains pathogenic for guinea pigs (CA, CL, CM,CP, CQ, CS, CU, BB, and Ai), which may indicate that thecorresponding epitopes were correlated with pathogenicityfor humans. This finding prompted us to try to inhibit bindingof 15 selected monoclonal antibodies to the MAVI strainantigen with antibodies contained in the sera from sevenpatients with BF, using one healthy blood donor and animmunized guinea pig as controls.Except for showing partial inhibition of CN, serum 28470

(from the patient from whom the MAVI strain was isolated)did not inhibit any of the tested monoclonal antibody super-natants. The one immunized guinea pig serum used inhibitedonly BD, CF (partially), and CM supernatants. Normalhuman serum inhibited none of the monoclonal antibodies,whereas the seven serum samples from BF patients pro-duced various patterns of inhibition. At present, we cannotsay whether the inhibition of reactivity observed was morelikely due to steric hindrance or to competition for a com-mon epitope recognized by polyvalent immune human serumand by the monoclonal antibodies.Taken together, these results can be attributed to several

factors, each warranting further investigation: (i) as is al-ready known, the hybridoma technique reveals epitopeseliciting antibodies that are less represented in a polyclonal

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response; (ii) epitopes are recognized differently in differentspecies; (iii) one patient (28470) responded poorly to thepathogen with respect to serum antibody production, a likelyresult since the IIF titer in this serum was 1:40.Another factor to be taken into account, reflected by the

low inhibition exerted on monoclonal antibody binding byimmune patient sera, is the likely difference between theantibody response elicited by immunization (monoclonalantibodies), which could reveal epitopes that are shieldedfrom the immune response due to pathogenetic mechanismsor are highly polymorphic, and the antibody response elic-ited by infection (patients and guinea pigs). This aspect isworth further study to determine relevant antigens to chosefor development of advanced vaccines or diagnostics.The results of this study, although obtained for a limited

number of serum samples and dilutions, indicate that thehuman immune response to SFG rickettsial antigens ishighly heterogeneous and may involve the prevalence ofantibodies to the BD and CF epitopes; no apparent correla-tion with epitopes present on rickettsial strains pathogenicfor guinea pigs was found.

This study, which is the first attempt to analyze theantigenic polymorphism of local isolates of SFG rickettsiaeby using a panel of monoclonal antibodies raised by immu-nization with another local isolate, indicates a marked poly-morphism among five strains. Analysis of additional SFGisolates, as well as extension of the use of this approach toother geographic areas, could give a measure of this poly-morphism. Such antigenic heterogeneity among rickettsialisolates in Sicily was predicted by biological studies ofpathogenicity in guinea pigs carried out by our group (14;Vitale, R. Librizzi, et al., in press; Vitale, Virga, et al., inpress). A similar heterogeneous response in biological as-says has also been observed in Latium (5), the United States(2), and eastern Europe (4).Other authors have reported a prevalence of IgG2a isotype

monoclonal antibodies to SFG rickettsiae (9). In contrast,our results indicate a higher frequency of IgM (46.6%)relative to that of IgG2a (33.3%); no IgGl monoclonalantibody was found. The frequency of the IgM isotype mayindicate a prevalence of monoclonal antibodies directed toT-cell-independent antigens (8). The MAF positivity titers inthe IIF assay that we found are lower than those reported byLange and Walker (9) for purified MAF fractions of mono-clonal antibodies to R. rickettsii. This result could also bedue to the high frequency of the IgM class among themonoclonal antibodies described in our study.One of the important questions that remain to be answered

is whether the strains showing low pathogenicity for exper-imental animals exert a protective action, on an epidemio-logical basis, for infections in humans that result in devel-opment of overt BF symptoms. Serological screenings basedon suitable monoclonal antibodies should be useful in thisrespect.

Finally, as has been the case for other intracellular patho-gens such as mycobacteria (6), monoclonal antibodies mayrepresent a useful tool for identifying the antigens andepitopes important both for the pathogenesis of the illnessand for a protective immune response. In the case of

mycobacteria, use of monoclonal antibodies, together withT-cell clones and recombinant DNA technology, has helpedto better define the relative roles of cellular and humoralimmune responses in mycobacterioses. The knowledge de-rived from a similar research strategy for rickettsia-causeddiseases could ultimately be used in the design of advancedvaccines for these illnesses.

LITERATURE CITED1. Aeschlimann, A., W. Burgdorfer, H. Matile, O. Peter, and R.

Wyler. 1979. Aspects nouveaux du role de vecteur jou parIxodes ricinus en Suisse. Acta Trop. 36:181-191.

2. Bell, E. J., G. M. Kohls, H. G. Stoenner, and D. B. Lackman.1963. Nonpathogenic rickettsiae related to the spotted fevergroup isolated from ticks Dermacentor variabilis and Derma-centor andersoni from eastern Montana. J. Immunol. 90:770-781.

3. Burgdorfer, W. 1970. Hemolymph test. A technique for detec-tion of Rickettsiae in ticks. Am. J. Trop. Med. Hyg. 19:1010-1014.

4. Burgdorfer, W., S. F. Hayes, and A. J. Mavros. 1981. Nonpath-ogenic Rickettsiae in Dermacentor andersoni: a limiting factorfor the distribution of Rickettsia rickettsii, p. 585. In W.Burgdorfer and R. L. Anacker (ed.), Rickettsiae and rickettsialdiseases. Academic Press, lnc., New York.

5. Cacciapuoti, B., L. Rivosecchi, E. Stella, L. Ciceroni, and C.Khoury. 1985. Osservazioni preliminari sulla prevalenza diRickettsiae del gruppo delle spotted fevers in Rhipicephalussanguineus catturati in aree sub-urbane. Boll. Ist. Sieroter.Milan. 64:77-81.

6. Damiani, G., A. Biano, A. Beltrame, D. Vismara, M. FilipponeMezzopreti, V. Colizzi, D. B. Young, and B. R. Bloom. 1988.Generation and characterization of monoclonal antibodies to28-, 35-, and 65-kilodalton proteins of Mycobacterium tubercu-losis. Infect. Immun. 56:1281-1287.

7. Fazekas de St. Groth, S., and D. Scheidegger. 1980. Productionof monoclonal antibodies: strategy and tactics. J. Immunol.Methods 35:1-21.

8. Feng, H. M., D. H. Walker, and J. G. Wang. 1987. Analysis ofT-cell-dependent and independent antigens of Rickettsia conoriiwith monoclonal antibodies. Infect. Immun. 55:7-15.

9. Lange, J. V., and D. H. Walker. 1984. Production and charac-terization of monoclonal antibodies to Rickettsia rickettsii.Infect. Immun. 46:289-294.

10. Mansueto, S., G. Tringali, and D. H. Walker. 1986. Widespread,simultaneous increase in the incidence of spotted fever grouprickettsioses. J. Infect. Dis. 154:539-540.

11. Mansueto, S., G. Vitale, G. Tringali, C. Pintagro, C. Occhino,and M. D. Miceli. 1983. Studi siero-immunologici nella FebbreBottonosa. 1. Valutazione di un kit del commercio per micro-immunofluorescenza nella diagnostic sierologica della FebbreBottonosa. Quad. Sclavo Diagn. Clin. Lab. 19:262-270.

12. Rehacek, J., V. Pospisil, and F. Ciampor. 1976. First record ofbacillary rickettsia-like organism in European tick Dermacentorinarginatus (Sulzer). Folia Parasitol. 23:301-308.

13. Sexton, D. J., W. Burgdorfer, L. Thomas, and B. R. Norment.1976. Rocky Mountain spotted fever in Mississippi: survey forspotted fever antibodies in dogs and for spotted fever grouprickettsiae in dog ticks. Am. J. Epidemiol. 103:192-197.

14. Tringali, G., V. Intonazzo, A. M. Perna, S. Mansueto, G. Vitale,and D. H. Walker. 1986. Epidemiology of Boutonneuse fever inwestern Sicily. Distribution and prevalence of spotted fevergroup rickettsial infection in dog ticks (Rhipicephalus san-guineus). Am. J. Epidemiol. 123:721-727.

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