INFECTION AND IMMUNITY, Aug. 1975, p. 398-403Copyright ( 1975 American Society for Microbiology

Vol. 12, No. 2Printed in U.S.A.

Serological Studies of Actinomyces israelii by CrossedImmunoelectrophoresis: Taxonomic and Diagnostic

ApplicationsKENNETH HOLMBERG,* CARL-ERIK NORD, AND TORKEL WADSTROM

Department of Bacteriology, the National Bacteriological Laboratory, Stockholm, Sweden

Received for publication 6 February 1975

Crossed immunoelectrophoresis (CIE) with intermediate gel was applied to theserological analysis of Actinomyces israelii to develop a test with high efficiencyin the laboratory diagnosis of human actinomycosis and classification of A.israelii. Recently developed standard antigen-antibody systems for A. israelii byCIE were used as reference. The reference systems were based on standardpreparations of cytoplasmic and whole cell-associated antigens of A. israelii anda standard immunoglobulin G pool purified from rabbit antisera to formalin-treated whole cells and cell lysates of A. israelii. The specificity of the standardantigens for A. israelii was evaluated in CIE studies by screening for antibodies tocomponents of the antigens in rabbit antisera raised against related bacteria. Thestandard system for A. israelii based on cytoplasmic antigens formed species-specif'ic precipitins, whereas antisera raised against A. naeslundii and/orPropionibacterium acnes precipitated components of' the other standard anti-gens. As a result of these analyses, the standard system for A. israelii based on10 cytoplasmic antigens was used as reference for CIE studies to detect humoralantibodies to A. israelii in sera from nine patients with actinomycosis. All thesera from the patients formed at the time of diagnosis one or more precipitins interms of the 10 reference precipitins. Up to five precipitins were found in singlesera. Follow-up studies covering a period of one-half year after treatment showeda gradually decreased precipitin response in the course of' time. In control serafrom patients with newly diagnosed tuberculosis, nocardiosis, deep Candidainfection, and aspergillosis, and in sera from healthy blood donors, no antibodieswere detected with specificity for the reference antigens.

Actinomyces israelii is the most commonetiological agent of actinomycosis in man (9,14, 21), although other species of the genusActinomyces and Arachnia propionica have alsobeen implicated (7, 10). Actinomycosis is proba-bly often missed in humans, since the labora-tory diagnosis of this disease depends on thedemonstration of A. israelii in exudates and/orthe isolation of this organism in culture (9).Diagnostic problems include the requirement ofspecial arrangements for sample taking and forculture conditions. In many cases antibiotictherapy is started before the true identity of thecausative organism is established. A reliableserological test would therefore be a usefuladjunct in the diagnosis and prediction of theprognosis of the disease. Moreover, it may oftenbe the only criterion of current or previousactinomycosis.

Antisera raised in rabbits against strains of A.israelii have in immunofluorescence, precipit in

tests, and cell wall agglutination tests alloweddifferentiation of a distinct serogroup of' A.israelii, as well as two serotypes (4, 6, 7, 8, 20,24).Even though considerable work has been done

on the production of' specific antisera to identifvand classify A. israelii, conflicting results are tobe found in the literature concerning serumtiters against A. israelii in patients with provedactinomycotic infections and concerning cross-reacting antibodies against antigens of A. isra-elii formed in other diseases (11, 15). However,little basic work has been done on the charac-terization of antigen preparations of A. israeliiand the development of generally acceptabletechniques for the conduct of serological teststo diagnose actinomycosis. The immunodiffu-sion tests, hemagglutination tests, and comple-ment fixation tests have been the basic sero-logical methods for studies on the serologicaldiagnosis of' actinomycosis.

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Crossed immunoelectrophoresis (CIE) withintermediate gel (1) proved to be a powerfulmethod in combination with a reference precipi-tin system for characterization of antibodies inmucocutaneous candidiasis (3), Pseudomonasaeruginosa infections (18), and Mycobacteriumleprae infections (2). The objectives of thisstudy were to apply CIE with intermediate geltechnique to the serological diagnosis of humanactinomycosis and classification of A. israelii,using recently developed standard antigen-anti-body systems as reference (17).

MATERIALS AND METHODS

Strains. For immunization of rabbits the following.type strains were used: A. israelii, serotype 1, ATCC12103, SBL 225/74; A. israelii, serotype 2, ATCC23036, WVU 307; A. viscosus, serotype 1, ATCC15987; A. viscosus, serotype 2, ATCC 19246; A.naeslundii, ATCC 12104; A. odontolvticus, serotype 1,ATCC 17982; A. odontolyticus, serotype 2, WVU 482;Arachnia propionica, serotype 1, ATCC 14157; A.propionica, serotype 2, WVU 346; P. acnes, serotype1, NCTC 737; P. acnes, serotype 2, ATCC 11828; P.avidum, ATCC 25277; P. granulosum, ATCC 25264;Nocardia asteroides, ATCC 19247, obtained fromAmerican Type Culture Collection (ATCC), WestVirginia University (WVU), and National Collectionof Type Cultures (NCTC).

Antisera. Rabbit antisera against Actinomycesspp., A. propionica, Propionibacterium spp., and N.asteroides were prepared by intravenous injections offormalin-killed whole cells by an immunizationschedule previously described (16). Two antiseraagainst each strain from different rabbits were pooled,and the immunoglobulin fraction was purified bysalting out with ammonium sulfate at 37% saturation.The antibody content of each pool was concentratedto 10 mg/ml. Also included in the study were commer-cial rabbit antiserum to Candida albicans (obtainedfrom Dakopatts A/S, Copenhagen, Denmark), rabbitantiserum to Aspergillus fumigatus (prepared in thisinstitution), and rabbit antisera to Aspergillus fumiga-tus, Thermoactinomyces vulgaris, and Micropolys-pora faeni (kindly supplied by D. W. R. MacKenzie,Mycological Reference Laboratory, Public HealthLaboratory Service, Colindale, England).Human sera were obtained from nine patients with

actinomycosis diagnosed on the basis of clinical andlaboratory findings. Five patients suffered from acervicofacial form and four from thoracic actinomyco-sis. In each case the clinical diagnosis was obtainedfrom the attending physician. The infecting orga-nisms were identified as A. israelii by the criteria of K.Holmberg and C.-E. Nord (J. Gen. Microbiol., inpress). Six patients were followed through remissionwith consecutive serum specimens. Serum specimenswere collected at the time of diagnosis, at 2 months, at4 months, at 6 months, and sometimes at 1 year afterestablishment of the diagnosis.Serum specimens, pooled in batches of 10 from 50

adult healthy donors selected at random, and pooled

serum specimens from 20 children aged 1 to 10 yearswere included as controls. Serum specimens from 10newly diagnosed tuberculosis patients, two nocardio-sis patients, two patients with deep candidosis, andthree patients with pulmonary aspergilloma weretested for precipitating antibodies to A. israelii. Thediagnosis of tuberculosis and nocardiosis were provenby isolation of Mycobacterium tuberculosis and N.asteroides, respectively. Candidosis was diagnosed onthe basis of isolation of C. albicans and positiveprecipitation reaction in double immunodiffusiontests. Aspergillosis was proven by isolation of A.fumigatus and positive immunodiffusion tests. Inaddition, sera from two patients with chronic acnesvulgaris from which P. acnes was isolated and tenpatients with chronic periodontal disease from whichA. israelii was isolated (Holmberg, Arch. Oral Biol.,in press) were included for tests. All sera were storedat -20 C with 15 mM NaN3 as preservative.

Standard antigen-antibody systems for A. is-raelii. Reference antigen-antibody systems for A. is-raelii, were prepared from antigen/water extracts onstandard preparations of sonic lysates, StAgSL, andchemical extracts from whole cells by 0.2 M hydro-chloric acid (StAgHCl), 4 M urea (StAgurea), and 10%(vol/vol) trichloroacetic acid (StAgTCA), and stan-dard rabbit antisera to formalin-treated whole cells(StAbI) and cell lysates (StAbII) of A. israelii (ATCC12103 and WVU 307). The procedure for preparingstandard antigens, standard antisera, and the immu-nochemical characteristics of the systems have beendescribed elsewhere (17).The standard system based on StAgSL and StAbI

comprised six reference precipitins, coded SL1 to SL6,the system based on StAgHCl comprised five refer-ence precipitins, coded HCll to HCl5, the systembased on StAgurea comprised five reference precipi-tins, coded ureal to urea5, and the system based onStAgTCA comprised one reference precipitin, codedTCA1. The reference precipitins coded SL1, SL2,SL4, SL5, HCl1, HCl2, HCl3, ureal, urea2, and urea-4were specific for their respective systems, whereasthe other reference precipitins of the systems showedreactions of partial or total identity to each other inCLE analysis (17). The system based on StAgSL andStAbII comprised 10 reference precipitates, coded 1to 10, of which six were identical to those revealed inthe system based on StAgSL and StAbI. None of theremaining precipitates was identical to any of theprecipitates in the other systems employed.CIE with intermediate gel. These immunoelectro-

phoreses were run by the procedures devised byAxelsen (1), using the same basic reagents as de-scribed previously (17). Antigen (5 ml) was submittedto the first-dimension electrophoretic run (9 to 10V/cm) for 50 min. The second-dimension electro-phoresis was run overnight, applying 2 V/cm. Thereference gel contained 5 gl of the standard prepara-tion of anti-A. israelii serum StAbII per cm2. Theintermediate gels contained 15 gl of the test serumper cm2. A negative control plate, containing the sameamount of saline instead of antiserum in the interme-diate gel, and a positive control plate, containing thereference antiserum, were always run with each setof six electrophoreses. First- and second-dimension

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400 HOLMBERG, NORD, AND WADSTROM

electrophoreses and the pressing, washing, andstaining of the plates were carried out as describedpreviously (17). Analyses of the immunoprecipitationpatterns were performed by comparison with the con-trol plates. Differences in precipitin pattern betweenthe test plates and the control plates were interpretedaccording to Axelsen (1).

RESULTSBy means of CIE with intermediate gel using

the standard antigen-antibody systems for A.israelii as a reference, the serological relation-ship between A. israelii and taxonomically re-lated bacteria was studied. Screening was per-formed for antibodies with specificities for theantigenic components of the different referencesystems for A. israelii in concentrated rabbitantibody pools to formalin-treated whole cells ofthe related bacteria (A. viscosus, serotype 1

[ATCC 15987]; A. viscosus, serotype 2 [ATCC19246]; A. naeslundii [ATCC 12104]; A. odon-tolyticus, serotype 1 [ATCC 17982]; A. odon-tolyticus, serotype 2 [WVU 482]; A. propionica,serotype 1 [ATCC 14157]; A. propionica, sero-type 2 [WVU 346]; P. acnes, serotype 1 [NCTC737] and serotype 2 [ATCC 11828]; P. avidum[ATCC 25577]; P. granulosum [ATCC 25564];and N. asteroides [ATCC 19247]). By compari-son with the control plates, no reference anti-gens of A. israelii StAgSL were retained byimmunoprecipitation in the intermediate gel bythese test sera. Antibodies with specificities forcomponents of the standard preparations of thechemical extracts, StAgHCl and StAgurea of A.israelii, were detected in antisera raised againstA. naeslundii by deflections of the referenceprecipitins coded HCll and urea4 compared tothe control plates. Antibodies against the anti-genic components urea4 of the antigen standardStAgurea were also revealed in sera raisedagainst P. acnes, serotypes 1 and 2. No precipi-tins were detected in these experiments thatindicated the presence of antibodies with speci-

ficities to other antigenic components of thestandard antigens than the reference antigens.No antibodies with specificity for the compo-nents of the different standard antigens of A.israelii were detected in rabbit antisera to C.albicans, A. fumigatus, Thermoactinomycesvulgaris, and Micropolyspora faeni.The identification of antibodies in the sera of

patients to A. israelii was conducted by meansof CIE with intermediate gel containing thesesera by the standard antigen-antibody systemfor A. israelii based on StAgSL and StAbIl asreference.

In sera from nine patients with actinomyco-sis, precipitating antibodies were detectedagainst this reference standard antigen. Theprecipitin response varied from patient to pa-tient. The number of retained reference anti-gens in the intermediate gel containing theserum of the patient ranged from one to four atthe time of diagnosis (Table 1). All precipitinswere identified in terms of the reference systemfor A. israelii. Antibodies specific for one of thecytoplasmic antigen components of A. israeliiwere present in sera from four patients. Two ofthese patients exhibited thoracic actinomyco-sis, and two patients exhibited a cervicofacialform of actinomycosis. This precipitin was iden-tified as antigen coded 7 or 10 (17). The serafrom two patients with cervicofacial ac-tinomycosis had antibodies to two of the refer-ence antigens, identified as precipitin codednumbers 7 and 10 and numbers 8 and 10,respectively. Antibodies to four reference anti-gens, identified as precipitins 6, 7, 8, and 10,were detected in sera from one patient withcervicofacial actinomycosis (Fig. 1). No at-tempts were made to quantitate the immuneresponses. No immunoprecipitation reactionswere obtained that indicated the presence offree antigens of A. israelii in these sera.

Follow-up studies of the sera from six patientsrevealed that the precipitin responses demon-

TABLE 1. Serological follow-up of patients with proven actinomNcosis

Precipitins at time of testingPatient Time of diagnosis 2 months _ 4months 66m onths _ 1 year

No. ] Identified as: No. Identified as: No. Identified as: No. Identified as: No. Identified as:GP 1 ioa 1 10 1 10 0 NDbGH 2 8,10 2 8,10 1 10 1 10 NDMF 1 10 1 10 1 10 0 NDAA 1 7 1 7 ND 0 NDSK 2 7,10 1 10 1 10 1 10 NDNZ 4 6, 7, 8, 10 4 6, 7, 8, 10 4 6, 7, 8, 10 4 6, 7, 8, 10 5 6, 7, 8, 9, 10a Precipitin codes, see text.b ND, Not done.

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PRECIPITATING ANTIBODIES TO A. ISRAELII 401

AStAblA+

5

BRef.gel StAb II

034Il

3x. ^,-e

-A2

... w

4

Inter-mediate /gely

.,.

StAgSL

StAbl.1 Ab-X

8 _6

I Icm

C

FIG. 1. CIE with intermediate gel using a cytoplasmic fraction of A. israelii (StAgSL) as antigen and rabbitanti-A. israelii serum (StA bII) as reference. (A) The standard antigen-antibody system for A. israelii by meansof CIE; (B) positiue control plate with the reference antibody standard in the intermediate gel; (C) serum from apatient with proven actinomycosis. The patient's precipitin response is indicated by the precipitates coded 6,7, 8, 9, and 10 in the intermediate gel.

strable at the time of diagnosis were graduallylost in the course of time after substantialclinical improvement following treatment. Sixmonths after establishment of the diagnosis, thesera which initially contained antibodies to oneof the reference antigens had lost their precipi-tating reactivity. The two patients with anti-body specificities to two reference antigens hadlost antibodies with specificity for antigenscoded 7 and 8, respectively. The sera from theremaining patient maintained the initial pre-cipitin pattern. A follow-up study of consecu-tive sera from this patient during exacerbations,covering a period of 1.5 years, showed anincreased number of precipitating antibodies toA. israelii. After 1 year, antibodies with specific-ity for the reference antigen component coded9 were also demonstrable.

In control experiments, neither the sera fromblood donors nor from patients with tuberculo-sis, nocardiosis, deep candidosis, chronic acnesvulgaris, or chronic periodontal disease pre-cipitated the reference standard antigen.

DISCUSSIONCIE with intermediate gel (1) in conjunction

with a reference antigen-antibody system for A.israelii (17) appeared to be useful for serologicaltaxonomic studies of A. israelii and for thestudy of circulating antibodies to A. israelii inhuman actinomycosis.

Analysis of the precipitin reactions, obtainedby CIE against standard antigens of A. israeliiwith antisera to some related bacteria, sup-ported current classification of a separate sero-logical group of A. israelii (5, 16, 22-24). Theserological reaction of the components of thecytoplasmic standard antigen of A. israelii dis-played species specificity. The distinction of a

species-specific serogroup of A. israelii by pre-cipitation tests seems to be less apparent withother antigenic materials of A. israelii. In thepresent study, this was indicated by the cross-reactivities found between antisera producedagainst whole cells of type strains of A. naes-lundii and P. acnes and some components ofthe whole cell-associated antigens of A. israelii,obtained by hydrochloric acid, trichloroaceticacid, and urea extractions. The findings ofcommon antigens between A. israelii and A.naeslundii are in agreement with those foundpreviously in agar gel diffusion studies withsupernatant culture fluid as the antigen. Pre-cipitating antigens, derived by acid extractionof cells of A. israelii, include species-specificantigens and cross-reacting components.Werner et al. (25) recently found that rabbitantisera against Propionibacterium (Coryne-bacterium) acnes strains in double immunodif-fusion tests formed one or two precipitationlines with A. israelii polysaccharide antigens,obtained by formamide extraction, and theclear supernatant of autoclaved whole cell sus-pensions of A. israelii. These findings are inagreement with the cross-reactions found in thepresent study between anti-P. acnes sera andone component of the standard preparation ofurea extract, StAgurea.The precipitating components of the cyto-

plasmic standard antigen of A. israelii appearedspecifically diagnostic for A. israelii. They alsopossessed immunochemical properties whichmade them more suitable for CIE studies (17)than the components of the standards extractedfrom whole cells of A. israelii. The antigen-anti-body system for A. israelii, based on the cyto-plasmic antigen standard and the standardantiserum, raised against crude cell lysates of A.

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402 HOLMBERG, NORD, AND WADSTROM

israelii, was therefore employed as reference inassays to estimate the humoral immunore-sponse to A. israelii in patients with ac-tinomycosis caused by this organism.

Using the technique of CIE with sera frompatients in the intermediate gel, it was possibleto detect antibodies to the reference antigens ofA. israelii in sera from patients with ac-tinomycosis at the time of diagnosis. Analysis ofthe immunoprecipitation patterns obtained in-dicated that there appeared to be a correlationbetween the time of active disease and thehumoral immune response to the referenceantigens. Thus, in one patient with rapidlyfulminating thoracic actinomycosis with aknown history of disease for about 2 months anda short period of remission after successfultreatment, the immune response was surpris-ingly weak at the time of diagnosis, with anti-bodies to only one component of the referenceantigens of A. israelii compared to that evokedin sera from a patient with a persistent activecervicofacial actinomycosis covering a period of1.5 years, which during that time developedantibodies to six components of the referencesystem.Although significant differences in the im-

munoresponse of individual patients may occur,these observations provide evidence for an in-creased humoral immunoresponse to the refer-ence antigens of A. israelii in chronic actinomy-cotic infections. The presence of one or twoprecipitins could indicate any form of ac-tinomycosis. The examination of serial serumspecimens provided information about the clini-cal course of the disease. After successful treat-ment there was a reduction in the number ofprecipitins.No titration of the positive sera with A.

israelii precipitins was made in the presentstudy. Titration might be useful in following theclinical course of the disease. It would beexpected that titration would reveal a lowerlevel of antibody prior to the time of disappear-ance of the precipitin reaction and, therefore,would provide an earlier guide for response totherapy.The role of the humoral antibodies in ac-

tinomycosis is still unclear. It is assumed thatinfections with A. israelii raise protective hu-moral antibodies to prevent dissemination ofthe infection to parietal organs. Mere coloniza-tion with A. israelii, which is a common com-mensal of tonsillar crypts in chronic tonsillitis(13) or of periodontal pockets in chronic perio-dontal disease (K. Holmberg, Arch. Oral Biol.,in press), does not seem to evoke detectablehumoral responses to the reference antigens ofA. israelii as indicated by the absence of precip-

itating antibodies in sera from patients withadvanced periodontal lesions from which A.israelii had been isolated and in sera fromhealthy blood donors.Although extensive studies were carried out,

cross-reacting antibodies with specificity for thereference antigens were not detectable in serumspecimens from patients with other diseasesthan actinomycosis. This contrasts with theconsiderable cross-reactions with A. israeliifound with heterogeneous sera from other dis-eases, such as tuberculosis, nocardiosis, strep-tococcal infections, and some fungal infections,and also in sera from healthy blood donors inprevious serological surveys of actinomycosis(11, 15). However, these studies used as anti-gen chemical fractions of A. israelii (15) andcrude antigens of A. israelii prepared by sonictreatment, formamide extraction of cells or ace-tone precipitation of supernatant fluids frombroth cultures (17) in conventional complementfixation, hemagglutination, and gel diffusiontests.CIE with intermediate gel using a monospe-

cific standard antigen-antibody system for A.israelii as reference permitted the serodiagnosisof actinomycosis. The value of the test rests onthe monospecificity of the reference antigens ofA. israelii and the fact that the primary produc-tion of antibodies is directed to these antigens inthe course of natural infections. The high sensi-tivity and the resolving power of CIE permitsthe determination of antibody specificities incomplex sera to the reference antigens of thecrude antigen mixtures. The test seems to be apromising method for routine laboratory prac-tice, with a major advantage being that antigenproduction is easy and purification of the refer-ence antigens is unnecessary. The test fulfillsthe need for efficiency of a laboratory test withregard to sensitivity and specificity. The possi-bility for standardization would be introducedby the test. A standardized test used in prospec-tive clinical trials would yield comparable infor-mation from different laboratories and, in turn,clarify the problem concerning the effectivenessof the test in diagnostic A. israelii serology.

LITERATURE CITED1. Axelsen, N. H. 1973. Intermediate gel in crossed and in

fused rocket immunoelectrophoresis. Scand. J. Immu-nol. 2:71-77.

2. Axelsen, N. H., M. Harboe, 0. Closs, and T. Godal. 1974.BCG antibody profiles in tuberculoid and lepromatousleprosy. Infect. Immun. 9:952-958.

3. Axelsen, N. H., C. H. Kirkpatrick, and R. H. Buckley.1974. Precipitins to Candida albicans in chronic muco-cutaneous candidiasis studied by crossed immunoelec-trophoresis with intermediate gel. Clin. Exp. Immunol.17:385-394.

4. Bowden, G. H., and J. M. Hardie. 1970. Commensal and

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pathogenic Actinomyces species in man, p. 277-299. InG. Sykes and F. A. Skinner (ed.), Actinomycetales.characteristics and practical implications. AcademicPress Inc., New York.

5. Brock, D. W., and L. K. Georg. 1969. Determination andanalysis of Actinomyces israelii serotypes by fluores-cent-antibody procedures. J. Bacteriol. 97:581-586.

6. Brock, D. W., and L. K. Georg. 1969. Characterization ofActinomyces israelii, serotypes 1 and 2. J. Bacteriol.97:587-589.

7. Brock, D. W., L. K. Georg, J. M. Brown, and M. D.Hicklin. 1974. Actinomycosis caused by Arachnia pro-pionica. Am. J. Clin. Pathol. 59:66-77.

8. Cummins, C. S. 1970. Actinomyces israelii, type 2, p.29-34. In H. Prauser (ed.), The Actinomycetales. VEBGustav Fischer Verlag, Jena.

9. Georg, L. K. 1970. Diagnostic procedures for the isolationand identification of the etiologic agents of actinomyco-sis, p. 71-80. In Proceedings Int. Symp. Mycoses.World Health Organization, Washington, D.C.

10. Georg, L. K., and C. M. Coleman. 1970. Comparativepathogenicity of various Actinomyces species, p. 35-45.In H. Prauser (ed.), The Actinomycetales. VEB GustavFischer Verlag, Jena.

11. Georg, L. K., R. M. Coleman, and J. M. Brown. 1968.Evaluation of an agar gel precipitin test for thesero-diagnosis of actinomycosis. J. Immunol.100:1288-1292.

12. Georg, L. K., G. W. Robertstad, and S. A. Brinkman.1964. Identification of species of Actinomyces. J. Bac-teriol. 88:477-490.

13. Gruner, 0. P. N. 1969. Actinomyces in tonsillar tissue.Acta Pathol. Microbiol. Scand. 76:239-244.

14. Holm, P. 1950. Studies on the aetiology of humanactinomycosis. Acta Pathol. Microbiol. Scand.27:736-751.

15. Holm, P., and J. B. Kwapinski. 1959. Studies on thedetection of Actinomvces antibodies in human sera byusing pure antigenic fractions of Actinomyces israelii.

Acta Pathol. Microbiol. Scand. 45:107-112.16. Holmberg, K., and U. Forsum. 1973. Identification of

Actinomyces, Arachnia, Bacterionema, Rothia andPropionibacterium species by defined immunofluores-cence. J. Bacteriol. 25:834-843.

17. Holmberg, K., C.-E. Nord, and T. Wadstrom. Serologicalstudies of ActinomYces israelii by crossed immunoelec-trophoresis: standard antigen-antibody system for A.israelii. Infect. Immun. 12:387-397.

18. Hojby, N., and N. H. Axelsen. 1973. Identification andquantitation of precipitins against Pseudomonasaeruginosa in patients with cystic fibrosis by means ofcrossed immunoelectrophoresis with intermediate gel.Acta Pathol. Microbiol. Scand. Sect. B 81:298-308.

19. King, S., and E. Meyer. 1963. Gel diffusion technique inantigen-antibody reactions of Actinomyces species and"anaerobic diphtheroides." J. Bacteriol. 85:186-190.

20. Lambert, F. W., J. M. Brown, and L. K. Georg. 1967.Identification of Actinomyces israelii and Actinomycesnaeslundii by fluorescent-antibody and agar gel diffu-sion techniques. J. Bacteriol. 94:1287-1295.

21. Slack, J. 1942. The source of infection of actinomycosis. J.Bacteriol. 43:193-209.

22. Slack, J. M., and M. A. Gerencser. 1966. Revision ofserological grouping of Actinomyces. J. Bacteriol.91:2107.

23. Slack, J. M., and M. A. Gerencser. 1970. Two newserological groups of Actinomyces. J. Bacteriol.103:265-266.

24. Slack, J. M., S. Landfried, and M. A. Gerencser. 1969.Morphological, biochemical, and serological studies on64 strains of Actinomyces israelii. J. Bacteriol.97:873-884.

25. Werner, H., G. Rintelen, and G. Bohm. 1974. Undersu-chungen zur Frage der Antigen-gemeinschaft zwischenActinomyces israelii and Corvnebacterium acnes. Zen-trabl. Bakteriol. Parasitenkd. Infektionskr. Hyg. Abt.1 Orig. Reihe A 226:264-271.

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