APPLIED MICROBIOLOGY, May 1973, p. 834-843 Vol. 25, No. 5 Copyright 0 1973 American Society for Microbiology Printed in U.S.A. Identification of , Arachnia, Bacterionema, , and Propionibacterium Species by Defined Immunofluorescence

KENNETH HOLMBERG AND URBAN FORSUM Department of Clinical Bacteriology, Central Hospital, Visterds, Sweden, and Institute of Medical Microbiology, University of Uppsala, Uppsala, Sweden Received for publication 13 February 1973

Fractionated fluorescein-isothiocyanate (FITC)-conjugated immunoglobulin G (dye-to-protein ratio < 10), produced against whole cells of Actinomyces spp., Arachnia, Bacterionena, Rothia, and Propionibacterium spp., give species- specific conjugates with controlled nonspecific staining reactions when appropri- ately diluted on the basis of their antibody content (10 mg/ml). Using this standardization in immunofluorescence, serotype-specific conjugates are also available after dilution for all serotypes of these organisms except for Actino- myces viscosus type 2, and Propionibacterium acnes type 1. Adequately ad- sorbed conjugates could be used to differentiate these serotypes from A. viscosus type 1 and P. acnes type 2, respectively. A serological classification in defined immunofluorescence corresponded to species and serotype designation proposed on the basis of other serological analysis and biochemical characteristics. This includes a separation in immunofluorescence of two serotypes of Propionibacte- rium acnes. The detection of certain actinomycetes of the family Actinomy- cetaceae and Propionibacterium species by the defined immunofluorescence in direct smears prepared from clinical specimens agreed to 88% with parallel cul- turing when including a prereduced (PRAS) medium technique for isolation. Qualitative studies revealed that single cells of these organisms could be spe- cifically identified by immunofluorescence when admixed with morphologically similar and a large number of other contaminants.

A rather substantial literature is developing (46). However, new applications of immuno- which suggests the importance of strains of fluorescent techniques pose special problems gram-positive filamentous or diphtheroidal bac- and require their evaluation and characteriza- teria, or both, in the pathogenesis of periodontal tion with respect to specific staining titer, disease. The pathogenicity of Actinomyces spp. fluorescein concentration, protein concentra- has recently been demonstrated in experimen- tion, fluorescein-to-protein ratio (F/P ratio), tal animals (11, 12, 20, 31, 50). However, the and usable diagnostic titer (cf. 3, 10, 24). etiological role of these organisms in human The aim of this investigation was to deter- remains to be determined. mine the optimal criteria for fluorescein-isothi- This is due in part to the fact that cultivation ocyanate (FITC) conjugated antisera against and isolation of these fastidious organisms re- certain genera of the family quire specific cultural techniques and are time- and certain species of Propionibacterium in consuming procedures (e.g., 19, 27, 29). Thus, direct immunofluorescence. The evaluations the need for specific and rapid identification were made using type species and oral isolates. procedures in ecological and epidemiological The accuracy of identifying these organisms by work has become important. defined immunofluorescence in direct smears Immunofluorescent techniques have been was determined by parallel cultures. found to be of value for detection of Actinomyces spp. in tissue impression smears MATERIALS AND METHODS from experimentally infected mice (34), in tis- Strains. Reference strains were obtained from sue or exudate smears from human tonsillar American Type Culture Collection (ATCC), Rock- materials (4), and in human dental calculus ville, Md., National Collection of Type Cultures 834 VOL. 25, 1973 SPECIES IDENTIFICATION BY IMMUNOFLUORESCENCE 835

(NCTC), Colindale, London, and National Center for (pH 6.3), further purified by chromatography on a Disease Control (CDC), Atlanta, Georgia. Two strains, diethylaminoethyl (DEAE)-cellulose column equili- serotype 2 (WVU 307) and brated and eluted with the dialysis buffer, and finally Arachnia propionica serotype 2 (WVU 346), were concentrated with an ultrafiltration cell (Diaflo model kindly supplied by M. Gerencser, Department of 51, Amicon Corp.) to a protein concentration of 18 to Microbiology, West Virginia University Medical 20 mg/ml. The protein concentration was determined School, Morgantown, W. Va. One strain of Coryne- by measuring the optical density at 280 nm. The IgG bacterium acnes group 2 (D 34) (Propionibacterium preparations gave a single IgG line in immunoelectro- granulosum) was supplied by J. G. Voss, The Proctor phoresis against anti-rabbit plasma protein serum & Gamble Co., Miami Valley Lab., Cincinnati, Ohio. (Behringwerke AG, Germany) (42). Immunization. Type strains (Table 1) were grown Conjugation. Rabbit IgG was conjugated with in Trypticase soy broth (Difco) for 48 to 72 h. After FITC (BDH Biochemicals, Poole, England). FITC centrifugation the cultures were suspended in 0.15 M was added to IgG in the proportion of 30 leg of dye per NaCl with 5% Formalin (vol/vol) for 1 h and washed mg of protein under vigorous stirring at room temper- twice in 0.15 M NaCl. One-milliliter cell suspensions ature for 1 h (pH 9.5). Nonreactive FITC was removed containing roughly 106 organisms per ml were injected by passing the solution through a Sephadex (Phar- intravenously in rabbits every third to fourth day for 4 macia AB, Sweden) G-25 column equilibrated with weeks. The rabbits were bled 7 days after the last phosphate-buffered saline (PBS) (35). injection. Slide agglutination titers of the sera ranged Fractionation of conjugates. To obtain fractions from 1/64 to 1/128 with living cells as antigens. For giving minimal nonspecific staining (24, 51), the each strain, two rabbits were immunized. conjugates were chromatographed on a DEAE-cell- Preparation of IgG. Rabbit immunoglobulin G ulose column equilibrated with 0.0175 M NaHPO4 (IgG) was prepared from sera by precipitation with (pH 6.3). After elution of a small amount of nonconju- ammonium sulfate at 37% saturation. The precipitate gated material with the starting buffer, a stepwise was dissolved in distilled water, and the procedure gradient was applied using 0.125 M NaCl, 0.250 M was repeated twice. The precipitated and redissolved NaCl, and 0.500 M NaCl in 0.0175 M NaHPO4 (pH material was dialyzed against 0.0175 M NaHPO4 6.3), respectively. The fractions were concentrated by TABLE 1. Specific staining titers for homologous strains and nonspecific staining of gingival tissue cells by fractions of FITC conjugates with different F/P ratios Spe- No-Spe- Non- FrIC-labeled antisera F/P cific FITC-labeled antisera | cf ratio specific ratioaP specific (10 mg/ml) (,Ug/mg)g tan- Stain- (10 mg/ml) nstag- stain- Ing Cgng ~ n titer' titer ig Rothia dentocariosa (ATCC 9.0 128 Arachnia proprionica type 1 10.0 16 17931) 13.0 32 (ATCC 14157) 12.0 8 20.0 4 ++ 20.0 4 + Bacterionema matruchotii 7.5 64 Arachnia propionica type 2 5.5 64 (ATCC 14265) 16.0 2 (+) (WVU 346) 12.5 8 20.0 +++ 18.0 1 +++ Actinomyces viscosus type 1 4.0 64 Propionibacterium acnes 7.5 64 (ATCC 15987) 13.0 8 type 1 (NCTC 737) 15.0 4 + 21.0 +++ 21.0 +++ Actinomyces viscosus type 2 4.0 64 Propionibacterium acnes 7.0 64 (ATCC 19246) 12.0 4 type 2 (ATCC 11828) 12.0 4 + 20.0 +++ 17.0 ++ Actinomyces naeslundii 8.0 64 Propionibacterium avidum 9;0 32 (ATCC 12104) 15.0 16 (ATCC 25577) 15.0 4 + 19.0 +++ 20.0 +++ Actinomyces israelii type 1 6.0 64 Propionibacterium 10.0 16 (ATCC 12103) 13.5 2 + granulosum (ATCC 25564) 13.0 8 20.0 +++ 19.0 +++ Actinomyces israelii type 2 5.0 128 Propionibacterium jensenii 10.0 32 (WVU 307) 12.0 8 (ATCC 4867) 16.0 4 + 20.0 2 ++ 18.0 ++ Actinomyces odontolyticus 9.0 32 (ATCC 17982) 15.0 4 + 20.0 1 ++ aExpressed as reciprocal of titer. 836 HOLMBERGjAND FORSUM APPL. MICROBIOL. ultrafiltration (Diaflo model 50, Amicon Corp.) to a acetone for 10 min. For estimation of specific staining final concentration of 10 mg/ml and dialyzed against reactions of type organisms and for identification of PBS (pH 7.2). The protein content and F/P ratio isolates, smears were prepared from 24- to 48-h pure (gg/mg) were estimated as described by Wells et al. cultures. Nonspecific staining of leukocytes were eval- (52). All conjugates were stored at 0 to 5 C or frozen in uated in smears prepared from freshly drawn human several small portions for storage. venous blood. After coagulation for 10 min at room Preparation of FITC-labeled F(ab'), fragments. temperature and then for 20 min at 37 C, the clot was To obtain F(ab')2 fragment of IgG from antisera to removed by washing with 0.15 M NaCl. Rothia dentocariosa, pepsin digestion was performed Staining of smears and recording procedures. (17, 36) using 1 mg of enzyme per 50 mg of IgG in 0.1 One drop of conjugate was placed on a smear and M sodium acetate (pH 3.0) for 7 h. The F(ab'), incubated for 20 min at 37 C. The smear was washed fragments were conjugated with FITC as described with PBS for 15 min and mounted under a cover glass above and fractionated on Sephadex G-25 equili- with phosphate-buffered glycerin (pH 7.2). brated with 0.0175 M Na,PHO4 (pH 4.7) into frac- The preparations were read under a Zeiss fluores- tions with different F/P ratios (17). The first peak to cence microscope equipped with an Osram HBO 200 emerge from the column was collected, and its pH was mercury lamp, oil immersion dark-field condensor, adjusted to 7.2. Conjugated F(ab')2 fragments with and a 4-mm BG 12 filter as primary and a Zeiss 50 high F/P ratios, which showed firmer adsorption to filter as secondary filter. Tissue specimens were read the gel and which therefore eluted in a second peak, with a Zeiss 44 filter as secondary filter. All observa- were discharged. The F/P ratio was calculated accord- tions were made under x500 magnification. The ing to Forsum (17). following criteria were used for recording the specific Dilution and adsorption of conjugates. The frac- fluorescent staining of microorganisms: 3+, intensely tionated conjugates were diluted in PBS (pH 7.2), and fluorescent margins, well marked edges; 2+, faintly the highest dilution giving 3+ staining of the homolo- fluorescent margins, edges usually diffuse; 1+, barely gous organism was recorded as the specific titer of distinguishable fluorescent margins with diffuse each fraction. edges. Nonspecific staining of gingival tissue cells and Adsorption of antisera was performed with equal leukocytes was recorded using the following criteria: volumes of conjugated antiserum and packed living 3+, brilliant nonspecific staining; 2+, clear-cut non- cells from a 48-h culture of the heterologous, cross- specific staining; 1+, doubtful nonspecific staining. reacting organism at 37 C for 2 h. Cultural and isolation procedures. The clinical Sampling ofclinical materials. Five patients with specimens were cultured aerobically and anaerobi- severe periodontal disease with pockets (Periodontal cally on conventional and prereduced media. The Index [PI], according to Russel [411, 4.7 to 5.8) were FTA buffer and the prereduced salt solution suspen- selected for this study. sions were diluted serially and 10-fold in cold (4 C) The subjects were instructed to refrain from tooth PBS, pH 7.2, and prereduced salt solution, respec- brushing for 24 h before collection of dental plaque tively. Samples of the PBS dilutions 10-2, 10- 4, and and calculus materials. Supragingival specimens were 10-1 were plated in duplicate on freshly prepared collected from the buccal surfaces of the upper molars NAYS agar (14) and on NAYS agar plates supple- with a sterile McCall curette (Sandvikens Coromant mented with 25 IE/ml of polymyxin-P-sulfate (Novo AB, Sweden) during preoperative periodontal treat- Industri A/S, Copenhagen, Denmark). One plate of ment. Subgingival dental plaque, calculus, and gingi- each medium was incubated aerobically with 10% val tissue specimens were taken during surgical treat- CO2 and one in an anaerobic jar using the Gas-Pak ment. Specimens were immediately transferred both anaerobic system (BBL). Samples from the dilutions to tubes containing 1.5 ml of sterile FTA-hemaggluti- 10-1, 10-8, and 10-6 of the prereduced salt solutions nation buffer (BBL) supplemented with 0.05% cys- were cultured on prereduced (PRAS) PY agar me- teine-hydrochloride, and to sterile, CO,-filled, stop- dium (27) and in prereduced PY agar roll tubes (Scott pered tubes, containing 1.5 ml of prereduced salt Laboratories Inc.) using the VPI Anaerobic Culture solutions according to the Anaerobe Laboratory Man- System (Bellco Inc.). All plates and tubes were ual (27). All samples were ground in an all-glass incubated at 37 C for 3 to 5 days before examination tissue grinder (Grave AB, Stockholm, Sweden) and and were then examined at a magnification of approx- dispersed in a mixer (Vortex-Genie, Scientific Indus- imately x10 using a stereoscopic zoom microscope tries Inc.) for 30 s. Samples of excised gingival tissue (Zeiss AG, Germany) with transmitted or reflected and granulation tissue were directly refrigerated, and lighting, or both. On the basis of colonial morphology, within 3 h after operation they were frozen to - 76 C. a Gram-stained smear was prepared from each colo- Preparation of smears. Smears from each clinical nial type. Two colonies of each type identified as specimen were Gram stained, and the morphotypes gram-positive diphtheroidal or filamentous orga- present were recorded. nisms, or both, were selected. One colony was trans- Smears for immunofluorescent staining were made ferred to an aerobic NAYS-agar plate and one colony from the suspensions in FTA-hemagglutination buffer was transferred to an anaerobic, prereduced PY- and prereduced salt solution by streaking 0.01 ml of agar plate. Organisms growing aerobically were sub- the suspension on a slide. For immunofluorescent jected to the diagnostic keys for this group of orga- staining of gingival tissue and granulation tissue, nisms proposed by Holmberg and Hallander (29). frozen samples were cut in a cryostat chamber (Lab.- Anaerobic isolates were identified by tests and media Tek., Sweden) maintained at about -20 C. Sections devised by the Anaerobe Laboratory Manual (27) as 4 gm thick were transferred to slides and fixed in diagnostic keys for facultative to anaerobic Ac- VOL. 25, 1973 SPECIES IDENTIFICATION BY IMMUNOFLUORESCENCE 837 tinomyces spp., A. propionica, and Propionibacte- cross-reactions in immunofluorescence with rium spp. Isolates of A. odontolyticus were identified various, presumably related, type species. by the diagnostic tests for this species devised by A limitation of type-specific immunofluores- Georg (19) and Gerencser and Slack (22). cence to a 3+ reaction as endpoint yielded type specificity for the individual species except for RESULTS anti-Actinomyces viscosus type 2 sera, which cross-reacted with A. israelii type 1 antigens, Characterization of conjugates. The FITC- and for some antisera to serotypes that cross- labeled and fractionated conjugates were char- reacted with the other serotypes within the acterized with respect to F/P ratios, specific same species. Dilution of antisera abolished the staining of homologous strains, and extraneous cross-reactions with 3+ fluorescent intensity at fluorescence. low titers and provided serotype-specific conju- Specific staining reaction. The staining ti- gates for all species except for anti-A. viscosus ters for homologous strains obtained with three type 2 and Propionibacterium acnes type fractions of each conjugate (10 mg/ml) with 1-conjugates which, at high titers near the various F/P ratios are presented in Table 1. The specific staining titers for these conjugates, still highest dilution giving 3+ fluorescent staining stained A. viscosus type 1 and P. acnes type 2 was recorded as the specific titer. The highest antigens with a 3+ reaction. The results are specific titers were consistently obtained with given in Table 2. the fractionated conjugates having F/P ratios Since dilution in providing serotype-specific less than 10. These fractions stained 3+ at conjugates for A. viscosus type 2 and P. acnes dilutions of 1: 32 to 1: 128. Fractions with F/P type 1 was inefficient because of cross-reactions ratios around 20 usually failed to give specific at high titers, adsorption of these conjugates staining. with cross-reacting antigens was performed. Nonspecific staining reactDn. The non- This procedure rendered the reagents serotype specific staining of gingival tissue smears was specific, although it resulted in weakened con- negligible with fractions with low F/P ratios jugates. (Table 1). As expected, nonspecific staining An extension of the type-specific fluorescence increased in direct proportion to the F/P ratios. to 1+ reactions gave a scattering of cross-reac- A marked nonspecific staining which interfered tions between the conjugate prepared and sev- with titration and impeded accurate recording eral species, indicating a broad spectrum of of the specific staining appeared when fractions antigenic similarities (Table 3). Dilution of with F/P ratios around 20 were used. In leuko- conjugates eliminated most cross-reactions at cytes an intense nonspecific cytoplasmatic low titers and rendered the conjugates species fluorescence was observed with all fractions of specific at dilutions of 1: 32 to 1: 64; homologous the conjugates with F/P ratios above 10. Low antigens were still stained with a 2+ to 3+ F/P ratios reduced this fluorescence to 2+, but reaction. in no instance was it eliminated. To confirm the cross-reactions found between Cross-reactions with Staphylococcus species, another set of type-species received as aureus. Since S. aureus with high protein A the following: R. dentocariosa ( sali- content (1,500-3,000 ng/101 bacteria) reacts vae), NCTC 10207; Leptotrichia dentium (Bac- with all FITC-labeled antisera at the same terionema matruchotii), ATCC 14919; A. dilutions as do the homologous strains, giving viscosus type 1, ATCC 15988; Actinomyces an unwanted cell margin fluorescence due to a naeslundii, NCTC 10301; A. israelii type 1, reaction between protein A and the Fc-part of ATCC 10048; P. acnes type 1, CDC 554; P. IgG (16). FITC-labeled F(ab')2 fragments were acnes, type 2, CDC 605; and P. avidum, CDC prepared from sera against R. dentocariosa, 9064, were tested in immunofluorescence with known to form coccoidal cells. When smears the conjugates prepared. No other relationships were prepared from suspensions of pure 18-h were detected with these strains as antigens, cultures of staphylococci and R. dentocariosa and the cross-reactions found were of the same and each was stained with anti-R. dentocariosa degree as in the primary tests, IgG and anti-R. dentocariosa F(ab')2 fragments The sera were also tested with a number of of IgG, nonspecific staining of the staphylococci oral isolates biochemically identified as R. den- was obtained with IgG but not with the F(ab')2 fragments. Both IgG and F(ab')2 fragment prep- tocariosa, B. matruschotii, A. viscosus, A. naes- arations stained the type species of R. lundii, A. israelii type 1 and 2, Actinomyces dentocariosa at their specific staining titers. odontolyticus, Arachnia propionica, P. acnes Cross-reactions. Fractions of each conjugate type 1 and 2. The pattern of cross-reactivity with optimal staining properties were tested for obtained with the type species was reproduced, 838 HOLMBERG AND FORSUM APPL. MICROBIOL. TABLE 2. Highest dilutions of FITC-labeled antisera against Actinomyces, Arachnia, Bacterionema, Rothia and Propionibacterium species giving 3+ fluorescence of homologous and heterologous antigens Antigen

Fractionated - C FITC-conjugated c : -. . c s > antisera a _ 3:a. (10mg/mi: 64__

A.israelioty 10- 4 6 2 .:. < E- F' - F A.ViSCOSUS~typ 14~ ~ ~~~~ot-a64 z 0 R. dentocariosaa 64 B. matruchotii 64 A. viscosustypel1 64 A. viscosus type 2 32 128 2 A. naeslundii 64 A. israelii type 1 64 2 A. israelii type 2 128 A. odontolyticus 32 A. propionica type 1 32 1 A. propionica type 2 64 P. acnes typel1 64 32 P. acnes type 2 1 64 P. avidum 32 P. granulosum 16 P.jensenii 32 a FITC-labeled F(ab')2 fragments of IgG. TABLE 3. End point titers ofFITC-labeled antisera against Actinomyces, Arachnia, Bacterionema, Rothia and Propionibacterium species giving 1 + fluorescence of homologous and heterologous antigens Antigen

Fractionated S1' CI4 FITC-conjugated a a- antisera a .a (10mg/mi: .tj &a V F/Pratio <10) m

Identified by | Sources immunofluores- .0 2 cence/culture o

Supragingival Plaque +/+ 4 1 3 2 0 0 1 0 +1- 0 2 0 0 1 2 0 1 -1+ ~~0 0 2 1 0 0 1 0 _/_ 1 2 0 2 4 3 3 4 Calculus +/+ 2 3 5 4 3 2 3 1 +/- 1 0 0 0 0 1 1 1 -/+ 0 1 0 0 0 0 0 0 -I- 2 1 0 1 2 2 1 3 Subgingival Plaque +/+ 0 2 0 4 5 0 2 2 +1- ~~1 0 1 0 0 0 0 0 -1+ 2 1 1 1 1 0 1 0 -I- 2 2 3 0 0 5 2 3 Granulation +/+ 0 1 1 5 5 0 2 2 tissue ±/- 0 0 0 0 0 2 0 1 -/+ 0 0 1 0 0 0 1 0 -I- 5 4 3 0 0 3 2 2 Total +/+ 6 7 9 15 13 2 8 5 65/160 (40.6%) +/- 2 2 1 0 1 5 1 3 15/160 (9.4%) -/+ 2 2 4 2 0 0 3 0 13/160 (8.1%) -/- 10 9 6 3 6 13 8 12 67/160 (41.9%) pionibacterium avidum, P. granulosum, and P. tain actinomycetes (Actinomyces, Bacte- jensenii were encountered. ionema, Rothia, Arachnia) and Propionibacte- Strains of R. dentocariosa, B. matruchotii, A. rium species. Earlier studies on detection of viscosus, A. naeslundii and A. odontolyticus these types of organisms in direct smears have appeared in specimens from supragingival sites been seriously disturbed by nonspecific staining whereas A. naeslundii, A. israelii, Arachnia pro- (4, 34, 40). A detailed evaluation of the degree pionica, and P. acnes were found mostly in of standardization in previous studies is, how- subgingival specimens. ever, impossible. High specific staining titers with brilliant staining of the cell margins of ho- DISCUSSION mologous strains and effective reduction of non- The usefulness of immunofluorescence as a specific staining was obtained if fractionated rapid and simple technique for detection of conjugates with F/P ratios below 10 were used. bacteria in pure cultures as well as on direct Nonspecific staining increased in direct pro- smears is well documented (10, 24). Increasing portion to the F/P ratios of the fractions, which attention has been paid to standaridzation in is in accordance with observations from other immunofluorescence with respect to criteria for applications, where an F/P ratio of approxi- preparation of conjugates and to a quality mately 1 to 14 has been found optimal (e.g., control of the conjugates to define their specific 39). Another problem of unwanted fluorescence and nonspecific staining properties (3, 26). arises due to the reaction between the Fc part This study presents standards for optimal of IgG and protein A of S. aureus (16, 17). This FITC-conjugated antisera to whole cells of cer- may cause diagnostic difficulties if coccoidal VOL. 25, 1973 SPECIES IDENTIFICATION BY IMMUNOFLUORESCENCE 841 variants of R. dentocariosa (7) and spherical genus Actinomyces, A. odontolyticus occurs as a cells of Arachnia propionica (9, 22, 38) appear rather distinct serological entity. In the present mixed with S. aureus. Using F(ab')2 fragments study no isolate was recovered which could be of the IgG preparations, this nonspecific im- referred to as serotype 2, cross-reacting with V. munological activity is completely abolished, viscosus type 1 sera as described by Slack, but the specific immunological activity is re- Landfried, and Gerencser (46). The other tained. Cross-reactions due to antigenic similar- Actinomyces species appear to be serologically ities between these organisms could be foreseen complex, with overlapping of antigens. There from results reported in earlier papers (30, 37, was a scattering of low-level cross-reactions 44). between A. viscosus, A. naeslundii, and A. The active antigenic components of Ac- israelii strains. The degree of relationship, how- tinomyces, Arachnia, Rothia, and Propionibac- ever, differed between examined strains. Dilut- terium species have been found to be polysac- ed, species-specific antisera gave serological charides in the cell walls or cytoplasm, which groups of the Actinomyces which corresponded seem to possess rather narrow serological type to the serological classification designed by specificity (30, 33). The data obtained should Slack and Gerencser (44). A subdivision of A. make it possible to determine the extent to viscosus in animal strains, referred to as sero- which cross-reactivity may be a problem in type 1 and human strains referred to as serotype defined immunofluorescence studies of actino- 2, with a one-way cross-reaction between sero- mycetes, Arachnia, and propionibacteria. In type 1 antibodies and serotype 2 antigens sug- diagnostic work, a 3+ immunofluorescent re- gested by Gerenscer and Slack (23) and Bellack action is essential for reliable species identifica- and Jordan (2) was confirmed in this study. tion. Appropriate dilutions of sera on the basis Two types of A. israelii have been described (6, of their protein content per milliliter, which 28). These two types agree with cell wall antigen effectively eliminate cross-reactions with a 3+ constituents (13). However, the antigenic ho- fluorescent intensity, can be determined. This mogeneity among the serotype 1 strains has is a major advantage since adsorption, which been found to vary. The low-titer cross-reactiv- often results in weakened specific staining re- ity we observed between the serotypes parallels actions, can be avoided. Defined immunoflu- the findings of Slack, Landfried, and Gerencser orescence of lower intensity than 3+ is of (45) but is in contrast to the one-way cross-reac- doubtful diagnostic value in differentiating be- tion between the serotypes, with serotype 1 tween morphologically and serologically similar conjugate staining serotype 2 antigens as re- organisms. Dilutions could also be used for ported by Blank & Georg (4). preparation of serotype-specific antisera. Ad- Questions have been raised about the serolog- sorption was necessary only for preparing sero- ical relationships of Actinomyces to type-specific sera against type 2 of A. viscosus Propionibacterium. A relationship in immuno- and type 1 of P. acnes. In practice, serological fluorescence between Actinomyces species and identification of species in mixed specimens will Corynebacterium (Propionibacterium) acnes be facilitated by the use of pools of conjugates was reported by Slack et al. (47), which induced containing sera against species serotypes. these authors to propose that P. acnes be Various techniques have been used for sero- included in the genus Actinomyces. As in later logical classification of the actinomycetes and reports of Slack and Gerencser (43, 44), P. acnes propionibacteria. Immunofluorescence seems to strains formed a rather homogenous group in be the best approach (44), since agglutination, immunofluorescence with the exception that P. immunodiffusion, and complement-fixation acnes sera, in full concentration, cross-reacted tests have given considerable cross-reactions with A. naeslundii and A. israelii strains. There (21, 48). were also low-level cross-reactions between the R. dentocariosa has a distinct reaction in P. acnes strains and Arachnia propionica, P. immunofluorescence, probably due to a species- avidum, and P. granulosum. This may be specific, fructose-containing cell wall antigen explained by the presence of some combination which distinguishes it from other gram-positive of galactose, glucose, and mannose in the cell filamentous organisms (25, 48). walls of all actinomycetes, Arachnia and pro- The taxonomic position of B. matruchotii is pionibacteria strains (5, 30, 37). uncertain (29, 37), but it seems to constitute a Two groups of P. acnes strains could be quite distinct serological group in immuno- defined by immunofluorescence. The reactions fluorescence, thereby confirming the results of obtained support the group designation pro- earlier serological analyses by agglutination and posed on the basis of biochemical reactions (8), immunodiffusion tests (32, 48). Within the susceptibility to phage, and gel-diffusion anal- 842 HOLMBERG AND FORSUM APPL. MICROBIOL. yses of soluble antigens in whole broth cultures the time involved in anaerobic culturing and (53). The existence of two serotypes, 1 and 2, the attendant risk of contamination (1, 30) has recently also been confirmed by cell wall emphasize the advantages of direct immuno- analyses and cell wall agglutination tests (30). fluorescence. Additional information emerging Arachnia propionica was recognized by im- from the present study pertains to the presence munofluorescence methods to be a homogene- of Rothia, Bacterionema, Actinomyces spp., ous group quite distinct from other genera of the Arachnia, and Propionibacterium acnes in the Actinomycetaceae. It is also quite distinct from resident microflora of supra- and subgingival the Propionibacterium species studied. Pine plaques and calculus and in granulomatous (37) has recently suggested that A. propionica tissues in severe periodontal disease. In the light should be transferred to the family of the possible etiological role of these orga- Propionibacteriaceae on the basis of cell wall nisms in periodontal disease (49) the defined composition. However, on the basis of a study of immunofluorescence techniques described ap- deoxyribonucleic acid (DNA) compositions of pears to be an excellent diagnostic tool for use in Arachnia to Propionibacterium species, John- further ecological and epidemiological studies. son and Cummins (30) state that A. propionica shows no DNA homology with the two major LITERATURE CITED groups of anaerobic coryneforms. For the pres- 1. Beerens, H. 1953. Etude comparative de six souches de bacteries anaerobies non sporul6es. Ann. Inst. Pasteur ent time the phylogenetic position of Arachnia 84:1026-1032. is difficult to determine; however, it will still be 2. Bellack, S., and H. V. Jordan. 1972. Serological identifi- included in the Actinomycetaceae in the new cation of rodent strains of Actinomyces viscosus and edition of Bergey's Manual. their relationship to Actinomyces of human origin. Archs oral Biol: 17:175-182. Serological classification of the ac- 3. Beutner, E. H. 1971. Defined immunofluorescent stain- tinomycetes and propionibacteria, by defined ing: past progress, present status, and future prospects immunofluorescence based on whole cell anti- for defined conjugates, Defined immunofluorescent gens, allows the establishment of distinct anti- staining, Annu. N.Y. Acad. Sci. 177:506-523. 4. Blank, C. H., and L. K. Georg. 1968. The use of genic determinants for each species and sup- fluorescent antibody methods for the detection and ports classifications based on morphological identification of Actinomyces species in clinical mate- and biochemical criteria as well as cell wall rial. J. Lab. Clin. Med. 71:283-293. analyses (29, 30, 37). 5. Boone, C. J., and L. Pine. 1968. Rapid method for characterization of Actinomycetes by cell wall compo- It is evident that defined immunofluores- sition. Appl. Microbiol. 16:279-284. cence is a method for detection of species of 6. Brock, D. W., and L. K. Georg. 1969. Determination and Actinomyces, Arachnia, Rothia, Bacterionema, analysis of Actinomyces israelii serotypes by fluores- and Propionibacterium in direct smears as spe- cent-antibody procedures. J. Bacteriol. 97:581-588. as The 7. Brown, J. M., L. K. Georg, and L. C. Waters. 1969. cific and sensitive cultural techniques. Laboratory identification of Rothia dentocariosa and two methods agreed to 88%, an agreement its occurrence in human clinical materials. Appl. comparable to that observed for other bacteria Microbiol. 17:150-156. (10, 18). Cross-reactions or inadequate cultural 8. Brzin, B. 1964. Studies on the Corynebacterium acnes. Acta Pathol. Microbiol. Scand. 60:599-608. and isolation techniques may not have con- 9. Buchanan, B. B., and L. Pine. 1962. Characterization of a tributed to the observed disagreement between propionic acid producing actinomycete, Actinomyces the methods; such factors seriously disturbed propionicus. Sp. Nov. J. Microbiol. 28:305-323. an earlier attempt to evaluate immunofluores- 10. Cherry, W. B., and M. D. Moody. 1965. Fluorescent-anti- body techniques in diagnostic bacteriology. Bacteriol. cence for detection of Actinomyces species and Rev. 29:222-250. some related bacteria in direct smears (46). The 11. Coleman, R. M., and L. K. Georg. 1969. Comparative cultural techniques employed appear to be pathogenicity of Actinomyces naeslundii and Ac- optimal for isolation of these types of organisms tinom-1ces israelii. Appl. Microbiol. 18:427-432. 12. Coleman, R. M., L. K. Georg, and A. R. Rozzell. 1969. (15, 19, 27). Failure to isolate an organism Actinomyces naeslundii as an agent of human ac- identified by immunofluorescence probably re- tinomycosis. Appl. Microbiol. 18:420-426. flects the effect of the dilution procedures and 13. Cummins, C. S. 1970. Actinomyces israelii, type 2, p. confluent growth of admixed bacteria. The 29-34. In H. Prauser (ed.), The . VEB Gustav Fischer Verlag, Jena. overwhelming majority of the negative im- 14. Davis, G. H. G., and J. H. Freer. 1960. Studies upon an munofluorescence but positive cultural results oral aerobic actinomycete. J. Gen. Microbiol. can be assigned to the dilution factor inherent 23:163-178. in preparing smears. 15. Finegold, S. M. 1970. Isolation of anaerobic bacteria, p. use of immuno- 265-279. In J. Z. Blair, E. H. Lennett, and J. P. Truant Several factors favor the (ed.), Manual of clinical microbiology. American So- fluorescence over that of culturing for identifi- ciety for Microbiology, Bethesda, Md., U.S.A. cation of these organisms. The difficulties and 16. Forsgren, A., and U. Forsum. 1970. Role of protein A in VOL. 25, 1973 SPECIES IDENTIFICATION BY IMMUNOFLUORESCENCE 843

nonspecific immunofluorescence of Staphylococcus 34. Lambert, F. W., Jr., J. M. Brown, and L. K. Georg. 1967. aureus. Infect. Immunity 2:307-391. Identification of Actinomyces israelii and Actinomyces 17. Forsum, U. 1972. Characterization of FITC-labeled naeslundii by fluorescent-antibody and agar-gel diffu- F(ab'),-fragments of IgG and a rapid technique for sion techniques. J. Bacteriol. 94:1287-1295. separation of optimally labeled fragments. J. Immunol. 35. Nairn, R. C. 1964. Fluorescent protein tracing. E&S Meth. 2:183-195. Livingstone Ltd., Edinburgh, England. 18. Forsum, U., and Holmberg. 1973. Identification of Strep- 36. Nisonoff, A. 1964. X. Enzymatic digestion of rabbit tococcus sanguis by defined immunofluorescence. Sub- gamma globulin and antibody and chromotography of mitted for publication. digestion products. Methods Med. Res. 10:134-141. 19. Georg, L. K. 1970. Diagnostic procedures for the isolation 37. Pine, L. 1970. Classification and phylogenetic relation- and identification of the etiologic agents of actinomyco- ship of microaerophilic actinomycetes. Intern. J. Syst. sis. In Proc. Intern. Symp. on Mycoses, Scientific Bacteriol. 20:445-474. Publication no. 205. Pan American Health Organiza- 38. Pine, L., and L. K. Georg. 1968. Reclassification of tion, Washington, D.C. Actinomyces propionicus. Intern. J. Syst. Bacteriol. 20. Georg, L. K., and R. M. Coleman. 1970. Comparative 19:267-272. pathogenicity of various Actinomyces species, p. 35-45. 39. Pittman, B., G. A. Herbert, W. B. Cherry, and G. C. In H. Prauser (ed.), The actinomycetales. VEB Gustav Taylor. 1967. The quantitation of nonspecific staining Fischer Verlag, Jena. as a guide for improvement of fluorescent antibody 21. Georg, L. K., R. M. Coleman, and J. M. Brown. 1968. conjugates. J. Immunol. 98:1196-1203. Evaluation of an agar gel precipitin test for the sero- 40. Ritz, H. L. 1969. Fluorescent antibody staining of Neis- diagnosis of actinomycosis. J. Immunol. 100:1288- seria, Streptococcus and Veillonella in frozen sections 1292. of human dental plaque. Arch. Oral Biol. 14:1073-1083. 22. Gerencser, M. A., and J. M. Slack. 1967. Isolation and 41. Russel, A. L. 1956. System of classification and scoring characterization of Actinomyces propionicus. J. Bac- for periodontal disease. J. Dent. Res. 35:350-359. teriol. 94:109-115. 42. Scheidegger, I. 1955. Une microm6thode de l'immunoe- 23. Gerencser, M. A., and J. M. Slack. 1969. Identification of lectrophorese. Int. Arch. Allergy Appl. Immunol. human strains of Actinomyces viscosus. Appl. Micro- 7:103-110. biol. 18:80-87. 43. Slack, J. M., and M. A. Gerencser. 1966. Revision of 24. Goldman, M. 1968. Fluorescent antibody methods. Aca- serological grouping of Actinomyces. J. Bacteriol. demic Press Inc., U.S.A. 91:2107. 25. Hammond, B. F. 1970. Isolation and serological charac- 44. Slack, J. M., and M. A. Gerencser. 1970. Two new terization of a cell wall antigen of Rothia dentocariosa. serological groups of Actinomyces. J. Bacteriol. J. Bacteriol. 103:634-640. 103:266-267. 26. Holborrow, E. J. 1970. Standardization in immuno- 45. Slack, J. M., S. Landfried, and M. A. Gerencser. 1969. fluorescence. Blackwell Scientific Publ., Oxford and Morphological, biochemical, and serological studies on Edinburgh. 64 strains of Actinomyces israelii. J. Bacteriol. 27. Holdeman, L. V., and W. E. C. Moore. 1972. Anaerobe 97:873-884. Laboratory Manual. The Virginia Polytechnic Institute 46. Slack, J. M., S. Landfried, and M. A. Gerencser. 1971. and State University Anaerobe Laboratory, Blacks- Identification of Actinomyces and related bacteria in burg, Virginia, U.S.A. dental calculus by the fluorescent antibody technique. 28. Holm, P. 1948. Some investigations into the J. Dent. Res. 50:78-82. sensitivity of human-pathogenic actinomycetes. Acta 47. Slack, J. M., A. Winger, and D. W. Moore. 1961. Pathol. Scand. 25:376-404. Serological grouping of Actinomyces by means of 29. Holmberg, K., and H. 0. Hallander. 1973. Numerical fluorescent antibodies. J. Bacteriol. 82:54-65. tazonomy and laboratory identification of Bacteri- 48. Snyder, M. L., M. S. Slawson, W. Bullock, and R. B. onema matruchotii, Rothia dentocariosa, Actinomyces Parker. 1967. Studies on oral filamentous bacteria. II. viscosus, A. naeslundii and some related bacteria. J. Serological relationships within the genera Ac- Gen. Microbiol. 76:43-63. tinomyces, Nocardia, Bacterionema and Leptotrichia. 30. Johnson, J. L., and C. S. Cummins. 1972. Cell wall J. Infect. Dis. 117:341-345. composition and deoxyribonucleic acid similarities 49. Socransky, S. S. 1970. Relationship of bacteria to the among the anaerobic coryneforms, classical pro- etiology of periodontal disease. J. Dent. Res. pionibacteria, and strains of Arachnia propionica. J. 49:203-222. Bacteriol. 109:1047-1066. 50. Socransky, S. S., C. Hubersak, and D. Propas. 1970. 31. Jordan, H. V., P. H. Keyes, and S. Bellack. 1972. Induction of periodontal destruction in gnotobiotic rats Periodontal lesions in hamsters and gnotobiotic rats by human oral strain ofActinomyces naeslundii. Archs infected with actinomyces of human origin. J. Peri- Oral Biol. 15:993-995. odont. Res. 7:21-28. 51. The, T. H., and T. E. W. Feltkamp. 1970. Conjugation of 32. Kroeger, A. V., and L. R. Sibal. 1961. Biochemical and fluorescein isothiocyanate antibodies. Immunol. serological reactions of an oral filamentous organism. J. 18:865-874. Bacteriol. 81:581-585. 52. Wells, P. F., C. E. Miller, and M. K. Nadel. 1966. Rapid 33. Kwapinski, J. B. G. 1969. Analytical serology of Ac- fluorescein and Protein assay method for fluorescent- tinomycetales. p. 86. In J. B. G. Kwapinski (ed.), anbitody conjugates. Appl. Microbiol. 14:271-275. Analytical serology of microorganisms, John Wiley & 53. Voss, J. G. 1970. Differentiation of two groups of Coryne- Sons Inc., U.S.A. bacterium acnes. J. Bacteriol. 101:392-397.