JOUflAL OF CLINICAL MICROBIOLOGY, Sept. 1978, p. 329-3380095-1137/78/0008-0329$02.00/0Copyright © 1978 American Society for Microbiology

Vol. 8, No. 3

Printed in U.S.A.

Detection of Legionnaires Disease Bacteria by DirectImmunofluorescent Staining

WILLIAM B. CHERRY,* BERTIE PITTMAN, PATRICIA P. HARRIS, G. ANN HEBERT, BERENICEM. THOMASON, LEROY THACKER, AND ROBERT E. WEAVER

Center for Disease Control, Atlanta, Georgia 30333

Received for publication 31 March 1978

Antisera and fluorescein isothiocyanate conjugates prepared for five strains ofthe Legionnaires bacteria were tested in both homologous and heterologousstaining reactions with 10 isolates of the organism from patients in seven geo-graphic areas. The strains were related but not identical as judged by the resultsof direct immunofluorescence staining. The conjugates were successfully used todetect Legionnaires disease bacteria in Formalin-fixed lung scrapings, in histolog-ical sections, and in fresh lung tissue obtained at biopsy or autopsy. In addition,the labeled antibodies are valuable for staining suspected cultures of the bacte-rium and for searching for the source of these organisms in soil, water, and otherenvironmental niches. The reagents are highly specific for detecting the Legion-naires organism in clinical specimens.

The clinical, pathological, and epidemiologicalfeatures of Legionnaires disease were detailed inprevious publications (5, 7, 12). McDade et al.(12) first demonstrated the pathogen in the yolksacs of embryonated eggs that had been inocu-lated with macerated tissue from guinea pigsinjected with infected human lung tissue ob-tained at autopsy. They used indirect immuno-fluorescence (IF) to obtain convincing evidencethat these organisms were the cause of the dis-ease. A high titer (1:128) against the Legion-naires bacteria in a single serum specimen or afourfold rise (to 1:64 or more) in the titer ofconvalescent-phase serum relative to acute-phase serum indicated that infection had oc-curred (12). One of us (R.E.W.) isolated thebacterium on nonliving media. Yolk sac isolateswere cultured on Mueller-Hinton agar supple-mented with 1% hemoglobin and 1% (vol/vol)IsoVitaleX (BBL) enrichment. Isolation of theorganism from clinical material by passagethrough guinea pigs and embryonated eggs is aslow and tedious process, although a very sen-sitive one (12). Direct isolation on the laboratorymedia that are presently available is uncertainbecause a large inoculum is required, and signif-icant growth does not usually occur for severaldays. The indirect IF test is valuable for seroe-pidemiological surveys and for retrospective di-agnosis of the disease, but does not fuiflil thecritical need for a rapid and specific diagnostictest that can guide the physician in managementof the infection. This unmet need was the stim-ulus for developing the direct IF test describedin this paper.

MATERIALS AND METHODS

Cultures. The following strains of the Legionnairesbacteria were used in this study.

Philadelphia 1, 2, 3, and 4, isolated by McDade andShepard from the lungs of four persons with fatalLegionnaires disease or Broad Street pneumonia con-tracted in Philadelphia, Pa., during July and August1976 (12). The organisms were isolated by passage oflung tissue through guinea pigs and into the yolk sacof embryonated eggs.

Pontiac, isolated by McDade from the frozen lungtissue of a sentinel guinea pig that had been exposedto the interior environment of the Oakland County,Mich., Department of Health during an outbreak of amild respiratory disease among the occupants of thatbuilding in 1968 (1). The tissue had been maintainedat -70°C since that time. The five strains listed abovewere later grown in pure culture on enriched Mueller-Hinton medium by one of us (R.E.W.).

Knoxville, isolated by McDade on enriched Muel-ler-Hinton medium after guinea pig and egg passageof tissue from the lung of a patient who died of thedisease in Knoxville, Tenn., in February 1977.

Albuquerque, isolated by McDade on enrichedMueller-Hinton agar following guinea pig and eggpassage of lung tissue from a patient who died ofpneumonia in New Mexico in November 1977.

Flint 1, isolated directly from pleural fluid inocu-lated onto enriched chocolate agar. It was submittedfor identification by Morris Dumoff.

Bellingham, isolated on enriched chocolate agar byBeth Hayhow at St. Joseph's Hospital in Bellingham,Wash. It was submitted for identification by M.McDowell of the Washington State Laboratory. Thesource of the culture was pleural fluid from a patientwith bronchopneumonia.

Burlington, isolated directly on enriched Mueller-Hinton agar by one of us (W.B.C.) from the lung of a

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330 CHERRY ET AL.

patient who died of pneumonia during the epidemic inVermont in the summer of 1977 (4).

Ail cultures (374) of non-Legionnaires bacteria wereobtained from stocks or were freshly isolated referencecultures.

Note. Enriched Mueller-Hinton agar was Mueller-Hinton agar (BBL or Difco) to which was added 1%dehydrated hemoglobin and either 1 or 2% IsoVitaleX(BBL), or in which 0.05% cysteine hydrochloride was

substituted for the IsoVitaleX. Enriched chocolateagar was one of the commercial GC base media en-

riched with heated blood and IsoVitaleX.Safety procedures. Ail work with living cultures

of Legionnaires disease bacteria or with clinical spec-

imens known or suspected of containing living bacteriawas performed in a biological safety cabinet underClass 2 conditions (2). These included geographic iso-lation and controlled access, negative air pressure,

protective equipment, and disinfection of the workarea and of materials leaving that area.

Antigen preparation. Immunogens were pre-

pared with the following cultures: Philadelphia 1 and2, Flint 1, Pontiac, and Knoxville. Mueller-Hinton agar

plates supplemented with 1% hemoglobin and 1%(vol/vol) IsoVitaleX (BioQuest Laboratories, Cock-eysville, Md.) were inoculated heavily using sterileswabs to scrape cells from young cultures of the abovestrains. The plates were incubated in a candle extinc-tion jar for 72 h at 35 to 37°C. The growth from threeto five plates was collected and suspended in approx-imately 3.0 ml of 0.85% NaCl solution containing 0.5%Formalin (37% formaldehyde). The suspensions were

refrigerated overnight and tested for viability by add-ing several drops to a fresh plate of the growth me-

dium. No growth was observed with any strain. How-ever, preliminary data from others suggest that 0.5%Formalin may not kill all Legionnaires bacteria (per-sonal communication, G. Gorman and J. McDade).Smears from the cell suspensions were tested for pu-rity with Gram staining. The cells were centrifuged,resuspended in the same menstruum, and concen-

trated to give a turbidity reading of 18 IU, relative tothat obtained with an international substandard ofPyrex glass beads (11). At this concentration, thesuspension contained approximately 2 x 109 bacteriacells per ml, estimated from the cell concentration ofa no. 7 McFarland standard for bacteria of comparablesize.

Immunization. Young adult New Zealand whiterabbits were immunized according to two injectionschedules. The intravenous injection schedule isshown in Table 1. Note that the concentration of theantigen was doubled to 36 to 40 IU on day 24 ofinjection and used at that concentration for the re-

mainder of the schedule. Boosters were given on days54 and 56.The other immunization method consisted of an

injection into the "footpad" of the rabbit followed byan intramuscular booster. The Formalin-saline sus-

pension of whole cells at a density of 18 IU was mixedwith an equal amount of Freund complete adjuvant(Difco) and emulsified by shaking. One milliliter ofthis mixture was injected into each front foot. Theanimals were bled on day 35 to monitor antibodyresponse. On day 42, each animal was given 2.0 ml of

TABLE 1. Protocol for intravenous immunization ofrabbits with antigensa of Legionnaires disease

bacteriaDay of injection Vol of antigen at (concn)b

1 0.1 (18-20)3 0.2 (18-20)5 0.5 (18-20)7 0.75 (18-20)9 1.0 (18-20)

11 2.0 (18-20)13 3.0 (18-20)15 4.0 (18-20)17 5.0 (18-20)24, 26, 28, 35, 3.0 (36-40)

37, 39, 4154, 56 5.0 (36-40)

a Cell suspensions in 0.85% NaCI containing 0.5%Formalin. Main bleedings were on days 34, 48, and 63.

b Volume is expressed in milliliters, concentrationin international units (IU) of turbidity. Total volumeinjected per rabbit (of 36-40 IU concentration orequivalent volume of 18-20 IU concentration), 39.3 ml.

the above antigen intramuscularly in the hip. Serumwas harvested on days 49 and 56.

Preimmunization serum for labeling with fluores-cein isothiocyanate (FITC) was obtained from eachanimal and served as a control to assess the specificityof the staining reaction.

Preparation of conjugates. Immunization prog-ress was followed by preparing whole serum conju-gates as McKinney et al. prescribed (13). This is arapid preliminary method for determining relative IFtiters and consists of reacting a constant amount ofFITC with 1.0 ml of whole serum and separating theunreacted dye from the labeled protein on a SephadexG-25 or G-50 column. When the direct IF stainingtiters appeared to be high enough (1:16 to 1:64), serumwas obtained from the rabbits, fractionated by(NH4)2SO4, and labeled with FITC. If these trial con-jugates had satisfactory titers (3+ to 4+ at dilutions of1:40 or more), several successive bleedings were done,and the serum was pooled for preparation of the finalproduct.

Sera were fractionated by three successive precipi-tations in 35% saturated (NH4)2SO4 (final concentra-tion in the mixture) (9). The globulin fraction waslabeled with FITC certified by the Biological StainCommission to be of 99% purity (International Biolog-icals, Inc., Melbourne, Florida), based on its labelingefficiency for bovine serum albumin (6).The amount of FITC used for labeling was calcu-

lated to produce fluorescein-to-protein ratios of ap-proximately 25 to 30 ,g of FITC per mg of protein(13). The protein content of most conjugates wasadjusted to approximately 10 mg of protein per ml wasmeasured by the method of Gornall et al. (8).The fractionation and labeling procedures used

were those detailed by Hebert et al. (9).Testing of conjugates. All conjugates were tested

with their homologous antigens and with the heterol-ogous antigens of all available Legionnaires cultures.Antigens consisted of suspensions of cells in 0.85%

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DIAGNOSIS OF LEGIONNAIRES DISEASE BY IF 331

NaCI containing 1% Formalin. The cells were har-vested from cultures incubated on enriched Mueller-Hinton medium at 35°C for 72 h. All cell suspensionswere adjusted to a turbidity approximating that of aMcFarland no. 1 standard. Twofold dilutions of theimmune conjugates were made in 0.01 M phosphate(pH 7.1) containing 0.1% NaN3 as a preservative. Theinitial dilution was 1:20. Conjugates of preimmuneserum were tested at a single dilution only (1:20),although the protein level of two conjugates, Flint andKnoxville (Table 2), was less than 10 mg/ml. Ailreagents were stored in the refrigerator at 4°C. Thetiter of the conjugates was defined as the end pointdilution factor of the highest dilution giving at least3+ staining. The working dilution was defined as thedilution factor of one doubling dilution less than thatof the titer. Observations were made by incident illu-mination with a 50-W halogen lamp utilizing thePloem illuminating system on a Leitz Dialux micro-scope. The primary filters were 2-KP 490; the dichroicbeam-splitting mirror, TK 510, was coupled with abuilt-in K 515 suppression filter to form "filter systemH for wide-band, blue-light excitation"; the ocularfilter was K 510.

Ail conjugates were analyzed by physicochemicalcharacterization using the methods of Hebert et al.(9). These included protein and FITC measurementsfor determination of the fluorescein-to-protein ratioand cellulose acetate strip electrophoresis for quanti-tation of the immunoglobulin.

Application of conjugates to the detection ofLegionnaires bacteria. Detection in scrapings ofFormalin-fixed tissue. At least one area of the lungor other tissue was tested. In the lung, areas of denseconsolidation were selected. Each block of tissue wastransferred to a sterile petri dish. A sharp scalpel wasused to cut through these areas and expose new tissuefaces. While the tissue was held with forceps, a scalpelwas used to scrape off a fine puree of tissue particles.(The lung tissue of victims of Legionnaires disease isusually quite friable.) The tissue particles weresmeared with the scalpel blade onto two 1.5-cm-di-ameter circular areas on a microscope slide. Thesmears were air dried and heat fixed. One smear wasstained with the working dilution of the Legionnairesconjugate and another with preimmune conjugate ofapproximately the same fluorescein-to-protein ratioand protein content. Smears were stained for 20 minat room temperature in a moist chamber, quicklyrinsed in phosphate-buffered saline (0.01 M phosphatebuffer, pH 7.6, containing 0.85% NaCl), and immersedin a container of phosphate-buffered saline for 5 min.They were then rinsed with distilled water, air dried,and mounted with a cover slip and a drop of glycerolbuffered at pH 8.0 to 9.0 (10).The stained preparations were examined first with

the x10 objective of the fluorescence microscope andthen under oi immersion at x1,250.

Detection in fresh or fresh-frozen tissue (au-topsy or biopsy). If the tissue was to be cultured,this was done before the impression smears weremade. All fresh or fresh-frozen tissue was processed ina safety cabinet. Sterile instruments were used to holdand to cut a fresh face of tissue and to press andsqueeze the tissue first onto the surface of an agar

plate for culture and then against a clean slide, makingat least two smears. If the tissue was very moist, it wasblotted on sterile gauze before touching it to the slide.Smears were air dried and fixed for 10 min by coveringthem with 10% neutral Formalin and placing them ina moist chamber to retard evaporation of the For-malin. The smears were rinsed in distilled water, airdried, gently heat fixed, and stained as described abovefor tissue scrapings.

Homogenates of fresh lung tissue were prepared bythe method described by McDade et al. (12). Smearswere processed as described above for the tissue im-prints.

Detection in tissue sections. Sections from par-affin blocks cut as thin as possible (4 ,pm or less) werefixed for approximately 15 min at 58 to 60°C and freedfrom paraffin by two passages through xylol followedby two passages each through absolute ethanol, 95%ethanol, and water. They were stained as describedabove.

Detection in exudates from the lungs. Sputum,transtracheal aspirates, bronchial washings, pleuralfluids, or other specimens from the lower respiratorytract were examined for Legionnaires bacteria whensuitable material was available. Rather thick smearswere prepared and processed as outlined above.

RESULTS

Characteristics of the conjugates. Thecharacteristics of the conjugates for the Legion-naires bacteria are shown in Table 2. The resultson fmal bleedings were obtained from conjugatesprepared from a pool of two or more specimenstaken near the end of the immunization period.Results on trial bleedings were obtained fromconjugates of earlier single bleedings taken dur-ing the course of immunization.The immunoglobulin content in the immune

conjugates ranged from 84 to 93%; that in thepreimmune, from 65 to 72%. Fluorescein-to-pro-tein ratios ranged from 19 to 33 ,ug/mg of protein.The protein content of immune conjugates var-ied between 3 and 12 mg/ml, and that of thepreimmune conjugates ranged between 4 and 10mg/ml. Direct staining titers on the conjugatesfrom the trial and final bleedings varied from 1:8to 1:1,280, being considerably higher in some ofthe earlier bleedings than in the final. This rangeprobably reflects the effects of the timing ofsome of the final bleedings and of the pooling ofthe serum from several bleedings.Homologous and heterologous staining

titers of conjugates. In Table 3, homologousand heterologous titers are shown for six conju-gates tested against 10 isolates of the Legion-naires bacterium. The Bellingham strain can bedifferentiated from the other nine strains on thebasis of its generally weak reactions with theconjugates tested. Nevertheless, it stained wellenough (3+ fluorescence) to permit its detectionby the Knoxville conjugate at a high dilution

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TABLE 2. Characteristics of conjugates prepared for five strains of Legionnaires bacteria by two routes ofinoculation

Strain Rabbit no. Injection Bleeding no. Percent F/P ratio Protein Homologousroute gammaa (mg/ml) 3+ titercPhiladelphia 1 338-39 i.V.f id NDe 28 10

3 92 33 8 64Finals 85 29 10 20

Philadelphia 2 341 i.v. 1 ND 27 103 90 19 3 8g

Finals 90 28 12 320

Philadelphia 2 344 Footpad 1 ND 30 103 93 20 5 329

Finals 88 26 10 320

Flint 1 342-43 i.v. 1 65 32 43 91 31 il 128

Finals 88 25 8 160

Pontiac 348 Footpad 1 ND 28 102 84 29 10 1,280

Finals 87 29 9 640

Knoxville 350 Footpad 1 72 32 42 90 25 10 320

Finals 91 28 il 160a Calculated from the cellulose acetate strip electrophoresis profile. Percent gamma is the ratio of the area

under the gamma curve to the total area under the protein profile (9).b The ratio of FITC, determined by reference to a fluorescein diacetate standard, to total protein, determined

by the biuret method (9).c End point dilution factor of highest dilution giving 3+ homologous staining.d Bleeding no. 1, Preimmunization bleeding. All preimmune conjugates were negative when tested.e ND, Not done.f i.v., Intravenous injection.g If titers were adjusted to correspond to 10 mg of protein per ml, they would be approximately 32 and 64,

respectively.

TABLE 3. Homologous and heterologous titers of six Legionnaires strain conjugatesConjugate titersb of:

StrainPhil. 1 (i.v.) Flint (i.v.) Phil. 2 (i.v.) Phil. 2 (fp) Pontiac (fp) Knoxville (fp)

Phil. 1 20 80 40 320 320 320Phil. 2 40 160 320 320 320 320Phil. 3 <20c 160 80 320 640 320Phil. 4 <20c 160 160 320 320 160Flint 1 20 160 80 320 160 320Pontiac 20 160 40 320 640 640Knoxville 40 40 40 320 80 160Bellingham 20 20 <20C 20 <20c 160Burlington <20c 40 <20C 320 320 640Albuquerque <20c 160 160 640 640 160a Suspension of cells in 0.85% NaCl containing 1% Formalin; turbidity = McFarland no. 1 standard. Phil.,

Philadelphia strain.b End point dilution factor of highest dilutions giving 3+ or greater staining reactions, iv., Intravenous

inoculation; fp, footpad inoculation.c End point dilution factor of lowest dilutions tested which did not give a 3+ staining reaction.

(1:160> and by three other conjugates at a lowdilution (1:20) (Table 3). It is evident that Phil-adelphia 1 was the weakest conjugate tested,although a conjugate from an earlier bleeding

had given a homologous titer of 1:64 (Table 2).Table 3 shows that the conjugates giving the

highest titers against heterologous antigens werethose obtained from the animals receiving "foot-

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DIAGNOSIS OF LEGIONNAIRES DISEASE BY IF 333

pad" and intramuscular injections.Preimmune conjugates comparable in every

other way to the immune conjugates usuallygave no staining of any antigens at the 1:20dilution. Weak staining (+- to 1+) of all cellswas observed occasionally, and strong (3+)staining of a few cells was seen rarely.Detection in scrapings of Formalin-fixed

tissues, tissue sections, and fresh tissue im-prints ofhomogenates. The direct IF stainingprocedure was used to study specimens of lungtissue from 143 patients with suspected Legion-naires disease. The conjugate for the Knoxvillestrain was diluted 1:80 in phosphate-bufferedsaline at pH 7.6 and used for the examinations.The tissues consisted ofwet Formalin-fixed lung,paraffin-embedded lung, and either fresh orfresh-frozen lung. Most of the specimens exam-ined were scrapings of Formalin-fixed lung, fol-lowed in order by tissue sections and by imprintsor homogenates of unfixed fresh lung. The cri-teria for determining IF positivity of lung speci-mens were those shown in Table 4, except thatearly in our experience with direct IF staining afew specimens were reported positive when lessthan 25 Legionnaires bacteria were seen persmear. In fluid specimens from the lower respi-ratory tract, however, the Legionnaires bacteriaare seldom numerous. Thus, the presence of fiveor more brightly stained bacteria per smear,with morphology typical of the Legionnairesbacteria, is considered a positive result withthese specimens.Approximately two-thirds of the patients

whose tissues were positive by direct IF staining,and about half of those whose tissues were neg-ative, were involved in outbreaks ofLegionnairesdisease in either Philadelphia, Pa., in 1976 (7),or Columbus, Ohio (3), Burlington, Vt. (4), or

Kingsport, Tenn., in 1977 (4).The criteria for determining tissue positivity

(John A. Blackmon, personal communication)were based (i) on the examination of hematox-ylin and eosin-stained sections for tissue path-ology that was distinctive for Legionnaires dis-ease and (ii) on the detection of Legionnaires-like bacteria in sections stained by the Dieterlesilver impregnation method (5).

In Table 5 the results of IF tests on a series ofLegionnaires disease tissues are compared withthe pathological evaluation of the same speci-mens.

In Fig. 1 the Legionnaires bacteria (Knoxvillestrain) are seen in a smear of scrapings of For-malin-fixed lung stained with the IF conjugate.The organisms in deparaffined sections of lungtissue from the same patient are shown in Fig.2. The enormous number of bacteria in the lungtissue is a striking feature of Legionnaires dis-ease. Frequently, from 50 to several hundredbacteria per oil immersion field are present inlung tissue from patients dying of the disease.Chains or filaments of more than two bacterialcells are seldom observed in tissues. The orga-nisms are found both intracellularly and extra-cellularly but, as demonstrated by comparingFig. 1 and 2, they are more easily visualized inthe lung scrapings than in sections.The appearance of IF-stained pure cultures of

the Knoxville strain of the Legionnaires bacte-rium is shown in Fig. 3 and 4. The smear pho-tographed in Fig. 3 was from a young (48-h)culture; that in Fig. 4 was from an older culture(5 day) on a medium of somewhat different com-position. The bacteria in Fig. 4 are typical ofthose usually seen in IF-stained culture smears;those in Fig. 3 are more like the tissue-phaseorganisms, although perhaps somewhat larger.

TABLE 4. Criteria for reporting results of direct IF tests for Legionnaires bacteria in lung tissues and fluidsResult Report

>50 strongly fluorescing bacteria per field ............... IF + (many)

2-50 strongly fluorescing bacteria per fielda b IF + (moderate)

>25 strongly fluorescing bacteria per smear b but>1 strongly fluorescing bacterium per fielda b.......... IF + (few)

<25 strongly fluorescing bacteria per smeara b Report numbers only

0 strongly fluorescing bacteria per smeara b IF -

>5 strongly fluorescing bacteria per smearbc IF +; report numbers

aIncludes scrapings of Formalin-fixed tissues, fresh tissue imprints and homogenates, and histologicalsections.

b Oil immersion objective (x1,250).C Applicable only to fluid specimens from the lower respiratory tract because Legionnaires bacteria are

seldom numerous in these specimens.

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Detection in exudates from the lungs.Twenty-five respiratory tract specimens re-ceived before 11 November 1977, and ratherpoorly documented as to site of origin or method

TABLE 5. Comparison of results of IF tests andpathological' examination of Legionnaires disease

tissues'

Test category No. of specimens (pa-tientsh)IF +; pathology + 38"IF +; pathology ? 5"IF +; pathology? 24IF-; pathology + 41IF -; pathology? 10IF -;pathology .-.......... 22

" Refers to the pathologist's diagnosis based onexamination of the tissues by hematoxylin and eosinstaining and by the Dieterle silver stain.

' Lung scrapings, homogenates, and sections fromautopsy or biopsy specimens.

Including patients from whose tissues six of thestrains discussed in this paper were isolated.

<l'issue examined by the pathologist may havebeen from a different area of the lung.

" Three of these four specimens were fixed in Zenkermercury solution. Tissue pathology was compatiblewith Legionnaires disease, but the Dieterle stains anddirect IF tests were negative possibly because of thefixative. One of these patients had an indirect If titerof 1:1,024. Sixty-two additional specimens (13 IF +; 49IF -) were not examined by the pathologist.

of collection, were examined by direct IF stain-ing. Only two specimens were positive. One ofthese was from a patient whose lung biopsysection was judged questionable for Legion-naires disease by the pathologist. By the indirecttest the patient had a significant titer rise from1:32 to 1:1,024 by the criteria of McDade et al.(12). The other patient also had a significanttiter, but no pathological data were available.For none of the 23 IF-negative specimens were

any histological data available. However, 12 ofthe 23 patients had significant indirect IF titers.Three patients were serologically negative, andspecimens from eight others were either notavailable or inappropriate for testing.Two pleural fluids were positive by direct IF

staining. No other confirmatory data were avail-able from these patients. Pleural fluids from fivepatients were negative. One of these patientshad a significant indirect test titer, but no tissuewas examined. No data were available on theother four patients with suspected Legionnairesdisease.

Specificity and reliability of direct IFstaining reactions. The preimmune sera ofrabbits used in our studies did not contain sig-nificant antibody reactive with Legionnaires an-tigens.To obtain data on the specificity of the con-

jugates and to search for possible serologicalrelationships, we screened smears of 374 pure

FIG. 1. Smear of scrapings of Formalin-fixed lung tissue of a patient who died of Legionnaires disease inKnoxville, stained with a 1:80 dilution of the homologous conjugate. Photographed on 135-mm Ektachrome200, EPD film. Magnification, X400.

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FIG. 2. Histological section of Formalin-fixed and paraffin-embedded lung of patient from Knoxville,stained andphotographed as in Fig. 1. Note relative difficulty of visualization of the bacteria in comparisonto smear of lung scrapings.

FIG. 3. Smear of 48-h culture of Knoxville strain of Legionnaires bacteria, stained with a 1:80 dilution ofhomologous conjugate. Culture was grown in modified Mueller-Hinton medium and suspended in phosphate-buffered saline containing 1% Formalin. Photographed as in Fig. 1. Note that these cells resemble those seenin tissue (Fig. 2).

cultures of bacteria by staining them with theworking dilution (1:80) of the Knoxville conju-gate. The cultures represented 25 known generaand 59 known species (Table 6). Also included

were 54 unidentified cultures. No significantstaining of any of these cultures was noted.

In general, the results of direct IF stainingreactions for detecting Legionnaires bacteria in

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FIG. 4. Smear of 5-day-old culture of Knoxville Legionnaires strain grown on a different modification ofMueller-Hinton medium. Stained andphotographed as in Fig. 1.

tissues correspond to the pathologist's assess-ment of the tissue appearance and the results ofapplying the Dieterle silver impregnation stain(Table 5).

In another series of examinations of Formalin-fixed lung tissues from 13 pneumonia patients,we found that 10 tissues were positive by directIF tests. Because of the high percentage of pos-itive results, we sought confirmation. A pathol-ogist examined sections of these tissues and re-ported that his diagnosis agreed with the IFresult for the eight specimens which we observedto contain "many," "moderate," or "few" flu-orescent organisms per field (xlOO oil immersionobjective). Of the other two specimens that wefound to be positive, one was from a patient withpneumococcal pneumonia with bacteremia, butonly six cells typical of Legionnaires bacteriawere seen in the entire smear. In the otherpositive specimen we saw 6 to 10 typical orga-nisms per smear. The pathologist rated thisspecimen as questionable and the former speci-men as negative for Legionnaires disease. Later,scrapings from a second block of Formalin-fixedtissue from the questionable specimen werestrongly positive by direct IF staining.An additional experiment was performed in

which the pathologist selected a group of 10Formalin-fixed lung specimens from patientswith pneumonia or pulmonary infection. Thespecimens were coded and submitted for directIF examination for Legionnaires disease bacte-ria. The Legionnaires bacteria were correctly

TABLE 6. Pure cultures (374) of bacteria of 25genera and 59 species giving negative results when

examined for relationship to Legionnairesbacterium by direct IF staining"Bacteria (no. of species) No. of cultures

Achromobacter (1) ... .. ..

Acinetobacter calcoaceticus; 31 serotypes ...

Actinobacillus actinomycetem-comitans ....Agrobacterium radiobacterAlcaligenes (3) .................. ....

Bacteroides ochraceus .............. ..

Bordetella (3) ............................

Brucella (1) ............. ................

Campylobacter fetus .....................

Cardiobacterium hominis .................

Corynebacterium (3) ......................

Flavobacterium (3) ...................Francisella tularensis .. ...

Haemophilus influenza .. .. ..

Klebsiella pneumoniae; 30 capsular types ...

Lactobacillus (7) .........................

Moraxella (1) .........................

Mycobacterium rhodochrousNeisseria (2) ...........................

Pasteurella (1) ...... .................

Pseudomonas (18)Serratia (1)Streptococcus pneumoniae; 30 capsular typesVibrio (3) ... ......... ..... ....

Yersinia pestis ........ .... ...

Unknown

75635

il482215756

30il4151

881

45102

54

'The diagnostic dilution (1:80) of the Knoxville conjugatewas used to examine these cultures. AUl were negative.

detected in three specimens; all others werenegative. The pathological assessment and theIF test results agreed in all 10 specimens.

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DISCUSSION

Conjugates prepared from sera raised in rab-bits by injection of Formalin-treated cells werevaluable in detecting the Legionnaires bacteriain both Formalinized and fresh autopsy andbiopsy lung tissue and in cultures.We had assumed that cells of the Legionnaires

bacteria might be quite toxic to rabbits becauseof the frequency of shock seen in human victims(7), but saw no evidence of toxic reactions.Treating the cells with 0.5% Formalin may havedetoxified them.

In immunizing rabbits with depot antigens forthe Legionnaires bacteria we recommend givingthe inoculation intracutaneously rather thaninto the "footpad." The former route results inless trauma to the animals and yields conjugatesof comparable titers (Richard George, personalcommunication).Because of the extremely large number of

Legionnaires bacteria present in the lung tissuesof many victims of the disease, each specimenmust be processed separately to prevent cross-

contamination of tissues by "wash over" ofstained organisms from a positive to a negativesmear. The degree to which "wash over" is aproblem can be estimated by examining knownnegative control smears appropriately spaced ona slide with known IF-positive smears. Also, themicrobiologist has no way of knowing whetherthe tissue he examines was contaminated beforehe received it. For these reasons we do notclassify tissues containing less than 25 well-stained organisms per smear (3- to 5-min search)as positive or negative (Table 4). The clinicianmust make the diagnosis on the basis of all theavailable clinical, epidemiological, pathological,serological, and microbiological data.The results that have been presented indicate

good agreement between the direct IF tests andthe pathologist's diagnosis based on the tissuereaction and the Dieterle silver impregnationstain. When disagreement occurred, it was likelyto be with lung specimens in which very fewLegionnaires bacteria were seen either with theDieterle stain or with IF tests or when thepathologist was uncertain about the tissue re-action. In these specimens the results depend toa large extent on the particular area of the organ

that is selected for study. When Legionnairesdisease is suspected, it is desirable to select grosstissue for study in consultation with a patholo-gist. Even if the initial IF test results are nega-tive, the pathologist should examine the tissuemicroscopically, because appropriate areas forfurther study by IF staining may be found.The serological specificity ofthe IF stain when

compared with the Dieterle stain is clearly ad-vantageous. Additional data may be obtainedfrom indirect IF tests for antibody in the sera ofpatients with evidence of Legionnaires disease.We have not presented serological data in ourreport because appropriate sera were availablefrom only a few of the patients. Relevance ofindirect IF titers to the results of direct IFexaminations of tissues or lower respiratory tractspecimens from Legionnaires disease patientsdepends upon factors such as the timing,method, and type of specimen collection, treat-ment regimen, and severity of the disease proc-ess.The Legionnaires bacteria were readily

stained in imprints of fresh tissue. At times,however, they were not as easily discernible inthe imprints as they were in Formalin-fixedscrapings of the same tissue. In imprints theorganisms may be surrounded by an envelopethat tends to obscure their outline; this did notoccur in the Formalin-fixed tissue. Also, scrapingthe tissue appeared to liberate intracellular bac-teria from macrophages and polymorphonuclearcells.Smears to be examined for Legionnaires bac-

teria by IF staining should not be fixed withKirkpatrick fixative because it depresses thestaining reaction. We have not used acetone tofix this organism, although it has been usedsuccessfully by others (12).

In embedded and deparaffined tissue sections,the organisms were shrunken in size and ob-scured to some extent by the thickness andcomplexity of the section, but they were highlyfluorescent and easily seen if present in moder-ate numbers. The integrity of the antigenic sur-face was maintained throughout the histologicalprocessing.Only a small number of fluid specimens from

the lower respiratory tract of people suspectedof having Legionnaires disease have been avail-able for study. Results indicate that generallyvery few Legionnaires organisms are found inthese specimens. Thus, the method and timingof specimen collection may prove to be the crit-ical factors in successfully demonstrating thesebacteria by direct IF staining. Research isneeded to evaluate the use of mucolytic agentsto liquefy and homogenize the specimens, todevise appropriate methods for concentratingthe organisms that are present, and to study theuse of counterstains for improving visualizationof fluorescent Legionnaires bacteria. Since theorganism grows slowly and is difficult to isolate,direct and indirect IF staining are currently theonly diagnostic tests available for rapid and spe-cific diagnosis.

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338 CHERRY ETALJ

In reasonably extensive testing of pure cul-tures of known and of unidentified bacteria as-sociated with humans we encountered only oneorganism, a strain of Pseudomonas fluorescens,that can confound the diagnosis of Legionnairesdisease by direct IF staining. This organism wasactively growing in a solution of commercial1:5,000 Evans blue which contained 3% bovineserum albumin and Merthiolate at a dilution of1:10,000. It was brilliantly and specificallystained by the diagnostic dilution (1:80) of theconjugate for the Knoxville Legionnaires strainand to a lesser extent by conjugates for the otherfour strains. In stained cultures the cells wereconsiderably wider and somewhat longer thanthose of the Legionnaires disease bacteria.Twenty-two additional cultures of this specieswere examined, but none fluoresced. Thus, carein handling IF reagents and in the interpretationof results is essential, because we do not knowthe frequency with which such serologically re-lated bacteria may occur. Preliminary data in-dicate that organisms serologically identical toor related to the Legionnaires bacterium arecommon in soils and soil animals from severalgeographic locations. None of these organismshas been isolated or identified.

ACKNOWLEDGMENTSWe thank Claire Broome, Gregory A. Storch, Ted F. Tsai,

and David W. Fraser for assistance in obtaining many of thespecimens studied and in accumulating relevant clinical andserological data on the patients. We are grateful to John A.Blackmon for guidance in choosing appropriate tissues forstudy and for sharing with us his pathological evaluation ofmany of the specimens. We thank Joseph E. McDade, JamesC. Feeley, George W. Gorman, and J. J. Farmer III forfurnishing some of the specimens examined and for their manyhelpful suggestions.

LITERATURE CITED

1. Center for Disease Control. 1968. Epidemic of obscure

illness-Pontiac, Mich. Morbid. Mortal. Weekly Rep.17:315-320.

2. Center for Disease Control. 1976. Classification of eti-ologic agents on the basis of hazard. U.S. Departmentof Health, Education, and Welfare, Center for DiseaseControl, Atlanta, Ga.

3. Center for Disease Control. 1977. Legionnaires' Dis-ease-Ohio. Morbid. Mortal. Weekly Rep. 26:300.

4. Center for Disease Control. 1977. Legionnaires' Dis-ease-Tennessee, Vermont. Morbid. Mortal. WeeklyRep. 26:336.

5. Chandler, F. W., M. D. Hicklin, and J. A. Blackmon.1977. Demonstration of the agent of Legionnaires' dis-ease in tissue. N. Engl. J. Med. 297:1218-1220.

6. Cherry, W. B., R. M. McKinney, V. M. Emmel, J. T.Spillane, G. A. Hebert, and B. Pittman. 1969. Eval-uation of commercial fluorescein isothiocyanates usedin fluorescent antibody studies. Stain Technol.44:179-186.

7. Fraser, D. W., T. Tsai, W. Orenstein, W. E. Parkin,H. J. Beecham, R. G. Sharrar, J. Harris, G. F.Mallison, S. M. Martin, J. E. McDade, C. C. Shep-ard, P. S. Brachman, and the Field InvestigationTeam. 1977. Legionnaires' disease. 1. Description of anepidemic of pneumonia. N. Engl. J. Med.297:1189-1197.

8. Gornall, A. G., C. J. Bardawill, and M. M. David.1949. Determination of serum proteins by means of thebiuret reaction. J. Biol. Chem. 177:751-766.

9. Hebert, G. A., B. Pittman, R. M. McKinney, and W.B. Cherry. 1972. The preparation and physicochemicalcharacterization of fluorescent antibody reagents. U.S.Department of Health, Education, and Welfare, Centerfor Disease Control, Atlanta, Ga.

10. Jones, G. L., G. A. Hebert, and W. B. Cherry. 1978.Fluorescent antibody techniques and bacterial applica-tions. U.S. Department of Health, Education, and Wel-fare, Center for Disease Control, Atlanta, Ga.

11. Maaloe, O. 1955. The international reference preparationfor opacity-notes and description. Bull. W.H.O.12:769-775.

12. McDade, J. E., C. C. Shepard, D. W. Fraser, T. R.Tsai, M. A. Redus, W. R. Dowdle, and the Labo-ratory Investigation Team. 1977. Legionnaires' dis-ease. Isolation of a bacterium and demonstration of itsrole in other respiratory disease. N. Engl. J. Med.297:1197-1203.

13. McKinney, R. M., L. Thacker, and G. A. Hebert. 1976.Conjugation methods in immunofluorescence. J. Dent.Res. (Special Issue A) 55:A38-A44.

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