APPLIED MICROBIOLOGY, Feb. 1974, p. 368-374Copyright 0 1974 American Society for Microbiology

Vol. 27, No. 2Printed in U.S.A.

Slide Agglutination Method for the Serological Identification ofNeisseria gonorrhoeae with Anti-Gonococcal Antibodies

Adsorbed to Protein A-Containing StaphylococciDAN DANIELSSON AND GORAN KRONVALL

Department of Clinical Bacteriology, Central County Hospital, 5rebro, Sweden, and Department ofMedicalMicrobiology, University of Lund, Lund, Sweden

Received for publication 24 October 1973

A rapid slide agglutination test has been developed for the identification ofNeisseria gonorrhoeae that are primarily detected as oxidase-positive colonies ingonococcal cultures. The technique is based on the specific nonimmunereactivity between the Fc portion of immunoglobulin (Ig)G and staphylococcalprotein A. IgG molecules adsorbed to stabilized staphylococci will therebybecome oriented with their antigen-reactive sites that are directed outwards.Protein A-containing staphylococci with unabsorbed anti-gonococcal antibodiesgave positive co-agglutination reactions with gonococci but also with menin-gococci, some Moraxella, Haemophilus, and Pseudomonas strains. These cross-reactions were eliminated by absorption of the anti-gonococcal antiserum withmeningococcal and Moraxella organisms prior to the coating of reagent staphylo-cocci. In the routine culture diagnosis of N. gonorrhoeae the use of specificgonococcal reagent staphylococci gave concordant results with fermentationprocedures and immunofluorescent techniques.

The identification of Neisseria gonorrhoeae inlaboratory cultures is primarily based on thepresence of oxidase-positive colonies of typicalmorphology. Colonies of other microorganismsmay also react with the oxidase reagent. Someof these strains are easily distinguished fromgonococcal colonies by an experienced exam-iner. Oxidase-positive colonies of some speciesof Moraxella, Haemophilus, Pseudomonas, andalso Neisseria other than gonococci may, how-ever, be impossible to distinguish on the basis ofcolony form. Confirmatory tests are thereforenecessary. For the correct laboratory diagnosisof N. gonorrhoeae, the criteria should alsoinclude the demonstration of gram-negativediplococci and fermentation tests showing acidproduced from glucose but not from maltose orlevulose (18, 20). Pure cultures are usuallyneeded for the fermentation tests which alsorequire media of high quality (10, 18, 20). Thesetests are laborious and time consuming. Inmany laboratories the fermentation reactionshave therefore been replaced by immunofluo-rescence tests when specimens from the genito-urinary tract are analyzed (18, 20; D. Daniel-sson, Ph.D. thesis, Univ. of Uppsala, Sweden,1965). In specimens from other sites, i.e., throatspecimens, blood, cerebrospinal fluid or jointexudate, the identification of gonococcal-like

colonies by immunofluorescence tests must beconfirmed by fermentation procedures andsometimes also by other bacteriological tests (3,4, 9, 20, 22).

Recently, a co-agglutinating serological rea-gent was described by Kronvall (14), and its usewas exemplified in the capsule typing of pneu-mococci. This reagent was also utilized for theserological grouping of streptococci (5) as wellas for mycobacterial typing (11). Reagent parti-cles consist of formaldehyde and heat-treatedCowan I staphylococci which are subsequentlycoated with specific antibody via the gammaglobulin Fc-protein A reaction. The antibodymolecules will thereby become oriented with theantigen-reactive Fab parts that are directedoutwards (14).

In the present paper the co-agglutinationmethod has been adopted for the identificationof N. gonorrhoeae. The results obtained in theroutine identification of gonococci using N.gonorrhoeae-specific reagent are compared withthe results obtained using immunofluorescenceand fermentation tests.

MATERIALS AND METHODSGonococcal strains. Ninety strains of N.

gonorrhoeae were studied in the experiments. Sevenof them were laboratory strains representing colonial

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morphology types T1 through T4 (12). Two of thesewere originally isolated at the Department of ClinicalBacteriology, Central County Hospital, Orebro, Swe-den, and the other five were isolated at the StateSerum Institute, Copenhagen, Denmark, at the Cen-ter for Disease Control, Atlanta, Ga., at the Instituteof Medical Microbiology, Uppsala University, Upp-sala, Sweden, and at the Department of ClinicalBacteriology, Sbdersjukhuset, Stockholm, Sweden.The other eighty-three strains were freshly isolatedgonococcal strains, predominantly of colonial mor-

phology types T1 or T2, obtained from routine cul-tures of genito-urinary specimens sent for gonococcalculture to the Department of Clinical Bacteriology,Central County Hospital, Orebro, Sweden. Thestrains were identified by oxidase testing, Gram stain,and immunofluorescent or fermentation procedures(18, 20; Danielsson, Ph.D. thesis). They were main-tained on colonial morphology typing medium by themethod of White and Kellogg (21) and on GC medium(BBL) (15). Slide agglutination tests with co-

agglutinating reagents were performed on coloniesfrom both of these media.

Other bacterial strains. Fifty oxidase-positivebacterial strains other than N. gonorrhoeae were alsostudied in the experiments. They represented thefollowing bacterial species: 10 strains of N.meningitidis belonging to the serogroups A, B, C, andD; 15 strains of N. pharyngis; 7 strains of Moraxellaspecies; 3 strains of Haemophilus influenzae; 3 strainsof Haemophilus parainfluenzae; 12 strains ofPseudomonas species. The strains were maintainedon ordinary blood agar and GC agar (BBL). Slideagglutination tests were performed with bacteriagrown on GC agar plates. A meningococcal strain ofserogroup B and a Moraxella strain (laboratory iden-tification number 100101) were used for the absorp-tion of anti-gonococcal sera and anti-gonococcal con-

jugates as described below.Routine gonococcal cultures. Three thousand one

hundred two rectal-genito-urinary and 173 tonsillo-pharyngeal specimens, sent for gonococcal cultureanalysis to the Department of Clinical Bacteriology,Central County Hospital, Orebro, Sweden, weregrown on selective and nonselective gonococcal cul-ture and media as described elsewhere (8). Routineidentification of N. gonorrhoeae was made as follows.The GC agar plates were subjected to oxidase testing,and smears from oxidase-positive colonies were

stained with fluorescein-labeled anti-gonococcal glob-ulin (20; Danielsson, Ph.D. thesis). Genito-urinaryspecimens with oxidase-positive colonies and fluores-cent antibody-positive smears showing gonococci withtypical morphology were considered positive for N.gonorrhoeae. Tonsillo-pharyngeal specimens and rec-tal specimens with oxidase-positive and fluorescentantibody-positive smears were further subjected tofermentation procedures and other bacteriology tests(4, 18, 20, 22). All cultures showing oxidase-positivecolonies were also subjected to slide agglutinationtests with protein A-containing staphylococci coatedwith specific anti-gonococcal antibodies as describedbelow. Unsensitized staphylococci or staphylococcicoated with normal rabbit gamma globulin were usedas control reagents.

Anti-gonococcal antisera. Anti-gonococcal anti-sera were obtained from rabbits immunized withformalin-treated, whole gonococcal organisms as de-scribed elsewhere (6). The sera were stored frozen at-62 C or -40 C. After thawing, the sera to be used forsensitization of reagent staphylococci were preservedby adding 1% (vol/vol) merthiolate (1: 100) and thenwere kept at 4 C. Anti-gonococcal antiserum K022was selected for absorption experiments. Absorptionswere carried out as follows. One volume of anti-gonococcal antiserum was mixed with 1 volume ofMoraxella organisms and/or meningococcal group Borganisms packed by centrifugation at 2,000 x g for 20min. The mixture was incubated at 37 C for 4 h andovernight at 4 C (7). The absorbed serum was re-covered by two centrifugations at 2,000 x g for 20 min.

IF procedures. The globulin portion of anti-gono-coccal antiserum was precipitated by 45% saturationwith (NH.),SO4 and then dissolved in distilled water.After dialysis against tap water and saline, the globulinwas labeled by standard procedures using 0.0375 mg offluorescein isothiocyanate (FITC) per mg of protein(16). A conjugate prepared from anti-gonococcal an-tiserum K022 which was also used for the preparationof reagent staphylococci was selected for immunofluo-rescence (IF) tests. This conjugate had an F/P ratio of14.7 x 10' (2). Absorption of the conjugated gammaglobulin was carried out as described above foranti-gonococcal antiserum. Preparation and stainingof smears followed standard procedures (6, 16).Fluorescence microscopy was performed with a Zeissmicroscope equipped with a high-pressure mercurylamp (Osram HBO 200) and an oil dark-field conden-sor. A BG12 was used as a primary filter and a Zeiss 47or Zeiss 50 was used as the secondary filter. Thepreparations were read at a magnification of x420.Positive reactions were graded 1+ through 4+ asdescribed before (6).

Preparation of co-agglutinating staphylococci.The stabilization and coating of protein A-containingstaphylococci to make co-agglutinating reagent fol-lowed procedures described previously (14). TheCowan I strain of Staphylococcus aureus (NCTC8530) was grown overnight in CCY broth (1), and thewashed bacteria were treated for 3 h with 0.5%formaldehyde, washed in phosphate-buffered saline(PBS), pH 7.4, and exposed to 80 C for 4 min asdescribed elsewhere (14). After additional washingsthe bacteria were suspended to 10% in PBS containing0.1% sodium azide and kept at 4 C until use.

For coating of the protein A-containing staphylo-cocci, 1 ml of the 10% suspension was added to 0.1 mlof unabsorbed or absorbed rabbit anti-gonococcalantiserum. After mixing the suspension, the staphylo-cocci were washed twice and suspended to 1% in PBScontaining 0.1% sodium azide. Control reagent con-

sisted of uncoated staphylococci or staphylococcicoated with gamma globulin from normal rabbitserum. The suspensions were stable for more than 6months when stored at 4 C.

RESULTS

Co-agglutination reactions of oxidase-pos-itive organisms with reagent staphylococci

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370 DANIELSSON AND KRONVALL

coated with unabsorbed anti-gonococcalantibodies. One or more colonies of the oxidase-positive strains listed in Table 1, including 90strains of N. gonorrhoeae. were emulsified in 2drops of the 1% suspension of reagent staphylo-cocci coated with unabsorbed anti-gonococcalantibodies. The slide was then tilted back andforth for approximately 30 s and observed withthe naked eye. Controls were performed withreagent consisting of uncoated staphylococci or

staphylococci coated with normal rabbitgamma globulin. The results obtained are sum-

marized in Table 1.All 90 gonococcal strains tested, regardless of

colony morphology types, co-agglutinated withreagent staphylococci coated with unabsorbedanti-gonococcal antibodies (Table 1). In mostinstances a positive reaction occurred within 10s and was fully developed within the next 20 to30 s. Moderate (2+) to strong (3+) or verystrong (4+) co-agglutinations were registered as

well as some minor variations between variousstrains in the appearance of the agglutinates.No reactions occurred with uncoated staphylo-cocci or with staphylococci coated with normalrabbit gamma globulin except on a few occa-sions where a weak auto-agglutination of a fewgonococcal strains of colony morphology types 1

or 2 was observed. These reactions had a finelygranular appearance not involving reagentstaphylococci and was quite different from a

true co-agglutination.

APPL. MICROBIOL.

N. meningitidis strains of various serogroupsgave weak (1+), moderate (2+), or strong (3+)co-agglutination reactions with reagent staphy-lococci coated with unabsorbed anti-gonococcalantibodies. A few apathogenic Neisseria strainsspontaneously gave heavy aggregates whenmixed with uncoated staphylococci or staphylo-cocci coated with normal rabbit gamma globu-lin or anti-gonococcal antibodies. These aggre-

gates did not involve reagent staphylococci andwere easily distinguished by their characterfrom true co-agglutination reactions. A fewapathogenic Neisseria strains gave weakly posi-tive (1+) co-agglutination reactions with thetest reagent whereas some others remainedhomogeneous when mixed with control as wellas test reagent.

Five strains of Moraxella species also formedheavy aggregates with both control and testreagent. The other two Moraxella strains in-cluded in the studies gave, with the test rea-

gent, weakly to moderately positive reactionsthat were indistinguishable from a positivereaction with meningococci or gonococci. Thiswas also true for some strains of H. influenzae.Negative, 1+, or 2+ reactions were noted withstrains representing H. parainfluenzae andPseudomonas species.Comparison between results of co-agglu-

tination tests with reagent staphylococcicoated with absorbed anti-gonococcal anti-bodies and results of IF staining. The results

TABLE 1. Coagglutination reactions of Neisseria gonorrhoeae and other oxidase-positive organisms with proteinA -containing staphylococci, uncoated or coated with unabsorbed anti-gonococcal antibodies or normal rabbit

gamma globulin

Co-ag-lutination reactions with reagent staphylococcia

Coated with:

Bacterial strains No. ofstrains Uncoated rabbit Unabsorbed anti-gonococcal antibodies

glaobmumlia K622 R85 R105

Neiserria gonorrhoeae 90Colony types T,-T, I OWAOWA + +,A+ + -4 +, t+. + + +.4 + +++

Colon1ytypes T,-T, _ ++++ ++ + + + +t+ 4 ++ ++,+++.+ ++ +

Neisseria meningitidis 10 _ +, +,I+ + + +, + +, ++,+ +, + + +(serogroups A, B, C. D)

Neisseria pharvngis 11 -, occasional - occasional - -, occasionalNeisseria pharvngis 4 SC SC SC SC SCMoraxella species 5 SC SC SC SC SCMoraxella species 1 + + + + +Moraxella species 1 _ _ + +Haemophilus influenzae 3 -_-- - ++ , ±+,+ +-

Haemophilus 3 _ _ -.+ -. + -+parainfluenzae

Pseudomonas species 12 _, - ++ , ,X, +,

Symbols: +, weak co-agglutination; + ±, moderate co-agglutination: ++ strong co-agglutination: + - + +. very strongco-agglutination. OWA, Occasional weak auto-agglutination; SC, spontaneous clumping.

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obtained in co-agglutination tests with reagentstaphylococci coated with anti-gonococcal anti-bodies, unabsorbed and absorbed withMoraxella and/or meningococcal organisms, are

summarized in Table 2.Staphylococci coated with anti-gonococcal

antibodies absorbed with meningococci stillgave co-agglutination reactions with gonococcibut not with meningococci or apathogenicNeisseria strains. Co-agglutination occurred,however, with Moraxella, H. influenzae, andPseudomonas. After absorption of the an-tiserum used for making reagent with bothmeningococcal and Moraxella organisms, co-

agglutination was only obtained with gonococci(Table 2, Fig. 1). It should be noted, however,that the co-agglutination reactions developedmore slowly with these absorbed reagent staph-ylococci. It usually took 20 to 30 s for theagglutination to appear whereas the samestrains gave positive reactions within 5 to 10 swith the unabsorbed reagent. The reactionswere also somewhat weaker with the absorbedreagent. Usually 2+ or 3+ reactions but occa-sional 1+ reactions were noted. By observingthe slide for another 2 to 3 min the weak 1 +reactions developed, however, at least to 2+reactions.

Fluorescent staining reactions obtained withFITC-labeled anti-gonococcal globulin from thesame serum (K022) used for coating of staphy-lococci are also summarized in Table 2. Theunabsorbed conjugate gave 3+ to 4+ reactionswith all gonococci tested. Reactions graded 2+to 4+ were noted with meningococci. The

FIG. 1. Co-agglutination patterns obtained with N.gonorrhoeae, N. meningitidis, and a Moraxella strainusing protein A-containing staphylococci coated withanti-gonococcal antibodies, unabsorbed or absorbedwith N. meningitidis and Moraxella organisms. Rea-gent staphylococci coated with unabsorbed antibodiesshow a 3+ reaction with N. gonorrhoeae (R1), a 1+reaction with N. meningitidis (R2), and a 2+ reactionwith a Moraxella strain (R3). Staphylococci coatedwith antibodies absorbed with N. meningitidis andMoraxella show a 3+ reaction with N. gonorrhoeae(T1), and negative reactions with N. meningitidis(T2) and Moraxella (T3).

Moraxella strain that gave a 2+ co-agglutina-tion reaction with unabsorbed reagent staphylo-cocci gave a 1+ to 3+ reaction with the anti-gonococcal conjugate. No reactions were ob-served with apathogenic Neisseria orPseudomonas species, but a rather strong solidstain reaction with H. influenzae was noted.

TABLE 2. Results of testing Neisseria gonorrhoeae and other oxidase-positive organisms with co-agglutinatinggonococcal reagent staphylococci and with FITC-labeled anti-gonococcus globulin

Co-agglutinat ion reactions of reagent Stainina reactions withstaphylococci coated with ant i-gonococcal FITC-labeled anti-gono-

antibodies coccal globulin

No. of Absorbed with: AbsorbedBacterial strains strains with N.Not absorbed N. menin- menin-

N. menin- .d d Not absorbed .iii andgitidis gitidis and (diluted 1:32) dandMoraxella Moraxella

(diluted1:8)

Neisseria gonorrhoeae (colony 50 +++, ++++ ++, +±++ ++, +++ 3to4+ 3+types T1, T2, T3, T4)

Neisseria meningitidis (sero- 10 +, ++, +++ 2 to 4+ -

groups A, B, C, D)Neisseria pharyngis 6 + _ _ -

Moraxella species 1 + + + + _ 1-3+Moraxella species 1 + + _ -

Haemophilus influenzae 3 +, ++ +, ++ Solid stainPseudomonas species 4 +, ++ ±, ++

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DANIELSSON AND KRONVALL

After absorption of the conjugate with menin-gococci and Moraxella organisms, staining reac-

tions were obtained only with gonococci. The IFtiter of the conjugate dropped, however, twotwofold dilution steps. The reactions obtainedwith the unabsorbed and absorbed conjugatescorresponded well with co-agglutination resultsusing unabsorbed and absorbed reagent staphy-lococci.

Identification of N. gonorrhoeae with spe-cific reagent staphylococci in routine gono-coccal cultures: comparison with IF stainingand fermentation test. A total of 249 oxidase-positive colonies from 3,175 routine gonococcalcultures representing 3,102 specimens from therectal-genito-urinary tract (urethra in males,and urethra, cervix, and rectum in females) and173 tonsillo-pharyngeal specimens obtainedfrom both males and females were tested inslide agglutination tests with reagent staphylo-cocci coated with anti-gonococcal antibodiesabsorbed with meningococcal and Moraxellaorganisms. Control reagent consisted of un-coated staphylococci or staphylococci coatedwith normal rabbit gamma globulin. The re-sults were compared with those obtained withIF tests or with Gram stain and fermentationprocedures. The results are summarized inTable 3.Reagent staphylococci coated with absorbed

anti-gonococcal antibodies gave positive co-agglutination reactions only with N.gonorrhoeae and differentiated them from me-ningococci, apathogenic Neisseria, and other

oxidase-positive bacteria as confirmed with IFtests or with Gram stain and fermentationprocedures. This was true for gonococci isolatedfrom the rectal-genito-urinary tract as well asfrom the tonsillo-pharyngeal region. A weakauto-agglutination of a few gonococcal strainsoccurred when the strains were emulsified incontrol reagent. It had the same character asthe weak auto-agglutination observed withsome gonococcal strains in experimental tests.It appeared as fine granules not involving rea-

gent staphylococci and was quite different fromthe true co-agglutination, mostly grade 3-+,shown by these strains. Examination of thesecolonies on colonial morphology typing mediumindicated that they represented mainly type 2colonies found to be rough in PBS or saline. Aspontaneous formation of heavy aggregates of adifferent nature than auto-agglutination oc-curred with some apathogenic Neisseria strainsand also with a few other oxidase-positivecolonies representing gram-negative rods. Thisspontaneous clumping of some bacteria withuncoated as well as coated reagent staphylo-cocci never interfered with the reading of thetrue co-agglutination reactions with gonococci.

DISCUSSIONA new serological technique based on the use

of protein A-containing staphylococci coatedwith specific antibodies (14) has been adoptedand utilized for the identification of N. gonor-rhoeae, primarily detected as oxidase-positivecolonies in gonococcal cultures. The use of

TABLE 3. Comparison of results obtained with co-agglutination tests using specific gonococcal reagentstaphylococci and with immunofluorescence or Gram stain and fermentation procedures in tests of

oxidase-positive colonies from 3175 routine gonococcal cultures

Co-agglutination reactions of reagentstaphylococcia

No. of cul- Coated with: Diagnosis arrived at withtureswith

immunofluorescence orSource of specimen oxidase- Anti-gonococcal Gram stain and

positive Not Normal antibodies ab- frettocolonies coated rabbit sorbedwith N.fermentation

gma meningiti~dis andglobulin Moraxella

Urethra, cervix, rectum 138 _ _ + ++,± + N. gonorrhoeae12 WA WA + + + N. gonorrhoeae

Pharynx, tonsils 9 _ _ +, + +++ N. gonorrhoeae21 _ _ - N. meningitidis

Urethra, cervix, rectum 18 _ _ _ Organisms other6 SC SC SC than Neisseria

Pharynx, tonsils 32 _ _ _ N. pharyngis or13 SC SC SC gram-negative rods

a WA, Weak auto-agglutination; SC, spontaneous clumping.

372 APPL MICROBIOL .

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reagent staphylococci coated with specific anti-gonococcal antibodies gave completely concord-ant results with Gram stain and fermentationprocedures, and with specific FITC-labeledanti-gonococcal antibodies (Tables 2 and 3).The described slide agglutination test consti-

tutes a simple and rapid method for the labora-tory culture diagnosis of N. gonorrhoeae. Ascompared to fermentation procedures it has theadvantage of being more rapid and less labori-ous, and does not require complicated fermen-tation media. A positive diagnosis can be ar-rived at within a few minutes using specificgonococcal reagent staphylococci whereas 1 ormore days are required for fermentation proce-dures.The use of IF tests, basically also a serological

technique, offers a morphological dimensionand is nearly as rapid as the co-agglutinationtest. The latter one has the advantage of notrequiring a fluorescence microscope. An IF testrequires, however, less gonococcal material thana co-agglutination test. The IF method is there-fore of advantage when only one or a fewoxidase-positive colonies are available. On theother hand, in rectal and tonsillo-pharyngealgonorrhoea, two well-documented clinical enti-ties (4, 13, 22), IF tests cannot be used alone butmust, when positive, be supplemented withfermentation tests and perhaps other bacterio-logical examinations, since in many of thesespecimens gonococci have to be differentiatedfrom meningococci, apathogenic Neisseria, andsome other oxidase-positive organisms (4, 18,20, 22). The results of the present investigationshow that co-agglutination tests are of greatvalue on these occasions.

It is well known that colony morphology typesT1 and T2 of N. gonorrhoeae become auto-agglutinated when grown in liquid media orwhen suspended in saline or PBS, a reactionwhich seems to be mediated by zones of adhe-sion on the gonococcal cell walls and possiblyaccentuated by the presence of pili (12, 19). Theauto-agglutination in saline or PBS was alsodemonstrated with T1 and T2 gonococcal colo-nies used in this study, although most of thesestrains were homogeneous when they weremixed with a suspension of nonreacting staphy-lococci. On a few occasions a slight auto-agglutination was observed with T2 colonieswhich, however, contrasted to the true andusually strong co-agglutination of these strainswith specific reagent. The advantage of theco-agglutination method for analyses of auto-agglutinating strains was pointed out by Chris-tensen et al. (5) and by Juhlin and Winblad(11).

A spontaneous clumping with specific reagentstaphylococci as well as with uncoated andcontrol staphylococci occurred with some apath-ogenic Neisseria, some Moraxella strains, andalso some other oxidase-positive bacteria. Theformation of such aggregates, some of whichwere rather heavy, also contrasted in characterto the true co-agglutination of gonococci withspecific reagent staphylococci.The results of our experiments show that

reagent staphylococci coated with unabsorbedanti-gonococcal antibodies gave moderate tostrong reactions with N. meningitidis and alsowith some Moraxella, Haemophilus, andPseudomonas strains. Cross-reactions with me-ningococci were expected since it is well knownthat gonococci and meningococci share manyantigens (6, 7, 20, 23). Absorption of the an-tiserum with meningococci eliminated thecross-reactions with these strains but not thosewith Moraxella, Haemophilus, or Pseudomonasorganisms until anti-gonococcal antibodies werealso absorbed with Moraxella organisms. Fur-ther investigation has shown that the sameresults are arrived at by absorption with H.influenzae or Pseudomonas organisms insteadof Moraxella. The observed serological cross-reactions with these organisms and the elimina-tion of these reactions by absorption indicate anantigenic relationship between these bacteriaand gonococci. These findings were supportedby results using IF tests and will be subjected tofurther study.

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