JOURNAL OF BACTERIOLOGYVol. 87, No. 4, p. 900-909 April, 1964Copyright © 1964 American Society for Mlicrobiology

Printed in U.S.A.

SEROLOGY OF THE MIMA-HERELLEA GROUPAND THE GENUS MORAXELLA1

PAUL D. MITCHELL AND ROBERT G. BURRELLDepartment of llicrobiology, West Virginia University M1edical Center, M1organtown, West Virginia

Received for publication 4 December 1963

ABSTRACT

MITCHELL, PAUIL 1). (West Virginia UJniversityNMedical Center, Morgantown), AND ROBERT G.BURRELL. Serology of tile Mima-Herellea groupand the genus M11oraxella. J. Bacteriol. 87:900-909.1964.-The identity of organisms which have beenassigned to the llinia-Herellea group of the tribeMimeae and their taxonomic position are uncer-tain. In considering the possible relationship ofthe l1imna-Herellea grouip to the genus 3loraxella,the approach used was the isolation and identifi-cation of representative antigenic constituents ofstrains designated Mlina polyniorpha, 31. poly-niorpha var. oxidans, Herellea vaginicola, 7lorax-ella lwoffi, 1ll. liquefaciens, M. non-liquefaciens,and Bacteriuni anitratuni. Cell-free extracts ofsonically disrupted cells were used in these studies,and the precipitinogens derived by this methodwere considered to be of capsular and somaticorigin. Identity among tlhe strains was establishedby immunodiffusion, with immune sera and anti-genic extracts. Heterologous and homologousreactions were performed with adsorbed andunadsorbed seia to verify the cases of identity.In every instance of homologous reaction, atleast five distinct antigen-antibody systems werediscernible within the Minta-Herellea group.However, as evidenced by heterologous studies,the demonstrable antigenic composition variedamong the species and, in some instances, amongthe strains. Al. polynliorpha var. oxidans appearedto be serologically distinct from H. vaginicolaand M. polyrnorpha, whereas strains of the lattertwo organisms were theorized to be closely relatedserologically. Serological cross-reactions in heter-ologous studies and reciprocal adsorption testsrevealed the existence of a serological relationshipbetween members of the Mimina-Herellea groupand the genus M1-or-axella. A tentative scheme ofidentity is postulated on the basis of these cross-reactions and cultural and biochemical reactionsamong the designated strains of Herellea, Mlinia,Moraxella, and Bacterimn.

I Presented in part at the Annual Meeting ofthe American Society for Microbiology, Cleve-land, Ohio, 6 M\ay 1963.

'Throughout this paper the designations J,limapolymorplha, 11. polyn?orpha var. oxida s1.s, Herelleavaginicola, Bacterittiin anitratoni, and Moraxellaiwoffi appear frequently. These designations atpresent are not recognized as legitimate taxonsbut, nonetheless, appear frequently in tile litera-ture and are regardled by many workers as validdesignations. The l)Iresent study is directly con-cerned with the taxonomy and nomenclature ofthe organi.sms represented by- these designations.These organisms are ietained and exa.liiied asthese designations to give additional evidence intheir classification ancd identity.

It wras recently suggested by CouItieu, Chass-ingnol, andl Longeray (1961) that IIinia poly-morpha and H. vaginicola should be includled in anew tribe, Mloraxelleae, with two genera: illorax-ella, which would include the oxidase-positiveorganisnis, and Herellea, to include those that areoxidase-negative. Rosebury (1962) and Lindbergand AIoschides (1962) suggested that tile genusHerellea, as proposed above, be changed to Mi?nainstead. The terms J1. vaginicola, to include theoxidase-negative forms which possess oiii(e degreeof fermentative ability, and lll. polyniorplta, com-prised of the oxidase-negative inactive forms,were suggested by these workers. In addition, Ill.polyntiorpha var. oxidans was suggested as being adesignation which is a synonym for Mloraxellanon-liquefaciens (Henriksen, 1963).There is little agreement as to the absolute

criteria for differentiating and identifying theorganisnms recognized as belonging to the tribeMimeae and the genus Moraxella. Also, there ap-pears to be insufficient evidence to justify theunification of these organisms into one tribe. It isthe opinion of several wA-orkers (Brisou andMlorichau-Beauchant, 1952; Henriksen, 1951;Murray and Truant, 1954) that these organismsare related, but what generic designations andepithets (leserve valid, taxonomic stattus is ofmain concern.The majority of serological studies directly con-

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SEROLOGY OF MIMA-HERELLEA AND MORAXELLA

cerned with these organisms have establishedserotypes, relationships, and identities on thebasis of the isolation of one antigenic component,primarily of capsular or somatic origin, which isconsidered strain-specific (Ferguson and Roberts,1950; Cary, Lindberg, and Faber, 1956; Schauband Hauber, 1948; Stuart, Formal, and McGann,1949; Cary et al., 1958; Ewing, 1949; Daly,Postic, and Cass, 1962; Aiken, Ward, and King,1956; Eveland et al., 1959; Biegeleisen et al.,1962; Cary, 1961). The approach of the studies tobe presented employed cell-free extracts of soni-cally disrupted cells in an attempt to establishspecies relationships of these organisms based uponthe isolation and identification of representativeantigenic constituents of each strain. In the ex-

periments to be described, advantage was taken ofthe independent diffusion and precipitation ofantigens and antibodies in gels. It is the purpose

of these studies to re-evaluate the serological like-nesses within the 3Mina-Herellea group and withinthe gentus Mloraxella, to make a serological com-

parison of the two groups and to establish a sero-

logical basis for stating whether the organisms inthese two groups merit a closer taxonomic status.

MIATERIALS AND METHIODS

Antigents. Antigenic extracts were preparedfrom each of the strains listed in Table 1.The growth from 24-hr slant cultures of each

organism wvas susp)ended in 4.0 ml of TrypticaseSoy Broth (BBL). Large Pyrex dishes of Trypti-case Soy Agar were inoculated with 3.0 ml of thissuspension. After 48 hr of incubation at 37 C, thecells were w-ashed from the agar with bufferedsaline ail(l centrifuged. Supernatant fluids were

discarded, and the cell sediments from each latevere resuslended in 40 ml of buffered saline.These suspensions were then disrupted for 35 minwith a Raytheon I 0-keiultrasonic generator. Aftersterilizing each susp)ension by centrifugation andSeitz filtration, Merthiolate (Eli Lilly & Co.,Indianapolis, Tnd.) was added as a preservative togive a final concentration of 1:5,000. Sterilitytests were performed on all extracts before use.

Antisera. A 0.5-ml amount of each of the anti-gen prel)arations was mixed with an equal amountof 4%, sodium alginate adjuvant (Colab Labora-

tories, Inc., Chicago Heights, Ill.) and injectedsubcutaneously into rabbits. The injection was

followe(d by 1.0 ml of a solution containing 600meq of calcium chloride. Booster doses of antigen

TABLE 1. Sources of strains*

Designation Strain no. Source Key no.

Herellea 7788 CDC Hivaginicola 7820 CDC H2

7822 CD)C H3Mimna poly- 7827 CDC Ml?norpha 7833 CDC M2

7821 CDC M37345 CD)C M47374 CDC M5

M. poly- 7603 CI)C 01niorpha 7349 CDC 02var. 7608 CDC 03oxidans 7427 CD)C 04

Moraxella 4810/61 BI NON-Linon-lique- 826/61 BI NON-L2faciens

M. iwoffi 881/57 BI LW1270/56 BI LW2

Bacterium 2459/62 BI ANIT1anitratnm

M. liquefa- 7911 BI LIQ1ciens

*-Cultures of these organisms were obtainedthrough the courtesy of E. 0. King of the Com-municable Disease Center (CDC), Atlanta, Ga.,and S. D. Henriksen of the Bakteriologiske Insti-tutt (BI), Oslo, Norway. The key numbers in theright-hand column are used when discussing theexperimental data.

alone consisting of a 1.0-ml intraperitoneal injec-tion on the 11th day and 2.0-ml subcutaneousinjections on the 19th, 26th, and 33rd days gaveoptimal results. A trial bleeding obtained bycardiac puncture was performed on the 46th dayafter the first injection. After satisfactory titerswere obtained, the animals were then exsangui-nated. The resulting sera were stored in the frozenstate until use.

Immunodiffusion. The immunodiffusion meth-od used in this study was a modification ofthe Ouchterlony (1948) technique. The agarused in the plates (12.0 ml per plate) contained(in g per liter of distilled water): lonagar 2(Oxoid), 8.0; NaCI, 8.5; glycine, 75; crystallineMerthiolate, 0.1. According to the lpattern usedin the various studies, 8-mm assay cylinders werearranged in siliconized petri dishes so that a dis-tance of 8 mm separated the wells to be used todemonstrate antigen-antibody systems. After theaddition of the reactants, all plates were incu-bated at 4 C.

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Cultural studies. All strains used in this studywere subjected to cultural and biochemical testsfor purposes of verifying identification and corre-lating the biochemical activities with the serologi-cal results obtained. An isolated, single colonywas used in each instance for purposes of study.

All media were prepared from the appropriateDifco products unless otherwise specified. Tlhebasal media employed in the tests were as follows:citrate utilization, Simmons Citrate Agar; nitratereduction, 2% peptone pilus 0.2%; KNO3 ; indoleproduction, 2% Tryptone; methyl red reaction,MR-VP Medium (Difco); acetylmethyl carbinolproduction, Coblentz rapid mnethod; gelatin lique-faction, Heart Infusion Broth plus 12c%, gelatin;motility, Motility Medium; action on litmus milkand oxidase reaction, 0.5 %O aqueous tetramethyl-p-phenylene diamine dihydrochloride; hemolysis,5%to rabbit blood-agar employing Trypticase SoyAgar base medium (BBL); catalase reaction, 3%Ghydrogen peroxide; metabolic oxidations, purl)lebroth base plus 1.0%( l)articular carbohydratesand alcohols employed; carbohydrate utilizationand hydrogen sulfide production, Triple SugarIron Agar.

RESULTS

Cultural and biochemical reactions. In smearsstained by Gram's method, the organisms ap-peared as small, gram-negative diplococcoid rods.Degrees of pleomorphism were existent, withdefinite bacillary and coccobacillary forms andswollen cells observed. A tendency towards reten-tion of the crystal violet, p)reviously noted by DeBord (1942), was noted in a limited number of theMima strains only. Bipolar staining was observedbut was never consistent, even within a givenstrain. Large clear zones around the stained, dip-lococcoid rods suggested the encapsulated state.Rough colonies were noted after several days ofincubation and after a number of transfers.

Isolated colonies, after 48 hr of growth on 5%blood-agar plates, were approximately 2 to 4 mmin diameter, circular, convex, entire, smooth,opaque, grayish-yellow to grayish-white in color,and mucoid in consistency. Of the strains, 50%were beta-hemolytic, without any limitations toany one genus.The organisms grew in Trypticase Soy Broth

(BBL) with an even turbidity and produced aslight sediment. A pellicle or surface ring wasnoted after 72 hr of incubation at 37 C. However,abundant growth could not be obtained in liquid

media. Growth was noted in all media employedin the complement of cultural and biochemicaltests.

Certain characteristics group these organisms:(i) aerobic, gram-negative, encapsulated, pre-dominantly diplococcoid in morphology, andfrequently pleomorl)hic; (ii) negative in the ni-trate, gelatin, indole, motility, methyl red, andVoges-Proskauer tests; and (iii) catalase-positive.The tests for oxidase p)roduction, citrate utiliza-

tion, action of litmus milk, and carbohydratemetabolism gave more variable results. [Theterms "oxidizer" or "nonfermenter" have beenassigned to this group of organisms. Negativeproduction of indole, and failure to reduce nitratesto nitrites are common characteristics among thenonfermenters (Hugh and Leifson, 1953).]

Organisms received as H. vaginicola and B.anitratun were noted to produce acid, demon-strate reductase activity, and in some cases tocoagulate litmus milk. All other organisms,strains of the genus Mloraxella and of the genusMfima, gave an alkaline reaction.

Tests demonstrating the oxidative dissimila-tion of carbohydrates were performed with thecarbohy-drates incorporated in an infusion broth.Species of the genera lIoraxella and Minia failedto acidify any of the carbohydrates tested,whereas the other organisms received as H. vagi-nicola and B. anitratbim acidified glucose consist-ently but attacked xylose with irregularity. Noneof the organisms studied showed any action ondulcitol nor mannitol, which is in direct contrastto De Bord's (1942) findings.

Serological re-evaluiation of the tribe 3Mimeae. Un-diluted antisera and antigens were usually foundto be of optimal concentration for use in agardiffusion tests, when analyzed by these conven-tional techniques. In almost all instances, thesharpest reactions were obtained after 2 or 3 daysof incubation in the cold. Prolonged incubationresulted in diffuse bands that obscured interpre-tation.The strains of organisms used in these studies

are given in Table 1. For simplicity of presenta-tion, we will refer to the organisms by the keynumbers in the right hand column.When homologous studies were done employing

antigenic extracts of IH. vaginicola (HI, H2, andH3) and their corresponding immune sera, fivedistinct antigen-antibody systems were observed.However, when reciprocal analyses were donewith antigenic extracts and immune sera of these

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strains, only four antigen-antibody systems werediscernible, demonstrating a specific antigenicconstituent for each strain. This was confirmedwhen testing each particular system (Fig. 1 and2). Five distinct bands are observed between eachcorresponding antiserum and antigenic extract.There are four bands which gave no "spurring" atthe angle of intersection and may be termed as"lines of identity." However, a fifth band wasdiscernible in each system which represents thedistinct antigenic constituent of each strain.When similar homologous studies were done

employing immune sera and antigenic extracts oftwo strains of .1. polymorpha (MI and M2), fivedistinct antigen-antibody bands were observedbetween each corresponding system. However,when one 11. polymorpha antigenic extract (Ml)was tested against MA2 immune serum, and viceversa, five lines were again present but only twolines of identity were observed. The three remain-ing lines demonstrated spurring or reactions ofpartial or nonidentity (Fig. 3).

Identity reactions among three strains of theoxidans variety of .11. polymorpha (02, 03, and04), revealed a very close, if not identical, sero-logical relationshil, whereas one strain (01)shared only one antigen of five with these threestrains.To determine the presence or absence of any

serological relationships among these organisms,heterologous studies were undertaken. One speciesof M. polymorpha (Ml) was shown to be veryclosely related to all strains of H. vaginicolastudied, sharing at least four group antigens. A

H I

HI

HA HI

42FIG. 2. Another type of diagranmmatic repre-

sentation of the precipitin lines demonstrating thereactivity of species-specific group antigens andexistent heterogeneity of Herellea vaginicola.

ml

M2 M Ie:aH3

-ee

Hi HZ 13FIG. 1. Diagrammatic representation of the

precipitin lines demonstrating the reactivity ofspecies-specific group antigens and existent hetero-

geneity of Herellea vaginicola.

M2FIG. 3. Diagramlmatic representation of the

precipitin lines demonstrating the reactivity ofspecies-specific group antigens and existent hetero-geneity of Mima polymorpha.

second strain of 31. polymorpha (MI2) appearedto be more distantly related to these strains ofHerellea, sharing only two or three groul) antigens.M. polymorpha var. oxidans demonstrated very

little cross-reactivity with either H. vaginicola or31. polymorpha. No bands of identity or partialidentity were observed when HI, H2, H3, andMI antigenic extracts and immune sera weretested separately with the oxidans immune seraand antigenic extracts. However, one strain of H.vaginicola (HI) and one strain of Ilf. polymorpha

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(M2) reacted with three of the antioxidans sera,

giving one or two bands of identity.To confirm the serological relationships be-

tween H. vaginicola and 31. polymorpha, furtheragar diffusion analyses were carried out afterselected adsorptions.The adsorptions were limited to reacting

samples of H. vaginicola (Hi) antiserum sepa-

rately with samples, of equal volume, of five Al.

polymorpha (MI-M\5) antigenic extracts and thehomologous (HI) antigenic extract. After an in-cubation period of 48 hr at 4 C, each adsorbedserum was centrifuged, and the supernatants were

filtered through a Swinney filter prior to use toassure removal of precipitated antigen-antibodycomplexes. Each adsorbed serum was then reactedwith the antigenic extracts employed in the tech-nique of adsorption. The results of these studiesindicated that ,1I. polymorpha (M11) and H. vagi-nicola (HI) were identical, whereas the otherstrains of lll. polymorpha, being homogenous intheir antigenic complement, were more distantlyrelated to H. vaginicola (HI), sharing only two or

three group antigens.Serological comiparison studies of the gennis 3-Io-

raxella, the tribe 1limeae, and closely resemblingorganisms. For reasons l)resented above, thefollowing experiments were designed to test cer-

tain hypotheses that have appeared in the litera-ture in regard to the relationship of the organismsrepresenting the tribe Mimeae to the genus

MIoraxella. In these studies, one additional strainof Il. polymorpha (AM5) and one strain designatedas B. anitraturn were included in these studies. Itwas the purpose in examining a strain labeled as

B. anitratum to evaluate the possibility of itsbeing used si-nonomously with H. vaginicola.

In every instance of reaction, whether homolo-gous or heterologous, a distinct band of identityadjacent to the serum well was observed witheverv representative strain. For purposes of our

discussion, this band will be excluded whenreferral is made to the number of bands a particu-lar system gave.

Both strains of Ml loraxella lwoffi (LWI andLW2) appeared to be homogenous. LW1, in itshomologous reaction of corresponding antigenicextract and immune serum, gave six separatebands of antigen-antibody precipitation, whereasLW2 in its homologous study gave five bands.When LWi immune serum was tested againstLW2 antigenic extract, six bands were observed.

When the systems were reversed (LW1 antigenicextract tested against LW,2 serum), five bandswere discernible.The two strains of 3I. non-liquefaciens exam-

ined (NON-LI and NON-L2) displayed a possibleheterogeneity within the species. Homologousreactions with NON-Li gave six bands, whereasNON-L2 gave only three bands. However, as evi-denced bv reciprocal reactions, (i) NON-L2 anti-genic extract was shown to possess four antigenicconstituents with anti-NON-LI, and (ii) NON-Liantigenic extract gave two bands when testedagainst anti-NON-L2. In the first instance, it maybe said that the immune serum to NON-L2lacked the antibodies for this particular antigenwhich NON-Li immune serum provided.Whether the results of the latter study reveal atrue heterogeneity or not is doubtful, owing tothe isolation of the organism's incomplete anti-genic lproperties or incomplete antibody forma-tion in the particular animals used.The one strain of Mloraxella liquefaciens (LIQ1)

gave evidence of five distinct antigen-antibodysystems when subjected to homologous studies.

31. polymorpha (A12) immune serum whentested against AM2 antigenic extract gave fivebands and .11. polymorpha (1\15) gave four lines inits homologous reaction. When M2 and M5 weretested heterologously (anti-M2 vs. Mf5 antigenicextract and vice versa), three bands were ob-served in each of the two instances. This variationcorresponds to the previously mentioned differ-ence within this species.

Since the organisms labeled as H. vaginicola andB. anitratum appeared to be identical on the basisof the cultural and biochemical reactions, theywere tested in a series of homologous and heterol-ogous reactions. The results of these studies gaveevidence for their being identical (representingone organism) in that they shared five to sevengroul) antigens.

Table 2 I)resents the complete data of the heter-ologous examinations of the genus Mforaxella, Al.polymorpha, 11M. polymorpha var. oxidans, H. vagi-nicola, and B. anitratumn.

In regard to the relationship of lloraxellaiwoffi. to the other organisms in question, it can beseen that both strains, appearing to be identical,possess a degree of heterogeneity when analyzedwith .1. non-liquefaciens (NON-LI and NON-L2). When these antigenic extracts were sepa-ratelv analyzed with if. lvoffi (LWI) antiserum,

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TABLE 2. Heterologous studies of Moraxella iwoffi (LW1 and LW2), M. non-liqllefaciens (nion-Liand non-L2), Minia polymorpha (M2 and M5), M. polymorpha var. oxidans (02, 03, and 04),

Mloraxella liquefaciens (LIQI), and Bacterium anitratum (ANITi1)

Antigenic extractsAntisera

LWl LW2 NON-LI NON-L2 M2 M5 03 02 04 LIQI Hi ANIT1

LW1....... ...6* 6 1 1 2 4 1 2 2 3 4 4LW2..... 5 5 3 3 2 3 1 1 1 3 3 3NON-Li 1 0 6 4 2 2 2 3 2 4 0 1NON-L2...0...0 0 2 3 1 0 0 2 0 3 0 0M2 .......... 3 3 2 2 5 3 1 1 1 3 3 3M5 ......... 4 4 2 1 3 4 1 2 1 3 5 403 ......... 2 1 1 1 1 1 4 4 3 2 1 102 ......... 2 1 1 2 1 2 4 5 5 2 2 304 . ..... 1 1 1 1 1 1 6 6 6 2 1 1kIQl .......... 1 2 3 4 1 3 1 1 1 5 2 2Hi ......... 3 3 3 2 3 3 1 2 0 2 5 5ANIT1 ....... 3 3 1 0 4 4 1 1 1 1 5 6

* Numbers refer to number of gel diffusion bands, excluding the one band common to all reactions.

one line was observed. However, when the anti-genic extracts were separately analyzed withLW2 antiserum, three lines were discernible.An existent, homologous condition was ob-

served within the two strains of M. iwoffi (LW1and LW2) when antigen extracts of them weretested separately with 31. non-liquefaciens (NON-LI and NON-L2) antiserum. Zero to three lineswere observed.

There is evidence that M1oraxella iwoffi and3lima polymorpha are closely related and that theorganisms designated B. anitratum and H. vagi-nicola represent an even closer serological rela-tionship to Moraxella iwoffi. As can be seen, theseorganisms share a serological relationship whichmay not merit the division into four differentgeneric designations.When Moraxella non-liquefaciens (NON-Li

and NON-L2) antigenic extracts were testedindividually with Ill. liquefaciens (LIQ1) anti-serum, three or four bands were observed, respec-tively. Similar results were obtained when recip-rocal reactions were performed. These studiesindicated a very close serological relationshipbetween 31. liquefaciens and M. non-liquefaciens.

In regard to the earlier-mentioned identity ofM. non-liquefaciens and Mima polymorpha var.oxidans, it was evident from heterologous analysesthat there is a basis of differentiation in that theseorganisms as a group shared only one to threecommon antigens.From these results, it can be seen that there is a

distinct serological relationship existing amongthese organisms, with some apparent cases ofidentity and cases in which the relationship ismore distant.As will be recalled, H. vaginicola and B. anitra-

tum, as a distinct group, evidently share with themajority of the strains (if not all) at least onecommon antigenic component. However, aspointed out earlier, it may be considered moreclosely related to Mima polymorpha, MlIoraxellaIwoffi, M1. liquefaciens, and Al. non-liquefaciens,while distantly related to M1ima poly?norpha var.oxidans.From the above heterologous reactions, a tenta-

tive grouping of the organisms in question washypothesized. They are given as follows: group 1,H. vaginicola and B. anitratum; group 2, 31.polymorpha and Moraxella iwoffi; group 3, 31.liquefaciens and M. non-liquefaciens; group 4,Mima polymorpha var. oxidans.With an evident overlapping of some of the

previously designated groups and evidence forperhaps identity of certain strains, it was decidedto attempt to resolve these questions bv means ofreciprocal adsorption studies.

In each case of homologous adsorption, anoptimal adsorption was observed in that no bandsof antigen-antibody precipitation were observedwhen resubjected to immunodiffusion.

In the majority of instances of heterologousadsorptions, it was observed that an incompleteadsorption may be existent, since reciprocal

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studies among certain alpparently identicalstrains revealed an evident condition of noniden-tity. For example, 1 ml of antiserum of H. vagini-cola (Hi) was adsorbed with an equal amount ofB. anitratum antigenic extract. The resultingserum, when tested against the antigenic extracts,revealed three lines to H. vaginicola but no lines toB. anitratum. However, when antiserum to thelatter was adsorbed with H. vaginicola antigenicextract and in turn analyzed, no lines of H. vagi-nicola were present, but four lines were present tothe homologous antigenic extract.Owing to the large number of antigen-antibody

combinations present in any given system, thevarying concentration of each antigen in a givenextract, and similar variations of antibody in eachserum, validity of such reciprocal adsorptions wasquestioned. To determine whether optimal pro-portions existed under the conditions used foradsorptions, the following experiment was carriedout. A 1-ml amount of lloraxella iwoffi (LW1)antiserum was adsorbed with 1 ml of 7lIinia poly-morpha (Ml) antigenic extract, in accord with thepreviously described technique of adsorption. Theresulting antiserum was then tested against anti-genic extracts of if1oraxella lwoffi (LWI and LW2)and .f irna polymorpha (M\2 andl M\15). No bandsof precipitate were ol)served with A12 and M5,but three bands were (liscernible with LWI andLW2. From the previously described relationshipof Mloraxella iwoffi to il[iioa polymiiorpha, theseresuilts were most confu-sing. However, if optimalconditions of adsorption w+ere not l)resent, thiswould explain the bands given with the twostrains of Mloraxella iwoffi ancd no bands to the twostrains of M1ima polyioorpl a.To determine wlhethleir optimal con(litions of

adsorlption were existent, tylpical adsorbed anti-sera wvere tested against dilutions of either ho-mologous or heterologous antigens and vice versa.This tVpe of experiment rev-ealed that the individ-ual quantities of each (listinet antigen varied frompre)aration to prepaaration. Thus, even thoughthe antigenic extracts of Mloraxella iwoffi andMimiia polyrnorpha wvere qcualitativelv similar, theinclividual antigens were seen to varY quantita-tively. For this reason, (lifficulty was encouinteredin p)erforming compilete serum adsorptions withthese soluble antigens, thus explaining the aboveinconsistent results. Additional l)reliminary ex-I)eriments have demonstrated that rep)eated ad-sor)tions are necesslary to completely remove all

of the identical antigens. Such studies are now inprogress.

DISCUSSION

From the results of the biochemical and cul-tural studies, it is apparent that there are certainunifying characteristics of the genus Moraxellaand the organisms which have been assigned tothe tribe Mimeae: (i) typically small, coccobacil-lary organisms, predominantly diplococci andwith a tendency towards pleomorphism; (ii)gram-negative with a tendency to retain thecrystal violet dye; (iii) aerobic and not producinggas from carbohydrates; (iv) not producing indoleor acetylmethylcarbinol and giving a negativereaction for the methyl red and gelatin liquefac-tion tests; and (v) nonmotile. Certain character-istics such as nitrate reduction, citrate utilization,hemolysis, and the oxidase reaction were subjectto occasional, and perhaps doubtful, variation.However, recognizing with the group such varia-tions, certain distinctions could be made thatallowed us to see a continuous gradient of suchdistinguishing features, even though such differ-entiation is seldom based on more than a singlecharacter.From the preliminary investigations re-evaluat-

ing the serological properties existent within thetrible Mimeae, cell-free extracts appeared to beexcellent precipitinogens. Also, the methods ofimmunization and of demonstrating antibodieshave greatly aided in studying serological rela-tionships in a manner not available with theclassical agglutination techniques. The evidenceshowed that the study of serological relationshipsis more readilv accomplished by this method;namely, such techniques allow direct visualiza-tion of the antigen-antibody reactions rather thaninferring that a particular system demiionstratesserological relationships.

In addition, the isolation of complex antigeniccomplements presents definitive data which mayfacilitate identification and differentiation pro-cedures for these organisms. With direct concernfor the taxonomy and nomenclature of theseorganisms, the cultural, biochemical, and sero-logical data provide certain apparent conclusions.When observing the variations of a single char-

acter, one group of organisms was seen that pro-duced acid from glucose, one that was oxidase-positive, and one that was oxidase-negative. Inreslective order, the following groups were al)par-

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ent: group 1, H. vaginicola and B. anitratum;group 2, Moraxella non-liquefaciens, Moraxellaliquefaciens, and Mlima polymorpha var. oxidans;group 3, Mima polymorpha and Moraxella iwoffi.From the apparent consistency of the charac-

teristics within these groups, only group 2 couldbe broken down further. Moraxella liquefaciensliquefied Loeffler's blood serum medium, whereas31. non-liquefaciens and Mima polymorpha var.oxidans did not. The other variables which maybe seen within these groups, i.e., citrate utilizationand hemolysis, did not necessarily merit separa-tion into further subgroups.From serological studies, it is feasible to assume

that, from the standpoint of antigenic relation-ship, (i) the examined, typical species of H. vagi-nicola and Mima polymorpha were serologicallyclosely related and the differentiation into twogenera may not be fully justified, and (ii) thethird species, Mima polymorpha var. oxidans, wasnot antigenically related closely enough to theother two species and thus appears to be out ofplace taxonomicallv when considering these or-ganisms per se.On the basis of the presented homologous,

heterologous, and reciprocal adsorption studies,the organisms used in these studies have beenassigned to a group of identity, to groups sharinga close serological identity, and to a group as beingdistinct: group 1, H. vaginicola and B. anitratum,serologically identical; group 2, Moraxella lique-faciens and .11. non-liquefaciens, related; group 3,Mima polymorpha and Moraxella iwoffi, related;and group 4, Mlima polymorpha var. oxidans,distinct.However, heterologous studies did show that

these groups can be unified in that there arecross-reactions existent among all the organismsstudied. Consequently, we have one heterologousgroup of serologically related organisms, whichcan be separated into serological subgroups.

Results of our studies established an apparentidentity among the organisms designated H.vaginicola and B. anitratum, owing to evidentidentical antigenic complements. Such studies ofheterologous and reciprocal adsorption reactionsdemonstrated group antigens among these organ-isms which were consistent in establishing one

hypothesized group.Whether or not Mlima polymorpha var. oxidans

is a synonym of Mloraxella non-liquefaciens isquestionable. There is an apparent relationship,

but the heterologous studies revealed this to be anextremely distant relationship, and, thus, wereconsidered strong evidence for these organismsnot being identical. Therefore, two organisms,Mllima polymorpha var. oxidans and Mlloraxellanon-liquefaciens, give the same cultural and bio-chemical reactions but are apparently serologi-cally heterogeneous. Thus, it would appear thatthe only valid means of identification and separa-tion into two species would be the isolation of agroup-specific antigen(s) from each particularorganism.When considering the possible identity of Mima

polymorpha and Moraxella Iwoffi, it was demon-strated that at least four group antigens maycontribute to such an identity. These two organ-isms showed an indistinguishable, consistent abil-ity to react in heterologous studies, demonstratinga serological relationship that would not confirmthe establishment of two separate species. Therelationshil) is further clarified when noting thenondifferentiating biochemical and cultural char-acteristics of these two organisms.The cross-reactions observed among the organ-

isms designated H. vaginicola, B. anitratum, M1imapolymorpha, and M-foraxella iwoffi are not in agree-ment with the use of four generic designations.Since we do have a serologically related groupthat can be separated into two subgroups basedupon glycolytic activity, it would appear benefi-cial to assign two species designations for theorganisms in this particular group.

Immunodiffusion studies permitted us to es-tablish a serological relationship between Mlorax-ella non-liquefaciens and 31. liquefaciens, eventhough at least one group antigen was foundwithin each particular species. Since these organ-isms may be differentiated on the basis of actionon Loeffler's blood serum medium, the problem ofidentification is restricted. The serological differ-ence may thus be explained on the basis of thedifferent enzymatic activities of these two organ-isms.The reciprocal adsorption studies did confirm

certain heterologous observations, but could notbe used to verify the exact identity of the organ-isms under study. Optimal conditions of adsorp-tion apparently cannot be obtained for eachantigen by this method. This may be explainedwhen considering two organisms of apparentidentity which we will label as X and Y. If bothof these organisms are identical, in having at least

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MITCHELL AND BURRELL

five common antigens in their antigenic structure,each will have antigens a, b, c, d, and e in theircorresponding extracts. As is well known, to ob-tain optimal adsorption the antigen must be inoptimal proportions to completely adsorb thehomologous precipitins. However, when workingwith antigenic extracts and antisera, each con-taining a large number of antigens and antibodies,the probability that each reactant is present inthe same amount of each extract or serum isremote.

Since we are working with five to seven antigen-antibody systems and cannot reach optimal con-ditions for each system using only one adsorptionof a mixture of antigens, the reciprocal adsorptionstudies were considered invalid for establishingtrue identities. It is suggested that, for purposesof demonstrating true identities, the antigenicextracts be purified and identifiedThe following scheme of identity is offered to

summarize the results of these studies: oxidizep-aminodimethylaniline monohydrochloride, Mlo-raxella; liquefy Loeffler's serum agar, Moraxellaliquefacierns; do not liquefy Loeffler's serum agar,Moraxella non-liquefaciens; do not oxidizep-aminodinmethylaniline monohydrochloride,Mima; ferment certain carbohydrates, Mimaanitraturn; do not ferment certain carbohydrates,M1ima liwofi.The species Mlforaxella non-liquefaciens would

include Moraxella non-liquefaciens and Alimapolymorpha var. oxidans, distinguished on thebasis of distinct serotypes. Mima iwoffi wouldinclude the organisms previously designated asMIoraxella iwoffi and Mima polymorpha, and Mimaanitratuni would include the designations H. vagi-nicola, B. aniitratum, and other glycolytic organ-isms given synonomous status with H. vaginicola.

ACKNOWLEDGMENT

This study was made possible by training grant2G-509 from the Division of Medical Services,National Institutes of Health, U.S. Public HealthService.

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