Purified native haptens of Brucella abortus B19 and B. melitensis 16M reveal the lipopolysaccharide origin of the antigens

~) ELSEVIER Paris 1988

Ann. Inst. Pasteur/Microbiol. 1988, 139, 42 t -433

PURWIED NATIVE HAPTENS OF B R U C E L L A A B O R T U S BI9 AND 11. M E L I T E N S I S 16M REVEAL THE LIPOPOLYSACCHARIDE

ORIGIN OF THE ANTIGENS M.S. Zygmunt (J), G. Dubray (l), D.R. Bundle (2) and M.P. Perry (2) O) lnstitut National de la Recherche Agronomique, Station de Pathologie de la Reproduction, 37380 Nouzilly (France), and (2) Division o f Biological Sciences, The National Rc~cearch Council o f Canada, Ottawa, Ontario KIA OR6

SUMMARY Purifcation of the Brucella polysaccharide referred to as native hapten (NH) and extracted from cells by the autoclaving procedure, was accomplished by ultrafiltration, followed by repetitive gel filtration using highperformance liquid chromatography on a <>column. The purified NH was analysed by SDS-PAGE, gas-liquid chromatography mass spectroscopy, and 13C and tH NMR spectroscopy. N i l from B. abortus BI9 (NH-A) was shown to have a structure identical to that of A polysaccharide from B. abortus 1119-3, a linear homopolymer of ~t-l,2-1inked-4,6-dideoxy4- formamido-D-mannopyrannosyl residues. The structure of the NH from B. melitensis 16M (NH-M) was identified as a linear homopolysaeeharide of the same sugar but composed of a pentasaccharide repeating unit in which four ~l,2-1inked-4,6-didcoxy-4-formamido-D-ma~opyrannosyl residues are linked a-1,3 to the last mono~aecharide of the sequence. This structure is similar to that determined for the Brucelta M polysaeeharide from B. mefitensis 16M. The dLccovery in highly purified N i l preparations of covalently bound monosaecharides characteristic of lipopolysaceharide inner core regions e.g., quinovosamine, mannose and 3-deoxy-D-manno-octulosonate (KDO), indicates that this polysaccharide is derived from lipopolysaccharides (LPS) by hydrolytic conditions fortuitously generated during the extraction protocol. The antigenieallyimportant polysaeeharides of Bruce!In are now established to be either A or M antigens. Polysaccharide B is a cyclic glucan with no strut-

Submitted Januaff 23, 19118,accepted June 11, 19~.

422

M.S. Z Y G M U N T A N D COLL.

tural or serological relationship to A or M polysaccharides, its apparent activity in diagnostic tests of infected cattle results from 0 polysaccharide contamination. This artefact, previously referred to as NH, results from LPS hydrolysis under the extraction conditions used to prepare polysaccharide B. KEY-WORDS:Brucella, Hapten, LPS; Purification, Structure, Antigenicity, Native hapten. INTRODUCTION Bruce/la organisms responsible for human and veterinary infections are detected mainly by the serological response of the host since isolation of the causative agent is difficult [1]. Antibodies against the immunodominant endotoxin from smooth cells (S-LPS) are the first to appear after infection [18] and these antibodies are detected by standard serological tests [1]. Vaccination of domestic animals by attenuated strains of Bruceila also induces such antibodies [11, 12], and their detection by standard serological tests impedes the differentiation of vaccinated from infected animals. A test using the Brucella polysaccharide fraction called native hapten (NH) was claimed to be a means of discriminating between infected and vaccinated animals in serological assays as anti-NH antibodies were demonstrated to be in the sera of infected animals by radial immunodiffusion [12, 13, 18, 20]. The detection of such antibodies which appear to be related to active infection, indicates that a clearer understanding of the chemical nature of this antigen and its relation to the other Brucella cell wall polysaccharides is highly desirable. The immunochemistry of Brucella cell wall polysaccharides has been complicated by numerous reports that described a variety of methods for extraction of the 3 cell wall polysaccharides, S-LPS, polysaccharide B and NH [12, 13, 18, 20]. The serological activity of such preparations has, in addition, been the subject of extensive discussion and speculation [11, 13, 16-18,

201. The generally accepted interpretation of such data prior to definitive structural studies of these antigens was that the polysaccharide component of the S-LPS was the structural entity bearing the classical A and M epitopes [11];

Btr/ GLC HIP3

= Brucella melitensis. = gas-liquid chromatograph),. = fibre cartridge with nominal cut-off o f 3 Kd. H I P 3 0 = fibre cartridge with nominal cu~-~ff o f 30 Kd. H P L C = high-performance liquid chromatography. KDO = 3-deoxy-D-manno-octuiosonate. LPS = lipopolysaccharide. mAb = monoclonal antibody,

NH NH-A NH-M

NMR

= native hapten. = native hapten from B. abortus BI9. = native hapten from B. melitensis 16M. =

nuclearmagneticresonance.

SDS-PAGE = polyac~.amidegeldeclroplmresis in presence o f sodium dodecyl sulphate. S-LPS = smooth 5popolysaccha6Ae. Ys = ¥¢rsinia enterocolitica.

BRUCELLA A A N D M N A T I V E H A P T E N S T R U C T U R E

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a conclusion recently confirmed using chemically-defined antigens and monoclonal antibodies (mAb) [10]. NH, the product of unbalanced synthesis of O polysaccharide was presumed to be accumulated in the cell wall, periplasm or cytoplasm [20]. Poly~ccharide B was thought to represent a third distinctive component with crossreactivity for both A and M antigens and NH [12, 17, 20]+ The successful application of high resolution NMR spectroscopy to the structural elucidation of A and M antigens [5, 9] and polysaccharide B [3, 4], and the consequent ease of antigen identification and characterization by such methods, offers a potenual solution to the question of NH structure and activity [19]. The O polysaccharides of Brucella serovars A and M are both homopolymers of 4,6-dideoxy-4-formamido-D-mannopyranose [2, 4, 5, 9]. Whilst A antigen is a linear 1,2-1inked polymer with an essentially monomer repeating unit [9], the M antigen is composed of a linear pentasaccharide sequence in which 4 sugar residues are linked a-l,2 and the fifth is linked a-l,3 [5]. Polysaccharide B by comparison is a cyclic polymer composed only of glucopyranose residues linked ~-1,2 [3, 4]. Its reported cross-serological reactivity with antibodies of infected cow sera [13, 18] was therefore interpreted as being due to the presence of contaminating O polysaccharide which was found to be present in polysaccharide B prepared by the published extraction methods [4, 12, 13, 17, 20]. These findings demonstrate that it is imperative to firmly establish the composition and structural basis as criteria for antigen purity. In this paper, we report the purification, analysis and structural characterization of NH extracted from Bruceila serovar A and M strains by application of those physical and chemical criteria established in earlier work on the Brucella and related polysaccharides [2-5, 7-9].

MATERIALS AND METHODS Bacterial strains. Smooth serovar strains were kept freeze-dried in the Bruc~e//aculture collection held at INRA-Nouzilly, France, and described in detail by Verger et ale [22]+ Preen

of NH extracts.

NH extract from B. abortus (NH-A) was prepared by autoclaving ceils, treating the supernatant with ribonudease, deoxyribonuclease and proteinase K, followed by ultrafiltration with 2 stirred ~ Amicon~, cells in ~ 123|+~ ~ was 6+6 % of cell dry weight. (HIP30 and HIP3). The yield was 3.82 % of cell dry weight.

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M.S. Z Y G M U N T A N D COLL.

Purification of NH-A and NH-M.

NH-A and NH-M were purified by HPLC using size exclusion, TSK-G2000-SW ~ analytical (600× 7.5 ram) or preparative (600 × 25 ram) columns. The eluant was 0.250 M ammonium acetate pH 7 and the flow rates were 1 ml/min and 5 ml/min for the analytical and preparative columns, respectively. The eluate was monitored at 220 nm and fractions were automatically collected and pooled with the <
UV spectroscopic analysis was perf~,naed on purified NH-A and NH-M samples using a 2140 rapid spectral detector and the LKB wavescan analysis program. SDS-PAGE analysis was done as previously described with silver staining for proteins [14] and S-LPS [14, 15]. NMR spectroscopy. Proton and t3C NMR spectra were recorded at 37°C using a -Bruker AM 500~ spectrometer for samples (20-30 mg) dissolved in deuterium oxide in a 5-mm precistun NMR tube [5, 9]. Gated decoupling was employed for acquisition of protoncoupled 13C spectra. Detailed .o~rating and experimental conditions are described in related publications from this laboratory [5, 9]. Monosaccharide analysis. Monosaccharide analysis was performed on the component monosaccharides released from NH-A and NH-M samples by acid hydrolysis as previously Oescribed [8, 9]. Alditol acetates wereprepared from the liberated m o n o s a c c ~ d e s by reduction with sodium borohydride followed by acetylafion according to standard conditions [8]. These derivatives were analysed by GLC-mass spectroscopy as previously described [5, 8, 9]. Colorimetric assays for monosaccharides were performed as previously described [8, 9]. Immunodiffnsion experiment. Antigens dissolved in water at 0.5-1.0 mg/ml were used for immunodiffusion ex~ periments c_onduct.ed, in p l a t e s . p ~ from 1 % w(v a~arose (Ftshe[ Scientific). mAO spechtc tor either or ooth the Bruce//a A arA M antigens UOl aria manuscript in preparation) were used as undiluted ascites fluid or diluted in PBS I11. Plates were read at 8 and 24 h.

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RESULTS Purification, The NH-enriched fraction from both Brucella strains under investigation were prepared by ultrafiitration following recovery of cell extract by the published procedure involving autoclaving whoI¢ c:ils [13, 23], The NH peaks (first peak) were easily separated and automa~cally collec!ed (fig. 1). The purified fractions showed a symmetrical peak when reanalysed (fig. 2). Protein and nucleicacid contaminants were undetected by absorption measurements conducted at 260 and 280 nm. Absence of T~roteLns was confirmed after silver staining of a 20 Izg sample run on a p~lyaerylamide gel, which corresponds to protein contamination below the 1 % level. In SDS-PAGE, characteristic LPS banding patterns were not seen after periodic

1.28 A

E 0 v

0 O

2J~

etutt~ F~G. L ~ HPL C a ~ M ~ ' o f N i t . A ~

~ 1 o f

B.

60

time ( a i n )

frem I ~

426

M.S. Z Y G MU N T AND COLL.

o

0

0

2O

etugion time (min) FIG. 2 . . . . Chrom¢lographic profile of purified NH-A~ Chromatographic profile of purified NH-M is similar. Chromatographic conditions were a~ ~ b c d in legend to figure I.

actd/sd er st~xmmgwhich corr~ponded to an upper limit of LI~ contamination of 150 ng or less than 0.75 %, w/w. Structure.

Proton and t3C NMR spectra were recorded for the two NH samples dissolved in deuterium oxide (t'~. 3 and 4). The spectra of the material purified from the serovar A ~rain B!9 were similar to ~ published for the O p o ~ charide of B. abor~us I 119~3. Both spectra (fiB- 3 and 4) indicate that the NH from B. abortus BI9 has a structure identical to that of the A antigen, a 1,2-1inked linear homo~lymer of 4 ~ x y - 4 - f o r m a m i d o ~ - D . mannopymxtose [91. In addition, the low itttensity, near ~ , resottatg~ were corrected with those found, in sampl~ of amhemic A polymcchaHde

BRUCELLA A A N D M NATIVE HAPTEN STRUCFURE

427

which has been liberated from S-LPS by mild hydrolysi~ [7, 9]. T h ~ resonances were imerpreted as resulting from the unique monoseccharide com,. ponents of the LPS core oh'gosaccharide through which the O polysaccharide is linked to lipid A. The IH and l~C spectra of the NH B. melitensis 16M (fig. 3 and 4)were in similar fashion identical to those repo~led for the M polysaccharide released from the 16M S-LPS by mild hydrolysis [4, 5]. Consequently, this material is also seen to be u ~ u r a l l y idemicat m its corresponding O polysaccharide. in order to establish the presence of core monosaccharides in the NH samples, acid hydrolysates and GLC a n a l ~ of the component sugar as their alditol acetate derivatives was performed. Thh data revealed the presence of KDO, q n i n o v ~ e and mannose, ead~ of which was detec~ted in quantitative amounts similar to those found in the cores of purified O p o l y s a c ~ x i d e preparations (table I). Immunodiffnsion ~ y s i s experiments were performed with the Nil samples and two mAb. :lhese had been previously shown to bind e i ~ both ~he A and M antigens, gs n ~ 2, or only the Brucelle M antigen antibody Ben n ~ 28 [6]. In these experiments (not shown), the NH-A from Bt9 precipitated only with mAb n ~' 2. NH-M from 16M precipitmed with both Ys n ~ 2 and Bin n ° 28. These results indicate that the latier prepanUion was s~roiogicat!y M-acti~, whils! that from ~rain BI9 possessed A activity. DISC~f~ION

NH from B. aborlus BI9 and B. melilensis 16M :strains expressing the A and M antigens were purified by a novel HPLC-based ~ filtration syaer., which was ataenable to automated operation. In Lhis way i| was possible to

Tse~,~ I. -- Glycose gmml~mitkm~~ the NH ~ I J ~ O ded'~ed from B. n b o n ~ s ~ BI9 amd Bo me~em~ B. ~

G|y~

D-,~

Bl9

NH LPS O ~

(*)

0..91

0.:94

B. me~ee~ t~M PS NH LPS O<9~. PS

iA~

0,~

428

M~S. Z Y G M U N T A N D COLL.

ensure a very high level of purity of the rcspcctiv¢ NH preparations, NH~A and NH-M. Th~s wa~ p3rt~culariy important to the ~ub~ucm discussion,, because h was ncces~ry to place a h~h |c~el of con~dcncc in the ab~ncc of LPS or polysaccharide B, components which could be prc--~-~min NH preparations [14, 1"7,20]. in this regard, the HPLC-purifi~ NH*A and N H . ~ were judged to be pure based on the ~iteria of UV spectra and SDS-PAGE

NH-M L,,

NH-A

8~0

~0

.from ¢ NH.M xH NMR QmL~W

4~

2~O

fe#p~ . ~ i . ~

BRUCELLA A A N D M N A T I V E H A P T E N STRUCTURE

429

analysis, techniques which reveal the ~ of p ~ e i n or LPS coniaminams. The a 'bsenc~ of polysaccharidc B was confirmed by ~H NMR s p ~ r ~ .

~C~ ~r~.~:(:~aCCha~TidC~lYe the ~[iC~p~i|~lJ profile of immu~KfuSion ~ |ions with mAb ~ f ' ~ c for the M all|ri~ll, o r both the A and M amige.. The results of the NMR mcasuremems not only required i ~ ~ch p o | ) ~ . charide sample was pure, but furthc¢ unequivocally eslabli:shed [ ~ r ~ r ~ c a l

idcn~Li~yand s[ructur¢. Thus NH-A :ga~¢ ~H and ~C ~pectra (fig. 3 and ,4)

!i

NH-M

J i

NH - A

I

,- i,. ij

430

M.S. Z Y G M U N T A N D COLL.

identical to those published for the A polysaccharide obtained from B. abuttus I 119-3 [7, 9]. In a similar manner, NH-M was seen to give complex t H and 13C NMR spectra highly characteristic of and identical to those of the M polysaceharide obtained from B. melitensis !6M [4, 5]. It is concluded that NH are structurally indistinguishable from the cor.respondin~ A and M polysaccharides. Furthermore, baseline resonances which are partic~Jlarly easy to verify in the relatively simple NMR spectra of A antigen are consistent with the presence of monosaccharides characteristic of LPS core. This conclusion was supported by subsequent component analysis for the indiv.;dtal monosaccharides. Acid hydrolysis of the NH-A and NH-M samples liberated component monosaccharides, which were analysed as their alditol acetates by GLC-mass spectrometry. The results of this analysis showed the presence of 2 mono~lccharides which are indicative of the presence of LPS inner core in NH preparations. Thus, quinovosamine, mannose and 2-keto-3-deoxy-D-octulosonic acid are reliable markers of the inner core region, especially when they are detected simultaneously and in quantitative agr~ment with their presence in purified O polysaccharide derived from LPS by mild acid hydrolysis. Each monosaccharide was identified on the basis of retention dine compared to authentic samples and by characteristic mass-fragmentation patterns. These conclusion regarding the presertce of monc-saccharides which can only derive from the LPS inner core component of S-LPS require that native hapten preparations are O polysaccharides attached to core oligosaccharide. Since the S-LPS molecule is biosynthesized from the lipid A end of the LPS , molecule, and preassemhled O polysaccharide is transferred to the core lipid A assembly [211, NH preparations mud derive from hydrolytic cleavage of S-LPS. Therefore, the nomenclature which suggests that. this NH polysaccharid¢ is somehow distinct, from A and M antigens should he corrected to indicate that the preparations previously referred to as NH are, in fact, artifacts of the extraction procedures. In this regard, we have consisles~tly failed to observe the presence of true

NH in any LPS worked-up by the phenol/water procedure. Con~uently, the conclusions drawn here are in agreementwith such unpublished observations (M.B. Perry, unpublished results). It is now possible to ~ previously proposed interpretations of the serological activity of so-called NH preparations. Since these are now seen to be identical to either the A or M antigen, suggestions that the antibodies recognizing ~ NH ~, are distinct from those which bind the O p o l y s a c c ~ should be reevaluated. It is most likely that the distinction between S * - L ~ a t e d A or M epitopes and those of the lipid-A-free polysaccharides are related m functional affinities (avidity) which derive from both the physical state of the antigen, ~aggregales,,, and the stage of antibody maturation. Thus, infected animals may be considered to be exposed to a constantly immunizing dose of antigen and pro~ duce antibodies with a h i ~ avidity than those produced at an early stage of infection or immunization.

BRUCELLA A A N D M N A T I V E H A P T E N S T R U C T U R E

431

The conclusions reached here are consistent with the contention thai polysaccharide B is a serologically i~active cyclic glucan [3, 4]. Its activity derives from O polysaccharide liberated from LPS during the autoclaving pro. cedure and co-extracted with the glucan. Discrimination of vaccinated from infected cattle may nevertheless be accomplished by lhe published procedures [12, 16, 18], although the active antigen is O poly~ccharide, previously referred to as NH [20]. Mixtures of the 2 polysaccharides may be resolved by HPLC or G-50 gel fihralion. In order to avoid the perpetuation of the confusing nomenclature associated with these historical interpretations of Brucella polysaccharide serology, we propose that the terms NH and polysaccharide B be abandoned. The only serologically significant polysaccharide antigens of Brucello are the A and M lipopolysaccharides or their lipid-A-free form, the O polysaccharide.

LEs HAPTI~NES NATIF$ PURIF|[S A PARTIR DE Bruc~LLa a~oRrus BI9 ~t DE B. meure~s~s 16M RI~,VELE~T L'ORIG!~qELIPOPOL¥OSIDIQ!dE DE CE$ ~TIGI~NES

La purification du polyoside de Brucella appel~ hapt~ne natif (HN), est effectuate par ultrafiltration apr~s autoclavage des cellules suivie d'une chromatographie d'exclusion par HPLC sur colonne TSK-G 2000-SW. Le HN purifi~ a t~t~analyse par ~lectrophor~¢ en gel de polyac~amide en pr~-sce de sodium dodecyl sulfate, par chromatographic gaz-liquide, par spectrometric de masse el r~onnance magn~tique nucl~ire. La structure du HN de B. aborrus Bl9 est identique ~t celle du polyoside A de B. aborlus 1119-3. C'est un homopolym~e iin~tire constitu~ de r~idus 4,6-did~,oxy~4.forma. mido-D*mannopyrannosyl lib en ~-1,2. La structure du HN de B. meliten_~is 16M est un homopolym~re du mihne sucre mais constitu~ d'une unit~ r~bl~irive pentaosidique dans laquelle 4 r~sidus de 4,6-did~oxy-4-formamido.Dmannopyrannosyl li~s en e=-l,2 son! | ~ au dernier monoside en ~-1,3. Cette structure est identique ~ celle du polyoside M de B. meli~ensis t6M. Dans le HN purifi~, la pt~ence de monosides charact~stiques de la partie regtteuse du lipopolyoside, e.g. quinovosamine, mannose et KDO, indique que c~ polyoside provient du lipopolyoside. Le HN est lib~r~ par .hydrolyse au coats de i'extraction. Les antig~'nes portant !es sp~cif'tcit~s A ~ M sont tes principaux polyosides antigAhtiques des Bncce//u. Le po!yoside B ¢s! un glycanne ~clique n'ayant aucune relation ~ructura~e et ~'rolosjque avec ~ polyosides A el M. Son activit~ clans les tests de diagno~ic r~ulte d'une contamination des extraits par la chaine O polyosidique. Mol:~'=~: Bruce//a, Hapt~r¢, LPS; Purifkation, Suxtcture, A n t ~ ~ , Hapt~ne natiL

432

M.S. Z Y G M U N T A N D COLL.

REFERENCES [1] ALTON, J.C., JONES, L.M. & PIETZ, D.E., in Laboratory techniques in brucellosis 2rid Ed. W.H.O. Monogr. Ser. No. 55. [2] BUNDLE,D.R., CHERWONOGRODZKY,J.W., CAROFF,M. & PERRY, M.B., The lipopolysaccharides of Brucella abortus and B. melitensis. Ann. Inst. Pasteur/Microbiol., 1987, 138, 92-98. [3] BUNDLE,D.R., CHERWONC~aRODZKY,J.W. & PERRY,M.B., Characterization of Brucella polysaccharide B. Infect. Immun., 1988, 56, 1101-1106. [4] BUNDLE,D.R., CHERWONOGRODZKY,J.W. & PERRY,M.B., The structure of the lipopolysaccharide O-chain (M antigen) and polysaccharide B produced by Brucella melitensis 16M. FEBS Letters, 1987, 216, 261-264. [5] BUNDLE,D.R., CHERWONOGRODZKY,J.W. & PERRY, M.B., Structural elucidation of the Brucella melitensis M antigen by high resolution NMR at 500 MHz. Biochemistry, 1987, 26, 8717-8726. [6] BUNDLE,D.R., GIDNEY,M.A.J., PERRY,M.B., DUNCAN,J.R. & CHERWONOGRODZKY, J.W., Serological confirmation of Brucella abortus and Yersinia enterocolitica 0:9 O-antigens by monoclonal antibodies. Infect. Immun., 1984, 46, 389-393. [7] BUNDLE,D.R. & PEARY,M.B., Structure and serology of the Brucella abortus O-antigen. Biochem. Soc. Trans., 1985, 13, 980-982. [8] CAROFF,M., BUNDLE,D.R. & PERRY, M.B., Structure of the O-chain of the phenol-phase soluble cellular lipopolysaccharide of Yersinia enterocolitica serotype 0:9. Europ. J. Biochem., 1984, 139, 195-200. [9] CAROFF,M., BUNDLE,D.R., PERRY,M.B., CHERWONOGRODZKY,J.W. & DUNCAN, J.R., Antigenic S-type iipopolysaccharide of Brucella abortus 1119-3. Infect. lmmun., 1984, 46, 384-388. [10] CHERWONOGRODZKY,J.W., BUNDLE,D.R. & PEggY, M.B., Identification of the A and M antigens of Brucella as the O polysaccharide of smooth lipopolysaccharides. Canad. J. Microbiol., 1987, 33, 979-981. [11] DIAz, R., JONES,L.M., LEONG,D. & WILSON,J.B., Surface antigens of smooth brucellae. J. Bact., 1968, 96, 893-901. [12] DIAZ, R., GARErA,P., JONES, L.M. & MORIYON,I., Radial immunodiffusion test with a Brucella polysaccharide antigen for differentiating infected from vaccinated cattle. J. clin. Microbiol., 1979, 10, 37-41. [13] DIAZ,R., ToYos, J., SALVO,M.D. & PARDO,M.L., A simple method for the extraction of polysaccharide B from Brucella cells for use in the radial immunodiffusion test diagnosis of bovine brucellosis. Ann. Rech. Vet., 1981, 12, 35-39. [14] DUeRAY,G. & BEZARD,G., A highly sensitive periodic acid-silver stain for 1,2-diol groups of glycoproteins and polysaccharides in polyacrylamide gels. Analyt. Biochem., 1982, 119, 325-329. [IS] DURRAY,G. & LIMET,J.N., Evidence of heterogeneity of lipopolysaccharides among Brucella biovars in relation to A and M specificities. Ann. Inst. Pasteur/MicrobioL, 1987, 138, 27-37. [16] FERNANDEZ-LAoo,L. & Dt~x, R., Demonstration of antibodies against Brucella melitensis 16M lipopolysaccharide and nattve hapten in human sera by enzyme-hnked immunosorbent assay. J. clin. Microbiol.~ 1986, 24, 76-80~ [17] FEaN^ND~-LAoo,L., MORIYON,l., TOYOS,J. & DtAz, R., Inununological identity of Brucella native hapten, polysaccharide B, and Yersinia enterocolitica serotype 9 native hapten, infect. Immun., 1982, 38, 778-780. [18] JONES,L.M., BERM~, D.T., MoxI~o, E., pEYO.E,B.L., GILOOIU~,M.J., Hung, J.D. & NICOLErn, P., Evaluation of radial immunodiffusion test with polysaccharide B antiRen for the dmgnosis of bovine brucellosis. J. clin. Microblol., 1980, 12, 753-760.

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[19] MORENO,E., MAYER,H. & MORIYON,I., Characterization of a native polysac-

charide hapten from B. melitensis. Infect. lmmun., 1987, 55, 2850-2853. [20] MORENO, E., SPETH, S.L., JONES, L.M. & BERMAN, D.T., lmmunochemical characterization of Brucella lipopolysaccharideand polysaccharides. Infect. lmmun., 1981, 31, 214-222. [21] NIKA!DO,H., Biosynthesis and assembly of lipopo!ysaccharide and the outer membrane layer of Gram-negative cell wall, in bacterial membranes and walls (L. Lieve). Marcel Dekker, New York, 1973. [22] VERGER,J.M., GRIMONT,F., GRIMONT, P.A.D. & GRAVON, M., Bruceila, a monospecific genus as shown by deoxyribonucleic acid hybridization. Int. J. syst. Bact., 1985, 35, 292-295. [23] ZYGMUNT,M.S. & DUBnAY,G., Preparation by uRrafiltration and control by high performance liquid chromatography of the native hapten of Bruceila abortus for use in radial immunodiffusion test. J. clin. Microbiol., 1987, 25, 1860-1863.